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Polycystic ovary syndrome (PCOS) is a complex endocrine and metabolic disorder with unclear etiology and unsatisfactory management. Effects of diets on the phenotype of PCOS were not fully understood. In the present study, we applied 45 and 60% high-fat diets (HFDs) on a rat model of PCOS induced by postnatal DHEA injection. We found that both DHEA and DHEA+HFDs rats exhibited reproductive abnormalities, including hyperandrogenism, irregular cycles and polycystic ovaries. The addition of HFDs, especially 60% HFDs, exaggerated morphological changes of ovaries and a number of metabolic changes, including increased body weight and body fat content, impaired glucose tolerance and increased serum insulin levels. Results from qPCR showed that DHEA-induced increased expression of hypothalamic androgen receptor and LH receptor were reversed by the addition of 60% HFDs. In contrast, the ovarian expression of LH receptor and insulin receptor mRNA was upregulated only with the addition of 60% HFDs. These findings indicated that DHEA and DHEA+HFDs might influence PCOS phenotypes through distinct mechanisms: DHEA affects the normal function of hypothalamus–pituitary–ovarian axis through LH, whereas the addition of HFDs exaggerated endocrine and metabolic dysfunction through ovarian responses to insulin-related mechanisms. We concluded that the addition of HFDs yielded distinct phenotypes of DHEA-induced PCOS and could be used for studies on both reproductive and metabolic features of the syndrome.

Decidualization is required for the successful establishment of pregnancy in rodents and primates. Fatty acid desaturase 3 (Fads3) belongs to the fatty acid desaturase family, which is a crucial enzyme for highly unsaturated fatty acid biosynthesis. However, the expression, regulation and function of Fads3 during early pregnancy in mice are still unknown. In this study, we examined Fads3 expression, regulation and function during mouse decidualization. The expression of Fads3 is detected in the subluminal stromal cells at implantation site on day 5 of pregnancy, but not at inter-implantation site and in day 5 pseudopregnant uteri. Compared to delayed implantation, Fads3 is strongly expressed after delayed implantation is activated by estrogen treatment. From days 6 to 8, Fads3 mRNA signals are significantly detected in the decidua. In ovariectomized mice, estrogen significantly stimulates Fads3 expression. However, estrogen has no effect on Fads3 expression in ovariectomized ERα-deficient mice, suggesting that estrogen regulation on Fads3 expression is ERα dependent. When ovariectomized mice were treated with progesterone, Fads3 expression is significantly increased by progesterone. Progesterone stimulation on Fads3 expression is also detected in cultured stromal cells, which is abrogated by RU486 treatment. These data indicate that progesterone upregulation on Fads3 expression is progesterone receptor-dependent. Fads3 knockdown by siRNA reduces in vitro decidualization of mouse stromal cells. Taken together, Fads3 may play an important role during mouse decidualization.

SRC family kinases (SFKs) are known regulators of multiple cellular events, including cell movement, differentiation, proliferation, survival and apoptosis. SFKs are expressed virtually by all mammalian cells. They are non-receptor protein kinases that phosphorylate a variety of cellular proteins on tyrosine, leading to the activation of protein targets in response to environmental stimuli. Among SFKs, SRC, YES and FYN are the ubiquitously expressed and best studied members. In fact, SRC, the prototypical SFK, was the first tyrosine kinase identified in mammalian cells. Studies have shown that SFKs are regulators of cell junctions, and function in endocytosis and membrane trafficking to regulate junction restructuring events. Herein, we briefly summarize the recent findings in the field regarding the role of SFKs in the testis in regulating spermatogenesis, particularly in Sertoli–Sertoli and Sertoli–germ cell adhesion. While it is almost 50 years since the identification of the oncogene v-Src encoded by Rous sarcoma transforming virus, the understanding of SFK involvement during spermatogenesis in the testis remains far behind that in other epithelia and tissues. The goal of this review is to bridge this gap.

Recent studies implicate the regulatory function of microRNAs (miRNAs) in oocyte maturation and ovarian follicular development. Differentially expressed miRNAs are found in the plasma of premature ovarian failure (POF) patients and normal cycling women. In this study, miRNA-regulated signaling pathways and related genes were described using Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes pathway analysis. The effect of mir-23a on granulosa cell apoptosis was also studied by examining the protein expression of X-linked inhibitor of apoptosis protein (XIAP) and caspase-3, followed by subsequent counting of apoptotic cells after Hoechst 33258 staining. Both GO analysis and pathway analysis suggested that many signaling pathways, including the AKT signaling pathway, steroid hormone receptor signaling pathways, and others, were regulated by this group of differentially expressed miRNAs. A decrease in XIAP expression (mRNA and protein level) and caspase-3 protein levels and an increase in cleaved caspase-3 protein were observed in human ovarian granulosa cells transfected with pre-mir-23a, along with an increased occurrence of apoptosis. In conclusion, differentially expressed miRNAs in the plasma of POF patients may have regulatory effects on proliferation and apoptosis of granulosa cells by affecting different signaling pathways. Mir-23a may play important roles in regulating apoptosis via decreasing XIAP expression in human ovarian granulosa cells.

It has been shown that both prostaglandin I2 (PGI2) and PGE2 are essential for mouse implantation, whereas only PGE2 is required for hamster implantation. To date, the expression and regulation of cyclooxygenase (COX) and prostaglandin E synthase (PGES), which are responsible for PGE2 production, have not been reported in the rat. The aim of this study was to examine the expression pattern and regulation of COX-1, COX-2, membrane-associated PGES-1 (mPGES-1), mPGES-2 and cytosolic PGES (cPGES) in rat uterus during early pregnancy and pseudopregnancy, and under delayed implantation. At implantation site on day 6 of pregnancy, COX-1 immunostaining was highly visible in the luminal epithelium, and COX-2 immunostaining was clearly observed in the subluminal stroma. Both mPGES-1 mRNA and protein were only observed in the subluminal stroma surrounding the implanting blastocyst at the implantation site on day 6 of pregancy , but were not seen in the inter-implantation site on day 6 of pregnancy and on day 6 of pseudopregnancy. Our data suggest that the presence of an active blastocyst is required for mPGES-1 expression at the implantation site. When pregnant rats on day 5 were treated with nimesulide for 24 h, mPGES-1 protein expression was completely inhibited. cPGES immunostaining was clearly observed in the luminal epithelium and subluminal stromal cells immediately surrounding the implanting blastocyst on day 6 of pregnancy. mPGES-2 immunostaining was clearly seen in the luminal epithelium at the implantation site. Additionally, immunostaining for prostaglandin I synthase (PGIS) was also strongly detected at the implantation site. In conclusion, our results indicate that PGE2 and PGI2 should have a very important role in rat implantation.

The micromolar calcium-activated neutral protease gene (CAPN1) is a physiological candidate gene for sperm motility. However, the molecular mechanisms involved in regulating the expression of the CAPN1 gene in bulls remain unknown. In this study, we investigated the expression pattern of CAPN1 in testis, epididymis, and sperm at the RNA and protein levels by qRT-PCR, western blot, immunohistochemistry, and immunofluorescence assay. Results revealed that the expression of CAPN1 levels was higher in the sperm head compared with that in other tissues. Moreover, we identified a novel single-nucleotide polymorphism (g.-1256 A>C, ss 1917715340) in the noncanonical core promoter of the CAPN1 gene between base g.-1306 and g.-1012. Additionally, we observed greater sperm motility in bulls with the genotype CC than in those with the genotype AA (P<0.01), indicating that different genotypes were associated with the bovine semen trait. Furthermore, a higher fluorescence intensity of the C allele than that of the A allele at g. -1256 A>C was revealed by transient transfection in MLTC-1 cells and luciferase report assay. Finally, CAPN1 was highly expressed in the spermatozoa with the CC genotype compared with that with the AA genotype by qRT-PCR. This study is the first report on genetic variant g.-1256 A>C in the promoter region of CAPN1 gene association with the semen quality of Chinese Holstein bulls by influencing its expression. g.-1256 A>C can be a functional molecular marker in cattle breeding.

The production of haploid gametes by meiosis is a cornerstone of sexual reproduction and maintenance of genome integrity. Zfp38 mRNA is expressed in spermatocytes, indicating that transcription factor ZFP38 has the potential to regulate transcription during meiosis. In this study, we generated Zfp38 conditional knockout mice (Zfp38 ^flox/flox, Stra8-Cre, hereafter called Zfp38 cKO) and found that spermatogenesis did not progress beyond meiosis prophase I in Zfp38 cKO mice. Using a chromosomal spread technique, we observed that Zfp38 cKO spermatocytes exhibited a failure in chromosomal synapsis observed by SYCP1/SYCP3 double staining. Progression of DNA double-strand breaks (DSB) repair is disrupted in Zfp38 cKO spermatocytes, as revealed by γ-H2AX, RAD51 and MLH1 staining. Furthermore, the mRNA and protein levels of DSB repair enzymes and factors that guide their loading onto sites of DSBs, such as RAD51, DMC1, RAD51, TEX15 and PALB2, were significantly reduced in Zfp38 cKO spermatocytes. Taken together, our data suggest that ZFP38 is critical for the chromosomal synapsis and DSB repairs partially via its regulation of DSB repair-associated protein expression during meiotic progression in mouse.

Decidualization is essential for the successful establishment of pregnancy, and the dysregulated decidualization may lead to early pregnancy loss. It was previously reported by us that miR-3074-5p could promote apoptosis but inhibit invasion of human extravillous trophoblast (EVT) cells in vitro, and the expression level of miR-3074-5p in villus tissues of recurrent miscarriage (RM) patients was significantly increased. The aim of this study was to preliminarily explore the role of miR-3074-5p played in the decidualization of human endometrial stromal cells (ESCs). It was found that the decidual expression level of miR-3074-5p in RM patients was remarkably higher than that in the control group. The overexpression of miR-3074-5p in the immortalized human ESC line, T-HESCs, showed suppressive effects not only on the cell proliferation, as well as the intracellular expression levels of cyclin B1 (CCNB1), CCND1 and CCNE1 but also on the in vitro-induced decidualization. CLN8 mRNA, encoding an endoplasmic reticulum (ER)-associated membrane protein, was validated to be directly targeted by miR-3074-5p. And, the expression level of CLN8 was continuously increased along with the decidualization process, whereas down-regulated CLN8 expression could inhibit the decidualization of T-HESCs in vitro. Furthermore, contrary to the increased expression level of miR-3074-5p, a significantly decreased CLN8 expression was observed in decidual tissues of RM patients. Collectively, these data suggested that an increased miR-3074-5p expression in ESCs might cause early pregnancy failure by disturbing decidualization of ESCs via the miR-3074-5p/CLN8 pathway, providing a potential diagnostic and therapeutic target for RM.

In cloned animals where somatic cell nuclei and oocytes are from the same or closely related species, the mitochondrial DNA (mtDNA) of the oocyte is dominantly inherited. However, in nuclear transfer (NT) embryos where nuclear donor and oocyte are from two distantly related species, the distribution of the mtDNA species is not known. Here we determined the levels of macaque and rabbit mtDNAs in macaque embryos reprogrammed by rabbit oocytes. Quantification using a real-time PCR method showed that both macaque and rabbit mtDNAs coexist in NT embryos at all preimplantation stages, with maternal mtDNA being dominant. Single NT embryos at the 1-cell stage immediately after fusion contained 2.6 × 10⁴ copies of macaque mtDNA and 1.3 × 10⁶ copies of rabbit mtDNA. Copy numbers of both mtDNA species did not change significantly from the 1-cell to the morula stages. In the single blastocyst, however, the number of rabbit mtDNA increased dramatically while macaque mtDNA decreased. The ratio of nuclear donor mtDNA to oocyte mtDNA dropped sharply from 2% at the 1-cell stage to 0.011% at the blastocyst stage. These results suggest that maternal mtDNA replicates after the morula stage.