A preclinical evaluation of pronuclear formation by microinjection of human spermatozoa into human oocytes

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1 FERTILITY AND STERILITY Copyright c 1988 The American Fertility Society Vol. 49, No.5, May 1988 Printed in U.S.A. A preclinical evaluation of pronuclear formation by microinjection of human spermatozoa into human oocytes Susan E. Lanzendorf, Ph.D.*t Mary K. Maloney, B.S.* Lucinda L. Veeck, M.L.T.* James Slusser, M.S.:\: Gary D. Hodgen, Ph.D. * Zev Rosenwaks, M.D.* Eastern Virginia Medical School, Jones Institute for Reproductive Medicine, Norfolk, Virginia In vitro fertilization (IVF) is recognized as an accepted treatment for male infertility. However, the fertilization rate is significantly lower than the fertilization rate of other IVF patient groups. Some male factor infertility patients still have a basic semen quality too poor for treatment by IVF. Microinjection of a spermatozoon directly into ooplasm has been recommended to assist fertilization in this subfertile population. This study found that oocytes from 5 of 11 patients microinjected with human spermatozoa demonstrated successful pronuclear formation and correlated with the incidence of pregnancy in these patients transferred with same-source oocytes inseminated by standard protocols. This initial evidence promotes the supposition of clinical feasibility of assisted fertilization by sperm microinjection. Fertil Steril 49:835, 1988 The use of in vitro fertilization (IVF) has become an accepted treatment for couples with male factor infertility. It has been established that the recovery of 1.5 X 10 6 motile spermatozoa results in a favorable IVF fertilization outcome.1 If fertilization is achieved and cleavage proceeds normally, the pregnancy rate is comparable to that seen in other groups of infertility patients. However, there is still a population of male factor infertility patients with suboptimal semen parameters who cannot be treated successfully with conventional IVF. This may be due to insufficient concentrations of functional spermatozoa necessary for the dispersion of cumulus cells, penetration of the zona pellucida, and/or fusion with the oocyte plasma membrane. In these instances, microinjection of a spermato- Received November 12, 1987; revised and accepted January 28,1988. * Jones Institute for Reproductive Medicine, Department of Obstetrics and Gynecology. t Reprint requests: Susan E. Lanzendorf, Ph.D., Eastern Virginia Medical School, Lewis Hall, Room 2006, 700 Olney Road, Norfolk, Virginia * Department of Anatomy and Cell Biology. Vol. 49, No.5, May 1988 zoon directly into the ooplasm, bypassing the zona pellucida and plasma membrane, may be a means of assisting fertilization in vitro in certain cases of male factor infertility.1-4 In 1976, Uehara and Yanagimachi 5 found that unfertilized hamster ova surgically injected with isolated sperm nuclei were capable of undergoing egg activation with the successful formation of both male and female pronuclei. Since that time, this procedure has been repeated by a number of investigators whose studies demonstrated that surgical fertilization of mammalian ova need not impair their functional capacity during early development.6-9 Extensive studies have already been performed at the Jones Institute lo - 12 which reveal that human spermatozoa that are incapable of penetrating and fertilizing ova in an in vitro system are capable of decondensation and pronuclear formation following their direct injection into the cytoplasm of the hamster oocyte. One might conclude from these results that the defect(s) in the spermatozoa are restricted to fertilization events preceeding successful incorporation into the ooplasm. These ob- Lanzendorf et ai. Microinjection: a preclinical evaluation 835

2 servations may indicate that such defective sperm, when mechanically introduced into the oocyte, may be able to participate in the subsequent events of fertilization. The purpose of the study presented here was to perform a preliminary evaluation of the human oocyte's ability to survive the sperm microinjection procedure and participate in the early events of fertilization. This study was limited to the donation of surplus immature (prophase 1)13 oocytes and discarded human sperm pellets to eliminate any possibility of adversely affecting patients' successful attempts at pregnancy through IVF. Patients MATERIALS AND METHODS IVF patients in series 28 (July 1987 through September 1987) at the Jones Institute for Reproductive Medicine were asked to sign an informed consent donating their surplus immature oocytes to this study. Ovarian stimulation was accomplished by administration of human menopausal gonadotropin (hmg; Pergonal, Serono Laboratories, Inc., Randolph, MA), human urinary folliclestimulating hormone (FSH; Metrodin, Serono Laboratories, Inc.), or a combination of hmg and FSH.14 Laparoscopy with oocyte retrieval was performed 34 to 36 hours after human chorionic gonadotropin (hcg) was given. The majority of 00- cytes harvested were at metaphase I (MI) and metaphase II (MII)13 stages of maturation and excluded from use in this study. Twenty-three immature oocytes undergoing germinal vesicle breakdown in vitro were donated to this study by 13 patients. Twenty of 23 oocytes were micro injected, while the remaining oocytes were fixed for transmission electron microscopy as controls. The study was limited to pronuclear development of the surgically fertilized oocytes because the Institute was not prepared to transfer human zygotes from this technique without more evidence of normalcy. Sperm Microinjection Procedure Sperm Preparation Sperm cells used were either those of the husband of the oocyte donor or a proven-fertile semen donor. If husband sperm cells were used, they were obtained following the sperm preparation performed by the IVF laboratory in the form of the 836 Lanzendorf et a1. Microinjection: a preclinical evaluation sperm pellets remaining after the washing procedure.15 The semen sample obtained from the donor was collected by masturbation, allowed to liquefy, and then twice washed by centrifugation with phosphate-buffered saline (PBS) at 270 X g for 10 minutes to remove seminal plasma. Pellets from both the patients and the sperm donor were resuspended in 1.0 ml PBS and frozen at C as both a form of storage and as a means of immobilizing the sperm cells prior to microinjection. Utilization of these samples in the microinjection procedure was preceded by diluting the pellets 1:5 in a solution of 10% polyvinyl pyrrolidone (PVP, MW = 90,000) in PBS to facilitate handling and to prevent the sperm cells from sticking to the microinjection pipette during the procedure.5-8 Oocyte Preparation Human oocytes used for the microinjection procedure were obtained at the germinal vesicle-bearing stage. Oocytes were cultured (37 C, 5% CO 2 ) in a modified16 Ham's F-10 medium (Gibco, Grand Island, NY) supplemented with 7.5% human fetal cord serum (HF % HFCS) in vitro until extrusion of the first polar body occurred, at which time the oocyte was said to be at a metaphase II stage of development.13 If present, cumulus cells were removed from oocytes with 0.1% hyaluronidase in HF % HFCS supplemented with HEPES buffer to give a final concentration of 10 mm. Cumulus cells were removed to allow visualization of the ooplasm during the microinjection procedure. Microinjection of Spermatozoa into Oocytes Micropipettes for the injection of spermatozoa were prepared from thin-walled glass capillary tubes (Drummond Scientific, Broomall, P A, outer and inner diameter of 0.9 mm and 0.6 mm, length 150 mm) using a Narishige (Narishige USA, Inc., Greendale, NY) PP 83 micropipette puller and MF 79 micropipette forge. The micropipette tips were opened by dipping them into 25% hydrofluoric acid followed by washes in distilled water and acetone. The inside diameter of the microinjection pipette tips were between 5 and 8 J.l,m. Holding micropipettes were prepared in a similar manner, but with an inner diameter of between 20 and 30 J.l,m. To achieve a smooth, blunt surface, the tips of the holding micropipettes were fire-polished using a microburner. Fertility and Sterility

3 cropipette and then released back into the oocyte along with the spermatozoon. The injection pipette was withdrawn and the oocyte was released from the egg-holder, washed three times, and cultured (37 C, 5% CO2 ) in HF % HFCS. All injection procedures were carried out at a magnification of 250X on the Nikon Diaphot Inverted Microscope with phase-contrast optics. Great care was taken to minimize the time the oocyte spent outside the incubator to under 5 minutes. Evaluation of Surgically Injected Oocytes Figure 1 Photographs illustrating the microinjection of a human oocyte. The oocyte is held firmly in place by the eggholding micropipette (a) and the injection micropipette is inserted through the zona pellucida and plasma membrane (b) directly into the ooplasm (c). After injection of the spermatozoon, the injection micropipette is withdrawn (d) (original magnification was X200). The fine movements of the micropipettes were controlled by two Narishige MO 202 micromanipulators in combination with two Narishige MN2 three-dimensional manipulators and two N arishige IM5B microinjectors. The micromanipulators and support system were mounted with a Narishige NSB mount on a Nikon Diaphot Microscope (Nikon Inc., Garden City, NY). ThE? medium used during the injection procedure was HEPES buffered HF-lO + 7.5% HFCS. Drops of the injection medium and the prepared sperm suspensions in PVP were placed inside a prepared well and the entire well was filled with heavy mineral oil as a cushion against evaporation and rapid ph changes. Prior to the injection procedure, the tip of the injection micropipette was snapped off inside the holding micropipette to create the sharp bevelled point required for penetration of the zona pellucida and plasma membrane. The injection micropipette was moved to the drop containing the sperm cells and a single spermatozoon was sucked into the tip of the micropipette. Both micropipettes were moved to the drop of medium containing the oocyte. An oocyte was picked up and held securely by suction on the holding micropipette. The injection of the sperm cell was achieved by firmly pushing the injection pipette through the zona pellucida and deep into the oocyte cytoplasm (Fig. 1). A small amount of cytoplasm was sucked up into the mivol. 49, No.5, May 1988 Following a 6-hour incubation period, injected oocytes were examined for activation as demonstrated by the extrusion of the second polar body. Culture then was extended an additional 7 hours and oocytes were again examined. All injected 00cytes were visually assessed by light microscopy for the following: (1) oocyte activation as shown by extrusion of the second polar body, (2) transformation of the meiotic apparatus into a female pronucleus, and (3) transformation of the injected spermatozoan into a decondensed sperm nucleus or male pronucleus. All oocytes were fixed at 13 hours postinjection by addition of 2.5% glutaraldehyde in 0.1 M cacodylate buffer. Following fixation, oocytes were either stained with 1% acetolacmoid stain or prepared for transmission electron microscopy. Transmission Electron Microscopy Injected oocytes exhibiting pronuclear formation following the 13-hour culture period were fixed and prepared for transmission electron microscopy as previously described by Sandow and N amy P 00cytes were fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer for 30 minutes. Fixed oocytes were placed on a glass slide with very little excess fluid. Melted agar (1.5%) was placed over the 00cytes and allowed to harden. A clean razor blade was used to cut the agar into a rectangle approximately 0.5 mm X 1.0 mm in which the oocyte was located. A notch was placed in the upper right corner of the rectangle for orientation. The agar block containing the oocyte was postfixed with 1% osmium tetroxide in 0.1 M cacodylate buffer, dehydrated with alcohol, and embedded in Poly IBed 812 (Polysciences, Inc., Warrington, PA). Thick sections were cut on an LKB Ultratome III ultramicrotome (LKB Instruments, Inc., Rockville, MD) and stained with Toluidine blue. Thin sections were cut with a diamond knife, stained with Lanzendorf et a!. Microinjection: a preclinical evaluation 837

4 Table 1 Results of Microinjection of Human Spermatozoa into Human Eggs Semen sample Patient Egg D-donor IH -husband Retained sperma 1 1 H 4 H 8 H 2 3 H H H D +b., 6 7 H + 10 H H + 7 H 13 H H + 13 H H 10 5 D H + 26 H +' 29 D + 30 D + a +, positive reaction; - negative reaction. b Egg degeneration believed to be due to mechanical injury during injection procedure., Injection of excess medium into egg. Pronuclear Decondensed Egg formation sperma activateda (PN-pronucleus)a + + (1 PN) _b.d _h,e d Egg fixed in anaphase 2 stage of meiosis II and exhibited degenerate morphology. e Egg spontaneously arrested in anaphase 2 of meiosis. lead citrate and uranyl acetate, and viewed with a Philips EM-301 electron microscope (Philips Electronic Instruments, Inc., Mahwah, NJ). RESULTS Human spermatozoa were microsurgically injected directly into the cytoplasm of 20 human 00- cytes. Data are summarized in Table 1. Approximately 5 hours following each sperm injection' an early assessment of the success of the procedure was made. Injected oocytes were examined for color and regularity of cytoplasm, shape of the plasma membrane and its relationship to the zona pellucida, volume of perivitelline space, and signs of degeneration. It was found that, if insufficient mechanical force was used to penetrate through the oolema to ensure adequate placement of the spermatozoon within the ooplasm, the injected sperm cell would not be retained and, upon examination of the oocyte, would be found within the perivitelline space. Loss of the injected spermatozoon was found to occur in five of the injected eggs. Placement of excess medium into the ooplasm upon release of the spermatozoa occurred in two of the injected oocytes. In these instances, medium continues to be released following release of the spermatozoon into the oocyte and prior to withdrawal of the microinjection pipette. The excess medium surrounds the injected spermatozoon and, upon light-microscopic examination, a large vacuole encloses the injected sperm head. The injection of excess medium was found to result in the failure of spermatozoon decondensation. This was believed to be due to the failure of the sperm head to come into contact with the cytoplasmic elements of the oocyte responsible for decondensation. Degeneration of injected oocytes, as demonstrated by a dark, granular-appearing cytoplasm and shrinking of the cell mass, occurred in eight of the studied oocytes. Because of the poor quality of some of the oocytes prior to the procedure, it is uncertain whether cell death can be attributed in all instances to mechanical disruption during the injection. However, injection procedures did occur in which disruption of the ooplasm was known to be too severe and believed to result in the death of five of the eight degenerating oocytes. Oocyte activation, as demonstrated by the resumption of meiosis, occurred in nine of the injected oocytes. Upon examination, one injected oocyte was found to have spontaneously arrested in anaphase II of the second meiotic division. An ad- 838 Lanzendorf et al. Microinjection: a preclinical evaluation Fertility and Sterility

5 Figure 2 Light micrograph of human oocyte, 13 hours after microinjection of human spermatozoon, showing two pronuclei (arrows; original magnification was X400). ditional oocyte was fixed after only 3 hours in culture due to a degenerate morphology and was found, upon staining, to also be in anaphase II of meiosis II. Extrusion of the second polar body from activated oocytes was seen approximately 6 hours following injection of the spermatozoon. One oocyte, activated by the injection procedure, failed to retain the injected spermatozoon, but did go on to develop a normal-appearing female pronucleus. Formation of both male and female pronuclei occurred in six ofthe injected oocytes (Fig. 2). In all instances, pronuclei were visible within 13 hours of the injection of the spermatozoon. The majority of the injection procedures used spermatozoa obtained from the husband of the oocyte donor (16 of 20 oocytes). The remaining 400- cytes were injected with spermatozoa obtained from a proven-fertile semen donor. At the time of this study, obtaining sufficient numbers of oocytes for examination of normal and abnormal semen parameters in the human was not possible. However, no apparent differences were detected in the fertilization rate when either normal, fertile spermatozoa, or spermatozoa obtained from a male factor patient was used in this initial investigation. In the 5 patients (seven oocytes) in whom successful pronuclear formation occurred following the microinjection procedure, there was also the successful establishment of pregnancy by transfer of same-source oocytes by standard IVF protocols (Table 2). Likewise, where the microinjection procedure was not found to result in oocyte activation Vol. 49, No.5, May 1988 and pronuclear formation, pregnancy was not achieved (6 of 11 patients). Ultrastructural examination of microinjected oocytes demonstrating successful formation of two pronuclei was performed by transmission electron microscopy. Oocytes, cultured for a total of 13 hours following the injection of the spermatozoon, were considered to be in the early pronuclear stage of development. For control purposes, morphologic comparisons were made between microinjected 00- cytes, unfertilized oocytes, and oocytes fertilized in vitro and then cultured to the pronuclear stage. Control oocytes and in vitro fertilized oocytes were excess material not required for transfer and donated by patients not participating in the microinjection study. Electron micrographs revealed normal-appearing organelles and pronuclei in all six of the injected oocytes (Figs. 3 and 4). Mitochondria, endoplasmic reticulum, Golgi complex, and annulate lamellae were present in normal distributions. The plasma membranes of these oocytes were smooth with few microvilli. The two pronuclei were located eccentrically and in close proximity to each other in all injected oocytes. Cortical granules were not observed within the cortical regions of serial sections of five of the six injected oocytes, suggesting that the cortical granule reaction had taken place (Fig. 3). It is not known why cortical granules were present within the cortex of an oocyte that did exhibit activation with the formation of two pronuclei. This suggests that the cortical granule reaction is not required for pronuclear formation. Table 2 Successful Pronuclear Formation by Microinjection and the Establishment of Pregnancy by Transfer of Same-Source Eggs Fertilized In Vitro Activated Pronuclear eggs by formation by Pregnancies Patient microinjection a microinjection b established 1 1/3 1 (1 PN) Yes 2 Oil No 3 Oil No (2 PN) Yes 5 Oil No No 7 2/3 1 (2 PN) Yes 8 0/2 No 9 Oil No (2 PN) Yes 11 3/4 3 (2 PN) Yes a Number of activated eggs by microinjection over the total number of eggs injected. b Total number of patient's eggs demonstrating formation of pronuclei following injection. Lanzendorf et al. Microinjection: a preclinical evaluation 839

6 Figure 3 Transmission electron micrograph of the cortical region of a fertilized human oocyte fixed 13 hours after microinjection of human spermatozoon. Note the absence of cortical granules in the region of the cortex (C); zona pellucida (ZP) (original magnification was X4500). Sites of degeneration were observed within the cytoplasm of one injected oocyte as demonstrated by the presence of large lysosomal vacuoles lying within the vicinity of the two pronuclei. All other organelles appeared normal. Mechanical injury at the time of the injection may have resulted in the degeneration of the oocyte. DISCUSSION The process of fertilization requires that various changes occur to spermatozoa, rendering them capable of fertilizing an oocyte. These changes include capacitation and the acrosome reaction. The acrosome reaction results in the fusion of the sperm plasma membrane and outer acrosomal membrane, thereby releasing acrosomal enzymes that facilitate sperm penetration through the oocyte investments. Binding takes place between the sperm surface and receptors on the zona pellucida, followed by penetration of the spermatozoon through the zona into the perivitelline space. The equatorial and postacrosomal regions of the sperm head then fuse with the oocyte plasma membrane, and the sperm nucleus is transported into the ooplasm. Cytoplasmic components within the oocyte reduce the disulfide bonds of the sperm chromatin and the sperm nucleus decondenses The association of the spermatozoon with the surface of the oocyte initiates a process called activation in which meiosis is completed, the second polar body extruded, and formation of the female 840 Lanzendorf et ai. Microinjection: a preclinical evaluation pronucleus initiated. The male pronucleus is formed from the decondensed sperm nucleus. During activation, the cortical granules found lining the cortex of the oocyte discharge their contents into the perivitelline space, an event believed to provide the major block to polyspermia Some spermatozoa are incapable of participating in the early events of fertilization involving the interaction with and penetration of the oocyte (i.e., absence of adequate motility, abnormal morphology). Activation of the oocyte, also a prerequisite for normal fertilization, is accomplished by the interaction between spermatozoon and oocyte. Microinjection of a spermatozoon into an oocyte can bypass all the potential barriers of the oocyte, as well as activate its development In a study designed to investigate whether mammalian sperm cells or sperm nuclei can form male pronuclei following their injection, Uehara and Yanagimachi5 microinjected hamster sperm nuclei, isolated by tissue homogenization, into unfertilized hamster oocytes. Fresh, frozen-thawed, and freeze-dried human spermatozoa also were injected into hamster oocytes to determine whether sperm nuclei can develop into pronuclei within the cytoplasm of an oocyte of another species and to determine the stability of sperm nuclei following various forms of storage. It was found that, even without the oocyte/sperm interactions required for normal fertilization, hamster oocytes injected with both hamster or human sperm nuclei, regardless of prior Figure 4 Transmission electron micrograph of a human oocyte fixed 13 hours following microinjection of a human spermatozoon. One of the two pronuclei is visible in this section; pronuclear envelope (PE), pronucleus (PN) (original magnification was X3400). Fertility and Sterility

7 storage conditions, were capable of undergoing resumption of meiosis with the successful formation of both male and female pronuclei. Exocytosis of the cortical granules in these oocytes also were reported. These investigators concluded from this study that: (1) the nuclei of mammalian sperm are very stable, as demonstrated by their ability to develop into male pronuclei following freeze-thawing or freeze-drying and mechanical disruption; (2) transformation of a sperm nucleus into a male pronucleus is not species-specific; and (3) surgical fertilization results in the activation of the oocyte, as demonstrated by the exocytosis of the cortical granules, the extrusion of the second polar body, and the formation of the female pronucleus. It also was postulated that it was the mechanical stimulation of the injection pipette on the plasma membrane or ooplasm that induced oocyte activation. 5 This was later demonstrated when mature hamster oocytes were "pricked" with a glass needle in an attempt to induce activation. 21 The results showed that activation occurred in approximately 80% of eggs by mechanical stimulation of the needle. The authors concluded from this study that the mechanical stimulus provided by the injection procedure is what initiates the activation ofthe oocyte. 21 In this study, we have demonstrated that human oocytes were capable of surviving the mechanical insertion of a spermatozoon directly into the ooplasm followed by activation and the formation of both male and female pronuclei. These results support the findings reported by investigators in animal models. 5-9 The successful use of microsurgical injection of spermatozoa into human oocytes has advanced the feasibility of incorporating this technique into standard IVF use. The potential value of this procedure is obvious in the treatment of male factor fertility disorders. It is interesting to note that there was a correlation between successful pronuclear development in microinjected oocytes and the successful establishment of pregnancy by transfer in the same patient where oocytes were inseminated by standard IVF protocols. The significance of this finding remains to be elucidated. This protocol used oocytes matured in vitro and spermatozoa obtained as discard from swim-up pellets. The deliberate use of the lowest quality human oocytes and sperm cells available may in some way have prejudiced the outcome of this study. However, the fact that success was achieved with the use of this grade of material is very valu- able, allowing generation of information without compromising the clinical needs of the patients donating the material. The selection of individual sperm for injection was random within a given sample and determined by gross morphology and ease of handling. Concern over the genetic normality of individually selected sperm for microinjection is legitimate and the need for further study in this area is obvious. The technique for direct analysis of chromosomes of microinjected spermatozoa is currently being developed and may assist in determining the correct usage of microsurgical fertilization in the human. The microinjection of human oocytes has provided important scientific and clinical information. It may yield critical data on the precise relationship of sperm/oolema contact, oocyte activation, sperm decondensation, and pronuclear formation. Microinjection may serve as a potential clinical test for sperm-oocyte interaction abnormalities (intraooplasmic). Further study with this technique in the human system can only increase our knowledge regarding the treatment of infertility. REFERENCES 1. Van Uem JFHM, Acosta AA, Swanson RJ, Mayer J, Ackerman S, Burkman LJ, Veeck L, McCowell JS, Bernardus RE, Jones HW Jr: Male factor evaluation in in vitro fertilization: Norfolk experience. Fertil Steril 44:375, Battin D, Vargyas JM, Sato F, Brown J, Marrs RP: The correlation between in vitro fertilization of human oocytes and semen profile. Fertil Steril 44:835, Cohen J, Edwards R, Fehilly C, Fishel S, Hewitt J, Purdy J, Rowland G, Steptoe P, Webster J: In vitro fertilization: a treatment for male infertility. Fertil Steril 43:422, Jones HW: Indications for in vitro fertilization. In In Vitro Fertilization-Norfolk, Edited by HW Jones, GS Jones, GD Hodgen, Z RosenV'laks. Baltimore, Williams & Wilkins, 1986, p 1 5. 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8 (Abstr). Presented at the Vth World Congress on In Vitro Fertilization and Embryo Transfer, Norfolk, VA, April 5-10, Published by the International Federation of Fertility and Sterility Society in the preliminary program, p Lanzendorf SE, Maloney M, Rosenwaks Z, Hodgen GD: The fertilizing potential of human spermatozoa microinjected into hamster ova: a study of semen samples utilized for human in vitro fertilization (Abstr). Presented at the forty-third annual meeting of The American Fertility Society, Reno, NV, September 28-30, Published by The American Fertility Society in the preliminary program, p Lanzendorf SE, Maloney M, Ackerman S, Acosta A, Hodgen GD: Fertilizing potential of acrosome-defective sperm following microsurgical injection into eggs. Gamete Res. In press 13. Veeck LL: Morphological estimation of mature oocytes and their preparation for insemination. In In Vitro Fertilization -Norfolk, Edited by HW Jones, GS Jones, GD Hodgen, Z Rosenwaks. Baltimore, Williams & Wilkins, 1986, p Rosenwaks Z, Muasher SJ: Recruitment of fertilizable eggs. In In Vitro Fertilization-Norfolk, Edited by HW Jones, GS Jones, GD Hodgen, Z Rosenwaks. Baltimore, Williams & Wilkins, 1986, p McDowell JS: Preparation of spermatozoa for insemination in vitro. In In Vitro Fertilization-Norfolk, Edited by HW Jones, GS Jones, GD Hodgen, Z Rosenwaks. Baltimore, Williams & Wilkins, 1986, p Veeck LL, Maloney M: Insemination and fertilization. In In Vitro Fertilization-Norfolk, Edited by HW Jones, GS Jones, GD Hodgen, Z Rosenwaks. Baltimore, Williams & Wilkins, 1986, p Sandow BA, Namy CA: Light and electron microscopic analysis of human oocytes. In In Vitro Fertilization-Norfolk, Edited by HW Jones, GS Jones, GD Hodgen, Z Rosenwaks. Baltimore, Williams & Wilkins, 1986, p Yanagimachi R: Mechanisms of fertilization in mammals. In Fertilization and Embryonic Development In Vitro, Edited by L Mastroianni, JD Biggers. New York, Plenum Press, 1981, p Bedford JM: Fertilization. In Reproduction in Mammals, Germ Cells and Fertilization, Edited by CR Austin, RV Short. New York, Cambridge University Press, 1982, p Longo FG: Pronuclear events during fertilization. In Biology of Fertilization, Vol 3, Edited by CB Metz, A Monroy. Orlando, Academic Press, 1985, p Uehara T, Yanagimachi R: Activation of hamster eggs by pricking. J Exp Zool 199:269, Lanzendorf et al. Microinjection: a preclinical evaluation Fertility and Sterility