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Endometrial cell co-culture (ECC) with single embryo may reflect endometrium responses in vivo. Bovine Day-6 in vitro-produced morulae were cultured until Day-8 in modified synthetic oviductal fluid (mSOF), or on the epithelial side of ECC. Expression of epithelial- and stromal-cell transcripts was analyzed by RT-PCR in ECC with one male (ME) or female embryo (FE). Concentrations of ARTEMIN (ARTN) and total protein were determined in epithelial cell-conditioned medium. ECCs yielded embryos with more cells in the inner cell mass than embryos cultured in mSOF. Embryos altered transcript expression only in epithelial cells, not in stromal ones. Thus, ME induced larger reductions than FE and controls (i.e., no embryos cultured) in hexose transporter solute carrier family 2 member 1 (SLC2A1) and member 5 (SLC2A5), connective tissue growth factor (CTGF), artemin (ARTN), and interferon alpha and beta receptors subunit IFNAR1 and IFNAR2. FE reduced SLC2A1 and SLC2A5, and increased ARTN expression with respect to controls. ME tended to reduce total protein concentration (P < 0.082) in ECC-conditioned medium, while ARTN protein and gene expressions strongly correlated (R > 0.90; P < 0.05) in the group of ME or FE, but not in controls (without embryo). Isolated male and female embryos may differentially release signaling factors that induce sexually dimorphic responses in endometrial cells.

Artemin a member of the glial cell line-derived neurotrophic factor (GDNF) family is present in mice and human preimplantation embryos, and reproductive tract, during early pregnancy promoting embryo development in vitro. The presence of artemin in cattle embryos and reproductive tract, however, is unknown. In the present work we identified for first time artemin in bovine uterine fluid (UF) (Western blot), endometrium (RT-PCR, Western blot and immunohistochemistry) and embryos (RT-PCR and immunohistochemistry) during early preimplantation development. In addition, GFRalpha3, a component of the artemin receptor was localized in blastocysts produced in vitro. Individually developing embryos released ARTEMIN in culture medium and triggered ARTEMIN mRNA down-regulation in epithelial cells from endometrial cell cultures. Our results suggest that ARTEMIN derived from early embryos and maternal reproductive tract may exert important roles during early development in cattle. © 2016 Elsevier Inc.

In cattle, individual in vitro embryo culture after Day 6 benefits development, allowing non-invasive analysis of culture medium. However, undefined supplements in culture reduce analytical reliability. In this study we assayed the short- and long-term performance of embryos after bovine serum albumin removal over a 24-h period in individual culture. The absence of protein decreased embryo development and cell counts in the inner cell mass without affecting blastocyst sex ratio. However, the absence of protein produced embryos with an improved tendency to survive vitrification after 24h in culture (P≤0.07). After transfer to recipients, birth rates of embryos that had been cultured with protein tended to decrease (P<0.06) mostly as a result of a higher number of miscarriages (P<0.013), reflecting lower viability. Birthweight, gestation length, height and thorax circumference did not differ between embryos cultured with or without protein. In fresh blastocysts cultured without protein, gene expression analysis showed higher abundance (P<0.05) of insulin-like growth factor 2 receptor (IGF2R; imprinting) and activating transcription factor 4 (ATF4) and DNA-damage-inducible transcript 3 (DDIT3; endoplasmic reticulum stress) transcripts, with DNA methyltransferase 3A (DNMT3A; imprinting) tending to increase (P≤0.062). However, in hatched blastocysts that survived cryopreservation, glucose-6-phosphate dehydrogenase (G6PD) was overexpressed in embryos cultured without protein (P<0.01). The absence of protein results in fewer blastocysts but improved long-term viability after cryopreservation. © 2017 CSIRO.

Hepatoma-derived growth factor (HDGF) is present in the endometrium of cows and other mammals. Recombinant HDGF (rHDGF) improves bovine blastocyst development in vitro. However, specific culture conditions and essential aspects of HDGF uterine physiology are yet unknown. In this work we quantified total HDGF protein in uterine fluid (UF) by multiple reaction monitoring (MRM), and analyzed effects of rHDGF on specific embryonic stages with Day-6 bovine embryos cultured in vitro with and without BSA, and on pregnancy viability and calf phenotypes after embryo transfer to recipients. In addition, mRNA abundance of HDGF in endometrial cells co-cultured with one male or one female embryo was quantified. In the presence of BSA, rHDGF had no effect on blastocyst development; however, in BSA-free culture rHDGF mainly promoted development of early blastocysts in contrast with morulae. As the presence of HDGF contained in commercial BSA replacements was suspected, western blot confirmed HDGF identification in BSA both with and without fatty acids. Total HDGF quantified by MRM tended to increase in UF without vs. UF with embryos (P = 0.083). Pregnancy and birth rates, birth weight and calf measurements did not differ between embryos cultured with rHDGF and controls without rHDGF. However, HDGF abundance in cultured epithelial, endometrial cells tended to increase (P < 0.08) in culture with one male embryo. rHDGF acts selectively on specific embryonic stages, but care should be taken with specific macromolecular supplements in culture. The endometrial expression of HDGF can be regulated by the embryonic sex. The use of rHDGF is compatible with pregnancy and birth of normal calves.

Sex determination in bovine embryos is a useful tool in reproductive biotechnology. This work compares two techniques of embryo sexing by polymerase chain reaction (PCR). Embryos were produced in vitro with sex-sorted sperm and two techniques of DNA lysis were tested (proteinase K versus heat shock). Subsequently, halves of each lysed sample was amplified both by amelogenin and BRY4a/SAT1 primers. Male embryos treated by both digestion methods and amplified by BRY4a/SAT1 gave higher rates of false negatives. Amelogenin amplification failed with embryos previously digested by proteinase K. In contrast, the lysis method allowed obtaining the best accuracy in terms of sex verification when using amelogenin amplification.

Mammalian oocytes can undergo artificial parthenogenesis in vitro and develop to the blastocyst stage. In this study, using real-time PCR, we analyzed the expression of genes representative of essential events in development. In vitro matured oocytes were either fertilized or activated with ionomycin + 6-DMAP and cultured in simple medium. The pluripotency-related gene Oct3/4 was downregulated in parthenotes, while the de novo methylation DNMT3A gene was unchanged. Among the pregnancy recognition genes, IFN-t was upregulated, PGRMC1 was downregulated and PLAC8 was unchanged in parthenotes. Among the metabolism genes, SLC2A1 was downregulated, while AKR1B1, COX2, H6PD and TXN were upregulated in parthenotes; there was no difference in SLC2A5. Among the genes involved in compaction/blastulation, GJA1 expression increased in parthenotes, but no differences were detected within ATP1A1 and CDH1. Expression of p66shc and the Bax/Bcl2 ratio were higher in parthenotes, and there was no difference in p53. Parthenotes and embryos may differ in the way they stimulate apoptosis, with a preponderant role for p66shc within parthenotes. Differentially affected functions may also include pluripotency, de novo methylation and early embryonic signalling.

Parthenotes may represent an alternate ethical source of stem cells, once biological differences between parthenotes and embryos can be understood. In this study, we analyzed development, trophectoderm (TE) differentiation, apoptosis/necrosis, and ploidy in parthenotes and in vitro produced bovine embryos. Subsequently, using real-time PCR, we analyzed the expression of genes expected to underlie the observed differences at the blastocyst stage. In vitro matured oocytes were either fertilized or activated with ionomycin +6-DMAP and cultured in simple medium. Parthenotes showed enhanced blastocyst development and diploidy and reduced TE cell counts. Apoptotic and necrotic indexes did not vary, but parthenotes evidenced a higher relative proportion of apoptotic cells between inner cell mass and TE. The pluripotence-related POU5F1 and the methylation DNMT3A genes were downregulated in parthenotes. Among pregnancy recognition genes, TP-1 was upregulated in parthenotes, while PGRMC1 and PLAC8 did not change. Expression of p66shc and BAX/BCL2 ratio were higher, and p53 lower, in parthenotes. Among metabolism genes, SLC2A1 was downregulated, while AKR1B1, PTGS2, H6PD, and TXN were upregulated in parthenotes, and SLC2A5 did not differ. Among genes involved in compaction/blastulation, GJA1 was downregulated in parthenotes, but no differences were detected within ATP1A1 and CDH1.Within parthenotes, the expression levels of SLC2A1, TP-1, and H6PD, and possibly AKR1B1, resemble patterns described in female embryos. The pro-apoptotic profile is more pronounced in parthenotes than in embryos, which may differ in their way to channel apoptotic stimuli, through p66shc and p53 respectively, and in their mechanisms to control pluripotency and de novo methylation. © 2009 Society for Reproduction and Fertility.

High follicular testosterone levels have been associated with a skew in the sex ratio in favor of males following in vitro fertilization, whereas egg incubation temperature has been found to influence sex ratio in some reptiles. The incubation temperature interferes with the aromatase activity, resulting in a sex determination mechanism thought to be lost in mammals. In this work we aimed to test the effects of testosterone on sex ratio of bovine embryos produced in vitro and to determine whether effects of sex and temperature are effectively decoupled in mammals. Bovine oocytes were in vitro matured for 22hr in TCM199, PVA, FSH and LH after a 22hr meiotic arrest in TCM199, PVA and roscovitine 25mM. Matured oocytes were in vitro fertilized and cultured up to Day 3, and embryos having three or more cells were sexed. In the first experiment, testosterone (0, 30, 300 and 1,500 nM), present both during meiotic inhibition and subsequent in vitro maturation (IVM), did not affect development rates or embryonic sex ratio. In the second experiment, increasing incubation temperatures (38, 39 or 40°C) during meiotic inhibition and subsequent IVM, reduced embryo development, but did not change the sex ratio. Under our experimental conditions, testosterone does not promote a preferential selection of Y-chromosome bearing spermatozoa by the oocyte, and temperature and sex ratio seems to be decoupled in mammals. J. Exp. Zool. 311A448-452, 2009. © 2009 Wiley-Liss, Inc.