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In vitro follicle growth is a potential approach to preserve fertility for young women who are facing a risk of premature ovarian failure (POF) caused by radiation or chemotherapy. Our two-step follicle culture strategy recapitulated the dynamic human follicle growth environment in vitro . Follicles developed from the preantral to antral stage, and, for the first time, produced meiotically competent metaphase II (MII) oocytes after in vitro maturation (IVM).

Background The potentially detrimental role of progesterone during the follicular phase has been a matter of controversy for several years; however, few studies have analyzed the effects of combined raised estradiol and progesterone levels on pregnancy outcomes. The aim of the present study was to determine the influence of high progesterone levels on clinical outcomes in the context of high ovarian response. Methods We performed a retrospective cohort study that included 2850 women classified as high responders. The women were subdivided into six groups depending on their progesterone concentration on the day of human chorionic gonadotropin (hCG) administration: <0.5 ng/ml (1.81 ng/ml (>p90). Ovarian response was classified as high when > =20 oocytes were retrieved or when estradiol was > =3000 pg/ml. Clinical outcomes of each subgroup were analyzed. We also examined data from frozen-thawed embryo transfers. Results were analyzed with Student’s t- test to compare continuous variables and chi-squared test to compare proportions. A p -value of < =0.05 was considered statistically significant. Results The progesterone concentration increased with ovarian response, and elevated progesterone did not show a significant clinical impact on implantation rate and pregnancy rates. These data provide evidence that progesterone levels higher than 1.8 ng/ml do not have detrimental effect on oocyte quality or endometrial receptivity. Conclusions These data allow us to conclude that high progesterone levels correlate significantly with high estradiol levels and that in high responder women; progesterone levels do not show a significant clinical impact on results.

Sperm chemotaxis in mammals have been identified towards several female sources as follicular fluid (FF), oviduct fluid, and conditioned medium from the cumulus oophorus (CU) and the oocyte (O). Though several substances were confirmed as sperm chemoattractant, Progesterone (P) seems to be the best chemoattractant candidate, because: 1) spermatozoa express a cell surface P receptor, 2) capacitated spermatozoa are chemotactically attracted in vitro by gradients of low quantities of P; 3) the CU cells produce and secrete P after ovulation; 4) a gradient of P may be kept stable along the CU; and 5) the most probable site for sperm chemotaxis in vivo could be near and/or inside the CU. The aim of this study was to verify whether P is the sperm chemoattractant secreted by the rabbit oocyte-cumulus complex (OCC) in the rabbit, as a mammalian animal model. By means of videomicroscopy and computer image analysis we observed that only the CU are a stable source of sperm attractants. The CU produce and secrete P since the hormone was localized inside these cells by immunocytochemistry and in the conditioned medium by enzyme immunoassay. In addition, rabbit spermatozoa express a cell surface P receptor detected by western blot and localized over the acrosomal region by immunocytochemistry. To confirm that P is the sperm chemoattractant secreted by the CU, the sperm chemotactic response towards the OCC conditioned medium was inhibited by three different approaches: P from the OCC conditioned medium was removed with an anti-P antibody, the attractant gradient of the OCC conditioned medium was disrupted by a P counter gradient, and the sperm P receptor was blocked with a specific antibody. We concluded that only the CU but not the oocyte secretes P, and the latter chemoattract spermatozoa by means of a cell surface receptor. Our findings may be of interest in assisted reproduction procedures in humans, animals of economic importance and endangered species.

Granulosa cell proliferation and differentiation are key developmental steps involved in the formation of the dominant follicle eligible for ovulation. This process is, in turn, regulated by spatiotemporally emerging molecular events. MicroRNAs (miRNAs) are one of the molecular signatures believed to regulate granulosa cell function by fine-tuning gene expression. Previously, we showed that the miR-17-92 cluster was differentially expressed in granulosa cells from subordinate and dominant follicles at day 19 of the estrous cycle. However, the role of this miRNA cluster in bovine follicular cell function is not known. Therefore, in the present study, we investigate the role of the miR-17-92 cluster in granulosa cell function by using an in vitro model. Target prediction and luciferase assay analysis revealed that the miR-17-92 cluster coordinately regulated the PTEN and BMPR2 genes. Overexpression of the miR-17-92 cluster by using a mimic promoted granulosa cell proliferation and reduced the proportion of differentiated cells. However, cluster inhibition resulted in decreased proliferation and increased differentiation in granulosa cells. This was further supported by expression analysis of marker genes of proliferation and differentiation. The role of the miR-17-92 cluster was cross-validated by selective knockdown of its target genes by the short interfering RNA technique. Suppression of the PTEN and BMPR2 genes revealed similar phenotypic and molecular alterations as observed when the granulosa cells were transfected with the miR-17-92 cluster mimic. Thus, the miR-17-92 cluster is involved in granulosa cell proliferation and differentiation by coordinately targeting the PTEN and BMPR2 genes.

Successful pregnancy requires coordination of embryo development, decidualization of endometrium, and placenta formation. Decidualization denotes the transformation of endometrial stromal cells into specialized secretory cells, a process further characterized with influx of specialized immune cells into stroma, predominantly uterine natural killer cells and macrophages, and vascular remodeling. This differentiation process depends on the convergence of the cyclic adenosine monophosphate and progesterone signaling pathways. The decidual process is indispensable for the formation of a functional feto-maternal interface as it controls tissue homeostasis during endovascular trophoblast invasion and bestows tissue resistance to environmental stress signals, including protection against oxidative cell death. FOXO proteins have emerged as key mediators of cell fate because of their ability to regulate either pro-apoptotic genes or genes involved in differentiation, cell cycle arrest, oxidative defenses, and DNA repair. In the endometrium, FOXO1 is of particular importance as a critical regulator of progesterone-dependent differentiation, menstrual shedding, and protection of the feto-maternal against oxidative damage during pregnancy.

The goal of the present study was to investigate the action of TBBPA on PPARγ protein expression in vitro in human choriocarcinoma-derived placental JEG-3 cells. We also analyzed TBBPA for its action on placental secretion of progesterone and β-hCG, cell viability, and apoptosis. Our results showed that after TBBPA treatment at 10 nM and 10 µM, PPARγ protein expression increased in a time-dependent manner until 48 h and then slightly decreased at 72 h but was still above the control level. This alteration in PPARγ protein expression was accompanied by a decreased β-hCG level. Interestingly, co-treatment with the PPARγ antagonist GW9662 reversed the TBBPA-mediated changes in PPARγ protein expression but, according to β-hCG secretion, potentiated an inhibitory effect of TBBPA. Additionally, in our study, we assessed the ability of TBBPA to increase progesterone levels in JEG-3 cells compared with those of controls. Finally, in the present study, we demonstrated that TBBPA at all of the tested doses significantly increased caspase-3 activity compared with that of the vehicle control. The apoptotic action of TBBPA was also confirmed by Hoechst 33342 staining. These results showed the up-regulation of PPARγ protein expression after TBBPA exposure in human placental cells. Although co-treatment with antagonist of PPARγ reversed the TBBPA-mediated increase in this protein expression and restored it to the control level, it did not reverse the effect on β-hCG secretion. This indicated that the mechanism of TBBPA-induced changes in β-hCG secretion is PPARγ-independent.

Brucella has been reported to impair placental trophoblasts, a cellular target where Brucella efficiently replicates in association with the endoplasmic reticulum (ER), and ultimately trigger abortion in pregnant animals. However, the precise effects of Brucella on trophoblast cells remain unclear. Here, we describe the infection and replication of Brucella suis vaccine strain 2 (B.suis.S2) in goat trophoblast cells (GTCs) and the cellular and molecular responses induced in vitro. Our studies demonstrated that B.suis.S2 was able to infect and proliferate to high titers, hamper the proliferation of GTCs and induce apoptosis due to ER stress. Tunicamycin (Tm), a pharmacological chaperone that strongly mounts ER stress-induced apoptosis, inhibited B.suis.S2 replication in GTCs. In addition, 4 phenyl butyric acid (4-PBA), a pharmacological chaperone that alleviates ER stress-induced apoptosis, significantly enhanced B.suis.S2 replication in GTCs. The Unfolded Protein Response (UPR) chaperone molecule GRP78 also promoted B.suis.S2 proliferation in GTCs by inhibiting ER stress-induced apoptosis. We also discovered that the IRE1 pathway, but not the PERK or ATF6 pathway, was activated in the process. However, decreasing the expression of phosphoIRE1α and IRE1α proteins with Irestatin 9389 (IRE1 antagonist) in GTCs did not affect the proliferation of B.suis.S2. Although GTC implantation was not affected upon B.suis.S2 infection, progesterone secretion was suppressed, and prolactin and estrogen secretion increased; these effects were accompanied by changes in the expression of genes encoding key steroidogenic enzymes. This study systematically explored the mechanisms of abortion in Brucella infection from the viewpoint of pathogen invasion, ER stress and reproductive endocrinology. Our findings may provide new insight for understanding the mechanisms involved in goat abortions caused by Brucella infection.

Two of the unanswered questions in mammalian developmental biology are when and where the fate of the germ cell is specified. Here, we report that stem cells isolated from the skin of porcine fetuses have the intrinsic ability to differentiate into oocyte-like cells. When differentiation was induced, a subpopulation of these cells expressed markers such as Oct4, Growth differentiation factor 9b (GDF9b), the Deleted in Azoospermia-like ( DAZL ) gene and Vasa — all consistent with germ-cell formation. On further differentiation, these cells formed follicle-like aggregates that secreted oestradiol and progesterone and responded to gonadotropin stimulation. Some of these aggregates extruded large oocyte-like cells that expressed oocyte markers, such as zona pellucida, and the meiosis marker, synaptonemal complex protein 3 (SCP3). Some of these oocyte-like cells spontaneously developed into parthenogenetic embryo-like structures. The ability to generate oocyte-like cells from skin-derived cells may offer new possibilities for tissue therapy and provide a new in vitro model to study germ-cell formation and oogenesis.

Folate is an essential B vitamin required for de novo purine and thymidylate synthesis, and for the remethylation of homocysteine to form methionine. Folate deficiency has been associated with placenta-related pregnancy complications, as have SNP in genes of the folate-dependent enzymes, methionine synthase (MTR) and methylenetetrahydrofolate dehydrogenase 1 (MTHFD1). We aimed to determine the effect of altered folate metabolism on placental cell proliferation, viability and invasive capacity and on progesterone and human chorionic gonadotropin (hCG) secretion. Human placental choriocarcinoma (JEG-3) cells cultured in low folic acid (FA) (2 nm) demonstrated 13 % (P<0·001) and 26 % (P<0·001) lower proliferation, 5·5 % (P=0·025) and 7·5 % (P=0·004) lower invasion capacity, and 5 to 7·5 % (P=0·004–0·025) lower viability compared with control (20 nm) or supplemented (100 nm) cells, respectively. FA concentration had no effect on progesterone or hCG secretion. Small interfering RNA (siRNA) knockdown of MTR gene and protein expression resulted in 17·7 % (P<0·0001) lower proliferation and 61 % (P=0·014) higher progesterone secretion, but had no effect on cell invasion and hCG secretion. siRNA knockdown of MTHFD1 gene expression in the absence of detectable changes in protein expression resulted in 10·3 % (P=0·001) lower cell proliferation, but had no effect on cell invasion and progesterone or hCG secretion. Our data indicate that impaired folate metabolism can result in lower trophoblast proliferation, and could alter viability, invasion capacity and progesterone secretion, which may explain in part the observed associations between folate and placenta-related complications.

Cytochrome P450 aromatase 19A1 (CYP19A1) is a critical enzyme in estrogen synthesis. However, the effect of CYP19A1 on cell growth and hormone secretion of buffalo follicular granulosa cells (BFGCs) is poorly understood. The objective of this study was to assess the role of CYP19A1 in cell proliferation and hormone secretion of BFGCs by knocking down CYP19A1 mRNA expression. The mRNA expression level of CYP19A1 gene was knocked down in BFGCs using the siCYP19A1-296 fragment with the best interference efficiency of 72.63%, as affirmed by real-time quantitative PCR (qPCR) and cell morphology analysis. The CYP19A1 knockdown promoted the proliferation of BFGCs through upregulating the mRNA expression levels of six proliferation-related genes (CCND1, CCNE1, CCNB1, CDK2, CDKN1A, and CDKN1B). Moreover, CYP19A1 knockdown increased ( P < 0.05) the concentrations of progesterone secretion (P4) in BFGCs through increasing the mRNA expression levels of three steroidogenic genes (HSD17B1, HSD17B7, and CYP17A1). Our data further found that the FSH could inhibit the mRNA expression level of CYP19A1 in BFGCs, while LH obtains the opposite effect. These findings showed that the CYP19A1 knockdown had a regulatory role in the hormone secretion and cell proliferation in BFGCs.