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Yufen Zhao, Boyang Yu, Xinyu Liu, Jitu Hu, Yanyan Yang, Erge Namei, Bingxue Yang, Yue Bai, Yinghong Qian, and Haijun Li

Although urokinase-type plasminogen activator (PLAU) and urokinase-type plasminogen activator receptor (PLAUR) have been reported to play key roles in ovarian function, their precise contribution to mammalian follicular development remains unclear. In this study, we first observed that PLAU and PLAUR were present in bovine granulosa cells (GCs). Following culture of granulosa cells with PLAU (0.5 ng/mL) and PLAUR antibody (10 µg/mL) separately and together for 24 or 48 h, a proliferation assay showed that interaction between PLAU and PLAUR contributes to bovine GC proliferation. To study the potential pathways involved in PLAU/PLAUR-induced cell proliferation, ELISA and Western blotting were performed. We found that PLAU significantly increased the ratio of phosphorylated to non-phosphorylated ERK1/2 through PLAUR signaling. Further treatment with U0126, a specific ERK1/2 phosphorylation inhibitor, markedly suppressed PLAU/PLAUR-induced ERK1/2 phosphorylation and cell proliferation. In addition, we found that PLAU and PLAUR significantly increased the intracellular cAMP level and the use of Rp-cAMP, a specific PKA inhibitor, prevented PLAU/PLAUR from promoting activation of the ERK1/2 pathway and GC proliferation. Therefore, the interaction between PLAU and PLAUR may be involved in accumulating cAMP signals and enabling MAPK/ERK1/2 activation, affecting GC proliferation. Here, we provide new mechanistic insights into the roles of PLAU and PLAUR on promoting bovine GC proliferation. The finding that potential cross-points between PLAU/PLAUR-induced intracellular signals affect GC proliferation will help in understanding the mechanisms regulating early follicular development.

Open access

Yizi Wang, Minghui Chen, Jian Xu, Xinyan Liu, Yuwei Duan, Canquan Zhou, and Yanwen Xu

Luteinization is the event of corpus luteum formation, a way of follicle cells transformation and a process of steroidogenesis alteration. As the core clock gene, Bmal1 was involved in the regulation of ovulation process and luteal function afterwards. Till now, the underlying roles of luteinization played by Bmal1 remain unknown. To explore the unique role of Bmal1 in luteal steroidogenesis and its underlying pathway, we investigated the luteal hormone synthesis profile in Bmal1 knockout female mice. We found that luteal hormone synthesis was notably impaired, and phosphorylation of PI3K/NfκB pathway was significantly activated. Then, the results were verified in in vitro cultured cells, including isolated Bmal1 interference granulosa cells (GCs) and theca cells (TCs), respectively. Hormones levels of supernatant culture media and mRNA expressions of steroidogenesis-associated genes (star, Hsd3β2, cyp19a1 in GCs, Lhcgr, star, Hsd3β2, cyp17a1 in TCs) were mutually decreased, while the phosphorylation of PI3K/NfκB was promoted during in vitro luteinization. After PI3K specific-inhibitor LY294002 intervention, mRNA expressions of Lhcgr and Hsd3β2 were partially rescued in Bmal1 interference TCs, together with significantly increased androstenedione and T synthesis. Further exploration in TCs demonstrated BMAL1 interacted directly but negatively with NfκB p65 (RelA), a subunit which was supposed as a mediator in Bmal1-governed PI3K signaling regulation. Taken together, we verified the novel role of Bmal1 in luteal steroidogenesis, achieving by negative interplay with RelA-mediated PI3K/NfκB pathway.

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Yi Sun, Xinping Sun, Lianming Zhao, Zhe Zhang, Yupeng Wang, Zhenfeng Dai, Xin Zhao, and Xiaoping Pu

Asthenozoospermia (AS), defined as low-motility spermatozoa in the ejaculate, is a frequent cause of human male infertility. DJ-1 (also known as PARK7), a protein highly associated with male sterility, binds to the mitochondrial complex I subunit to protect mitochondrial function. However, its involvement in spermatogenesis has not been fully elucidated. Previously, the levels of DJ-1 were shown to be significantly decreased in testicular tissues of rats with ornidazole (ORN)-induced AS. Here, we used a rat model to investigate the localization and expression levels of DJ-1 and its interacting NDUFS3 and NDUFA4 mitochondrial complex I subunits, as well as AS-induced metabolic alterations in testicular tissues. ORN significantly reduced the levels of DJ-1 in the nucleus of secondary spermatocytes, while increasing the expression of NDUFS3 in the cytoplasm of primary spermatocytes. Further, NDUFA4 showed higher expression after treatment with ORN. The principal ORN-induced changes in metabolic small molecules related to the accumulation of glucose, glutamine, and N-acetyl aspartate, enhancement of purine pathway, increase of the phosphatidic acid (PA) (18:0/18:1), phosphatidylethanolamine (PE) (16:0/18:1), and PA (18:0/20:4) lipid metabolites, and imbalance in the concentrations of Na+ and K+. However, we did not observe any abnormalities of certain small metabolic molecules and metal ions in semen samples from patients with AS. In conclusion, these results suggest that DJ-1 deficiency in testicular tissues might be closely related to the localization of NDUFS3 and content of NDUFA4, thus causing abnormalities in the mitochondrial energy metabolism and multiple other metabolic pathways.

Free access

Yohanes N S Nursalim, Cherie Blenkiron, Katie M Groom, and Lawrence W Chamley

Trophoblasts are unique epithelial cells found only in the placenta. It has been possible to isolate and maintain human trophoblasts in in vitro culture for many decades. During this period there have been a vast array of media and supplements reported for trophoblast culture and often the reasons for using the media and specific supplements employed in any given laboratory have been lost in the ‘mists of time’. After a gradual development over many years this field has recently changed, with the publication of several reports of the isolation, growth and differentiation of human trophoblast stem or stem-like cells. This advance was made largely because of a greater understanding of the molecular pathways that control human trophoblasts and availability of media supplements that can be used to manipulate those pathways. We have searched the literature and here summarise many of the different media and supplements and describe how and why they were developed and are used to culture human trophoblasts.

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Riley E Thompson, Aime K Johnson, Pouya Dini, Margherita Y Turco, Tulio M Prado, Christopher Premanandan, Graham J Burton, Barry A Ball, Brian K Whitlock, and Budhan S Pukazhenthi

The endometrium, the inner uterine lining, is composed of cell layers that come in direct contact with an embryo during early pregnancy and later with the fetal placenta. The endometrium is responsible for signals associated with normal reproductive cyclicity as well as maintenance of pregnancy. In the mare, functionally competent in vitro models of the endometrium have not been successful. Furthermore, the ability to study various reproductive processes in vitro may allow critical evaluation of signaling pathways involved in the reproductive diseases of animals that cannot be handled frequently, such as various wildlife species. Here we report the establishment of organoids, 3D structures, derived from fresh and frozen–thawed equine endometrium (Equus ferus caballus and E. f. przewalskii). Although organoids from domestic mares responded to exogenous hormonal stimuli, organoids from Przewalski’s horse failed to respond to exogenous hormones. The present study represents a ‘first’ for any large animal model or endangered species. These physiologically functional organoids may facilitate improved understanding of normal reproductive mechanisms, uterine pathologies, and signaling mechanisms between the conceptus and endometrium and may lead to the development of novel bioassays for drug discovery.

Free access

Sachiko Matsuzaki and Michael W Pankhurst

Serum anti-Müllerian hormone (AMH) levels decrease after surgical treatment of ovarian endometrioma. This is the main reason that surgery for ovarian endometrioma endometriosis is not recommended before in vitro fertilization, unless the patient has severe pain or suspected malignant cysts. Furthermore, it has been suggested that ovarian endometrioma itself damages ovarian reserve. This raises two important challenges: (1) determining how to prevent surgical damage to the ovarian reserve in women with ovarian endometrioma and severe pain requiring surgical treatment and (2) deciding the best treatment for women with ovarian endometrioma without pain, who do not wish to conceive immediately. The mechanisms underlying the decline in ovarian reserve are potentially induced by both ovarian endometrioma and surgical injury but the relative contribution of each process has not been determined. Data obtained from various animal models and human studies suggest that hyperactivation of dormant primordial follicles caused by the local microenvironment of ovarian endometrioma (mechanical and/or chemical cues) is the main factor responsible for the decreased primordial follicle numbers in women with ovarian endometrioma. However, surgical injury also induces hyperactivation of dormant primordial follicles, which may further reduce ovarian reserve after removal of the endometriosis. Although further studies are required to elucidate the mechanisms underlying diminished ovarian reserve in women with ovarian endometrioma, the available data strongly suggests the need to prevent/minimize hyperactivation of dormant primordial follicles, regardless of whether surgery is performed, for better clinical management of ovarian endometrioma.

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Rafael R Domingues, O J Ginther, Mateus Z Toledo, and Milo C Wiltbank

Understanding the impacts of nutrition on reproductive physiology in cattle are fundamental to improve reproductive efficiency for animals under different nutritional conditions. Starting on Day 0 (day of ovulation) until next ovulation, Holstein heifers (n = 24) were fed: low energy diet (ad libitum grass hay; LED) and high energy diet (ad libitum grass hay + concentrate supplement; HED). Heifers on HED gained more weight (average daily gain: 0.824 ± 0.07 vs 0.598 ± 0.09 kg/day) and had increased insulin concentrations. The dominant follicle of wave 1 in HED had greater growth rate overall from Days 0 to 8 and on Days 6–7 and 8–9 and started atresia later. The dominant follicle of wave 2 in HED had greater growth rate overall from Day 9 to 18 and on Days 14–15 and 15–16. In two-wave patterns, there was no difference in estradiol or progesterone concentrations but concentrations of FSH were lower in HED on Days 15 and 16. Estradiol concentrations increased earlier in two-wave patterns in association with earlier luteolysis. The frequency of two follicular waves was greater in HED than LED (11/12 vs 6/11; 92.7% vs 54.5%). In conclusion, an acute increase in dietary energy altered not only growth rate of the dominant follicle but also follicular wave pattern in heifers by increasing frequency of two follicular waves. The hypotheses were supported that an acute increase in dietary energy (1) prolongs growth period of dominant follicles and (2) alters follicular wave pattern in heifers.

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Meng-Chieh Hsu, Leang-Shin Wu, De-Shien Jong, and Chih-Hsien Chiu

Kisspeptin and its receptor KISS1R have been proven as pivotal regulators on controlling the hypothalamus–pituitary–gonad axis. Inactivating mutations in one of them cause idiopathic hypogonadotropic hypogonadism in human as well as rodent models. Notably, gonadotropin insensitivity, failure in hCG response, was presented in the male patients with loss-function-mutations in KISS1R gene; this reveals the essential role of KISS1R signaling in regulating testosterone production beyond the hypothalamic functions of kisspeptin. In this study, we hypothesized that the autocrine action of kisspeptin on Leydig cells may modulate steroidogenesis. Based on the mouse cell model, we first demonstrated that the cAMP/protein kinase A (PKA)/cAMP response element-binding protein (CREB) signaling pathway mediated gonadotropin-induced kisspeptin expression. By using siRNA interfering technique, knockdown of Kiss1r in MA-10 cells, a mouse Leydig tumor cell line, significantly reduced progesterone productions in both basal and hCG-treated conditions. Integrating the results from both quantitative real-time PCR and steroidogenic enzyme-activity assay, we found that this steroidogenic defect was associated with decreased luteinizing hormone/choriogonadotropin receptor (Lhcgr) and StAR protein (Star) expressions. Furthermore, exogenous expression of human LHCGR completely rescued hCG-stimulated progesterone production in the KISS1R-deficient cells. In conclusion, we proposed that the reproductive functions of KISS1R signaling in Leydig cell include modulating Lhcgr and steroidogenic gene expressions, which may shed the light on the pathophysiology of gonadotropin insensitivity.

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Yuan Yuan, Ling Zhao, Xiaoying Wang, Feng Lian, and Yan Cai

Preeclampsia (PE), a serious complication of pregnancy, is associated with abnormal trophoblast cell differentiation and autophagy. Herein, we investigated the molecular mechanism underlying the function of ligustrazine (2,3,5,6-tetramethylpyrazine, TMP), a constituent of the traditional Chinese plant medicine Ligusticum wallichii, in PE. Lipopolysaccharide (LPS) was applied to induce a PE rat model, followed by tail vein injection of TMP or lentiviral vector overexpressing microRNA-16-5p (miR-16-5p). Human trophoblast cell line JEG3 was cultured in vitro to construct a PE cell model, followed by t he treatment with different concentrations of TMP, miR-16-5p mimic/inhibitor, or shRNA (shRNA) against insulin growth factor-2 (IGF-2) (sh-IGF-2). Formation of autophagosomes and autophagy-related proteins were then examined. Cell counting kit-8 (CCK-8) and Transwell assays were applied to measure trophoblast cell viability and migration. The binding affinity between miR-16-5p and IGF-2 was verified by dual luciferase report assay. After TMP treatment, autophagosome formation was reduced in trophoblast cells of placental tissue of PE rats, along with downregulation of autophagy-related proteins Light Chain 3 (LC3)-II/LC3-I, Beclin1 (BECN1), and SQSTM1. Moreover, TMP repressed JEG3 cell autophagy, promoted viability and migration concentration-responsively. MiR-16-5p was upregulated in PE, and TMP inhibited miR-16-5p expression. Besides, miR-16-5p downregulated IGF-2 expression to promote cell autophagy and inhibit the viability and migration of JEG3 cells. Further, in vivo experiments validated that TMP impeded PE progression in rats by regulating the miR-16-5p/IGF-2 axis. In summary, TMP inhibits trophoblast cell autophagy and promotes its viability and migration in PE rat model through regulating the miR-16-5p/IGF-2 axis.

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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.