Molecular human reproduction最新文献

EXOC5 is a crucial component of a large multi-subunit tethering complex, the exocyst complex, that is required for fusion of secretory vesicles with the plasma membrane. Exoc5 deleted mice die as early embryos. Therefore, to determine the role of EXOC5 in follicular and oocyte development, it was necessary to produce a conditional knockout (cKO), Zp3-Exoc5-cKO, in which Exoc5 was deleted only in oocytes. The first wave of folliculogenesis appeared histologically normal and progressed to the antral stage. However, after IVF with normal sperm, oocytes collected from the first wave (superovulated 21-day-old cKO mice) were shown to be developmentally incompetent. Adult follicular waves did not progress beyond the secondary follicle stage where they underwent apoptosis. Female cKO mice were infertile. Overall, these data suggest that the first wave of folliculogenesis is less sensitive to oocyte-specific loss of Exoc5, but the resulting gametes have reduced developmental competence. In contrast, subsequent waves of folliculogenesis require oocyte-specific Exoc5 for development past the preantral follicle stage. The Zp3-Exoc5-cKO mouse provides a model for disrupting folliculogenesis that also enables the separation between the first and subsequent waves of folliculogenesis.

Placental growth is most rapid during the first trimester (FT) of pregnancy, making it vulnerable to metabolic and endocrine influences. Obesity, with its inflammatory and oxidative stress, can cause cellular damage. We hypothesized that maternal obesity increases DNA damage in the FT placenta, affecting DNA damage response and trophoblast turnover. Examining placental tissue from lean and obese non-smoking women (4-12 gestational weeks), we observed higher overall DNA damage in obesity (COMET assay). Specifically, DNA double-strand breaks were found in villous cytotrophoblasts (vCTB; semi-quantitative γH2AX immunostaining), while oxidative DNA modifications (8-hydroxydeoxyguanosine; FPG-COMET assay) were absent. Increased DNA damage in obese FT placentas did not correlate with enhanced DNA damage sensing and repair. Indeed, obesity led to reduced expression of multiple DNA repair genes (mRNA array), which were further shown to be influenced by inflammation through in vitro experiments using tumor necrosis factor-α treatment on FT chorionic villous explants. Tissue changes included elevated vCTB apoptosis (TUNEL assay; caspase-cleaved cytokeratin 18), but unchanged senescence (p16) and reduced proliferation (Ki67) of vCTB, the main driver of FT placental growth. Overall, obesity is linked to heightened non-oxidative DNA damage in FT placentas, negatively affecting trophoblast growth and potentially leading to temporary reduction in early fetal growth.

Pronuclear transfer has been successfully used in human-assisted reproduction to suppress the adverse effects of a defective oocyte cytoplasm or to bypass an idiopathic developmental arrest. However, the effects of the initial parental genome remodelling in a defective cytoplasm on the subsequent development after pronucleus transfer have not been systematically studied. By performing pronuclear transfer in pre-replication and post-replication mouse embryos, we show that the timing of the procedure plays a critical role. Although apparently morphologically normal blastocysts were obtained in both pre- and post-replication pronuclear transfer groups, post-replication pronuclear transfer led to a decrease in developmental competence and profound changes in embryonic gene expression. By inhibiting the replication in the abnormal cytoplasm before pronuclear transfer into a healthy cytoplasm, the developmental potential of embryos could be largely restored. This shows that the conditions under which the first embryonic replication occurs strongly influence developmental potential. Although pronuclear transfer is the method of choice for mitigating the impact of a faulty oocyte cytoplasm on early development, our results show that the timing of this intervention should be restricted to the pre-replication phase.

Advanced maternal age is associated with a decline in oocyte quality, which often leads to reproductive failure in humans. However, the mechanisms behind this age-related decline remain unclear. To gain insights into this phenomenon, we applied plexDIA, a multiplexed data-independent acquisition, single-cell mass spectrometry method, to analyze the proteome of oocytes from both young women and women of advanced maternal age. Our findings primarily revealed distinct proteomic profiles between immature fully grown germinal vesicle and mature metaphase II oocytes. Importantly, we further show that a woman's age is associated with changes in her oocyte proteome. Specifically, when compared to oocytes obtained from young women, advanced maternal age oocytes exhibited lower levels of the proteasome and TRiC complex, as well as other key regulators of proteostasis and meiosis. This suggests that aging adversely affects the proteostasis and meiosis networks in human oocytes. The proteins identified in this study hold potential as targets for improving oocyte quality and may guide future studies into the molecular processes underlying oocyte aging.

Acephalic spermatozoa syndrome (ASS) is a severe teratospermia with decaudated, decapitated, and malformed sperm, resulting in male infertility. Nuclear envelope protein SUN5 localizes to the junction between the sperm head and tail. Mutations in the SUN5 gene have been identified most frequently (33-47%) in ASS cases, and its molecular mechanism of action is yet to be explored. In the present study, we generated Sun5 knockout mice, which presented the phenotype of ASS. Nuclear membrane protein LaminB1 and cytoskeletal GTPases Septin12 and Septin2 were identified as potential partners for interacting with SUN5 by immunoprecipitation-mass spectrometry in mouse testis. Further studies demonstrated that SUN5 connected the nucleus by interacting with LaminB1 and connected the proximal centriole by interacting with Septin12. The binding between SUN5 and Septin12 promoted their aggregation together in the sperm neck. The disruption of the LaminB1/SUN5/Septin12 complex by Sun5 deficiency caused separation of the Septin12-proximal centriole from the nucleus, leading to the breakage of the head-to-tail junction. Collectively, these data provide new insights into the pathogenesis of ASS caused by SUN5 deficiency.

The role of cumulus cells (CCs) in the acquisition of oocyte developmental competence is not yet fully understood. In a previous study, we matured cumulus-denuded fully-grown mouse oocytes to metaphase II (MII) on a feeder layer of CCs (FL-CCs) isolated from developmentally competent (FL-SN-CCs) or incompetent (FL-NSN-CCs) SN (surrounded nucleolus) or NSN (not surrounding nucleolus) oocytes, respectively. We observed that oocytes cultured on the former could develop into blastocysts, while those matured on the latter arrested at the 2-cell stage. To investigate the CC factors contributing to oocyte developmental competence, here we focused on the CCs' release into the medium of extracellular vesicles (EVs) and on their miRNA content. We found that, during the 15-h transition to MII, both FL-SN-CCs and FL-NSN-CCs release EVs that can be detected, by confocal microscopy, inside the zona pellucida (ZP) or the ooplasm. The majority of EVs are <200 nm in size, which is compatible with their ability to cross the ZP. Next-generation sequencing of the miRNome of FL-SN-CC versus FL-NSN-CC EVs highlighted 74 differentially expressed miRNAs, with 43 up- and 31 down-regulated. Although most of these miRNAs do not have known roles in the ovary, in silico functional analysis showed that seven of these miRNAs regulate 71 target genes with specific roles in meiosis resumption (N = 24), follicle growth (N = 23), fertilization (N = 1), and the acquisition of oocyte developmental competence (N = 23). Overall, our results indicate CC EVs as emerging candidates of the CC-to-oocyte communication axis and uncover a group of miRNAs as potential regulatory factors.

Uterine glands are branched, tubular structures whose secretions are essential for pregnancy success. It is known that pre-implantation glandular expression of leukemia inhibitory factor (LIF) is crucial for embryo implantation; however, the contribution of uterine gland structure to gland secretions, such as LIF, is not known. Here, we use mice deficient in estrogen receptor 1 (ESR1) signaling to uncover the role of ESR1 signaling in gland branching and the role of a branched structure in LIF secretion and embryo implantation. We observed that deletion of ESR1 in neonatal uterine epithelium, stroma, and muscle using the progesterone receptor PgrCre causes a block in uterine gland development at the gland bud stage. Embryonic epithelial deletion of ESR1 using a Müllerian duct Cre line, Pax2Cre, displays gland bud elongation but a failure in gland branching. Reduction of ESR1 in adult uterine epithelium using the lactoferrin-Cre (LtfCre) displays normally branched uterine glands. Unbranched glands from Pax2Cre Esr1flox/flox uteri fail to express glandular pre-implantation Lif, preventing implantation chamber formation and embryo alignment along the uterine mesometrial-antimesometrial axis. In contrast, branched glands from LtfCre Esr1flox/flox uteri display reduced expression of ESR1 and glandular Lif resulting in delayed implantation chamber formation and embryo-uterine axes alignment but mice deliver a normal number of pups. Finally, pre-pubertal unbranched glands in control mice express Lif in the luminal epithelium but fail to express Lif in the glandular epithelium, even in the presence of estrogen. These data strongly suggest that branched glands are necessary for pre-implantation glandular Lif expression for implantation success. Our study is the first to identify a relationship between the branched structure and secretory function of uterine glands and provides a framework for understanding how uterine gland structure-function contributes to pregnancy success.

Persistent and intense uterine contraction is a risk factor for preterm labor. We previously found that methyl-CpG-binding protein 2 (MeCP2), as a target of infection-related microRNA miR-212-3p, may play an inhibitory role in regulating myometrium contraction. However, the molecular mechanisms by which MeCP2 regulates myometrial contraction are still unknown. In this study, we found that MeCP2 protein expression was lower in myometrial specimens obtained from preterm labor cases, compared to those obtained from term labor cases. Herein, using RNA sequence analysis of global gene expression in human uterine smooth muscle cells (HUSMCs) following siMeCP2, we show that MeCP2 silencing caused dysregulation of the cholesterol metabolism pathway. Notably, MeCP2 silencing resulted in the upregulation of CYP27A1, the key enzyme involved in regulating cholesterol homeostasis, in HUSMCs. Methylation-specific PCR, chromatin immunoprecipitation, and dual luciferase reporter gene technology indicated that MeCP2 could bind to the methylated CYP27A1 promoter region and repress its transcription. Administration of siCYP27A1 in a lipopolysaccharide (LPS)-induced preterm labor mouse model delayed the onset of preterm labor. Human preterm myometrium and the LPS-induced preterm labor mouse model both showed lower expression of MeCP2 and increased expression of CYP27A1. These results demonstrated that aberrant upregulation of CYP27A1 induced by MeCP2 silencing is one of the mechanisms facilitating inappropriate myometrial contraction. CYP27A1 could be exploited as a novel therapeutic target for preterm birth.