Environmental pollution is an inevitable ecological issue accompanying the process of socialization, with increasing attention to its impacts on individual organisms and ecological chains. The reproductive system, responsible for transmitting genetic material in animals, is one of the most sensitive systems to environmental toxins. Research reveals that Sertoli cells are the primary target cells for the action of environmental toxins. Different environmental toxins mostly affect the blood–testis barrier and lead to male reproductive disorders by disrupting Sertoli cells. Therefore, this article provides an in-depth exploration of the toxic mechanisms of various types of environmental toxins on the male testes. It reveals the dynamic processes of tight junctions in the blood–testis barrier affected by environmental toxins and their specific roles in the reconstruction process.

Summary Sentence: Environmental pollution is an inevitable ecological issue during social development. The reproductive system, responsible for the transmission of genetic material in animals, is one of the systems most sensitive to environmental toxins. Various environmental toxins primarily affect the blood-testis barrier by damaging Sertoli cells, leading to male reproductive disorders. Investigating the toxic mechanisms of different environmental toxins on male testes will aid in developing strategies in the future to mitigate the burden of environmental factors on males and their negative impacts on health and fertility.

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Miscarriage poses a significant threat to both maternal and fetal health. Its etiology remains unknown, and there are no established effective identification or prevention strategies. A low-oxygen environment in early pregnancy is a physiological necessity for embryonic and placental growth. Hypoxia-inducible factors are a family of classic hypoxia signaling molecules whose expression level may fluctuate abnormally because of an imbalance in oxygen levels. Its unusual fluctuations initiate multiple signaling pathways at the maternal womb. Hypoxia-inducible factors are a family of classic hypoxia-signaling molecules and immune tolerance. Notably, aberrant regulation of these processes may lead to miscarriage. This review aims to clarify how the hypoxia-inducible factor-1α mediates the aberrant regulation of biological processes, including autophagy, metabolic reprogramming, et al., and how these effects impact trophoblasts and other cells at the maternal-fetal interface. These findings provide new insights into potential therapeutic and preventive strategies for miscarriage.

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.

Summary Sentence

We profile the transcriptome of two widely used stem cell-based TB models, compare to TB s from peri-implantation human embryos, and validate several genes in peri-implantation human embryos with immunofluorescence.

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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.

Summary Sentence

We report that ZNHIT1 and ZNHIT2 are indispensable for mouse embryonic development with knockout embryos dying during peri-implantation stages. Embryos lacking ZNHIT1 or ZNHIT2 result in overlapping phenotypes but through distinct mechanisms.

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Interactions between female metabolic status, immune response, and reproductive system functioning are complex and not fully understood. We hypothesized that chemerin, considered a hormonal link between the above-mentioned processes, influences endometrial functions, particularly cytokine secretion and signaling. Using porcine endometrial explants collected during early pregnancy and the estrous cycle, we investigated chemerin effects on the secretion of interleukins (IL-1β, IL-6, and IL-8), leukemia inhibitory factor, tumor necrosis factor α, transforming growth factor α, and protein abundances of their respective receptors. Our results demonstrate chemerin modulation of cytokine secretion and receptor expression, with effects dependent on the stage of pregnancy and dose of chemerin. Furthermore, chemerin influences the phosphorylation of stress-activated protein kinase/Jun-amino-terminal kinase and nuclear factor kappa-light-chain-enhancer of activated B cells in the endometrium. Chemerin multifaceted actions, such as involvement in immune response, cell proliferation, and tissue remodeling, seem to be mediated by cytokines, at least in the endometrium. These findings underscore the potential crosstalk between chemerin and hormonal signaling pathways, providing insights into the complex mechanisms underlying early pregnancy establishment and maintenance.

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Objective: Endocervical mucus production is a key regulator of fertility throughout the menstrual cycle. With cycle-dependent variability in mucus quality and quantity, cervical mucus can either facilitate or block sperm ascension into the upper female reproductive tract. This study seeks to identify genes involved in the hormonal regulation of mucus production, modification, and regulation through profiling the transcriptome of endocervical cells from the non-human primate, the rhesus macaque (Macaca mulatta).

Intervention: We treated differentiated primary endocervical cultures with estradiol (E2) and progesterone (P4) to mimic peri-ovulatory and luteal-phase hormonal changes. Using RNA-sequencing, we identified differential expression of gene pathways and mucus-producing and mucus-modifying genes in cells treated with E2 compared to hormone-free conditions and E2 compared to E2-primed cells treated with P4.

Main Outcome Measures: We pursued differential gene expression analysis on RNA-sequenced cells. Sequence validation was done using quantitative PCR (qPCR).

Results: Our study identified 158 genes that show significant differential expression in E2-only conditions compared to hormone-free control and 250 genes that show significant differential expression in P4-treated conditions compared to E2-only conditions. From this list, we found hormone-induced changes in transcriptional profiles for genes across several classes of mucus production, including ion channels and enzymes involved in post-translational mucin modification that have not previously been described as hormonally regulated.

Conclusion: Our study is the first to use an in vitro culture system to create an epithelial cell–specific transcriptome of the endocervix. As a result, our study identifies new genes and pathways altered by sex steroids in cervical mucus production.

Summary Sentence

In vitro hormonal regulation of mucus production, modification, and secretion was profiled using primary epithelial endocervical cells.

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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.

Summary Sentence

Extracellular vesicles (EVs) from the caput epididymis influenced gene expression, with 1233 differentially expressed genes identified, in corpus epididymis explants after 3 days of exposure to EVs using a reproducible in vitro culture system.

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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.

Summary Sentence

Knockout model male mouse of 18 genes mainly expressed in the testis does not lose fertility.

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Kisspeptin is a major regulator of gonadotropin secretion in pigs. Previously, CRISPR/Cas9 knockout of KISS1 was used to develop a mosaic parental line of pigs to generate offspring that would not need castration due to loss of kisspeptin. The current goal was to characterize growth and reproductive development of F1 pigs from this parental line. Body weights, gonadotropin concentrations and gonadal development were measured from birth through development (boars to 220 days of age, n = 42; gilts to 160 days of age, n = 36). Testosterone, skatole, and androstenone were also measured in boars. Blood samples were collected by jugular venipuncture for quantification of serum hormones, gonadal tissues were collected for gross morphology and histology, and a fat biopsy was collected (boars) for skatole and androstenone analysis. Body weight did not differ with genotype. There were no differences between KISS1^+/+ and heterozygote KISS1⁺/– animals for most parameters measured. Gonadotropin concentrations were reduced in KISS1–/– boars and gilts compared with KISS1^+/+ and KISS1⁺/– animals (P < 0.05). Concentrations of testosterone in serum and both androstenone and skatole in adipose were less in KISS1–/– boars than in KISS1^+/+ and KISS1⁺/– boars (P < 0.05). Hypogonadism was present in all KISS1–^/– gilts and boars. These data indicate that knocking out KISS1 causes hypogonadotropic hypogonadism but does not negatively affect growth in pigs. Only one KISS1 allele is needed for normal gonadotropin secretion and gonadal development, and accumulation of compounds in adipose leading to boar taint.

Summary Sentence

Knocking out kisspeptin in pigs causes hypogonadotropic hypogonadism and reduces accumulation of skatole in fat but does not affect growth.

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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.

Summary Sentence

Deletion of the catalytic subunit of glutamate cysteine ligase, the rate-limiting enzyme in glutathione synthesis, in granulosa cells of growing ovarian follicles causes decreased fertility in mice, while oocyte-specific deletion has no such effects.

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Previous studies have suggested that adamts9 (a disintegrin and metalloprotease with thrombospondin type-1 motifs, member 9), an extracellular matrix (ECM) metalloprotease, participates in primordial germ cell (PGC) migration and is necessary for female fertility. In this study, we found that adamts9 knockout (KO) led to reduced body size, and female-to-male sex conversion in late juvenile or adult zebrafish; however, primary sex determination was not affected in early juveniles of adamts9 KO. Overfeeding and lowering the rearing density rescued growth defects in female adamts9 KO fish but did not rescue defects in ovarian development in adamts9 KO. Delayed PGC proliferation, significantly reduced number and size of Stage IB follicles (equivalent to primary follicles) in early juveniles of adamts9 KO, and arrested development at Stage IB follicles in mid- or late-juveniles of adamts9 KO are likely causes of female infertility and sex conversion. Via RNAseq, we found significant enrichment of differentially expressed genes involved in ECM organization during sexual maturation in ovaries of wildtype fish; and significant dysregulation of these genes in adamts9 KO ovaries. RNAseq analysis also showed enrichment of inflammatory transcriptomic signatures in adult ovaries of these adamts9 KO. Taken together, our results indicate that adamts9 is critical for development of primary ovarian follicles and maintenance of female sex, and loss of adamts9 leads to defects in ovarian follicle development, female infertility, and sex conversion in late juveniles and mature adults. These results show that the ECM and extracellular metalloproteases play major roles in maintaining ovarian follicle development in zebrafish.

Summary Sentence

Arrested primary ovarian follicles and female to male sex conversion in Adamts9 KO zebrafish are due to dysregulation of extracellular matrix.

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Preeclampsia (PE) is a pregnancy-specific disease that causes maternal symptoms such as high blood pressure and adverse pregnancy outcomes. 2-methoxyestradiol (2-MeO-E₂), an endogenous metabolite of 17β-estradiol (E₂) formed by catechol-O-methyltransferase (COMT), plays an important role in pregnancy. Our earlier studies have shown that polyphenols present in coffee can inhibit COMT activity, which may inhibit the formation of 2-MeO-E₂ and contribute to PE. Therefore, the current study aims to investigate the possible effect and mechanism of coffee intake during pregnancy on PE in rats. Coffee is administered with or without the co-treatment of 2-MeO-E₂ to pregnant rats from the10^th to the18^th day of pregnancy. The results show that pregnant rats with coffee intake had prominent fetal growth restriction, hypertension, and proteinuria, which can be ameliorated by co-treatment of 2-MeO-E₂. In addition, coffee treatment leads to significantly decreased serum 2-MeO-E₂. Therefore, the PE symptoms induced by coffee treatment are probably mediated by decreased 2-MeO-E_2. In sum, our findings provide a new mechanistic insight into how coffee intake could lead to increased risk of PE, and demonstrate the effectiveness of 2-MeO-E₂ supplementation as a potential therapeutic agent for PE.

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Impaired extravillous trophoblast (EVT) invasion and resulted poor placentation play a vital role in the development of preeclampsia (PE). However, the underlying mechanisms of dysregulated EVTs remain unclear. This study aimed to explore the role of poly (C)-binding protein 2 (PCBP2), a multifunctional RNA-binding protein, in the pathogenesis of PE and to investigate the detailed signaling pathway. Using qRT-PCR, western blot, and immunohistochemistry, we confirmed that the expression of PCBP2 significantly decreased in placentas from 18 early-onset PE and 30 late-onset PE in comparison to those from 30 normotensive pregnancies. Besides, more significant suppression of PCBP2 was observed in the early-onset type. After transfection of HTR-8/SVneo with small-interfering RNA specific to PCBP2, the cellular biological behaviors including vitality, immigration, invasiveness, and apoptosis were evaluated by CCK-8 assay, wound-healing assay, transwell assay, and flow cytometry respectively. RNA-seq was applied to screen differentially expressed genes in HTR-8/SVneo upon PCBP2 silencing. GO and KEGG analysis indicated that WNT signaling pathway and the related processes such as extracellular matrix remodeling and cell adhesion were among the most enriched pathways or processes. Meanwhile, the alternative splicing of WNT5A regulated by PCBP2 was also identified by RIP-seq. Based on HTR-8/SVneo and villous explant, the regulatory roles of PCBP2 on trophoblast were confirmed to be mediated by WNT5A. Besides, it revealed that ROR2/JNK/MMP2/9 pathway was a vital pathway downstream WNT5A in trophoblast cells. In conclusion, this study suggests that down-regulated PCBP2 impaired the functions of EVTs via suppression of WNT5A-mediating ROR2/JNK/MMPs pathway, which may eventually contribute to the development of PE.

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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.

Summary Sentence

Viable eggs are produced by in vitro formation of reconstructed granulosa cell–oocyte complexes using denuded oocytes from early preantral follicles.

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