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
ABSTRACT
Objective: We recently demonstrated spatial regionalization of maternal transcripts and proteins within unfertilized ovine oocyte. Here, we investigated the likelihood of oocyte polarity for the first time in bovine
Materials and Methods: In this experimental study, in vitro matured bovine oocytes were used for manual bisection [into oocyte halve that were near-to [HNS] and far-from [FS] spindle] or trisection [into MII-spindle [S], the spindle-side half [NS], and the distal half unassociated with the spindle [FS]]. Prepared pools of oocyte substructures were used for comparative quantitative real-time polymerase chain reaction [RT-qPCR]. To map the possible preferential sperm entry point [SEP], the spatial relationship between SEP and MII-spindle was measured 5 hours post-fertilization
Results: The proportional amount of maternal mRNA in S oocyte fragment was estimated to be 6 to 11-fold higher than NS and FS counterparts. The relative abundances of Nanog, Oct4, Fgf4 and Tead4 were significantly higher in HNS oocyte fragment compared t0 FS. The relative abundances of Ctnb, Carm1, Rex1, Sox2 and Cdx2 were comparable between HNS and NS oocyte fragments. FS oocyte fragment possessed significantly higher transcripts of Gata4 compared to HNS. The distribution of certain transcripts related to pluripotency and lineage commitment were different depending upon the region of the oocyte; either enriched at S [Tead4, Nanog, Ctnb and Sox2], NS [Oct4], or FS [Gata6]. The SEP in almost [90%] fertilized oocytes was located in MII-hemisphere
Conclusion: The observation of spatial restriction of mRNAs and SEP within MII-oocyte may indicate that the principal forces of oocyte polarity are evolutionary conserved. This may in turn highlight the need for refinements in the methodology of intracytoplasmic sperm injection [where a sperm is injected far from the MII-spindle] and somatic cell nuclear transfer [where a major amount of regulative mRNAs that are associated with MIIspindle is removed during enucleation]
ABSTRACT
Testis tissue xenografting and the resultant sperm in a xenograft may provide a unique approach to rescue the genetic material of males that die prematurely and is a model for the study of human spermatogenesis and can represent an alternative approach for fertility preservation in cancer patients. This study was aimed to evaluate the xenogenic dog sperm in formation of male pronucleus following injection into the sheep oocytes. The in vitro matured slaughterhouse derived sheep oocytes were subjected to Intracytoplasmic Sperm Injection [ICSI] with epididymal, testicular, and xenogenic dog sperm. The ICSI was performed after scoring of the sperm midpiece using an IX71-Olympus inverted microscope with Nomarsky optics. Within 1 hr after injection, the injected oocytes in activated group were exposed to 5 microM ionomycin for 5 min. The data were analyzed by Chi-square and ANOVA using Sigma Stat, version 3.5, and p<0.05 was considered significant. The formation of female pronuCIeus after ICSI of xenogenic sperm was higher than epididymal and testicular sperm in non-activated oocytes. The corresponding rate in activated oocytes was higher or comparable with testicular and epididymal sperm. The rate of male pronucleus formation after ICSI of xenogenic sperm was comparable with injection of two other sperm sources. Oocyte activation had an inductive role in female and male pronuclear formation. Dog xenogenic sperm was capable to induce oocyte activation and proportion of male pronucleous formation was comparable to the testicular and epididymal sperm