Talk:Fertilization: Difference between revisions

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PMID 17307906
PMID 17307906
==2002==
===Time from insemination to first cleavage predicts developmental competence of human preimplantation embryos in vitro===
Hum Reprod. 2002 Feb;17(2):407-12.
Fenwick J, Platteau P, Murdoch AP, Herbert M.
Source
Reproductive Medicine, BioScience Centre, ICFL, Times Square, Newcastle upon Tyne NE1 4EP, UK.
Abstract
BACKGROUND:
The absence of reliable markers for the identification of viable embryos for transfer at the early cleavage stage is likely to contribute to the generally low implantation rates and high incidence of multiple gestation in IVF treatment. In this study, we investigate the relationship between timing of first cleavage and the incidence of blastocyst formation in vitro.
METHODS:
Couples (n = 70) with at least one embryo remaining after transfer were included in the analyses. All embryos (n = 579) were examined for early cleavage at 25 h after insemination. Following embryo transfer, the remaining embryos (n = 426) were cultured until day 7 of development, and assessed for blastocyst formation.
RESULTS:
Eighty-five embryos (14.7%) cleaved to the 2-cell stage within 25 h of insemination; 26 of these were selected for transfer on day 2. Of the 59 embryos remaining in culture, 19 (32.2%) developed to the blastocyst stage; this was a significantly higher number than was observed in embryos (61/367; 16.6%) that failed to cleave within 25 h of insemination (P < 0.01). Within these two groups of embryos the proportion of hatched blastocysts was 11/59 (18.6%) and 26/367 (7.1%) respectively (P < 0.005).
CONCLUSIONS:
These findings indicate that early cleavage is indicative of increased developmental potential in human embryos and may be useful as an additional criterion in the selection of embryos for transfer.
PMID 11821286


==1999==
==1999==

Revision as of 09:16, 20 April 2012

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Cite this page: Hill, M.A. (2024, March 28) Embryology Fertilization. Retrieved from https://embryology.med.unsw.edu.au/embryology/index.php/Talk:Fertilization

2011

CD9 tetraspanin generates fusion competent sites on the egg membrane for mammalian fertilization

Proc Natl Acad Sci U S A. 2011 Jun 20. [Epub ahead of print]

Jégou A, Ziyyat A, Barraud-Lange V, Perez E, Wolf JP, Pincet F, Gourier C. Source Laboratoire de Physique Statistique, Ecole Normale Supérieure de Paris, Université Pierre et Marie Curie, Université Paris Diderot, Centre National de la Recherche Scientifique, 24 rue Lhomond, 75005 Paris, France.

Abstract

CD9 tetraspanin is the only egg membrane protein known to be essential for fertilization. To investigate its role, we have measured, on a unique acrosome reacted sperm brought in contact with an egg, the adhesion probability and strength with a sensitivity of a single molecule attachment. Probing the binding events at different locations of wild-type egg we described different modes of interaction. Here, we show that more gamete adhesion events occur on Cd9 null eggs but that the strongest interaction mode disappears. We propose that sperm-egg fusion is a direct consequence of CD9 controlled sperm-egg adhesion properties. CD9 generates adhesion sites responsible for the strongest of the observed gamete interaction. These strong adhesion sites impose, during the whole interaction lifetime, a tight proximity of the gamete membranes, which is a requirement for fusion to take place. The CD9-induced adhesion sites would be the actual location where fusion occurs.

PMID 21690351

Most fertilizing mouse spermatozoa begin their acrosome reaction before contact with the zona pellucida during in vitro fertilization

Proc Natl Acad Sci U S A. 2011 Mar 22;108(12):4892-6. Epub 2011 Mar 7.

Jin M, Fujiwara E, Kakiuchi Y, Okabe M, Satouh Y, Baba SA, Chiba K, Hirohashi N.

Department of Biological Sciences, Department of Genetic Counseling, and Glycoscience Institute, Ochanomizu University, Tokyo 112-8610, Japan. Abstract To fuse with oocytes, spermatozoa of eutherian mammals must pass through extracellular coats, the cumulus cell layer, and the zona pellucida (ZP). It is generally believed that the acrosome reaction (AR) of spermatozoa, essential for zona penetration and fusion with oocytes, is triggered by sperm contact with the zona pellucida. Therefore, in most previous studies of sperm-oocyte interactions in the mouse, the cumulus has been removed before insemination to facilitate the examination of sperm-zona interactions. We used transgenic mouse spermatozoa, which enabled us to detect the onset of the acrosome reaction using fluorescence microscopy. We found that the spermatozoa that began the acrosome reaction before reaching the zona were able to penetrate the zona and fused with the oocyte's plasma membrane. In fact, most fertilizing spermatozoa underwent the acrosome reaction before reaching the zona pellucida of cumulus-enclosed oocytes, at least under the experimental conditions we used. The incidence of in vitro fertilization of cumulus-free oocytes was increased by coincubating oocytes with cumulus cells, suggesting an important role for cumulus cells and their matrix in natural fertilization.

PMID 21383182

Sperm Proteasomes Degrade Sperm Receptor on the Egg Zona Pellucida during Mammalian Fertilization

PLoS One. 2011 Feb 23;6(2):e17256.

Zimmerman SW, Manandhar G, Yi YJ, Gupta SK, Sutovsky M, Odhiambo JF, Powell MD, Miller DJ, Sutovsky P.

Division of Animal Science, and Departments of Obstetrics, Gynecology, and Women's Health, University of Missouri-Columbia, Columbia, Missouri, United States of America. Abstract Despite decades of research, the mechanism by which the fertilizing spermatozoon penetrates the mammalian vitelline membrane, the zona pellucida (ZP) remains one of the unexplained fundamental events of human/mammalian development. Evidence has been accumulating in support of the 26S proteasome as a candidate for echinoderm, ascidian and mammalian egg coat lysin. Monitoring ZP protein degradation by sperm during fertilization is nearly impossible because those few spermatozoa that penetrate the ZP leave behind a virtually untraceable residue of degraded proteins. We have overcome this hurdle by designing an experimentally consistent in vitro system in which live boar spermatozoa are co-incubated with ZP-proteins (ZPP) solubilized from porcine oocytes. Using this assay, mimicking sperm-egg interactions, we demonstrate that the sperm-borne proteasomes can degrade the sperm receptor protein ZPC. Upon coincubation with motile spermatozoa, the solubilized ZPP, which appear to be ubiquitinated, adhered to sperm acrosomal caps and induced acrosomal exocytosis/formation of the acrosomal shroud. The degradation of the sperm receptor protein ZPC was assessed by Western blotting band-densitometry and proteomics. A nearly identical pattern of sperm receptor degradation, evident already within the first 5 min of coincubation, was observed when the spermatozoa were replaced with the isolated, enzymatically active, sperm-derived proteasomes. ZPC degradation was blocked by proteasomal inhibitors and accelerated by ubiquitin-aldehyde(UBAL), a modified ubiquitin protein that stimulates proteasomal proteolysis. Such a degradation pattern of ZPC is consistent with in vitro fertilization studies, in which proteasomal inhibitors completely blocked fertilization, and UBAL increased fertilization and polyspermy rates. Preincubation of intact zona-enclosed ova with isolated active sperm proteasomes caused digestion, abrasions and loosening of the exposed zonae, and significantly reduced the fertilization/polyspermy rates after IVF, accompanied by en-mass detachment of zona bound sperm. Thus, the sperm borne 26S proteasome is a candidate zona lysin in mammals. This new paradigm has implications for contraception and assisted reproductive technologies in humans, as well as animals.

PMID 21383844

2010

Mechanisms of sperm-egg interactions: between sugars and broken bonds

Sci Signal. 2010 Oct 5;3(142):pe35.

Visconti PE, Florman HM.

Department of Veterinary and Animal Sciences, University of Massachusetts, Amherst, MA 01003, USA. pvisconti@vasci.umass.edu Abstract A model of the early events of mammalian fertilization has emerged during the past 30 years. However, studies during the past decade have used newly available mouse models to readdress these processes. Here, we will consider these new data in light of the existing model and point to areas of reconciliation and of controversy.

PMID 20923932

http://stke.sciencemag.org/cgi/content/full/sigtrans;3/142/pe35

Paper suggests the following 3 models for Mouse Models of sperm binding to the ZP.

1. The glycan model - Sperm bind by interacting selectively with O-glycans conjugated to ZP3 at Ser332, Ser334, or both residues. Sperm fail to bind after fertilization because of the enzymatic modification of the adhesion glycan by an egg glycosidase.

2. The supermolecular structure model - The sperm binding site is not specified (shown as including elements of ZP2 and ZP3). Access to this binding site is regulated by proteolytic cleavage of ZP2, such that after fertilization sperm are restricted from this site and cannot bind to the ZP.

3. A hybrid model - Sperm bind to an O-glycan that is conjugated to ZP3 at a site other than Ser332 or Ser334. Sperm access to this glycan is regulated by the proteolytic cleavage state of ZP2.

The role and regulation of sperm gelsolin prior to fertilization

J Biol Chem. 2010 Oct 11.

Finkelstein M, Etkovitz N, Breitbart H.

Bar-Ilan University, Israel. Abstract In order to acquire fertilization competence, spermatozoa should undergo several biochemical changes in the female reproductive tract, known as capacitation. The capacitated spermatozoon can interact with the egg zona-pellucida resulting in the occurrence of the acrosome reaction, a process which allowed its penetration into the egg and fertilize it. Sperm capacitation requires actin polymerization, while F-actin must disperse prior to the acrosome reaction. Here we suggest that the actin severing protein, gelsolin, is inactive during capacitation and is activated prior to the acrosome reaction. The release of bound gelsolin from phosphatidylinositol 4,5-bisphosphate (PIP(2)) by PBP10, a peptide containing the PIP2-binding domain of gelsolin, or by activation of PLC which hydrolyses PIP(2), caused rapid Ca(2+)-dependent F-actin depolymerization as well as enhanced acrosome reaction. Using immunoprecipitation assays, we showed that the tyrosine kinase SRC and gelsolin coimmunoprecipitate, and activating SRC by adding 8Br-cAMP enhanced the amount of gelsolin in this precipitate. Moreover, 8Br-cAMP enhanced tyrosine phosphorylation of gelsolin and its binding to PIP(2(4,5)), both of which inactivated gelsolin, allowing actin polymerization during capacitation. This actin polymerization was blocked by inhibiting the Src-family-kinases, suggesting that gelsolin is activated under these conditions. These results are further supported by our finding that PBP10 was unable to cause complete F-actin breakdown in the presence of 8Br-cAMP or vanadate. In conclusion, inactivation of gelsolin during capacitation occurs by its binding to PIP(2) and tyrosine phosphorylation by SRC. The release of gelsolin from PIP(2) together with its dephosphorylation enables gelsolin activation, resulting in the acrosome reaction.

PMID 20937821

Sperm head binding to epithelium of the oviduct isthmus is not an essential preliminary to mammalian fertilization - review

Zygote. 2010 Jul 21:1-5.

Hunter RH.

Institute for Reproductive Medicine, Hannover Veterinary University, Bünteweg 15, D-30559 Hannover, Germany. Abstract SummaryIn endeavouring to understand the nature of sperm-oviduct interactions in mammals, attention was focused on experimental models in which fertilization can occur without a preliminary phase of sperm head binding to the isthmus epithelium. The ovarian endocrine milieu imposed on the oviduct tissues plays an important role in the binding phenomenon, although less so after the time of ovulation. Nonetheless, a sperm suspension introduced into the peritoneal cavity or surgical insemination directly into the oviduct ampulla before ovulation can result in fertilization, as can a surgical model in which the isthmus has been resected and the remaining portions of the duct reanastomosed. Mating or artificial insemination after ovulation in pigs permits rapid sperm transport to the site of fertilization, and the frequency of polyspermic penetration increases with the post-ovulatory age of eggs.Strategies underlying sperm binding were considered, especially in terms of preovulatory sperm storage and suppression of full membranous maturation. These, in turn, raised the problem of how sperm binding in vitro to oviduct cells from prepuberal animals or to cells harvested during the luteal phase of the estrous cycle, or to cells from the ampulla or even the tracheal epithelium, can act to regulate sperm storage and maturation with precision. In an evolutionary perspective, preovulatory binding of diverse populations of cells to the endosalpinx may have developed as a form of fine tuning to assist in sperm selection, to synchronize completion of capacitation with the events of ovulation, and to promote monospermic fertilization by a controlled release of competent gametes.

PMID 20663263

2009

Reduction of mouse egg surface integrin alpha9 subunit (ITGA9) reduces the egg's ability to support sperm-egg binding and fusion

Biol Reprod. 2009 Apr;80(4):833-41. Epub 2009 Jan 7.

Vjugina U, Zhu X, Oh E, Bracero NJ, Evans JP.

Department of Biochemistry and Molecular Biology, Division of Reproductive Biology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland 21205, USA. Abstract The involvement of egg integrins in mammalian sperm-egg interactions has been controversial, with data from integrin inhibitor studies contrasting with evidence from knockouts showing that specific integrin subunits are not essential for fertility. An alpha(4)/alpha(9) (ITGA4/ITGA9) integrin subfamily member has been implicated in fertilization but not extensively examined, so we tested the following three hypotheses: 1) an ITGA4/ITGA9 integrin participates in sperm-egg interactions, 2) short-term acute knockdown by RNA interference of integrin subunits would result in a fertilization phenotype differing from that of chronic depletion via knockout, and 3) detection of a fertilization phenotype is sensitive to in vitro fertilization (IVF) assay conditions. We show that mouse and human eggs express the alpha(9) integrin subunit (ITGA9). RNA interference-mediated knockdown resulted in reduced levels of Itga9 mRNA and surface protein in mouse eggs. RNA interference attempts to knockdown ITGA9's likely beta partner, beta(1) (ITGB1), resulted in reduced Itgb1 mRNA but no reduction in ITGB1 surface protein. Therefore, studies using a function-blocking anti-ITGB1 antibody tested the hypothesis that ITGB1 participates in gamete interactions. Analyses of sperm-egg interactions with Itga9-knockdown eggs and anti-ITGB1 antibody-treated eggs in IVF assays using specific sperm:egg ratios revealed the following: 1) a reduction, but not complete loss, of sperm-egg binding and fusion was observed and 2) the reduction of sperm-egg binding and fusion was not detected in inseminations with high sperm:egg ratios. These data demonstrate that ITGA9 and ITGB1 participate in sperm-egg interactions but clearly are not the only molecules involved. This also shows that careful design of IVF parameters allows detection of deficiencies in gamete interactions.

PMID 19129508

Etiology of sperm immunity in women

Fertil Steril. 2009 Feb;91(2):639-43. Epub 2008 Feb 20.

Clarke GN.

Andrology Unit, Royal Women's Hospital, Melbourne, Victoria, Australia. gary.clarke@rch.org.au

Abstract Sperm immunity in females can reduce the likelihood of natural conception, and sperm antibodies from female sera have been shown to inhibit IVF in humans and in several animal models. The etiology of sperm immunity in human females is unknown, but several possible mechanisms have been proposed, including cross-reactivity with microbial antigens and interferon gamma-mediated potentiation of the antisperm immune response in women whose male partners have sperm autoantibodies in their semen. This article reviews these ideas and postulates a novel hypothesis based on the potential for the generation of anti-idiotype antibodies in women whose partners have sperm antibodies in their semen.

PMID 18281044

Egg coat proteins activate calcium entry into mouse sperm via CATSPER channels

Xia J, Ren D. Biol Reprod. 2009 Jun;80(6):1092-8. Epub 2009 Feb 11. PMID: 19211808

2008

IJDB

Vol. 52 Nos. 5/6 (2008) Fertilization

http://www.ijdb.ehu.es/web/contents.php?vol=52&issue=5-6

Participation of cysteine-rich secretory proteins (CRISP) in mammalian sperm-egg interaction

Int J Dev Biol. 2008;52(5-6):737-42.

Cohen DJ, Busso D, Da Ros V, Ellerman DA, Maldera JA, Goldweic N, Cuasnicu PS.

Instituto de Biología y Medicina Experimental (IBYME-CONICET), Buenos Aires, Argentina. Abstract Mammalian fertilization is a complex multi-step process mediated by different molecules present on both gametes. CRISP1 (cysteine-rich secretory protein 1) is an epididymal protein thought to participate in gamete fusion through its binding to egg-complementary sites. Structure-function studies using recombinant fragments of CRISP1 as well as synthetic peptides reveal that its egg-binding ability resides in a 12 amino acid region corresponding to an evolutionary conserved motif of the CRISP family, named Signature 2 (S2). Further experiments analyzing both the ability of other CRISP proteins to bind to the rat egg and the amino acid sequence of their S2 regions show that the amino acid sequence of the S2 is needed for CRISP1 to interact with the egg. CRISP1 appears to be involved in the first step of sperm binding to the zona pellucida, identifying a novel role for this protein in fertilization. The observation that sperm testicular CRISP2 is also able to bind to the egg surface suggests a role for this protein in gamete fusion. Subsequent experiments confirmed the participation of CRISP2 in this step of fertilization and revealed that CRISP1 and CRISP2 interact with common egg surface binding sites. Together, these results suggest a functional cooperation between CRISP1 and CRISP2 to ensure the success of fertilization. These observations contribute to a better understanding of the molecular mechanisms underlying mammalian fertilization.

PMID 18649285

Laparoscopic observation of spontaneous human ovulation

Lousse JC, Donnez J.

Fertil Steril. 2008 Sep;90(3):833-4. Epub 2008 Apr 28.

Department of Gynecology, Université Catholique de Louvain, 1200 Brussels, Belgium. Abstract We report laparoscopic observation of spontaneous human ovulation, illustrated by protrusion of the mature follicle and oocyte release into the peritoneal cavity.

PMID: 18440526


Sperm binding to the zona pellucida is not sufficient to induce acrosome exocytosis.

Baibakov B, Gauthier L, Talbot P, Rankin TL, Dean J. Development. 2007 Mar;134(5):933-43. PMID: 17293534

ZP2 and ZP3 traffic independently within oocytes prior to assembly into the extracellular zona pellucida

Hoodbhoy T, Avilés M, Baibakov B, Epifano O, Jiménez-Movilla M, Gauthier L, Dean J. Mol Cell Biol. 2006 Nov;26(21):7991-8. PMID: 17047254


inactive X chromosome

Cell cycle-dependent localization of macroH2A in chromatin of the inactive X chromosome. Chadwick BP, Willard HF. J Cell Biol. 2002 Jun 24;157(7):1113-23. Epub 2002 Jun 24. PMID: 12082075 \ JCB

Histone modifications and nuclear architecture: a review.

Bártová E, Krejcí J, Harnicarová A, Galiová G, Kozubek S.

J Histochem Cytochem. 2008 Aug;56(8):711-21. Epub 2008 May 12. Review.PMID: 18474937

2007

Establishment of the mammalian membrane block to polyspermy: evidence for calcium-dependent and -independent regulation

Reproduction. 2007 Feb;133(2):383-93.

Gardner AJ, Williams CJ, Evans JP.

Division of Reproductive Biology, Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Room W3606, 615 N. Wolfe St., Maryland, USA. Abstract One crucial result of egg activation is the establishment of blocks on the zona pellucida and the egg plasma membrane to prevent fertilization by additional sperm. The mechanism(s) by which a mammalian egg regulates the establishment of the membrane block to polyspermy is largely unknown. Since Ca(2+) signaling regulates several egg activation events, this study investigates how sperm-induced Ca(2+) transients affect the membrane block to polyspermy, building on our previous work (Biology of Reproduction 67:1342). We demonstrate that mouse eggs that experience only one sperm-induced Ca(2+) transient establish a membrane block that is less effective, than in eggs that experience normal sperm-induced Ca(2+) transients but that is more effective than in eggs with completely suppressed [Ca(2+)](cyt) increases. Sperm-induced increases in [Ca(2+)](cyt) regulate the timing of membrane block establishment, as this block is established more slowly in eggs that experience one or no sperm-induced Ca(2+) transients. Finally, our studies produce the intriguing discovery that there is also a Ca(2+)-independent event that is associated with fertilization in the pathway leading to membrane block establishment. Taken together, these data indicate that Ca(2+) plays a role in facilitating membrane block establishment by regulating the timing with which this change in egg membrane function occurs, and also that the membrane block differs from other post-fertilization egg activation responses as Ca(2+) is not the only stimulus. The membrane block to polyspermy in mammalian eggs is likely to be the culmination of multiple post-fertilization events that together modify the egg membrane's receptivity to sperm.

PMID 17307906

2002

Time from insemination to first cleavage predicts developmental competence of human preimplantation embryos in vitro

Hum Reprod. 2002 Feb;17(2):407-12.

Fenwick J, Platteau P, Murdoch AP, Herbert M. Source Reproductive Medicine, BioScience Centre, ICFL, Times Square, Newcastle upon Tyne NE1 4EP, UK.

Abstract

BACKGROUND: The absence of reliable markers for the identification of viable embryos for transfer at the early cleavage stage is likely to contribute to the generally low implantation rates and high incidence of multiple gestation in IVF treatment. In this study, we investigate the relationship between timing of first cleavage and the incidence of blastocyst formation in vitro. METHODS: Couples (n = 70) with at least one embryo remaining after transfer were included in the analyses. All embryos (n = 579) were examined for early cleavage at 25 h after insemination. Following embryo transfer, the remaining embryos (n = 426) were cultured until day 7 of development, and assessed for blastocyst formation. RESULTS: Eighty-five embryos (14.7%) cleaved to the 2-cell stage within 25 h of insemination; 26 of these were selected for transfer on day 2. Of the 59 embryos remaining in culture, 19 (32.2%) developed to the blastocyst stage; this was a significantly higher number than was observed in embryos (61/367; 16.6%) that failed to cleave within 25 h of insemination (P < 0.01). Within these two groups of embryos the proportion of hatched blastocysts was 11/59 (18.6%) and 26/367 (7.1%) respectively (P < 0.005). CONCLUSIONS: These findings indicate that early cleavage is indicative of increased developmental potential in human embryos and may be useful as an additional criterion in the selection of embryos for transfer.

PMID 11821286

1999

Magnetic resonance imaging of male and female genitals during coitus and female sexual arousal

BMJ. 1999 Dec 18-25;319(7225):1596-600.

Schultz WW, van Andel P, Sabelis I, Mooyaart E.

Department of Gynaecology, University Hospital Groningen, PO Box 30 001, 9700 RB Groningen, Netherlands. w.c.m.weymar.schultz@oprit.rug.nl Abstract OBJECTIVE: To find out whether taking images of the male and female genitals during coitus is feasible and to find out whether former and current ideas about the anatomy during sexual intercourse and during female sexual arousal are based on assumptions or on facts.

DESIGN: Observational study.

SETTING: University hospital in the Netherlands.

METHODS: Magnetic resonance imaging was used to study the female sexual response and the male and female genitals during coitus. Thirteen experiments were performed with eight couples and three single women.

RESULTS: The images obtained showed that during intercourse in the "missionary position" the penis has the shape of a boomerang and 1/3 of its length consists of the root of the penis. During female sexual arousal without intercourse the uterus was raised and the anterior vaginal wall lengthened. The size of the uterus did not increase during sexual arousal.

CONCLUSION: Taking magnetic resonance images of the male and female genitals during coitus is feasible and contributes to understanding of anatomy.

PMID: 10600954 http://www.ncbi.nlm.nih.gov/pubmed/10600954

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC28302

http://www.bmj.com/content/319/7225/1596.full

1998

Post-ovulatory ageing of the human oocyte and embryo failure

Hum Reprod. 1998 Feb;13(2):394-7.

Wilcox AJ, Weinberg CR, Baird DD. Source Epidemiology Branch, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709, USA. Abstract We carried out a prospective study of 221 healthy women who were attempting pregnancy. During the study, women collected daily urine samples and kept daily records of intercourse. Ovulation and early pregnancy losses were later identified by immunoassays of urinary human chorionic gonadotrophin and steroid metabolites. We have used these data to examine whether the risk of early pregnancy loss was higher with post-ovulatory ageing of the oocyte. 192 pregnancies were ranked by the probability that the oocyte might have aged before fertilization. There was a statistically significant increase in the risk of early loss as the likelihood of oocyte ageing increased (P < 0.05). No similar risk was observed for clinical miscarriages. Post-ovulatory ageing of the oocyte prior to fertilization may cause early pregnancy failure in humans as it does in several other mammalian species.

PMID 9557845

1997

Preliminary observations on polar body extrusion and pronuclear formation in human oocytes using time-lapse video cinematography

Hum Reprod. 1997 Mar;12(3):532-41.

Payne D, Flaherty SP, Barry MF, Matthews CD. Source Reproductive Medicine Unit, The University of Adelaide, The Queen Elizabeth Hospital, South Australia.

Abstract

In this study, we have used time-lapse video cinematography to study fertilization in 50 human oocytes that had undergone intracytoplasmic sperm injection (ICSI). Time-lapse recording commenced shortly after ICSI and proceeded for 17-20 h. Oocytes were cultured in an environmental chamber which was maintained under standard culture conditions. Overall, 38 oocytes (76%) were fertilized normally, and the fertilization rate and embryo quality were not significantly different from 487 sibling oocytes cultured in a conventional incubator. Normal fertilization followed a defined course of events, although the timing of these events varied markedly between oocytes. In 35 of the 38 fertilized oocytes (92%), there were circular waves of granulation within the ooplasm which had a periodicity of 20-53 min. The sperm head decondensed during this granulation phase. The second polar body was then extruded, and this was followed by the central formation of the male pronucleus. The female pronucleus formed in the cytoplasm adjacent to the second polar body at the same time as, or slightly after, the male pronucleus, and was subsequently drawn towards the male pronucleus until the two abutted. Both pronuclei then increased in size, the nucleoli moved around within the pronuclei and some nucleoli coalesced. During pronuclear growth, the organelles contracted from the cortex towards the centre of the oocyte, leaving a clear cortical zone. The oocyte decreased in diameter from 112 to 106 microm (P < 0.0001) during the course of the observation period. The female pronucleus was significantly smaller in diameter than the male pronucleus (24.1 and 22.4 microm respectively, P = 0.008) and contained fewer nucleoli (4.2 and 7.0 respectively, P < 0.0001). After time-lapse recording, oocytes were cultured for 48 h prior to embryo transfer or cryopreservation. Embryo quality was related to fertilization events and periodicity of the cytoplasmic wave, and it was found that good quality embryos arose from oocytes that had more uniform timing from injection to pronuclear abuttal and tended to have a longer cytoplasmic wave. In conclusion, we have shown that time-lapse video cinematography is an excellent tool for studying fertilization and early embryo development, and have demonstrated that human fertilization comprises numerous complex dynamic events.

PMID 9130755

A role for the disintegrin domain of cyritestin, a sperm surface protein belonging to the ADAM family, in mouse sperm-egg plasma membrane adhesion and fusion

J Cell Biol. 1997 Apr 7;137(1):105-12.

Yuan R, Primakoff P, Myles DG.

Molecular and Cellular Biology, University of California, Davis 95616, USA. Abstract Sperm-egg plasma membrane fusion is preceded by sperm adhesion to the egg plasma membrane. Cell-cell adhesion frequently involves multiple adhesion molecules on the adhering cells. One sperm surface protein with a role in sperm-egg plasma membrane adhesion is fertilin, a transmembrane heterodimer (alpha and beta subunits). Fertilin alpha and beta are the first identified members of a new family of membrane proteins that each has the following domains: pro-, metalloprotease, disintegrin, cysteine-rich, EGF-like, transmembrane, and cytoplasmic domain. This protein family has been named ADAM because all members contain a disintegrin and metalloprotease domain. Previous studies indicate that the disintegrin domain of fertilin beta functions in sperm-egg adhesion leading to fusion. Full length cDNA clones have been isolated for five ADAMs expressed in mouse testis: fertilin alpha, fertilin beta, cyritestin, ADAM 4, and ADAM 5. The presence of the disintegrin domain, a known integrin ligand, suggests that like fertilin beta, other testis ADAMs could be involved in sperm adhesion to the egg membrane. We tested peptide mimetics from the predicted binding sites in the disintegrin domains of the five testis-expressed ADAMs in a sperm-egg plasma membrane adhesion and fusion assay. The active site peptide from cyritestin strongly inhibited (80-90%) sperm adhesion and fusion and was a more potent inhibitor than the fertilin beta active site peptide. Antibodies generated against the active site region of either cyritestin or fertilin beta also strongly inhibited (80-90%) both sperm-egg adhesion and fusion. Characterization of these two ADAM family members showed that they are both processed during sperm maturation and present on mature sperm. Indirect immunofluorescence on live, acrosome-reacted sperm using antibodies against either cyritestin or fertilin beta showed staining of the equatorial region, a region of the sperm membrane that participates in the early steps of membrane fusion. Collectively, these data indicate that a second ADAM family member, cyritestin, functions with fertilin beta in sperm-egg plasma membrane adhesion leading to fusion.

PMID 9105040