How does a sperm fertilise a human egg in vitro?

Transcription

1 How does a sperm fertilise a human egg in vitro? A. Henry Sathananthan & Christopher Chen Monash University, Melbourne & Flinders Medical Centre, S. Australia & Singapore Sperm-oocyte fusion is the key event of fertilization in the human, as well as in animals. It is a classic example of spontaneous membrane fusion between 2 cells without the intervention of agents such as viruses or chemicals. The process of gamete fusion has been extensively studied in various animals, including mammals. This is the very first report in the human we now revisit.

2 Sperm- egg membrane fusion original publication (Sathananthan & Chen 1986) Abstract Sperm-oocyte membrane fusion has been observed during monospermic fertilization of a human oocyte in vitro. Women were stimulated with both clomiphene citrate and human menopausal gonadotropin and were given human chorionic gonadotropin before a LH-surge. Twelve oocytes, collected at laparoscopy from six women who became pregnant by IVF, were allowed to mature for 7 14 hours in vitro and inseminated with preincubated sperm, fixed between 1 3 hours after insemination, and examined by transmission electron microscopy. Membrane fusion had occurred in one ovum 2 hours after insemination, and the oocyte had resumed maturation and was at anaphase II of meiosis. Cortical granules had been exocytosed, and some of their contents were visible at the surface close to the oolemma all around the oocyte. The sperm that fused with this oocyte was acrosome-reacted and had been partly incorporated into the ooplasm, while the anterior two-thirds of its head was phagocytosed by a tongue of cortical ooplasm. Membrane fusion had occurred between the oolemma and the plasma membrane overlying the postacrosomal segment of the sperm head, posterior to the equatorial vestige. Sperm chromatin had not decondensed, and serial sections revealed a midpiece attached to the basal plate and a tail located deeper in the ooplasm, all devoid of plasma membrane. Supplementary sperm penetrating the inner zona, approaching the perivitelline space, had undergone the acrosome reaction but had a persistent vestige of the equatorial segment of the acrosome with intact plasma membrane. Evidence of sperm chromatin decondensation was seen in other oocytes, 3 hours after insemination, which were at telophase II of meiosis. Eight oocytes penetrated by sperm were monospermic, while four were unfertilized. The general pattern of sperm fusion and incorporation appears to conform to that seen in most other mammals. The study also reveals that sperm have to complete the acrosome reaction before fusing with the egg. Gamete Research (1986)

3 SPERM PENETRATION & ACROSOME REACTION - TEXT Before the sperm fuse with the oocyte they need to penetrate the egg vestments, the cumulus and the ZP, during normal fertilization. Hyperactivated motility and the AR are thought to be involved in the process of penetration. TEM studies show that the AR could be either initiated in between the cumulus cells or more commonly at the surface of the zona between the CR and the ZP. Both acrosome-intact and acrosome-reacted sperm are found in these two locations (figures 22-33) but only acrosome-reacted sperm could penetrate through the thickness of the ZP and finally enter the PVS. Acrosome-intact sperm may be firmly bound to the surface of the ZP via their PM before penetration, while others are closely associated with CR cells. Vesiculation of surface membranes usually occurs at the surface of the zona and the sperm digests a pathway through the thickness of the ZP whilst the IAM is in close contact with the zona material. Three glycoproteins have been identified in mouse zonae and one of these, ZP3, induces the sperm AR. These glycoproteins have also been identified and categorized.in the human ZP The mechanics of the sperm AR is shown in the following diagram. To fuse with the egg the sperm must undergo capacitation and complete the acrosome reaction (AR). Sperm capacitation is a physiological process that occurs somewhere in the female reproductive tract in vivo. The AR is usually completed at the surface of the ZP just prior to fusion with the egg in vitro. The AR is the morphological manifestation of sperm capacitation where sperm acquire the ability to penetrate the egg vestments, cumulus oophorus and the ZP aided by hyperactivated motility. During capacitation molecular changes are thought to occur on the sperm PM which have been demonstrated by freeze - fracture electron microscopy, especially along the mid-segment of the sperm head. The AR usually takes place by vesiculation of surface membranes when multiple fusions occur between the outer PM and the outer acrosome membrane (OAM). The AR may occur in 2 stages, the acrosome cap region reacting first followed by the equatorial segment. The AR that occurs on the surface of the zona is regarded as the physiological AR and it is induced by both the cumulus and the glycoproteins that compose the ZP. Three glycoproteins are found in the zona pellucida - ZP1, ZP2 and ZP3. ZP3 is known to induce the AR during sperm penetration. Up to 60% of the sperm have been observed to undergo the AR at the surface of the ZP in normally fertilised and polyspermic ova, three hours after insemination. Only fully acrosome reacted sperm are capable of penetrating the inner ZP. Once the AR has taken place the PM in the anterior half of the PAS becomes receptive to egg fusion. Various methods have been used to assess the sperm AR by LM. Amongst these, monoclonal antibodies and lectins such as peanut agglutinin, have been used by fluorescent microscopy. However, the most reliable method to date has been TEM where all stages of the sperm AR can be visualised including the early stages prior to vesiculation, which are not easily detectable by LM (Sathananthan et al.1993)

4 Sperm penetration of mature egg - diagram Sperm binding Acrosome-intact Acrosome-reacting AR in Cumulus Acrosomereacted Sperm-egg fusion CR=corona radiata Sperm incorporation PVS=perivitelline space ZP=zona

5 Sperm penetration of cumulus and zona TEM The acrosome has reacted exposing the post acrosomal fusogenic segment (F) intact cumulus cumulus zona F F

6 Sperm penetration of the zona pellucida and perivitelline space (pvs) TEM The sperm literally eats it way through the gelatinous zona using its acrosomal enzymes. The oocyte (o) extends its microvilli toward the sperm equatorial segment (e) so to speak kissing the sperm before fusion. zona PVS PVS

7 Sperm egg membrane fusion and sperm incorporation TEM The whole spermhead, midpiece and tail are progressively phagocytosed (swallowed) by the egg, lock, stock and barrel, soon after fusion. C = centriole in black box, PP=phagocytic process. zona tail PP sperm C midpiece midpiece midpiece oocyte

8 Sperm egg membrane fusion - diagram Midpiece C Ooplasm Fusogenic zone Oscillin Oop[asm a) Drawn 3h postinsemination. C=centriole in black box, I=inner acrosome membrane, O=oolemma, P=postacrosomal segment (anterior Tail region), S=SER, T=phagocytic process of egg, V=equatorial vestige, Fusogenic zone=p + V (modified Sathananthan et al,1986)

9 Progressive incorporation of sperm midpiece & tail into the ooplasm. Note peculiar filaments at point of incorporation TEM. zona tail filaments midpiece Golgi tail Oop[asm

10 Incorporation of the sperm tail into the ooplasm Note peculiar filaments at point of incorporation & cortical granule exocytosis into perivitelline space beneath the zona TEM filaments zona tail ooplasm filaments

11 Formation of male pronuclei after sperm incorporation TEM The spermhead expands chromatin to form the male pronucleus and introduces the male centriole from its neck that initiates cleavage & development, the true meaning of activation. This is the first report of a centriole in humans which led us to rethink and review its role in mammalian development, rewriting the text books in Ooplasm centriole pronucleus midpiece pronucleus Golgi mitochondria

12 Fertilization events diagram fulfilled by oocytes in this study A sperm-egg fusion. B second maturation division. C cortical reaction. D zona reaction. E association of male & female pronuclei. (Sperm aster forms around centriole soon after sperm incorporation)

13 References Familiari, G., Notolla, S.A, Micara, G. et al. (1988). Is sperm binding capability of the zona pullucida linked to its surface structure? A scanning electron microscopic study of human in vitro fertlization. J.In vito Fert. Embryo Transfer 5: Makabe, S., Nottola, A. and Motta, P.M. (1989) Life history of the female germ cells ultrastructural aspects. In: Ultrastructure of Human Embryology and Early Gametogenesis. J. Van Blerkom and P.M. Motta, eds. Kluwer Academic Publishers, Boston, pp Motta, P.M., Nottola, S.A, Familiari, G., et al. (1995) Ultrastructure of human reproduction from folliculogenesis to early embryo development. A review. It. J. Anat. Embryol., 100: 9 Sathananthan AH et al Human Sperm-Oocyte Fusion Mechanism of Fertilization: Plants to Humans Volume 45 of the series NATO ASI Series pp Sathananthan A H, Kola I, Osborne J, Trounson A, Ng S C, Bongso A, Ratnam S S (1991). Centrioles in the beginning of human development. Proc Natl Acad Sci, USA 88: Sathananthan A H (1985). Maturation of the human oocyte in vitro: nuclear events during meiosis (an ultrastructural study). Gamete Res 12: Sathananthan A H (ed) (1996) Visual atlas of human sperm structure and function for assisted reproduction, Serono,Singapore, pp279. Sathananthan A.H. (1997): Mitosis in the human embryo: the vital role of the sperm centrosome (centriole). Histol. Histopathol. 12: Sathananthan, A.H. (1998) Human fertilization.in: Modern Reproductive Medicine (eds) Suzuki et al, Medical View, Tokyo pp Sathananthan A.H. (1998): Paternal centrosomal dynamics in early human development. J. Assist. Reprod. Genet. 15: Sathananthan A.H. (2000): Ultrastructure of human gametes, fertilization and embryo development. In Trounson AO and Gardner DK (eds), Handbook of In Vitro Fertilization, 2nd ed., CRC Press, Boca Raton, Florida, pp Sathananthan, A.H. (2003) Morphology and pathology of the human oocyte. In Biology and Pathology of the Human Oocyte. A. Trounson and R. Gosden, eds. Cambridge University Press (in proofs). Sathananthan A H & Trounson A O (1982). Cortical granule release and zona interaction in monospermic and polyspermic human ova fertilized in vitro. Gamete Res. 6: Sathananthan A H, Ng S C, Edirisinghe R, Ratnam S S, Wong P C (1986). Sperm-oocyte interaction in the human during polyspermic fertilization in vitro. Gamete Res 15: Sathananthan AH & Chen C (1986) Sperm-oocyte membrane fusion in the human during monospermic fertilization. Gamete Res 15: Chen C and Sathananthan AH (1986) Early penetration of human sperm through the vestments of human eggs in vitro. Arch Androl 16, Sathananthan AH, Deen F eds Microscopic images of human sperm, oocytes and embryos for assisted reproductive technology, including embryonic stem cells. Google, E book of atlases.