Biology of Reproduction最新文献

In vitro culture of ungrown oocytes in preantral follicles is one of the intriguing subjects being pursued to produce viable eggs in assisted reproductive technology. Previous studies have succeeded in obtaining mature eggs after in vitro culture of preantral follicles, while denuded undeveloped oocytes, which are obtained occasionally when collecting preantral follicles, seem to be almost useless. Moreover, methods to culture them efficiently to produce viable eggs have not been established yet. The present study was conducted to demonstrate in vitro culture of mouse denuded undeveloped oocytes by reconstructing granulosa cell-oocyte complexes, and to analyze cellular communication in reconstructed granulosa cell-oocyte complexes. Single denuded undeveloped oocytes were aggregated with 1 × 104 granulosa cells in wells with U-shaped bottoms in a low-binding cell culture plate for 8 days under either 20% or 5% O2, and then the reconstructed granulosa cell-oocyte complexes formed were cultured on a collagen-coated culture membrane insert for 4 days under 5% O2. At day 8 of culture, the rates of reconstructed granulosa cell-oocyte complexes formation were significantly higher in the culture group under 5% O2 (64.9%) than that under 20% O2 (42.3%; P < 0.001); furthermore, the formation of transzonal projections was observed. After maturation and fertilization, we produced matured eggs and blastocysts at higher rates (>90% and 61.9%, respectively) in the group cultured under 5% O2. After transferring 126 two- to four-cell stage embryos, six live pups were obtained. This is the first report that demonstrates production of viable eggs after in vitro culture of denuded undeveloped oocytes from preantral follicles by reconstruction of granulosa cell-oocyte complexes.

Mammalian preimplantation development culminates in the formation of a blastocyst that undergoes extensive gene expression regulation to successfully implant into the maternal endometrium. Zinc-finger HIT domain-containing (ZNHIT) 1 and 2 are members of a highly conserved family, yet they have been identified as subunits of distinct complexes. Here, we report that knockout of either Znhit1 or Znhit2 results in embryonic lethality during peri-implantation stages. Znhit1 and Znhit2 mutant embryos have overlapping phenotypes, including reduced proportion of SOX2-positive inner cell mass cells, a lack of Fgf4 expression, and aberrant expression of NANOG and SOX17. Furthermore, we find that the similar phenotypes are caused by distinct mechanisms. Specifically, embryos lacking ZNHIT1 likely fail to incorporate sufficient H2A.Z at the promoter region of Fgf4 and other genes involved in cell projection organization resulting in impaired invasion of trophoblast cells during implantation. In contrast, Znhit2 mutant embryos display a complete lack of nuclear EFTUD2, a key component of U5 spliceosome, indicating a global splicing deficiency. Our findings unveil the indispensable yet distinct roles of ZNHIT1 and ZNHIT2 in early mammalian embryonic development.

There are approximately 20 000 protein-coding genes in humans and mice. More than 1000 of these genes are predominantly expressed in the testis or are testis-specific and thought to play an important role in male reproduction. Through the production of gene knockout mouse models and phenotypic evaluations, many genes essential for spermatogenesis, sperm maturation, and fertilization have been discovered, greatly contributing to the elucidation of their molecular mechanisms. On the other hand, there are many cases in which single-gene knockout models do not affect fertility, indicating that tissue-specific genes are not always critical. Here, we selected 18 genes whose mRNA expression is restricted to the testis or higher than in other tissues, but whose function in male reproduction is unknown. We then created single-gene KO mouse models using the CRISPR/Cas9 system. The established KO males were subjected to mating tests and screened for effects on fecundity, revealing that these genes were not essential for spermatogenesis and male fertility. This knowledge will contribute to understanding the functions of genes characteristic of the testis and identify the cause of male infertility.

Mechanisms controlling trophoblast (TB) proliferation and differentiation during embryo implantation are poorly understood. Human trophoblast stem cells (TSC) and BMP4/A83-01/PD173074-treated pluripotent stem cell-derived trophoblast cells (BAP) are two widely employed, contemporary models to study TB development and function, but how faithfully they mimic early TB cells has not been fully examined. We evaluated the transcriptomes of TB cells from BAP and TSC and directly compared them with those from peri-implantation human embryos during extended embryo culture (EEC) between embryonic days 8 to 12. The BAP and TSC grouped closely with TB cells from EEC within each TB sublineage following dimensional analysis and unsupervised hierarchical clustering. However, subtle differences in transcriptional programs existed within each TB sublineage. We also validated the presence of six genes in peri-implantation human embryos by immunolocalization. Our analysis reveals that both BAP and TSC models have features of peri-implantation TB s, while maintaining minor transcriptomic differences, and thus serve as valuable tools for studying implantation in lieu of human embryos.

Sperm maturation depends on exposure to microenvironments within the different segments of the epididymis, but mechanisms underlying how these microenvironments are produced or maintained are not well understood. We hypothesized that epididymal extracellular vesicles could play a role in the process of maintaining microenvironments in different regions of the epididymis. Specifically, we tested whether the extracellular vesicles from different regions of the epididymis can ensure paracrine communication between cells in different segments. Domestic cat tissues were used to develop a reproducible in vitro culture system for corpus epididymis explants that were then exposed to extracellular vesicles collected from upstream (i.e., caput) segments. Impacts of different culture or exposure conditions were compared by analyzing the morphology, apoptosis, transcriptional activity, and gene expression in the explants. Here, we report the development of the first in vitro culture system for epididymal tissue explants in the domestic cat model. Using this system, we found that extracellular vesicles from the caput segment have a significant effect on the transcriptional profile of tissue from the corpus segment (1233 differentially expressed genes due to extracellular vesicle supplementation). Of note, expressions of genes associated with regulation of epithelial cell differentiation and cytokine signaling in the epididymis were influenced by the presence of extracellular vesicles. Together, our findings comprise the first report in any species of paracrine control of segmental gene regulation by epididymal extracellular vesicles. These results contribute to a better understanding of epididymis biology and could lead to strategies to enhance or suppress male fertility.

Prior studies showed that mice deficient in the modifier subunit of glutamate cysteine ligase (Gclm), the rate-limiting enzyme in synthesis of the thiol antioxidant glutathione, have decreased ovarian glutathione concentrations, chronic ovarian oxidative stress, poor oocyte quality resulting in early preimplantation embryonic mortality and decreased litter size, and accelerated age-related decline in ovarian follicle numbers. Global deficiency of the catalytic subunit of this enzyme, Gclc, is embryonic lethal. We tested the hypothesis that granulosa cell- or oocyte-specific deletion of Gclc recapitulates the female reproductive phenotype of global Gclm deficiency. We deleted Gclc in granulosa cells or oocytes of growing follicles using Gclc floxed transgenic mice paired with Amhr2-Cre or Zp3-Cre alleles, respectively. We discovered that granulosa cell-specific deletion of Gclc in Amhr2Cre;Gclc(f/-) mice recapitulates the decreased litter size observed in Gclm-/- mice but does not recapitulate the accelerated age-related decline in ovarian follicles observed in Gclm-/- mice. In addition to having lower glutathione concentrations in granulosa cells, Amhr2Cre;Gclc(f/-) mice also had decreased glutathione concentrations in oocytes. By contrast, oocyte-specific deletion of Gclc in Zp3Cre;Gclc(f/-) mice did not affect litter size or accelerate the age-related decline in follicle numbers, and these mice did not have decreased oocyte glutathione concentrations, consistent with transport of glutathione between cells via gap junctions. The results suggest that glutathione deficiency at earlier stages of follicle development may be required to generate the accelerated follicle depletion phenotype observed in global Gclm null mice.

Anti-Müllerian hormone (AMH) is widely used in the clinic as a biomarker for ovarian reserve and to predict ovarian response to gonadotropin stimulation. Patients with higher AMH levels tend to yield more oocytes and have better outcomes from assisted reproductive technology (ART) procedures. The goal of this study is to determine if AMH can be used to predict the outcome of controlled ovarian stimulation in rhesus macaques, which are commonly used in biomedical research, to refine animal use while maximizing oocyte yield. We hypothesized that pre-stimulation AMH values can be used to predict oocyte yield and quality. Regularly cycling adult macaques underwent controlled ovarian stimulation and baseline (pre-stimulation) plasma AMH levels were determined using an AMH-specific enzyme-linked immunoassay. Oocytes were collected by laparoscopic or ultrasound-guided aspiration, then counted and evaluated for quality and stage of meiosis. Sperm from established fertile males were used to inseminate the oocytes in vitro with fertilization success checked 14 - 16 hours later. Females were grouped by oocyte yield: low ≤ 17; mid = 18 - 41; high ≥ 42. We found that high and mid yielders had significantly higher AMH than low yielders (p<0.0001) and the percent of mature oocytes was greater in the high and mid yielders. There were no significant differences in oocyte quality or ova fertilization rate. These data suggest that AMH is a useful measure for controlled ovarian stimulation success in rhesus macaques and can be used to identify suitable animals for oocyte donation before entering them into a stimulation protocol.

Bisphenols are a family of chemicals used in the manufacture of consumer products containing polycarbonate plastics and epoxy resins. Studies have shown that exposure to bisphenol A (BPA) may disrupt steroidogenesis and induce adverse effects on male and female reproduction, but little is known about BPA replacements. We determined the effects of six bisphenols on the steroidogenic function of MA-10 Leydig cells and KGN granulosa cells by measuring the levels of progesterone and estradiol produced by these cells as well as the expression of transcripts involved in steroid and cholesterol biosynthesis. MA-10 and KGN cells were exposed for 48 hours to one of six bisphenols (0.01-50 μM): BPA, bisphenol F, bisphenol S, bisphenol AF, bisphenol M, or bisphenol TMC under both basal and dibutyryl cAMP (Bu2cAMP)-stimulated conditions. In MA-10 cells, most bisphenols increased the Bu2cAMP-stimulated production of progesterone. In KGN cells, there was a general decrease in progesterone production, while estradiol levels were increased following exposure to many bisphenols. qRT-PCR analyses revealed that all six bisphenols (≥1 μM) upregulated the expression of STAR, a cholesterol transporter, in both cell lines after stimulation. Key transcripts directly involved in steroid and cholesterol biosynthesis were significantly altered in a cell line, chemical, and concentration-dependent manner. The expression of upstream genes was also differentially affected. Thus, BPA and five of its analogs can disrupt steroid production in two steroidogenic cell lines and alter the levels of transcripts involved in this process. Importantly, BPA replacements do not appear to have fewer effects than BPA.

The female reproductive system ages before any other physiological system, making it a sensitive indicator of aging. Early reproductive aging is associated with the early onset of infertility and an increased risk of several diseases. During aging, systemic and reproductive oxidative stress and inflammation levels increase through inflammasome activation, leading to ovarian follicle loss. Other markers of reproductive aging include increased fibrosis and shortening of telomeres in ovarian cells. The factors that accelerate reproductive aging are unclear, but likely involve exposure to endocrine-disrupting chemicals such as phthalates. Di(2-ethylhexyl) phthalate (DEHP) is a widely used phthalate and humans are exposed to it daily. Several studies show that DEHP induces reproductive toxicity by affecting estrous cyclicity, follicle numbers, and hormone levels. However, little is known about the mechanisms underlying DEHP-induced early onset of reproductive aging. Thus, this study tested the hypothesis that dietary exposure to DEHP induces early reproductive aging by affecting inflammation, fibrosis, and the expression of telomere regulators and antioxidant enzymes. Adult CD-1 female mice were exposed to vehicle (corn oil) or DEHP (0.5, 1.5, or 1500 ppm) via the chow for six months. Exposure to DEHP increased the expression of antioxidant enzymes and Casp3, increased expression of telomere-associated genes, and increased fibrosis levels in the ovary. In addition, DEHP exposure for 6 months altered ovarian and systemic inflammatory status. Collectively, our novel data suggest that 6-month dietary exposure to DEHP may accelerate reproductive aging by affecting several reproductive aging markers in female mice.