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OBJECTIVE: To investigate the effects of peritoneal endometriosis on rat ovaries. METHODS: A rat model of peritoneal endometriosis was established by autologous transplantation. qPCR was performed to measure mRNA levels of steroid hormone and steroid synthesis-related genes in the ovaries of endometriosis rats. Immunohistochemistry was performed to characterize the distribution of FSHR in the ovaries of endometriosis rats. RNAseq was performed to find pathological changes in the ovaries of endometriosis rats. RESULTS: By qPCR, it was revealed that mRNA levels of steroid hormone synthesis-related genes were decreased in the ovaries of rats with endometriosis; With IHC, observed that FSHR expression was significantly decreased in the antral follicles of rats with endometriosis. RNAseq revealed that endometriosis affected transcription of the genes related to the microtubule structure and tight junctions of rat ovarian cells. CONCLUSION: Peritoneal endometriosis decreased the genic expression of ovarian steroid hormone synthetases and FSHR protein level in granulosa cells of antral follicles, and reduced the mRNA levels of the microtubule structure and tight junctions in rat ovarian cells, which contribute to the impairment of ovarian function.

Successful human reproduction initiates from normal gamete formation, fertilization and early embryonic development. Abnormalities in any of these steps will lead to infertility. Many infertile patients undergo several failures of IVF and intracytoplasmic sperm injection (ICSI) cycles, and embryonic developmental arrest is a common phenotype in cases of recurrent failure of IVF/ICSI attempts. However, the genetic basis for this phenotype is poorly understood. The subcortical maternal complex (SCMC) genes play important roles during embryonic development, and using whole-exome sequencing novel biallelic mutations in the SCMC genes TLE6, PADI6 and KHDC3L were identified in four patients with embryonic developmental arrest. A mutation in TLE6 was found in a patient with cleaved embryos that arrested on day 3 and failed to form blastocysts. Two patients with embryos that arrested at the cleavage stage had mutations in PADI6, and a mutation in KHDC3L was found in a patient with embryos arrested at the morula stage. No mutations were identified in these genes in an additional 80 patients. These findings provide further evidence for the important roles of TLE6, PADI6 and KHDC3L in embryonic development. This work lays the foundation for the genetic diagnosis of patients with recurrent IVF/ICSI failure.

Caulis Sargentodoxae prescription has been confirmed by the gynecological clinical observation to be effective in the treatment of endometriosis (EMs), and inflammatory cytokines were involved in EMs. In this paper, animal experiments were designed to explain anti-inflammatory mechanisms of Caulis Sargentodoxae prescription on endometriosis. The EMs model was established by autoplastic transplantation, and rats were randomly divided into seven groups: normal control group, model group, ovariectomized group, gestrinone (Western medicine) group, Caulis Sargentodoxae prescription (Chinese medicine) group, celecoxib (inhibitor) group, and combination (Chinese medicine + inhibitor) group. After oral administration for 21 days, the growth inhibitory rates of the ectopic endometria in treatment groups were evaluated, and the levels of inflammatory cytokines in the serum and peritoneal fluid were determined by ELISA, as well as the expression of prostaglandin endoperoxide synthase 2 (PTGS2) in the ectopic endometrial tissues was detected by real-time polymerase chain reaction, immunohistochemistry, and western blotting. The growth inhibitory rates of the ectopic endometria were significantly higher in the Caulis Sargentodoxae prescription group and gestrinone group, in comparison with the model group p<0.05. In the Caulis Sargentodoxae prescription group, the levels of inflammatory cytokines including IL-1, IL-2, IL-6, TNF-α, and PGE2 were all reduced in the serum and peritoneal fluid p<0.05. In addition, the specific expression of PTGS2 in the ectopic endometrial tissues significantly decreased in the Caulis Sargentodoxae prescription group and PTGS2 inhibitor celecoxib group both at mRNA and protein levels, but in the steroid hormone drug gestrinone group not at the mRNA level. So, Caulis Sargentodoxae prescription has a reliable therapeutic effect on the EMs by its comprehensive anti-inflammatory roles, possibly in a way different from gestrinone.

Aim: To investigate the association of LEPR polymorphisms and plasma leptin and soluble leptin receptor (sOB-R) levels with polycystic ovary syndrome (PCOS) in Chinese women. Patients & methods: LEPR Lys109Arg (rs1137100) and Gln223Arg (rs1137101) polymorphisms of PCOS patients and the controls were genotyped. Plasma leptin and sOB-R levels of two groups were measured. Results: The genotypic distributions of Lys109Arg (rs1137100) differed between the PCOS and control groups. Plasma sOB-R levels increased significantly in PCOS patients and were associated with PCOS independent of BMI. Furthermore, luteinizing hormone, triglyceride and fasting blood glucose correlated significantly to PCOS patients’ sOB-R levels. Conclusion: LEPR Lys109Arg (rs1137100) was associated with PCOS susceptibility and genotype AA was deduced to be a protective factor for PCOS; sOB-R levels might be recognized as a new indicator for the severity of PCOS.

MicroRNAs (miRNAs) are critical regulators of gene expression; they have emerged as new players in the pathophysiology of reproductive disorders such as preeclampsia and recurrent miscarriage (RM). In this study, miRNA expression profiles were determined by deep sequencing analysis in placental villi obtained from women with RM and with gestational age-matched normal pregnancy (NP). A total of 69 miRNAs were found to be aberrantly expressed in RM. Five of these human (Homo sapiens) miRNAs (hsa-miRNAs), including hsa-miR-3074-5p, -486-3p, -1269b, -6765-3p and -144-3p, were validated by qRT-PCR in 12 RM and 10 NP placental villi. Hsa-miR-3074-5p expression was significantly higher, while hsa-miR-486-3p expression was significantly lower, in RM compared to NP. Subsequently, the expression of mouse (Mus musculus) miR-486-3p (mmu-miR-486-3p) and mmu-miR-3074-5p, which are identical to hsa-miR-486-3p and hsa-miR-3074-5p, respectively, were examined by qRT-PCR in the uterus during early pregnancy. Mmu-miR-486-3p expression was significantly increased during the peri-implantation period, and the levels were significantly higher at implantation than at non-implantation sites. In contrast, the expression of mmu-miR-3074-5p was markedly decreased at implantation compared to non-implantation sites. These data suggest that miR-486-3p and miR-3074-5p may be involved in embryo implantation and that their aberrant expression is associated with RM. Larger studies are warranted to follow up this pilot study.

Objective To examine whether sequence variants within the FSHR and CYP19A1 genes are related to the ovarian response to controlled ovarian stimulation (COS). Design Genetic association study using both single-gene and combined analyses of women with sequence variants undergoing in vitro fertilization treatment. Setting Academic research institute hospital. Patient(s) Seven hundred and five women undergoing ovarian stimulation with recombinant follicle-stimulating hormone (FSH). Intervention(s) Peripheral blood extraction, DNA purification, and FSHR c.919G>A (rs6165, p.Thr307Ala) and CYP19A1 c.*19C>T (rs10046) sequence variants analyses. Main Outcome Measure(s) Single-gene statistical analysis and combined statistical analysis with the SPSS17.0 software; FSHR c.919G>A and CYP19A1 c.*19C>T sequence variant genotypes and clinical parameters related to the COS response as oocyte retrieval and hormone levels, doses of exogenous FSH. Result(s) Women with genotype Ala/Ala at FSHR position 307 had higher basal levels of FSH and were more likely to have a low ovarian response compared with other genotypes. Women with genotype TT at CYP19A1 yielded fewer oocytes after ovarian stimulation. The combined analysis of these two sequence variants revealed that these two single-nucleotide variants have a synergistic effect in conferring the risk of a low ovarian response. Conclusion(s) Our results support an association of sequence variants in the genes that participate in estrogen synthesis, notably the FSHR and CYP19A1 genes, with the outcome of COS.

RGS2 is a negative regulator of G protein signaling that contains a GTPase-activating domain and a β-tubulin binding region. This study aimed to determine the localization and function of RGS2 during mouse oocyte maturation in vitro. Immunofluorescent staining revealed that RGS2 was widely expressed in the cytoplasm with a greater abundance on both meiotic spindles and first/second polar bodies from the fully-grown germinal vesicle (GV) stage to the MII stages. Co-expression of RGS2 and β-tubulin could also be detected in the spindle and polar body of mouse oocytes at the MI, AI, and MII stages. Inhibition of the binding site between RGS2 and β-tubulin was accomplished by injecting anti-RGS2 antibody into GV-stage oocytes, which could result in oocytes arrest at the MI or AI stage during in vitro maturation, but it did not affect germinal vesicle breakdown. Moreover, injecting anti-RGS2 antibody into oocytes resulted in a significant reduction in the rate of first polar body extrusion and abnormal spindle formation. Additionally, levels of phosphorylated MEK1/2 were significantly reduced in anti-RGS2 antibody injected oocytes compared with control oocytes. These findings suggest that RGS2 might play a critical role in mouse oocyte meiotic maturation by affecting β-tubulin polymerization and chromosome segregation.

Background TUBB8 is a primate-specific β-tubulin isotype whose expression is confined to oocytes and the early embryo. We previously found that mutations in TUBB8 caused oocyte maturation arrest. The objective was to describe newly discovered mutations in TUBB8 and to characterise the accompanying spectrum of phenotypes and modes of inheritance. Methods and results Patients with oocyte maturation arrest were sequenced with respect to TUBB8 . We investigated the effects of identified mutations in vitro, in cultured cells and in mouse oocytes. Seven heterozygous missense and two homozygous mutations were identified. These mutations cause a range of folding defects in vitro, different degrees of microtubule disruption upon expression in cultured cells and interfere to varying extents in the proper assembly of the meiotic spindle in mouse oocytes. Several of the newly discovered TUBB8 mutations result in phenotypic variability. For example, oocytes harbouring any of three missense mutations (I210V, T238M and N348S) could extrude the first polar body. Moreover, they could be fertilised, although the ensuing embryos became developmentally arrested. Surprisingly, oocytes from patients harbouring homozygous TUBB8 mutations that in either case preclude the expression of a functional TUBB8 polypeptide nonetheless contained identifiable spindles. Conclusions Our data substantially expand the range of dysfunctional oocyte phenotypes incurred by mutation in TUBB8 , underscore the independent nature of human oocyte meiosis and differentiation, extend the class of genetic diseases known as the tubulinopathies and provide new criteria for the qualitative evaluation of meiosis II (MII) oocytes for in vitro fertilization (IVF).

SummaryThe goal of this project was to determine whether the originating strain of mouse embryonic stem (ES) cells affects the maintenance of their pluripotency under uniform culture conditions. ES cells from two strains of mice, E14 and C2J, were tested. Both ES cell lines were cultured in KOSR + 2i medium and then injected into C57BL/6J blastocysts. Our results demonstrate that this medium could support both E14 and C2J ES cells to keep their pluripotency, though E14 ES cells were found to have a higher chimeric rate than C2J ES cells. However, analysis by backcrossing revealed that C2J and E14 ES cells have the same ability for germline transmission. Our results demonstrate that ES cells derived from E14 and C2J cells have the same capacity for germline transmission when injected into C57BL/6J blastocysts; however, due to the limitation of mixed genetic background between E14 cells and host C57BL/6J embryos, C2J ES cells are preferable to E14 ES cells for use in gene-targeting and should become the cell line of choice for the generation of genetically engineered mutant mouse lines.

Background Proprotein convertase 5/6 (PC6) is critical for endometrial epithelial receptivity and stromal cell decidualization for embryo implantation in women. We hypothesized that inhibiting PC6 could block implantation for contraception. The aim of this study was to prove this concept using human cell models and rabbits. Study Design A potential PC6 inhibitor, C1239-PEG-Poly R, was biochemically confirmed to be a potent PC6 inhibitor. The potential contraceptive action of the inhibitor was then tested in decidualization of primary human endometrial stromal cells in a human trophoblast spheroid attachment model and in vivo in rabbits. Results The PC6 inhibitor C1239-PEG-Poly R inhibited in a dose-dependent manner both decidualization and spheroid attachment. Vaginal delivery of 200 μL of the inhibitor at a final concentration of 5 mM to rabbits over a 3-day period starting 6 days after mating resulted in a 60% decrease in implantation and, hence, pregnancy. Conclusions This study presents proof of concept that PC6 inhibition has the potential to block embryo implantation, providing nonhormonal contraception for women.

Background This study was conducted to observe the in vivo effect of 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF) on embryo implantation in rats and its in vitro effect on cell adhesion. Study Design The anti-implantation efficacy of AEBSF in rats was determined by counting the number of visible implanted embryos on day 8 of pregnancy following intrauterine (5 mg and 10 mg AEBSF per horn) or tail vein (10 mg AEBSF per rat) administration on day 3 of pregnancy. The effects of AEBSF on cell adhesion were detected, respectively, by using the mouse blastocysts–endometrial cells or the human umbilical vein endothelial cells (HUVECs)–HeLa cells co-culture model. The alteration in protein secretion pattern of HUVECs and HeLa cells was detected by the proteome analysis. Results 4-(2-Aminoethyl)benzenesulfonyl fluoride hydrochloride showed an in vivo inhibitory effect on embryo implantation in rat. In vitro, AEBSF could disturb the growth of blastocysts on endometrial cells and inhibit the adhesion of HeLa cells on HUVECs. The treatment of AEBSF could alter the protein secretion pattern of co-cultured HUVEC–HeLa cells. Conclusion 4-(2-Aminoethyl)benzenesulfonyl fluoride hydrochloride might be a potential leading compound for novel contraceptives, and its inhibitory effect on implantation might result from the interference in extracellular matrix remodeling process.

Objective To assess the predictive value of endocrine gland–derived vascular endothelial growth factor (EG-VEGF) concentrations in follicular fluid (FF) and serum for ovarian hyperstimulation syndrome (OHSS) in patients undergoing controlled ovarian hyperstimulation. Design Retrospective, case-control study. Setting University hospital, IVF center. Patient(s) Seventeen women with OHSS and 61 controls. Intervention(s) None. Main Outcome Measure(s) FF and serum EG-VEGF and VEGF concentrations, IVF outcome. Result(s) FF and serum EG-VEGF concentrations showed a significant negative correlation with serum E 2 concentration on the day of hCG administration. FF, but not serum, VEGF concentrations also showed a significant negative correlation with serum E 2 concentrations on hCG day. The FF EG-VEGF, FF VEGF, and serum EG-VEFG concentrations were significantly lower in the OHSS group than in the non-OHSS group. There was no significant difference in serum VEGF concentrations. Among FF and serum EG-VEGF and VEGF concentrations, only FF EG-VEGF concentrations were significantly lower in patients with moderate OHSS than in those with mild OHSS. Conclusion(s) FF and serum EG-VEGF concentrations may predict OHSS occurrence. Furthermore, FF EG-VEGF concentrations were significantly correlated with OHSS severity; thus, EG-VEGF appears to be more valuable than VEGF for predicting OHSS.

The aim of this study was to identify a novel implantation-related molecule and to examine EMO2 expression in the mouse uterus during the peri-implantation period.Experimental study.Research laboratory.Adult ICR mice aged 6-8 weeks.Adult female mice were mated with fertile males to achieve pregnancy. Implantation was delayed by ovariectomizing pregnant mice on day 4 and administering P during days 5-7; implantation was then initiated by administering E(2). Pseudopregnant mice were obtained by mating females with vasectomized males.The tissue distribution of EMO2 mRNA was detected by reverse transcriptase-polymerase chain reaction, and the uterine expression pattern of the EMO2 protein was determined by immunohistochemistry.The full cDNA sequence of EMO2 was registered in GenBank (AY372183). EMO2 mRNA expression was observed in all mouse tissues tested. The expression of the EMO2 protein was predominately localized in decidual cells at the implantation site during days 5-6 of pregnancy, and its expression was induced by the active blastocyst and artificially induced decidualization.Our data indicate that EMO2 may play a key role in the mouse embryo implantation process.

A novel nonviral gene transfer vector was developed by modifying nanostructured lipid carrier (NLC) with cetylated polyethylenimine (PEI). Polycation nanostructured lipid carrier (PNLC) was prepared using the emulsion-solvent evaporation method. Its in vitro gene transfer properties were evaluated in the human lung adenocarcinoma cell line SPC-A1 and Chinese Hamster Ovary (CHO) cells. Enhanced transfection efficiency of PNLC was observed after the addition of triolein to the PNLC formulation and the transfection efficiency of the optimized PNLC was comparable to that of Lipofectamine 2000. In the presence of 10% serum the transfection efficiency of the optimal PNLC was not significantly changed in either cell line, whereas that of Lipofectamine 2000 was greatly decreased in both. Thus, PNLC is an effective nonviral gene transfer vector and the gene delivery activity of PNLC was enhanced after triolein was included into the PNLC formulation.

Copyright © 2016 Massachusetts Medical Society. All rights reserved. BACKGROUND: Human reproduction depends on the fusion of a mature oocyte with a sperm cell to form a fertilized egg. The genetic events that lead to the arrest of human oocyte maturation are unknown. METHODS: We sequenced the exomes of five members of a four-generation family, three of whom had infertility due to oocyte meiosis I arrest. We performed Sanger sequencing of a candidate gene, TUBB8, in DNA samples from these members, additional family members, and members of 23 other affected families. The expression of TUBB8 and all other β-tubulin isotypes was assessed in human oocytes, early embryos, sperm cells, and several somatic tissues by means of a quantitative reverse- transcriptase-polymerase-chain-reaction assay. We evaluated the effect of the TUBB8 mutations on the assembly of the heterodimer consisting of one α-tubulin polypeptide and one β-tubulin polypeptide (α/β-tubulin heterodimer) in vitro, on microtubule architecture in HeLa cells, on microtubule dynamics in yeast cells, and on spindle assembly in mouse and human oocytes. RESULTSL: We identified seven mutations in the primate-specific gene TUBB8 that were responsible for oocyte meiosis I arrest in 7 of the 24 families. TUBB8 expression is unique to oocytes and the early embryo, in which this gene accounts for almost all the expressed β-tubulin. The mutations affect chaperone-dependent folding and assembly of the α/β-tubulin heterodimer, disrupt microtubule behavior on expression in cultured cells, alter microtubule dynamics in vivo, and cause catastrophic spindle-assembly defects and maturation arrest on expression in mouse and human oocytes. CONCLUSIONS: TUBB8 mutations have dominant-negative effects that disrupt microtubule behavior and oocyte meiotic spindle assembly and maturation, causing female infertility.

Leptin exerts many biological functions, such as in metabolism and reproduction, through binding to and activating the leptin receptor, LepRb, which is expressed in many regions of the brain. To better understand the roles of LepR downstream signaling pathways, Y123F mice, which expressed mutant leptin receptors with phenylalanine (F) substituted for three tyrosines (Y) (Tyr985, Tyr1077 and Tyr1138), were generated. The body weight and abdominal fat deposits of Y123F homozygous mice (HOM) were higher than those of wild-type mice (WT). HOM ovaries were atrophic and the follicles developed abnormally; however, the HOM ovaries did not exhibit polycystic phenotypes. Moreover, Y123F HOM adults had no estrous cycle and the blood estrogen concentration remained stable at a low level below detection limit of 5 pg/ml. LepR expression in HOM ovaries was higher than in WT ovaries. Using cDNA Microarrays, the mRNA expressions of 41 genes were increased, and 100 were decreased in HOM vs. WT ovaries, and many signaling pathways were evaluated to be involved significantly. The expressions of 19 genes were validated by real-time quantitative PCR, most of which were consistent with the microarray results. Thus, Y123F HOM mice were suggested as a new animal model of PCOS for research that mainly emphasizes metabolic disorders and anovulation, but not the polycystic phenotype. Meanwhile, using the model, we found that JAK-STAT and hormone biosynthesis pathways were involved in the follicular development and ovulation disorders caused by LepR deficiency in ovaries, although we could not exclude indirect actions from the brain.

Background N-myc down-regulated gene 2 (NDRG2) is a tumor suppressor involved in cell proliferation and differentiation. The aim of this study was to determine the uterine expression pattern of this gene during early pregnancy in mice. Methods Uterine NDRG2 mRNA and protein expression levels were determined by RT-PCR and Western blot analyses, respectively, during the peri-implantation period in mice. Immunohistochemical (IHC) analysis was performed to examine the spatial localization of NDRG2 expression in mouse uterine tissues. The in vitro decidualization model of mouse endometrial stromal cells (ESCs) was used to evaluate decidualization of ESCs following NDRG2 knock down by small interfering RNA (siRNA). Statistical significance was analyzed by one-way ANOVA using SPSS 19.0 software. Results Uterine NDRG2 gene expression was significantly up-regulated and was predominantly localized to the secondary decidual zone on days 5 and 8 of pregnancy in mice. Its increased expression was associated with artificial decidualization as well as the activation of delayed implantation. Furthermore, uterine NDRG2 expression was induced by estrogen and progesterone treatments. The in vitro decidualization of mouse ESCs was accompanied by up-regulation of NDRG2 expression, and knock down of its expression in these cells by siRNA inhibited the decidualization process. Conclusions These results suggest that NDRG2 might play an important role in the process of decidualization during early pregnancy.

Maternal immune tolerance to the semi-allogenic fetus is required for successful pregnancy in mammals. Monoclonal nonspecific suppressor factor beta (MNSFB) is an immunosuppressive factor present in uterine epithelial and stromal cells, as well as in macrophages and T cells. Although the functional neutralization of MNSFB using specific antibodies against it lead to failed embryo implantation in mice, the exact role of MNSFB at the fetal-maternal interface remains unclear. The present study generated conditional heterozygous Mnsfb-deficient (Mnsfb(+/) (-) ) mice using the LoxP/Cre system. Western-blot analyses showed that uterine MNSFB protein in Mnsfb(+/-) mice was remarkably down-regulated compared to that in the wild-type (Mnsfb(+/+) ) mice. The litter size of female Mnsfb(+/-) mice was significantly reduced, which corresponded to developmental failure of embryos beyond Day 11 of pregnancy. The expression level of MNSFB protein was also lower in the failing compared to the normal embryos. An aberrant interaction between the embryos of Day-4 pregnant wild-type mice and endometrial stromal cells of female Mnsfb(+/-) mice was observed in vitro. The uterine Day-5 abundance of P53, BAX, and BCL-G in pregnant Mnsfb(+/-) mice was significantly decreased compared to that of wild-type mice, whereas the expression of P27 and tumor necrosis factor alpha (TNFA) was elevated. By comparison, the levels of MNSFB and BAX proteins in human decidual tissues obtained from recurrent spontaneous miscarriage patients were significantly reduced compared to those obtained from legal medial abortion, highlighting the involvement of MNSFB in the pathogenesis of recurrent spontaneous miscarriage. Together, these results demonstrated that a deficiency in MNSFb is associated with pregnancy loss, probably through reduced P53 and/or increased TNFA production at the fetal-maternal interface.

Background Proprotein convertase 5/6 (PC5/6) is critical for embryo implantation in women, regulating both uterine epithelial receptivity and stromal cell decidualization. PC5/6 is likewise essential for implantation in mice, but involved only in decidualization. An alternative animal model is required to address the function of PC5/6 in the uterine epithelium. This study aimed to establish whether PC5/6 is associated with embryo implantation in rabbits. Methods Virgin New-Zealand white rabbits aged 3-4 moths were mated with males of the same strain, or pseudo-pregnancy induced. After mating, uterine tissues were collected over a 10 day (d) period (n = 3 per time point) for RNA, protein and histological analyses to determine the temporal and spatial uterine expression pattern of PC5/6 during the initial stages of pregnancy or induced pseudo-pregnancy. Results PC5/6 mRNA was up-regulated just prior to embryo attachment on d6, and the elevated expression was maintained throughout implantation on d6.5-10. Western analysis revealed a preferential up-regulation of PC5/6 in the implantation sites. Immunohistochemical analysis identified that both the amount and cellular localization of PC5/6 changed with increasing pregnancy stages. Before embryo attachment, PC5/6 was low and localised in the luminal and glandular epithelium. It increased on d6.5 in the basal glands and mucosal folds, and then strongly intensified on d7-10 in the multinucleated luminal symplasma and decidual cells at the site of embryo implantation. In contrast, the pseudo-pregnant uterus displayed relatively low and static PC6 mRNA expression throughout the 10 days, with no obvious changes in either PC5/6 level or cellular localization. Conclusions These findings demonstrate that embryo implantation in the rabbit is closely associated with dynamic expression of uterine PC5/6, and that the rabbit may be an appropriate model to investigate the function of PC5/6 in the uterine epithelium during embryo attachment.

Background Ovarian granulosa cells are the predominant source of estradiol and progesterone biosynthesis in vivo. Rosiglitazone, a synthetic agonist of the peroxisome proliferator-activated receptor gamma (PPAR gamma), is applied as the treatment of insulin resistance including women with PCOS. The aim of the study was to investigate the direct effects of rosiglitazone on steroidogenesis and proinflammatory factor production in human granulosa-lutein cells (GLCs). Methods Primary human GLCs were separated during in vitro fertilization and cultured in the presence of rosiglitazone, GW9662 (an antagonist of PPAR gamma) and hCG. The mRNA expression of key steroidogenic factors including 3beta- hydroxysteriod dehydrogenase (3beta-HSD), cytochrome P-450 scc (CYP11A1), cytochrome P-450 aromatase (CYP19A1), and steroidogenic acute regulatory protein (StAR) were detected by quantitative real-time PCR. Estradiol and progesterone levels in GLCs cultures were measured by chemiluminescence immunoassay, and the proinflammtory factors (TNFalpha and IL-6) in conditioned culture media were measured by ELISA. Results PPAR gamma mRNA levels increased up to 3.24 fold by rosiglitazone at the concentration of 30 microM compared to control (P < 0.05). hCG alone or hCG with rosiglitazone had no significant effects on PPAR gamma mRNA levels. The CYP19A1 mRNA level at exposure to rosiglitazone alone showed a drop, but was not significantly reduced comparing to control. The expression levels of enzymes 3beta-HSD and CYP11A1 in all treatments did not alter significantly. The StAR mRNA expression at exposure to rosiglitazone was significantly increased comparing to control (P < 0.05). The media concentrations of E2 and progesterone by rosiglitazone treatment showed a declining trend comparing to control or cotreatment with hCG, which did not reach significance. Most importantly, treatment with rosiglitazone decreased TNFalpha secretion in a statistically significant manner compared with control (P < 0.05). The concentration of IL-6 following rosiglitazone exposure did not significantly decrease comparing to control. Conclusion In cultured GLCs, rosiglitazone stimulated StAR expression, but did not significantly affect steroidogenic enzymes, as well as E2 and progesterone production. Moreover, rosiglitazone significantly decreased the production of TNFalpha in human GLCs, suggesting that PPAR gamma may play a role in the regulation of GLCs functions through inhibiting proinflammatory factors.

Background Sec63 is a key component of the protein translocation machinery in the mammalian endoplasmic reticulum (ER), and involved in the post-translation processing of secretory proteins. The aim of this study was to determine the expression pattern of SEC63 gene in mouse uterus during the early pregnancy. Methods Real-time quantitative PCR and Western blot analyses were used to evaluate the alteration in levels of uterine SEC63 gene expression during the peri-implantation period in mice. Further, both in situ hybridization and immunohistochemical analyses were performed to examine the spatial localization of SEC63 gene expression in mouse uterine tissues. The presence of Sec63 protein in human uterine tissue was also detected by immunohistochemical analysis. Statistical analysis was carried out using Tukey test. Results Uterine SEC63 gene expression was up-regulated and predominantly localized in mouse decidual cells during days 5–8 of pregnancy. More interestingly, Sec63 protein was also detected in human decidua of 10-week pregnancy, whereas was not observed in human endometrial tissues both at proliferative and secretory phases of menstrual cycle. Conclusion The pattern of SEC63 gene expression is consistent with a possible role for SEC63 in decidualization.

Regulator of G-protein signalling 2 (Rgs2) is involved in G-protein-mediated signalling by negatively regulating the activity of the G-protein α-subunit. In the present study, the expression patterns of Rgs2 in mouse ovarian tissues and early embryos were determined by semiquantitative reverse transcription–polymerase chain reaction, immunohistochemistry and immunofluorescent analyses. Rgs2 expression was observed in the ovarian tissues of adult female mice, with an almost equal expression levels during different stages of the oestrous cycle. Rgs2 was abundant in the cytoplasm, membrane, nuclei and spindles of intact polar bodies in mouse early embryos at different developmental stages from the zygote to blastocyst. The effect of Rgs2 knockdown on early embryonic development in vitro was examined by microinjecting Rgs2-specific short interfering (si) RNAs into mouse zygotes. Knockdown of endogenous Rgs2 expression led to abnormal embryonic development in vitro, with a considerable number of early embryos arrested at the 2- or 4-cell stage. Moreover, mRNA expression of three zygotic gene activation-related genes (i.e. Zscan4, Tcstv1 and MuERV-L) was decreased significantly in 2-cell arrested embryos. These results suggest that Rgs2 plays a critical role in early embryo development.