High expression of CFTR in cumulus cells from mature oocytes is associated with high-quality of oocyte and subsequent embryonic development.

OBJECTIVE: The purpose of this study was to explore the association of expression of cystic fibrosis transmembrane conductance regulator (CFTR) in cumulus cells (CCs) from mature oocytes with oocyte quality and embryonic development. METHODS: A total of 338 infertile women who underwent ovarian stimulation cycle of oocyte retrieval in Zhejiang University School of Medicine were retrospectively enrolled in this study. The relative mRNA expression levels of CFTR, bone morphogenetic protein 15 (BMP15), and growth differentiation factor 9 (GDF9) in CCs were detected by qPCR technology. ROC curve was applied for the diagnosis of oocyte maturation. The serum levels of anti-Müllerian hormone (AMH), E2, follicle-stimulating hormone (FSH), luteinizing hormone (LH), and androstenedione were measured. Oocyte maturation rate, fertilization rate, cleavage rate, high-quality embryo formation rate, and implantation rate after embryo transfer were also determined. RESULTS: The mRNA expression levels of CFTR in CCs were significantly increased in metaphase II (MII) oocytes compared to that in metaphase I (MI) or germinal vesicle (GV) oocytes. The ROC curve analysis illustrated that CFTR mRNA expression could efficiently discriminate MII oocytes from MI or GV oocytes (AUC = 0.954), and revealed that 0.695 RQU is the optimal cut-off value for diagnosis. So the cut-off value of 2-ΔΔCT = 0.70 was used to divide the patients into two groups: low- (n = 114) and high-CFTR group (n = 224). The mRNA expression of CFTR in CCs was positively correlated with the antral follicular count (AFC), number of oocytes retrieved, number of MII oocytes, serum E2 level on hCG day, and BMP15 and GDF9 expression in CCs. Under continuous stimulation with the same dose of recombinant follicle-stimulating hormone (rFSH), the number of follicles, average recovered oocytes, recovered oocytes, MII oocytes, as well as the oocyte recovery rate, fertilization rate, oocyte cleavage rate, high-quality embryo formation rate, and implantation rate were decreased in patients with lower CFTR. CONCLUSIONS: This study suggests that CFTR expression in CCs is associated with the developmental potential of human oocytes.

Follicle stimulating hormone promotes production of renin through its receptor in juxtaglomerular cells of kidney.

BACKGROUND: Post-menopausal hypertension has been attributed solely to declining estrogen levels. The purpose of the research is to elucidate the mechanism by which follicle stimulating hormone(FSH) increases renin production involved in the regulation of blood pressure. METHODS: The expression of follicle stimulating hormone receptors (FSHRs) in renal juxtaglomerular cells and a As4.1 juxtaglomerular mouse cell line was evaluated. We established a mouse model by ovariectomy (OVX). Ovariectomized mice were treated with gonadotropin-releasing hormone agonist (GnRHa) (OVX + GnRHa). Ovariectomized mice initially received physiological doses of estrogen and were then injected with recombinant FSH (OVX + E + FSH). RESULTS: We found that FSHR was expressed in mouse renal juxtaglomerular cells labeled by renin antibody and in As4.1 cells. FSH promoted renin synthesis via Gsα-coupled FSHRs that activated protein kinase A, cyclic adenosine monophosphate(cAMP) response element-binding protein, extracellular signal-regulated kinase (Erk1/2), Protein kinase B(AKT), and c-Jun N-terminal kinase signaling pathways in As4.1 cells. We found increased serum FSH levels in the ovariectomized mouse with concurrent increases in renin, angiotensin II, heart rate (HR), systolic blood pressure (SBP), diastolic blood pressure (DBP), and mean arterial blood pressure (MAP). Additionally, increases in serum renin, angiotensin II, HR, SBP, DBP, and MAP were reduced by the additional injection of GnRHa. Exogenous FSH administration completely reversed decreases in renin, angiotensin II, HR, SBP, DBP, and MAP even in mice that received physiological doses of estrogen to maintain normal estradiol levels. CONCLUSIONS: Elevated FSH stimulates renin production involving a mechanism that may be relevant to the expression of FSH receptors in renal juxtaglomerular cells.

High Maternal Serum Estradiol in First Trimester of Multiple Pregnancy Contributes to Small for Gestational Age via DNMT1-Mediated CDKN1C Upregulation.

High maternal serum estradiol (E2) levels in the first trimester of pregnancy are associated with a high incidence of low birth weight (LBW) and small for gestational age (SGA). This study aimed to investigate the effect of first-trimester high maternal serum E2 levels on fetal growth and the underlying mechanisms in multiple pregnancies. Maternal serum E2 levels of women at 8 weeks of gestation were measured. The expression levels of imprinted genes and DNMT1 were determined by RT-qPCR, and KvDMR1 methylation in embryo tissue, placenta, and newborn cord blood samples was examined by bisulfite sequencing PCR. The effect of E2 on CDKN1C expression was investigated in HTR8 cells. The incidence of SGA was significantly higher in multiple pregnancies reduced to singleton than that in primary singleton pregnancies (11.4% vs. 2.9%) (P < 0.01) and multiple pregnancies reduced to twins than primary twins (38.5% vs. 27.3%) (P < 0.01). The maternal serum E2 level at 8 weeks of gestation increased with the number of fetuses and was negatively correlated with offspring birth weight. CDKN1C and DNMT1 expression was significantly upregulated in embryo tissue, placenta, and cord blood from multiple pregnancies. Furthermore, there was a positive correlation between CDKN1C mRNA expression and KvDMR1 methylation levels. In HTR8 cells, DNMT1 mediated the estrogen-induced upregulation of CDKN1C, which might contribute to SGA. To minimize the risks of LBW and SGA, our findings suggest that abnormally high maternal serum E2 levels should be avoided during the first trimester of multiple pregnancies from assisted reproductive technology (ART).

Involvement of Cl(-)/HCO3(-) exchanger SLC26A3 and SLC26A6 in preimplantation embryo cleavage.

Bicarbonate (HCO3(-)) is essential for preimplantation embryo development. However, the mechanism underlying the HCO3(-) transport into the embryo remains elusive. In the present study, we examined the possible involvement of Cl(-)/HCO3(-) exchanger in mediating HCO3(-) transport into the embryo. Our results showed that depletion of extracellular Cl(-), even in the presence of HCO3(-), suppressed embryo cleavage in a concentration-dependent manner. Cleavage-associated HCO3(-)-dependent events, including increase of intracellular pH, upregulation of miR-125b and downregulation of p53, also required Cl(-). We further showed that Cl(-)/HCO3(-) exchanger solute carrier family 26 (SLC26) A3 and A6 were expressed at 2-cell through blastocyst stage. Blocking individual exchanger's activity by inhibitors or gene knockdown differentially decreased embryo cleavage and inhibited HCO3(-)-dependent events, while inhibiting/knocking down both produced an additive effect to an extent similar to that observed when CFTR was inhibited. These results indicate the involvement of SLC26A3 and A6 in transporting HCO3(-) essential for embryo cleavage, possibly working in concert with CFTR through a Cl(-) recycling pathway. The present study sheds light into our understanding of molecular mechanisms regulating embryo cleavage by the female reproductive tract.

Small-conductance, calcium-activated potassium channel 3 (SK3) is a modulator of endometrial remodeling during endometrial growth.

BACKGROUND: Small-conductance, Ca(2+)-activated K(+) channel 3 (SK3) has been shown to be expressed in porcine endometrium. However, the roles of SK3 in human endometrium during the menstrual cycle and early pregnancy are unknown. OBJECTIVE: The objective of the study was to investigate the expression and function of SK3 in human endometrium and the mechanism involved. METHODS: We determined the expression of SK3 in human endometrium by RT-PCR, Western blotting, and immunofluorescence. Using electrophysiological and fluorescent imaging techniques, we investigated the effects of SK3 on the membrane potential and the concentrations of cytosolic calcium, respectively. The effects of SK3 on endometrial thickness and pregnancy outcome were also investigated. Knockdown of endometrial SK3 was used to examine the effects of SK3 on cell migration, cytoskeleton formation, and calcium concentration in the cytosol. RESULTS: SK3 channels are present in human endometrium. In vivo experimental and clinical data demonstrated that the reduced expression of SK3 was associated with a thin endometrium and unsuccessful pregnancy outcomes. Knockdown of human endometrial SK3 attenuated the rise in cytosolic calcium and membrane hyperpolarization induced by thapsigargin, a Ca(2+)-ATPase inhibitor, cell migration, and F-actin assembly. Knockdown of endometrial SK3 in mice also resulted in a thin endometrium and unsuccessful pregnancy outcome. CONCLUSIONS: These observations demonstrate that SK3 channels are expressed in human endometrial cells. Reduced SK3 expression attenuates endometrial cell migration and is associated with unsuccessful pregnancy outcomes.

Co action of CFTR and AQP1 increases permeability of peritoneal epithelial cells on estrogen-induced ovarian hyper stimulation syndrome.

BACKGROUND: Ovarian hyper stimulation syndrome (OHSS) is an iatrogenic complication associated with fertility drugs. It is characterized by increased vascular permeability and substantial fluid shift with accumulation in the body cavity. The pathogenesis of OHSS remains obscure, and no definitive treatments are currently available. RESULTS: Using western blot and short-circuit current (Isc) techniques, we investigate the potential coactions of analysis in cystic fibrosis transmembrane conductance regulator (CFTR) and aquaporin 1 (AQP1) on the hyper permeability of body cavity peritoneal epithelial cells in the pathogenesis of OHSS. The rats develop OHSS symptoms, with the up regulation of both CFTR and AQP1 expression and enhanced CFTR channel activity in peritoneal epithelial cells, can also be mimicked by administration of estrogen, alone in ovariectomized rats. Administration of progesterone suppresses CFTR activity, OHSS symptoms as well as CFTR and AQP1 expression. Besides, AQP1 inhibitor, HgCl(2), can suppress CFTR channel activity. Therefore, antisera against CFTR or AQP1 to OHSS animals may result in alleviation of the symptom. CONCLUSION: This study confirms the coactions of CFTR and AQP1 play a critical role in the development and progression of increased peritoneal epithelial permeability in severe OHSS. These findings may provide grounds for ameliorating assisted reproduction treatment strategy to reduce the risk of OHSS in in vitro fertilization (IVF).

CFTR mediates bicarbonate-dependent activation of miR-125b in preimplantation embryo development.

Although HCO(3)(-) is known to be required for early embryo development, its exact role remains elusive. Here we report that HCO(3)(-) acts as an environmental cue in regulating miR-125b expression through CFTR-mediated influx during preimplantation embryo development. The results show that the effect of HCO(3)(-) on preimplantation embryo development can be suppressed by interfering the function of a HCO(3)(-)-conducting channel, CFTR, by a specific inhibitor or gene knockout. Removal of extracellular HCO(3)(-) or inhibition of CFTR reduces miR-125b expression in 2 cell-stage mouse embryos. Knockdown of miR-125b mimics the effect of HCO(3)(-) removal and CFTR inhibition, while injection of miR-125b precursor reverses it. Downregulation of miR-125b upregulates p53 cascade in both human and mouse embryos. The activation of miR-125b is shown to be mediated by sAC/PKA-dependent nuclear shuttling of NF-κB. These results have revealed a critical role of CFTR in signal transduction linking the environmental HCO(3)(-) to activation of miR-125b during preimplantation embryo development and indicated the importance of ion channels in regulation of miRNAs.

Impaired CFTR-dependent amplification of FSH-stimulated estrogen production in cystic fibrosis and PCOS.

CONTEXT: Estrogens play important roles in a wide range of physiological and pathological processes, and their biosynthesis is profoundly influenced by FSH that regulates the rate-limiting enzyme aromatase-converting estrogens from androgens. Abnormal estrogen levels are often seen in diseases such as ovarian disorders in polycystic ovarian syndrome (PCOS), an endocrine disorder affecting 5-10% of women of reproductive age, and cystic fibrosis (CF), a common genetic disease caused by mutations of the cystic fibrosis transmembrane conductance regulator (CFTR). OBJECTIVES: We undertook the present study to investigate the mechanism underlying these ovarian disorders, which is not well understood. RESULTS: FSH-stimulated cAMP-responsive element binding protein phosphorylation, aromatase expression, and estradiol production are found to be enhanced by HCO3- and a HCO3- sensor, the soluble adenylyl cyclase, which could be significantly reduced by CFTR inhibition or in ovaries or granulosa cells of cftr knockout/ΔF508 mutant mice. CFTR expression is found positively correlated with aromatase expression in human granulosa cells, supporting its role in regulating estrogen production in humans. Reduced CFTR and aromatase expression is also found in PCOS rodent models and human patients. CONCLUSIONS: CFTR regulates ovarian estrogen biosynthesis by amplifying the FSH-stimulated signal via the nuclear soluble adenylyl cyclase. The present findings suggest that defective CFTR-dependent regulation of estrogen production may underlie the ovarian disorders seen in CF and PCOS.

Functional expression of large-conductance calcium-activated potassium channels in human endometrium: a novel mechanism involved in endometrial receptivity and embryo implantation.

BACKGROUND: Large-conductance calcium-activated potassium channels (BK(Ca) channels) mediate physiological processes in nonexcitable cells. OBJECTIVE: The aim of the study was to determine BK(Ca) channel expression in human endometrium and its role in endometrial receptivity and embryo implantation. METHODS: BK(Ca) channel expression in human endometrium is described at different phases of the menstrual cycle using quantitative real time-PCR and Western blot techniques. Their effects on embryo implantation were examined using JAr spheroid attachment assays and in vivo mouse model. We examined their effects on endometrial receptivity factors, nuclear factor-κB (NF-κB) activity using quantitative real time-PCR, Western blot, and EMSA analyses. Changes in electrophysiological properties and cytosolic free Ca(2+) were measured in endometrial cells with or without specific BK(Ca) blocker or transfected with BK(Ca) small interfering RNA using patch-clamp and fluorescence analyses, respectively. RESULTS: BK(Ca) channels are expressed in human endometrial cells in a phase-related fashion during the menstrual cycle (proliferative, 0.20 ± 0.02, vs. mid-secretory, 0.72 ± 0.07; P < 0.01). Blocking BK(Ca) channel function or knockdown of endogenous BK(Ca) channel expression not only decreased JAr spheroid attachment rate and embryo implantation rate in mice but also significantly reduced the expression levels of endometrial receptive factors, including leukemia inhibitory factor, integrin ß3, claudin-4, and DKK-1, in human endometrial cells. Blocking BK(Ca) channels also reduced BK(Ca)-regulated NF-κB activity, cytosolic Ca(2+) concentrations, and membrane potentials in human endometrial cells. CONCLUSIONS: These observations demonstrate that BK(Ca) channels: 1) are expressed in endometrial cells; 2) affect embryo implantation by mediating endometrial receptive factors; and 3) alter the activity of NF-κB and homeostasis of Ca(2+) in the human endometrial cells.

Involvement of CFTR in oviductal HCO3- secretion and its effect on soluble adenylate cyclase-dependent early embryo development.

BACKGROUND: The cystic fibrosis transmembrane conductance regulator (CFTR) plays a critical role in electrolyte and fluid transport in epithelial cells, and women with cystic fibrosis (CF), caused by CFTR gene mutations, have a higher incidence of infertility. METHODS: In the present study, we investigated the expression of CFTR in porcine oviduct and its functional role in oviductal HCO(3)(-) secretion and embryo development with RT-PCR, western blot, patch-clamp, short-circuit current (I(sc)), pH measurement and embryo culture. RESULTS: RT-PCR and western blot analysis showed the expression of CFTR mRNA and protein in the oviduct with its localization demonstrated by immunohistochemistry. The whole-cell patch-clamp recording revealed a forskolin (FSK)-activated current with electrophysiological and pharmacological characteristics of CFTR. The I(sc) measurement showed that FSK-stimulated an increase in the I(sc), which could be significantly reduced by CFTR inhibitor or removal of both CO(2) and HCO(3)(-). pH measurement showed a FSK stimulated alkalization at the apical surface, which could be inhibited by CFTR inhibitor, indicating CFTR-mediated HCO(3)(-) secretion. Mouse embryo development from 2-cell to morula or blastocyst stage was significantly inhibited in the absence of HCO(3)(-) or when co-cultured with HCO(3)(-) secretion-deficient CFTR mutant cells as compared with the wild-type. RT-PCR, western blot and immunostaining showed the expression of soluble adenylate cyclase (sAC), the known HCO(3)(-) sensor, in embryos. Treatment with its inhibitors, 2-hydroxyestradiol and KH7, prevented the HCO(3)(-) dependent embryo development. CONCLUSION: The present results suggest that CFTR-mediated oviductal HCO(3)(-) secretion may be vital for sAC-dependent early embryo development, a defect of which may contribute to the reduced fertility seen in women with CF.

[Distribution of Fas and FasL in the central nervous system of adult rhesus].

OBJECTIVE: To investigate the distribution of Fas and FasL in the CNS of adult rhesus. METHODS: Frozen sections were incubated in polyclonal anti-Fas and anti-FasL antibody by the immunohistochemical SP method. RESULTS: The Fas and FasL immunopositive neurons were observed in many areas. Fas immunoreactivity could be seen in the cytoplasm and processes of Purkinje cells and in the brain stem nuclei, including vestibular nucleus, dorsal nucleus of vagus and spinal nucleus of trigeminal nerve. FasL immunopositive neurons were observed in cerebral cortex, especially in pyramidal neurons of lamina I and V, cerebellar nuclei, diencephalon, and brain stem nuclei involving pontine nucleus, vestibular nucleus, cochlear nucleus, spinal nucleus of trigeminal nerve, hypoglossal nucleus, nucleus ambiguous and reticular formation. Fas and FasL immunoreactivity mainly distributed in motor neurons of spinal ventral horn and neural fibers and glia cells in white matter. They all took on brown staining in the cytoplasm and process. CONCLUSION: The distribution profiles of Fas and FasL in various areas of CNS indicate that they may fill some roles in the immune and physical function of the aforesaid anatomic