Cyclic cell proliferation and endometrial remodeling during the estrous cycle are important for maintaining normal endometrial function. However, the regulatory mechanisms underlying this cell proliferation have not yet been elucidated. In this study, the function of matrix metalloproteinase 3 (MMP3) on endometrial cell proliferation was analyzed. Gene expression in bovine endometrial stromal (BES) and epithelial (BEE) cells was analyzed using qPCR. The protein expression of MMP3 and heparin-binding EGF-like growth factor (HB-EGF) was analyzed using casein zymography and western blotting, respectively. Cell proliferation was analyzed using an automated cell counter. The results revealed that MMP3 was highly expressed at the follicular stage compared to that at the luteal and implantation stages. Estrogen (E2) increased the gene expression and release of MMP3 protein in BES in vitro, whereas progesterone (P4) and interferon alpha (IFNα) decreased mRNA and protein expression. E2 also increased the proliferation of BES, but the inhibitors of MMP3 and epidermal growth factor receptor (EGFR) inhibited the proliferation induced by E2. Furthermore, E2 increased the release of HB-EGF from BES, whereas the MMP3 inhibitor suppressed this release. The effect of E2 on BEE cell proliferation was not reported. However, the conditioned medium of BES treated with E2 increased BEE cell proliferation but was inhibited by an EGFR inhibitor. E2 induced MMP3 protein expression and promotes HB-EGF release from BES. These results suggest that MMP3 is involved in endometrial cell proliferation during the follicular stage.

In vitro fertilization (IVF) is crucial for livestock reproduction; however, pregnancy rates after embryo transfer vary depending on the developmental speeds of the embryos. Although quantitative PCR (qPCR) is used to predict developmental potential, its reliability depends on the selection of appropriate reference genes (RGs) for normalization. To determine suitable RGs in bovine blastocysts with different developing speed, we evaluated the stability of eight candidate RGs (18S, ACTB, GAPDH, HMBS, PPIA, TBP, HPRT1, and SDHA) in early-, mid-, and late-developing IVF blastocysts (E-BL, M-BL, and L-BL, respectively) using RefFinder. Despite morphological similarities, E-BL, M-BL, and L-BL exhibited different biological features, including significantly lower pregnancy rates in L-BL than in the other groups, and less abundant transcript levels of five candidate RGs in L-BL than in E-BL. RefFinder revealed that ACTB was the most stable RG, whereas TBP was the least stable. To emphasize the critical importance of selecting stable RGs, we analyzed the expression of key developmental markers including those of the inner cell mass (ICM; OCT4, SOX2) and trophectoderm (TE; CDX2, GATA3, IFNτ), using various RGs for normalization. For ICM markers, normalization with ACTB showed results consistent with pregnancy rates, whereas moderately stable (18S) and less stable (TBP) RGs yielded contradictory outcomes. Normalization with unstable RGs produced inconsistent TE marker expression patterns (CDX2, GATA3) and overestimated (IFNτ) results across groups, compared with the results of ACTB. These results demonstrate that selecting inappropriate RGs for qPCR normalization can lead to misinterpretation, highlighting the necessity of proper RG evaluation to ensure accurate results in bovine embryo research.

Spermatogenesis is a complex process that is required for sperm production. Multiple RNA-binding proteins participate in regulating spermatogenesis. Y-box-binding protein 1 (YBX1) is involved in transcriptional regulation, mRNA stabilization, and translational repression. However, its specific role in spermatogenesis remains unclear. This study investigated the role of YBX1 in spermatogenesis using a Ybx1 conditional knockout (Ybx1 cKO) mouse model. By analyzing the phenotype of Ybx1 cKO mice, we investigated the role of YBX1 in spermatogenesis and male fertility. The morphology and weight of Ybx1 cKO mouse testes were similar to those of wild-type (WT) testes. Sperm count and motility were lower in Ybx1 cKO mice than in WT mice. Histological analysis showed reduced numbers of elongated spermatids in seminiferous tubules and spermatozoa in tubules of the epididymis in Ybx1 cKO mice. Although YBX1 was highly expressed in the cytoplasm of spermatocytes, meiosis progressed normally in Ybx1 cKO spermatocytes. Finally, the fertilization potential of spermatozoa from Ybx1 cKO epididymis was decreased. In conclusion, our results indicate that YBX1 participates in the regulation of spermatid development but is dispensable for meiosis.

Climate change is causing heat stress (HS) in dairy cattle. This study aimed to compare the clinical efficacy of two GnRH synthetic analogs, dephereline and busereline, as ovulation inducers under HS conditions. The study population comprised 1,000 lactating dairy cows showing signs of spontaneous estrus which were assigned to the groups: DEPH (489 cows receiving 100 µg of dephereline) and BUS (511 cows receiving 10 µg of busereline) at the time of insemination. Cows were included only once in the study. Treatment with busereline increased the risk of multiple ovulations and twin pregnancies, with an odds ratio (OR) of 1.6, and twin pregnancies, with an OR of 2.8, when compared with dephereline. The likelihood of pregnancy in multiple-ovulating cows was significantly higher in the DEPH group than the BUS group. Collectively, our results comparing two ovulation inducers showed that dephereline treatment may improve the fertility of lactating dairy cows under HS conditions.

Ethanolamine plasmalogens (EPls) and choline plasmalogens (CPls), unique glycerophospholipids may play important roles in milk production and reproduction in postpartum dairy cows. While CPls are more abundant in bovine blood, EPls are predominant in the brain. Brain EPls are the only recognized ligands of G protein-coupled receptor 61 (GPR61), a receptor that co-localizes with GnRH receptors on gonadotrophs. We hypothesized that chemosynthetic CPls stimulate gonadotropin secretion from bovine gonadotrophs, similar to the reported effects of chemosynthetic EPls. Anterior pituitary cells from healthy, post-pubertal heifers, were cultured for 3.5 days and then treated with increasing concentrations (0, 0.7, 7, 70, or 700 pM) of EPl with vinyl-ether-bonded stearic acid and ester-bonded oleic acid (C18:0-C18:1EPl) as a positive control, or CPls with vinyl-ether-bonded stearic acid and ester-bonded oleic acid (C18:0-C18:1CPl), arachidonic acid (C18:0-C20:4CPl), or docosahexaenoic acid (C18:0-C22:6CPl). After 2 h, the medium samples were harvested for FSH and LH assays. C18:0-C18:1EPl (7–700 pM) stimulated basal FSH and LH secretion (P < 0.01). None of the tested CPl concentrations stimulated LH secretion. Only 700 pM of C18:0-C18:1CPl, but not lower concentrations, stimulated FSH secretion (P < 0.05), an effect that was inhibited by a SMAD pathway inhibitor. However, both C18:0-C18:1CPl and C18:0-C20:4CPl synergized with GnRH to stimulate FSH secretion. In silico molecular-docking simulations using the deep-learning algorithm ColabFold revealed that CPls bind to the three-dimensional structural model of GPR61. In conclusion, C18:0-C20:4CPl stimulated FSH secretion exclusively in the presence of GnRH, whereas C18:0-C18:1CPl weakly stimulated FSH secretion and showed potential interaction with the GnRH signaling pathways.

Bovine blastocysts cultured under mild hypothermia (MH) can be maintained with non-hatching viable embryos compared to normothermic controls (38.5°C). However, the mechanism by which mildly hypothermic culture delays embryonic growth has not yet been elucidated. This study evaluated the number of cells in embryos cultured under MH conditions and the expression of genes involved in embryonic differentiation. Bovine blastocysts cultured under MH conditions exhibited reduced cell numbers and interferon-tau mRNA expression. Both forkhead box O3 (FOXO3) mRNA expression and FOXO3 protein level in blastocysts cultured under MH conditions were higher than those in normothermic controls (P < 0.05). On the phosphorylated FOXO3 protein level, there was no significant difference between blastocysts cultured under MH and normothermic conditions. In contrast, no significant difference was observed in the ATP content of blastocysts between the MH and normothermic groups. In blastocysts cultured under MH conditions, cold-inducible RNA-binding protein (CIRP) and RNA-binding motif protein 3 (RBM3) mRNA expression increased, and heat shock protein 70 (HSP70) mRNA expression decreased compared to that in normothermic controls (P < 0.05). Considering that HSP70 is involved in preventing apoptosis, these results suggest that MH retards embryonic development via apoptosis induced by HSP70 downregulation during the culture period.