We aimed to evaluate in the bovine endometrium whether (1) key genes involved in endometrial extracellular matrix (ECM) remodeling are regulated by the endocrine peri-ovulatory milieu and (2) specific endometrial ECM-related transcriptome can be linked to pregnancy outcome. In Experiment 1, pre-ovulatory follicle growth of cows was manipulated to obtain two groups with specific endocrine peri-ovulatory profiles: the Large Follicle-Large CL group (LF-LCL) served as a paradigm for greater receptivity and fertility and showed greater plasma pre-ovulatory estradiol and post-ovulatory progesterone concentrations compared to the Small Follicle-Small CL group (SF-SCL). Endometrium was collected on days 4 and 7 of the estrous cycle. Histology revealed a greater abundance of total collagen content in SF-SCL on day 4 endometrium. In Experiment 2, cows were artificially inseminated and, six days later, endometrial biopsies were collected. Cows were retrospectively divided into pregnant and non-pregnant (P vs NP) groups after diagnosis on day 30. In both experiments, expression of genes related to ECM remodeling in the endometrium was studied by RNAseq and qPCR. Gene ontology analysis showed an inhibition in the expression of ECM-related genes in the high receptivity groups (LF-LCL and P). Specifically, there was downregulation of TGFB2, ADAMTS2, 5 and 14, TIMP3 and COL1A2, COL3A1, COL7A1 and COL3A3 in the LF-LCL and P groups. In summary, the overlapping set of genes differently expressed in both fertility models: (1) suggests that disregulation of ECM remodeling can impair receptivity and (2) can be used as markers to predict pregnancy outcome in cattle.
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Saara Carollina Scolari, Guilherme Pugliesi, Ricardo de Francisco Strefezzi, Sónia Cristina da Silva Andrade, Luiz Lehmann Coutinho, and Mario Binelli
Mariana Sponchiado, Waleed F A Marei, Gerrit T S Beemster, Peter E J Bols, Mario Binelli, and Jo L M R Leroy
In cattle, pre-implantation embryo development occurs within the confinement of the uterine lumen. Current understanding of the bi-lateral molecular interactions between embryo and endometrium that are required for a successful pregnancy is limited. We hypothesized that the nature and intensity of reciprocal embryo-endometrium interactions depend on the extent of their physical proximity. Bovine endometrial epithelial cells (bEECs) and morulae were co-cultured in juxtacrine (Contact+) or non-juxtacrine (Contact−) apposition. Co-culture with bEECs improved blastocyst rates on day 7.5, regardless of juxtaposition. Contact+ regulated transcription of 1797 endometrial genes vs only 230 in the Contact− group compared to their control (no embryos) counterparts. A subset of 50 overlapping differentially expressed genes (DEGs) defined embryo-induced effects on bEEC transcriptome irrespective of juxtaposition. Functional analysis revealed pathways associated with interferon signaling and prostanoid biosynthesis. A total of 175 genes displayed a graded expression level depending on Contact+ or Contact−. These genes were involved in interferon-related and antigen presentation pathways. Biological processes enriched exclusively in Contact+ included regulation of cell cycle and sex-steroid biosynthesis. We speculate that, in vivo, embryonic signals fine-tune the function of surrounding cells to ultimately maximize pregnancy success.
Felipe A. C. C. Silva, Thiago Martins, Mariana Sponchiado, Cecilia C. Rocha, Nadia Ashrafi, Stewart F. Graham, Ky Pohler, Francisco Peñagaricano, Angela Gonella-Diaza, and Mario Binelli
In cattle, the concentration of sex steroids modulates uterine function, which is reflected in the composition of the luminal metabolome. Ultimately, the uterine luminal metabolome influences embryonic growth and development. Our objectives were (1) to compare the luminal metabolome 4, 7, and 14 days after estrus of cows that were exposed to greater (HP4; n = 16) vs. lower (LP4; n = 24) concentrations of progesterone before displaying estrus and ovulating spontaneously and (2) to identify changes in the luminal concentration of metabolites across these time points. Luminal epithelial cells and fluid were collected using a cytology brush and gene expression and metabolite concentrations were assessed by RNAseq and targeted mass spectrometry, respectively. Metabolome profile was similar between treatments within each of days 4, 7, and 14 (FDR ≥ 0.1). Concentrations of 53 metabolites changed, independent of treatment, across the diestrus. Metabolites were mostly lipids (40 out 53) and the greatest concentrations were at d 14 (FDR ≤ 0.1). On d 7, the concentration of putrescine and the gene expression of ODC1, PAOX, SLC3A2, and SAT1 increased (P ≤ 0.05). On d 14, the concentration of three ceramides, four glucosylceramides, and 12 sphingomyelins and the expression of SGMS2 were increased, in addition to the concentration of choline and 20 phosphatidylcholines. Collectively, the post-estrus concentration of luminal metabolites changed dynamically, independent of the concentration of sex steroids on the previous cycle, and the greatest magnitude changes were on day 14, when lipid metabolism was the most enriched pathway.
Daniela F da Silva, Thaís A Rodrigues, Juliano C da Silveira, Angela M Gonella-Diaza, Mario Binelli, Juliana V Lopes, Marcelo T Moura, Weber B Feitosa, and Fabíola F Paula-Lopes
Elevated temperatures disturbed sperm physiology. Bovine sperm cells exposed to heat shock led to diminished mitochondrial activity, fertilizing ability, increased oxidative stress and caspase activity concomitant with a delay in embryonic developmental kinetics and modulation of sperm-borne microRNAsmiRNAs.
Sperm function is susceptible to adverse environmental conditions. It has been demonstrated that in vivo and in vitro exposure of bovine sperm to elevated temperature reduces sperm motility and fertilizing potential. However, the cascade of functional, cellular, and molecular events triggered by elevated temperature in the mature sperm cell remains not fully understood. Therefore, the aim of this study was to determine the effect of heat shock on mature sperm cells. Frozen-thawed Holstein sperm were evaluated immediately after Percoll purification (0 h non-incubation control) or after incubation at 35, 38.5, and 41°C for 4 h. Heat shock reduced sperm motility after 3–4 h at 41°C while mitochondrial activity was reduced by 38.5 and 41°C when compared to the control. Heat shock also increased sperm reactive oxygen species production and caspase activity. Heat-shocked sperm had lower fertilizing ability, which led to diminished cleavage and blastocyst rates. Preimplantation embryo developmental kinetics was also slowed and reduced by sperm heat shock. The microRNA (miR) profiling identified >300 miRs in bovine sperm. Among these, three and seven miRs were exclusively identified in sperm cells exposed to 35 and 41°C, respectively. Moreover, miR-181d was enriched in sperm cells exposed to higher temperatures. Hence, elevated temperature altered the physiology of mature sperm cells by perturbing cellular processes and the miR profile, which collectively led to lower fertilizing ability and preimplantation development.