Abstract. This study was designed to investigate whether freeze-dried (FD) bull spermatozoa maintained the function of the microtubule-organizing center (MTOC) after rehydration and intracytoplasmic sperm injection (ICSI). In a preliminary attempt, the cleavage and blastocyst formation rates in FD-ICSI zygotes (36 and 1%, respectively) were found to be considerably lower than those in control ICSI zygotes (67 and 21%, respectively) or in IVF zygotes (78 and 43%, respectively). An alkaline comet assay indicated that the DNA fragmentation index (length of comet tail × % DNA liberated) was not significantly different between fresh and FD spermatozoa. In the main experiment, formation of sperm-asters in the FD-ICSI oocytes 7 h postinsemination occurred at a similar rate when compared with the control ICSI oocytes (41 vs. 49%). Among the oocytes exhibiting sperm aster formation, the extent of microtubule network assembly was comparable between the FD-ICSI and control ICSI groups. However, the MTOC of the ICSI oocytes was not as functional as that of IVF oocytes in terms of the aster formation rate (97%) and the fluorescent intensity of the microtubule network (2.0 folds). These results suggest that the freeze-drying process per se had no adverse effect on maintaining the MTOC function in bull spermatozoa. Key words: Bovine Intracytoplasmic sperm injection (ICSI), Freeze-drying, Microtubule-organizing center (MTOC), Sperm-aster (J. Reprod. Dev. 57: [428][429][430][431][432] 2011) centrosome is composed of a pair of centrioles surrounded by pericentriolar materials such as γ-tubulin, centrin and pericentrin. Since an interphase network of microtubules and the mitotic bipolar spindle are nucleated from the centrosome, the centrosome is considered to be the microtubule-organizing center (MTOC) [1]. During fertilization in most mammalian species, the centrosome brought into an oocyte by a spermatozoon plays a critical role in assembly of the microtubule network that brings both male and female pronuclei to the center of the newly formed zygote, as reported in humans [2], rhesus monkeys [3], rabbits [4], pigs [5] and cattle [6]. Interestingly, paternal inheritance of the MTOC does not occur in the mouse [7] and rat [8], and the microtubule network developed from multiple cytoplasmic asters, instead of a single sperm-aster, is involved in the migration of pronuclei [9].Freeze-drying has been proposed as an alternative method to preserve mammalian spermatozoa [10], although freeze-dried (FD) spermatozoa after rehydration lose their motility and application of intracytoplasmic sperm injection (ICSI) technique is necessary. Rodent spermatozoa can be stored practically at refrigeration temperatures [11][12][13]. However, in large domestic species, in vitro production of blastocysts derived from FD-ICSI is still considered to be a challenging endeavor [14][15][16]. We have recently reported that the freeze-drying protocol slightly reduced the ability of bull spermatozoa to induce calcium oscillations [17] and that it had no a...

Abstract. Epigenetic reprogramming in early preimplantation embryos, that refers to erasing and remodeling epigenetic marks such as DNA methylation, is essential for differentiation and development. In many species, paternal genome is subjected to genome-wide active demethylation before the DNA replication commences, while maternal genome maintains its methylation status until being demethylated passively during the subsequent cleavage divisions. The purpose of this manuscript was to review the available knowledge about the paternal genome active demethylation process concerning the possible mechanisms, species variation and the factors affecting the active demethylation dynamics such as in vitro protocols for production of pronuclear-stage zygotes. Better understanding the mechanisms by which the epigenetic reprogramming is occurred may contribute to clarify the biological significance of this process. Key words: Active demethylation, Epigenetics, Paternal genome, Pronuclear zygotes (J. Reprod. Dev. 55: [356][357][358][359][360] 2009) ene expression is regulated by both genetic and epigenetic mechanisms. Epigenetics includes modifications of DNA itself (methylation of cytosine in the CpG dinucleotide) and/or the associated protein (phosphorylation, acetylation and methylation of histone) [1]. These modifications can regulate the gene expression without changing the DNA consequences [2]. Each cell type in our body has its own epigenetic signature which reflects different gene expression, and subsequently results in different structure and function among genetically homogenous cells. DNA methylation is one of the most-studied epigenetic mechanisms, and it is recognized as a chief contributor to the stability of gene expression state [3]. DNA Methylation: Nature, Function and ReprogrammingDNA methylation is a process that includes transferring a methyl group from S-adenosylmethionine to C5 positions of the cytosine residues in the CpG dinucleotides by different categories of methyltransferase enzymes (Dnmt1, Dnmt1o, Dnmt2, Dnmt3a, Dnmt3b and Dnmt3L) [4,5]. The CpG dinucleotides are mainly present in a cluster called CpG islands, which are associated with genes, and mostly located in promoters and first exons [6]. These islands are defined as the initiation sites for both transcription and DNA replication, and may represent genomic footprints for replication initiation [7]. Methylation of the CpG dinucleotides in the gene promoter may repress gene expression by interfering with the access of the DNA binding proteins and subsequently blocking the transcription or by binding to the transcriptional repressor MeCP2 [4,8,9]. In spite of great correlation between gene methylation status and gene expression, it is not yet clear whether DNA methylation is the cause of or a subsequence to the transcriptional repression [10]. In an another view for the correlation between gene methylation status and its expression, methylation can increase or decrease the level of gene transcription depending on whether the methylation in...

Regardless of the presence of sperm-borne oocyte-activating factors, activation of bovine oocytes with exogenous activation stimuli is required for further development after intracytoplasmic sperm injection (ICSI). The present study was designed to develop a new activation regimen for improving the blastocyst yield after ICSI of bovine oocytes harvested from ovaries stored at 10-12 oC for 24 h. Following ICSI, oocytes were treated with 5 μM ionomycin for 5 min, 7% ethanol for 5 or 10 min, ionomycin followed by ethanol (5 or 10 min), ionomycin followed by 10 µg/mL Cycloheximide for 5 h, or ionomycin followed by 1.9 mM 6-dimethylaminopurine for 3 h. Across the activation regimens, the cleavage rates of ICSI oocytes (45-77%) were higher than those of parthenogenetically activated oocytes (11-21%; P<0.05). Activating the ICSI oocytes with ionomycin plus ethanol improved the blastocyst yield (29-30%) comparing to the non-treated oocytes (12%; P<0.05) but the other regimens did not (9-18%; P>0.05). The higher blastocyst yields were due to increasing the proportion of ICSI oocytes that passed through the early postfertilization events until cleavage. None of the regimens have any adverse effect on the quality of the blastocysts regarding the total cell number or the proportion of the inner cell mass cells. Thus, a new activation regimen composed from two triggers for single calcium increase has been proven effective to improve the blastocyst yield after bovine ICSI using oocytes harvested from stored ovaries.Dear Dr. John P. Kastelic, Thank you very much for prompt reply and accepting our manuscript for publication in Theriogenology. We have replied for each comment from Co-editor and the reviewer-2 in pointby-point basis.Sincerely yours, Shinichi Hochi (Corresponding author)Hany Abdalla (First author)Co-editor 1-P value has been added to the Abstract (Lines 27,29).2-The number of the references in Introduction section has been reduced. Reviewer #21-Regarding omitting the two groups (5 min ethanol group and ionomycin followed by 5 min ethanol group); We do not think that the presence of these two groups causes any difficulty to recognize the superiority of ionomycin and ethanol combination. Since activation of 7% ethanol for 5 min 4 hr after ICSI is a commonly applied method for bovine ICSI, presence of such group must be important to declare the superiority of the new combination (ionomycin and ethanol) over the methods of ethanol alone under the same experimental circumstance regarding oocyte quality, injection skill, culture condition, and so on. For the ionomycin followed by 5 min ethanol, the efficiency of this method to improve the cleavage and the blastocyst yield was similar to that of ionomycin followed by 10 min ethanol, making it possible to choose the method in which oocytes are exposed to minimum exogenous stimuli. Tables 1 and 2. 2-The time of 2 nd polar body detection has been added in footnote of 15Running head: Bovine oocyte activation after ICSI * Manuscript 2 AbstractRegardless of the pres...

Abstract. This study was designed to investigate the dynamics of the paternal genome demethylation in pronuclearstage bovine zygotes produced either by in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) using freeze-thawed (FT) as well as freeze-dried (FD) bull sperm stored at +4 or -196 C for one year. Zygotes were fixed and immunostained using anti-5-methyl-cytosin at 8, 10, 14 and 18 h post IVF (hpi) and at 6 and 12 h post ICSI (hpic). In conventional IVF-derived zygotes, the overall average of the relative methylation (RM; male/female) decreased from 0.92 at 8 hpi to 0.69 at 10 hpi (P<0.05) without any additional decrease at 14 and 18 hpi (0.67 and 0.64, respectively; P>0.05). This was accompanied by higher proportions of zygotes showing RM<0.6 (45.5, 37.5 and 38.2% at 10, 14 and 18 hpi, respectively; P<0.05) compared with 3.7% at 8 hpi. The overall averages of the RM in the FT-ICSI derived zygotes (0.79 and 0.66 at 6 and 12 hpic, respectively) were similar to those in the corresponding IVF-derived zygotes (8 and 14 hpi), but a higher proportion of the 6 hpic zygotes (37.8%; P<0.05) showed an RM<0.6 compared with the 8 hpi zygotes (3.7%). The proportions of FD-ICSI derived zygotes at 12 hpic showing an RM<0.6 (60.6 and 62.4% for +4 and -196 C storage, respectively) were higher than that of the FT-ICSI derived zygotes (39.4%; P<0.05). Thus, the bovine paternal genome rapidly demethylated within 10 h after IVF and 6 h after ICSI, and the freeze-drying and/or the storage process had no adverse effect on demethylation of the paternal genome. The extent of demethylation in the pronuclear-stage bovine zygotes was moderate, with 0.4≤RM<0.6. Key words: Bovine zygotes, Demethylation, Freeze-drying, Intracytoplasmic sperm injection (ICSI), In vitro fertilization (IVF) (J. Reprod. Dev. 55: [433][434][435][436][437][438][439] 2009) pigenetic modification of DNA itself (methylation of cytosine in the dinucleotide CpG) and/or associated proteins (phosphorylation, acetylation and methylation of histone) are responsible for regulation of gene expression without changing the DNA consequences [1]. Methylation of the DNA is one of the best-studied epigenetic mechanisms, and it is recognized as a principle contributor to the stability of gene expression state [2]. The genomic methylation pattern is generally stable and heritable in differentiated somatic cells; however, genome-wide reprogramming has been reported to occur in germ cells and preimplantation embryos [3]. The reprogramming process includes erasing the existing epigenetic marks and re-establishing new cell-specific marks to generate cells with nuclear totipotency and broad developmental potential [3,4]. The biological importance of DNA methylation is still unclear [5], but aberrant epigenetic reprogramming, which has been reported in cattle and buffalo zygotes reconstructed with somatic cells [6][7][8], has been considered as a possible cause for higher incidence of gestational and neonatal fetal anomalies or deaths of such zygotes. Moreover,...

Three-hundred-and-twenty-five patients on an assisted conception programme underwent 378 cycles of oocyte retrieval (OPU) following ovarian stimulation using a GnRH analogue and human menopausal gonadotrophins (HMG), a regimen which allows programmed cycles and delayed oocyte retrieval. Eighteen cycles were excluded (failed OPU in three and failure of fertilization in 15). In 360 cycles, patients completed their treatment with either in-vitro fertilization/embryo transfer (IVF/ET) (116) or gamete intra-Fallopian transfer (GIFT) (244), of which 241 took place at the normal time and 119 were delayed for 24 h or more to avoid weekend operating. The overall pregnancy rate per OPU was 29.5%, with the IVF group being 24.1% and the GIFT group being 32.8%. In the group of patients in whom OPU was delayed, the pregnancy rate was significantly higher in each sub-group than in the corresponding non-delayed sub-group (overall, 37.0 versus 25.7%; IVF/ET, 38.5 versus 16.9%; GIFT, 36.3 versus 31.1%). There was a significantly higher number of oocytes collected, gametes/embryos transferred in the group whose OPU had been delayed. In patients receiving GnRH analogue and HMG for ovarian stimulation, delaying oocyte retrieval is not harmful, may result in an improved outcome and allows OPU to be performed on routine operating lists. This facility, together with the improved pregnancy rates associated with this protocol of ovarian stimulation should improve the cost-effectiveness of assisted conception programmes.

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This study was carried out to investigate the potential effects of Chlorella vulgaris (CV) on testicular function and oxidant/antioxidant status in normal and deltamethrin‐intoxicated rats. Forty adult male rats were drenched either with normal saline, CV (50 mg/kg), deltamethrin (DM) (3 mg/kg), or CV combined with DM, daily for 8 weeks. At the end of the protocol, the epididymal sperm quality was evaluated and the testicular superoxide dismutase (SOD), catalase enzyme (CAT) and malondialdehyde (MDA), and the serum testosterone levels were estimated. Normal rats treated with CV showed a significant increase in the total sperm number/epididymal tail, testicular SOD, and CAT levels with a significant decrease in the testicular MDA. Deltamethrin intoxication significantly decreased the proportions of motile and live sperm, the testosterone concentration, the testicular SOD and CAT levels, whereas it significantly increased the proportion of abnormal sperm and the testicular MDA. Chlorella vulgaris treatment significantly ameliorated the adverse effects of DM‐intoxication and restored most of the parameters to levels that are comparable to those of the control group. In conclusion, CV administration improved the testicular function of normal rats and ameliorated the effect of severe oxidative stress conditions.

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