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

Original Article Male Pronuclear Formation and Embryo Development Following Intracytoplasmic Injection of Ovine Pretreated Sperm Abolfazl Shirazi 1,2*, Arefeh Golestanfar 2, Masomeh Bashiri 2, Ebrahim Ahmadi 2, and Naser Shams-Esfandabadi 2 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 Abstract * 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: Received: 24 Jan 2017 Accepted: 15 Apr 2017 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. Avicenna J Med Biotech 2018; 10(1): 41-48 Keywords: Intracytoplasmic sperm injection (ICSI), Male, Ovine, Pronucleus Introduction Intracytoplasmic Sperm Injection (ICSI) as an integral part of assisted reproduction has become increasingly popular in human male patients with certain infertility problems 1. In animal species, this technique has several applications such as avoidance of the polyspermy problem (e.g. porcine) 2, extending the sperm vector system for transgenic animal production, and preserving the endangered species. ICSI also provides an opportunity for research into cell cycle control and mechanisms involved in sperm-induced oocyte activation 3. A problem commonly encountered in ICSI technique is the low rate of viability of the microinjected oocytes and inconsistent level of male pronuclear for- mation after microinjection. The inadequate sperm chromatin decondensation and its transformation into the Male Pronucleus (MPN) together with a failure to activate the oocyte seem to be the major causes behind the poor ICSI efficiency in some species 4,5. In this context, while ICSI alone in some species such as mice, humans, hamsters, and rabbits is sufficient to activate the oocytes for further embryonic development 6-10, in other species such as cattle, sheep, pig 11,12, buffalo 13, and horse 14,15, an additional parthenogenetic activation is necessary to activate the oocytes after ICSI 16. The nuclei of mammalian spermatozoa are genetically inactivated and structurally stabilized by association of sperm DNA with protamines. Copyright © 2018, Avicenna Journal of Medical Biotechnology. All rights reserved. Vol. 10, No. 1, January-March 2018 41 Male Pronuclear Formation Under in vitro conditions, decondensation of mammalian sperm nuclei can be induced by sperm pretreatment with a disulfide-reducing agent alone 17 or together with neutral detergents 18, anionic detergents 19, proteases 20, salts 21 or mechanical demembranation 22. In mice, the success of ICSI could be improved by the removal of both sperm membranes and acrosome before injection 23. Moreover, sperm pretreatment can affect the ability of oocytes to activate 12. Among mechanical pre-treatments, sonication 24 and repetitive freezing/thawing without cryoprotectants 25 have been reported to improve MPN formation after ICSI. However, sperm pre-treatment with DTT 26 and Triton X-100 26 or sperm freezing 27 has been documented to cause reduction of oocyte-activating capacity of porcine oocytes following ICSI. These treatments were aimed to remove sperm membrane so that to improve male pronuclear formation and to speed up the availability of sperm-borne oocyte activation factor to the oocyte 5,28. Regarding the strategies applied on the sperm to improve normal fertilization and embryo development after ICSI, several treatment regimens have been tested to disrupt or even remove the sperm plasma membrane to allow a more direct interaction of sperm with the oocyte cytoplasm. In line with our previous studies 12 indicating the low efficiency of ICSI in sheep, the purpose of this study was to evaluate the influence of several strategies to improve male pronuclear formation and normal fertilization following ICSI in this species. Materials and Methods Except where otherwise indicated, all chemicals were obtained from the Sigma (St. Louis, MO, USA). Oocyte collection and in vitro maturation Abattoir obtained sheep ovaries were transported to the laboratory in saline (25-30C) in a thermos flask within 2-3 hr. Ovaries were washed three times with prewarmed fresh saline (37C), and all visible follicles with a diameter of 2-6 mm were aspirated using gentle vacuum (30 mmHg) via a 20 gauge short beveled needle connected to a vacuum pump. Prior to aspiration, the collecting tube was filled with 2 ml preincubated hepes-modified TCM, supplemented with 50 IU/ml heparin. After aspiration, only oocytes surrounded by more than three layers of unexpanded cumulus cells (COCs: cumulus oocyte complexes) were selected for in vitro maturation (IVM). Before culturing, oocytes were washed in Hepes-buffered TCM199 (HTCM199) supplemented with 5% FBS (Fetal Bovine Serum, Gibco 10270), and 2 mM glutamine. The oocyte culture medium (OCM) consisted of bicarbonate buffered TCM 199 with 2 mM L-glutamine supplemented with 0.02 mg/ml cysteamine, 1 IU/ml hCG, 0.1 IU/ml FSH, 100 ml/ml penicillin, 100 mg/ml streptomycin, 10% FBS (Fetal bovine serum, Gibco 10270), and 0.2 mM 42 42 Na-Pyruvate. The medium osmolarity was (...truncated)