Genetic and environmental factors produce different levels of DNA damage in spermatozoa. Usually, DNA-fragmented spermatozoa (DFS) are used with intra

Although genetically identical for autosomal Chrs (Chr), male and female preimplantation embryos could display sex-specific transcriptional regulation. To illustrate sex-specific differences at the mRNA level, we compared gene-expression patterns between male and female blastocysts by DNA microarray comparison of nine groups of 60 bovine in vitro-produced blastocysts of each sex. Almost one-third of the transcripts detected showed sexual dimorphism (2,921 transcripts; false-discovery rate, P < 0.05), suggesting that in the absence of hormonal influences, the sex Chrs impose an extensive transcriptional regulation upon autosomal genes. Six genes were analyzed by qPCR in in vivo-derived embryos, which displayed similar sexual dimorphism. Ontology analysis suggested a higher global transcriptional level in females and a more active protein metabolism in males. A gene homolog to an X-linked gene involved in network interactions during spliceosome assembly was found in the Y-Chr. Most of the X-linked-expressed transcripts (88.5%) were up-regulated in females, but most of them (70%) exhibited fold-changes lower than 1.6, suggesting that X-Chr inactivation is partially achieved at the blastocyst stage. Almost half of the transcripts up-regulated in female embryos exhibiting more than 1.6-fold change were present in the X-Chr and eight of them were selected to determine a putative paternal imprinting by gene expression comparison with parthenogenetic embryos. Five ( BEX , CAPN6 , BEX2 , SRPX2 , and UBE2A ) exhibited a higher expression in females than in parthenotes, suggesting that they are predominantly expressed by the paternal inherited X-Chr and that imprinting may increase the transcriptional skew caused by double X-Chr dosage.

Elevated concentrations of serum non-esterified fatty acids (NEFA), associated with maternal disorders such as obesity and type II diabetes, alter the ovarian follicular micro-environment and have been associated with subfertility arising from reduced oocyte developmental competence. We have asked whether elevated NEFA concentrations during oocyte maturation affect the development and physiology of zygotes formed from such oocytes, using the cow as a model. The zygotes were grown to blastocysts, which were evaluated for their quality in terms of cell number, apoptosis, expression of key genes, amino acid turnover and oxidative metabolism. Oocyte maturation under elevated NEFA concentrations resulted in blastocysts with significantly lower cell number, increased apoptotic cell ratio and altered mRNA abundance of DNMT3A, IGF2R and SLC2A1. In addition, the blastocysts displayed reduced oxygen, pyruvate and glucose consumption, up-regulated lactate consumption and higher amino acid metabolism. These data indicate that exposure of maturing oocytes to elevated NEFA concentrations has a negative impact on fertility not only through a reduction in oocyte developmental capacity but through compromised early embryo quality, viability and metabolism.

Epigenetic differences between male and female bovine blastocysts provide a plausible link between physiological and gene transcription differences observed between male and female embryos. The aim of this study was to examine sex-related epigenetic differences in bovine blastocysts produced in vitro. Oocytes were matured in vitro and inseminated with frozen-thawed sex-sorted (X or Y) and unsorted (control) bull sperm. Zygotes were cultured to blastocyst stage and were analyzed for embryo sexing, mtDNA content, telomere lengths, methylation analysis, and quantification of mRNA transcripts of DNA methyltransferases (Dnmt1, Dnmt3a, Dnmt3b) HMT1 hnRNP methyltransferase-like 2 (Hmt1), and interleukin enhancer binding factor 3 (Ilf3). There was a difference (P < 0.05) in the mean mtDNA copy number between male (410,000 ± 23,000) and female (360,000 ± 21,000) blastocysts. Telomere length was shorter in male blastocysts (P < 0.01). The level of methylation in a sequence near a variable number of tandem repeats minisatellite region [variable number of tandem repeats (VNTR)] in males (39.8% ± 4.8) was higher than in females (23.7% ± 3.1) (P < 0.05); however, no differences were found in other regions analyzed. Moreover, transcription differences between sexes were observed for Dnmt3a, Dnmt3b, Hmt1, and Ilf3. These results provide evidence of epigenetic differences between male and female bovine in vitro produced embryos and suggest that before initiation of gonadal differentiation, epigenetic events may modulate the difference between speed of development, metabolism, and transcription observed during preimplantation development between male and female embryos.

The fusion of gamete membranes during fertilization is an essential process for sexual reproduction. Despite its importance, only three proteins are known to be indispensable for sperm-egg membrane fusion: the sperm proteins IZUMO1 and SPACA6, and the egg protein JUNO. Here we demonstrate that another sperm protein, TMEM95, is necessary for sperm-egg interaction. TMEM95 ablation in mice caused complete male-specific infertility. Sperm lacking this protein were morphologically normal exhibited normal motility, and could penetrate the zona pellucida and bind to the oolemma. However, once bound to the oolemma, TMEM95-deficient sperm were unable to fuse with the egg membrane or penetrate into the ooplasm, and fertilization could only be achieved by mechanical injection of one sperm into the ooplasm, thereby bypassing membrane fusion. These data demonstrate that TMEM95 is essential for mammalian fertilization.