Number Of Sertoli Cells Research Articles

Neonatal undernutrition induced by litter size expansion alters testicular parameters in adult Wistar rats.

Several models of maternal undernutrition reveal impairment of testicular development and compromise spermatogenesis in male offspring. The expansion of the litter size model, valuable for studying the impact of undernutrition on early development, has not yet been used to evaluate the consequences of early undernutrition in the adult male reproductive system. For this purpose, pups were raised in either normal litter (ten pups/dam) or large litter (LL; sixteen pups/dam). On postnatal day 90, sexual behaviour was evaluated or blood, adipose and reproductive tissues were collected for biochemical, histological and morphological analysis. Adult LL animals were lighter and thinner than controls. They showed increased food intake, but decrease of retroperitoneal white adipose tissue weight, glycaemia after oral glucose overload and plasma concentration of cholesterol. Reproductive organ weights were not altered by undernutrition, but histopathological analysis revealed an increased number of abnormal seminiferous tubules and number of immature spermatids in the tubular lumen of LL animals. These animals also showed reduction in total spermatic reserve and daily sperm production in the testes. Undernutrition decreased the number of Sertoli cells, and testosterone production was increased in the LL group. Mitochondrial activity of spermatozoa remained unchanged between experimental groups, suggesting no significant impact on the energy-related processes associated with sperm function. All animals from both experimental groups were considered sexually competent, with no significant difference in the parameters of sexual behaviour. We conclude that neonatal undernutrition induces histological and physiological testicular changes, without altering sperm quality and sexual behaviour of animals.

8786 Novel TGFbeta Ligand Regulated Genes in WT and FSTL3 KO Testis Identified by ChIP-Seq Analysis: Implications for Signalling Cross-talk

Abstract Disclosure: R. Ballesteros: None. A. Mukherjee: None. Folistatin Like 3 (FSTL3) is a known antagonist of activin and myostatin action that when deleted from the mouse genome displayed a set of phenotypic changes that are directly linked to glucose metabolism and reproductive function. Although our previous work showed that β-cell hyperplasia, increased pancreatic islet number and size and reduced visceral fat seen in FSTL3 knockout mice (FSTL3 KO) could be explained by enhanced activin and myostatin action, the molecular bases of the changes in the reproductive function seen in this mouse model remain elusive. In contrast to testicular degeneration produced by INHBA (activin A) transgenic overexpression in mice, deletion of FSTL3 results in increased testicular size, with increased Sertoli cell numbers and early meiotic entry of the first spermatogenic wave. This suggests a possibility that the phenotype in FSTL3 deleted mice might involve alteration of additional transcripts other than those induced by activin alone in the testis. To identify transcripts induced in FSTL3 deleted testis as a consequence of induced TGFβ ligand action we performed sequencing of Smad4 Chromatin immunoprecipitation (ChIP-Seq) from postnatal (3d) and post-pubertal (56d) whole testis. Our results show high activity of Smad4 signalling during postnatal stages compared to post-pubertal stages in both genotypes (Fstl3-KO vs wild-type). Interestingly, postnatal testis from FSTL3 KO contains significantly less activity from Smad4 signalling (69 peaks) compared to wild type (775 peaks). By contrast, on the whole, Smad4 signalling in post-pubertal stages is less active with 34 peaks in wild-type and 29 peaks in Fstl3-KO of which 8 genes are SMAD activated in both genotypes. To identify whether SMAD-dependent signalling produces significant differences in RNA expression, we then used the list of genes that generated peaks in the Smad4-ChIP-Seq experiment to filter our RNA-Seq data generated in a parallel experiment followed by differential gene expression analysis of GO terms by GSEA in postnatal testis from both genotypes. We found that Smad4-interacting genes in posnatal FSTL3 KO testis generate enrichment of 13 pathways including the androgen receptor signalling pathway. Taken together so far, our findings reveal a novel catalogue of SMAD-regulated genes in the testis, identifies a set of genes specifically upregulated in the testis in a SMAD-dependent manner upon FSTL3 deletion and also identify possible cross-talk between TGFβ and 13 other signalling pathways. Currently we are further analysing our data to identify whether there are SMAD regulated genes and pathways common in postnatal and postpubertal testes. We are also undertaking experiments to investigate expression pattern in FSTL3 KO testis to identify which pathways might specifically affect Sertoli cell proliferation and activity. Presentation: 6/2/2024

8786 Novel TGFbeta Ligand Regulated Genes in WT and FSTL3 KO Testis Identified by ChIP-Seq Analysis: Implications for Signalling Cross-talk

Abstract Disclosure: R. Ballesteros: None. A. Mukherjee: None. Folistatin Like 3 (FSTL3) is a known antagonist of activin and myostatin action that when deleted from the mouse genome displayed a set of phenotypic changes that are directly linked to glucose metabolism and reproductive function. Although our previous work showed that β-cell hyperplasia, increased pancreatic islet number and size and reduced visceral fat seen in FSTL3 knockout mice (FSTL3 KO) could be explained by enhanced activin and myostatin action, the molecular bases of the changes in the reproductive function seen in this mouse model remain elusive. In contrast to testicular degeneration produced by INHBA (activin A) transgenic overexpression in mice, deletion of FSTL3 results in increased testicular size, with increased Sertoli cell numbers and early meiotic entry of the first spermatogenic wave. This suggests a possibility that the phenotype in FSTL3 deleted mice might involve alteration of additional transcripts other than those induced by activin alone in the testis. To identify transcripts induced in FSTL3 deleted testis as a consequence of induced TGFβ ligand action we performed sequencing of Smad4 Chromatin immunoprecipitation (ChIP-Seq) from postnatal (3d) and post-pubertal (56d) whole testis. Our results show high activity of Smad4 signalling during postnatal stages compared to post-pubertal stages in both genotypes (Fstl3-KO vs wild-type). Interestingly, postnatal testis from FSTL3 KO contains significantly less activity from Smad4 signalling (69 peaks) compared to wild type (775 peaks). By contrast, on the whole, Smad4 signalling in post-pubertal stages is less active with 34 peaks in wild-type and 29 peaks in Fstl3-KO of which 8 genes are SMAD activated in both genotypes. To identify whether SMAD-dependent signalling produces significant differences in RNA expression, we then used the list of genes that generated peaks in the Smad4-ChIP-Seq experiment to filter our RNA-Seq data generated in a parallel experiment followed by differential gene expression analysis of GO terms by GSEA in postnatal testis from both genotypes. We found that Smad4-interacting genes in posnatal FSTL3 KO testis generate enrichment of 13 pathways including the androgen receptor signalling pathway. Taken together so far, our findings reveal a novel catalogue of SMAD-regulated genes in the testis, identifies a set of genes specifically upregulated in the testis in a SMAD-dependent manner upon FSTL3 deletion and also identify possible cross-talk between TGFβ and 13 other signalling pathways. Currently we are further analysing our data to identify whether there are SMAD regulated genes and pathways common in postnatal and postpubertal testes. We are also undertaking experiments to investigate expression pattern in FSTL3 KO testis to identify which pathways might specifically affect Sertoli cell proliferation and activity. Presentation: 6/2/2024

8796 Increased Sertoli Cell Proliferation in FSTL3 Deleted Mouse Testis

Abstract Disclosure: R. Ballesteros: None. A. Mukherjee: None. Activin, a TGFβ ligand, plays key roles in Sertoli cell proliferation. Follistatin like 3 (FSTL3) is a glycoprotein inhibitor of activin expressed strongly in the testis. We have shown that FSTL3 deletion (FSTL3 KO) in mice increases testicular size, likely caused by increased spermatogenesis supported by the increased Sertoli cell numbers seen in FSTL3 KO mouse testes. It is not clear, however, whether there is increased proliferation of Sertoli cells postnatally. We, therefore, investigated Sertoli cell proliferation, and molecular pathways that might be activated upon FSTL3 deletion that lead to increased Sertoli cell proliferation in mice. Paired testes weight comparison indicates that there is significant increase in testicular mass in FSTL3 KO between 28d and 56d coinciding with the onset of continued spermatogenesis. Immunofluorescence studies using PCNA as marker for proliferation and SOX9 to identify Sertoli cells, however, found no significant differences between proliferation of Sertoli cells in WT and FSTL3 KO. BrdU injection of mice followed by immunofluorescence labelling of proliferating Sertoli cells also showed no difference between the two genotypes by immunofluorescence. We then proceeded to assess whether there is an alteration of differentiation in FSTL3 KO testes to test the possibility that instead of proliferation there is an increased differentiation of a pre-Sertoli like cell in FSTL3 KO mice using WT1 and SOX9 as markers for early or late Sertoli cells. This experiment revealed that there was no difference between the ratio of WT1 and SOX9 expression in the two genotypes. Flow cytometric analyses using testicular dispersates from mice using Sox9 and Ki67 antibodies to identify proliferating Sertoli cells, however, revealed an approximately two-fold increase in proliferating Sertoli cells in FSTL3 KO compared to WT at 7d. At 56d there were very few proliferating Sertoli cells in either genotype and there was no difference in this number in FSTL3 deleted mice compared to WT. mRNA sequence analysis showed that approximately 1000 genes are differentially regulated between WT and FSTL3 KO at 3d. Gene ontology and pathway analysis revealed that a constellation of meiosis related genes and activin signalling pathway, among others, are enriched in FSTL3 KO testis at this stage. Data presented here demonstrates that FSTL3 deletion induces activin receptor signalling pathway, genes crucial for meiosis and therefore the spermatogenic process, and Sertoli cell proliferation. This opens the possibility that the early arrest of Sertoli cell proliferation seen in mammals including mice and humans might be circumvented by inactivating FSTL3. This could then lead to a therapeutic angle in male infertility that are based on paucity of Sertoli cells. It will be important to address why FSTL3 deletion induces Sertoli cell proliferation only postnatally but not post pubertally. Presentation: 6/1/2024

8796 Increased Sertoli Cell Proliferation in FSTL3 Deleted Mouse Testis

Abstract Disclosure: R. Ballesteros: None. A. Mukherjee: None. Activin, a TGFβ ligand, plays key roles in Sertoli cell proliferation. Follistatin like 3 (FSTL3) is a glycoprotein inhibitor of activin expressed strongly in the testis. We have shown that FSTL3 deletion (FSTL3 KO) in mice increases testicular size, likely caused by increased spermatogenesis supported by the increased Sertoli cell numbers seen in FSTL3 KO mouse testes. It is not clear, however, whether there is increased proliferation of Sertoli cells postnatally. We, therefore, investigated Sertoli cell proliferation, and molecular pathways that might be activated upon FSTL3 deletion that lead to increased Sertoli cell proliferation in mice. Paired testes weight comparison indicates that there is significant increase in testicular mass in FSTL3 KO between 28d and 56d coinciding with the onset of continued spermatogenesis. Immunofluorescence studies using PCNA as marker for proliferation and SOX9 to identify Sertoli cells, however, found no significant differences between proliferation of Sertoli cells in WT and FSTL3 KO. BrdU injection of mice followed by immunofluorescence labelling of proliferating Sertoli cells also showed no difference between the two genotypes by immunofluorescence. We then proceeded to assess whether there is an alteration of differentiation in FSTL3 KO testes to test the possibility that instead of proliferation there is an increased differentiation of a pre-Sertoli like cell in FSTL3 KO mice using WT1 and SOX9 as markers for early or late Sertoli cells. This experiment revealed that there was no difference between the ratio of WT1 and SOX9 expression in the two genotypes. Flow cytometric analyses using testicular dispersates from mice using Sox9 and Ki67 antibodies to identify proliferating Sertoli cells, however, revealed an approximately two-fold increase in proliferating Sertoli cells in FSTL3 KO compared to WT at 7d. At 56d there were very few proliferating Sertoli cells in either genotype and there was no difference in this number in FSTL3 deleted mice compared to WT. mRNA sequence analysis showed that approximately 1000 genes are differentially regulated between WT and FSTL3 KO at 3d. Gene ontology and pathway analysis revealed that a constellation of meiosis related genes and activin signalling pathway, among others, are enriched in FSTL3 KO testis at this stage. Data presented here demonstrates that FSTL3 deletion induces activin receptor signalling pathway, genes crucial for meiosis and therefore the spermatogenic process, and Sertoli cell proliferation. This opens the possibility that the early arrest of Sertoli cell proliferation seen in mammals including mice and humans might be circumvented by inactivating FSTL3. This could then lead to a therapeutic angle in male infertility that are based on paucity of Sertoli cells. It will be important to address why FSTL3 deletion induces Sertoli cell proliferation only postnatally but not post pubertally. Presentation: 6/1/2024

Near-stop in spermatogonia progression and reduced sperm motility in two testis forms of captive North African catfish in Kalasin, Thailand

North African catfish (Clarias gariepinus) is integral to aquaculture, particularly in hybrid production with bighead catfish (Clarias macrocephalus). Recent declines in their production output have been linked to the quality of the parental sperm. This study aimed to evaluate the state of spermatogenesis in two morphological testis forms, normal white testes (CGA_A) and black-brown testes (CGA_B), in captive populations to understand their differences in the cytological processes of spermatogenesis and impact on sperm quality and fertility. We performed computer-assisted sperm analysis (CASA), meiotic chromosome analysis, gene expression profiling, histological examination, and identification of apoptotic cells using the terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL). Additionally, microsatellite genotyping was conducted to assess genetic diversity of the populations, showing that similar gene pool patterns were observed between the two populations with the CGA_A and the CGA_B testes. The average sperm motility was 11.15 %. Histologically, the CGA_A testes predominantly contained spermatozoa. By contrast, the CGA_B testes, characterized by black-brown color and harder texture, were composed mainly of spermatocytes and exhibited a higher incidence of apoptotic spermatogonia. Additionally, the CGA_B testes displayed reduced expression of genes essential for normal testicular development. Notably, the CGA_A testes showed significantly higher expression of GtH I, GtH II, Dazl, and Cxcr4b genes and lower levels of Caspase-3 and p53 (p < 0.05). The selective removal of deficient spermatogonia through early apoptosis in CGA_B testes suggest a mechanism contributing to male infertility due to imbalances between the number of germ cells and Sertoli cells. Despite the use of CGA_A testis for fertilization, a hatching rate in the captive populations was 36.43 % over the last three years (2021−2023), substantially lower than that previously reported, indicating the presence of genetic and environmental factors that influence reproductive success in North African catfish. Differences in spermatogenesis between testis forms highlight the need for genetic and environmental strategies to enhance reproductive outcomes in aquaculture.

Silymarin mitigates toxic effects of cyclophosphamide on testicular tissue and sperm parameters in mice

Cyclophosphamide, a chemotherapy drug, increases oxidative stress in sperm and testicular tissue. This study evaluated the effect of silymarin, a potent antioxidant, on the quality of sperm and testicular tissue in mice treated with cyclophosphamide. NMRI adult male mice were divided into four groups: control; cyclophosphamide (intraperitoneal injection, 100 mg/kg, once a week); cyclophosphamide + silymarin; and silymarin (intraperitoneal injection, 200 mg/kg, every other day). After a 35-day treatment period, the caudal region of the epididymis was examined for sperm parameters, the right testis was used for stereological studies, and the left testis was used to assess biochemical factors. The data were statistically analyzed using SPSS software, one-way ANOVA and Tukey's test. In the cyclophosphamide group, there was a significant reduction in the mean total volume of testicular tissue, the average volume of seminiferous tubules and their components, and the average volume of interstitial tissue. Additionally, there was a notable decrease (p < 0.001) in the average number of Leydig cells, Sertoli cells, and sperm parameters. The mean concentration of testosterone hormone (p < 0.05) and total antioxidant capacity (TAC) level (p < 0.01) also significantly decreased, while the malondialdehyde (MDA) level increased significantly (p < 0.05). However, these adverse changes were mitigated in the cyclophosphamide + silymarin group compared to the cyclophosphamide group. Our results showed that silymarin as an antioxidant can mitigate the adverse effects of cyclophosphamide on testicular tissue and sperm parameters.

Subnutrition in different pregnancy stages affects IGF-1 serum concentration, its receptor expression and testicular morphology in pubertal male rats

In order to study the effects of subnutrition during different pregnancy stages on testicle development and its impact on both, IGF-1 serum concentration and its receptor production in Sertoli, Leydig and myoid cells, twenty-four primiparous female Wistar rats (331.9 ± 12.4 g body weight) were mated by the same male rat. Four groups were established: (CC) fed ad libitum during the entire pregnancy, (RC) restricted for days 0–10 of pregnancy, restricted from day 11 of pregnancy till parturition and (CR) restricted during the entire pregnancy (RR). All males from all groups were fed without restrictions since birth until puberty at day 40. During restriction stages, animals were offered 50% of the ad libitum daily food intake. Both, body and testicular weight were affected depending on which pregnancy stage the treatment was applied. On the other hand, IGF-1 production and the total Sertoli cell number was lower at day 40 if the treatment had been applied during either the entire pregnancy or in the second half. IGF-1 receptor expression was affected in Leydig cells if restriction was applied, regardless of which restricted group was studied. However, in myoid cells IGF-1 receptor expression increased when treatment was applied during the entire pregnancy. In conclusion, subnutrition in the different pregnancy stages affects body development, testicular weight and testicular morphology in pubertal male rats.

Role of L-glutamine in the regulation of rat Sertoli cell proliferation by FSH.

FSH leads to glutamine dependence, which is required for mTORC1 activation and in consequence Sertoli cell proliferation. The spermatogenic capacity of adult individuals depends on, among other factors, the number of Sertoli cells (SCs) that result from the proliferative waves during development. FSH upregulates SC proliferation at least partly, through the activation of the PI3K/Akt/mTORC1 pathway, among other mechanisms. It is widely known that mTORC1 is a sensor of amino acids. Among amino acids, glutamine acquires relevance since it might contribute to cell cycle progression through the modulation of mTORC1 activity. It has not been studied yet whether glutamine intervenes in FSH-mediated regulation of SC proliferation and cell cycle progression, or if FSH has any effect on glutamine metabolism. Eight-day-old rat SCs were incubated in culture media without glutamine or with glutamine in the absence or presence of a glutamine transporter inhibitor or a glutaminase activity inhibitor under basal conditions or stimulated with FSH. The results obtained show that FSH does not promote SC proliferation and mTORC1 activation in the absence of glutamine. Also, FSH modulates glutamine metabolism increasing glutaminase isoform 2 and reducing glutamine synthetaseexpression. FSH did not promote SC proliferation and mTORC1 activation when glutaminase activity was inhibited. The results suggest that glutamine or its metabolites might cooperate with FSH in the upregulation of SC proliferation through mTORC1. In addition, as FSH modulates glutamine metabolism through the induction of glutaminase isoform 2, the hormonal control of glutamine metabolism might be part of the intricate signaling network triggered by FSH, which is crucial to establish the population of mature SCs that supports the reproductive function.

Icariin alleviates the injury of Sertoli cell junction function by upregulating PKR pathway via ERα/c-fos signaling in aged mice

Ethnopharmacological relevenaceSertoli cells are vital to maintain spermatogenesis and their function decline during aging. Epimedium has the effects of tonifying kidney-yang, strengthening bones and muscles, and expelling wind and dampness, and is commonly used in the treatment of kidney-yang deficiency, impotence and spermatorrhea. Icariin is the main active ingredients from Epimedium exhibiting delaying aging effects and improving male reproductive dysfunction. Whereas, it remains poorly understood how icariin alleviates age-associated decline in testicular function by protecting against the damage of junction function of Sertoli cells. Aim of the studyThis study aimed to evaluate the improvement effect of icariin on Sertoli cell junction function damage and explore the underlying mechanisms. Materials and methodsMale C57BL/6 mice and mouse Sertoli cell line TM4 cells were utilized to assess the improvement effect of icariin on aging-associated Sertoli cell junction function injury. H&E staining, transmission electron microscopy, qPCR, Western blot, molecular docking, siRNA transfection, and immunofluorescence were performed in this study. ResultsDietary administration of icariin remarkly attenuated age-associated deterioration in spermatogenic function as evidenced by elevated testicular weight and index, sperm concentration and sperm viability. In addition, icariin protected Sertoli cell junction function from age-associated damage as proven by increased Sertoli cell numbers, improved tight junction ultrastructure, and upregulated junction-related proteins (ZO-1, Occludin and β-Catenin). Moreover, icariin significantly upregulated ERα/c-fos signaling and PKR pathway in testicular Sertoli cells. Similarly, in vitro studies revealed that deletion of ERα, c-fos or PKR abolished the improvement effects of icariin on Sertoli cell junction function damage. ConclusionsIcariin effectively mitigates age-associated decline in testicular function by diminished Sertoli cell junction function damage through upregulating PKR pathway via ERα/c-fos signaling. Therefore, attenuating Sertoli cell junction function injury by the upregulation of PKR pathway via ERα/c-fos signaling probably indicates an effective target for the prevention and treatment of testicular spermatogenic function with aging.

Maternal exposure to phenanthrene induces testicular apoptosis and Sertoli cell dysfunction in F1 adult male mice: a histological and molecular study.

Phenanthrene, a polycyclic aromatic hydrocarbon, is found in abundance in environmental pollutants, food, and drinking water. This substance can accumulate in body tissues and exert harmful effects. Moreover, phenanthrene can cross the placental barrier, potentially impacting fetal development. We aimed to explore the impacts of maternal exposure to phenanthrene on testicular tissue and Sertoli cell function in F1 mice. Female rats with vaginal plugs were randomly assigned to one of three groups: control, sham, or phenanthrene. The control group received no intervention during pregnancy. In the sham and phenanthrene groups, corn oil and a phenanthrene solution, respectively, were administered via gavage once every 2 days. Offspring were separated by sex 21 days after birth. At 56 days postnatal, male F1 offspring were euthanized, and their testes were harvested for histological and molecular analyses. Phenanthrene exposure was associated with a lower testicular weight and volume, a smaller diameter of the seminiferous tubules, and a relative thinning of the germinal epithelium. These changes were associated with increased cellular apoptosis, as shown by the upregulation of caspase 3 expression. Additionally, we observed an increase in vacuolization and residual bodies within the tissue. Conversely, the number of Sertoli cells and expression levels of Sox9, as well as the Ocln and Itgb1 genes, were found to be lowered. Maternal exposure to phenanthrene impacts both germ cells and Sertoli cells, disrupting their function and leading to fertility disorders in male F1 offspring mice.

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-1β 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.

Role of Anti-Müllerian Hormone in Male Reproduction and Sperm Motility.

Anti-Müllerian hormone (AMH) is secreted by Sertoli cells and is responsible for the regression of Müllerian ducts in the male fetus as part of the sexual differentiation process. Serum AMH concentrations are at their lowest levels in the first days after birth but increase after the first week, likely reflecting active Sertoli cell proliferation. AMH rises rapidly in concentration in boys during the first month, reaching a peak level at ∼6 months of age, and it remains high during childhood, then they will slowly decline during puberty, falling to low levels in adulthood. Serum AMH measurement is used by pediatric endocrinologist as a specific marker of immature Sertoli cell number and function during childhood. After puberty, AMH is released especially by the apical pole of the Sertoli cells toward the lumen of the seminiferous tubules, resulting in higher levels in the seminal plasma than in the serum. Recently, AMH has received increasing attention in research on male fertility-related disorders. This article reviews and summarizes the potential contribution of serum AMH measurement in different male fertility-related disorders.

Atrazine disorganises laminin formation and reduces cell numbers in the tammar testis during early differentiation.

Atrazine, like oestrogen, disorganises laminin formation and reduces the number of germ cells and Sertoli cells in the developing testes of the tammar wallaby. This study suggests that interfering with the balance of androgen and oestrogen affects the integrity of laminin structure and testis differentiation. The herbicide atrazine was banned in Europe in 2003 due to its endocrine disrupting activity but remains widely used. The integrity of the laminin structure in fetal testis cords requires oestrogen signalling but overexposure to xenoestrogens in the adult can cause testicular dysgenesis. However, whether xenoestrogens affect laminin formation in developing testes has not been investigated. Here we examined the effects of atrazine in the marsupial tammar wallaby during early development and compare it with the effects of the anti-androgen flutamide, oestrogen, and the oestrogen degrader fulvestrant. The tammar, like all marsupials, gives birth to altricial young, allowing direct treatment of the developing young during the male programming window (day 20-40 post partum (pp)). Male pouch young were treated orally with atrazine (5 mg/kg), flutamide (10 mg/kg), 17β-oestradiol (2.5 mg/kg) and fulvestrant (1 mg/kg) daily from day 20 to 40 pp. Distribution of laminin, vimentin, SOX9 and DDX4, cell proliferation and mRNA expression of SRY, SOX9, AMH, and SF1 were examined in testes at day 50 post partum after the treatment. Direct exposure to atrazine, flutamide, 17β-oestradiol, and fulvestrant all disorganised laminin but had no effect on vimentin distribution in testes. Atrazine reduced the number of germ cells and Sertoli cells when examined at day 40-50 pp and day 20 to 40 pp, respectively. Both flutamide and fulvestrant reduced the number of germ cells and Sertoli cells. Atrazine also downregulated SRY expression and impaired SOX9 nuclear translocation. Our results demonstrate that atrazine can compromise normal testicular differentiation during the critical male programming window.

Heat stress upregulates arachidonic acid to trigger autophagy in sertoli cells via dysfunctional mitochondrial respiratory chain function

As a key factor in determining testis size and sperm number, sertoli cells (SCs) play a crucial role in male infertility. Heat stress (HS) reduces SCs counts, negatively impacting nutrient transport and supply to germ cells, and leading to spermatogenesis failure in humans and animals. However, how HS affects the number of SCs remains unclear. We hypothesized that changes in SC metabolism contribute to the adverse effects of HS. In this study, we first observed an upregulation of arachidonic acid (AA), an unsaturated fatty acid after HS exposure by LC-MS/MS metabolome detection. By increasing ROS levels, expression of KEAP1 and NRF2 proteins as well as LC3 and LAMP2, 100 µM AA induced autophagy in SCs by activating oxidative stress (OS). We observed adverse effects of AA on mitochondria under HS with a decrease of mitochondrial number and an increase of mitochondrial membrane potential (MMP). We also found that AA alternated the oxygen transport and absorption function of mitochondria by increasing glycolysis flux and decreasing oxygen consumption rate as well as the expression of mitochondrial electron transport chain (ETC) proteins Complex I, II, V. However, pretreatment with 5 mM NAC (ROS inhibitor) and 2 µM Rotenone (mitochondrial ETC inhibitor) reversed the autophagy induced by AA. In summary, AA modulates autophagy in SCs during HS by disrupting mitochondrial ETC function, inferring that the release of AA is a switch-like response, and providing insight into the underlying mechanism of high temperatures causing male infertility.

Single-Cell RNA Sequencing Analysis of Steroidogenesis and Spermatogenesis Impairment in the Testis of db/db Mice.

The mechanism of steroidogenesis and spermatogenesis impairment in men with type 2 diabetes remains unclear. We aimed to explore the local changes of steroidogenesis and spermatogenesis in the testis of db/db mice. Research Design and Methods. We performed single-cell RNA sequencing analysis in the testis of db/db and C57BL/6J mice. The differentially expressed genes were then confirmed by real-time PCR. The histopathological characteristics of testis in db/db mice and C57BL/6J control were also performed. The 20-week-old db/db mice had significantly higher blood glucose and body weight (both p < 0.001). The serum testosterone levels (4.4 ± 0.8 vs. 9.8 ± 0.7 ng/ml, p=0.001) and weight of the testis (0.16 ± 0.01 vs. 0.24 ± 0.01 g, p < 0.001) were significantly lower in db/db mice than that in C57BL/6J controls. db/db mice had a lower cross-sectional area of seminiferous tubules and thickness of the cell layer (both p < 0.05). The numbers of Sertoli cells and Leydig cells decreased in db/db mice (both p < 0.01). Single-cell RNA sequencing analysis showed that compared with the control group, the percentage of spermatogonia was significantly higher in the db/db mouse (p < 0.001), while the proportions of spermatocytes, round and elongating spermatids, and sperms were all lower in the db/db mouse (p all < 0.001). The most differentially expressed genes were found in round spermatids (n = 86), which were not found in spermatogonia, spermatocyte, and sperm. Igfbp5 was the most significantly decreased gene in Leydig cells of the db/db mouse, while the expression of Cd74, H2-Aa, and H2-Eb1 was elevated. Ccl7 and Ptgds were the most significantly increased and decreased genes in Sertoli cells of the db/db mouse. The present study indicates spermiogenesis and steroidogenesis defects in db/db mice. The mechanism of steroidogenesis impairment in the testis of db/db mice deserves further investigation.

Endosulfan induces reproductive & genotoxic effect in male and female Swiss albino mice

BackgroundToxicity by pesticide has become a global health issue and leaves a harmful impact on human health via various ways. The people exposed to pesticides in the rural population get affected by the harmful effects of it as they enter the human body system through skin, inhalation, oral administration, food chain and many more ways. The present work is designed to study the toxic effect of endosulfan in male (n=30) and female (n=30) Swiss albino mice. Endosulfan was administered by oral gavage (oral administration) method, at the dose of 3.5 mg/Kg body weight daily for period of 3 weeks, 5 weeks and 7 weeks. After the completion of the treatment, the mice were sacrificed and their ovary and testis tissues were dissected out to check the degeneration. The blood was collected for karyotyping, biochemical and hormonal analysis of pesticide induced genotoxicity. After 7 weeks of administration with Endosulfan, various abnormalities were observed in male and female mice.ResultsTreatment with endosulfan at the dose of 3.5 mg/Kg body weight caused a higher degree of degeneration in the reproductive organ of Swiss albino mice . Treatment by this pesticide generated degeneration in long duration of dosage for 3,5 and 7 weeks. Ovaries of endosulfan administered groups showed degenerated germinal epithelium, Graffian follicles and corpus luteum. In testis of endosulfan treated mice, microscopic examination showed that there is significant damage and reduction in the tissue of seminiferous tubules and primordial germ cells. High degree of degeneration caused the disarrangement and deformation of spermatogonia with the decrease in the number of Sertoli cells. Biochemical and hormonal properties was also affected by endosulfan treatment. There was significant 5 folds decrease in the testosterone value of endosulfan in 7 weeks treated mice in comparison to control (p < 0.0001) and similarly there was significant elevation in the estrogen levels found in 7th week endosulfan treated mice. It also influenced the level of free radicals as there was significant decrease (p < 0.0001) in the value in catalase levels in 7 weeks endosulfan treated male and female mice, while significant (p < 0.0001) increase in the values of lipid peroxidation levels as 8 folds and 10 folds in 7 weeks endosulfan treated male and female Swiss albino mice respectively. This study hence speculates that the endosulfan exposed population are at the risk of reproductive health hazards.ConclusionsThe present study thus concludes that, endosulfan after 7 weeks of exposure caused significant reproductive damage to both male and female Swiss albino mice groups. Moreover, the karyotyping study also correlated the genotoxic damage in the mice.

Protective effects of diosmin on doxorubicin-induced testicular toxicity in rat.

Doxorubicin (DOX) can be applied to treat several cancers. DOX-induced oxidative stress causes testicular damage. Diosmin (DIO), as a potent antioxidant, reduces many drugs' side effects. We determined DIO therapeutic effects on DOX-related testicular toxicity. Forty rats were assigned to five groups as control, DOX (2.5mg/kg six i.p. injections at equal intervals over two weeks), DOX + DIO (25, 50, 100mg/kg, orally, daily, for two weeks) groups. Oxidative and antioxidant markers, fertility parameters levels, sperm parameters, and a histopathological examination were analyzed. DOX group showed a significant decrease in the number of spermatogonia, primary spermatocytes, and sertoli cells, seminiferous tubular diameter, seminiferous luminal diameter, and seminiferous epithelial height. Moreover, testosterone levels, glutathione (GSH) levels, catalase (CAT), glutathione peroxidase (GPx), and superoxide dismutase (SOD) activities showed a significant decrease. Furthermore, nitric oxide (NO) and malondialdehyde (MDA) contents and also follicle-stimulating hormone (FSH) and luteinizing hormone (LH) levels showed a significant increase in the DOX group compared to the control group. DIO improved DOX-related alterations in levels of hormones, spermatogonia, spermatocytes, and sertoli cell number, and seminiferous diameters (tubular, luminal, and epithelial height). Furthermore, GSH level, SOD, GPx, and CAT activities showed a significant increase, and MDA and NO contents showed a significant decrease in the DOX + DIO group than the DOX group. The results indicate that DIO mitigate DOX-induced testicular toxicity by its anti-oxidant activity.

Protective Effect of The Extract of Dayak Onions (Eleutherine palmifolia) on Sertoli and Leydig Cell Necrosis in Mice (Mus Musculus) Induced with Monosodium Glutamate

This study aims to determine the effect of the extract of Dayak onions (Eleutherine palmifolia) on the number of necrotic Sertoli and Leydig cells in mice (Mus musculus) induced with monosodium glutamate (MSG). This study involved 25 male mice aged 11 weeks and weighing approximately 20 g. The mice were divided into five groups, namely C- (0.5% CMC-Na), C+ (4 mg/g BW of MSG and 0.5% CMC-Na), T1 (4 mg/g BW of MSG and 30 mg/kg BW of Dayak onion extract), T2 (4 mg/g BW of MSG and 60 mg/kg of Dayak onion extract), and T3 (4 mg/g BW of MSG and 120 mg/kg BW of Dayak onion extract). All treatments were administered for 52 days. The mice were euthanized on day 53 of the experiment. Their testicles were removed and used to prepare histological specimens with the H&amp;E staining method. The results showed significant differences (p &lt; 0.05) in the number of necrotic Sertoli and Leydig cells between the C+ group and the T1, T2, and T3 groups with gradually decreasing values. The results suggested that the administration of the extract of Dayak onions can prevent Sertoli and Leydig cell necrosis in mice induced with MSG at an optimal dose of 120 mg/kg BW.

Single-cell insights into mouse testicular toxicity under peripubertal exposure to di(2-ethylhexyl) phthalate.

Male fertility depends on normal pubertal development. Di-(2-ethylhexyl) phthalate (DEHP) is a potent antiandrogen chemical, and exposure to DEHP during peripuberty can damage the developing male reproductive system, especially the testis. However, the specific cellular targets and differentiation processes affected by DEHP, which lead to testicular toxicity, remain poorly defined. Herein, we presented the first single-cell transcriptomic profile of the pubertal mouse testis following DEHP exposure. To carry out the experiment, 2 groups (n = 8 each) of 3-week-old male mice were orally administered 0.5% carboxymethylcellulose sodium salt or 100 mg/kg body weight DEHP daily from postnatal day 21-48, respectively. Using single-cell RNA sequencing, a total of 31 distinct cell populations were identified, notably, Sertoli and Leydig cells emerged as important targets of DEHP. DEHP exposure significantly decreased the proportions of Sertoli cell clusters expressing mature Sertoli markers (Sox9 and Ar), and selectively reduced the expression of testosterone synthesis genes in fetal Leydig cells. Through cell-cell interaction analyses, we observed changed numbers of interactions in Sertoli cells 1 (SCs1), Leydig cells 1 (LCs1), and interstitial macrophages, and we also identified cell-specific ligand gene expressions in these clusters, such as Inha, Fyn, Vcam1, and Apoe. Complementary in vitro assays confirmed that DEHP directly reduced the expression of genes related to Sertoli cell adhesion and intercellular communication. In conclusion, peripubertal DEHP exposure reduced the number of mature Sertoli cells and may disrupt testicular steroidogenesis by affecting the testosterone synthesis genes in fetal Leydig cells rather than adult Leydig cells.