Male Pronuclear Formation and Embryo Development Following Intracytoplasmic Injection of Ovine Pretreated Sperm

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Abstract

Background:

Failure of Male Pronucleus (MPN) formation is a major concern in the success of Intracytoplasmic Sperm Injection (ICSI) in some species. Despite the conducted unsuccessful efforts to improve ICSI efficiency in ovine, the present study was aimed to improve MPN formation and embryo development in ovine ICSI procedure through accompaniment of sperm pretreatment with co-injection of some compounds.

Methods:

In experiment 1, sperm were treated with either 2-mercaptoethanol (2ME), glutathione (GSH), or DTT (dithiothreitol) in combination with Heparin (Hep). Following DNA integrity and fragmentation assessments, the best sperm pretreatment approach in induction of sperm head decondensation was applied for the second and third experiments. In experiment 2, in vitro matured oocytes were subjected to ICSI using pretreated sperm with or without GSH and Sperm Extract (SE) co-injection. In experiment 3, the procedure was followed as experiment 2 with acrosome reacted sperm.

Results:

The highest percentages of oocyte activation were observed in Hep+GSH and Hep+2ME groups. The greatest MPN formations were also observed in the same groups when ICSI procedure was accompanied with GSH co-injection. Despite the higher percentage of MPN formation and oocyte activation in Hep+GSH and Hep+2ME groups, none of the employed strategies could increase the cleavage and blastocyst rates compared to the control.

Conclusion:

In our study condition, despite the lack of significant increase in embryo development in treated groups, the significant increase in MPN formation in Hep+ GSH+co.GSH and Hep+2ME+co.GSH groups indicates the lower chance of parthenote formation that means a higher chance of normal fertilization compared with control.

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Most cited references 43

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Glutathione concentration during maturation and after fertilization in pig oocytes: relevance to the ability of oocytes to form male pronucleus.

M Yoshida, M Chikyu, Taku Nagai … (1993)

The present study examined the kinetics of glutathione (GSH) concentration during maturation and after fertilization in pig oocytes and its relevance to the ability of pig oocytes to form a male pronucleus after in vitro fertilization. The GSH concentration was significantly higher in pig oocytes matured in Waymouth medium than in pig oocytes matured in either modified (m) TCM-199 or mTLP media. The addition of 0.04-0.57 mM cysteine (CySH) to mTLP significantly increased both the GSH concentrations in oocytes matured in vitro and the rate of male pronucleus formation as compared to those in oocytes cultured in mTLP alone. When pig oocytes were cultured 12, 24, or 36 h in mTLP plus 0.14 mM CySH, their GSH concentrations were significantly higher than in uncultured oocytes. After fertilization, the GSH concentration in pig oocytes declined significantly. GSH concentrations in oocytes matured in vivo did not differ from those in oocytes matured in mTLP plus 0.14 or 0.57 mM CySH. The results indicate that 1) the composition of maturation medium affects the GSH concentration in pig oocytes; 2) the addition of CySH to maturation medium permits GSH synthesis by the pig oocytes; 3) GSH levels in pig oocytes change during maturation and after fertilization; and 4) GSH synthesis during oocyte maturation is an important factor for promoting their ability to form a male pronucleus after fertilization.

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Depletion of glutathione during bovine oocyte maturation reversibly blocks the decondensation of the male pronucleus and pronuclear apposition during fertilization.

G Schatten, P Sutovsky (1997)

Oocyte-produced glutathione (the tripeptide gamma-glutamyl-cysteinyl-glycine; GSH) has been implicated in the reduction of disulfide bonds in the sperm nucleus during fertilization and thus in the development of the male pronucleus (PN). In this study, we show that the depletion of endogenous glutathione by 10 mM buthionine sulfoximine (BSO; specific inhibitor of GSH synthesis) during bovine oocyte maturation (24 h in vitro; represents prophase I to metaphase II transition in this species) blocks the formation of a male PN in > 85% of treated oocytes (vs. 6.8% in controls) and prevents the assembly of the sperm aster microtubules in approximately 35%. Consequently, the pronuclear migration and apposition do not occur. Ultrastructural observations suggest that the effect of BSO on pronuclear apposition might be due to incomplete disassembly of the sperm tail connecting piece, which normally leads to the release of the sperm centriole and to the reconstitution of the zygotic centrosome during fertilization. The sperm nucleus decondensation and migration blocks were reversed by the treatment of the GSH-depleted oocytes with 1-10 mM dithiothreitol (a disulfide bond-reducing agent) applied 8 h after insemination: 82% of these oocytes exhibited a normal male PN and pronuclear apposition 20 h after insemination. The pool of glutathione seems to be generated during oocyte maturation since > 80% of oocytes that were matured in the absence of BSO displayed a normal male PN, as apposed to a female PN, when inseminated and cultured in the presence of 10 mM BSO. These data suggest that the reduction of disulfide bonds in the sperm after incorporation is important for the formation of the male PN, as well as for the disassembly of the sperm tail connecting piece and pronuclear apposition. The lack of disulfide-reducing power in the GSH-depleted oocytes can be reversed by treatment with disulfide bond-reducing agents.

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Simultaneous removal of sperm plasma membrane and acrosome before intracytoplasmic sperm injection improves oocyte activation/embryonic development.

R Yanagimachi, Shunichi Miyazaki, T Shikano … (2006)

Direct injection of a single spermatozoon into an oocyte (ICSI) can produce apparently normal offspring. Although the production of normal offspring by ICSI has been successful in mice and humans, it has been less successful in many other species. The reason for this is not clear, but could be, in part, due to inconsistent activation of oocytes because of delayed disintegration of sperm plasma membrane within oocytes and incorporation of the acrosome containing a spectrum of hydrolyzing enzymes. In the mouse, the removal of sperm plasma membrane and acrosome was not a prerequisite to produce offspring by ICSI, but it resulted in earlier onset of oocyte activation and better embryonic development. The best result was obtained when spermatozoa were demembranated individually immediately before ICSI by using lysolecithin, a hydrolysis product of membrane phospholipids.

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Author and article information

Journal

Journal ID (nlm-ta): Avicenna J Med Biotechnol

Journal ID (iso-abbrev): Avicenna J Med Biotechnol

Journal ID (publisher-id): AJMB

Journal ID (pmc): AJMB

Title: Avicenna Journal of Medical Biotechnology

Publisher: Avicenna Research Institute

ISSN (Print): 2008-2835

ISSN (Electronic): 2008-4625

Publication date (Print): Jan-Mar 2018

Volume: 10

Issue: 1

Pages: 41-48

Affiliations

[1. ]Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR, Tehran, Iran

[2. ]Department of Gametes and Cloning, Research Institute of Animal Embryo Technology, Shahrekord University, Shahrekord, Iran

Author notes

[* ] Corresponding author: Abolfazl Shirazi, Ph.D., Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR, Tehran, Iran, Tel: +98 21 22404144, Fax: +98 21 22404145, E-mail: shiraziabbas@ 123456yahoo.com

Article

Publisher ID: ajmb-10-41

PMC ID: 5742653

SO-VID: d8ad946e-c2c8-4265-8b32-2559cc7c4af1

License:

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Male Pronuclear Formation and Embryo Development Following Intracytoplasmic Injection of Ovine Pretreated Sperm

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Abstract

Background:

Methods:

Results:

Conclusion:

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Most cited references 43

Glutathione concentration during maturation and after fertilization in pig oocytes: relevance to the ability of oocytes to form male pronucleus.

Depletion of glutathione during bovine oocyte maturation reversibly blocks the decondensation of the male pronucleus and pronuclear apposition during fertilization.

Simultaneous removal of sperm plasma membrane and acrosome before intracytoplasmic sperm injection improves oocyte activation/embryonic development.

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