Systems Biology in Reproductive Medicine最新文献

There is a need to understand the molecular basis of testes under Non-Obstructive Azoospermia (NOA), a state of failed spermatogenesis. There has been a lack of attention to the transcriptome at the level of alternatively spliced mRNAs (iso-mRNAs) and the mechanism of gene expression regulation. Hence, we aimed to establish a reliable iso-mRNA profile of NOA-testes, and explore molecular mechanisms - especially those related to gene expression regulation. We sequenced mRNAs from testicular samples of donors with complete spermatogenesis (control samples) and a failure of spermatogenesis (NOA samples). We identified differentially expressed genes and their iso-mRNAs via standard NGS data analyses. We then listed these iso-mRNAs hierarchically based on the extent of consistency of differential quantities across samples and groups, and validated the lists via RT-qPCRs (for 80 iso-mRNAs). In addition, we performed extensive bioinformatic analysis of the splicing features, domains, interactions, and functions of differentially expressed genes and iso-mRNAs. Many top-ranking down-regulated genes and iso-mRNAs, i.e., those down-regulated more consistently across the NOA samples, are associated with mitosis, replication, meiosis, cilium, RNA regulation, and post-translational modifications such as ubiquitination and phosphorylation. Most down-regulated iso-mRNAs correspond to full-length proteins that include all expected domains. The predominance of alternative promoters and termination sites in these iso-mRNAs indicate their gene expression regulation via promoters and UTRs. We compiled a new, comprehensive list of human transcription factors (TFs) and used it to identify TF-'TF gene' interactions with potential significance in down-regulating genes under the NOA condition. The results indicate that RAD51 suppression by HSF4 prevents SP1-activation, and SP1, in turn, could regulate multiple TF genes. This potential regulatory axis and other TF interactions identified in this study could explain the down-regulation of multiple genes in NOA-testes. Such molecular interactions may also have key regulatory roles during normal human spermatogenesis.

This review provides details on the role of centrin in human spermatozoa and in various forms of male infertility. Centrin is a calcium (Ca²⁺)-binding phosphoprotein that is located in the centrioles - which are typical structures of the sperm connecting piece and play a key role in centrosome dynamics during sperm morphogenesis - as well as in zygotes and early embryos during spindle assembly. In humans, three different centrin genes encoding three isoforms have been discovered. Centrin 1, the only one expressed in spermatozoa, seems to be lost inside the oocyte after fertilization. The sperm connecting piece is characterized by the presence of numerous proteins including centrin, that deserves particular attention because, in humans, it is enriched during maturation of the centrioles. In normal sperm, centrin 1 is visible as two distinct spots in the head-tail junction; however, in some defective spermatozoa, centrin 1 distribution is altered. Centrin has been studied in humans and animal models. Its mutations may lead to several structural alterations, such as serious defects in the connective piece and, subsequently, fertilization failure or incomplete embryonic development. However, the effects of these abnormalities on male fertility have not been fully studied. Because the presence and the function of centrin in the sperm connecting piece appears important for reproductive success, additional studies are needed to bring medical benefits in resolving some cases of idiopathic infertility.

Oxytocin (OXT) plays a significant role during pregnancy, especially toward the end of pregnancy. Some studies have reported that OXT is involved in the stimulation of steroidogenesis in several organs. However, the effects of OXT on placental steroidogenesis have not yet been established. In this study, we investigated the regulation of steroid hormones and steroidogenic enzymes by OXT-associated signaling in vitro and in vivo. OXT increased the gene expression of steroidogenic enzymes, which convert pregnenolone to progesterone and dehydroepiandrosterone (DHEA) in vitro. In OXT-administered pregnant rats, pregnenolone and DHEA levels were significantly enhanced in the plasma and the expression of the enzymes synthesizing DHEA, testosterone, and estradiol (E2) was increased in placental tissues. Furthermore, OXT was found to affect placental cell differentiation, which is closely related to steroid hormone synthesis. After treatment of the pregnant rats with atosiban, an antagonist of the OXT receptor, the concentration of E2 in the plasma and the expression of E2-synthesizing enzyme were reduced. This regulation may be due to OXT-mediated differentiation, because OXT increases the expression of corticotropin-releasing hormone, which is a biomarker of placental cell differentiation. Our findings suggest that OXT contributes to maintaining pregnancy by regulating the differentiation of placental cells and steroidogenesis during pregnancy.

Multiple effects of stress on health have been reported; however, reproductive alterations in oocytes and cumulus cells have not been fully described. In females, chronic stress has been shown to produce alterations in the estrous cycle, to decrease oocyte in vivo maturation, and to increase the percentage of abnormal oocytes. The aim of this study was to evaluate whether the oocytes from chronically stressed female rats could recover and mature in vitro by providing them with all the necessary culture conditions, as well as to evaluate the functionality of the GAP junctions, and the viability and DNA integrity of the cumulus cells, which are crucial for the complete maturation and development of the oocyte. For this, rats were stressed daily by cold water immersion (15 °C) during 15 min for 30 consecutive days. Corticosterone serum levels in rats increased as an indicator of stress. Chronic stress decreased the percentage of in vitro matured oocytes because the cumulus cells presented irreparable damage to their DNA that led to their death, being unable to establish bidirectional communication with the oocyte for its meiotic resumption through the GAP junctions, which were also damaged. These findings could partially explain an association between stress and infertility.

Artificial oocyte activation (AOA) is considered an effective method to improve clinical outcomes in patients with some forms of male factor infertility and does not increase the risk of birth defects. However, the effects of AOA on patients with multiple morphological abnormalities of the sperm flagella (MMAF) caused by a DNAH1 mutation are still unknown. To explore the effects, our study analyzed a case with MMAF due to DNAH1 homozygous mutation that underwent testicular sperm extraction (TESE) combined with intracytoplasmic sperm injection (ICSI). The case had 28 MII oocytes. The 28 oocytes were divided randomly and equally into AOA and non-AOA groups. Ionomycin was used for AOA. We compared the clinical outcomes of two groups and selected three blastulation failure embryos from each group for transcriptome analysis (Data can be accessed through GSE216618). Differentially expressed genes (DEGs) were determined with an adjusted p-value <0.05 and a |log2-fold change| ≥1. The comparison of clinical outcomes showed that the two pronuclei (2PN) rate and grade 1-2 embryo rate at day 3 were not significantly different between the two groups. Transcriptome analyses of blastulation failed embryos showed that the use of AOA had potential risks of chromosome structure defects, transcriptional regulation defects, and epigenetic defects. In conclusion, when the case with MMAF due to DNAH1 mutation underwent TESE-ICSI, ionomycin-induced oocyte activation could not improve the clinical outcomes and introduced the risks of chromosome structure defect, transcriptional regulation defect, and epigenetic defect.

Polycystic ovary syndrome (PCOS) is a disease characterized by metabolic disorders. This study aimed to examine the effects of resveratrol treatment on ovulation in the PCOS rat model. Quantitative real-time PCR and immunohistochemistry were used to determine the mRNA and protein expression levels. TNUEL assay was used to evaluate cell apoptosis in ovary. The metabolites were evaluated by liquid chromatography with tandem mass spectrometry. Resveratrol alleviated disrupted estrous cycle and improved granular cell layers, and reversed the decreased proliferation and increased cell apoptosis of granulosa cells in the ovarian tissues of PCOS rats. Resveratrol restored the changes in the mRNA expression levels in the rate-limiting genes of glycolysis in the PCOS ovary. The expression of lactate dehydrogenase A (LDH-A), pyruvate kinase isozyme M2 (PKM2), and sirtuin 1 (SIRT1) was significantly downregulated in ovarian tissues of the PCOS rats; while the resveratrol treatment significantly increased the expression of LDH-A, PKM2, and SIRT1 in the ovarian tissues of PCOS rats. Collectively, the protective effects of resveratrol in the PCOS rats may be associated with the regulation of glycolysis-related mediators including PKM2, LDH-A, and SIRT1. Resveratrol may represent a good candidate in alleviating the development of PCOS.

Prostate cancer (PCa) is as a serious threat to male's health around the world. Recent studies have indicated that long non-coding RNAs (lncRNAs) occupy an important position in various human cancers. However, the function and mechanism of lncRNA DNMBP antisense RNA 1 (DNMBP-AS1) in PCa is rarely investigated. RT-qPCR analysis was used to test gene expression. CCK-8, colony formation, EdU staining and transwell assays were conducted to assess the function of DNMBP-AS1 on PCa cell behaviors. RNA pull down, RIP and luciferase reporter assays were implemented to verify the mechanism of DNMBP-AS1. DNMBP-AS1 was obviously up-regulated in PCa cell lines. Functionally, DNMBP-AS1 knockdown weakened cell proliferation, migration and invasion of PCa. Mechanistically, DNMBP-AS1 sponged microRNA-6766-3p (miR-6766-3p) to regulate lysocardiolipin acyltransferase 1 (LCLAT1) expression. Furthermore, DNMBP-AS1 could stabilize LCLAT1 expression by recruiting ELAV like RNA binding protein 1 (ELAVL1). Consequently, rescue assays demonstrated that DNMBP-AS1 regulated PCa cell proliferation, migration and invasion through enhancing LCLAT1 expression. Collectively, we elucidated the function and regulatory mechanism of DNMBP-AS1 and provided the first evidence of DNMBP-AS1 as a driver for PCa.

Both vitamin A and E support female reproduction and embryonic development. These vitamins have been associated with decreased fertility or failure to end the pregnancy in animals. An observational study was conducted on follicular fluid (FF) samples to determine the concentrations of fat-soluble vitamins of women undergoing in vitro fertilization and its correlation with assisted reproductive technology characteristics and pregnancy outcomes. Moreover, the effects of all-trans-retinoic acid (atRA) and alpha-tocopherol on granulosa cell viability, apoptosis, autophagy and hormonal production were evaluated. No association was identified between fat-soluble vitamin concentrations in FF and infertility aetiology, body mass index or woman's age. There were differences in follicular antioxidant profiles and ovarian response stimulation. In vitro evaluation of atRA and alpha-tocopherol reveals that, at physiological concentrations, both compounds may affect the viability of granulosa cells. In addition, these compounds are able to protect granulosa cells from oxidative stress, as well as to affect estradiol and progesterone production. Our data suggest that atRA and alpha-tocopherol levels should be well controlled as they may have implications in the function and viability of granulosa cells and highlights retinol as a marker of the oxidative defenses within ovary environment.

The purpose of this study is to investigate the optimal framerate (FR) and the use of different counting chambers for improving CASA-Mot technology use in Andrology. Images were captured at 500 fps, then segmented and analyzed in several ranges of FRs (from 25 to 250) to define the asymptotic point that as an optimal FR. This work was replicated using counting chambers based in capillarity (disposable) or drop displacement (reusable) to study their effects on the motility results and kinematic values of the samples under the different experimental conditions. The α value (asymptote corresponding to FR_o) of the exponential curve was 150.23 fps, corresponding to a VCL of 130.58 mm/s, far from the value of 98.89 mm/s corresponding to 50 fps (the highest FR used by most current CASA-Mot systems). Our results have shown that, when using reusable counting chambers, type and depth have influence. In addition, different results were obtained depending on the area of image captured inside the different counting chamber types. To have reliable results in human sperm kinematic studies, almost 150 fps should be used for capturing and analyzing and differences between chambers should be considered by sampling from different areas, to obtain a representative value of the whole sample.

There is evidence of the existence of an intraovarian gonadotropin-releasing hormone (GnRH) system. There are also reports about the influence of extrinsic ovarian innervation in gonadal function. Therefore, it is interesting to study the relationship between ovarian sympathetic innervation and GnRH to shed light on possible physiological and pathophysiological implications. This work aimed to investigate whether noradrenergic stimulation of the superior mesenteric ganglion (SMG) can modify the levels of ovarian GnRH and cause functional and morphological changes in the gonad through the ovarian plexus nerve (OPN), during estrus and diestrus II in rats. The SMG-OPN-Ovary system and an ovary without extrinsic innervation were removed from Holtzman rats in estrus and diestrus II stages and placed in specially designed cuvettes containing Krebs-Ringer buffer. In the experimental groups, SMGs and denervated ovaries were stimulated with 10^-6 M noradrenaline (NA). GnRH and progesterone levels (in the ovarian incubation medium) and the mRNA expression of 3beta-hydroxysteroid dehydrogenase (Hsd3b3), 20alpha-hydroxysteroid dehydrogenase (Akr1c18), Bax, and Bcl2 were analyzed. Histological studies of the ovaries were performed. In estrus, NA decreased GnRH levels in both experimental schemes. Furthermore, progesterone levels increased while the Akr1c18 expression and Bax/Bcl2 ratio decreased, without causing changes in ovarian morphology. In diestrus, the noradrenergic stimulation of the ganglion increased GnRH levels, decreased progesterone levels, and increased Akr1c18 expression and Bax/Bcl2 ratio. Follicles with histoarchitecture alterations and corpus luteum with signs of cell death were observed. In denervated ovaries, NA increased the levels of GnRH and progesterone. Furthermore, NA decreased the Bax/Bcl2 ratio and histological studies revealed signs compatible with a possible atretogenic effect. In conclusion, noradrenergic stimulation of the SMG-OPN pathway regulates ovarian cyclicity. The SMG modulates the cross-talk between NA and ovarian GnRH, protecting the ovary from atretogenic effects and luteal apoptosis during estrus while inducing luteal regression in the diestrus II.