Molecular human reproduction最新文献

Recently, the expression of bitter taste receptors and their downstream taste signaling cascade has been widely found outside the gustatory system, indicating important physiological functions of bitter taste receptors in various extraoral organs, including testis, but little is known about their functions in spermatogenesis. Here, we describe the localization and expression pattern of taste signaling transduction molecules in the testis. Genetic mutation of bitter signaling transduction molecules decreased the litter size, the IVF rate, and the diameter of seminiferous tubules and even resulted in empty seminiferous tubules. Transmission electron microscopy observations further revealed that overdeveloped acrosomes adhered to atrophic round spermatids in double-mutant mice. Mutant mice lacking bitter taste receptor signaling exhibited a dysfunction of adenosine 5'-monophosphate-activated protein kinase and inducible nitric oxide synthase signaling pathways, and a decreased expression of zonula occludens-1 and β-catenin in testis, indicating a disruption for the structure and functions of the blood-testis barrier. Transcriptome analysis further showed that bitter taste signaling deficiency can alter the expression profile of transcripts related to signal pathways, hormone synthesis, cell adhesion molecules, the chemokine signaling pathway, and cell metabolism in the testis, which finally contribute to impaired male fertility. In short, our work provides previously unidentified in vivo evidence that bitter taste signaling plays a critical role in the maintenance of normal spermatogenesis. These data further support the concept that bitter taste receptors exert functions outside the gustatory system and may have implications for the diagnosis and management of human male infertility.

It is common to sex-stratify analyses of omics data and to report effects as 'sex-specific' when they are significant in only one sex. However, when analysing hundreds or thousands of molecules, this approach will yield many spurious 'sex-specific' effects if not supported by significant interactions. I illustrate this problem using an RNA sequencing dataset showing almost no significant sex by treatment interactions, but where sex-stratified analyses yield hundreds of 'sex-specific' effects of treatment. These 'sex-specific' effects could be spurious or could be real but not show interactions due to low statistical power. To distinguish these possibilities, I describe permutation tests, which provide an intuitive way to determine if a pattern of observations differs from what would be expected due to chance. For this dataset, assigning sex at random often generates more 'sex-specific' effects than the real data, demonstrating that there is little evidence of sex differences. Next, I simulate an RNA sequencing dataset that includes genes modelled to have sex-specific effects of a condition. As expected, analysis of this simulated dataset yields both significant interactions and sex-specific effects in sex-stratified analyses. While stratified analyses detect a higher number of sex-specific effects than the analysis of interactions, they erroneously identify genes not modelled to show sex-specific effects more often than interactions. A permutation test confirms that the number of sex-specific effects observed in the simulated dataset is greater than expected due to chance. Permutation tests can be applied to omics studies of sex differences, simultaneously providing (i) a clear and simple demonstration of the problems of sex-stratified analyses, and (ii) additional evidence of sex-specific effects where these are present. R code is provided for permutations, simulations, and plots to visualize potential sex-specific effects, which can be adapted to other types of data.

Katanins are microtubule severing enzymes that play roles in diverse cell functions including meiotic and mitotic spindle formation. To address the role of Katanin p60 isozymes in mammalian oocytes, we have used the ZP3-CreLox approach to specifically delete Katanin A1 (KATNA1) and Katanin A-like 1 (KATNAL1) from the start of oocyte growth. Here, we show that KATNAL1 is not required for normal fertility, but deletion of KATNA1 causes a 50% decrease in fertility. Further investigation in Katna1-/- oocytes revealed no effect on MI spindle morphology but a modest effect on the morphology of MII spindles. This was accompanied by a decreased rate of fertilization, but Katna1-/+ heterozygous embryos that reached the 2-cell stage developed at normal rates to the blastocyst stage. Parthenogenetic activation of Katna1-/- oocytes to generate diploid homozygous embryos revealed a reduced rate of blastocyst formation. Further, the Katna1-/- parthenogenetic blastocysts had a reduced diameter, decreased cell number, and increased nuclear size. Taken together, our data indicate KATNA1, but not KATNAL1, plays a role in MII spindle function and mitotic cell divisions of the preimplantation embryo. The ability of the paternal allele to rescue preimplantation development suggests the origin of the decrease in the fertility of conditional Katna1-/- mice lies in abnormalities arising in the egg to embryo transition prior to embryonic genome activation.

Leiomyomas are benign proliferations of uterine smooth muscle found in 60% of women. A spatial redistribution of ecto-5'-nucleotidase (CD73, NT5E) that results in reduced extracellular concentrations of adenosine has recently been described in leiomyomas. However, the mechanisms by which altered extracellular adenosine levels contribute to leiomyoma growth remain poorly understood. To address this deficiency, a series of tissue specimens and primary cultures generated from matched specimens of myometrium and leiomyoma were used. Overexpression of Type 1 adenosine receptors (ADORA1) was observed when matched specimens and primary cultures were interrogated by RT-qPCR and western blot. By immunohistochemistry, ADORA1 expression was diffusely observed in myocytes in the leiomyoma complex, with only limited expression in vascular and other structures. Overexpression of ADORA1 was also observed in fibroblasts and multiple smooth muscle subtypes in the leiomyoma complex when single-cell transcriptomics data were interrogated. Incubation with N6-cyclopentyladenosine (CPA), a selective ADORA1 agonist, resulted in decreased proliferation of primary leiomyoma cultures, accompanied by decreased intracellular cAMP and enhanced cyclin D1 and phospho-AKT1 expression. To confirm the specificity of this observation, ADORA1 expression was directly targeted by siRNA, resulting in decreased proliferation, increased intracellular cAMP, and lower levels of cyclin D1 and phospho-AKT1. Collectively, these data indicate that overexpression of the ADORA1 receptor is a robust feature of uterine leiomyomas, where its activation by residual levels of extracellular adenosine potentially contributes to tumor growth by regulating AKT1-mediated signaling.

Current interventions for endometriosis mainly involve hormone therapies but have limited efficacy and unacceptable side effects due to the lack of selectivity to distinguish between endometriosis and endometrial tissues. Elucidating the molecular mechanism underlying the rapid growth of endometrial-like stromal cells, one of the main components of endometriotic lesions, will pave a path for more effective treatment of endometriosis. In the current study, we utilized transcriptome sequencing to compare the transcriptional profiles of endometrial-like stromal cells from endometriosis and endometrial tissues and demonstrated that Homeobox C4 (HOXC4) is preferentially expressed in endometriotic lesions. HOXC4 is indispensable for the proliferation of stromal cells from endometriosis, but not those from endometrial tissues. Mechanistically, HOXC4 acts as a transcription factor to promote the expression of Slit Guidance Ligand 2 (SLIT2) and thereby increases the p38 MAPK activity via the SLIT2 receptor roundabout guidance receptor 1 (ROBO1). Considering the essential role of the p38 MAPK activity in facilitating the development of ectopic endometrium, our findings strongly support the idea of HOXC4, as well as the SLIT2-ROBO1 axis, being potential therapeutic targets for endometriosis.

The Wolffian duct (WD) is the embryonic primordium that gives rise to the epididymis, vas deferens, and seminal vesicle. The androgen action in the mesenchyme is the predominant driver for fetal WD maintenance, which is essential for male fertility. However, the androgen's capability of promoting WD maintenance was completely lost in the absence of Wnt9b in mice. In this study, we followed up with this interesting phenomenon and revealed cellular and molecular mechanisms whereby Wnt9b facilitates WD maintenance in male embryos. Wnt9b belongs to the WNT family of secreted proteins and is expressed in the WD epithelium. We found that WD degeneration in Wnt9b-/- male embryos was accompanied by decreased cell proliferation in the epithelium but not in the mesenchyme during sexual differentiation. Wnt9b deletion did not impair testicular androgen synthesis but altered androgen receptor (AR) expression pattern. The percentage of AR-positive cells in the mesenchyme was significantly reduced, which can be the cause of decreased epithelial proliferation. Wnt9b actions can be transduced by both β-catenin-dependent and β-catenin-independent pathways in the context of target cells. Transcriptomic analysis of embryonic day (E) 12.5 Wnt9b+/+ and Wnt9b-/- mesonephroi revealed that expression of multiple WNT/β-catenin-target genes was reduced in the absence of Wnt9b. Deletion of mesenchymal β-catenin led to caudal WD degeneration and cystic formation in the cranial region. Taken together, our study uncovers the important WNT9B-AR signaling axis that mediates the epithelial-mesenchymal interaction in WD development.

The ability to grow undifferentiated oocytes in vitro from primordial follicles would increase the availability of fully grown oocytes in fertility preservation programs and other downstream applications. To date, the development of living offspring in vitro from the primordial follicle reserve has only been achieved in mice, proving the principle of the potential value of follicle culture as a source of competent oocytes. In certain pathophysiological conditions, such as polycystic ovarian syndrome, premature ovarian failure, or ovarian and blood cancer, where the ovarian tissue cannot be reintroduced into the patient, it is essential to isolate these follicles from the surrounding tissue and culture them in vitro. However, the culture systems that produce mature oocytes from isolated primordial follicles are still under investigation. Upon isolation from the ovarian microenvironment, a critical limiting factor is follicle death after a short period of culture. Previous studies suggest that glycine, a key component of glutathione (GSH), plays a protective role against the programmed cell death mechanism, ferroptosis, in in vitro matured porcine oocytes via the System Xc-/GSH/glutathione peroxidase 4 (GPX4) axis. Employing a previously developed high-yielding primordial follicle mechanical isolation strategy and a defined culture system, we used RNA-seq to advance the knowledge of the main transcriptional events and molecular factors determining follicle fate in a 2D culture system. Our transcriptome analyses identified genes involved in ferroptosis that may bring about primordial follicle death. To suppress ferroptosis, glycine supplementation maintained the viability of primordial follicles at ∼85% for 16 h. Future improvements to the culture system should inhibit programmed cell death mechanisms and ensure the physiological compliance of the genes regulating primordial follicle activation and transition to the primary stage, along with effective supplementation media to develop isolated primordial follicles in vitro.

The spatiotemporal turnover of metabolites is essential for oocyte maturation, embryonic development, and cell lineage differentiation. Here, we analyzed the metabolic profiles of individual living mouse oocytes and studied how bisphosphoglycerate mutase (BPGM), an important maternal factor, influences metabolite regulation during oocyte maturation. We found that BPGM is expressed in mouse follicles, oocytes, and embryos, as well as in human embryos. Notably, deletion of Bpgm significantly reduced the rate of oocyte maturation and reduced mouse fertility, which was observed as reduced pups per litter. Also, the expression levels for meiosis-related genes and genes related to glucose metabolic pathways (glycolysis, tricarboxylic acid cycle, and pentose phosphate pathway) were altered in BPGM-deficient mouse oocytes. We used a highly sensitive, live-cell sampling approach to carry out metabolite assays using induced nanoelectrospray-ionization mass spectrometry technology on 1 picolitre of aspirated cytoplasm from oocytes. BPGM gene disruption impaired glucose metabolism pathways, tyrosine metabolism, and amino acid biosynthesis. Together, our findings indicate that Bpgm participates in oocyte and embryo development, and we demonstrate the feasibility of studying metabolite composition and other phenotypic features of single oocytes.