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Magdalena Kotlarska, Dawid Winiarczyk, Wiesława Florek, Marta Ziętek, Jolanta Pęczkowicz-Szyszka, Adrian Mateusz Stankiewicz, Rafał Radosław Starzyński, Roberta Arena, Gaspare Drago, Silvestre Sampino, and Jacek Andrzej Modlinski

Preimplantation embryos are particularly vulnerable to environmental perturbations, including those related to assisted reproductive technologies. Invasive embryo manipulations, such as blastomere biopsy, are applied worldwide in clinical settings for preimplantation genetic testing. Mouse models have previously shown that blastomere biopsy may be associated with altered phenotypes in adult offspring. The aim of the present study was to investigate the specific contribution of blastomere removal to the physiological, behavioral, and molecular regulators of energy homeostasis, as compared to sham manipulation (re-introducing the blastomere into the embryo after its removal) and in vitro culture. Mice derived from 8-cell embryos subjected to blastomere removal displayed: (i) higher body weight and adiposity, (ii) increased food intake and sucrose preference, (iii) decreased time of immobility in the tail suspension test, and (iv) resistance to weight loss after social isolation or following 3 days of physical exercise – compared to mice derived from sham biopsy or from in vitro-cultured embryos. Mice generated after blastomere removal also had increased circulating leptin and leptin gene expression in adipose tissue, as well as increased ghrelin receptor gene expression in the hypothalamus, compared to control mice. The effects of blastomere biopsy on offspring phenotype were sexually dimorphic, with females not being affected. These results indicate that blastomere deprivation, rather than other perturbations of the blastomere biopsy procedure, programs male embryos to develop physiological, behavioral, and molecular dysregulation of energy homeostasis, leading to postnatal obesity.

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Deepak S Hiremath, Fernanda B M Priviero, R Clinton Webb, CheMyong Ko, and Prema Narayan

Timely activation of the luteinizing hormone receptor (LHCGR) is critical for fertility. Activating mutations in LHCGR cause familial male-limited precocious puberty (FMPP) due to premature synthesis of testosterone. A mouse model of FMPP (KiLHRD582G), expressing a constitutively activating mutation in LHCGR, was previously developed in our laboratory. KiLHRD582G mice became progressively infertile due to sexual dysfunction and exhibited smooth muscle loss and chondrocyte accumulation in the penis. In this study, we tested the hypothesis that KiLHRD582G mice had erectile dysfunction due to impaired smooth muscle function. Apomorphine-induced erection studies determined that KiLHRD582G mice had erectile dysfunction. Penile smooth muscle and endothelial function were assessed using penile cavernosal strips. Penile endothelial cell content was not changed in KiLHRD582G mice. The maximal relaxation response to acetylcholine and the nitric oxide donor, sodium nitroprusside, was significantly reduced in KiLHRD582G mice indicating an impairment in the nitric oxide (NO)-mediated signaling. Cyclic GMP (cGMP) levels were significantly reduced in KiLHRD582G mice in response to acetylcholine, sodium nitroprusside and the soluble guanylate cyclase stimulator, BAY 41-2272. Expression of NOS1, NOS3 and PKRG1 were unchanged. The Rho-kinase signaling pathway for smooth muscle contraction was not altered. Together, these data indicate that KiLHRD582G mice have erectile dysfunction due to impaired NO-mediated activation of soluble guanylate cyclase resulting in decreased levels of cGMP and penile smooth muscle relaxation. These studies in the KiLHRD582G mice demonstrate that activating mutations in the mouse LHCGR cause erectile dysfunction due to impairment of the NO-mediated signaling pathway in the penile smooth muscle.

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Mengdie Li, Xiandong Peng, Jinfeng Qian, Fengrun Sun, Chunqin Chen, Songcun Wang, Jianping Zhang, and Meirong Du

To obtain a successful pregnancy, trophoblasts must provide a physical barrier, suppress maternal reactivity, produce immunosuppressive hormones locally, and enhance the production of blocking factors that are able to bind to several antigenic sites. Inadequate placental perfusion has been closely associated with several pregnancy-associated diseases. Galectin-9 (Gal-9) has a wide variety of regulatory functions in innate and adaptive immunity during infection, tumor growth, and organ transplantation. We utilized immortalized human first-trimester extravillous trophoblast cells (HTR8/SVneo) for our functional study and examined the effects of Gal-9 on apoptosis, cytokine production and angiogenesis of HTR8/SVneo cells. Gal-9 inhibited the apoptosis and IFN-γ and IL-17A production, promoted IL-4 production, and coordinated the crosstalk between HTR8/SVneo cells and human umbilical vein endothelial cells via its interaction with Tim-3. Blockade of JNK signaling inhibited Gal-9 activities in HTR8/SVneo cells. In addition, we detected a correlation between low levels of Gal-9 and spontaneous abortion. So Gal-9 could inhibit the apoptosis and proinflammatory cytokine expression, and promote the angiogenesis and IL-4 production in HTR8/SVneo cells via Tim-3 in a JNK dependent manner to help the maintenance of normal pregnancy. These findings possibly identify Gal-9 as a key regulator of trophoblast cells and suggest its potential as a biomarker and target for the treatment of recurrent pregnancy loss.

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Hsiu-Chi Lee, Shih-Chieh Lin, Meng-Hsing Wu, and Shaw-Jenq Tsai

Endometriosis is a common gynecological disease in reproductive-age women. Although the hormone-dependent therapy is the first line treatment for endometriosis, it is not a curative regimen and associated with severe side-effects, which significantly decrease the life quality of affected individuals. To seek a target for treatment of endometriosis, we focused on plasma membrane proteins that are elevated in ectopic cells and exert beneficial effects in cell growth and survival. We performed bioinformatics analysis and identified the neurotrophic receptor tyrosine kinase 2 (NTRK2) as a potential candidate for treatment. The expression levels of NTRK2 were markedly upregulated in the lesions of clinical specimen as well as in the mouse endometriotic-like lesion. Mechanistic investigation demonstrated that upregulation of NTRK2 is induced by hypoxia in a hypoxia-inducible factor 1 alpha-dependent manner. Knockdown of NTRK2 or administration of ANA-12, a selective antagonist of NTRK2, significantly induced endometriotic stromal cells death, suggesting it may be a potential therapeutic agent. In vivo study using surgery-induced endometriosis mice model showed ANA-12 (1.5 mg/kg body weight) treatment induced apoptosis of endometriotic cells and caused the regression of ectopic lesions. Taken together, our findings suggest a possible mechanism responsible for the aberrant expression of NTRK2 in endometriotic lesions and this may be involved in the pathogenesis of endometriosis.

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Griselda Irusta

Although ovarian cancer mortality rates have slightly declined in the last 40 years, ovarian cancer continues to be the eighth cause of cancer death in women. Ovarian cancer is characterized by its high response to treatments but also by its high rate of recurrence. Although treatments are limited to cytoreductive surgery and platinum-based chemotherapy, other therapies using antiangiogenic agents and poly (ADP-ribose) polymerase inhibitors are being tested. Nevertheless, these therapeutic strategies have had poor results and new potential targets and approaches are thus needed. The present review focuses on the recent evidence on antiangiogenic strategies in ovarian cancer cells and on the mechanisms governed by Notch and β-catenin proteins. It also describes the concept of ‘vascular normalization’ by using the platelet-derived growth factor, PDGFB, molecule as a tool to regulate ovarian tumor angiogenesis and thus improve ovarian tumor treatment. It has been reported that alterations in the Notch system components and changes in the canonical Wnt/β-catenin signaling, the other pathway of our interest, are relevant to molecular events that contribute to ovarian cancer development. Thus, in this review, we consider these aspects of the ovarian tumor biology as potential new therapeutic strategies for the treatment of this disease.

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Fabiola Zambrano, Liliana Silva, Pamela Uribe, Ulrich Gärtner, Anja Taubert, Mabel Schulz, Raúl Sánchez, and Carlos Hermosilla

Human spermatozoa activate neutrophil extracellular traps (NETs) in vitro. NETosis is an efficient mechanism through which polymorphonuclear neutrophils (PMN) capture sperm in vitro. The objective of this study was to establish the role of store-operated Ca+2 entry (SOCE) in human sperm-triggered NETs and its impact on sperm integrity and oocyte binding capacity. PMN isolated from donors were exposed to spermatozoa isolated from normozoospermic donors using the swim-up technique and were divided into the following groups: (1) sperm, (2) PMN, (3) PMN + sperm, (4) PMN (pretreated with 2-APB, SOCE inhibitor) + sperm, (5) (PMN + DNase) + sperm, and (6) (PMN + PMA) + sperm (positive control). NETs were quantified using PicoGreen® and visualised by scanning electron microscopy and immunofluorescence of extracellular DNA and neutrophil elastase. Plasma membrane, acrosome, and DNA integrity were analysed by flow cytometry, and oocyte binding was evaluated using the hemizona pellucida assay. Sperm-triggered NETosis negatively affected the sperm membrane and acrosome integrity and decreased the oocyte binding capacity. These effects were negated by an SOCE inhibitor, thus improving sperm function and achieving high oocyte binding capacity. The SOCE inhibitor significantly reduced NET formation compared with that in control PMN/sperm (P < 0.05). Collectively, these results advance the knowledge about the role of PMN in reproduction and will allow the development of strategies to block NET formation in situations of reduced fertilisation success.

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Liselot J W Thunnissen, Cindy G J Cleypool, and Bernadette S de Bakker

Summary

Although it is commonly accepted that fertilisation in humans occurs in the ampulla of the fallopian tube, the peritoneal cavity might represent an alternative fertilisation site. Studies substantiating both fertilisation sites were reviewed and new insights on the fertilisation site in humans are discussed, including their implications for reproductive medicine.

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Anthony M Carter

The mouse is often criticized as a model for pregnancy research as gestation is short, with much of organ development completed postnatally. There are also differences in the structure and physiology of the placenta between mouse and human. This review considers eight alternative models that recently have been proposed and two established ones that seem underutilized. A promising newcomer among rodents is the spiny mouse, which has a longer gestation than the mouse with organogenesis complete at birth. The guinea pig is also recommended both because it has well-developed neonates and because there is a wealth of information on pregnancy and placentation in the literature. Several smaller primates are considered. The mouse lemur has its advocates yet is less suited as a model for human pregnancy as its young are altricial, placentation very different from that of humans, and husbandry requirements not fully assessed. In contrast, the common marmoset, a New World monkey, has well-developed neonates and is kept at many primate centres. Marmoset placenta has some features that closely resemble human placentation, such as the interhaemal barrier, although it is uncertain if invasion of the uterine arteries occurs in this species. In conclusion, pregnancy research would benefit greatly from increased use of alternative models such as the spiny mouse and common marmoset.

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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.