Reproduction最新文献

Today, male contraceptive options remain limited to condoms and vasectomy, highlighting the urgent need for alternative methods. In this context, discovering and characterizing reproductive-tract-specific proteins that can be targeted by natural or chemical molecules is of particular interest. Recent studies on the sperm protein glutamine-rich protein 2 (QRICH2) show that it could represent a promising candidate. Indeed, it has been genetically confirmed to be essential for male fertility in mice, bulls, and humans, with gene knockout and loss-of-function mutations leading to defective sperm and complete infertility without evident accompanying symptoms. However, information on human QRICH2 remains limited. In this study, we aimed to better characterize human QRICH2 to assess its potential as a target for male contraceptive development. Using mass spectrometry, we assessed which of the QRICH2 isoforms described in databases might be expressed in human sperm. Through in silico analyses, we showed that QRICH2 has no paralogs in humans, and is conserved across mammals, particularly in a region containing two functional domains, suggesting their importance for QRICH2 function. Finally, using immunodetection methods and proteomic dataset analyses, we investigated the tissue specificity of QRICH2 by examining its protein expression across 12 human organs. Our results show that QRICH2 is restricted to the testes, where it localizes to different cellular compartments throughout spermatogenesis, and acts as a cytoskeletal component in mature sperm, both in the head and flagellum. We conclude that QRICH2 represents a promising candidate for further investigation as a potential target for male contraception and we propose different strategies that could be explored for its inhibition.

Mitochondrial purification, including mitophagy, during oogenesis is critical for ensuring accurate mitochondrial DNA transmission, but it remains unclear whether meiosis plays a role in eliminating defective mitochondria. Here, we show that mitochondria in the first polar body (PB1) exhibit reduced membrane potential compared with those retained in oocytes. TUNEL and cytochrome c assays suggest that mitochondrial dysfunction in PB1 precedes nuclear fragmentation. Notably, the second polar body (PB2) exhibits heterogeneous membrane potentials, with both functional and dysfunctional populations present. By injecting dysfunctional and functional mitochondria, isolated respectively from the cytoplasts of the PB1and the oocyte, into germinal vesicle-stage mouse oocytes, we found that dysfunctional mitochondria were preferentially extruded into PB1, while functional mitochondria were retained after meiosis I. Interestingly, mitochondria from PB1 exhibit low membrane potential even after being transferred into a new, healthy oocyte, whereas oocyte-derived mitochondria maintain normal membrane potential following the same procedure. Immunofluorescence analysis further shows that PB1-derived mitochondria lack colocalization with motor protein Rho T1, in contrast to their oocyte-derived counterparts. Furthermore, PB1 transfer combined with mitochondrial probe and fast-NGS demonstrated that meiosis II also contributes to the extrusion of dysfunctional mitochondria into PB2. By comparison, spindle transfer revealed that most functional mitochondria were retained in the oocyte, with only minimal amounts detected in PB2. Predictably, PB2 and pronuclear transfer failed to extrude foreign mitochondria. Collectively, these findings identify meiosis as a distinctive safeguard for mitochondrial quality during oogenesis, with implications that warrant further investigation in humans.

In brief: The non-genomic factors responsible for developmental arrest in SCNT embryos remain poorly understood. Using live-cell fluorescence imaging, we revealed that autophagic activity is impaired in preimplantation SCNT embryos, possibly due to ectopic activation of the mTORC1 signaling pathway, providing new insights into cytoplasmic barriers to cloning efficiency.

Abstract: Activation of autophagy after fertilization is essential for mammalian embryonic development, as it supplies embryos with nutrients and energy. Somatic cell nuclear transfer (SCNT) embryos frequently exhibit developmental arrest, largely because of incomplete genomic reprogramming; however, the role of non-genomic factors remains unclear. Here, we investigated autophagy dynamics in mouse SCNT embryos using immunostaining and live-cell fluorescence imaging. In fertilized embryos, autophagy increased markedly from the late 2-cell stage and peaked at the morula stage. SCNT embryos followed a similar timeline but consistently showed reduced autophagic activity. Notably, the autophagic activity levels varied among SCNT embryos and positively correlated with their developmental potential. Attempts to enhance genomic reprogramming, including the removal of somatic histone methylation, did not restore autophagy. Instead, transcriptome analysis revealed ectopic activation of mTORC1 signaling as a likely cause of impaired autophagy. Consistently, treatment with an mTORC1 inhibitor successfully rescued autophagic activity in SCNT embryos. These findings identify a persistent autophagy defect during preimplantation development in SCNT embryos and suggest that modulation of non-genomic pathways, such as mTORC1 signaling, could improve SCNT efficiency.

In brief: With an automated image analysis approach using artificial intelligence and machine learning, cytoarchitectural and spatial characteristics of prepartum cervix remodeling occur before term and preterm birth with loss of response to progesterone. Evaluation of local features of inflammation associated with prepartum cervix remodeling may distinguish pathophysiological from physiological processes to reduce risk of adverse pregnancy outcomes.

Abstract: The cervix functions as a gatekeeper barrier to maintain pregnancy and virtually vanishes for birth. Cervix remodeling occurs well before term while progesterone (P4) is at or near peak in maternal circulation. The inflammatory ripening process is associated with reduced cell nuclei (CN) density, increased density of resident macrophages (Mφs), and reduced cross-linked collagen in stroma. Whether a functional or actual loss of response to P4 regulates cytomorphological characteristics associated with prepartum ripening at term was the focus of the present study. In a murine model, ovariectomy on pregnancy day 16 produced preterm birth (<24 h, Ovx) in mice with an oil-filled implant, while P4 treatment (Ovx + P4) and controls (PP) birthed at term. Cytoarchitecture of cervix remodeling (reduced CN/area and cross-linked collagen degradation) was advanced in Ovx compared to control mice, but forestalled by sustained plasma P4 in Ovx + P4 mice. Loss of response to P4 was also evident at term by increased resident Mφs in controls and Ovx + P4 PP mice from initial responses. By contrast, births were delayed or did not occur in ovary-intact mice given P4. Adverse pregnancy outcomes and dystocia were associated with arrest of prepartum cervix ripening. A novel spatial morphometric approach found Mφ-stain area/Mφ/CN increased during prepartum ripening versus that on preterm or term birth. This biomarker for local inflammation was blocked in intact + P4 mice. Mφ area, as indicative of alternative activation versus smaller-sized inflammatory phenotypes, collectively suggests loss of prepartum cervix P4 responses in the final common pathway for term and preterm parturition.

Infertility is a global health issue affecting a significant portion of couples, estimated at ∼10-15% of reproductive-aged couples worldwide, with the World Health Organisation (WHO) suggesting roughly one in six (∼17.5%) of the adult population experiences infertility. Male causes of infertility are attributable as a sole or leading cause in 40-50% of cases. Furthermore, sperm/semen counts have plummeted by ∼60% over the past 50-60 years in males attending fertility clinics. There is thus an urgent need to understand the causes behind these numbers to address such worrying trends. However, human male infertility is a heterogeneous and often idiopathic condition, with genetic factors increasingly recognised as major contributors. In this review, we examine known and emerging genetic causes of male infertility, highlighting how knockout mouse models have been leveraged to understand not only male reproductive biology and sperm physiological function, but also to illustrate how specific genetic disruptions correspond to particular reproductive failures, discussing how such mouse models are illuminating the causes of human idiopathic male infertility and guiding the discovery of novel infertility genes. We compare the similarities and differences between human and mouse infertility, not only identifying areas of further investigation that require urgent attention, but also potential novel avenues of therapeutic treatment.

In brief: Molecular changes during human sperm in vitro capacitation were investigated through RNA sequencing and bioinformatic analysis. The ubiquitin-dependent protein catabolic process emerged as a key pathway, and proteomic analysis supported that the ubiquitin-proteasome system plays a regulatory role in human sperm capacitation.

Abstract: Capacitation involves a series of biochemical and physiological changes that spermatozoa undergo during their transit along the female reproductive tract, which are essential for fertilizing the oocyte. While several processes associated with capacitation, such as increased tyrosine phosphorylation, have been extensively studied, the molecular mechanisms regulating this process remain unclear. This study aimed to identify biological processes associated with human sperm in vitro capacitation using next-generation RNA sequencing. Our findings revealed that sperm capacitation is associated with transcriptomic changes, characterized by 337 differential gene transcripts. Notably, one of the primary biological processes associated with capacitation was the ubiquitin-dependent protein catabolic process. To explore this further, we compared the ubiquitinated protein profiles of non-capacitated and capacitated spermatozoa using Western blot analysis after protein separation by denaturing gel electrophoresis and two-dimensional electrophoresis. The results showed increased protein ubiquitination during capacitation, which paralleled the expected increase in tyrosine phosphorylation. Interestingly, inhibition of proteasome activity with 50 μM MG132 avoided the degradation of ubiquitin conjugates, whereas tyrosine phosphorylation levels remained constant. These findings suggest that ubiquitin-conjugated sperm proteins and their subsequent degradation by the proteasome may play a role in sperm capacitation. Further investigation of ubiquitin-mediated mechanisms during capacitation could provide valuable insights into the signaling pathways essential for fertilization.

In brief: Generalized anxiety disorder, major depressive disorder and their treatment selective serotonin reuptake inhibitors (SSRIs) impact 4-17% of pregnancies worldwide and alter the epigenome of numerous tissues, but their effects on the ovarian follicle are unknown. This study profiles the methylome of granulosa cells, revealing novel epigenetic pathways and molecular mechanisms altered by mental health conditions and their treatments.

Abstract: Generalized anxiety and major depressive disorders (GAD/MDD) impact 4-17% of pregnancies worldwide. GAD/MDD and SSRIs alter the epigenome of numerous tissues; however, their effect on the ovarian follicular niche is unknown. In this study, we determined SSRI concentrations in the follicular fluid and matched patients by clinical and stimulation characteristics, and then grouped them into three groups: i) treated GAD/MDD (n = 10), ii) untreated GAD/MDD (n = 4), and iii) control (n = 10). DNA methylation sequencing was performed on granulosa cells using the Illumina TruSeq Methyl Capture EPIC kit. For patients with untreated GAD/MDD, we identified 3,829 differentially methylated sites (DMSs). Pathway analysis revealed an enrichment in genes involved in catabolism and immune response for the hypomethylated DMSs and hypermethylated DMSs were associated with protein localization and cellular transport. When assessing the effect of SSRI treatment, we identified 3,690 DMSs. Hypomethylated DMSs were associated with genes involved in cytoskeleton organization and cellular transport, whereas hypermethylated DMSs were associated with apoptosis and cell cycle. This is the first study profiling the methylome of human granulosa cells from patients with treated or untreated GAD/MDD. This study provides a valuable dataset describing the effects of SSRI on cells in the ovarian follicular niche.

In brief: Proper degradation of maternally inherited proteins is a prerequisite for successful embryonic development. This study shows the species-specificity of this process.

Abstract: The mechanism of targeting maternal proteins for degradation during preimplantation development is an unexplored process. Only a few proteins that need to be degraded for the proper course of the maternal-to-zygotic transition have been described in mice, and a few more in non-mammalian species. However, it is not well known whether the need for degradation is conserved across species or if it is driven in a species-specific way. Therefore, we selected six proteins that need to be degraded for the proper course of the maternal-to-zygotic transition in mice or Xenopus, and thoroughly characterized their expression at both the mRNA and protein level during bovine embryogenesis. Further, we analysed the protein expression in mice and pigs and compared it to bovine embryos. Thus, we provide a unique interspecies comparison of three mammalian representatives. We found that the degree of conservation between species is low and does not depend on the evolutionary relatedness of the species. This paper suggests that protein degradation during preimplantation development is controlled by a combination of species-specific factors from the embryo and the sequences of protein homologues.

In brief: This manuscript shows that the Na+ and K+ transporter Na+,K+-ATPase α4, specific to sperm, is expressed on the surface of the sperm head and flagellum in a very structured manner. This is also true for Na+,K+-ATPase α1, the other Na+,K+-ATPase isoform present in sperm and also in all cells. However, Na+,K+-ATPase α4 distribution changes when the cells are capacitated, an event necessary for fertilization. This dynamic remodeling, along with the distinct functional properties of Na+,K+-ATPase α4 and α1, provides evidence for the refined level of specialization that sperm have developed to achieve the amazing goal of fertilizing the oocyte.

Abstract: Na+,K+-ATPase α4 is a unique Na+ and K+ transporter of the plasma membrane of spermatozoa, which is essential for male fertility. While previous studies have found Na+,K+-ATPase α4 to be mainly expressed in the sperm flagellum, less is known about its localization in the sperm head. Moreover, the spatial arrangement of Na+,K+-ATPase α4 at the subcellular level and its relationship to the functional state of the cells are unclear. We studied this using stimulated emission depletion (STED) super-resolution microscopy. We show that, under non-capacitated conditions, Na+,K+-ATPase α4 is distributed in a trilinear pattern along the midpiece and as a scattered single line along the principal piece of the sperm flagellum. Under capacitated conditions, Na+,K+-ATPase α4 pattern undergoes remodeling and its distribution shifts to a single line along the flagellum. On the other hand, Na+,K+-ATPase α1, the somatic isoform of Na+,K+-ATPase also present in sperm, exhibits a similar trilaminar localization at the flagellar midpiece but a bilinear pattern in the principal piece. This distribution, unlike that of Na+,K+-ATPase α4, does not change during sperm capacitation. We also found Na+,K+-ATPase α1 and α4 in the sperm head, where they present a complex distribution under both non-capacitated and capacitated conditions. These differences in the localization pattern and spatial dynamics of Na+,K+-ATPase isoform expression, along with their different functional properties, highlight the distinct roles that both isoforms play to support sperm function.

In brief: There is no ideal animal model of adenomyosis, which reflects the imperfect understanding of complex human pathogenesis. In this study, we successfully induced adenomyosis in a mouse model via sharp-blunt trauma, which more closely mimics clinical observations in humans than previous models.

Abstract: Adenomyosis is a common gynecological disease in women of reproductive. To date, a satisfactory animal model of adenomyosis has not been established. In this study, 51 female mice were divided into four groups: negative control, an abdominal skin incision was made and sutured without uterine injury; puncture, the uterine horn was punctured using a needle; dilation & curettage, a self-made curette was used to simulate D&C; puncture + dilation & curettage, the uterine horn was punctured, and dilation & curettage were performed. The mice were euthanized 2 weeks, 1 month, or 2 months post-surgery, and the uteruses were harvested. Validity was assessed by histopathological examination. The levels of EMT markers were also detected among the groups. The success rate of adenomyosis induction was higher in the Punct + D&C group than in other groups at all three time points. The highest success rate was observed in the Punct + D&C group 2 months post-surgery. Significantly increased VIM expression was observed in ectopic lesions compared with eutopic endometrium in the Punct + D&C group 2 weeks post-surgery. In addition, VIM expression in eutopic endometrium in the Punct + D&C group was significantly higher than that in the sham group 2 months post-surgery. CDH1 expression was downregulated in the Punct + D&C group compared with the sham group at 2 weeks and 2 months post-surgery. In this study, we successfully established a mouse model of adenomyosis based on invagination theory, which is low cost, quick to establish, and does not interfere with hormone secretion.