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Open access

Ram Prakash Yadav, Sini Leskinen, Lin Ma, Juho-Antti Mäkelä, and Noora Kotaja

In brief

Proper regulation of heterochromatin is critical for spermatogenesis. This study reveals the dynamic localization patterns of distinct chromatin regulators during spermatogenesis and disrupted sex chromatin status in spermatocytes in the absence of DICER.

Abstract

Heterochromatin is dynamically formed and organized in differentiating male germ cells, and its proper regulation is a prerequisite for normal spermatogenesis. While heterochromatin is generally transcriptionally silent, we have previously shown that major satellite repeat (MSR) DNA in the pericentric heterochromatin (PCH) is transcribed during spermatogenesis. We have also shown that DICER associates with PCH and is involved in the regulation of MSR-derived transcripts. To shed light on the heterochromatin regulation in the male germline, we studied the expression, localization and heterochromatin association of selected testis-enriched chromatin regulators in the mouse testis. Our results show that HELLS, WDHD1 and BAZ1A are dynamically expressed during spermatogenesis. They display limited overlap in expression, suggesting involvement in distinct heterochromatin-associated processes at different steps of differentiation. We also show that HELLS and BAZ1A interact with DICER and MSR chromatin. Interestingly, deletion of Dicer1 affects the sex chromosome heterochromatin status in late pachytene spermatocytes, as demonstrated by mislocalization of Polycomb protein family member SCML1 to the sex body. These data substantiate the importance of dynamic heterochromatin regulation during spermatogenesis and emphasize the key role of DICER in the maintenance of chromatin status in meiotic male germ cells.

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Jung-Chien Cheng, Yibo Gao, Jiaye Chen, Qingxue Meng, and Lanlan Fang

In brief

Although the pro-invasive role of epidermal growth factor (EGF) has been reported in human trophoblast cells, the underlying mechanism remains largely unexplored. This work reveals that EGF-induced downregulation of connective tissue growth factor (CTGF) mediates the EGF-stimulated human trophoblast cell invasion.

Abstract

During the development of the placenta, trophoblast cell invasion must be carefully regulated. Although EGF has been shown to promote trophoblast cell invasion, the underlying mechanism remains largely undetermined. Our previous study using RNA-sequencing (RNA-seq) has identified that kisspeptin-1 is a downstream target of EGF in a human trophoblast cell line, HTR-8/SVneo, and mediates EGF-stimulated cell invasion. In the present study, after re-analysis of our previous RNA-seq data, we found that the CTGF was also downregulated in response to the EGF treatment. The inhibitory effects of EGF on CTGF mRNA and protein levels were confirmed in HTR-8/SVneo cells by reverse transcription quantitative real-time PCR and western blot, respectively. Treatment with EGF activated both PI3K/AKT and ERK1/2 signaling pathways. Using pharmacological inhibitors, our results showed that EGFR-mediated activation of PI3K/AKT signaling was required for the EGF-downregulated CTGF mRNA and protein levels. Matrigel-coated transwell invasion assays demonstrated that EGF treatment stimulated cell invasion. In addition, the invasiveness of HTR-8/SVneo cells was suppressed by treatment with recombinant human CTGF. By contrast, siRNA-mediated knockdown of CTGF increased cell invasion. Notably, the EGF-promoted HTR-8/SVneo cell invasion was attenuated by co-treatment with CTGF. This study provides important insights into the molecular mechanisms mediating EGF-stimulated human trophoblast cell invasion and increases the understanding of the biological functions of CTGF in the human placenta.

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Xiaoheng Li, Lanlan Chen, Yiyan Wang, Huitao Li, Qiqi Zhu, and Ren-Shan Ge

In brief

Glucagon-like peptide-1 stimulates stem Leydig cell development. Glucagon-like peptide-1 stimulates stem Leydig cell differentiation without affecting its proliferation.

Abstract

The regulators of stem Leydig cell (SLC) development remain largely unknown. The effect of glucagon-like peptide-1 (GLP-1) on rat SLC proliferation and differentiation was investigated using a 3D tissue culture system and an ethane dimethane sulfonate (EDS)-treated in vivo LC regeneration model. RNA-seq analysis was performed to analyze pathways in which GLP-1 may be involved. GLP-1 (3 and 30 nmol/L) significantly increased medium testosterone abundances and upregulated the expression of Scarb1, Cyp11a1, and Hsd11b1. GLP-1 in vitro did not affect SLC proliferation by 5-Ethynyl-2’- deoxyuridine (EdU) incorporation assay. Intratesticular injection of GLP-1 (10 and 100 ng/testis) into the LC-depleted testis from day 14 to day 28 post-EDS significantly increased serum testosterone abundances and upregulated the expression of Cyp11a1, Hsd3b1, and Hsd11b1. It did not affect the number of HSD11B1+ and CYP11A1+ LCs. RNA-seq analysis revealed that GLP-1 upregulated several pathways, including cAMP-PKA-EPAC1 and MEK/ERK1/2. GLP-1 stimulates SLC differentiation without affecting its proliferation, showing its novel action and mechanism on rat SLC development.

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Tatiane Pereira Scarpelli, Eloisa Zanin Pytlowanciv, Maria Etelvina Pinto-Fochi, Sebastião Roberto Taboga, and Rejane Maira Góes

In brief

Maternal obesity plus high-fat diet in breastfeeding induces stromal hyperplasia and diffuse acinar atrophy in the rat prostate at aging, related to dyslipidemia and testosterone reduction. The high-lipid nutritional environment from intrauterine and throughout life favors the development of prostatic intraepithelial neoplasia and aggravated degenerative alterations in the gland.

Abstract

Maternal obesity and high-fat diet (HFD) affect permanently prostate histophysiology in adulthood, but the consequences during aging are unknown. Here, we evaluated the prostate alterations in middle-aged rats subjected to a high-lipid nutritional environment (HLE) in different ontogenetic periods. Wistar rats (56 weeks of age) were assigned into groups exposed to standard nutrition (C) or HLE during gestation (G), gestation and lactation (GL), from lactation onward (L), from weaning onward (W) and from gestation onward (AL). HLE in the periods after weaning consisted of HFD (20% fat), and during gestation and lactation it also included previous maternal obesity induced by the HFD. HLE increased total cholesterol and triglyceride levels in all groups and led to insulin resistance in GL and AL and obesity in L. Serum testosterone levels decreased ~67% in GL, ~146% in L and W, and ~233% in AL. Histological and stereological analysis revealed an increment of the stromal compartment and collagen fibers in the prostates of all HLE groups, as well as degenerative lesions, such as cell vacuolation and prostate concretions. HLE aggravated acinar atrophy in G, GL, and L, and in AL it reached more than 50% of the prostate area for most animals. The foci of prostatic intraepithelial neoplasia increased in AL. Tissue expression of androgen receptor did not vary among groups, except for a higher stromal expression for G and GL. Even when restricted to gestation and lactation, HLE induces diffuse acinar atrophy in the aging prostate and worsens degenerative and premalignant lesions when it continues throughout life.

Open access

Dalileh Nabi, Davide Bosi, Neha Gupta, Nidhi Thaker, Rafael Fissore, and Lynae M Brayboy

In brief

Oocyte quality remains the most important and unsolved issue in reproduction. Our data show that multidrug resistance transporters and oocyte mitochondria are involved in determining oocyte quality in a mouse model.

Abstract

Multidrug resistance transporter-1 (MDR-1) is a transmembrane ATP-dependent effluxer present in organs that transport a variety of xenobiotics and by-products. Previous findings by our group demonstrated that this transporter is also present in the oocyte mitochondrial membrane and that its mutation led to abnormal mitochondrial homeostasis. Considering the importance of these organelles in the female gamete, we assessed the impact of MDR-1 dysfunction on mouse oocyte quality, with a particular focus on the meiotic spindle organization, aneuploidies, Ca2+ homeostasis, ATP production and mtDNA mutations. Our results demonstrate that young Mdr1a mutant mice produce oocytes characterized by lower quality, with a significant delay in the germinal vesicle to germinal vesicle breakdown transition, an increased percentage of symmetric divisions, chromosome misalignments and a severely altered meiotic spindle shape compared to the wild types. Mutant oocytes exhibit 7000 more SNPs in the exomic DNA and twice the amount of mitochondrial DNA (mtDNA) SNPs compared to the wild-type ones. Ca2+ analysis revealed the inability of MDR-1 mutant oocytes to manage Ca2+ storage content and oscillations in response to several stimuli, and ATP quantification shows that mutant oocytes trend toward lower ATP levels compared to wild types. Finally, 1-year-old mutant ovaries express a lower amount of SIRT1, SIRT3, SIRT5, SIRT6 and SIRT7 compared to wild-type levels. These results together emphasize the importance of MDR-1 in mitochondrial physiology and highlight the influence of MDR-1 on oocyte quality and ovarian aging.

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Hui Li, Huan Wang, Jianmin Xu, Xinxin Zeng, Yingpu Sun, and Qingling Yang

In brief

Oocyte quality and its NAD+ level decrease with time during in vitro culture. This study shows that nicotinamide riboside (NR) supplementation improves early embryonic development potential in post-ovulatory oocytes by decreasing the reactive oxygen species (ROS) levels and reducing DNA damage and apoptosis which could potentially increase the success rate of assisted reproductive technology (ART).

Abstract

The quality of post-ovulatory oocytes deteriorates over time, impacting the outcome of early embryonic development during human ART. We and other groups have found that NAD+, a prominent redox cofactor and enzyme substrate, decreases in both aging ovaries and oocytes. In this study, we found that the NAD+ levels decreased in the post-ovulatory mouse oocytes during in vitro culture and this decrease was partly prevented by NR supplementation. NR treatmenty restored MII oocyte quality and enhanced the early embryonic development potential of post-ovulatory oocytes via alleviating mitochondrial dysfunction and maintaining normal spindle/chromosome structure. Also, treatment with NR decreased ROS levels and reduced DNA damage and apoptosis in post-ovulatory oocytes. Taken together, our findings indicated that NR supplementation increases the oocyte quality and early embryonic development potential in post-ovulatory oocytes which could potentially increase the success rate of ART.

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Rafael R Domingues, OJ Ginther, Victor Gomez-Leon, Polyana Nunes da Silva, Thadeu Castro, August Hoppmann, and Milo C Wiltbank

In brief

Endometrial and luteal synthesis of prostaglandin F2alpha (PGF2A) occurs before and during luteolysis and is critical for luteal regression. This study demonstrates that PGF2A stimulates further PGF2A synthesis (autoamplification) apparently from the corpus luteum.

Abstract

Understanding the endocrine profile of prostaglandin F2alpha (PGF2A) autoamplification is fundamental to comprehend luteal and endometrial responses to PGF2A. On day 10 of postovulation (preluteolysis), heifers (n  = 6/group) were treated intrauterine with saline or PGF2A (0.5 mg; hour 0). A third group received flunixin meglumine + PGF (FM+PGF) to prevent endogenous synthesis of PGF2A. Exogenous PGF2A was metabolized at hour 2 as measured by PGF2A metabolite (PGFM). From hours 5 to 48, concentrations of PGFM were greatest in the PGF group, smallest in the FM+PGF, and intermediate in the control suggesting endogenous synthesis of PGF2A only in PGF group. Progesterone (P4) concentrations decreased transiently between hours 0 and 1 in PGF and FM+PGF groups but rebounded to pretreatment concentrations by hours 6 and 4, respectively. No control or FM+PGF heifers underwent luteolysis during the experimental period. Conversely, in the PGF group, one heifer had complete luteolysis (P4 < 1 ng/mL), two heifers had partial luteolysis followed by P4 and CL resurgence by hour 48, and three heifers did not undergo luteolysis. Endogenous PGF2A appears to be of luteal origin due to the lack of pulsatile pattern of PGFM and lack of endometrial upregulation of oxytocin receptor (typical of endometrial synthesis of PGF2A), whereas luteal downregulation of PGF receptor and HPGD indicates a classic luteal response to PGF2A signaling although other specific mechanisms were not investigated. The hypothesis was supported that a single PGF2A treatment simulating the peak of a natural luteolytic pulse and the uteroovarian transport of PGF2A stimulates measurable endogenous PGF2A production.

Free access

Bridget M Arman, Natalie K Binder, Natasha de Alwis, Tu’uhevaha J Kaitu’u-Lino, and Natalie J Hannan

In brief

Preterm birth is the leading cause of perinatal morbidity and mortality; however, current therapies offer limited efficacy to delay birth and improve neonatal outcomes. This review explores the potential of repurposing drugs with known safety profiles to quench uterine contractions and inflammation, identifying promising agents for clinical trials.

Abstract

Preterm birth is the leading cause of neonatal morbidity and mortality globally. Despite extensive research into the underlying pathophysiology, rates of preterm birth have not significantly reduced. Currently, preterm labour management is based on optimising neonatal outcomes. Treatment involves administering drugs (tocolytics) to suppress uterine contractions to allow sufficient time for transfer to an appropriate facility and administration of antenatal corticosteroids for fetal lung maturation. Current tocolytics are limited as they are associated with adverse maternal and fetal effects and only delay delivery for a short period. There has been a serious lack of therapeutic development for preterm birth, and new approaches to protect against or delay preterm birth are urgently needed. Repurposing drugs for the prevention of preterm birth presents as a promising approach by reducing the time and costs associated with pharmaceutical drug development. In this review, we explore the evidence for the potential of therapies, specifically proton pump inhibitors, tumour necrosis factor inhibitors, prostaglandin receptor antagonists, aspirin, and statins, to be repurposed as preventatives and/or treatments for preterm birth. Importantly, many of these innovative approaches being explored have good safety profiles in pregnancy. We also review how delivery of these drugs can be enhanced, either through targeted delivery systems or via combination therapy approaches. We aim to present innovative strategies capable of targeting multiple aspects of the complex pathophysiology that underlie preterm birth. There is an urgent unmet need for preterm birth therapeutic development, and these strategies hold great promise for improving neonatal outcomes.

Free access

Xuesong Sui, Arne Klungland, and Lu Gao

In brief

RNA modifications play key roles in regulating various biological processes. This article discusses and summarizes the recent advances of RNA m6A modifications related to mammalian gametogenesis, early embryonic development, and miscarriage.

Abstract

The epitranscriptome is defined as the collection of post-transcriptional chemical modifications of RNA in a cell. RNA methylation refers to the chemical post-transcriptional modification of RNA by selectively adding methyl groups under the catalysis of a methyltransferase. The N6 methyladenosine (m6A) is one of the most common of the more than 100 known RNA modifications. Recent research has revealed that RNA m6A modifications are reversible. Additionally, m6A containing RNA can be selectively identified by immunoprecipitation followed by high-throughput sequencing (MeRIP-SEQ). These two developments have inspired a tremendous effort to unravel the biological role of m6A. The role of RNA m6A modifications in immune regulation, cell division, stem cell renewal, gametogenesis, embryonic development, and placental function has gradually emerged, which is of great significance for the study of post-transcriptional regulation of gene expression in reproductive biology. This review summarizes the current knowledge about RNA m6A modification in a variety of mammalian reproductive events.

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Olivia E Smith, Fanny Morin, Vickie Roussel, Micka C Bertucci, Alexandre Boyer, and Bruce D Murphy

In brief

The nuclear receptor steroidogenic factor 1 (SF-1) is essential for mature mouse gonad steroidogenic gene expression, for Leydig and Sertoli cell function, and depletion of SF-1 in steroidogenic cells of the testis compromises steroidogenesis, spermatogenesis and male fertility.

Abstract

Steroidogenic factor 1 (SF-1 or NR5A1) plays an essential role in the development of fetal gonads and regulates genes involved in steroid biosynthesis. Since SF-1 is expressed in multiple cell types in mouse gonads, we developed three novel conditional knockout (cKO) mouse models employing Cre-recombinase and floxed alleles of SF-1 (Nr5a1f/f) to identify its role in testes and ovaries of mature mice: Cytochrome P450 17α-hydroxylase (Cyp17Cre/+;Nr5a1f/f, Leydig and theca cell-specific), aromatase (Cyp19Cre/+;Nr5a1f/f, Sertoli and granulosa cell-specific), as well as a combination of both (Cyp17+Cyp19-Cre;Nr5a1f/f). Compared to control animals, Cyp19-Cre;Nr5a1f/f cKO males showed normal fertility and testicular function. The Cyp17Cre/+;Nr5a1f/f cKO males had smaller testis, with drastically reduced Leydig cell volumes and impaired steroidogenesis, though their reproductive performance remained comparable to controls. Some 50% of Cyp17Cre/++Cyp19Cre/+;Nr5a1f/f double-cKO (dKO) males were infertile, while the remaining 50% showed significantly reduced fertility. These dKO males also had smaller testis with degenerative seminiferous tubules, abnormal Leydig cell morphology and lower levels of intra-testicular testosterone. Abnormal Sertoli cell localization was noted in dKO testes, with increased Sox9, p27 and inhibin subunit ßb and decreased androgen receptor expression. Female mice from all genotypes showed normal reproductive capacity, though steroidogenic gene expression levels were significantly decreased in both Cyp17Cre/+;Nr5a1f/f cKO and dKO females. These results show the essential role of SF-1 in mature mouse gonad steroidogenic gene expression, for Leydig and Sertoli cell function, and that depletion SF-1 in all steroidogenic cells of the testis compromises steroidogenesis, spermatogenesis and male fertility.