Review Intracytoplasmic injection of spermatozoa and spermatogenic cells: its biology and applications in humans and animals

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1 RBMOnline - Vol 10. No Reproductive BioMedicine Online; on web 21 December 2004 Review Intracytoplasmic injection of spermatozoa and spermatogenic cells: its biology and applications in humans and animals Dr Ryuzo Yanagimachi Dr Ryuzo (Yana) Yanagimachi received his PhD degree from the Hokkaido University, Sapporo, Japan. After post-doctoral years ( ) at the Worcester Foundation for Experimental Biology, Shrewsbury, Massachusetts with Dr MC Chang as his mentor, he returned to Japan before joining the University of Hawaii Medical School in 1966, where he has been Professor of Anatomy of Reproductive Biology. His current research interests include gamete biology, assisted fertilization and cloning. He is a member of the National Academy of Sciences (USA) and has received various awards including: Research Award (1982) and Carl G Hartman Award (1999) from the Society for Study of Reproduction, Marshall Medal from the Society for the Study of Fertility (1994), International Prize of Biology (1996) and Distinguished Andrologist Award from the American Society of Andrology (1998). Ryuzo Yanagimachi Institute for Biogenesis Research, University of Hawaii Medical School, Honolulu, Hawaii 96822, USA Correspondence: Tel: ; Fax: ; yana@hawaii.edu Abstract Intracytoplasmic sperm injection (ICSI) has become the method of choice to overcome male infertility when all other forms of assisted fertilization have failed. Animals in which ICSI has produced normal offspring include many species. Success rate with normal spermatozoa is well above 50% in the mouse but ICSI success rates in other animals have been low, ranging from 0.3 to 16.5%. Mouse ICSI revealed that spermatozoa that cannot participate in normal fertilization can produce normal offspring by ICSI, provided their nuclei are genomically intact. Human ICSI using infertile spermatozoa has been highly successful perhaps because of the intrinsic instability of human sperm plasma membrane. The health of children born after ICSI and other assisted fertilization techniques is of major concern. Careful analyses suggest that higher incidences of congenital malformations and/or low birth weights after assisted fertilization are largely attributable to parental genetic background and increased incidence of multiple births, rather than to the techniques of assisted fertilization. Since the physiological and nutritional environments of developing embryos may cause persisting alteration in DNA methylation, extreme caution must be exercised in handling gametes and embryos in vitro. In the mouse, round spermatid injection (ROSI) has been routinely successful but its use in humans is controversial. Whether human ROSI and assisted fertilization involving younger spermatogenic cells are medically safe must be the subject of further investigations. Keywords: fertilization, infertile, intracytoplasmic injection, spermatid, spermatocyte, spermatozoa Introduction The microsurgical injection of a single spermatozoon into an oocyte, commonly called intracytoplasmic sperm injection or ICSI, has become the method of choice for the treatment of severe male factor infertility and unexplained total failure of fertilization (Van Steirteghem et al., 1993). By means of ICSI, 60 90% (sometime 100%) of the oocytes selected for good morphology are fertilized. Today, thousands of childless couples who are unable to conceive after unsuccessful IVF attempts have a fairly good chance of having their own children by means of ICSI. Although ICSI will not and should not replace conventional methods of insemination for both animals and humans, it is the last method of insemination in the foreseeable future when all other methods of insemination fail. ICSI is an aggressive method of insemination. One must be fully aware of the differences between normal fertilization and ICSI and possible risks to the health of newly born children and of future generations. Here, the outline of normal fertilization in mammals will be first described, followed by discussion of the current status of ICSI and other forms of assisted fertilization in mice, human and several other species. Unsolved problems and the future of various technologies involved in assisted fertilization will then be discussed. 247

2 248 Normal fertilization in mammals: how female and male germ cells develop and prepare themselves for fertilization (see Figure 1). Female and male germ cells develop from the primordial germ cells (PGC) (A and A ) that are first identified in an extraembryonic location of very early embryos soon after implantation. Multiplying PGC migrate, in an amoeboid fashion, into the developing gonads where they develop into oogonia (B) in the female and to spermatogonia (B ) in the male. In the female, multiplying oogonia stop mitosis shortly before or soon after birth, depending on the species. Shortly after the last mitosis, oogonia (C) replicate DNA and enter a long-lasting prophase of the first meiotic division. Homologous chromosomes are quickly paired, and parts of paternal and maternal genes are exchanged before the oocyte enters a long-lasting dictylate stage when chromosomes are diffused into the nucleus. Oocytes with large nuclei (commonly called germinal vesicles) grow during this stage to acquire a great complexity of cytoplasmic organelles. The mid-cycle surge of the gonadotrophin LH from the anterior pituitary then triggers the resumption of meiosis, with oocytes attaining the metaphase of the second meiosis (D) being released from follicles in the processes of ovulation. Ovulated oocytes, each surrounded by thousands of cumulus cells, are quickly transported into the upper region of the oviducts where they meet spermatozoa. In some animals, oocytes become competent for fertilization only when both divisions are completed (e.g. sea urchins), whereas in mammals fertilization occurs at the metaphase of the second meiotic division. Experimentally, oocytes in earlier stages can be penetrated by spermatozoa, but they are unable to respond and remain unactivated (Usui and Yanagimachi, 1976). Only after reaching the metaphase of the second meiosis do they become fully responsive to spermatozoa (Ducibella and Buetow, 1994; Shiraishi et al., 1995). According to a recent study by Johnson et al. (2004), adult mouse ovaries contain oogonia-like cells capable of initiating meiosis. Whether they produce functional oocytes remain to be determined. In males, the precursor of spermatogonia, i.e. the gonocytes, migrate to the basement membrane of seminiferous tubules to become spermatogonia (B ). In adults, spermatogonia retain their own numbers and cyclically produce the primary spermatocytes (C ) that undergo meiosis to produce secondary spermatocytes (D ), then haploid round spermatids (E ). Transformation of the spermatids into flagellated spermatozoa is called spermiogenesis. In young men, about of spermatozoa are produced in the testis each day (Johnson, 1982). Many germ cells degrade during spermatogenesis and meiosis. This may be a mechanism to limit germ cells to the number that can be sustained by the available Sertoli cells, rather than due to the elimination of chromosomally abnormal spermatogenic cells (Johnson, 1986, 1990). Spermatozoa leaving the testes are immotile or only weakly motile and are not ready for normal fertilization. They gain the ability to move actively while passing through the epididymis. Physical and biochemical changes of spermatozoa during transition through the epididymis are collectively called maturation. This includes alterations in the composition of plasma membrane glycoproteins and sterols (Eddy and O Brien, 1994; Yanagimachi, 1994) and extensive cross-linkings of nuclear protamines by disulfide bonds (Bedford and Calvin, 1974; Bedford and Hoskins, 1990; Yanagimachi, 1994). The luminal environment of the epididymis protects maturing spermatozoa from oxidative stress (Hinton et al., 1996). Some epididymal proteins integrated into the sperm plasma membrane may have piv otal roles in later sperm oocyte interactions (e.g. Cohen et al., 2000). At ejaculation, some seminal plasma components adsorbed onto the sperm plasma membrane (Yanagimachi, 1994) may regulate the behaviour of spermatozoa at fertilization (Fraser and Adeoya-Osiguwa, 2001). Spermatozoa become competent for fertilization after spending some time in the female genital tract. This can be achieved in vitro under proper conditions. Physiological changes that make spermatozoa capable of interacting with oocytes are called capacitation. This process includes molecular changes in the sperm plasma membrane and in the components of signal transduction (Visconti et al., 1998; Kopf et al., 1999) that enable spermatozoa to undergo hyperactivated motility and the acrosome reaction (Bedford, 1983; Yanagimachi, 1994). The acrosome reaction normally takes place immediately before or after contact with the zona pellucida, and makes spermatozoa capable of penetrating the zona pellucida and fusing with the oocyte s plasma membrane (Yanagimachi, 1994). Sperm oocyte fusion triggers an abrupt release of Ca 2+ from the oocyte s endoplasmic reticulum (ER). Ca 2+ re-enters ER, then is released again at more or less regular intervals for several hours (Miyazaki et al., 1993). Perhaps, the first intracellular Ca 2+ rise triggers the resumption of the meiosis of oocyte as well as the exocytosis of cortical granules (Schultz and Kopf, 1995; Abbott and Ducibella, 2001), which renders the zona pellucida impenetrable to excess spermatozoa. Whether repeated intracellular Ca 2+ rises are essential for normal embryonic development remains to be determined. All structural and biochemical changes invoked in oocytes by the fertilizing spermatozoon are collectively called activation. It is still a matter of debate whether activation is triggered by interactions between the surface ligands on spermatozoa and the surface receptors on oocytes (Foltz, 1995; Schultz and Kopf, 1995), or through the introduction of a sperm-born oocyte-activating factor (SOAF). SOAF is relatively non-species specific (Homa and Swann, 1994; Sasagawa et al., 1997; Kimura et al., 1998b; Perry et al., 1999b; Sakurai et al., 1999; Yazawa et al., 2000). Its localization (Kimura et al., 1998b; Perry et al., 1999b; Knott et al., 2003) and chemical nature are still the subject of debates. The most promising candidate for SOAF is thus far phospholipase C zeta (Parrington et al., 1998; Swann and Parrington, 1999; Parrington, 2001; Swann et al., 2004). During activation, the oocyte nucleus completes meiosis and transforms into a female pronucleus. Meantime, the compact sperm nucleus decondenses (E) and transform into a male pronucleus. DNA synthesis occurs during the mid-stage of pronuclear development. In most species, sperm centrosome that entered the oocyte forms a microtubular aster to bring the male and female pronuclei to close apposition, commonly at the centre of the egg (Schatten, 1994). The union of male and female pronuclei and mingling of their genomes (syngamy) (F) completes the processes of fertilization and signals the beginning of life of a new individual. For details of the processes and mechanisms of normal fertilization, readers are

3 Figure 1. Production of germ cells, fertilization and the initiation of embryo development. Both female and male germ cells originate from primordial germ cells (A and A ). They and their descendants (oogonia B and spermatogonia B ) multiply by mitosis. Oogonia and spermatogonia that have stopped mitosis enter the prophase of the first meiotic division (C and C ). Although fully mature female and male germ cells differ considerably in their structures, they are genetically equivalent. While male germ cells become fertilization-competent after two meiotic divisions and extensive structural and physiological modifications, female germ cells become fertilization-competent during the progression to the second meiotic division (D). Meiosis in female germ cells completes after sperm entry (E). An oocyte that completes two meiotic divisions should be called an ootid, but this term is seldom used in literature. Mingling of female and male genomes (F) is the end of fertilization and the beginning of a new individual. For more details, see text. 249

4 250 referred to reviews (e.g. Yanagimachi, 1994; Hardy, 2002; Florman and Ducibella, 2005). Figure 2 presents a diagram of the behaviour of the spermatozoon (more specifically, sperm head) before and after entry into the oocyte. Note that the sperm plasma membrane over the acrosomal cap, the outer acrosomal membrane, and the whole contents of the acrosomal cap are shed from the spermatozoon during the acrosome reaction. Plasma membrane remaining in the spermatozoon after the acrosome reaction mingles with the oocyte s plasma membrane to become a mosaic plasma membrane of the zygote. Intracellular components of the spermatozoon entering the oocyte include the nucleus and its envelope, perinuclear materials (theca), inner acrosomal membrane, mitochondria, proximal centrosome, axoneme complex, dense fibre and fibrous sheath. In the hamster and perhaps in common laboratory rodents, the nucleus is the only sperm component that survives after fertilization. All other components are destroyed sooner or later by multivesicular bodies, lysosomes (Hiraoka and Hirao, 1988) and ubiquitin-dependent proteasomes (Shitara et al., 2000; Sutovsky et al., 2003a,b). In other animals including humans, both the nucleus and centrosome survive after fertilization. An obvious, important difference arises between ICSI and normal fertilization. During ICSI, the entire spermatozoon is introduced into the ooplasm including the acrosome and sperm plasma membrane, whereas these never enter the oocyte during normal fertilization. ICSI certainly imposes extra work on the oocyte to handle these exotic materials. Early history of ICSI What happens if a spermatozoon or spermatozoa is/are injected into an egg? This simple question was asked by GL Kite in the beginning of the last century who injected 2 3 or >20 starfish spermatozoa into an egg of the same species. To his surprise, nothing happened. Citing Kite s unpublished work, Lillie (1914) inferred that normal penetration is necessary for fertilization if the spermatozoon is to be effective. Similar experiments were performed by Hiramoto (1962), who injected several live sea urchin spermatozoa into sea urchin eggs. Again, nothing happened. Some spermatozoa kept moving spasmodically for hours within the egg. Some hit the egg nucleus, but there was no sign of egg activation. When sperm-injected eggs were inseminated from outside, however, all were activated and underwent polyspermic cleavages. Hiramoto concluded that injected spermatozoa participate in the mitotic process only when the egg cytoplasm is activated by the fertilizing spermatozoon. Brun (1974) was the first to produce live offspring by sperm injection. He collected motile spermatozoa from frog testes and injected them individually into 562 oocytes. Four (0.7%) developed into normal, diploid metamorphosed frogs. Although the success rate was rather low, this clearly demonstrated that testicular spermatozoa had the ability to initiate and participate in embryo development. Uehara and Yanagimachi (1976, 1977) were the first to inject mammalian spermatozoa into oocytes. Using hamsters they homogenized an epididymal sperm suspension, and injected individual sperm heads (without acrosomes) into mature oocytes. Two to 3 h later, most oocytes contained both sperm (male) and egg (female) pronuclei. Testicular sperm heads also transformed into well-developed pronuclei after injection into oocytes. To their surprise, freeze-dried human sperm heads developed into pronuclei within hamster oocytes. Subsequently, other investigators reported that sperm heads (nuclei) of other species (e.g. the mouse, rat, baboon, rabbit and bovine) were able to develop into pronuclei when injected into the oocytes of homologous and heterogonous species (Thadani, 1980; Markert, 1983; Shaikh et al., 1984; Keefer and Brackett, 1987). Later, Iritani et al. (1989) and Goto et al. (1990) obtained live offspring (of the rabbit and cattle) by ICSI. While Iritani et al. used live spermatozoa from the cauda epididymis, Goto et al. used frozen thawed (killed) ejaculated spermatozoa. After a pioneering work by Lanzendorf et al. (1988), Palermo et al. (1992) succeeded in producing healthy human babies by ICSI. As of today the species in which live offspring were obtained by ICSI include mouse, hamster, rat, rabbit, cat, sheep, pig, cattle, horse, monkey and human. Here, ICSI in the mouse and human will be discussed first, followed by ICSI in other animals. Mouse ICSI History Mouse ICSI was first attempted by Markert (1983) who injected mature spermatozoa into (i) immature oocytes with germinal vesicle (GV), (ii) mature unfertilized oocytes and (iii) fertilized eggs with decondensing sperm heads or pronuclei. Injected sperm heads decondensed in all cases except when sperm heads were injected into fertilized eggs with pronuclei. According to Markert, some of the sperminjected GV oocytes developed into blastocysts, but as of today no one else confirmed this. The birth of live mouse offspring after ICSI was reported in 1995 by three research groups. Ahmadi et al. (1995) and Lacham-Kaplan and Trounson (1995) used the conventional injection pipette (about 10 µm in outer diameter) with its tip beveled at Kimura and Yanagimachi (1995a), on the other hand, used a blunt-ended pipette attached to a Piezo-electric actuator. This actuator can drive the injection pipette a very short distance (e.g. 0.5 µm) and at a very high speed. Oocyte survival rate after ICSI using the pipette attached to this actuator is excellent. Even though the piezo-actuator is not essential for mouse ICSI (Suzuki and Yanagimachi, 1997), the laboratory uses it routinely because (i) the preparation of injection pipettes is easy and (ii) the zona pellucida and plasma membrane of the oocyte are readily pierced by the pipette. This apparatus can be used for injection of spermatids, spermatocytes, ES cells and even adult somatic cells. The stepby-step instruction of mouse ICSI using the Piezo-electric actuator is in a chapter of the technical manual Manipulating Mouse Embryos edited by Nagy et al. (2003). ICSI using mature spermatozoa Even though ICSI can be applied to any strains and mutants of the mouse, it is expected that the oocytes of hybrid mice in general are more tolerant to micromanipulation (Markert, 1983) and develop better to live offspring (Kawase et al., 2001; Szczygiel et al. 2002) than those of inbred mice. Fully mature spermatozoa from the cauda epididymis have been used commonly for mouse ICSI, but testicular spermatozoa can also be used (Kimura and Yanagimachi 1995b). The

5 Figure 2. Diagrams showing normal processes of sperm entry in the oocyte. Longitudinal sections of the anterior part of the fertilizing spermatozoon are shown here. (A) Sperm head before the acrosome reaction: acp, the principal (acrosomal cap) region of acrosome; eq, the equatorial region of acrosome. (B C) The acrosome-reacted spermatozoon passes through the zona pellucida (zp) and the perivitelline space (pvs). (D E) Membrane fusion begins between sperm plasma membrane over the equatorial segment (eq) of acrosome and oocyte plasma membrane. Cortical granules (cg) are quickly exocytosed to modify both the oocyte surface and the zone pellucida to prevent polyspermic fertilization. (F G) The anterior portion of sperm head is engulfed into the oocyte in a phagocytic fashion, while the rest of the sperm head and tail enter the oocyte by plasma membrane fusion. Note that the contents of acrosomal cap never enter the oocyte. Sperm plasma membrane also does enter the oocyte; it mingles with the oocyte plasma membrane to become a mosaic plasma membrane of the zygote. (H) The inner acrosomal membrane (iam) separates from the decondensing sperm nucleus. Endoplasmic reticulum (er) gradually form the envelope of the male pronucleus. (B G) from Yanagimachi (1988), with permission of Academic Press. 251

6 252 efficiency of ICSI may vary from laboratory to laboratory, largely dependent on the skill of investigators. Typically in the laboratory, % of oocytes survive ICSI. The majority (90 95%) of survived oocytes are fertilized normally to become 2-cell embryos. Transfer of 2-cell embryos to surrogate mother results in the birth of normal pups, with overall success rates (the proportion of ICSI oocytes that develop into live offspring) reaching 50 60% (Yamazaki et al., 2001; Kishigami et al., 2004). ICSI is far more efficient than artificial insemination (AI) or IVF when only very few live spermatozoa are available for insemination. Hundreds to millions of spermatozoa are needed to fertilize one oocyte by AI or IVF, while only one good spermatozoon is needed for ICSI to fertilize each oocyte. Differences between normal fertilization and ICSI During normal fertilization, the sperm plasma membrane and contents of acrosome never enter the oocyte (Figure 2). During ICSI, in contrast, the entire spermatozoon (Figure 3) plus a small amount of extracellular medium are deposited in an oocyte. Obviously, ICSI oocytes must do extra work that normally fertilized oocytes do not need to do. This includes the elimination of (i) sperm plasma and acrosomal membranes, (ii) various macromolecules added onto the sperm plasma membrane during epididymal maturation and ejaculation, (iii) acrosomal contents with a spectrum of hydrolysing enzymes and glycoproteins, and (iv) medium components co-injected with spermatozoa during ICSI (such as polyvinyl pyrrolidone which makes spermatozoa within the injection pipette more maneuverable). Therefore, it is quite remarkable that ICSI oocytes survive the operation and develop into live offspring. It is also somewhat astonishing that the pattern of intracellular Ca 2+ oscillations following sperm injection is quite different from that after normal fertilization (Figure 4), yet ICSI oocytes, if not all, develop into apparently normal offspring. Importance of sperm immobilization prior to ICSI It is recommended to immobilize or kill the spermatozoon immediately before ICSI. When a live spermatozoon is placed inside the oocyte, it may keep moving for 30 min or longer before it stops. Until its plasma membrane disintegrates, the contents of the spermatozoon (including the nucleus and perinuclear materials) will never make direct contact with the oocyte s cytoplasm. For this reason, prior destruction or removal of the membrane is recommended to obtain better results in ICSI. Membrane disruption is commonly achieved by (i) scoring sperm tail against the glass (dish or pipette) surface (Kimura and Yanagimachi, 1995a) or (ii) separating the head from the tail by sonication or by the application of a few Piezo pulses to the neck tail junction of the spermatozoon (Kuretake et al., 1996a). Theoretically, complete removal of the sperm plasma membrane (and the acrosome) is preferable for ICSI, but the injection of a spermatozoon with locally disrupted plasma membrane is acceptable. The membrane of a killed spermatozoon will disintegrate gradually within the oocyte (Figure 3a). Membrane disruption (sperm killing) should be performed immediately before injection. Once the sperm plasma membrane is disrupted or removed in ordinary media, sperm nuclei will be exposed directly to medium components to invoke a rapid deterioration of the sperm nucleus. Thus, it is very important to minimize the time interval between the disruption of the sperm plasma membrane disruption and sperm injection into oocytes. Sperm components necessary for successful ICSI At least for the mouse and perhaps all other common laboratory rodents, the sperm head is the only component needed for successful ICSI. The sperm head separated from the tail by sonication in the presence of detergent Triton X-100 has nothing but the nucleus with the surrounding perinuclear materials (Kuretake et al., 1996a). This naked sperm head is fully capable of activating oocytes, and activated oocytes develop into healthy offspring. Perinuclear materials contain oocyte-activating proteins (Kimura et al., 1998a; Sutovsky et al., 2003a). If the perinuclear materials are defective or removed prior to ICSI, the oocytes need to be activated artificially by physical or chemical agents. The mouse and other common laboratory rodents are unique in that unlike most other mammals, they do not require sperm centrosome for fertilization. Centrosomes of rodent spermatozoa degenerate within the oocyte s cytoplasm. Numerous microtubule-organizing centres, which are scattered throughout the cytoplasm, construct a microtubule network to bring sperm (male) and oocyte (female) pronuclei to the centre of zygote (Maro, 1986; Schatten et al., 1986). In most other species, the sperm centrosome plays the central role in the formation of the microtubule network (sperm aster), which is necessary for the union of male and female pronuclei (Schatten, 1994). ICSI using spermatozoa with abnormal head morphology Not all spermatozoa in the ejaculate are normal. Some are abnormal in their motility, structure or both. Some strains of mice have a high incidence of abnormal spermatozoa. In BALB/c mice, for example, 60 80% of mature spermatozoa have deformed heads (Figure 5). Spermatozoa with highly deformed heads are unable to cross the zona pellucida and are therefore infertile. When injected into oocytes, however, these spermatozoa are able to participate in normal fertilization and embryonic development. Male offspring thus produced are all fertile (Burruel et al., 1996). Although the incidence of chromosomal abnormality is higher among deformed spermatozoa than among structurally normal spermatozoa, >50% of the spermatozoa with structurally abnormal heads are karyologically normal (Kishikawa et al., 1999a). Male mice homozygous for the azh gene are infertile. A large proportion of azh/azh spermatozoa are structurally abnormal (in the head, tail or both). The spermatozoa with highly deformed heads are not expected to fertilize oocytes by conventional IVF, but when injected into oocytes of normal female (+/+), they produced fertile offspring (azh/+) in which >74% of spermatozoa had normal head morphology (Akutsu et al., 2001).

7 Figure 3. Electron micrographs of mouse spermatozoa injected into oocytes. A single epididymal spermatozoon was sucked into an injection pipette and immobilized by applying a few piezo-pulses to the midpiece region before injection into each oocyte. (A) Thirty minutes after ICSI; decondensation of sperm nucleus has begun; note that the entire acrosome is within the oocyte. (A ) A cross-section of sperm tail with the plasma membrane partially missing (arrow). (B) About 60 min after ICSI. Sperm nucleus has extensively decondensed. Acrosome (left) and midpiece mitochondria and axoneme (left) are seen. Figure 4. Comparison of repetitive intracellular calcium oscillations in normally fertilized (A) and ICSI-fertilized (B) mouse oocytes. (A) When zona-free oocytes are inseminated in vitro, the first Ca 2+ transient occurs about 10 min after insemination (Hehlman and Kline, 1994); the time interval between sperm oocyte fusion and the first Ca 2+ transient is estimated to be about 70 s (Lawrence et al., 1997). (B) During ICSI a large Ca 2+ rise occurs almost immediately, which is believed to be an artefact. The real Ca 2+ transient begins 30 min after ICSI (Sato et al., 1999). Redrawn from Helman and Kline (1994) and Sato et al. (1999). With permission of the Society for the Study of Reproduction (A) and Harcourt Publishers Ltd (B). 253

8 Figure 5. BALB/c spermatozoa. This strain of mouse is commonly used for transgenesis and mutagenesis research, but it is one of the least fertile strains of the mouse. (A) Only about 25% of their spermatozoa have normal head morphology. (B and C) Others have various head deformities and are infertile. (D) ICSI using spermatozoa like B and C can produce normal fertile offspring. 254 ICSI using epididymal spermatozoa of infertile males Male mice carrying two t complementary haplotypes (t w5 /t w32 ) are totally infertile. Their spermatozoa have poor motility and fertilize neither in vivo nor in vitro (Olds, 1971; McGrath and Hillman, 1980). However, when injected into oocytes, they are able to participate in fertilization and normal embryonic development. Male offspring thus produced are fertile (Kuretake et al., 1996b). Some mutant and transgenic mice are infertile due to poor sperm production. Male mice homozygous for the qk gene mutation (qk/qk) are infertile. Only few highly deformed spermatozoa are seen in the epididymis. These spermatozoa, however, are able to produce offspring by ICSI (Yanagimachi et al., 2004). Some mutant or transgenic male mice are infertile. Their infertility may not be recognized for a long time, by which time the males may be too old or sick to mate or provide spermatozoa for IVF. In such cases ICSI may be a valuable alternative (Li et al., 2003). ICSI using preserved spermatozoa Spermatozoa in the epididymis do not die immediately after the death of the animal. When male mice were killed and kept at 4 C for 15 days, fewer than 10% of the spermatozoa from their cauda epididymides were motile. Although these spermatozoa fertilized only about 2% of oocytes by IVF, they could fertilize >50% of oocytes by ICSI, culminating in the birth of live offspring (Kishikawa et al., 1999b). Mouse spermatozoa cryopreserved in raffinose skim milk mixture fertilized oocytes more efficiently by ICSI than IVF (Szczygiel et al., 2002). Spermatozoa that had been snap frozen without cryoprotectants and those which were freeze-dried were all dead in the conventional sense (their plasma membranes were all broken), but they were able to produce live offspring by ICSI (Wakayama and Yanagimachi, 1998; Wakayama et al., 1998; Kusakabe et al., 2001; Kaneko et al., 2003a,b; Ward et al., 2003) (Figure 6). It was found that mouse spermatozoa stored in 80% ethanol for 1 day were able to produce live offspring by ICSI (Tateno et al., 1998). According to Cozzi at al. (2001), mouse spermatozoa heated at 60 C for 3 min are able to reduce live offspring by ICSI. More recently, Bhowmick et al. (2003) reported that mouse spermatozoa dried by blowing nitrogen at room temperature and stored at 4 C for 1 day produced mid-term fetuses after ICSI. These findings raise an interesting question as to the life and death of spermatozoa. Those spermatozoa with broken plasma membranes are obviously dead as cells, but they are able to produce normal offspring. In that sense, they must still be alive. Definitions of the life and death of spermatozoa are thus arbitrary. Importance of killing spermatozoa immediately before ICSI For ICSI, spermatozoa do not need to be alive. Killing spermatozoa immediately before injection is advisable for the reason already mentioned. However, it is important to know that sperm plasma membrane, unlike plasma membranes of most other cells, lacks the ability to heal due to the lack of

9 Figure 6. Freeze-dried mouse spermatozoa. (A) Glass ampoules with freeze-dried spermatozoa. Light (B) and electron (C and C ) micrographs of freeze-dried spermatozoa: heads and tails are often separated and sperm plasma membranes and acrosomes are missing or extensively damaged. (D) ICSI using such spermatozoa can produce fertile offspring. A C, with permission from Nature America, Inc. 255

10 256 underlying cytoplasm. Therefore, upon the disruption of the membrane, regardless of the size of disruption, Na +- and Ca 2+ - rich extracellular medium surges into the spermatozoon. This may activate endogenous nucleases, which cleave sperm DNAs. Although degradation of sperm nuclei is somewhat delayed in a K + -rich medium (Kuretake et al., 1996a) there are no media that keep the sperm genome (chromosomes) intact for many hours. Spermatozoa that have been freeze-dried or snap frozen without cryoprotection retain their chromosomes intact for many months (Ward et al., 2003), but once the spermatozoa are rehydrated or defrosted, their chromosomes start to degenerate because of the absence of intact sperm plasma membranes. It was thought that sonication is detrimental to sperm chromosomes (Martin et al., 1988), but it is the prolonged exposure of plasma membrane-disrupted spermatozoa to media, not sonication per se, that is detrimental to sperm chromosomes (Tateno et al., 2000). For ordinary mouse ICSI, a motile (live) spermatozoon is picked up, immobilized (killed) by breaking its neck or midpiece plasma membrane, and injected into an oocyte. As the mouse sperm tail is very long (about 120 µm), separation of the head from the tail and injection of only the head is recommended. It is important to select live spermatozoa for ICSI. Spermatozoa that are immotile when first examined may have been dead for just 1 min or 1 week. Since it is impossible to distinguish between these two, it is advisable to use only motile spermatozoa. As long as spermatozoa show signs of movement (even feeble, twitching movements), they are alive and have fairly good chances of producing live offspring by ICSI. Spermatozoa of some mutant mice are immotile, yet some of them (sometimes many) are alive. Live, immotile spermatozoa may be distinguished from dead ones by suspending them for min in a cell culture medium (e.g. TC 199 or IVF medium) containing 1% eosin Y. The heads of dead spermatozoa (with broken plasma membranes) are stained red, whereas those of live spermatozoa (with intact plasma membranes) remain unstained. Unstained spermatozoa can be used for ICSI after washing with eosinfree medium. ICSI using testicular spermatozoa of normal and infertile males Testicular spermatozoa of normal mice are as good as mature epididymal spermatozoa for ICSI. The majority of the oocytes injected with testicular sperm heads are activated and many develop into normal offspring (Kimura and Yanagimachi, 1995b). Testicular spermatozoa are better for ICSI than mature spermatozoa when they are unable to survive in the epididymis. Spermatozoa of male mice lacking genes for transition proteins die in the epididymis (Zhao et al., 2004). Those that are still in the testis are able to produce live offspring by ICSI (Zhao et al., 2004). Male mice with Steel mutation (SI/SId) are sterile even though they have a few spermatozoa in their testes. Kanatsu-Shinohara et al. (2002) produced one pup by ICSI using testicular spermatozoa of these mice. Mutant mice homozygous for the qk gene have deficient myelination in the central and peripheral nerve system and all males are sterile (Bennett et al., 1971). Testes contain only a few structurally abnormal spermatozoa that are nevertheless able to produce offspring after injection into oocytes of normal (wild-type) females (Yanagimachi et al., 2004). ICSI using spermatozoa developed in the testes or extra-gonadal sites of other male individuals In-vitro culture of spermatogonia to functional spermatozoa has been successful in fish (Miura et al., 1991; Sakai, 2002), but not in mammals. Mouse spermatogonia isolated from the testis of an individual and transplanted to the testis of another individual are able to develop into spermatozoa (Brinster and Zimmerman, 1994; Ogawa et al., 1997). Shinohara et al. (2002) transplanted small pieces of testis of 5 7 day old mice into the testes of immunodeficient mice without spermatogenic cells. Spermatozoa collected from these transplanted pieces 2 months later were able to produce normal offspring by ICSI. Honaramooz et al. (2002) transplanted a testis fragment of newborn mice under the skin of an immunodeficient mouse. Ten weeks later, fully developed spermatozoa were collected from the testis fragments. These spermatozoa produced normal fetuses by ICSI. The spermatozoa developed in such transplanted testes would never mature because they have no chance to enter the epididymis. ICSI is the only way to produce offspring until a way is found to induce in-vitro maturation of spermatozoa. Use of ICSI for infertile females CD9 is a plasma membrane-integrated protein associated with integrins and other membrane proteins. Male mice deficient in CD9 protein are completely fertile, while female counterparts are barely fertile. This is largely due to the failure of oocyte s plasma membrane to fuse with the fertilizing spermatozoon. ICSI circumvents this problem (Miyado et al., 2000). Use of ICSI for the examination of sperm and somatic cell chromosomes Since ICSI bypasses sperm oocyte fusion, oocytes of any species can be used to examine the chromosomes of spermatozoa of the same and other species. In fact, Lee et al. (1996a), Rybouchkin et al. (1997a), Araki et al. (1999) and Watanabe (2003) examined human sperm chromosomes after injection of human spermatozoa into mouse oocytes. Human and mouse chromosomes are easily distinguishable by their distinct morphology. According to Lee et al. (1996a), human spermatozoa with abnormal heads are not necessarily chromosomally abnormal; spermatozoa with normal head morphology are not necessarily chromosomally normal either, even though the incidence of chromosomally abnormal spermatozoa is higher among morphologically abnormal spermatozoa than normal ones. This is true for BALB/c mouse spermatozoa, which have a very high incidence of morphologically abnormal spermatozoa (Kishikawa et al., 1999a). There is no reason to believe that oocytes of species other than the mouse cannot be used for examination of sperm chromosomes of human and other species. In fact, Wakayama et al. (1997) described chromosomes of sea urchin spermatozoa after their injection into mouse oocytes. If the metaphase II chromosomes are removed from oocytes prior to ICSI, all chromosomes that appear during the first cleavage are of sperm origin. Using this approach, Osada et al. (2002) examined chromosomes of adult mouse neurons. The only

11 difference between spermatozoa and neurons is that the latter, unlike the former, cannot activate oocytes. Therefore, neuroninjected oocytes must be activated artificially. Tateno et al. (2003a) were able to examine bird erythrocyte chromosomes after injection of erythrocyte nuclei into mouse oocytes. Since sperm-borne oocyte-activating factors (SOAF) are not strictly species-specific, mouse oocytes can be used to assess biological activities of SOAF in spermatozoa and spermatogenic cells of the same and other species (Kimura et al., 1998b; Yazawa et al., 2000). ICSI-mediated transgenesis Transgenesis is most commonly achieved today by injection of a desirable gene into male pronuclei of zygotes, followed by the transfer of resulted embryos to surrogate mothers. Its efficiency (as determined by the proportion of transgenic offspring developed from all injected oocytes, is about at 3% at best. Some offspring show chimeric expression of transgenes, suggesting that transgenes are integrated both before and after the first cleavage of the zygotes. ICSImediated transgenesis can be achieved by injection of an unfertilized oocyte with a single spermatozoon (or an isolated sperm head) plus a gene of interest. In contrast to the pronuclear injection which uses a very small pipette (about 1 µm diameter). ICSI uses a much larger pipette (6 7 µm in diameter). The efficiency of ICSI-mediated transgenesis is up to 7% (Perry et al., 1999a) with few chimeric offspring. This suggests that in most cases a transgene of interest is integrated into the genome before the first cleavage. An advantage of ICSI-mediated transgenesis is that it allows the injection of high concentrations of a large transgene fragment (>170 kd) into oocytes without breaking it with a narrow pipette. Thus, laboratories wishing to perform transgenesis using large sized transgenes have found ICSI offers an alternative means of producing transgenic animals (Perry et al., 2001; Perry, 2002). Human ICSI Human ICSI was first reported by Lanzendorf et al. (1988) who obtained several pronuclear eggs following sperm injection into oocytes. It was a Belgian group that first reported the birth of healthy babies by ICSI (Palermo et al., 1992; Van Steirteghem et al., 1993). They washed the spermatozoa, sucked a single, motile spermatozoon into an injection pipette, one at a time, immobilized (killed) it by scraping its tail against the glass surface, and injected it into an oocyte. This simple procedure is still being widely used today. ICSI using morphologically normal and abnormal spermatozoa Theoretically, only one genomically normal spermatozoon and one normal oocyte are necessary to produce a healthy offspring. The presence of just a few weakly, twitching spermatozoa in semen is all that is required for ICSI (Silber et al., 1996). In fact, many babies were born following ICSI using extremely poor spermatozoa that failed to fertilize oocytes by conventional assisted fertilization technologies including IVF (Nagy et al., 1995; Svalander et al., 1995). Since structurally abnormal human spermatozoa do not necessarily contain an abnormal chromosome constitution (Lee et al., 1996a), it is not surprising that many normal babies were born after ICSI using deformed spermatozoa (De Vos et al., 2003). Examples include the birth of healthy babies after ICSI using round-headed (acrosome-less) spermatozoa (Lundin et al., 1995; Battaglia et al., 1997; Rybouchkin et al., 1997b; Khalili et al., 1998; Zeyneloglu et al., 2002), stump tail-spermatozoa (Stalf et al., 1995) and immotile spermatozoa of men with axonemal defects (Van Zumbusch et al., 1998; Okada et al., 1999a). Spermatozoa of some men are morphologically normal and highly motile, yet they are unable to fertilize any oocytes in vivo and even by IVF. This may be due to the inability of spermatozoa to undergo the acrosome reaction on the zona (Liu et al., 1995) or the presence of autoantisperm antibodies on sperm surfaces (Lahteenmaki et al., 1995; Clarke et al., 1997; Nagy et al., 1998a). ICSI overcomes these problems. Bartoov et al. (2003) compared fertilization and pregnancy rates after ICSI using spermatozoa selected for their good head morphology under the conventional (ca ) and the high power ( 6000) light microscopies. Although fertilization rates were about the same (about 60%) in both groups, the quality of developing preimplantation embryos and pregnancy rates were considerably higher when normallooking spermatozoa were used for ICSI after selection by higher power-microscopes as compared with their selection under conventional, low power microscope. Even though all structurally normal spermatozoa are not expected to be normal genomically, the incidence of genomically normal spermatozoa must be higher among normal-looking spermatozoa than among those with structural abnormalities (Lee et al., 1996a). Importance of the selection of live spermatozoa and their immobilization (killing) before ICSI Immotile spermatozoa are not necessarily dead, but under ordinary conditions they are most likely dead and therefore should not be used for ICSI. Regardless of the quality of motility, motile spermatozoa must have intact plasma membranes and their nuclei are likely to have undamaged DNAs. The selection of motile (live) spermatozoa is a key to successful ICSI. If the spermatozoa available for ICSI are all immotile, two methods can be used to discover if some are still alive. The first test of a living spermatozoon is the dye (e.g. 1% eosin Y) exclusion test. This is based on the principle that an intact plasma membrane does not allow the entry of dye into the cell, so the nuclei of live spermatozoa remain unstained (Dozortsev et al., 1995a; Barros et al., 1997a; Yanagida et al., 2001). The second test involves hypo-osmotic media and is based on the principle that an intact plasma membrane allows the entry of water from a hypotonic medium ( mosmol) into the cell such that the plasma membrane balloons and the tail axoneme curls within the swollen plasma membrane; in dead spermatozoa with broken/damaged plasma membranes, tails remain straight (Casper et al., 1996; Barros et al., 1997b; Tsai et al., 1997; Sallam et al., 2001). Thus, spermatozoa unstained by dye or with curled tail in hypotonic medium can be diagnosed alive. Spermatozoa from some men may be immotile due to low intracellular concentration of camp. Such spermatozoa may become motile by incubating them in media containing a plasma 257

12 258 membrane-permeable camp analogue or an agent like caffeine and pentoxyfelline (Tasdemir et al., 1998), which increases intracellular camp concentration by inhibiting phosphodiesterase. Babies were born after ICSI using such awakened spermatozoa (Terriou et al., 2000). ICSI works best when live spermatozoa are injected into oocytes immediately after immobilization ( killing ) (Dozortsev et al., 1995a; Gerris et al., 1995; Van den Bergh et al., 1995; Palermo et al., 1996). When a live spermatozoon is injected into an oocyte, it may keep moving inside the oocyte, sometimes for hours, before it stops. Its final arrest could mean that the sperm plasma membrane became damaged or broken at that moment. As long as the spermatozoon remains alive (its plasma membrane is intact), intracellular sperm components, including the nucleus, never mingle with oocyte cytoplasm, so the oocyte remains unactivated unless activated spontaneously by ageing. The most likely explanation of the beneficial effect of sperm immobilization (killing) is that sperm plasma membrane disintegrates much faster within the oocyte when the membrane is locally disrupted before ICSI than when it remains intact during ICSI. Kasai et al. (1999) studied how the presence or absence of sperm plasma membrane affects the onset of oocyte activation. They prepared human spermatozoa with intact or broken plasma membrane or no membranes at all. The plasma membrane was partially disrupted by application of a few piezo-electric pulses to the mid-region of the sperm tail. Both the plasma and acrosomal membranes were removed by treating spermatozoa with Triton X-100. Kasai et al. discovered that oocyte activation (resumption of meiosis and pronuclei formation) took place fastest when membrane-less spermatozoa were injected, followed by membrane-disrupted and membrane-intact spermatozoa. Takeuchi et al. (2004) reported that mechanically immobilized human spermatozoa possessed altered acrosomal regions such as a disrupted plasma membrane and even loss of the acrosome. Apparently, damage to the plasma membrane at the tail propagates quickly to the head region before and after injection into oocytes. Spermatozoa that were killed (or died) long before ICSI may be able to activate oocytes and initiate their development. However, the resulting embryos would not develop to term. Upon the disruption of sperm plasma membrane, it must be expected that sperm chromatin (DNAs) begins to degrade (Rybouchkin et al., 1997a). When this situation becomes irreparable, ICSI embryos are destined to die. Plasma membrane disruption is essential for successful ICSI, and its timing is of critical importance. Response of oocytes to injected spermatozoa During human ICSI the entire sperm components are injected into oocytes. Theoretically, sperm structures such as the plasma and acrosomal membranes and the contents of the acrosome should not be injected because these structures/materials never enter the oocyte under normal conditions. According to Lee et al. (1996b), oocytes injected with acrosome-reacted spermatozoa are fertilized and develop better than those injected with whole spermatozoa. This merits further investigations. Takeuchi et al. (2004) found that all human spermatozoa immobilized in a standard manner (scraping sperm tail) had a disrupted plasma membrane and/or acrosomes. No one knows how quickly human oocytes in vivo are activated by spermatozoa. When oocytes are injected with spermatozoa, the sperm nucleus begins to decondense about 30 min after ICSI. Oocyte activation, estimated by cortical granule exocytosis, begins between 60 and 90 min after ICSI, and male and female pronuclei become distinct after about 4 h (Bourgain et al., 1998). There is no difference between ejaculated and testicular spermatozoa with respect to the time of pronuclear formation (Nagy et al., 1998b). Although persisting acrosomal materials around the anterior portion of the sperm head after ICSI may slow down nuclear decondensation in this particular region (e.g. as reported in Rhesus monkey: Sutovsky et al., 1996), it does not seem to disturb the full development of the male (sperm) pronucleus (Bourgain et al., 1998) (Figure 7). The first distinct sign of oocyte activation in laboratory animals is a sudden increase in intracellular Ca 2+, which is repeated at regular intervals until the oocyte reaches the pronuclear stage (Jones et al., 1995). According to Tesarik et al. (1994) and Yanagida et al. (2001), there was a large Ca 2+ rise during insertion and withdrawal of the sperm-injection pipette. This must be a pseudo-ca 2+ rise due to the influx of extracellular Ca 2+ through the ruptured plasma membrane. The real Ca 2+ rise began in 18 ± 3 min after ICSI when spermatozoa were immobilized by mechanical squeezing. The intervals between ICSI and the first intracellular Ca 2+ must represent the time when the sperm-derived oocyte-activating factor is first exposed to the oocyte s cytoplasm after partial disintegration of the sperm plasma membrane. This notion is supported by the fact that human spermatozoa which have been completely freed from their plasma membranes by Triton X-100 treatment activate (mouse) oocytes faster than those immobilized by partial membrane-disruption, and much faster than those which have intact plasma membranes at ICSI (Kasai et al., 1999) (Figure 8). Causes of the failure of ICSI Some ICSI oocytes may remain unactivated. On rare occasions the majority or all oocytes are not activated. In some occasions, most activated oocytes may fail to develop beyond the pronuclear stage (Bergere et al., 1995; Dozortsev et al., 1995b,c; Flaherty et al., 1995a,b). Activation failure could be due to unsuccessful sperm injection, yet in many cases the spermatozoa were definitely placed inside the oocytes, which remained unactivated. In such oocytes, sperm nuclei remain condensed, partially decondensed or become prematurely condensed chromosomes without developing into a male pronucleus. As stated earlier, mammalian spermatozoa carry a factor that invokes oocyte activation. The failure of oocyte activation after ICSI may be due to the lack or deficiency of this sperm factor. Until the chemical nature of the human sperm factor is identified and its purified form becomes available for clinical use, ICSI-failed oocytes must be activated artificially. Electric stimulation and calcium ionophore have been used successfully to activate ICSI-failed oocytes, culminating in the birth of healthy offspring (Tesarik and Sousa, 1995; Yanagida

13 Figure 7. Pronuclei developed from human spermatozoa, 30 min (A) and 60 min (B) after ICSI. Remnant of the acrosome (arrows) still covers part of developing pronucleus at 30 min after ICSI. From Bourgain et al. (1998), with permission of Oxford University Press. Figure 8. Comparison of the speed of mouse oocyte activation after injection of human spermatozoa treated in different ways. (A) Triton treatment removed almost all plasma and other membranes from spermatozoa. (B) A single or few piezopulses applied to a sperm midpiece immobilized the spermatozoon instantly by damaging sperm plasma membrane of this region. (C) Intact spermatozoa denote the spermatozoa without any treatments; they were motile during ICSI. Oocytes that resumed the second meiotic division (from metaphase to anaphase/telophase) were recorded as activated. Tritontreated spermatozoa activated oocytes fastest, followed by immobilized and intact spermatozoa. 259

14 260 et al., 1999; Zhang et al., 1999; Eldar-Geva et al., 2003). ICSI failure in some cases could be due to defects in the sperm centrosome. The human sperm centrosome, located in the head and tail junction, plays a pivotal role in microtubule organization (aster formation) during normal fertilization (Simerly et al., 1995). Since defective centrosome and abnormal aster formation lead to abnormal fertilization (Asch et al., 1995), replacement of the defective centrosome with a normal one may rescue male infertility in certain instances (Van Blerkom and Davis, 1995). ICSI using epididymal and testicular spermatozoa Some men possess no spermatozoa in their semen. Others produce only dead spermatozoa in their semen. AI and IVF cannot help such men. Their fertility can be restored, however, if they have live spermatozoa in the testis and/or epididymis. The first pregnancies by injection of testicular and epididymal spermatozoa into oocytes were achieved by Schoysman et al. (1993) and Tournaye et al. (1994a,b). These spermatozoa were taken from men with the syndrome of congenital absence of vas deferens or failed vesectoepidymosectomy. The birth of many healthy babies following injection of testicular spermatozoa of azoospermic men (with or without obstructed vas deferens) was reported by Devroey et al. (1996) and De Croo et al. (2000). Risks of congenital malformation after ICSI using testicular, epididymal and ejaculated spermatozoa are about the same (Bonduelle et al., 2002; Ludwig and Katalinic, 2003). Causes of male infertility are not necessarily genetic Poor sperm production in the testis is not necessarily due to genetic causes. Here is an example in animals (Yanagimachi, unpublished experiments). Golden (Syrian) hamster males, at day 23 after birth (about the time of weaning), were anaesthetized and hemi-vasectomized by removing a large part of one vas deferens, leaving the vas deferens of the other side untouched. The testes and epididymides of the untouched side served as controls. When the males matured sexually (72 days after the operation), they were mated with normal females. Males proved to be fertile. They were killed and their testes and epididymides were fixed and histologically examined. Prior to fixation, spermatozoa, if any, were collected from the cauda epididymidis and examined for their motility in IVF medium. Testes in the control side all had active spermatogenesis (Figure 9A) and many spermatozoa were seen in the epididymis. Spermatozoa collected from fresh cauda epididymis displayed vigorous movement in IVF medium. The testes in the vasectomized side were almost always smaller (60 70% less in weight) than those of the control side. In 40% of the males, testes of the vasectomized side had local, limited spermatogenesis (Figure 9B). There were spermatozoa in the epididymis, but they were unable to move in IVF medium and were presumably all dead. In the remaining 60% males, spermatogenesis was largely disturbed (Figure 9C). Spermatogenic cells and spermatids free in the testicular lumen appeared to be in the process of degeneration. There were no spermatozoa in their epididymides. It should be noted that all the hemi-vasectomized males were fertile as they had functional testes and epididymides in one side of their bodies. Obviously, the cause of spermatogenetic problem in these animals is not genetic, endocrinological or immunological. A physical trauma must have affected the physiology of the testis and epididymis. It is possible that some fertile men have functional testis and epididymis on only one side of their body. The testis and epididymis of the other side may have become non-functional after infectious diseases and/or physical trauma, for instance. If both sides of the gonad and/or genital tract were damaged, men would surely be infertile. There is a tendency to assume that all infertile men have genetic problems, but many could be the victims of such non-genetic causes such as infectious diseases, physical trauma, food and drug poisoning, toxins, or environmental chemicals. In this case, there would be no medical problem in assisting such persons by ICSI and other assisted fertilization technologies. Fortunately, most genes affecting male infertility are recessive. As long as wives do not carry the same recessive genes, their offspring will be fertile. Chromosomal and genomic screening of couples will assist clinicians to estimate patients risks of having offspring with fertility problems, but screening may not benefit many couples as they would go ahead regardless of information given (Griffin et al., 2003). ICSI for men with chromosomal aberrations The frequency of men with somatic chromosome aberrations in the general population (i.e. males and females combined), as assessed by the examination of newborns after natural conception, is % (Nielsen and Wohlert, 1991; Jacobs et al., 1992). It is very high among oligospermic men (5 6%) and even higher among azoospermic men (12 15%) (Chandley, 1979; Van Assche et al., 1996; Schover et al. 1998; Cruger et al., 2003). According to Johnson (1998) and Tuerlings et al. (1998), % of all men attending ICSI programmes have sex chromosome aberrations, and % of all ICSI male candidates have autosomal aberrations. It is important to note that the large majority of infertile men, including azoospermic men, are chromosomally normal. It is also important to emphasize that women can be responsible for infertility. According to Gekas et al. (2001), the frequency of chromosomal aberrations in the couples attending their ICSI programmes was 6.1% for men and 4.8% for women respectively; figures reported by other clinics were (average 4.6%) for men and (average 4.8%) for women. Men with chromosomal aberrations may be identified by their physical appearance. Those with Klinefelter syndrome (47,XXY or 47XXY/46XY mosaic), for example, are characterized by small testis size. They are usually azoospermic, but some are oligozoospermic and even fertile. Using spermatozoa of oligospermic men with this syndrome, healthy male and female babies were produced by ICSI (Bourne et al., 1997; Cruger et al. 2001; Ulug et al., 2003; Komori et al., 2004). It is expected that their spermatozoa carry XX, XY, X or Y at about the same rates. Surprisingly, sperm chromosome analyses by FISH revealed that 93 97% of spermatozoa carried either X or Y chromosomes at a ratio of nearly 1:1 (Bourne et al., 1997). Perhaps, most spermatids

15 carrying XX, XY or no sex chromosome at all somehow do not develop into spermatozoa. Since there is still the possibility that chromosomally abnormal spermatozoa are injected using ICSI, preimplantation diagnosis of embryos must be performed. Men and women with Down syndrome (trisomy 21) are characterized by unique facial features and mental retardation. Females are fertile, but males are either infertile or have low fertility. Zuhlke et al. (1994) reported the birth of an unaffected girl after ICSI using spermatozoa of a man with this syndrome. The baby girl was brought up by her grandmother. Although both men and women with Down syndrome have a good chance of having normal children of their own, preimplantation diagnosis of their embryos should be performed to avoid the birth of children with Down syndrome for the sake of the children themselves, parents and their families. About 15% of azoospermic men and about 10% of severely oligospermic men carry mutations in the azoospermic factors (AZF) near the terminal end of the long arm of Y chromosome (Reijo et al., 1996). This region harbours genes necessary for spermatogonial multiplication, progression of meiosis and spermiogenesis (Vogt, 1995). Large deletions in the AZF region can be detected by regular karyotyping, whereas microdeletions are detected by molecular biology techniques alone. Of three major subregions (a,b,c) at the AZF locus, c is most frequently deleted in oligo- and azoospermic men (Oates et al., 2002). Men with deleted AZFc are healthy, but the use of their spermatozoa for ICSI results in the birth of boys without AZFc who, like their fathers, will grow up into infertile adults (Oates et al., 2002; Komori et al. 2002). A day may come when affected AZF regions are replaced with normal ones or a defective Y chromosome is replaced with a normal one from another individual. One should not be reluctant to perform such active therapies. Figure 9. Spermatogenesis in hemi-vasectomized hamsters. Hamster males shortly after weaning (22 25 days after birth) were hemi-vasectomized. Two months later, when males were sexually mature, the testes in the non-operated side had activated spermatogenesis (A), while those in the operated side had either reduced sperm production (B) or no sperm production at all (C). 261

16 262 ICSI using spermatozoa of men with single gene defects Table 1 lists some recessive and dominant genes affecting male fertility. Spermatozoa of men with Kartagener syndrome are either motile or immotile depending on the quantity of dynein arms (Eliasson et al., 1977; Kay and Irvine, 2000). A man with this syndrome fathered a healthy girl after ICSI (Cayan et al., 2001); another man fathered healthy twins, a girl and a boy (Van Zumbusch et al., 1998). Spermatozoa of men with polycystic kidney disease are immotile due to the absence of central axoneme (9 + 0 syndrome). A pregnancy was achieved by ICSI (Okada et al., 1999b), but the birth of a baby has not been reported. Much attention was paid to men with cystic fibrosis due to the gene mutation CFTR. Some men with this syndrome are fertile, while others are infertile (Van der Ven et al., 1996). Congenital absence of vas deference is a common feature among these infertile men. Spermatozoa collected from their testes, vasa efferentia or caput epididymis are morphologically normal (Asch et al., 1992). ICSI resulted in the birth of apparently healthy babies (Liu et al., 1994; Silber et al., 1995). Since CFTR gene is recessive, patients must be homozygous for this mutation to be affected, and mutant CFTR genes will be transmitted by these patients to the next generation. However, if their wives do not carry the CTFR mutation, none of their sons and daughters will be affected by this disease. If both husband and wife have CFTR mutation, up to 100% of their children will develop clinical cystic fibrosis and die young after a number of years of suffering. Thus, all infertile couples where men have idiopathic oligozoospermia or azoospermia should be offered genetic testing (In t Veld et al., 1997). For further reading on the genetic basis of male fertility and potential risks of assisted fertilization to the health and fertility of these patients offspring, readers are referred to: Vogt, 1995, 1997, 2004; Tuerlings et al., 1997; Meschede and Horst 1997; Wieacker and Jakubiczka, 1997; Patrizio and Broomfield, 1999; Szczygiel and Kurpise, 1999, 2001; Hargreave, 2000; Brugh et al., Incidence of children with congenital malformation born after assisted fertilization Since ICSI (and IVF) uses spermatozoa that normally do not participate in fertilization, medical safety of the future generation is the prime concern. Whether assisted fertilization increases the incidence of congenital malformation, low birth weight and neurological sequelae of children born as compared with natural birth has been controversial. This controversy seems to stem, in part, from the differences among investigators as to (i) the definition of major and minor malformations, (ii) the classification of male patients, and (iii) how control groups are chosen from the general population. According to Anthony et al. (2002), the incidence of children with one of more congenital malformations after IVF (3.2%) is slightly higher than that after natural conception (2.7%). Ludwig and Katalinic (2002) and Tournaye (2003) maintain that this is largely attributed to differences in maternal characteristics (such as the age of patients) and the genetic background of couples, and not to any aspects of the technological procedure itself. The risks of major congenital malformations in children born after ICSI is % (Schlegel, 1999; Tournaye, 2003). A major problem in both IVF and ICSI has been the substantial increase in multiple pregnancies (Wennerholm et al., 2000; Van Steirteghem et al., 2002a,b) which inevitably results in the competition between growing fetuses and low birth weights of many newborns. Until recently, twins, triplets and quadruplets constituted over 40% of all IVF and ICSI babies combined (Anthony et al., 2002; Bonduelle et al. 2002; Lambert, 2002; Nygren and Andersen, 2002). In the United States, for example, multiple births increased 5- fold since 1980, when assisted reproductive technologies started to become popular. It seems to be multiple pregnancy, not IVF/ICSI procedures themselves, that causes a slight, yet significant increase in birth defects and paediatric problems (Wennerholm et al., 2000). In many infertility clinics today, a single or two embryos are transferred to each mother (except for older mothers) to minimize the risk of multiple pregnancy (Lambert, 2002; De Sutter et al., 2003; Tiitinen et al., 2003) even though many IVF/ICSI mothers prefer twins as their first offspring (Pinborg et al., 2003). Why most babies born after ICSI are normal despite high frequencies of chromosomal/genomic abnormalities in gametes and zygotes It is rather surprising that 21 37% of human oocytes and 7 15% of human spermatozoa are chromosomally abnormal (Table 2). Meiotic errors responsible for chromosomal abnormalities of oocytes and spermatozoa increase with advancement of maternal and paternal age (Martin and Rademaker, 1987; Shi and Martin, 2000; Te Velde and Peason, 2000; Kuliev et al., 2003). Since the oocytes and spermatozoa with abnormal chromosomal/genomic constitutions are not discriminated against during fertilization (Almeida and Bolton, 1994; Meschede et al., 1995; Engel et al., 1996; Marchetti et al., 1999), one-third or even half of naturally fertilized human eggs and human early embryos could be chromosomally/genomically abnormal. High pregnancy loss even in fertile women has been known for many years (Hertig et al., 1959; Carr, 1971; Kerr, 1971; Edwards, 1986). An interesting study using mutant mice was reported by Gropp et al. (1983). When in-vivo development of trisomic mouse embryos was examined, all embryos except for trisomy 19 did not survive crucial phases of development; only embryos with trisomy 19 survived beyond birth (Figure 10). Such a stringent selection of embryos must be operating in humans as well. The data compiled in Table 3 support the view that most embryos and fetuses with major chromosomal and/or genomic abnormalities do not reach the prenatal stage of development (Wisanto et al., 1996). It is very likely that infertile men, in particular those who are attending ICSI programmes, have more chromosomally or genomically abnormal spermatozoa than fertile men (Martin, 1988; Moosani et al., 1995). However, as long as they have normal spermatozoa, even oligo-astheno-tetrazoospermic men, have chances to become fathers of normal children. Since parents and society must care for abnormal children over the rest of their lives, all

17 Table 1. Some gene mutations responsible for human male infertility a. Syndrome Phenotype Genotype, gene and location Kallman s Delayed puberty, small testis Recessive, KLA-1, chromosome Xp22.3 Androgen Testicular feminization Recessive, AR, chromosome Xq11 12 insensitivity Kartagener s Immotile sperm, bronchioectasis Recessive, p28, chromosome 1p35.1 Cystic fibrosis Respiratory infections, pancreatic insufficiency, Recessive, CFTR, chromosome 7q31.2 congenital absence of vas deferens Bardet Biedl s Obesity, mental retardation, hypogonadism Recessive, BBS2, BBS4, chromosome 16q2.1 Usher s Hearing loss, sperm axonemal defect Recessive, USH1A-E, chromosome, 11q13.5 Noonan s Short stature, congenital hearing problem, cryptorchidism Dominant, PTPN1, chromosome 12q22 Myotonic Muscle wasting, cataract,, testicular atrophy Dominant, chromosome 19q13.3 dystrophy Polycystic Multiple cysts in kidney, liver and epididymis Dominant, PKD1, PKD2, chromosome 16q13.3, and 4q disease a Cited from Patrizio and Broomfield (1999), Meschede and Horst (1997), Vogt (1995, 1997, 2004), Shah et al. (2003). Table 2. Incidence (%) of chromosomal abnormalities (aneuploidy, structural anomalies, polyploidy) in human gametes, zygotes, embryos, perinatal fetuses and newborns. Gametes, Frequency (%) of Investigators embryos, and chromosomal newborn abnormality Mature oocytes Zenzes et al. (1992); Kamiguchi et al. (1993); Plachot (2001); Pellestor et al. (2002) Mature 7 15 Kamiguchi et al. (1986); Martin et al. (1987); Plachot et al. (1988); Martin (1995); spermatozoa Guttenbach et al. (1997); Vidal et al. (2001); 15 versus 20 a Moosani et al. (1995) Zygotes 37 versus 87 b Pellestor (1995) Cleaving Plachot et al. (1987); Papadopoulos et al. (1989); Zenzes et al. (1992); Angell (1994) embryos 24 versus 83 b Edirisinghe et al. (1992) 34 versus 91 b Pellestor et al. (1995) 61 versus 52 c Munné et al. (1998) Perinatal and d Nielsen et al. (1991); Jacobs et al. (1992) newborn babies 2.5 e Bonduelle et al. (1998) 0.2 versus 2.1 c Bonduelle et al. (2002) 1.2 versus 1.2 g Wisanto et al. (1996) a Fertile men versus infertile men. b Good quality embryos versus poor quality embryos. c IVF embryos versus ICSI embryos. d Babies born after natural conception. e IVF babies unclassified. f ICSI babies of men > sperm per ml versus babies of man with < sperm per ml. g Inherited versus de-novo chromosomal abnormalities in ICSI babies. 263

18 Figure 10. Prenatal selection against monosomies and trisomies in the mouse. Embryos with monosomies die at very early stages of development. Embryos with trisomies all die at a characteristic prenatal stage. Only the trisomy 19 mice survived until birth. Redrawn from Gropp et al. (1983). 264 efforts must be made to reduce the risks of producing offspring with genetic abnormalities. Genetic consultation and screening of infertile couples and preimplantation diagnosis of ICSI-produced embryos are of critical importance particularly for patients with suspected genetic problems. Risks, benefits and future of human ICSI During the year 2000 alone, assisted fertilization (mostly IVF and ICSI) produced approximately 37,700 babies in the United States (SART and ASRM, 2004) and 22 European countries (Andersen et al., 2004). The number is expected to increase worldwide in the foreseeable future due to increasing maternal age and the development of assisted reproduction technologies. Demands for IVF and ICSI will increase, rather than decrease because the vast majority of couples are reluctant to utilize gamete donation since they desire their own children (Schover et al., 1998). Theoretical concerns for IVF and ICSI stems from the use of the spermatozoa that are unable to participate in normal fertilization (Djerassi, 1999; Sutcliffe et al., 2000; Maduro et al., 2003). Will babies born by assisted reproduction have serious medical or physical problems? Will ICSI and IVF transmit infertility genes to future generations? Will in-vitro manipulation of spermatozoa and oocytes/embryos have lasting effects on the health of offspring? Some congenital defects, which are not detected at birth, may become apparent as children grow (Hansen et al., 2002). IVF and ICSI are by no means free of risks, but normal pregnancy is not free of risks either. From time to time, laymen encounter articles in popular magazines and newspapers that babies conceived through IVF and ICSI are more than twice as likely as naturally conceived babies to suffer major birth defects and nearly three times as likely to be born small, with a significant risk factor for later cardiovascular and skeletal muscular problems. Such statements may scare IVF and ICSI candidates, but it is important to inform them that the vast majority of babies born after ICSI and IVF are normal and healthy (Bonduelle et al., 2002, 2003, 2004). The decision of whether or not to initiate therapy must be made by affected couples who are fully informed of the nature and magnitude of potential risks of therapy (Meschede et al., 1995). For most couples who desperately want to have their own babies, benefits of assisted fertilization overweigh their concerns (Tournaye and Van Steirteghem, 1997). One should not solely rely on nature, even though efficient mechanisms reduce the birth of defective babies. It would be ideal to use only genomically normal oocytes and spermatozoa for assisted fertilization, but at present there is no reliable, non-invasive method to distinguish genomically normal from abnormal gametes. Today, the preimplantation diagnosis of embryos prior to transfer to mothers is the only reliable method to determine normality and abnormality of embryos (Baschat et al., 1996). Embryos are sensitive to the environment where they grow. Mishandling of gametes and exposure of embryos to stressful conditions may affect the later development of embryos and fetuses and the health of offspring by disturbing the proper expression of imprinted genes (Cox et al., 2002; Schultz and Williams, 2002; Thompson et al., 2002; Maher et al., 2003). The birth of a baby girl with Angelman syndrome after ICSI with gametes of a couple who did not have a defective pattern of imprinted genes (Orstavik et al., 2003) gives us a warning to be very cautious about the handling of gametes and embryos throughout the procedures of any assisted fertilization. Will ICSI and other assisted fertilization technologies increase overall male and female infertility by spreading of genes involved in infertility? Faddy et al. (2001) and Engel et al. (1996) do not think so in view of the widespread propagation of spontaneous genetic mutations in general population. The male and female are equally responsible for infertility. The day may come when it is possible to correct defective genes or add missing genes to male and/or female gametes (and zygotes) so couples can have normal children without transmitting defective genes to future generations.

19 ICSI in animals other than the mouse and humans Species with successful ICSI At the time of this writing, animals other than human and mice in which ICSI have been successful in producing live offspring include golden hamster, rat, rabbit, cat, cattle, pig, sheep, horse and monkey (Table 3). This list will expand steadily with time. It is very difficult to compare the efficiency of ICSI in different animals because some investigators failed to report the numbers of ICSI oocytes utilized or the numbers surviving ICSI. Many papers also failed to describe whether all or only selected embryos were transferred to surrogate mothers. Overall ICSI efficiencies, listed in this table, are expressed by proportions (%) of live offspring developed from either ICSIsurvived oocytes or ICSI-fertilized oocytes. Let us assume that (i) 200 oocytes survived ICSI and 120 of them developed to the transferable stage (e.g. 2- to 4-cell embryos or morulae/blastocysts) and (ii) 100 embryos were selected randomly and transferred to surrogate mothers. If 10 live offspring were obtained, the overall efficiency is calculated as 120/200 (60%) 10/100 (10%) = 6.0%. Reservations must be expressed about figures shown in Table 3 since each investigator used a different experimental protocol. Some of them repeated experiments many times using many oocytes, while others performed only one or a few experiments using considerably fewer oocytes. The hamster was the first mammal in which ICSI was performed (Uehara and Yanagimachi, 1976, 1977), but it was not until very recently that live hamster offspring were obtained by ICSI (Yamauchi et al., 2002). The efficiency of this method in the hamster today is 9%, much lower than that in mice (55%) (Kusakabe et al., 2001; Yamazaki et al., 2001). Of all mammals the rabbit was the first to produce live offspring by ICSI (Iritani et al., 1989), but ICSI efficiency in this species is still rather low, i.e. 4% at best. Many investigators have tried pig ICSI because polyspermic fertilization was a frustrating problem for IVF of this species (Prather and Day, 1998; Coy and Roar, 2002). Injecting a single spermatozoon into an oocyte should solve this problem. Table 3. Comparison of ICSI efficiency in animals other than the human and mouse. Species Overall References efficiency Golden hamster 9.1 Yamauchi et al. (2002) Rat 11.6 Miyata et al. (2000) 3.8 Hirabayashi et al. (2002) <2.3 Said et al. (2003) Rabbit 0.8 Iritani et al. (1989) 4.1 Deng and Yang (2001) 1.8 Li et al. (2001a) 3.4 Shinohara et al. (2002) b Cat 2.5 Pope et al. (1998) 3.2 Gomez et al. (2000) Cattle 0.4 Goto et al. (1990) c 1.4 Hamano et al. (1999) d 11.3 Wei and Fukui (2002) 10.0 Horiuchi et al. (2002) Pig 0.3 Kolbe and Holtz (2000) 1.1 Martin (2000) 0.2 Nakai et al. (2003) 1.1 Lai et al. (2001) 1.8 Probst and Rath (2003) d Sheep 0.3 Catt et al. (1996) 0.8 Gomez et al. (1998a) Horse 5.3 Cochran et al. (1998) 2.4 Li et al. (2001b) Monkey 5.6 Chan et al. (2001) 3.6 Ng et al. (2002) 16.5 Hewitson et al. (2002) 10.0 Wolf et al. (2004) a The percentage of ICSI-survived or ICSI-fertilized oocytes that developed into live offspring. b These authors xeno-transplanted rabbit spermatogonia into mouse testis, allowing the spermatogonia to develop into spermatozoa. c They freeze thawed spermatozoa prior to ICSI. d They used X- or Y-sorted spermatozoa. 265

20 Table 4. Comparison of maximum efficiencies of natural and assisted fertilization in the laboratory mouse today. Fertilization method Efficiency (% of zygotes developed into live offspring) In-vivo fertilization and development 100 a Assisted fertilization and embryo transfer, at the best IVF ICSI ROS Spermatocyte injection Secondary spermatocyte b Primary spermatocyte 3 10 b a Although 100% of ovulated oocytes can be fertilized and develop into normal offspring, embryo loss occurs commonly under normal in-vivo conditions. According to Asdell (1946), numbers of corpus lutea, embryos, and young born have averaged 8.4, 6.4, 5.9 respectively, indicating that the considerable loss occurs from ovulation to birth, due to loss of oocytes/zygotes, implantation failure, and fetal resorption. In the mouse, the second litter is the largest, then there is a steady decrease afterwards. Some inbred and mutant mice have very low fertility. b Based on the number of oocytes injected with the nuclei of the secondary (2N) or primary (4N) spermatocytes. 266 Thus far, however, ICSI efficiency in the pig is disappointingly low (Table 4). No live offspring was born after ICSI in the goat (Keskintepe et al., 1997) and mink whale (Wei and Fukui, 2000). Attempts to obtain live offspring using freeze-dried spermatozoa have not been successful in cattle and pig (Keskintepe et al., 2002; Lee et al., 2003). Needless to say, animal ICSI today is far less efficient than human ICSI. it should be realized that human ICSI uses the spermatozoa that are unable to fertilize oocytes in vivo and by conventional IVF. If clinicians use normal, fertile spermatozoa for ICSI, the rates of successful fertilization and pregnancy are expected to be very high. High success rates in human ICSI using poor spermatozoa are undoubtedly due to hard work by pioneer researchers and high demand and enthusiastic cooperation of patients. How to improve animal ICSI The reason ICSI is not very successful in animals today is simply because relatively few researchers engage in this endeavour, and are still looking for technical tricks. Once tricks are learned, which will differ for each species, animal ICSI will become much easier than it is today. Targets for technical improvement include the following. Sperm plasma membrane It is expected that sperm plasma membranes of some species are much more stable than in others. When plasma membranes of spermatozoa of a given species are exceptionally stable, injected spermatozoa will retain their membranes intact for many hours. This will result in the complete failure of ICSI or considerable delay in the onset of oocyte activation. Spermatozoa of the species with poor ICSI results (e.g. the pig and sheep) may have very stable sperm plasma membranes. Failed or delayed disintegration of sperm plasma membrane within the ooplasm would result in no or incomplete oocyte activation, disorganized assembly of the cytoskeletal system, and/or the failure of syngamy. Higher success rates may be gained if sperm plasma membranes are removed initially, without damaging sperm nuclei. Since sperm nucleus begins to deteriorate once the plasma membrane is disrupted or removed, it is very important to damage or remove it immediately before ICSI. A K + -rich medium, called nucleus isolation medium or NIM (Kuretake et al. 1996a; Tateno et al., 2000) was used. This medium could keep mouse sperm nuclei genetically (chromosomally) intact significantly longer than ordinary cell culture media, yet it was still far from ideal. Media are required which retain the genetic integrity of sperm nuclei over a long period to avoid hurrying the preparation and injection of sperm nuclei. The acrosome The acrosome is a cap-like structure covering the anterior portion of the sperm head. It contains a spectrum of hydrolysing enzymes (Zaneveld and De Jonge, 1991). The bulk of its contents are released from fertilizing spermatozoa before their passage through the zona pellucida. The acrosomal components that enter the ooplasm during normal fertilization are the inner acrosomal membrane and part of the outer acrosomal membrane (see Figure 2). Conventional ICSI introduces the entire acrosome into the ooplasm. Unlike human and mouse oocytes, oocytes of some species (e.g. golden hamster) cannot withstand acrosomal materials. Acrosomes must be removed from spermatozoa to succeed with hamster ICSI (Yamauchi et al., 2002) (Figure 11). Why hamster oocytes cannot tolerate acrosome-intact spermatozoa is unknown, and acrosome size may be a factor. The hamster sperm acrosome has a much larger volume than in human and mouse. Sutovsky et al. (1996) examined monkey oocytes after ICSI using electron microscopy and discovered that acrosomal material persists at the anterior sperm head and hinders the decondensation of sperm nuclei. This does not seem to be a serious problem for the mouse (Kimura and Yanagimachi, 1995a) and human (Bourgain et al., 1998). Removing acrosomes may not be essential for successful ICSI with many

21 species, but is preferable theoretically. The ideal would be to inject physiologically acrosome-reacted spermatozoa, but distinguishing acrosome-reacted and non-reacted spermatozoa would be difficult in species with small acrosomes unless either the acrosomal contents or the inner acrosomal membranes are specifically labelled with fluorescent reagents (e.g. Nakanishi et al., 1999). Oocyte activation The slower oocyte activation in ICSI oocytes than in normally fertilized oocytes is puzzling (see Figure 4). Perhaps, it is partly due to a slow disintegration of the sperm plasma membrane in the ooplasm and partly due to the presence of culture media around the injected sperm head which delays interactions between intracellular sperm components and ooplasm. Thus, injecting plasma membrane-free spermatozoon with minimum amounts of culture medium would hasten oocyte activation. Although the spermatozoon itself contains oocyte-activating factor, additional stimulations may facilitate both oocyte activation and subsequent embryonic development. Reagents used to stimulate ICSI oocytes include ethanol, calcium ionophore, strontium, electric pulses and protein (MPF) synthesis inhibitor (Gomez et al., 1998a,b; Hwang et al., 2000; Suttner et al., 2000; Li et al., 2001b; Horiuchi et al., 2002; Nakai et al., 2003). Oocytes plasma membrane (oolemma) Oolemmae of immature oocytes at the germinal vesicle (GV) stage are extremely venerable to mechanical damages and therefore ICSI of GV oocytes is extremely difficult, if not impossible. Wound-healing ability increases dramatically in the oolemma after GV breakdown, which is why ICSI is effective with maturing and matured oocytes. It is expected that the wound-healing ability of the oolemma of a mature oocyte varies from species to species. If the oolemma of a particular species has a poor wound-healing ability, inclusion of a high concentration of serum in the oocyte-manipulation medium (Suzuki and Yanagimachi, 1997) may solve the problem. According to Kishigami et al. (2004), oolemmae of activated (mouse) oocytes are more resistant to mechanical damages than those of unactivated oocytes. Thus, oocyte activation prior to ICSI may increase the chance of successful ICSI. Centrosome and tail In most mammals, the sperm centrosome located at the junction of head and tail is pivotal in organizing cytoskeletal networks to bring male and female pronuclei to the centre of an egg (Schatten, 1994; Navara et al., 1995; Wu et al., 1996). Therefore, injection of a whole spermatozoon is recommended. If the species of interest has very large (long) spermatozoa, the middle and distal parts of sperm tail can be removed prior to ICSI. Sperm heads carrying neighbouring centrosomes are the only sperm components needed for successful ICSI. Common laboratory rodents such as the mouse, rat and hamster have small oocytes (70 80 µm in diameter) and long spermatozoa ( µm in length). Since sperm centrosomes are not necessary for their fertilization, it is more sensible to inject isolated sperm heads A B Figure 11. Result of hamster ICSI using acrosome-intact and acrosomeless spermatozoa. (A) Oocytes injected with acrosomeintact spermatozoa were all deformed in 3 h and died by 5 h after ICSI. (B) Oocytes injected with acrosomeless spermatozoa all survived and developed normally. In this experiment, acrosomes were disrupted by freeze thawing spermatozoa without cryoprotection. 267

22 268 only. In this way, the introduction of considerable amounts of culture media into small oocytes can be avoided. Light Hamster oocytes may be exceptional in being sensitive to short-wave visible light (< nm) emitted from ordinary fluorescent light sources (Hirao and Yanagimachi, 1978; Umaoka et al., 1992). The oocytes injected with spermatozoa under fluorescence lamps do not develop beyond the 2-cell stage (Yamauchi et al., 2002). The reason is not clear, but may be due to the generation of radical oxygen species, which harm oocytes. Oocytes of other species seem to be less sensitive (Bedford and Drobnis, 1989), yet their prolonged exposure to strong light should be avoided. Since mammalian oocytes and embryos have not been exposed to direct sun light for millions of years, they may have lost the essential protective mechanisms against strong light. ICSI for exotic animals When achieving conception, natural mating, AI and IVF should be tested in this order. If none is successful, ICSI should be attempted since it bypasses the need for optimal conditions for sperm capacitation and IVF. Attempts to fertilize bat oocytes by IVF were unsuccessful (Crichton, Krutzsch and Yanagimachi, unpublished). However, injecting individual bat spermatozoa into mouse oocytes resulted in 100% fertilization, each having well developed male (bat) and female (mouse) pronuclei (see Figure 90 in Yanagimachi, 1994). Perhaps, oocytes of any exotic animals can be fertilized by ICSI using spermatozoa of the same species. The mink whale is an example (Asada et al., 2001). Spermatid injection (ROSI) The injection of round spermatids into oocytes, commonly called round spermatid injection or ROSI, was first attempted in hamster oocytes to measure their capacity of transformation into pronuclei (Ogura and Yanagimachi, 1993). Live mouse ROSI offspring were first obtained after the electrofusion of round spermatids with oocytes (Ogura et al., 1994). As of today, other species in which live ROSI offspring were obtained include the rat, hamster, rabbit, Mastomys, pig, monkey and human. Mouse ROSI Mouse round spermatid, the smallest cells in the testis, are easily be recognized by their centrally located chromatin mass (Figure 12). Success rates with mouse ROSI (the proportion of live offspring developed from all ROSI oocytes) was <1.7% (Ogura et al., 1994). It rose to 12% when oocytes were electroactivated first, then mechanically injected with round sperm nuclei (Kimura and Yanagimachi, 1995b). Figure 13 shows three different ROSI methods. In the laboratory today, ROSI success rate using methods B and C is ca. 30% (Suganuma and Yanagimachi, unpublished data). Similar success rates were gained by Kishigami et al. (2004) with a method fundamentally similar to method B. With method C, Marh et al. (2003) obtained live offspring after injecting round spermatids developed in vitro from early pachytene spermatocytes. Unlike spermatozoa, mouse round spermatids (stages 1 7) cannot activate oocytes so ROSI oocytes must be stimulated with parthenogenic agents to initiate development. These agents include electric pulses, cyclohexamide, Sr 2+ and sperm extracts (Ogura et al., 1994; Kimura and Yanagimachi, 1995b; Sasagawa and Yanagimachi, 1996; Cummins et al., 1998). Routinely, a blunt-ended injection pipette connected to the Piezo-electric actuator drills the zona pellucida and breaks the oocyte s plasma membrane before spermatid nuclei are individually injected into oocytes. Conventional pipettes or electro-fusion of spermatids with oocytes can be used if a Piezo-actuator is not available (Liu et al., 1997; Ziyyat and Lefevre, 2001). Spermatid mitochondria introduced into oocytes by ROSI are selectively and continuously eliminated during embryo development, and do not pose a risk to offspring by the transmission of the mitochondrial genomes (Cummins et al., 1998; Shitara et al., 2000). A variety of methods are available to activate ROSI mouse oocytes, but SrCl2 in Ca 2+ -free medium is commonly used (Whittingham and Siracusas, 1978; Marcus, 1990; Cummins et al., 1998; Meng et al., 2002; Hayashi et al., 2003) because its induction of repetitive intracellular Ca 2+ oscillations resemble those arising after normal fertilization (Kline, 1996). Adenophostin, a potent agonist of IP3 receptor, also well activates mouse ROSI oocytes by releasing Ca 2+ repeatedly from the endoplasmic reticulum of the oocyte (Sato et al., 1998). The native sperm-borne oocyte-activating factor (SOAF) would be ideal to activate ROSI oocytes, provided it is characterized and becomes commercially readily available. In mice, SOAF appears or becomes active during spermiogenesis (Kimura and Yanagimachi, 1995b; Kimura et al., 1998b). In some animals and humans, SOAF may appear (or become active) before spermiogenesis (Yazawa et al., 2000, 2001). It is interesting to note that elongated spermatozoa cannot elicit the typical pattern of Ca 2+ oscillations, yet they can activate most oocytes to result in the birth of live offspring (Yazawa et al. 2001). Sasagawa et al. (1998b) obtained mouse offspring using round spermatids from prepubertal mice. ROSI also generated offspring of male mice that lacked spermatozoa or possessed only dead or infertile spermatozoa in their testes and epididymides (Ogura et al., 1996; Tanemura et al., 1997; Kanatsu-Shinohara et al., 2002; Meng et al., 2002; Yanagimachi et al., 2004). Genes essential for normal development (e.g. Oct 4, HPRT, elf-1a, HDAC1, ERV-L and LINE 1) show typical patterns of transcriptional activation in ROSI embryos as in normally fertilized preimplantation embryos (Hayashi et al., 2004). Paternal mrna introduced into oocytes by ROSI (e.g. mrnas of protamines 1 and 2 and transition proteins) are removed before the end of the 2-cell stage of embryos. The only exception is the endogenous retrovirus-like mobile elements (IAP) that is over-expressed in ROSI blastocysts. The meaning of this finding is unclear. Shamanski et al. (1999) find imprinted genes (such as Snrpn, Igf-2 and Peg1) are correctly expressed in post-implantation embryos after ROSI. Theses data were collected from surviving embryos and marked difference may emerge in arrested embryos. Tamashiro et al. (1999) produced five generations of mice by ROSI, propagating mice for five generations by bypassing

23 Figure 12. Mouse round spermatid and primary (pachytene) spermatocyte. The residual body is a small droplet of sperm cytoplasm released from the sperm body during discharge of spermatozoa from the testis. The nucleus of round spermatid is characterized by the presence of a centrally located chromatin mass. Figure 13. Production of mouse offspring by ROSI (round spermatid injection). Method A: (1) injection of a round spermatid first, (2) followed by oocyte activation in the presence of cytochalasin B. This is to prevent cytokinesis so that all oocyte and spermatid chromosomes stay inside an activated oocyte. (3) Removal of one oocyte pronucleus produces a pronuclear egg with diploid chromosomes (4), which develop into an offspring. Method B: (1) activation of an oocyte first, (2) followed by injection of a spermatid after emission of the first polar body (Pb). (3) The chance of successful ROSI is high when spermatid nucleus develops into a large male pronucleus. Method C: (1) Injection of spermatid first, (2) followed by oocyte activation. The chance of successful ROSI is high when spermatid nucleus develops into a large male pronucleus (3). 269

24 270 spermiogenesis, sperm maturation, capacitation and sperm oocyte membrane fusion, which are required for normal fertilization. No significant differences emerged between the growth and behaviour of the fifth generation of ROSI offspring with those of control animals (the second generation of normally fertilized mice). Infertility may be induced in males by homozygous recessive infertility genes including blind-sterile, quaking and CREM. ROSI can rescue such males (Yanagimachi et al., 2004) (Figure 14). Some transgenic male mice are infertile, and these are also rescued by ROSI (Meng et al., 2002; Kanatsu- Shinohara et al., 2002). Kanatsu-Shinohara et al. (2003) obtained live mouse offspring using round spermatids developed from spermatogenic cells that had been transplanted in the testes of infertile males. Mice homozygous for the gene juvenile spermatogonia depletion (Jsd) are infertile (Tohda et al., 2002). They have a very high intra-testicular level of testosterone, which can be modified by treatment with a gonadotrophin-releasing hormone (GnRH) antagonist followed by its withdrawal. This treatment allows spermatogonia to develop into round and elongated spermatids. Tohda et al. (2002) obtained live offspring using such spermatids. ROSI in animals other than the mouse Hirabayashi et al. (2002) performed rat ROSI using cryopreserved round spermatids and obtained four offspring after transferring cell embryos. They started with 527 oocytes and 485 of these survived ROSI, so overall success rate was <1%. Kato et al. (2004) increased the rate to 4.9% by activating ROSI oocytes with two electric pulses followed by treatment with 6-dimethylaminopurine. Rabbit ROSI was successful to an efficiency of 0.8% (Sofikitis et al., 1994). Two developing pig embryos, but not live offspring resulted from ROSI (Kim et al., 1999). Live pups were obtained in African rodent Mastomys by injecting elongated, but not round spermatids (Ogoniki et al., 2003a). One Rhesus monkey baby was born after injecting an elongated spermatid (Hewitson et al., 2002) and one mid-term fetus of a cynomogus monkey was obtained by round spermatid injection (Ogoniki et al., 2003b). The reason why ROSI is so inefficient in animals other than the mouse is not clear. First, the cells selected for ROSI might not have been round spermatids. Spermatogonia and small spermatocytes at the early stage of meiosis may resemble to round spermatids. Sutovsky et al. (1999) proposed to use mitochondria-specific fluorescent dye to distinguish spermatids from other cells. Spermatids carry many specific proteins such as transition proteins, protamines, and fibrous sheath proteins. Since they are all intracellular proteins, procedures using these as spermatid marker proteins may kill the cells. Antibodies or other markers specific to the surface of round spermatids are not available (Dr Norman Hecht, personal communication). Second, centrosomes of round spermatids may not be fully mature in non-rodent mammals, and may be incapable of organizing microtubular network in oocytes (Schatten, 1994). Ineffective spermatid centrosome may be replaced with those from a mature spermatozoon. Third, the gender-specific genomic imprinting essential for normal embryo development (Barlow, 1994, 1995; Mann, 2001; Kierszembaum, 2002) may not be fully formed in round spermatids. While this possibility is unlikely (Shamanski et al., 1999), the status of imprinted genes in round spermatids, elongated spermatids and spermatozoa must be re-assessed for each species. Lastly, the activation of ROSI oocytes could be a problem. Although ROSI oocytes may develop into apparently normal preimplantation embryos without any need for parthenogenetic stimulations (e.g. monkey; Ogonuki et al., 2003b), the quality of oocyte activation could be rather poor. Round spermatids may not be able to induce typical pattern of Ca 2+ oscillations (Yazawa et al., 2000; Ogonuki et al., 2001). An oocyte activation protocol that is the best for one species may not necessarily be the best for others. ROSI is still in its infantile stage. One should be aware that mouse ROS, for example, was very difficult in the beginning and became easier with technical improvements. Human ROSI Some infertile men have no spermatozoa in semen, epididymis and testes. Others possess a few spermatozoa in testes, although harvesting them from testis biopsies can be arduous and they may be dead or severely deformed. This is why ROSI caught clinical attention. Some clinicians obtained healthy children by ROSI (Tesarik et al., 1995, 1996; Vanderzwalmen et al., 1997; Barak et al., 1998; Gianaroli et al., 1999; Saremi et al., 2002), but others could not (Yamanaka et al., 1997; Kahraman et al., 1998; Ghazzawi et al., 1999; Balaban et al., 2000; Vicdan et al., 2001; Urman et al., 2002). The clinical value of human ROSI has been widely debated (Fishel et al., 1996; Sousa et al., 1998; Tesarik et al., 1998a; Vanderzwalmen et al., 1998; Prapas et al., 1999; Silber et al., 2000). Two of four babies born after intracytoplasmic injection of elongated spermatids had congenital abnormalities (Zech et al., 2000). Uncertainty over safety lead to a number of countries imposing a moratorium on human ROSI (cited from Aslam et al., 1998). The American Society for Reproductive Medicine (2003) recommends ROSI should not be performed when more mature sperm forms (elongated spermatids or spermatozoa) are available for ICSI. They recommend clinicians to consider ROSI as experimental and that candidates for ROSI should be informed of the potential risks of the procedure. According to Verheyen et al. (1998) and Silber et al. (2000), spermatogenesis arrest at the round spermatid stage is very rare. Whenever round spermatids are presented in a testis, elongated spermatids and spermatozoa are also present (Silber and Johnson, 1998). Elongated spermatids are well known to produce healthy children more efficiently than round spermatids (Fishel et al., 1995; Araki et al., 1997; Antinori et al., 1997; Kahraman et al., 1998; Sofikitis et al., 1998a,b). Prapas et al. (1999) stated if only round spermatids are found and if the patient s previous history has never shown elongated spermatids or mature spermatozoa in ejaculates or in the diagnostic testicular biopsy, then the couple should be advised about the very low possibility of obtaining a pregnancy using such cells, and thus IVF with donor spermatozoa should be offered as an alternative, with the final decision being made by the patients. Even though the initial enthusiasm of human ROSI has faded, this does not mean that human ROSI has no future. Demands remain. Several factors might improve human ROSI. First, many spermatogenic cells arrested at the round spermatid stage may be undergoing apoptosis (Jurisicova et al., 1999). Round spermatids of seven men with non-obstructive azoospermia had a high

25 Figure 14. Production of offspring from infertile CREM-null mice. The most advanced spermatogenic cells in the testis of CREM-null mice are round spermatids (A, B and B ). ROSI can produce normal offspring (C). 271

26 incidence (37 78%) of apoptotic DNA fragmentation (Jurisicova et al., 1999). They were unable to impregnate their wives by ROSI. In contrast, a man whose round spermatids had the lowest incidence (18%) of DNA fragmentation impregnated his wife by ROSI, although the resulting pregnancy was spontaneously terminated in the first trimester. Thus, stringent selection of nonapoptotic spermatids may increase the chance of successful ROSI. Second, the cells judged and used as round spermatids may not really be round spermatids. Spermatids are the smallest of all germ cells (Johnson et al., 1999), but some small cells released from blood vessels and/or intertubular tissues might be mistaken as round spermatids. Seminiferous tubules of men with nonobstructive azoospermia contained >90% of small round cells (5 6 µm in diameter) which were indeed haploid spermatids (Angelopoulous et al., 1997), a report awaiting confirmation. The presence of acrosomal granules and/or acrosomal vesicle, generally considered as a distinct feature of the spermatid (Mendoza et al., 1996; Columbero et al., 1997; Sofikitis et al., 1997), may be unreliable since acrosomes may form in the secondary spermatocytes under abnormal conditions (Mori et al., 1999). The distribution characteristics of mitochondria were proposed to differentiate round spermatids from other cells (Sutovsky et al., 1999), but its reliability has not been confirmed. Until reliable cell surface markers become available, simple morphological parameters and supportive biochemical, molecular and karyological supporting data must be used to identify round spermatids for ROSI. Claims that elongated spermatids develop from round spermatids or primary spermatocytes after a few days of culture in vitro (Tanaka et al., 1997; Aslam and Fishel, 1998; Tesarik et al., 1998a,b, 1999, 2000a; Cremades et al., 1999, 2001) are surprising in that spermatocytes can transform into elongated spermatids within 2 days of culture (Tesarik et al., 1999). This process normally takes >10 days in vivo (Heller and Clermont, 1964; Sharp, 1994). Despite such ambiguities, the in-vitro culture of spermatocytes and round spermatids may be helpful to distinguish healthy cells from damaged or apoptotic ones (Tesarik et al., 2000b; Tanaka et al., 2003). Third, human oocytes injected with round spermatids might not be fully activated without additional stimulations. Unlike mouse round spermatids, human spermatids activate oocytes (Sousa et al., 1996; Yamanaka et al., 1997), but not as effectively as elongated spermatids or mature spermatozoa (Yazawa et al., 2000). ROSI oocytes may need additional stimulation to secure full development of embryos. Native human sperm-borne oocyte activating factor would be ideal to activate ROSI oocytes. Agents such as adenophostin, which induces repetitive intracellular Ca 2+ oscillations, might offer a substitute for ROSI activation. Most ROSI oocytes remain unactivated (ca. 60%) or fail to develop beyond the pronuclear stage (ca. 23%) (Benkhalifa et al., 2004), perhaps due partly to incomplete oocyte activation. It is worthwhile using proper electrical pulse or combination of calcium ionophore and 6-DMAP (a protein kinase inhibitor) known to be effective after a failed ICSI (Yanagida et al., 1999) or after a failed cloning by somatic cell nucleus transfer (Hwang et al., 2004). Last, the sperm centrosome that becomes the centre of the microtubular network in fertilized oocyte (Schatten, 1994; Palermo et al., 1997) may be immature in human round spermatids. Replacing the immature centrosome with a mature centrosome is an intriguing possibility, but mature sperm centrosome might be vulnerable to mechanical manipulations (Palermo et al., 1997). More basic studies on animals are clearly needed before ROS is sage for humans. Spermatogenesis in some men is arrested at the early spermatid stage due to the defect of CREM-tau gene that produces proteins necessary for spermiogenesis (Weinbauer at al., 1998; Peri and Serio, 2002). Since ROSI can rescue CREM-null male mice (Yanagimachi et al., 2004), it may be able to rescue men with this gene defect. Spermatocyte injection Spermatogenesis arrest can occur at any stages, but in humans it is most frequent at the primary spermatocyte level (Martin-du Pan and Campana, 1993). Can spermatocytes be used for assisted fertilization? Kimura and Yanagimachi (1995c) were the first to obtain mouse pups by injecting nuclei of secondary spermatocytes into mature oocytes (Figure 15). The spermatocyte nucleus transforms into metaphase chromosomes without activating oocytes, resulting in the formation of two metaphase spindles within one oocyte. When activated by an electric shock, two polar bodies and two pronuclei were formed in each oocyte, followed by cleavages. Seven pups were obtained after transfer of 29 cleaving embryos. The overall success rate (i.e. the proportion of nucleus-injected oocytes that developing into live pups) was ~15%. Sofikitis et al. (1998a) reported the birth of a healthy boy after injecting six mature oocytes with nuclei of secondary spermatocytes. Live mouse offspring were also obtained by injecting nuclei of primary spermatocytes into mature oocytes (Kimura et al., 1998a; Ogura et al., 1998; Sasagawa et al., 1998a) (Figure 16). Success Figure 15. Production of offspring using secondary spermatocytes. (1) Injection of the nucleus of the secondary spermatocyte into a mature oocyte allows it to transform into metaphase chromosomes (2). When an oocyte with two metaphase chromosomes is activated, it emits two polar bodies (Pb) (3), and forms one female and one male pronucleus (4).

27 Figure 16. Production of offspring using primary spermatocytes. Method A: a germinal vesicle oocyte is fused with a primary (pachytene) spermatocyte (1), allowing the oocyte and spermatocyte chromosomes to mingle to form a large, octoploid metaphase chromosomes (2). This oocyte proceeds spontaneously to tetraploid metaphase chromosomes (3). Transfer the tetraploid chromosomes to a mature enucleated oocyte (4). When activated, this oocyte will have a diploid pronucleus (5) with both maternal and paternal genomes. Method B: injection of a primary spermatocyte into an oocyte during the first meiotic division of an oocyte (1) results in the formation of two tetraploid metaphase chromosomes (2). Soon, two first polar bodies (Pb) are emitted (3) and two diploid metaphase chromosomes appear within the oocyte (4). Transfer of the metaphase chromosomes (or polar body chromosomes) of spermatocyte origin into a mature oocyte (5) followed by oocyte activation results in the development of two pronuclei of maternal and paternal origin (6), leading to the development of offspring. Method C: injection of a primary spermatocyte into a mature oocyte (1), followed by oocyte activation (2) and transfer of polar body nucleus into another mature oocyte (3 4) results in an oocyte with two diploid metaphase chromosomes (5). When activated (5), this oocyte forms two pronuclei of maternal and paternal origin (6 7). 273