Biology of Reproduction最新文献

Glycine is a key regulator of cell volume in early preimplantation mouse embryos and supports embryo viability. Its accumulation is initiated when the GLYT1 glycine transporter (SLC6A9) is activated in oocytes at about the same time the oocyte is released from meiotic arrest at the germinal vesicle (GV) stage. The mechanism by which GLYT1 is maintained in an inactive state before ovulation is triggered is unknown. Here, we have shown that GLYT1 activity can remain suppressed in isolated cumulus oocyte complexes (COCs) under defined culture conditions that include keeping COCs physically separated and using the physiological mediator of GV arrest, Natriuretic Peptide Precursor C (NPPC). When GV arrest is instead maintained in oocytes within COCs by inhibiting phosphodiesterase 3A (PDE3A) or cyclin-dependent kinase 1 (CDK1), GLYT1 similarly remains inactive. However, GLYT1 becomes activated in isolated GV oocytes similarly maintained in GV arrest, indicating that cumulus cells are required for suppressing GLYT1 activity. This implied that meiotic arrest was necessary but not sufficient for preventing GLYT1 activation and that an inhibitory factor likely arising from the cumulus was also required. Finally, we found that pyrrophenone, a selective inhibitor of arachidonic acid production by cytoplasmic phospholipase A alpha (cPLAα), caused GLYT1 to become activated in oocytes within COCs despite maintenance of meiotic arrest of the oocyte. Since arachidonic acid levels decrease in oocytes after release from GV arrest, we propose that arachidonic acid may be a candidate for the inhibitory factor in COCs that regulates GLYT1 activity.

Over the past two decades, others and our group have explored the different signalling cascade in human spermatozoa and observed that their deregulation might lead to sperm dysfunctions, contributing to infertility. Studies of patients with infertility have reported variations in sperm features, such as asthenozoospermia, either alone or in combination with other pathologies, more specifically in patients with genital tract infections, sperm metabolic diseases, varicocele, and anatomic/molecular sperm anomalies. Infertility is idiopathic and multifactorial in most patients. Although several studies have elucidated many of the components and mechanisms involved in the human sperm signalling pathways, their role in defective spermatozoa or systemic pathologies remains nebulous. In this review, we focused on transducing signals, the underlying regulatory mechanisms in human spermatozoa and explored the known pro-survival and pro-apoptotic pathways as well as the factors involved.

In sheep, cervical artificial insemination (AI) of frozen-thawed semen is not practiced worldwide due to the unacceptably low pregnancy rates achieved (typically <30%). The exception to this is in Norway where farmers perform shot-in-the-dark vaginal AI with frozen-thawed semen and achieve pregnancy rates over 60%. Research in Ireland has shown that this is due to the breed of the ewe used in Norway and specifically the ability of sperm to traverse the cervix in some ewe breeds (such as those in Norway) but not others. This review summarises the published studies performed over the last two decades which have focused on characterising the biological differences in the cervix of ewe breeds of divergent fertility. It discusses how this unique ewe breed effect can be used to develop our understanding of how frozen-thawed semen can cross the ovine cervix but also provides new in vivo opportunities through the addition of specific biomolecules that will protect sperm, thus improving the success of cervical AI using frozen-thawed semen in sheep.

Early embryo loss affects all mammalian species, including humans and agriculturally important food-producing mammals such as cattle. The developing conceptus (embryo and extra-embryonic membranes) secretes factors which modify the endometrium and can be critical for early pregnancy processes such maternal recognition of pregnancy (MRP) and enhancing uterine receptivity to implantation. For example, a competent bovine conceptus secretes IFNT to initiate MRP. The bovine conceptus also secretes other proteins at the time of MRP, including CAPG and PDI, which are highly conserved among placental mammals. We have previously shown that these proteins act upon the endometrium to modulate receptivity, embryo development, and implantation in species with different implantation strategies (humans and cattle). We hypothesise that developing a novel 3D bovine endometrium on a chip system will enhance our understanding of the role of conceptus-derived factors in altering the endometrium and/or ULF secretion. Here we have developed a 3D bovine endometrium on a chip system, comprising both stromal and epithelial cell culture combined with culture medium flow better mimics the in vivo endometrium and exposure to conceptus-derived factors than conventional 2D endometrial cell culture. We have demonstrated that the conceptus-derived proteins CAPG and PDI modulate the endometrial transcriptome and secretory response to promote pathways associated with early pregnancy and alter ULF composition. This work highlights the critical need for more robust and in vivo-like culture systems to study endometrial-conceptus interactions in vitro to further investigate the role of conceptus derived factors for pregnancy success.

Liver receptor homolog 1 (LRH-1; Nr5a2) and steroidogenic factor 1 (SF-1; Nr5a1) are two closely related orphan nuclear receptors that bind to the same genomic motif. Conditional depletion of either of these receptors in the ovary results in infertility, but through different mechanisms, with SF-1 being critical early in ovarian development and LRH-1 regulating ovulation. We conditionally depleted both LRH-1 and SF-1 from the ovary, using two different models of conditional depletion, generating two lines of double conditional knockout (dko) mice. In one, we used the Amhr2Cre (Amhr2-dko) mouse, where depletion is initiated in the prenatal ovary before the stage of germ cell nest breakdown. In the other, we employed Cyp19a1Cre (Cyp19a1-dko)-mediated depletion, which is initiated following formation of the follicular antrum. Both models were completely anovulatory and infertile, and no ovulation occurred following administration of exogenous gonadotropins. The Amhr2-dko mouse had dramatically reduced follicular populations at every stage of development, as well as disrupted extracellular matrix, characterized by dysregulation of collagen and laminin expression in reproductively mature mice, reduced expression of steroidogenic genes, dysregulated lipid metabolism, and inhibited granulosa cell proliferation. The latter resulted in a phenotype of reduced ovarian size in this model. The Cyp19al dko mouse displayed dysregulation of luteinizing hormone (LH) response and ovulatory mechanisms and increased activation of the activin/inhibin signaling axis, suggesting impaired gonadotropin responsiveness. In summary, both dko models demonstrated a phenotype of complete infertility, confirming the critical importance of both LRH-1 and SF-1 in ovarian function.

Typically, DNA-damaging chemotherapy (CTx) regimens have a gonadotoxic effect and cause premature ovarian insufficiency (POI), characterized by infertility and estrogen deficiency. However, whether loss of granulosa cells killed directly by CTx contributes significantly to POI has not been determined. To address this issue, we used a previously established mouse model of CTx-induced POI. The alkylating drugs Busulfan (8.75 mg/kg) and Cyclophosphamide (100 mg/kg) were administered to 8-week-old FVB female mice by intraperitoneal (i.p.) injection three times at 48-hour intervals, after which ovarian tissues were harvested and examined by immunofluorescence. The number of primordial follicles was significantly reduced at day (d)6, whereas the number of growing follicles was relatively unchanged. CTx led to DNA double strand breaks in both oocytes and granulosa cells based on the presence of γH2AX foci. However, markers of apoptosis predominantly labeled granulosa cells in growing follicles. We next examined the effect of inhibiting apoptosis in growing granulosa cells by generating Bak-/-Baxfxfx; Cyp19a1Cre transgenic mice. On d10 after the first CTx, Bak-/-Baxfxfx; Cyp19a1Cre ovaries had fewer apoptotic granulosa cells and more surviving follicles than controls. Furthermore, Bak-/-Baxfxfx; Cyp19a1Cre mice showed better fertility than controls after CTx. Our data suggest that granulosa cell death is a significant contributor to follicle depletion and fertility loss after Cyclophosphamide and Busulfan.

In assisted reproduction, many factors in the culture environment, including light, temperature, pH, and culture media, can reduce preimplantation embryo viability. Laboratory glassware is also a known risk factor for in vitro embryos; however, the underlying mechanisms that disrupt embryonic development remain unclear. We identified Zn eluted from glassware as an embryotoxic substance. In mouse embryos, Zn induced delayed development, abnormalities in chromosome segregation, cytokinesis, zygotic gene activation (e.g. Zscan4a and murine endogenous retrovirus with leucine, also known as MERVL), and aberrantly upregulated developmental gene expression (e.g. Hoxa1, Hoxb9, T, and Fgf8) that could be mediated through metal regulatory transcription factors (e.g. Mtf1). Subsequently, Zn exposure led to significantly reduced blastocyst formation. Post-implantation, Zn-exposed embryos were associated with normal birth rates, however, the birth weight increased by an average of 18% compared with embryos cultured without Zn. Furthermore, Zn exposure affected the development of bovine and human embryos, with species-based variation in the strength and timing of these effects. To mitigate these embryotoxic effects, we identified a method to prevent glass toxicity using chelating agents. This research not only highlights the importance of risk control in embryo culture but also facilitates the development of safe and effective methods for assisted reproduction.

DMRT1 is a key factor in testis development, where it is involved in sex determination and fertility. Mutations in DMRT1 have been described in humans, with patients presenting 46, XY Disorders of Sex Development (46, XY DSD) or infertility. In a previous study, we demonstrated that DMRT1 is a testis-determining factor in rabbits, with DMRT1-/- rabbits exhibiting a male to female XY sex reversal. In this study, we show that DMRT1 haploinsufficiency induces secondary infertility, with XY rabbits presenting oligospermia or even azoospermia at two years of age. We observed that sperm concentration decreases and sperm anomalies increase in DMRT1+/- rabbits at adulthood. Furthermore, spermatogenesis is impacted as early as 4 months (the earliest stage where spermatozoa are detected), with dysregulation of genes involved in spermatid maturation and oocyte/spermatozoa fusion, as well as overexpression of genes involved in the mitosis/meiosis transition of spermatogonial stem cells (SSCs). Finally, DMRT1 haploinsufficiency impacts the earliest stages of germ cell differentiation, with persistent proliferation and pluripotency in the postnatal period. In conclusion, our findings underscore DMRT1 as a crucial factor at various stages of testicular development, and reinforce its role in the multiple phenotypes observed in humans.

Although uterine matrix metalloproteinases (MMPs) are known to play a role in the development of pre-eclampsia (PE), it remains unclear whether increased levels of serum MMPs are the pathogenesis underlying pregnancy-induced hypertension (PIH). We aimed to investigate whether the serum levels of MMP2 and MMP9 play a role in PIH using a specific inhibitor, SB-3CT. Twenty-five nine-week-old pregnant rats were randomly divided into five groups: normal pregnancy (SHAM) group, PE (RUPP) group, as well as low, middle, and high-dose intervention groups. The intervention groups received continuous intraperitoneal injections of SB-3CT at 25 mg, 50 mg, or 75 mg/kg/d doses. The SHAM and RUPP groups were injected with equal doses of solvent. Seven days later, the arterial pressure was tested at the carotid artery and rats were sacrificed. Serum MMP2, MMP9, tissue inhibitor of metalloproteinase-1 (TIMP1), endothelin-1 (ET-1), angiotensin II (AngII), and endothelial nitric oxide synthase (eNOS) levels were tested by ELISA. Vascular wall changes in cross-sections of the aorta abdominalis were observed using H&E-staining and the activities of MMP2 and MMP9 in the aorta abdominalis were tested using gelatin zymography. There were significant increases in blood pressure, serum MMP2 and MMP9 levels, and activities of MMP2 and MMP9 in the aorta abdominalis, along with notable vascular remodeling in the RUPP group. After the SB-3CT intervention, increased blood pressure was relieved and vascular remodeling improved, while MMP2 and MMP9 levels and activities were reduced. In summary, specific inhibition of MMP2 and MMP9 can decrease blood pressure in a PIH model. This indicates that increased MMP2 and MMP9 in maternal serum might contribute to the pathogenesis of PIH.

In dairy cows, detrimental effects of global warming and intensive genetic selection for high milk yield on reproductive performance have become increasingly relevant in cooler regions. Based on the current knowledge we hypothesized that elevated temperature during oocyte maturation affects mitochondria and endoplasmic reticulum in parallel with mitochondrial dysfunction representing the mechanistic link between ROS and ER Stress. To proof that, the present study aimed to uncover the consequences of heat stress during oocyte maturation on mitochondrial health, cellular oxidative stress response and its implications for endoplasmic reticulum stress (ER-stress). Immature bovine oocytes were matured either under routine temperatures (38.8°C, Control) or exposed to elevated temperatures (41°C, HS). MII oocytes as well as subsequent blastocysts were analysed in terms of developmental capacity, mitochondrial membrane potential, the bioenergetic profile, ROS level as well as expression of candidate genes playing a role in oxidative stress, endoplasmic reticulum stress and apoptosis. While no effect on matured oocytes became obvious, HS embryos demonstrated typical alterations of the mitochondrial bioenergetic profile in terms of higher mitochondrial membrane potentials not going along with higher ATP-linked oxygen consumptions and significantly lower maximum respiration and spare capacity rates implicating less efficient mitochondria accompanied with significantly higher ROS levels. Moreover, gene expression of HS embryos supported the assumption that mitochondria are the mechanistic link between oxidative stress and endoplasmic reticulum stress, impairing early embryo development by promoting apoptosis. In summary, the present study contributes to the elucidation of the multiple negative effects of heat stress during the maturation process.