Neonatal exposure to high D-galactose affects germ cell development in neonatal testes organ culture.

Excess exogenous supplementation of D-galactose (D-gal), a monosaccharide and reducing sugar, generates reactive oxygen species (ROS), leading to cell damage and death. ROS accumulation is critical in aging. Therefore, D-gal-induced aging mouse models are used in aging studies. Herein, we evaluated D-gal's effect on neonatal testis development using an in vitro organ culture method. Mouse testicular fragments (MTFs) derived from neonatal testes (postnatal day 5) were cultured with 500 mM D-gal for 5 days. D-gal-treated MTFs showed a significantly increased and decreased expression of undifferentiated and differentiated germ cell markers, respectively, with a substantial reduction in meiotic cells. In D-gal-exposed MTFs, expression levels of Sertoli cell markers (Sox9 and Wt1) increased, while those of StAR and 17ß-HSD3, whose expressions are abundant in D-Gal treated adult Leydig cells, decreased. Additionally, the enzyme 3 ß-HSD1, essential for steroidogenesis in Leydig cells, was significantly reduced in D-gal-exposed MTFs compared to that in controls.D-gal significantly increased the expression of Bad, Bax, and cleaved caspase-3 and -8. Via oxidative stress in MTF. Overall, D-gal negatively regulates germ cell and Leydig cell development in neonatal testes through pro-apoptotic mechanisms and ROS.

Transgenerational response of germline histone acetyltransferases and deacetylases to nanoplastics at predicted environmental doses in Caenorhabditis elegans.

Nanoplastics could cause toxic effects on organism and their offsprings; however, how this transgenerational toxicity is formed remains largely unclear. We here examined potential involvement of germline histone acetylation regulation in modulating transgenerational toxicity of polyetyrene nanoparticle (PS-NP) in Caenorhabditis elegans. At parental generation (P0-G), PS-NP (1-100 µg/L) decreased expressions of germline cbp-1 and taf-1 encoding histone acetyltransferases, as well as germline expressions of sir-2.1 and hda-3 encoding histone deacetylase. Decrease in these 4 germline genes were also observed in the offspring of PS-NP (1-100 µg/L) exposed nematodes. Germline RNAi of cbp-1, taf-1, sir-2.1 and hda-3 resulted in more severe transgenerational PS-NP toxicity on locomotion and brood size. Meanwhile, in PS-NP exposed nematodes, germline RNAi of cbp-1, taf-1, sir-2.1 and hda-3 increased expression of genes encoding insulin, FGF, Wnt, and/or Notch ligands and expressions of their receptor genes in the offspring. Susceptibility to transgenerational PS-NP toxicity in cbp-1(RNAi), taf-1(RNAi), sir-2.1(RNAi), and hda-3 (RNAi) was inhibited by RNAi of these germline ligands genes. Moreover, histone deacetylase inhibition served as molecular initiating event (MIE) leading to transgenerational toxicity in epigenetic adverse outcome pathway (AOP) for nanoplastics. Our data provided evidence that germline histone acetylation regulation functioned as an important mechanism for transgenerational toxicity of nanoplastics at predicted environmental doses (PEDs) by affecting secreted ligands in organisms.

Benzo(a)pyrene exposure during pregnancy leads to germ cell apoptosis in male mice offspring via affecting histone modifications and oxidative stress levels.

Infertility has gradually become a global health concern, and evidence suggests that exposure to environmental endocrine-disrupting chemicals (EDCs) represent one of the key causes of infertility. Benzo(a)pyrene (BaP) is a typical EDC that is widespread in the environment. Previous studies have detected BaP in human urine, semen, cervical mucus, oocytes and follicular fluid, resulting in reduced fertility and irreversible reproductive damage. However, the mechanisms underlying the effects of gestational BaP exposure on offspring fertility in male mice have not been fully explored. In this study, pregnant mice were administered BaP at doses of 0, 5, 10 and 20 mg/kg/day via gavage from Days 7.5 to 12.5 of gestation. The results revealed that BaP exposure during pregnancy disrupted the structural integrity of testicular tissue, causing a disorganized arrangement of spermatogenic cells, compromised sperm quality, elevated levels of histone modifications and increased apoptosis in the testicular tissue of F1 male mice. Furthermore, oxidative stress was also increased in the testicular tissue of F1 male mice. BaP activated the AhR/ERα signaling pathway, affected H3K4me3 expression and induced apoptosis in testicular tissue. AhR and Cyp1a1 were overexpressed, and the expression of key molecules in the antioxidant pathway, including Keap1 and Nrf2, was reduced. The combined effects of these molecules led to apoptosis in testicular tissues, damaging and compromising sperm quality. This impairment in testicular cells further contributed to compromised testicular tissues, ultimately impacting the reproductive health of F1 male mice.

Noncanonical inheritance of phenotypic information by protein amyloids.

All known heritable phenotypic information in animals is transmitted by direct inheritance of nucleic acids, their covalent modifications or histone modifications that modulate expression of associated genomic regions. Nonetheless, numerous familial traits and disorders cannot be attributed to known heritable molecular factors. Here we identify amyloid-like protein structures that are stably inherited in wild-type animals and influence traits. Their perturbation by genetic, environmental or pharmacological treatments leads to developmental phenotypes that can be epigenetically passed onto progeny. Injection of amyloids isolated from different phenotypic backgrounds into naive animals recapitulates the associated phenotype in offspring. Genetic and proteomic analyses reveal that the 26S proteasome and its conserved regulators maintain heritable amyloids across generations, which enables proper germ cell sex differentiation. We propose that inheritance of a proteinaceous epigenetic memory coordinates developmental timing and patterning with the environment to confer adaptive fitness.

Erasure of DNA methylation in rat fetal germ cells is sex-specific and sensitive to maternal high-fat diet.

In mammals, DNA methylation (DNAme) erasure and reinstatement during embryo development and germline establishment are sensitive to the intrauterine environment. Maternal intake of a high-fat diet (HFD), associated with excessive gestational weight gain, has transgenerational effects on offspring health, which may be mediated by changes in DNAme in the germline. Here, we tested the impact of a maternal HFD on embryonic germline DNAme erasure using a rat strain that expresses green fluorescent protein specifically in germ cells. DNAme was analysed by methyl-seq capture in germ cells collected from male and female F1 gonads at gestational day 16. Our data show that although HFD induced global hypomethylation in both sexes, DNAme erasure in female germ cells was more advanced compared to male germ cells. The delay in DNAme erasure in males and the greater impact of HFD suggest that male germ cells are more vulnerable to alterations by exogenous factors.

Cadmium exposure induces multigenerational inheritance of germ cell apoptosis and fertility suppression in Caenorhabditis elegans.

Cadmium is a significant environmental pollutant that poses a substantial health hazard to humans due to its propensity to accumulate in the body and resist excretion. We have a comprehensive understanding of the damage caused by Cd exposure and the mechanisms of tolerance, however, the intricate mechanisms underlying multigenerational effects resulting from Cd exposure remain poorly understood. In this study, Caenorhabditis elegans were used as a model organism to investigate Cd-induced multigenerational effects and its association with epigenetic modifications. The results showed that Cd exposure leads to an increase in germ cell apoptosis and a decrease in fertility, which can be passed down to subsequent generations. Further analysis revealed that transcription factors DAF-16/FOXO and SKN-1/Nrf2 play essential roles in responding to Cd exposure and in the transgenerational induction of germ cell apoptosis. Additionally, histone H3K4 trimethylation (H3K4me3) marks stress-responsive genes and enhances their transcription, ultimately triggering multigenerational germ cell apoptosis. This study provides compelling evidence that the detrimental effects of Cd on the reproductive system can be inherited across generations. These findings enhance our understanding of the multigenerational effects of environmental pollutants and may inform strategies for the prevention and control of such pollutants.

Role of PI3K/AKT signaling pathway involved in self-renewing and maintaining biological properties of chicken primordial germ cells.

Avian primordial germ cells (PGCs) are important culture cells for the production of transgenic chickens and preservation of the genetic resources of endangered species; however, culturing these cells in vitro proves challenging. Although the proliferation of chicken PGCs is dependent on insulin, the underlying molecular mechanisms remain unclear. In the present study, we explored the expression of the PI3K/AKT signaling pathway in PGCs, investigated its effects on PGC self-renewal and biological properties, and identified the underlying mechanisms. Our findings indicated that although supplementation with the PI3K/AKT activator IGF-1 failed to promote proliferation under the assessed culture conditions, the PI3K/AKT inhibitor LY294002 resulted in retarded cell proliferation and reduced expression of germ cell-related markers. We further demonstrated that inhibition of PI3K/AKT regulates the cell cycle and promotes apoptosis in PGCs by activating the expression of BAX and inhibiting that of Bcl-2. These findings indicated that the PI3K/AKT pathway is required for cell renewal, apoptosis, and maintenance of the reproductive potential in chicken PGCs. This study aimed to provide a theoretical basis for the optimization and improvement of a culture system for chicken PGCs and provide insights into the self-renewal of vertebrate PGCs as well as potential evolutionary changes in this unique cell population.

Nanoplastic at environmentally relevant concentrations induces toxicity across multiple generations associated with inhibition in germline G protein-coupled receptor CED-1 in Caenorhabditis elegans.

Nanoplastics at environmentally relevant concentrations (ERCs) could cause transgenerational toxicity on organisms. Caenorhabditis elegans is an important model for the study of transgenerational toxicology of pollutants. Nevertheless, the underlying mechanisms for the control of transgenerational nanoplastic toxicity by germline signals remain largely unclear. In C. elegans, exposure to 1-100 µg/L polystyrene nanoparticle (PS-NP) decreased expression of germline ced-1 encoding a G protein-coupled receptor at parental generation (P0-G). After PS-NP exposure at P0-G, transgenerational decrease in germline ced-1 expression could be detected. Meanwhile, the susceptibility to transgenerational PS-NP toxicity was observed in ced-1(RNAi) animals. After PS-NP exposure at P0-G, germline RNAi of ced-1 increased expressions of met-2 and set-6 encoding histone methylation transferases. The susceptibility of ced-1(RNAi) to transgenerational PS-NP toxicity could be inhibited by RNAi of met-2 and set-6. Moreover, in PS-NP exposed met-2(RNAi) and set-6(RNAi) nematodes, expressions of ins-39, wrt-3, and/or efn-3 encoding secreted ligands were decreased. Therefore, our results demonstrated that inhibition in germline CED-1 mediated the toxicity induction of nanoplastics at ERCs across multiple generations in nematodes.

Acute effects of atrazine on the immunoexpressions of sertoli and germ cells molecular markers, cytokines, chemokines, and sex hormones levels in mice testes and epididymides.

Atrazine is currently one of the most commonly used agrochemicals in the United States and elsewhere. Here, we studied the immunoexpression of molecular markers of mammalian testicular functions: androgen receptor (AR), promyelocytic leukemia zinc finger (PLZF), GDNF family receptor alpha-1 (GFRA1), VASA/DDX4 (DEAD-Box Helicase 4) as well as the levels of intratesticular and intra-epididymal estradiol (E2) and dihydrotestosterone (DHT), tumor necrosis factor-alpha (TNF-α), interferon-gamma (IFN-γ), interleukins (IL-1ß and IL-6, IL-10) and testicular chemokines (CXCL-1, CCL-2 and CCL3) in BalB/c mice after a sub-acute gavage treatment with a gonado-toxin, atrazine (50 mg/kg body wt.) for three days. We found high numbers of AR immunopositive Sertoli cells and low numbers of GFRA1, PLZF and VASA/DDX4-positive germ cells in the seminiferous tubule regions of the testes. While TNF-α level in the testes fell and remained unchanged in the epididymides, IFN-γ levels in the testes remained constant but increased in the epididymides. E2 and DHT concentrations remained unaltered in the testes but were changed in the epididymides. There were no significant changes in the levels of interleukins in the testis and epididymis. Intratesticular chemokines were also not significantly altered, except for CCL-4, which was increased in the testis. Light microscopy of the epididymis showed detached epithelium and some detached cells in the lumen. It is concluded that atrazine changed the inflammatory status of the gonads and highlighted Sertoli and undifferentiated spermatogonia as important targets for atrazine's toxic effects in the testis of mice. Concerning the epididymis, atrazine altered the epididymal hormonal concentrations and promoted histopathological modifications in its parenchyma.

Amino-modified nanoplastics at predicted environmental concentrations cause transgenerational toxicity through activating germline EGF signal in Caenorhabditis elegans.

In the real environment, some chemical functional groups are unavoidably combined on the nanoplastic surface. Reportedly, amino-modified polystyrene nanoparticles (PS-A NPs) exposure in parents can induce severe transgenerational toxicity, but the underlying molecular mechanisms remain largely unclear. Using Caenorhabditis elegans as the animal model, this study was performed to investigate the role of germline epidermal growth factor (EGF) signal on modulating PS-A NPs' transgenerational toxicity. As a result, 1-10 µg/L PS-A NPs exposure transgenerationally enhanced germline EGF ligand/LIN-3 and NSH-1 levels. Germline RNAi of lin-3 and nsh-1 was resistant against PS-A NPs' transgenerational toxicity, implying the involvement of EGF ligand activation in inducing PS-A NPs' transgenerational toxicity. Furthermore, LIN-3 overexpression transgenerationally enhanced EGF receptor/LET-23 expression in the progeny, and let-23 RNAi in F1-generation notably suppressed PS-A NPs' transgenerational toxicity in the exposed worms overexpressing germline LIN-3 at P0 generation. Finally, LET-23 functioned in neurons and intestine for regulating PS-A NPs' transgenerational toxicity. LET-23 acted at the upstream DAF-16/FOXO within the intestine in response to PS-A NPs' transgenerational toxicity. In neurons, LET-23 functioned at the upstream of DAF-7/DBL-1, ligands of TGF-ß signals, to mediate PS-A NPs' transgenerational toxicity. Briefly, this work revealed the exposure risk of PS-A NPs' transgenerational toxicity, which was regulated through activating germline EGF signal in organisms.

Tebuconazole Induces Mouse Fetal Testes Damage via ROS Generation in an Organ Culture Method.

The fungicide tebuconazole (TEB) poses risks to human and animal health via various exposure routes. It induces toxicity in multiple organs and disrupts reproductive health by affecting steroid hormone synthesis and fetal development. In this study, we investigated the impact of TEB on fetal testes using in vitro models, focusing on germ, Sertoli, and Leydig cells, and explored the mechanisms underlying cellular damage. The results revealed significant damage to germ cells and disruption of Leydig cell development. TEB exposure led to a decrease in germ cell numbers, as indicated by histological and immunostaining analyses. TEB induced the up- and down-regulation of the expression of fetal and adult Leydig cell markers, respectively. Additionally, TEB-treated fetal testes exhibited increased expression of oxidative-stress-related genes and proteins. However, co-treatment with the antioxidant N-acetylcysteine mitigated TEB-induced germ cell damage and prevented abnormal Leydig cell development. These findings suggest that administration of antioxidants can prevent the intratesticular damage typically caused by TEB exposure.

Spermatogenesis in mouse testicular organoids with testis-specific architecture, improved germ cell survival and testosterone production.

This study presents a biphasic approach to overcome the limitations of current testicular organoid (TO) cultures, including histological heterogeneity, germ cell loss and absence of spermatogenesis. Agarose microwells were utilized to create TOs from prepubertal C57BL/6 J testicular cells. First emphasis was on improving germ cell survival during the initial 2-week reorganization phase by comparingα-MEM + 10% knockout serum replacement (KSR) medium, known to support TO generation in mice, to three optimized media (1-3). Cell densities and culture dynamics were also tested to recreate histological resemblance to testes. After optimizing germ cell survival and cell organization, the effect of growth factors and immunomodulation through CD45+immune cell depletion or dexamethasone (DEX) supplementation were assessed for enhancing spermatogenesis during the subsequent differentiation phase. Testicular cells self-reorganized into organoids resembling the testicular anatomical unit, characterized by one tubule-like structure surrounded by interstitium. Media 1-3 proved superior for organoid growth during the reorganization phase, with TOs in medium 3 exhibiting germ cell numbers (7.4% ± 4.8%) comparable to controls (9.3% ± 5.3%). Additionally, 37% ± 30% demonstrated organized histology from 32 × 103cells under static conditions. Switching toα-MEM + 10% KSR during the differentiation phase increased formation efficiency to 85 ± 7%, along with elevated germ cell numbers, testosterone production (3.1 ± 0.9 ng ml-1) and generation ofγ-H2AX+spermatid-like cells (steps 8-11, 1.2% ± 2.2% of the total). Adding differentiation factors to theα-MEM increased spermatid-like cell numbers to 2.9% ± 5.9%, confirmed through positive staining for CREM, transition protein 1, and peanut agglutinin. Although, these remained diploid with irregular nuclear maturation. DEX supplementation had no additional effect, and immune cell depletion adversely impacted TO formation. The manipulability of TOs offers advantages in studying male infertility and exploring therapies, with scalability enabling high-throughput chemical screening and reducing animal usage in reproductive toxicity and drug discovery studies.

Pyriproxyfen enhances germline stem cell proliferation and reduces reproduction in Drosophila by up-regulating juvenile hormone signaling.

BACKGROUND: Pyriproxyfen is an insect growth regulator (IGR) that is effective against various types of insect pests. However, the molecular mechanism underlying pyriproxyfen effects on insect reproduction remains unclear. Thus, in this study, we attempted to uncover the mechanisms underlying the impact of pyriproxyfen on the reproductive system of the model organism Drosophila melanogaster. RESULTS: A significant decrease in Drosophila reproduction was observed after pyriproxyfen treatment. The juvenile hormone (JH) titer was significantly increased (120.4%) in the ovary samples of pyriproxyfen-treated flies. Likewise, the concentrations of key enzymes and the expression of key genes related to the JH signaling pathway were also increased in the pyriproxyfen-treated group compared with the control group. Furthermore, pyriproxyfen treatment significantly increased (15.6%) the number of germline stem cells (GSCs) and significantly decreased (17%) the number of cystoblasts (CBs). However, no significant differences were observed in the number of somatic cells. We performed RNA interference (RNAi) on five key genes (Met, Tai, gce, ftz-f1, and hairy) related to the JH signaling pathway in germ cells using the germ cell-specific Gal4 driver. Interestingly, RNAi of the selected genes significantly decreased the number of both GSCs and CBs in pyriproxyfen-treated transgenic flies. These results further validate that pyriproxyfen enhances GSC proliferation by up-regulating JH signaling. CONCLUSION: Our results indicate that pyriproxyfen significantly decreases reproduction by affecting germ cells in female adult ovaries. The effect of pyriproxyfen on germ cell proliferation and differentiation is mediated by an increase in JH signaling. This study has significant implications for optimizing pest control strategies, developing sustainable agriculture practices, and understanding the mechanism of insecticide action. © 2024 Society of Chemical Industry.

Characteristics of the Vasa Gene in Silurus asotus and Its Expression Response to Letrozole Treatment.

The identification and expression of germ cells are important for studying sex-related mechanisms in fish. The vasa gene, encoding an ATP-dependent RNA helicase, is recognized as a molecular marker of germ cells and plays a crucial role in germ cell development. Silurus asotus, an important freshwater economic fish species in China, shows significant sex dimorphism with the female growing faster than the male. However, the molecular mechanisms underlying these sex differences especially involving in the vasa gene in this fish remain poorly understood. In this work, the vasa gene sequence of S. asotus (named as Savasa) was obtained through RT-PCR and rapid amplification of cDNA end (RACE), and its expression in embryos and tissues was analyzed using qRT-PCR and an in situ hybridization method. Letrozole (LT) treatment on the larvae fish was also conducted to investigate its influence on the gene. The results revealed that the open reading frame (ORF) of Savasa was 1989 bp, encoding 662 amino acids. The SaVasa protein contains 10 conserved domains unique to the DEAD-box protein family, showing the highest sequence identity of 95.92% with that of Silurus meridionalis. In embryos, Savasa is highly expressed from the two-cell stage to the blastula stage in early embryos, with a gradually decreasing trend from the gastrula stage to the heart-beating stage. Furthermore, Savasa was initially detected at the end of the cleavage furrow during the two-cell stage, later condensing into four symmetrical cell clusters with embryonic development. At the gastrula stage, Savasa-positive cells increased and began to migrate towards the dorsal side of the embryo. In tissues, Savasa is predominantly expressed in the ovaries, with almost no or lower expression in other detected tissues. Moreover, Savasa was expressed in phase I-V oocytes in the ovaries, as well as in spermatogonia and spermatocytes in the testis, implying a specific expression pattern of germ cells. In addition, LT significantly upregulated the expression of Savasa in a concentration-dependent manner during the key gonadal differentiation period of the fish. Notably, at 120 dph after LT treatment, Savasa expression was the lowest in the testis and ovary of the high concentration group. Collectively, findings from gene structure, protein sequence, phylogenetic analysis, RNA expression patterns, and response to LT suggest that Savasa is maternally inherited with conserved features, serving as a potential marker gene for germ cells in S.asotus, and might participate in LT-induced early embryonic development and gonadal development processes of the fish. This would provide a basis for further research on the application of germ cell markers and the molecular mechanisms of sex differences in S. asotus.

Aged polystyrene microplastics cause reproductive impairment via DNA-damage induced apoptosis in Caenorhabditis elegans.

Although polystyrene microplastics (PS-MPs) could induce toxic effects on environmental organisms, the toxicity of aged PS-MPs with H2O2 on soil organisms remains unclear. Our study utilized Caenorhabditis elegans as model organism to examine the reproductive toxicity of pristine PS-MPs (pPS-MPs) and aged PS-MPs (aPS-MPs) at environmentally relevant concentrations (0.1-100 µg/L). Acute exposure to aPS-MPs could induce greater reproductive impairment compared to pPS-MPs, as evidenced by changes in brood size and egg release. Assessment of gonad development using the number of mitotic cells, length of gonad arm, and relative area of gonad arm as parameters revealed a high reproductive toxicity caused by aPS-MPs exposure. Furthermore, aPS-MPs exposure promoted substantial germline apoptosis. Additionally, exposure to aPS-MPs (100 µg/L) markedly altered the expression of DNA damage-induced apoptosis-related genes (e.g., egl-1, cep-1, clk-2, ced-3, -4, and -9). Alterations in germline apoptosis caused by aPS-MPs were observed in mutants of cep-1, hus-1, egl-1, ced-3, -4, and -9. Consequently, the augmentation of reproductive toxicity resulting from aPS-MPs exposure was attributed to DNA damage-triggered cellular apoptosis. Additionally, the EGL-1-CEP-1-HUS-1-CED-3-CED-4-CED-9 signaling pathway was identified as a key regulator of germline apoptosis in nematodes. Our study provides insights into potential environmental risk of aPS-MPs with H2O2 on environmental organisms.

The effects of a glyphosate-based herbicide on the bovine gametes during an in vitro embryo production model.

Roundup® (R), while it is the most used herbicide globally, and its residues are ubiquitous in urban and suburban areas, its impact on vertebrates' safety remains highly debated. Here, in three in vitro experiments, we investigated the effects of a very low dose (1 ppm) of R on the fertilization capacity and embryo development in cattle. In the first experiment, frozen-thawed bull semen exposed to R for 1 h exhibited reduced motility parameters but unaffected fertilization ability. However, after in vitro fertilization, the rates of embryo formation were significantly lower compared to the untreated controls. In the second experiment, oocytes exposed to R during in vitro maturation showed reduced cleavage rates, and the embryo yield on days 7, 8, and 9 of embryo culture was significantly lower than that of the controls. In the third experiment, oocytes were matured in the presence of R and in a medium containing both R and Zinc, chosen to offer antioxidant protection to the oocytes. Day-7 blastocysts were analyzed for the expression of genes associated with oxidative stress, apoptosis, and epigenetic reprogramming. Exposure to R markedly suppressed embryo formation rates compared to the controls. The combination of R with Zinc restored the blastocyst yield, which on days 8 and 9 was comparable to that of the controls and higher than the groups exposed only to R on all days. The gene expression analysis revealed that R promotes oxidative stress development, triggers apoptosis, and induces epigenetic changes in developing embryos, while zinc presence alleviates these adverse effects of R. These findings imply that even at very low doses, R could be highly toxic, leading to functional abnormalities in both gametes, potentially affecting fertility in both genders.

Long-term exposure to 6-PPD quinone reduces reproductive capacity by enhancing germline apoptosis associated with activation of both DNA damage and cell corpse engulfment in Caenorhabditis elegans.

Recently, 6-PPD quinone (6-PPDQ), a derivative of tire antioxidant 6-PPD, was reported to have acute toxicity for organisms. However, the possible reproductive toxicity of 6-PPDQ is still largely unclear. In this study, the reproductive toxicity of 6-PPDQ after long-term exposure was further investigated in Caenorhabditis elegans. Exposure to 1 and 10 µg/L 6-PPDQ reduced the reproductive capacity. Meanwhile, exposure to 1 and 10 µg/L 6-PPDQ enhanced the germline apoptosis, which was accompanied by upregulation of ced-3, ced-4, and egl-1 expressions and downregulation of ced-9 expression. The observed increase in germline apoptosis in 1 and 10 µg/L 6-PPDQ exposed nematodes was associated with the enhancement in DNA damage and increase in expressions of related genes of cep-1, clk-2, hus-1, and mrt-2. The detected enhancement in germline apoptosis in 1 and 10 µg/L 6-PPDQ exposed nematodes was further associated with the increase in expressions of ced-1 and ced-6 governing the cell corpse engulfment process. Molecular docking analysis indicated the binding potentials of 6-PPDQ with three DNA damage checkpoints (CLK-2, HUS-1, and MRT-2) and corpse-recognizing phagocytic receptor CED-1. Therefore, our data suggested the toxicity on reproductive capacity by 6-PPDQ at environmentally relevant concentrations by enhancing DNA damage- and cell corpse engulfment-induced germline apoptosis in organisms.

Follicle-stimulating hormone-mediated decline in miR-92a-3p expression in pubertal mice Sertoli cells is crucial for germ cell differentiation and fertility.

Sertoli cells (Sc) are the sole target of follicle-stimulating hormone (FSH) in the testis and attain functional maturation post-birth to significantly augment germ cell (Gc) division and differentiation at puberty. Despite having an operational microRNA (miRNA) machinery, limited information is available on miRNA-mediated regulation of Sc maturation and male fertility. We have shown before that miR-92a-3p levels decline in pubertal rat Sc. In response to FSH treatment, the expressions of FSH Receptor, Claudin11 and Klf4 were found to be elevated in pubertal rat Sc coinciding with our finding of FSH-induced decline in miR-92a-3p levels. To investigate the association of miR-92a-3p and spermatogenesis, we generated transgenic mice where such pubertal decline of miR-92a-3p was prevented by its overexpression in pubertal Sc under proximal Rhox5 promoter, which is known to be activated specifically at puberty, in Sc. Our in vivo observations provided substantial evidence that FSH-induced decline in miR-92a-3p expression during Sc maturation acts as an essential prerequisite for the pubertal onset of spermatogenesis. Elevated expression of miR-92a-3p in post-pubertal testes results into functionally compromised Sc, leading to impairment of the blood-testis barrier formation and apoptosis of pre-meiotic Gc, ultimately culminating into infertility. Collectively, our data suggest that regulation of miR-92a-3p expression is crucial for Sc-mediated induction of active spermatogenesis at puberty and regulation of male fertility.

The emerging risk of microplastics and nanoplastics on the microstructure and function of reproductive organs in mammals: A systematic review of preclinical evidence.

AIMS: Plastic particles (PP) pollution is a global environmental concern. Although the reproductive toxicity of PP is primarily understood for invertebrates, the evidence for mammals is still fragmented. We used a systematic review framework to investigate the reproductive impact of microplastics and nanoplastics (MNP) on mammals. MATERIALS AND METHODS: Research records were screened from Embase, Medline, Scopus and Web of Science. Twelve original papers were identified and reviewed. Immunological, oxidative and morphofunctional outcomes, and the risk of bias in all studies reviewed were analyzed. KEY FINDINGS: These studies indicated that PP can accumulate in the gonads, triggering seminiferous degeneration, Sertoli cells death, blood-testis barrier disruption, sperm degeneration, malformation, reduced number and mobility, ovarian cysts, reduced follicular growth and granulosa cells death. Gonadal damage was associated with upregulation of prooxidant mediators (oxygen reactive species, lipid and DNA oxidation), cell death, proinflammatory molecular pathways and cytokines, as well as inhibition of enzymatic and non-enzymatic antioxidant defense mechanisms. Spermatogenesis, folliculogenesis, testosterone, progesterone and estrogen levels were also impaired in PP-treated animals, which were potentially associated with down-regulation of molecules involved in germ cells microstructural organization (occludin, N-cadherin, ß-catenin and connexin 43) and steroidogenesis, such as hydroxysteroid dehydrogenases, steroidogenic acute regulatory proteins, follicle stimulating and luteinizing hormones. Selection, performance and detection bias were the main limitations identified. SIGNIFICANCE: Current evidence indicates that PP can induce dose-dependent microstructural and functional gonadal damage, which is orchestrated by pro-oxidant and pro-inflammatory mechanisms that disrupt genes, molecular effectors, and hormones that control spermatogenesis and folliculogenesis.

Intragenic recombination within the pun allele of the pink-eyed dilution locus in pre-melanocytes and primordial germ cells of embryonic mice treated with N-ethyl-N-nitrosourea.

The forward or reverse processes of intragenic recombination (IGR), which occur through the addition or deletion of duplicated homologous exons of the pun allele in Pun mice, was observed in vivo, after introducing an homozygous pun allele in a C57BL/6 background. We assessed the frequency of IGR upon N-ethyl-N-nitrosourea (ENU) treatment of pre-melanocytes (PMCs: somatic cells) and primordial germ cells (PGCs: germ cells) of embryonic mice at 10.5 days of development (E10.5). We simultaneously examined IGR and other mutations at the p locus of PMCs responsible for coat color in the offspring obtained by crossing pun/pun with pun/P mice. The frequencies of both spontaneous and ENU-induced IGR were markedly higher than that of the recessive mutation (RM) in PMCs obtained from crossing C57BL/6 and PW strains (Shibuya et al., 1982). ENU also induces IGR at a higher frequency in PGCs at E10.5, which was observed in the next generation. These results indicate that ENU, which preferentially induces gene mutations through base substitution, also induces IGR at a high frequency in the pun allele in both somatic and germ cells of embryonic mice at the E10.5 developmental stage.