Biology of Reproduction最新文献

For a brief but critical period post-fertilization, the mammalian embryo is entirely dependent on maternal products inherited from the oocyte. Previous research showed that oocyte-specific loss of Med12, an X-linked gene and Mediator complex subunit, leads to female sterility despite normal folliculogenesis and ovulation. Here, we show that loss of maternal Med12 has minimal effect on the oocyte transcriptome and does not manifest in embryonic lethality until post-implantation. Implants derived from Med12-null oocytes demonstrate abnormal placentation at E9.5, with an overabundance of trophoblast giant cells (TGC). This phenotype associates with early disruption of lineage markers at the blastocyst stage (e.g. Pou5f1 and Gata3), and later by downregulation of trophoblast pluripotency markers (e.g. Cdx2) and activation of drivers of TGC identity (e.g. Stra13) in the E7.5 extraembryonic ectoderm, revealing a previously undescribed role for Med12 in trophoblast pluripotency maintenance. Notably, we find consistently low Med12 expression in embryos derived from Med12-null oocytes, likely due to programmed paternal X chromosome inactivation (XCI). To isolate the consequences of maternal Med12 depletion, we introduced an autosomal Med12 transgene and show that embryonic expression of the transgene rescues development of Med12-null oocytes. We conclude that oocyte-specific deletion of Med12 produces a maternal-zygotic double knock-out in extraembryonic tissues due to paternal XCI, leading to loss of pluripotency in the trophoblast, placental malformation, and embryonic death.

The establishment of pregnancy in ruminants is dependent on the production of interferon-tau (IFNT) by the conceptus which stimulates the production of interferon stimulated gene 15 (ISG15) in maternal tissues. ISG15 covalently binds to (ISGylation) and regulates intracellular proteins processes in a manner like ubiquitin. The aim of this study was to identify ISGylated proteins and infer their specific functions and biochemical pathways within endometrium. Endometrium was collected from pregnant ewes on day 17 (P17, n=4), synchronized non-inseminated ewes on Day 10 (EC10, n=4) and Day 17 (EC17, n=4) after estrus (Day 0). In addition, bovine endometrial (BEND) cells were cultured with 10 ng/ml of recombinant bovine (rb)IFNT for 24 h. ISGylated proteins in protein extracts were immunoprecipitated with anti-bovine ISG15 antibody. The ISGylated proteins in eluates were identified using Liquid Chromatography-Tandem Mass Spectrometry and confirmed by reciprocal immunoprecipitation of selected ISGylated proteins. Seventy-five ISGylated proteins were differentially expressed in the endometrium of EC10, EC17 and P17 ewes. Ingenuity pathway analysis indicated that interferon α/β signaling, ISG15 antiviral mechanism, Glycolysis I, and Gluconeogenesis I pathways were the most highly significant pathways induced in the pregnant endometrium. Enrichment of the ISGylated proteins STAT1, MX1, and ENO1 was achieved by immunoprecipitation with specific antibodies and their conjugation to ISG15 was confirmed. The ISGylated endometrial proteins identified in pregnant ovine endometrium may function in IFN signaling, antiviral mechanisms and glucose metabolism with implications for these pathways in the preparation of endometrium for successful implantation and maintenance of pregnancy.

Senescent cells have been implicated in the pathogenesis of metabolic dysfunction-associated liver disease (MASLD), which can negatively affect female fertility. Senolytic drugs are reported to eliminate senescent cells in various tissues, including the ovary. However, the efficacy of senolytic drugs in reducing liver damage and preserving fertility in female mice with MASLD remains unclear. Therefore, this study aimed to evaluate the protective effect of senolytic drugs on liver damage and fertility in reproductive aged female mice with MASLD. Three-month-old female mice were fed a standard chow (SD) or Western diet (WD) to induce MASLD until nine months of age. Starting at six months of age, mice were also randomized to receive senolytic treatment (Dasatinib + Quercetin, D + Q) or vehicle within each diet. We observed that mice fed the WD exhibited liver damage characteristic of MASLD, with increased liver size, triglyceride accumulation and fibrosis. These mice also exhibited increased liver senescence and inflammation. Senolytic treatment slightly reduced liver mass and modulated some liver senescence and inflammation related genes, suggesting limited efficacy in controlling WD-induced liver damage. Pregnancy rates were reduced in mice with MASLD and improved by senolytic treatment. Mice with MASLD had increased ovarian senescence, inflammation, and fibrosis, which was attenuated by senolytic treatment, despite having no effect on the ovarian follicle reserve. We conclude that senolytic treatment has potential for improving reproductive function in aged female mice with MASLD, despite limited impact in liver and systemic indicators.

Mammalian spermatogonial stem cells (SSCs) sustain male fertility through continuous self-renewal and differentiation, leading to the production of haploid spermatozoa throughout adulthood. However, SSCs are vulnerable to genotoxic drugs, and patients receiving chemotherapy face a high risk of germline instability and infertility. The molecular mechanisms and cellular pathways that choreograph SSC recovery after chemotherapeutic insult remain unknown. Previously, we identified SPRY4 as an ERK-dependent negative feedback regulator of growth factor signaling that is critical for preservation of stem cell activity in cultured mouse SSCs. Here, we demonstrate that following alkylating agent busulfan (BU)-induced injury in adult mice, germline-specific Spry4 gene deletion (Spry4G-KO) reduces stem cell regeneration with an enhanced genotoxic stress response and differentiation with rapidly enhanced nuclear ERK1/2 activity in undifferentiated (Aundiff) spermatogonia (including SSCs). Genes essential for stem cell maintenance, including Id1 and Cxcl12, were dysregulated by loss of Spry4. Furthermore, the MEK1/2 inhibitor PD0325901, but not mTORC1 inhibitor rapamycin, was sufficient to promote spermatogonial proliferation in Spry4G-KO testis 10 days post-BU treatment. Notably, the restoration of both spermatogonia pool and fertility was delayed in adult Spry4G-KO males long-term after injury. In summary, germline-specific deletion of Spry4 results in hyper-activation of the MAPK/ERK pathway in Aundiff spermatogonia, reducing spermatogonial genome integrity, unleashing excessive spermatogenesis after germline damage, and ultimately impairing germline regeneration in adult males. Our study indicates an essential role for SPRY4-ERK signaling as a molecular checkpoint in securing SSC recovery upon chemotherapy drug-induced germline damage, revealing how stem cells normally withstand environmental stress.

Non-esterified fatty acids (NEFAs) influence ovarian cell function, but the underlying molecular mechanisms remain incompletely understood. Our previous work showed that NEFA induces lipid accumulation and inhibits glucose uptake in bovine granulosa cells. Here, we investigated the effect of NEFA on energy metabolism and the transcriptome, with a focus on pyruvate dehydrogenase kinase 4 (PDK4). NEFA treatment significantly increased ATP levels and mitochondrial membrane potential. Transcriptome analysis revealed differential expression of 176 genes, with downregulation of steroidogenesis and fatty acid synthesis pathways, and upregulation of apoptosis, fatty acid oxidation, and innate immune responses in NEFA treated cells. Specifically, genes involved in fatty acid oxidation (e.g. CPT1A, CPT1B, HADHA, and SLC25A20) were upregulated, whereas those related to glucose metabolism remained largely unchanged except for the marked upregulation of PDK4. Silencing PDK4 expression induced glucose utilization by upregulating glucose transporters (SLC2A1, SLC2A10) and glycolytic enzymes (GAPDH, ENO1, and LDHA). In contrast, genes associated with fatty acid oxidation (SLC27A1 and CPT1B) showed downregulation upon PDK4 silencing compared with controls, suggesting a metabolic shift favoring glucose oxidation even in the presence of NEFA. We observed that expression of PDK4, CPT1A, and CPT1B was significantly upregulated in large luteal cells compared to granulosa cells. PDK4 knockdown significantly downregulated steroidogenic genes (STAR, HSD3B1, and CYP11A1) and decreased progesterone production, suggesting that increased expression of PDK4 in luteal cells may supports steroidogenesis. Together, these findings identify PDK4 as a critical regulator of metabolic flexibility and progesterone production in granulosa cells.

ADAM5 is a testis-specific transmembrane protein whose role in male fertility remains poorly understood. In this study, we report that Adam5 knockout (KO) male mice are severely subfertile, despite exhibiting normal testicular morphology, sperm structure, and motility. Adam5 KO sperm failed to transit the uterotubal junction (UTJ) and displayed severe defects in zona pellucida (ZP) binding, phenotypes that resemble those observed in Adam2 and Adam3 knockout mice. Western blot analysis revealed a significant reduction in the levels of ADAM2 and ADAM3 in Adam5 KO spermatozoa, supporting the previous finding that ADAM5 interacts with these proteins to form a complex. Additionally, Adam5 KO spermatozoa exhibited reduced binding to extracellular matrix (ECM) components, including Laminin I and Fibronectin. These findings suggest that ADAM5 plays a crucial role in sperm ECM interaction, a process likely critical for successful UTJ transit and ZP binding. While ADAM5 is a pseudogene in humans, our results provide valuable insights into the function of ADAM family proteins in mammalian reproduction.

Embryonic implantation is a crucial developmental phase characterized by intricate molecular crosstalk between the embryo and endometrium, with emerging evidence implicating circular RNAs (circRNAs) as important regulators. This study elucidated the role of hsa_circ_0001550 in impairing embryo adhesion and evaluated recombinant human granulocyte colony-stimulating factor (rhG-CSF) as a therapeutic strategy. Overexpression of hsa_circ_0001550 in an in vitro adhesion model using Ishikawa cells reduced the adhesion capacity of BeWo or JAR spheroids and diminished the murine blastocyst implantation rate. Knocking down hsa_circ_0001550 increased the adhesive ability of BeWo spheroids. Crucially, administration of rhG-CSF downregulated hsa_circ_0001550 expression and upregulated homeobox A10 and leukemia inhibitory factor, thereby restoring the adhesive capacity. In an endometrial injury model, administration of rhG-CSF enhanced epithelial proliferation (as measured by Ki67), suppressed apoptosis (based on the TUNEL assay), and activated mesenchymal-to-epithelial transition pathways, ultimately improving the embryo implantation rate. Collectively, this study reveals that rhG-CSF improves implantation partly by downregulating hsa_circ_0001550. These findings provide new perspectives for understanding implantation mechanisms and developing therapeutic strategies.

Amniotic fluid (AF) profiling provides a minimally invasive window into early fetal physiology. We characterized the AF metabolome from first trimester (Day 68) Holstein dairy heifers (n = 45), considering fetal sex, conception method [in vitro fertilization vs. artificial insemination (AI)], and eventual pregnancy outcome as key variables. Multivariate statistics uncovered differentially abundant metabolites for each comparison-including markers that preceded spontaneous abortion-independently of recipient age, weight, gestation length, or fetal genetics. Thereafter, a machine learning algorithm using panels of six metabolites accurately predicted fetal sex (AUROC = 0.76; P = 0.023) and pregnancy viability (AUROC = 0.81; P = 0.018), while corroborating conception method (AUROC = 0.91; P = 0.001). External validation using AF (Day 42) from an independent cohort of beef heifers (n = 22) reproduced the fetal sex classifier with similarly high sensitivity and specificity (AUROC = 0.85, P = 0.029). These findings reveal metabolic signatures that forecast fetal sex and pregnancy viability, while confirming distinct metabolic imprints of assisted-conception modalities. These data lay the groundwork for next-generation AF prenatal diagnostics in veterinary and human obstetrics.

Many studies have examined the acute effects of chemotherapies and radiation on the ovary; however, few address the impact of long-term chemotherapeutic agents on future reproduction for cancer survivors. The purpose of our study was to determine the effect of long-term tamoxifen (TAM) treatment on follicle development, ovarian reserve, and embryogenesis in a murine model. Adult female mice were treated with TAM (250 mg/kg fed ad libitum in mouse chow) or control chow for three weeks, followed by 0-, 1-, or 3-weeks of TAM washout. Mice were then superovulated, mated, and their oviducts flushed to determine ovulation, fertilization, and in vitro embryo development rates. At specific timepoints, one ovary from each animal was harvested, sectioned, and stained with H&E, while the other ovary was evaluated for gene expression of targeted genes. Ovarian size was calculated based on average surface area over six slices. Follicles (primordial, primary, secondary, antral / pre-ovulatory), and corpora lutea (CL) were counted and averaged for each slice. All data were analyzed using mixed-effects analysis of variance (ANOVA), with treatment group as the fixed effect and ovary treated as a random effect nested within treatment. Ovaries of TAM treated mice were 36% smaller on average than control ovaries (P = 0.01). Primordial follicle counts were 35% lower than control ovaries (P = 0.007) and there were 3.4x more atretic follicles in the ovaries of TAM treated mice (P < 0.001). There were no significant differences in the number of primary, secondary, antral or pre-ovulatory follicles. The number of oocytes and embryos flushed from control mice (total ovulated) was 1.65x greater than TAM treated mice. However, fertilization rates and in vitro embryo blastulation rates were not significantly different between controls and TAM groups. In summary, for this murine model, long-term TAM negatively impacted ovarian reserve (primordial follicle number) without overtly affecting the in vitro embryo development to the blastocyst.