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Background LepR tyrosine site mutation mice (Y123F) exhibit decreased serum E2 levels, immature reproductive organs, infertility as well as metabolic abnormalities. Although the actions of leptin and lepR in the control of reproductive function are thought to be exerted mainly via the hypothalamic-pituitary-gonadal axis, relatively less is known regarding their local effects on the peripheral ovary, especially on steroid hormone synthesis. Meanwhile, whether the decreased fertility of Y123F mouse could be restored by gonadotropin has not been clear yet. Methods The serum levels of E2, P4, FSH, LH, T and leptin of Y123F and WT mice at the age of 12 weeks were measured by enzyme-linked immunosorbent assay. Immunohistochemistry was used to compare the distribution of hormone synthases (STAR, CYP11A1, CYP19A1, HSD17B7) and FSHR in adult mouse ovaries of two genotypes. Western blot and real-time PCR were used to detect the expression levels of four ovarian hormone synthases and JAK2-STAT3 / STAT5 signaling pathway in 4 and 12 weeks old mice, as well as the effects of exogenous hFSH stimulation on hormone synthases and FSHR. Results Compared with WT mice, the serum levels of FSH, LH and E2 in 12-week-old Y123F mice were significantly decreased; T and leptin levels were significantly increased; but there was no significant difference of serum P4 levels. STAR, CYP11A1, HSD17B7 expression levels and the phosphorylation levels of JAK2 and STAT3 were significantly decreased in adult Y123F mice, while the expression of CYP19A1 and phospho-STAT5 were significantly increased. No significant differences were found between 4-week-old Y123F and WT mice. After exogenous hFSH stimulation, E2 levels and expression of CYP19A1 and HSD17B7 were significantly higher than that in the non-stimulated state, but significant differences still existed between Y123F and WT genotype mice under the same condition. Conclusions Abnormal sex hormone levels of Y123F mice were due to not only decreased gonadotropin levels in the central nervous system, but also ovarian hormone synthase abnormalities in the peripheral gonads. Both FSH signaling pathway and JAK2-STAT3/STAT5 signaling pathway were involved in regulation of ovarian hormone synthases expression. Exogenous FSH just partly improved the blood E2 levels and ovarian hormone synthase expression.

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 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.

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.

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.

To evaluate multiple follicular development synchronization after estrogen stimulation in prepubertal mice, follicular responsiveness to gonadotropin superovulation, the prospective reproductive potential and ovarian polycystic ovary syndrome (PCOS)-like symptoms at adulthood, prepubertal mice were intraperitoneally injected with estrogen to establish an animal model with solvent as control. When synchronized tertiary follicles in ovaries, in vitro oocyte maturation and fertilization rates, blastocyst formation rate, developmental potential into offspring by embryo transfer, adult fertility and PCOS-like symptoms, and involved molecular mechanisms were focused, it was found that estrogen stimulation (10 μg/gBW) leads to follicular development synchronization at the early tertiary stage in prepubertal mice; reproduction from oocytes to offspring could be realized by means of the artificial reproductive technology though the model mice lost their natural fertility when they were reared to adulthood; and typical symptoms of PCOS, except changes in inflammatory pathways, were not remained up to adulthood. So in conclusion, estrogen can lead to synchronization in follicular development in prepubertal mice, but does not affect reproductive outcome of oocytes, and no typical symptoms of PCOS remained at adulthood despite changes related to inflammation.

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.

Successful pregnancy depends on the precise regulation of extravilloustrophoblast (EVT) invasion into the uterine decidua. SPARC (secreted protein acidic and rich in cysteine) is a matricellular glycoprotein that plays critical roles in the pathologies associated with obesity and diabetes, as well as tumorigenesis. The objective of this study was to investigate the role of SPARC in the process of trophoblast invasion which shares many similarities with tumor cell invasion. By Western blot, higher expression of SPARC was observed in mouse brain, ovary and uterus compared to other mouse tissues. Immunohistochemistry analysis revealed a spatio-temporal expression of SPARC in mouse uterus in the periimplantation period. At the implantation site of d8 pregnancy, SPARC mainly accumulated in the secondary decidua zone (SDZ), trophoblast cells and blastocyst. The expression of SPARC was also detected in human placental villi and trophoblast cell lines. In a Matrigel invasion assay, we found SPARC-specific RNA interference significantly reduced the invasion of human extravilloustrophoblast HTR8/SVneo cells. Microarray analysis revealed that SPARC depletion upregulated the expression of interleukin 11 (IL11), KISS1, insulin-like growth factor binding protein 4 (IGFBP4), collagen type I alpha 1 (COLIA1), matrix metallopeptidase 9 (MMP9), and downregulated the expression of the alpha polypeptide of chorionic gonadotropin (CGA), MMP1, gap junction protein alpha 1 (GJA1), et al. The gene array result was further validated by qRT-PCR and Western blot. The present data indicate that SPARC may play an important role in the regulation of normal placentation by promoting the invasion of trophoblast cells into the uterine decidua.