Transcription ceases upon stimulation of oocyte maturation and gene expression during oocyte maturation, fertilization, and early cleavage relies on translational activation of maternally derived mRNAs. Two key mechanisms that mediate translation of mRNAs in oocytes have been described in detail: cytoplasmic polyadenylation-dependent and -independent. Both of these mechanisms utilize specific protein complexes that interact with cis-acting sequences located on 3′-untranslated region (3′-UTR), and both involve embryonic poly(A) binding protein (EPAB), the predominant poly(A) binding protein during early development. While mechanistic details of these pathways have primarily been elucidated using the Xenopus model, their roles are conserved in mammals and targeted disruption of key regulators in mouse results in female infertility. Here, we provide a detailed account of the molecular mechanisms involved in translational activation during oocyte and early embryo development, and the role of EPAB in this process.

Summary Sentence

Regulation of maternally derived mRNAs' translation and presence of embryonic poly(A) binding protein (EPAB) are critical for gene expression profile in oocytes and early embryo.

Assisted reproductive technologies (ARTs) have been proposed as a means of overcoming the significant challenges of managing small, isolated populations of endangered species in zoos. However, efficient protocols for ARTs do not exist for most endangered species. This review will focus on research efforts to characterize unique reproductive mechanisms and develop species-specific ARTs. Central to these studies are assays to measure steroid metabolites in urine or feces and/or training programs to allow unrestrained blood collections and ultrasound evaluations. The resulting information about estrous cycle dynamics, combined with studies of semen collection and processing, provides the foundation for the development of artificial insemination (AI). In vitro fertilization and embryo transfer are also discussed in relation to the advantages these techniques could provide relative to AI, as well as the significant challenges involved with technologies that require oocytes and embryos. Finally, an argument is made for additional research of nontraditional model species (e.g., domestic cats and dogs) and the development of novel models representing unique taxa. Whether these species are studied by zoo-based researchers with the expressed intent of developing ARTs for conservation or academic scientists interested in basic biology, the resulting information will provide a unique, evolutionary perspective on reproduction that could have wide-reaching benefits. The more information we have available, the better our chances will be of developing effective ARTs and making a difference in conservation efforts for endangered species.

Summary Sentence

Reproductive biologists developing ARTs for wildlife face the challenge of developing sophisticated protocols requiring significant amounts of information for species that have never been studied before and are not readily available to study now.

Objective: To study the potential role of miR-30a-3p in embryo implantation and explore underlying mechanisms.

Methods: We first established normal pregnancy, pseudopregnancy, delayed implantation, and artificial decidualization mouse models. Next, we detected miR-30a-3p expression profiles of these models with real-time reverse transcription PCR(qRT-PCR), then predicted potential target genes through a dual-luciferase assay. Immunofluorescence-fluorescence in situ hybridization co-located miR-30a-3p and target genes. We then examined the effect of miR-30a-3p on embryo implantation in vivo and in vitro. Wound healing and transwell assays were employed to explore possible miR30a-3p effects on epithelial-mesenchymal transition (EMT), before molecules related to the latter process were examined with qRT-PCR.

Results: MiR-30a-3p expression decreased significantly on embryo implantation day, compared with the peri-implantation period (P < 0.05). Identified target gene Snai2 expression increased significantly during implantation (P < 0.05). In vivo and in vitro analysis showed that up-regulation of miR-30a-3p by agomir and mimics resulted in decreased implantation sites and embryo implantation rate. Transfection of miR-30a-3p mimics to HEC-1-b cells decreased expression of Snai2 and mesenchymal markers (Vimentin and N-cadherin). Furthermore, wound healing area decreased, as did migration and invasion capacity.

Conclusion: MiR-30a-3p is down-regulated in the embryo implantation period and might have some effect on embryo implantation by acting as a suppressor of EMT through targeting Snai2.

Summary Sentence

MiR-30a-3p is down-regulated on the embryo implantation day and suppresses the EMT through targeting Snai2, and up-regulation of miR-30a-3p weakens the capacity of migration and invasion.

Advanced age is a risk factor undermining women's fertility. Hence, the optimization of assisted reproduction techniques is an interdisciplinary challenge that requires the improvement of in vitro culture systems. Here, we hypothesize that supplementation of embryo culture medium with extracellular vesicles from endometrial-derived mesenchymal stem cells (EV-endMSCs) may have a positive impact on the embryo competence of aged oocytes. In this work, 24 weeks old B6D2 female mice were used as egg donors and in vitro fertilization assays were performed using males from the same strain (8–12 weeks); the presumptive zygotes were incubated in the presence of 0, 10, 20, 40, or 80 µg/ml of EV-endMSCs. The results from the proteomic analysis of EV-endMSCs and the classification by Reactome pathways allowed us to identify proteins closely related with the fertilization process. Moreover, in our aged murine model, the supplementation of the embryo culture medium with EV-endMSCs improved the developmental competence of the embryos as well as the total blastomere count. Finally, gene expression analysis of murine blastocysts showed significant changes on core genes related to cellular response to oxidative stress, metabolism, placentation, and trophectoderm/inner cell mass formation. In summary, we demonstrate that EV-endMSCs increase the quality of the embryos, and according to proteomic and genomic analysis, presumably by modulating the expression of antioxidant enzymes and promoting pluripotent activity. Therefore, EV-endMSCs could be a valuable tool in human assisted reproduction improving the developmental competence of aged oocytes and increasing the odds of implantation and subsequent delivery.

Summary Sentence

The supplementation of embryo culture medium with extracellular vesicles from endometrialderived mesenchymal stem cells increases the quality of aged embryos.

Maternal high-fat diet (HFD) during pregnancy is linked to cardiovascular diseases in postnatal life. The current study tested the hypothesis that maternal HFD causes myocardial changes through angiotensin II receptor (AGTR) expression modulation in fetal and neonatal rat hearts. The control group of pregnant rats was fed a normal diet and the treatment group of pregnant rats was on a HFD (60% kcal fat). Hearts were isolated from embryonic day 21 fetuses (E21) and postnatal day 7 pups (PD7). Maternal HFD decreased the body weight of the offspring in both E21 and PD7. The ratio of heart weight to body weight was increased in E21, but not PD7, when compared to the control group. Transmission electron microscopy revealed disorganized myofibrils and effacement of mitochondria cristae in the treatment group. Maternal HFD decreased S-phase and increased G1-phase of the cellular cycle for fetal and neonatal cardiac cells. Molecular markers of cardiac hypertrophy, such as Nppa and Myh7, were found to be increased in the treatment group. There was an associated increase in Agtr2 mRNA and protein, whereas Agtr1a mRNA and AGTR1 protein were decreased in HFD fetal and neonatal hearts. Furthermore, maternal HFD decreased glucocorticoid receptors (GRs) binding to glucocorticoid response elements at the Agtr1a and Agtr2 promoter, which correlated with downregulation of GR in fetal and neonatal hearts. These findings suggest that maternal HFD may promote premature termination of fetal and neonatal cardiomyocyte proliferation and compensatory hypertrophy through intrauterine modulation of AGTR1 and AGTR2 expression via GR dependent mechanism.

Summary Sentence

AGTR plays a vital role in maternal HFD-mediated cardiac changes in the developing heart, as a target of therapeutic intervention.

Many mammalian species undergo embryonic diapause and suspend development at the blastocyst stage before implantation, which is also known as delayed implantation. We studied the process of how mouse embryos enter a dormancy status at a cellular level. Immunofluorescent analysis of differentiation markers for epiblast, primitive endoderm, and trophectoderm suggested that cell differentiation status was maintained during 7 days in diapause. To understand the progression of cellular dormancy during diapause, we examined the expression of a transgenic cell cycle marker Fucci2 and Ki67 by antibody staining, in addition to direct counting of nuclei in embryos. From these analyses, embryos during diapause were categorized into four stages by cell number and cell cycle. Cell cycle arrest occurred from the ab-embryonic region and from the trophectoderm to the ICM in the embryonic side. We also observed cell cycle transition by live imaging of Fucci2 embryos during the reactivation in culture from dormant status. Cell cycle was initially recovered from the embryonic side of embryos and eventually spread throughout the whole embryo. We also found that embryos in later stages of diapause required a longer period of time for reactivation. From these observations, it was shown that entrance into and exit from dormant status varied depending on cell types and location of cells in an embryo. These results suggest that embryonic diapause includes multiple steps and the mechanisms involved in cellular dormancy may be distinct between embryonic regions.

Summary Sentence

Dormancy progression during mouse embryonic diapause was categorized into four stages by cell number and cell cycle. Entrance into and exit from dormant status varied depending on cell types and location of cells in an embryo.

A decellularized uterine scaffold (DUS) prepared from rats permits recellularization and regeneration of uterine tissues when placed onto a partially excised uterus and supports pregnancy in a fashion comparable to the intact uterus. The underlying extracellular matrix (ECM) together with an acellular, perfusable vascular architecture preserved in DUS is thought to be responsible for appropriate regeneration of the uterus. To investigate this concept, we examined the effect of the orientation of the DUS-preserving ECM and the vascular architecture on uterine regeneration through placement of a DUS onto a partially defective uterine area in the reversed orientation such that the luminal face of the DUS was outside and the serosal face was inside. We characterized the tissue structure and function of the regenerated uterus, comparing the outcome to that when the DUS was placed in the correct orientation. Histological analysis revealed that aberrant structures including ectopic location of glands and an abnormal lining of smooth muscle layers were observed significantly more frequently in the reversed group than in the correct group (70% vs. 30%, P < 0.05). Despite the changes in tissue topology, the uteri regenerated with an incorrectly oriented DUS could achieve pregnancy in a way similar to uteri regenerated with a correctly oriented DUS. These results suggest that DUS-driven ECM orientation determines the regenerated uterus structure. Using DUS in the correct orientation is preferable when clinically applied. The disoriented DUS may deteriorate the tissue topology leading to structural disease of the uterus even though the fertility potential is not immediately affected.

Summary Sentence

The disoriented placement of a decellularized uterine scaffold onto a partially defective uterine area results in the regeneration of uterus with aberrant structures including ectopic location of glands and an abnormal lining of smooth muscle layers in rats.

Angiogenesis is essential for cyclic endometrial growth, implantation, and pregnancy maintenance. Vasculogenesis, the formation of new blood vessels by bone marrow (BM)-derived endothelial progenitor cells (EPCs), has been shown to contribute to endometrial vasculature. However, it is unknown whether vasculogenesis occurs in neovascularization of the decidua during pregnancy. To investigate the contribution of BM-derived EPCs to vascularization of the pregnant uterus, we induced non-gonadotoxic submyeloablation by 5-fluorouracil administration to wild-type FVB/N female mice recipients followed by BM transplantation from transgenic mice expressing green fluorescent protein (GFP) under regulation of Tie2 endothelial-specific promoter. Following 1 month, Tie2-GFP BM-transplantedmice were bred and sacrificed at various gestational days (ED6.5, ED10.5, ED13.5, ED18.5, and postpartum). Bone-marrow-transplanted non-pregnant and saline-injected pregnant mice served as controls (n = 5–6/group). Implantation sites were analyzed by flow cytometry, immunohistochemistry, and immunofluorescence. While no GFP-positive EPCs were found in non-pregnant or early pregnant uteri of BM-transplanted mice, GFP-positive EPCs were first detected in pregnant uterus on ED10.5 (0.12%) and increased as the pregnancy progressed (1.14% on ED13.5), peaking on ED18.5 (1.42%) followed by decrease in the postpartum (0.9%). The percentage of endothelial cells that were BM-derived out of the total endothelial cell population in the implantation sites (GFP+CD31+/CD31+) were 9.3%, 15.8%, and 6.1% on ED13.5, ED18.5, and postpartum, respectively. Immunohistochemistry demonstrated that EPCs incorporated into decidual vasculature, and immunofluorescence showed that GFP-positive EPCs colocalized with CD31 in vascular endothelium of uterine implantation sites, confirming their endothelial lineage. Our findings indicate that BM-derived EPCs contribute to vasculogenesis of the pregnant mouse decidua.

Summary Sentence

Bone marrow-derived endothelial progenitor cells contribute to decidual neovascularization of the pregnant uterus via vasculogenesis.

X-linked α-thalassemia with mental retardation (ATRX) is a chromatin remodeling protein that belongs to the SWItch/sucrose non-fermentable (SWI2/SNF2) family of helicase/ATPases. During meiosis, ATRX is necessary for heterochromatin formation and maintenance of chromosome stability in order to ensure proper assembly of the metaphase II spindle. Previously, we established ATRX as a novel progesterone regulated protein during bovine meiotic maturation, in addition to being dynamically regulated in response to DNA damage in oocytes. In the present study, we utilize the Xenopus laevis model system to further elucidate the signaling pathways regulating ATRX expression within the oocyte. Here, we present an analysis of endogenous ATRX protein expression during oogenesis, oocyte meiotic maturation, and early embryonic development. ATRX expression is dynamically regulated as evidenced by loss of the protein in metaphase II of meiosis. The downstream activation of meiosis via protein kinase A inhibition resulted in a similar decrease in ATRX protein expression. We demonstrate that the ATRX protein is detected in ubiquitin immuno-precipitates from germinal vesicle oocyte extracts and experimentally demonstrate that proteosomal degradation is responsible for the decreased expression of ATRX during meiosis. ATRX expression is significantly increased in response to gamma-irradiation induced DNA damage in oocytes and embryos. This increased expression is independent of p53 protein expression in apoptotic embryos, as determined by the expression of active caspase-3. Thus, regulation of ATRX protein expression impacts on G2–M progression and ultimately has consequences for cell survival.

Summary Sentence

X-linked α-thalassemia withmental retardation (ATRX) protein regulation impacts meiotic progression and oocyte survival.

The physical arrangement of chromatin in the nucleus is cell type and species-specific, a fact particularly evident in sperm, in which most of the cytoplasm has been lost. Analysis of the characteristic falciform (“hook shaped”) sperm in mice is important in studies of sperm development, hybrid sterility, infertility, and toxicology. However, quantification of sperm shape differences typically relies on subjective manual assessment, rendering comparisons within and between samples difficult.

We have developed an analysis program for morphometric analysis of asymmetric nuclei and characterized the sperm of mice from a range of inbred, outbred, and wild-derived mouse strains. We find that laboratory strains have elevated sperm shape variability both within and between samples in comparison to wild-derived inbred strains, and that sperm shape in F1 offspring from a cross between CBA and C57Bl6J strains is subtly affected by the direction of the cross. We further show that hierarchical clustering can discriminate distinct sperm shapes with greater efficiency and reproducibility than even experienced manual assessors, and is useful both to distinguish between samples and also to identify different morphological classes within a single sample. Our approach allows for the analysis of nuclear shape with unprecedented precision and scale and will be widely applicable to different species and different areas of biology.

Summary Sentence

Subtle morphological differences in sperm nuclei can be detected with a new analysis technique; in mice, C57Bl6 and CBA crosses are intermediate to their parental shapes, and the direction of the cross matters.

Spermatozoa from three feline species—the domestic cat (Felis catus), the cheetah (Acinonyx jubatus), and the clouded leopard (Neofelis nebulosa)—were analyzed using metabolomic profiling and ¹³C-based fluxomics to address questions raised regarding their energy metabolism. Metabolic profiles and utilization of ¹³C-labeled energy substrates were detected and quantified using gas chromatography–mass spectrometry (GC-MS). Spermatozoa were collected by electroejaculation and incubated in media supplemented with 1.0 mM [U¹³C]-glucose, [U¹³C]-fructose, or [U¹³C]pyruvate. Evaluation of intracellular metabolites following GC-MS analysis revealed the uptake and utilization of labeled glucose and fructose in sperm, as indicated by the presence of heavy ions in glycolytic products lactate and pyruvate. Despite evidence of substrate utilization, neither glucose nor fructose had an effect on the sperm motility index of ejaculated spermatozoa from any of the three felid species, and limited entry of pyruvate derived from these hexose substrates into mitochondria and the tricarboxylic acid cycle was detected. However, pathway utilization was species-specific for the limited number of individuals (four to seven males per species) assessed in these studies. An inhibitor of fatty acid beta-oxidation (FAO), etomoxir, altered metabolic profiles of all three felid species but decreased motility only in the cheetah. While fluxomic analysis provided direct evidence that glucose and fructose undergo catabolic metabolism, other endogenous substrates such as endogenous lipids may provide energy to fuel motility.

Summary Sentence

Fluxomic analysis demonstrated that glucose, fructose, and pyruvate are metabolized by ejaculated spermatozoa of domestic cats (Felis catus), cheetahs (Acinonyx jubatus), and clouded leopards (Neofelis nebulosa), but pathway utilization is species-specific.

Diabetes is associated with poor oocyte quality and the dysregulation of ovarian function and is thus a leading contributor to the increasing prevalence of female reproductive pathologies. Accordingly, it is well-established that insulin fulfills a key role in the regulation of several facets of female reproduction. What remains less certain is whether proinsulin C-peptide,which has recently been implicated in cellular signaling cascades, holds a functional role in the female germline. In the present study, we examined the expression of insulin, C-peptide, and its purported receptor; GPR146, within the mouse ovary and oocyte. Our data establish the presence of abundant C-peptide within follicular fluid and raise the prospect that this bioactive peptide is internalized by oocytes in a G-protein coupled receptor-dependent manner. Further, our data reveal that internalized C-peptide undergoes pronounced subcellular relocalization from the ooplasm to the pronuclei postfertilization. The application of immunoprecipitation analysis and mass spectrometry identified breast cancer type 2 susceptibility protein (BRCA2), the meiotic resumption/DNA repair protein, as a primary binding partner for C-peptide within the oocyte. Collectively, these findings establish a novel accumulation profile for C-peptide in the female germline and provide the first evidence for an interaction between C-peptide and BRCA2. This interaction is particularly intriguing when considering the propensity for oocytes from diabetic women to experience aberrant meiotic resumption and perturbation of traditional DNA repair processes. This therefore provides a clear imperative for further investigation of the implications of dysregulated C-peptide production in these individuals.

Summary Sentence

Proinsulin C-peptide is internalized by the mouse oocyte and thereafter may play a key role in folliculogenesis and embryogenesis.

Intra-amniotic inflammation is strongly associated with spontaneous preterm labor and birth, the leading cause of perinatal mortality and morbidity worldwide. Previous studies have suggested a role for the NLRP3 (NLR family pyrin domain-containing protein 3) inflammasome in the mechanisms that lead to preterm labor and birth. However, a causal link between the NLRP3 inflammasome and preterm labor/birth induced by intra-amniotic inflammation has not been established. Herein, using an animal model of lipopolysaccharide-induced intra-amniotic inflammation (IAI), we demonstrated that there was priming of the NLRP3 inflammasome (1) at the transcriptional level, indicated by enhanced mRNA expression of inflammasome-related genes (Nlrp3, Casp1, Il1b); and (2) at the protein level, indicated by greater protein concentrations of NLRP3, in both the fetal membranes and decidua basalis prior to preterm birth. Additionally, we showed that there was canonical activation of the NLRP3 inflammasome in the fetal membranes, but not in the decidua basalis, prior to IAI-induced preterm birth as evidenced by increased protein levels of active caspase-1. Protein concentrations of released IL1β were also increased in both the fetal membranes and decidua basalis, as well as in the amniotic fluid, prior to IAI-induced preterm birth. Finally, using the specific NLRP3 inhibitor, MCC950, we showed that in vivo inhibition of the NLRP3 inflammasome reduced IAI-induced preterm birth and neonatal mortality. Collectively, these results provide a causal link between NLRP3 inflammasome activation and spontaneous preterm labor and birth in the context of intra-amniotic inflammation. We also showed that, by targeting the NLRP3 inflammasome, adverse pregnancy and neonatal outcomes can be significantly reduced.

Summary Sentence

Intra-amniotic inflammation induces the activation of the NLRP3 inflammasome in the fetal membranes and decidua basalis prior to preterm birth, which is significantly reduced by inhibiting such a pathway.

Sterile intra-amniotic inflammation is commonly observed in patients with spontaneous preterm labor, a syndrome that commonly precedes preterm birth, the leading cause of perinatal morbidity and mortality worldwide. However, the mechanisms leading to sterile intra-amniotic inflammation are poorly understood and no treatment exists for this clinical condition. Herein, we investigated whether the alarmin S100B could induce sterile intra-amniotic inflammation by activating the NLRP3 inflammasome, and whether the inhibition of this pathway could prevent preterm labor/ birth and adverse neonatal outcomes. We found that the ultrasound-guided intra-amniotic administration of S100B induced a 50% rate of preterm labor/birth and a high rate of neonatal mortality (59.7%) without altering the fetal and placental weights. Using a multiplex cytokine array and immunoblotting, we reported that S100B caused a proinflammatory response in the amniotic cavity and induced the activation of the NLRP3 inflammasome in the fetalmembranes, indicated by the upregulation of the NLRP3 protein and increased release of active caspase-1 and mature IL-1β. Inhibition of the NLRP3 inflammasome via the specific inhibitor MCC950 prevented preterm labor/ birth by 35.7% and reduced neonatalmortality by 26.7%. Yet, inhibition of the NLRP3 inflammasome at term did not drastically obstruct the physiological process of parturition. In conclusion, the data presented herein indicate that the alarmin S100B can induce sterile intra-amniotic inflammation, preterm labor/birth, and adverse neonatal outcomes by activating the NLRP3 inflammasome, which can be prevented by inhibiting such a pathway. These findings provide evidence that sterile intra-amniotic inflammation could be treated by targeting the NLRP3 inflammasome.

Summary Sentence

Intra-amniotic administration of the alarmin S100B, at clinically relevant concentrations, induces preterm labor/birth and adverse neonatal outcomes by activating the NLRP3 inflammasome, which can be prevented by targeting this pathway

Estradiol-17β (E₂) and 11-ketotestosterone (11KT) have been implicated in vitellogenesis and in regulating expression of the follicle-stimulating hormone receptor (fshr), respectively. To override the captivity-induced reproductive block in shortfinned eel, Anguilla australis, we hypothesized that in combination, 11KT and E₂ would stimulate ovarian uptake of vitellogenin (Vtg). Early pubertal eels received hormone implants containing varying concentrations of E₂ (0, 0.2, 2, 5 mg) with or without 11KT (1 mg). Vtg levels were determined in plasma, liver, and ovarian tissues by histological examination, qPCR, immunoblotting, or single radial immunodiffusion. The expression of gonadotropin-beta subunits and gonadotropin receptors in the pituitary and ovary, respectively, were analyzed to determine mechanisms by which steroid effects may be exerted. When administered alone, E₂ increased hepatic production and plasma levels of Vtg. In contrast, 11KT decreased plasma levels of Vtg, seemingly reducing its production. Neither 11KT nor E₂ could induce uptake of Vtg into oocytes, although E₂ treatment appeared necessary for uptake to occur. This was the case despite 11KT dramatically increasing both oocyte size and fshr mRNA levels. Astonishingly, the uptake of Vtg was successfully induced by co-treatment with 11KT and E₂, suggesting that 11KT might facilitate the incorporation of Vtg into the developing oocyte. These results highlight the potential of sex steroid co-treatment, an approach aimed at mimicking oogenesis in wild eels, to induce vitellogenesis, specifically ovarian yolk deposition, even in the absence of exogenous gonadotropin treatment.

Summary Sentence

Combined treatment with estradiol-17β and 11-ketotestosterone results in evident yolk accumulation in the shortfinned eel ovary, showing the potential of steroid co-treatment to induce vitellogenesis, even in the absence of exogenous gonadotropins.

We previously showed that rat, pig, sheep, and red deer oocytes express species-specific ratios of GDF9: BMP15 mRNA (3.7, 0.5, 1.26, and 0.1, respectively), and with the exception of the pig, they are directly correlated to litter size. The purpose of this study was to determine the alternative mechanism that enables pig oocytes to secrete low ratios whilst maintaining a large litter size. Herein, we performed same- and cross-species coincubations of oocytes with granulosa cells (GCs) of rat, pig, sheep, and red deer to compare the proliferation rate, mRNA expression levels of growth factor receptors, and downstream signalling pathways in GCs. A decreased proliferation rate, lower Bmpr1b and Bmpr2 mRNA expression levels, and higher SMAD1/5/8 protein levels were exhibited in rat GCs cocultured with red deer oocytes, compared to all other species. Pig GCs unequivocally expressed GDF9 mRNA, suggesting that, similar to rat GCs, the proliferation of pig GCs is regulated mainly by GDF9, despite lower intraoocyte expression of GDF9 mRNA. In support, a higher basal proliferation, and their ability to proliferate readily when coincubated with red deer oocytes, was observed in pig GCs. In contrast, red deer GC proliferation is likely to be mainly regulated by BMP15 in vivo with only red deer oocytes capable of altering SMAD1/5/8 and pSMAD2/3 levels, while both GDF9 and BMP15 appear important for sheep GC proliferation. In summary, this study strengthens our hypothesis that the ratio of GDF9: BMP15 in the intrafollicular milieu is directly correlated with litter size, and that the GCs of each species have evolved to respond to these unique ratios.

Summary Sentence

Species-specific responsiveness to and ratios of GDF9: BMP15 determines the litter size in mammals.

The Wilms tumor (WT) gene WT1 encodes the splicing variants WT1(+KTS) and WT1(-KTS). Recent data suggest that WT1 plays an important role in the development of mice follicles. However, the mechanism through which WT1 influences ovarian steroidogenesis remains unknown. This study identified WT1 and evaluated the impact of splicing variants WT1(+KTS) and WT1(-KTS) on steroidogenesis using adult bovine granulosa cells (GCs). Using RT-qPCR and western blotting, we found that the ratio between WT1(+KTS) and WT1(-KTS) was stabilized. WT1 expression, however, decreased gradually in bovine GCs in response to follicle enlargement or atresia. The downregulation of WT1 increased the secretion of basal and follicle-stimulating-hormone-induced progesterone (P4), but decreased the secretion of basal-induced estradiol (E2). This was associated with an increase in the expression of 3β-HSD, and a decrease in the expression of CYP19A1. In addition, WT1(-KTS) overexpression suppresses the secretion of E2 and P4 compared with WT1(+KTS) overexpression. This was associated with a decrease in the expression of CYP19A1, CYP11A1, and 3β-HSD in cultured bovine GCs. Of note, the downregulation of WT1 suppresses the phosphorylation levels of AKT and p-ERK1/2. However, WT1(-KTS) overexpression promotes the phosphorylation levels of AKT and suppresses p-ERK1/2 levels. LY294002 (AKT inhibitor) increases MKP3 mRNA expression levels but decreases the level of p-AKT and p-ERK1/2. Collectively, WT1 significantly suppresses the mRNA expression of CYP11A1 and 3β-HSD and the secretion of P4 in bovine GCs. Moreover, it regulates CYP19A1 mRNA expression and E2 secretion with complex networks, at least in part, by modulating AKT and ERK1/2 signaling. The effect of WT1(-KTS) was more pronounced than that exerted by WT1(+KTS).

Summary Sentence

This study showed the effect of WT1(+/-KTS) on the steroidogenesis of GCs, and elucidated the role of the PI3K/AKT and ERK1/2 pathways in WT1-regulated steroidogenesis.

We previously developed a model of gestational diabetes mellitus (GDM) in which dams exhibit glucose intolerance, insulin resistance, and reduced insulin response to glucose challenge only during pregnancy, without accompanying obesity. Here, we aimed to determine how lean gestational glucose intolerance affects offspring risk of metabolic dysfunction. One cohort of offspring was sacrificed at 19 weeks, and one at 31 weeks, with half of the second cohort placed on a high-fat, high-sucrose diet (HFHS) at 23 weeks. Exposure to maternal glucose intolerance increased weights of HFHS-fed offspring. Chow-fed offspring of GDM dams exhibited higher body fat percentages at 4, 12, and 20 weeks of age. At 28 weeks, offspring of GDM dams fed the HFHS but not the chow diet (CD) also had higher body fat percentages than offspring of controls (CON). Exposure to GDM increased the respiratory quotient (Vol CO₂/Vol O₂) in offspring. Maternal GDM increased adipose mRNA levels of peroxisome proliferator-activated receptor gamma (Pparg) and adiponectin (Adipoq) in 31-week-old CD-fed male offspring, and increased mRNA levels of insulin receptor (Insr) and lipoprotein lipase (Lpl) in 31-week-old male offspring on both diets. In liver at 31 weeks, mRNA levels of peroxisome proliferator-activated receptor alpha (Ppara) were elevated in CD-fed male offspring of GDM dams, and male offspring of GDM dams exhibited higher mRNA levels of Insr on both diets. Neither fasting insulin nor glucose tolerance was affected by exposure to GDM. Our findings show that GDM comprising glucose intolerance only during pregnancy programs increased adiposity in offspring, and suggests increased insulin sensitivity of subcutaneous adipose tissue.

Summary Sentence

Glucose intolerance in a lean model of GDM results in offspring with increased lipid storage mediated by insulin sensitive adipose tissue and decreased lipid energy utilization.

The change from the state of pregnancy to the state of parturition, which we call uterine transitioning, requires the actions of inflammatory mediators and results in an activated uterus capable of performing the physiology of labor. Interleukin (IL)-1β and prostaglandin (PG)F2α are two key mediators implicated in preparing the uterus for labor by regulating the expression of uterine activation proteins (UAPs) and proinflammatory cytokines and chemokines. To investigate this process, primary human myometrial smooth muscle cells (HMSMC) isolated from the lower segment of women undergoing elective cesarean sections at term (not in labor) were used to test the inflammatory cytokine and UAP outputs induced by PGF2α and IL-1β alone or in sequential combinations. PGF2α and IL-1β regulate mRNA abundance of the PGF2α receptor FP, the IL-1 receptor system, interleukin 6, and other UAPs (OXTR, COX2), driving positive feedback interactions to further amplify their own proinflammatory effects. Sequential stimulation of HMSMC by PGF2α and IL-1β in either order results in amplified upregulation of IL-6 and COX-2 mRNA and protein, compared to their effects individually. These profound increases were unique to myometrium and not observed with stimulation of human fetal membrane explants. These results suggest that PGF2α and IL-1β act cooperatively upstream in the birth cascade to maximize amplification of IL-6 and COX-2, to build inflammatory load and thereby promote uterine transition. Targeting PGF2α or IL-1β, their actions, or intermediates (e.g. IL-6) would be an effective therapeutic intervention for preterm birth prevention or delay.

Summary Sentence

PGF2α and IL-1β act cooperatively upstream in the birth cascade to maximize amplification of IL-6 and COX-2, contributing to increased inflammatory burden and promoting uterine transition.

To test the hypothesis that macrophages are essential for remodeling the cervix in preparation for birth, pregnant homozygous CD11b-dtr mice were injected with diphtheria toxin (DT) on days 14 and 16 postbreeding. On day 15 postbreeding, macrophages (F4/80+) were depleted in cervix and kidney, but not in liver, ovary, or other non-reproductive tissues in DT—compared to saline—treated dtr mice or wild-type controls given DT or saline. Within 24 h of DT-treatment, the density of cell nuclei and macrophages declined in cervix stroma in dtr mice versus controls, but birefringence of collagen, as an indication of extracellular cross-linked structure, remained unchanged. Only in the cervix of DT-treated dtr mice was an apoptotic morphology evident in macrophages. DT-treatment did not alter the sparse presence or morphology of neutrophils. By day 18 postbreeding, macrophages repopulated the cervix in DT-treated dtr mice so that the numbers were comparable to that in controls. However, at term, evidence of fetal mortality without cervix ripening occurred in most dtr mice given DT—a possible consequence of treatment effects on placental function. These findings suggest that CD11b⁺ F4/80⁺ macrophages are important to sustain pregnancy and are required for processes that remodel the cervix in preparation for parturition.

Summary Sentence

Conditional depletion of macrophages in CD11b-dtr mice during the critical period for cervix remodeling interfered with ripening and the progress of pregnancy.

Impaired decidualization has been considered a major cause of infertility in adenomyosis. However, the mechanism remains poorly understood. Recent studies suggest that microRNAs (miRNA) play a crucial role in embryo implantation. The aim of the present study was to identify the role of miR-21 in human endometrial stromal cell (hESC) decidualization in vitro. To explore the roles of miR-21 in decidualization, we detected the expression of miR-21 in the endometrium of fertile control and adenomyosis patients, and analyzed the effects of miR-21 on the biological behaviors of hESC decidualization. The results demonstrated that miR-21 was downregulated in the endometrium of adenomyosis patients compared with the control endometrium. miR-21 effectively promoted the expression of the 8Br-cAMP plus medroxyprogesterone acetate (MPA)-induced hESC decidualization marker genes PRL and IGFBP-1 and morphological transformation through the modulation of KLF12 and NR4A1 expression; conversely, inhibition of miR-21 expression compromised hESC decidualization in vitro. In addition, Luciferase reporter, western blotting, and quantitative real-time PCR (qRT-PCR) assays confirmed that miR-21 interacted with the 3′ untranslated region of the transcription factor KLF12 and downregulated KLF12 at the transcriptional and translational levels. KLF12 overexpression abolished miR-21-enhanced 8Br-cAMP plus MPA-induced decidualization. Taken together, these results illustrate that miR-21 promotes endometrial decidualization by inhibiting KLF12, and miR-21 overexpression reverses the poor decidual response of hESCs in patients with adenomyosis in vitro.

Summary Sentence

miR-21, which is decreased in the endometrium of adenomyosis, reverses impaired decidualization through modulation of KLF12 and NR4A1 expression.

In mammals, circadian clock regulates concentration of many reproductive hormones including testosterone. Previously, we characterized pattern of circadian transcription of core clock genes in testosterone-producing Leydig cells. Here, the potential role of luteinizing hormone receptor (LHR)-cAMP signaling in synchronization of Leydig cell's circadian clock and rhythmic testosterone production were examined. Results showed that activation of LHR-cAMP signaling in primary rat Leydig cell culture increased Star/STAR and changed expression of many clock genes (upregulated Per1/PER1, Dec1/2, and Rorb, and downregulated Bmal1 and Rev-erba/b). Inhibition of protein kinase A prevented LHR-triggered increase in transcription of Per1 and Dec1. Effect of stimulated LHR-cAMP signaling on Leydig cell's clock transcription was also confirmed in vivo, using rats treated with single hCG injection. To analyze in vivo effect of low LH-cAMP activity on rhythmical Leydig cell function, rats with experimental hypogonadotropic hypogonadism were used. Characteristics of hypogonadal rats were decreased LH and testosterone secretion without circadian fluctuation; in Leydig cells decreased arrhythmic cAMP and transcription of steroidogenic genes (Cyp11a1 and Cyp17a1) were observed, while decreased Star/STAR expression retains circadian pattern. However, expression of clock genes, despite changes in transcription levels (increased Bmal1, Per2, Cry1, Cry2, Rora, Rorb, Rev-erba/b/REV-ERBB, Dec1, Csnk1e, and decreased Npas2 and PER1) kept circadian patterns observed in control groups. Altogether, the results strengthened the hypothesis about role of LH-cAMP signaling as synchronizer of Leydig cell's clock. However, clock in Leydig cells is not sufficient to sustain rhythmicity of testosterone production in absence of rhythmic activity of LH-cAMP signaling.

Summary Sentence

LH-cAMP signaling is involved in synchronization of Leydig cell's circadian clock and rhythmic testosterone production