Molecular human reproduction最新文献

Protamines are considered among the most relevant sperm proteins because of their functional implications on paternal genome packaging and protection. Although the proteomic evaluation of protamines is technically challenging, mass spectrometry-based studies have shown a complex population of protamine proteoforms in the human sperm. This includes intact, truncated, and modified forms for protamine 1 (P1) and mature and immature components of the protamine 2 (P2) family. However, it is still unknown whether global or specific protamine proteoform levels may be unbalanced under conditions that may impair paternal chromatin maturity and epigenetic information. In this study, protamines from normozoospermic men stratified according to body mass index, age, and chromatin maturity (assessed through the P1/P2 ratio derived from acid-urea electrophoresis) were evaluated using a refined top-down mass spectrometry protocol for protamine proteoform quantification and comparative analysis. Accumulation of the P2 immature forms HPS1 and HPI2 was significantly associated with abnormally high P1/P2 ratios, suggesting either impaired eviction of P2 immature forms or defective P2 processing during spermatogenesis in these men clinically classified as normozoospermic. When considering weight and age as factors, P1 was the only affected protamine. Sperm from obese men, which were found to be exposed to high levels of oxidative damage derived from lipid peroxidation, showed mass shift(s) of +61 Da from the unmodified P1 protein sequence. Men of advanced age showed a specific loss of diphosphorylated P1, mainly on Ser 11 and 22. Our results allow the hypothesis that protamine proteoforms in the male gamete act as additional layers of epigenetic information, the alteration of which might be related to some cases of impaired sperm function.

It is hypothesized that male fetuses are more vulnerable to in utero insults than females due to different growth strategies, and that the placenta contributes to these sex differences. We examined sex differences in the fetal and placental responses to maternal food restriction (∼60% of ad libitum) beginning mid-gestation (Day 11.5). To dissect the roles of chromosomal and gonadal sex, we used the Four Core Genotypes mouse model, which combines deletion of the testis-determining Sry gene from the Y chromosome and autosomal insertion of the Sry gene, such that XX gonadal males and XY gonadal females are produced in addition to XX females and XY males. Food restriction reduced fetal and placental weights but had no effect on the number of viable conceptuses. However, this effect did not differ between gonadal male and female, or between XX and XY, conceptuses. Sex differences in gene expression in both the labyrinth and the combined junctional zone/decidua, as assessed by RNA sequencing, were due entirely to chromosomal sex and not gonadal sex. Food restriction affected the expression of 525 and 665 genes in the labyrinth and the junctional zone/decidua, respectively. However, these effects of food restriction did not differ by gonadal or chromosomal sex when assessed for statistical interactions. In contrast, when analyzing XX and XY placentas separately, hundreds of genes were affected by food restriction in one sex but not in the other, including hundreds of genes not found to be significant in the combined analyses. However, estimated effect sizes were generally similar for XX and XY placentas, suggesting that these sex-stratified analyses greatly exaggerated the extent of sex-dependent responses. Overall, we did not find evidence of the hypothesized sex differences in fetal growth strategy and found that sex differences in placental gene expression were largely due to chromosomal sex.

Cellular prion (PRNP) is a GPI-anchored extrinsic membrane glycoprotein, which has been implicated in mouse decidualization. However, the molecular function of this protein in mouse decidua is not known. In this article, we have characterized and elucidated the possible role of PRNP in mouse decidua. We demonstrated that PRNP expression is evident on embryonic day (E) 6.5 to E9.5 across the primary decidual zone (PDZ) in the mouse implantation site. As gestation progressed, PRNP continued to be expressed in the receding decidua, up to E17.5. shRNA-mediated knockdown of PRNP on E5.5 through E7.5 led to decreased expression of tight junction proteins (TJPs) in PDZ in vivo. These included Cingulin, Afadin, Catenin-α1, Lethal (2) giant larvae protein homolog 1, Claudin-5, and ICAM1. Furthermore, PRNP-positive decidual cells showed augmented expression of autophagic machinery. PRNP knockdown curtailed expression of autophagy-related genes in decidua in vivo. Our results highlight hitherto unknown novel functions of PRNP: (i) an inducer of TJPs at PDZ, which protects the developing embryo and (ii) a decision-maker protein between life and death in decidual cells.

Exposure to PM2.5 (particulate matter <2.5 μm) has been implicated in increasing the risk of endometriosis and worsening its symptoms. However, the molecular mechanisms and direct associations remain unclear. This study explored whether PM2.5 contributes to the onset or progression of endometriosis using in vitro and in vivo models. Endometrial (EM) cells from women without endometriosis were cultured to the second passages (P2) with or without exposure to PM2.5 at a concentration of 200 µg/ml (N = 5 for each group). Z-stack confocal imaging confirmed PM accumulation in the nucleus and cytoplasm of exposed EM cells. Initial PM exposure at the primary passage (P0) led to decreased proliferation, migration, anti-apoptosis, and oxidative stress, accompanied by downregulation of associated pathways. However, repeated PM exposure during subculturing to P2 led to increased proliferation, enhanced anti-apoptotic activity, and elevated oxidative stress. Given the similarity of these gene expression alterations to those observed in endometriosis, an endometriosis-induced mouse model was established to assess the potential of repeated PM exposure to exacerbate the condition in vivo. To investigate the in vivo effects, an endometriosis-induced mouse model was developed using female C57BL/6 mice exposed to low (10 mg/kg/day) or high (20 mg/kg/day) doses of PM2.5 for 4 weeks (n = 6 for each group). PM exposure significantly enlarged endometriotic lesions compared to controls (no PM exposure). Upregulated gene expression in endometriotic lesions included anti-apoptotic (Bcl2/Bax), proliferative (p-ERK), inflammatory (p-NF-κB, p-c-jun, IL-6, IL-1β), and migration (MMP-2, MMP-9) markers. PM exposure altered estrogen receptor (ER) expression, resulting in a decreased ERα/ERβ ratio in both dose groups. The control group exhibited a ratio of 1.03 ± 0.09, while the low-dose and high-dose mice had ratios of 0.57 ± 0.08 (P = 0.02) and 0.46 ± 0.26 (P = 0.03), respectively. In conclusion, PM2.5 exposure alters gene expression related to cell growth, survival, oxidative stress, and migration in EM cells and exacerbates endometriotic lesions in vivo, likely through ER modulation. These findings suggest PM2.5 may contribute to other estrogen-dependent conditions, such as leiomyoma or adenomyosis, by influencing ER pathways.

Endometriosis is a chronic inflammatory disorder in which immune cells, specifically macrophages, play a pivotal yet incompletely understood role. Aberrations within the eutopic endometrium are implicated in the initiation and progression of endometriosis. In this study, we reported a notable increase in the proportion of a distinct S100A9+ macrophage subpopulation undergoing ferroptosis in the eutopic endometrium of endometriosis patients compared with normal endometrium, as evidenced by single-cell RNA sequencing and experimental validation. Furthermore, we confirmed that Ammonium iron (III) citrate-treated macrophages upregulate S100A9 through the NF-κB pathway in vitro. Subsequent cell function experiments and endometriosis mouse models revealed that S100A9 promotes the development of endometriosis by facilitating angiogenesis. Notably, the S100A9 inhibitor Tasquinimod effectively reduced angiogenesis and thereby reduced ectopic lesions in mice. These results indicated that S100A9+ macrophages represent a potential therapeutic target for endometriosis.

Blastocyst mosaicism is increasingly detected due to advances in preimplantation genetic testing for aneuploidy (PGT-A). While some mosaic embryos result in a live birth, there are reports of altered implantation potential. Various approaches are needed to better understand their developmental uniqueness, including their transcriptomic profile. This study aimed to profile the transcriptomic signatures of mosaic embryos and investigate how mosaicism influences global gene expression. Utilizing a novel method enabling simultaneous sequencing of both genomic (g)DNA, for PGT-A, and mRNA, we analyzed 76 blastocysts from 60 IVF-ICSI patients, including 29 euploid, 23 mosaic, and 24 aneuploid embryos. Transcriptomic analysis revealed that 14% of annotated genes are differentially expressed (DE) between mosaic and euploid embryos, with the majority of genes being upregulated in mosaic embryos. We further divided the mosaic cohort into mosaic-gain (mosaic trisomy) and mosaic-loss (mosaic monosomy) embryos and identified a core set of 902 DE genes that are shared regardless of the direction of the mosaicism (gain or loss). Pathway analysis revealed significant upregulation in pathways associated with cell cycle regulation, mitochondrial respiration, DNA repair, and vesicle transport. Following leading-edge analysis, genes previously annotated to be involved in embryo implantation were downregulated in mosaic embryos. Separately, in a subset of aneuploid embryos, we identified gene dosage effects; while embryos with trisomies 18 and 21 exhibit transcriptional signatures similar to euploid embryos, those with trisomies 16 and 22 demonstrate more divergent profiles, correlating with their previously reported implantation and clinical outcomes. These findings enhance current literature on the transcriptomic consequences of mosaicism in the trophectoderm of embryos. They suggest that mosaic blastocysts exhibit transcriptional signatures that reflect their mixed chromosomal composition, potentially influencing their implantation efficiency and developmental potential. Despite the inherent cellular stress, a proportion of mosaics retain developmental resilience, underscoring the complexity of embryo selection in ART.

Reverse genetic approaches are the standard in molecular biology to determine a protein's function. Traditionally, nucleic acid targeting via gene knockout (DNA) and knockdown (RNA) has been the method of choice to remove proteins-of-interest. However, the nature of mammalian oocyte maturation and preimplantation embryo development can make nucleic acid-targeting approaches difficult. Gene knockout allows time for compensatory mechanisms and secondary phenotypes to develop which can make interpretation of a protein's function difficult. Furthermore, genes can be essential for animal and/or oocyte survival, and therefore, gene knockout is not always a viable approach to investigate oocyte maturation and preimplantation embryo development. Conversely, RNA-targeting approaches, i.e. RNA interference (RNAi) and morpholinos, rely on protein half-life and therefore are unable to knockdown every protein-of-interest. An increasing number of reverse genetic approaches that directly target proteins have been developed to overcome the limitations of nucleic acid-based approaches, including Trim-Away and auxin-inducible degradation. These protein-targeting approaches give researchers exquisite and fast control of protein loss. This review will discuss how Trim-Away and auxin-inducible degradation can overcome many of the challenges of nucleic acid-based reverse genetic approaches. Furthermore, it highlights the unique research opportunities these approaches afford, such as targeting post-translationally modified proteins.

Fibrosis constitutes the principal pathophysiological mediator of pain and infertility manifestations in endometriosis, and the inhibitory factor of the TGF-β pathway, MADH7, makes a vital impact on the progression of fibrosis. Ovarian tumor domain-containing protein 1 (OTUD1) deubiquitinase binds to the MADH7 protein, although its specific role in endometriosis needs to be investigated. This study is the first to explore the role of OTUD1 in endometriosis and to investigate its impact on the growth of endometriosis lesions in vitro and in vivo, using C57BL/6N female mice and human primary stromal endometriosis cells (HEMCs). Moreover, the obtained results demonstrated that OTUD1 inhibited the expression of fibrosis-related proteins in HEMCs in vitro, and the mechanistic execution of this phenotype was achieved via coordinated deubiquitination coupled with MADH7-mediated transcriptional reprogramming. These events stopped the growth of lesions in vivo and reduced abdominal inflammation. The study demonstrated the critical role of the deubiquitinating enzyme OTUD1 in endometriosis, indicating its potential therapeutic effect on endometriosis.

Stepwise decidualization of endometrial stromal cells (EnSC) mediates the ability of the endometrium to respond to embryo quality, determining whether to permit or restrict its implantation. Currently, epigenetic mechanisms are recognized as key regulators of transcriptional gene networks during this hormone-induced differentiation of EnSC. In this study, we focused on the potential role of histone methyltransferase SETD7 in regulating EnSC decidualization. Using SETD7 knockout EnSC, we demonstrated that SETD7 is essential for proper decidualization; however, its impact is rather associated with stabilization of the estrogen receptor protein than with its histone methylation activity. By analyzing publicly available single-cell RNA sequencing data, we found that activation of SETD7 expression precedes the emergence of decidual subpopulations during EnSC decidualization. Furthermore, we identified SETD7 as a critical regulator of cell fate divergence of decidualizing EnSC, as cells lacking SETD7 expression exhibit accelerated senescence and an impaired decidual response. Finally, through the combined analysis of the available single-cell RNA sequencing data and the bulk mRNA microarray data for endometrial tissue, we revealed lower levels of SETD7 expression in patients with recurrent pregnancy losses. This decrease in SEDT7 expression correlates well with a reduced ratio of SCARA5/DIO2 gene expression in the endometrial stroma of these patients. Our data uncover SETD7 as a novel molecular regulator of EnSC decidualization, required for the proper establishment of decidual subpopulations.

The activation of dormant primordial follicles is a promising method to improve the fertility of premature ovarian insufficiency (POI) patients. Many experiments from both human and animal studies suggest that human platelet-rich plasma (hPRP) may restore ovarian function and promote follicle growth. However, the underlying mechanisms remain unclear. In the current study, our results demonstrate that hPRP significantly increased the number of growing follicles and promoted the proliferation of granulosa cells in cultured mouse ovaries. hPRP also significantly increased the protein levels of phosphorylated protein kinase B (p-Akt) and forkhead box O3a (p-FOXO3a), as well as the number of oocytes with FOXO3a nuclear export in cultured mouse ovaries. Immunofluorescence results showed that in vitro treatment with hPRP significantly increased the fluorescence intensity of p-Akt in oocytes. The inhibition of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway by LY294002 blocked the hPRP-induced increase in the number of growing follicles in cultured mouse ovaries. Furthermore, hPRP injected i.p. or added to the medium significantly increased the number of growing follicles and the protein levels of p-Akt in the ovaries of newborn mice and in cultured human ovarian tissues. Taken together, our findings from mouse and human experiments indicate that hPRP promotes the activation of primordial follicles through the PI3K/Akt signaling pathway in oocytes.