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.gender | preimplantation | microarray | imprinting | X-inactivation

Genetic and environmental factors produce different levels of DNA damage in spermatozoa. Usually, DNA-fragmented spermatozoa (DFS) are used with intracytoplasmic sperm injection (ICSI) treatments in human reproduction, and use of DFS is still a matter of concern. The purpose of the present study was to investigate the long-term consequences on development and behavior of mice generated by ICSI with DFS. Using CD1 and B6D2F1 mouse strains, oocytes were injected with fresh spermatozoa or with frozen-thawed spermatozoa without cryoprotector. This treatment increased the percentage of TUNEL-positive spermatozoa, tail length as measured by comet assay, and loss of telomeres as measured by quantitative PCR. The ICSI-generated embryos were cultured for 24 h in KSOM, and 2-cell embryos were transferred into CD1 females. The DFS reduced both the rate of preimplantation embryo development and number of offspring. Immunofluorescence staining with an antibody against 5-methylcytosine showed a delay of 2 h on the active demethylation of male pronucleus in the embryos produced by ICSI. Moreover, ICSI affected gene transcription and methylation of some epigenetically regulated genes like imprinting, X-linked genes, and retrotransposon genes. At 3 and 12 mo of age, ICSI with DFS-produced animals and in vivo-fertilized controls were submitted to behavioral tests: locomotor activity (open field), exploratory/anxiety behavior (elevated plus maze, open field), and spatial memory (free-choice exploration paradigm in Y maze). Females produced by ICSI showed increased anxiety, lack of habituation pattern, deficit in short-term spatial memory, and age-dependent hypolocomotion in the open-field test (P<0.05). Postnatal weight gain of mice produced by ICSI with fresh or frozen sperm was higher than that of their control counterparts from 16 wk on (P<0.01). Anatomopathological analysis of animals at 16 mo of age showed some large organs and an increase in pathologies (33% of CD1 females produced with DFS presented some solid tumors in lungs and dermis of back or neck). Moreover, 20% of the B6D2F1 mice generated with DFS died during the first 5 mo of life, with 25% of the surviving animals showing premature aging symptoms, and 70% of the B6D2F1 mice generated with DFS died earlier than controls with different kind of tumors. We propose that depending on the level of DFS, oocytes may partially repair fragmented DNA, producing blastocysts able to implant and produce live offspring. The incomplete repair, however, may lead to long-term pathologies. Our data indicate that use of DFS in ICSI can generate effects that only emerge during later life, such as aberrant growth, premature aging, abnormal behavior, and mesenchymal tumors.

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.

The aim of this study was to evaluate the effect of extracellular vesicles (EV) from oviductal fluid (OF), either from the ampulla or isthmus, on the development and quality of -cultured bovine embryos. Zygotes were cultured in synthetic oviduct fluid (SOF + 3 mg/mL BSA) without calf serum (C- group), in the presence of 3 × 10 EV/mL from ampullary or isthmic OF at either 1 × 10 (10 K) or 1 × 10 (100 K), and compared with SOF + 5% FCS (C+ group). OF-EV size and concentration were assessed by electron microscopy and nanotracking analysis system. Embryo development was recorded on Days 7-9, and blastocyst quality was assessed through cryotolerance and gene expression analysis. Lower blastocyst yield was observed on Day 7 in the C- and OF-EV groups (12.0-14.3%) compared with C+ (20.6%); however, these differences were compensated at Days 8 and 9 (Day 9: 28.5-30.8%). Importantly, the survival rate of blastocysts produced with isthmic 100 K OF-EV was higher than that of C+ and C- group at 72 h after vitrification and warming (80.1 vs 34.5 and 50.5% respectively, < 0.05). In terms of gene expression, blastocysts produced in the presence of 100 K isthmic OF-EV upregulated the water channel and and transcripts compared with the C+, with the expression in C- being intermediate. The lipid receptor was downregulated in C+ compared with all other groups. In conclusion, the addition of oviductal fluid extracellular vesicles from isthmus, to culture of bovine embryos in the absence of serum improves the development and quality of the embryos produced.

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.

Despite major efforts directed at improving the yield of blastocysts from immature oocytes in vitro, the quality of such blastocysts continually lags behind that of blastocysts produced in vivo. These differences are manifested at the level of morphology, metabolism, gene expression and cryotolerance, and may have a knock-on effect further along the developmental axis. Evidence suggesting that in vitro culture conditions, while capable of producing blastocysts in relatively high numbers, are far from optimal with deficiencies being manifested in terms of abnormally large offspring. It is clear nowadays that modification of the post-fertilization culture environment in vitro can improve blastocyst quality to some extent.

In adult tissues, sexual dimorphism is largely attributed to sex hormone effects, although there is increasing evidence for a major role of sex chromosome dosage. During preimplantation development, male and female embryos can display phenotypic differences that can only be attributed to the transcriptional differences resulting from their different sex chromosome complements. Thus, all expressed Y-linked genes and those X-linked genes that totally or partially escape X-chromosome inactivation at each specific developmental stage display transcriptional sexual dimorphism. Furthermore, these differentially expressed sex chromosome transcripts can regulate the transcription of autosomal genes, leading to a large transcriptional sexual dimorphism. The sex-dependent transcriptional differences may affect several molecular pathways such as glucose metabolism, DNA methylation and epigenetic regulation, and protein metabolism. These molecular differences may have developmental consequences, including sex-selective embryo loss and sex-specific epigenetic responses to environmental hazards, leading to long-term effects. This review discusses transcriptional sexual dimorphism in preimplantation embryos, its consequences on sex ratio biases and on the developmental origin of health and disease, and its significance for transcriptional studies and adult sexual dimorphism.

The ratio of male/female embryos may be modified by environmental factors such as maternal diet in vivo and the composition of embryo culture media in vitro. We have used amino acid profiling, a noninvasive marker of developmental potential to compare the effect of sex on the metabolism of bovine blastocysts conceived in vivo and in vitro. Blastocysts were incubated individually for 24 hr in a close-to-physiological mixture of amino acids and the depletion or appearance of 18 amino acids measured using HPLC. Blastocysts were then sexed by PCR. Amino acid depletion by in vitro-produced blastocysts and expanded blastocysts was higher than in embryos conceived in vivo (P = 0.02). When cultured in vitro, female embryos exhibited increased depletion of arginine, glutamate, and methionine and appearance of glycine, while male embryos displayed increased depletion of phenylalanine, tyrosine, and valine. Overall, in vitro-produced blastocysts exhibited sex-specific differences in metabolic profiles of 7 out of 18 amino acids; in vivo-produced, in 2 out of 18. These differences had disappeared by the expanded blastocyst stages. We have also shown that amino acid metabolism can predict the ability of bovine zygotes to develop to the blastocyst stage, providing "proof of principle" for the use of this technology in clinical IVF to select single embryos for transfer and thereby avoid the problem of multiple births.