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In our previous study, we have demonstrated that the epithelial sodium channel (ENaC) mediates the embryo-derived signals leading to the activation of CREB and upregulation of cyclooxygenase type 2 (COX2) required for embryo implantation. This study aims to investigate whether microRNAs (miRNAs) are involved in the ENaC-induced upregulation of COX2 during embryo implantation. The results show that the levels of miR-101 and miR-199a-3p, two COX2 targeting miRNAs, are reduced by ENaC activation, and increased by ENaC inhibition or knock-down of ENaC subunit (ENaCα) in human endometrial surface epithelial (HES) cells or in mouse uteri during implantation. Phosphorylation of CREB is induced by the activation of ENaC, and blocked by ENaC inhibition or knockdown in HES cells. Knockdown of ENaCα or CREB in HES cells or in mouse uterus in vivo results in increases in miR-101 and miR-199a-3p, accompanied with decreases in COX2 protein levels and reduction in implantation rate. The downregulation of COX2 caused by knockdown of ENaC or CREB can be recovered by the inhibitors of miR-101 or miR-199a-3p in HES cells. These results reveal a novel molecular mechanism modulating COX2 expression during embryo implantation via ENaC-dependent CREB activation and COX2-targeting miRNAs.

Asthenozoospermia is one of the leading causes of male infertility owing to a decline in sperm motility. Herein, we determined if there is a correlation between RNASET2 content on human spermatozoa and sperm motility in 205 semen samples from both asthenozoospermia patients and normozoospermia individuals. RNASET2 content was higher in sperm from asthenozoospermia patients than in normozoospermia individuals. On the other hand, its content was inversely correlated with sperm motility as well as progressive motility. Moreover, the inhibitory effect of RNASET2 on sperm motility was induced by incubating normozoospermic sperm with RNase T2 protein. Such treatment caused significant declines in intracellular spermatozoa PKA activity, PI3K activity and calcium level, which resulted in severely impaired sperm motility, and the sperm motility was largely rescued by cAMP supplementation. Finally, protein immunoprecipitation and mass spectrometry identified proteins whose interactions with RNASET2 were associated with declines in human spermatozoa motility. AKAP4, a protein regulating PKA activity, coimmunoprecipated with RNASET2 and they colocalized with one another in the sperm tail, which might contribute to reduced sperm motility. Thus, RNASET2 may be a novel biomarker of asthenozoospermia. Increases in RNASET2 can interact with AKAP4 in human sperm tail and subsequently reduce sperm motility by suppressing PKA/PI3K/calcium signaling pathways.

Hormone-sensitive lipase-knockout (HSL−/−) mice exhibit azoospermia for unclear reasons. To explore the basis of sterility, we performed the following three experiments. First, HSL protein distribution in the testis was determined. Next, transcriptome analyses were performed on the testes of three experimental groups. Finally, the fatty acid and cholesterol levels in the testes with three different genotypes studied were determined. We found that the HSL protein was present from spermatocyte cells to mature sperm acrosomes in wild-type (HSL+/+) testes. Spermiogenesis ceased at the elongation phase of HSL−/− testes. Transcriptome analysis indicated that genes involved in lipid metabolism, cell membrane, reproduction and inflammation-related processes were disordered in HSL−/− testes. The cholesterol content was significantly higher in HSL−/− than that in HSL+/+ testis. Therefore, gene expression and cholesterol ester content differed in HSL−/− testes compared to other testes, which may explain the sterility of male HSL−/− mice.

Maternal diabetes adversely affects preimplantation embryo development and oocyte maturation. Thus, it is important to identify ways to eliminate the effects of maternal diabetes on preimplantation embryos and oocytes. The objectives of this study were to investigate whether islet transplantation could reverse the effects of diabetes on oocytes. Our results revealed that maternal diabetes induced decreased ovulation; increased the frequency of meiotic spindle defects, chromosome misalignment, and aneuploidy; increased the relative expression levels of Mad2 and Bub1; and enhanced the sensitivity of oocytes to parthenogenetic activation. Islet transplantation prevented these detrimental effects. Therefore, we concluded that islet transplantation could reverse the effects of diabetes on oocytes, and that this technique may be useful to treat the fundamental reproductive problems of women with diabetes mellitus.

FSH plays an essential role in processes involved in human reproduction, including spermatogenesis and the ovarian cycle. While the transcriptional regulatory mechanisms underlying its synthesis and secretion have been extensively studied, little is known about its posttranscriptional regulation. A bioinformatics analysis from our group indicated that a microRNA (miRNA; miR-361-3p) could regulate FSH secretion by potentially targeting the FSHB subunit. Herein, we sought to confirm these findings by investigating the miR-361-3p-mediated regulation of FSH production in primary pig anterior pituitary cells. Gonadotropin-releasing hormone (GnRH) treatment resulted in an increase in FSHB synthesis at both the mRNA, protein/hormone level, along with a significant decrease in miR-361-3p and its precursor (pre-miR-361) levels in time- and dose-dependent manner. Using the Dual-Luciferase Assay, we confirmed that miR-361-3p directly targets FSHB. Additionally, overexpression of miR-361-3p using mimics significantly decreased the FSHB production at both the mRNA and protein levels, with a reduction in both protein synthesis and secretion. Conversely, both synthesis and secretion were significantly increased following miR-361-3p blockade. To confirm that miR-361-3p targets FSHB, we designed FSH-targeted siRNAs, and co-transfected anterior pituitary cells with both the siRNA and miR-361-3p inhibitors. Our results indicated that the siRNA blocked the miR-361-3p inhibitor-mediated upregulation of FSH, while no significant effect on non-target expression. Taken together, our results demonstrate that miR-361-3p negatively regulates FSH synthesis and secretion by targeting FSHB, which provides more functional evidence that a miRNA is involved in the direct regulation of FSH.

Fertilization failure often occurs during in vitro fertilization (IVF) cycles despite apparently normal sperm and oocytes. Accumulating evidence suggests that mitochondria play crucial roles in the regulation of sperm function and male fertility. 3-Nitrophthalic acid (3-NPA) can induce oxidative stress in mitochondria, and melatonin, as an antioxidant, can improve mitochondrial function by reducing mitochondrial oxidative stress. The role of sperm mitochondrial dysfunction in fertilization failure during IVF is unclear. The present study revealed that spermatozoa with low, or poor, fertilization rates had swollen mitochondria, increased mitochondria-derived ROS, and attenuated mitochondrial respiratory capacity. 3-NPA treatment enhanced mitochondrial dysfunction in sperm. Spermatozoa with poor fertilization rates, and spermatozoa treated with 3-NPA, had reduced penetration ability. The concentration of melatonin was decreased in semen samples with low and poor fertilization rates. Melatonin, not only decreased excessive mitochondria-derived ROS, but also ‘rescued’ the reduced penetration capacity of spermatozoa treated with 3-NPA. Taken together, the study suggested that mitochondria-derived ROS and mitochondrial respiratory capacity are independent bio-markers for sperm dysfunction, and melatonin may be useful in improving sperm quality and overall male fertility.