Integration of hepatitis B virus into patients' sperm genome and its clinical risks.

BACKGROUND: Like the coronavirus disease 2019, the hepatitis B virus is also wreaking havoc worldwide, which has infected over 2 billion people globally. Using an experimental animal model, our previous research observed that the hepatitis B virus genes integrated into human spermatozoa can replicate and express after being transmitted to embryos. However, as of now, this phenomenon has not been confirmed in clinical data from patients. OBJECTIVES: To explore the integration of the hepatitis B virus into patients' sperm genome and its potential clinical risks. MATERIALS AND METHODS: Forty-eight patients with chronic hepatitis B virus infection were categorized into two groups: Test Group-1 comprised 23 patients without integration of hepatitis B virus DNA within the sperm genome. Test Group-2 comprised 25 patients with integration of hepatitis B virus DNA within the sperm genome. Forty-eight healthy male donors were included as control. The standard semen parameter analysis, real-time polymerase chain reaction, quantitative real-time polymerase chain reaction, sperm chromatin structure assay, fluorescence in situ hybridization, and immunofluorescence assays were utilized. RESULTS: The difference in the median copy number of hepatitis B virus DNA per mL of sera between Test Group-1 and Group-2 was not statistically significant. In Test Group-2, the integration rate of hepatitis B virus DNA was 0.109%, which showed a significant correlation with the median copy number of hepatitis B virus DNA in motile spermatozoa (1.18 × 103 /mL). Abnormal semen parameters were found in almost all these 25 patients. The integrated hepatitis B virus S, C, X, and P genes were detected to be introduced into sperm-derived embryos through fertilization and retained their function in replication, transcription, and translation. CONCLUSION: Our findings suggest that hepatitis B virus infection can lead to sperm quality deterioration and reduced fertilization capacity. Furthermore, viral integration causes instability in the sperm genome, increasing the potential risk of termination, miscarriage, and stillbirth. This study identified an unconventional mode of hepatitis B virus transmission through genes rather than virions. The presence of viral sequences in the embryonic genome poses a risk of liver inflammation and cancer.

A novel homozygous TSGA10 missense variant causes acephalic spermatozoa syndrome in a Pakistani family.

BACKGROUND: Acephalic spermatozoa syndrome is a rare type of teratozoospermia causing male infertility due to detachment of the sperm head and flagellum, which precludes fertilization potential. Although loss-of-function variations in several genes, including TSGA10, have been associated with acephalic spermatozoa syndrome, the genetic cause of many cases remains unclear. RESULTS: We recruited a Pakistani family with two infertile brothers who suffered from acephalic spermatozoa syndrome. Through whole-exome sequencing (WES) followed by Sanger sequencing, we identified a novel missense variant in TSGA10 (c.1112T > C, p. Leu371Pro), which recessively co-segregated with the acephalic spermatozoa syndrome within this family. Ultrastructural analyses of spermatozoa from the patient revealed that 98% of flagellar cross-sections displayed abnormal axonemal ultrastructure, in addition to the head-flagellum detachment. Real-time quantitative PCR analysis revealed almost no detectable TSAG10 mRNA and western blot analysis also failed to detect TSAG10 protein in patient's sperm samples while TSGA10 expression was clearly detected in control samples. Consistently, immunofluorescence analysis demonstrated the presence of TSGA10 signal in the midpiece of sperm from the control but a complete absence of TSGA10 signal in sperm from the patient. CONCLUSION: Altogether, our study identifies a novel TSGA10 pathogenic variant as a cause of acephalic spermatozoa syndrome in this family and provides information regarding the clinical manifestations associated with TSGA10 variants in human.

RéSUMé: CONTEXTE: Le syndrome des spermatozoïdes acéphaliques est un type rare de tératozoospermie provoquant une infertilité masculine en raison du détachement de la tête et du flagelle des spermatozoïdes, ce qui exclut une potentielle fécondation. Bien que des variations de perte de fonction dans plusieurs gènes, y compris TSGA10, aient été associées au syndrome des spermatozoïdes acéphaliques, la cause génétique de nombreux cas reste incertaine. RéSULTATS: Nous avons recruté une famille pakistanaise avec deux frères infertiles qui souffraient du syndrome des spermatozoïdes acéphaliques. Grâce au séquençage de l'exome entier (WES) suivi du séquençage Sanger, nous avons identifié un nouveau variant faux-sens dans TSGA10 (c.1112T > C, p. Leu371Pro), qui co-ségréguait de manière récessive avec le syndrome des spermatozoïdes acéphaliques au sein de cette famille. Les analyses ultrastructurales des spermatozoïdes des patients ont révélé que 98% des coupes transversales flagellaires présentaient une ultrastructure axonémiques anormales, en plus du décollement tête-flagelle. L'analyse quantitative par PCR en temps réel n'a révélé presque aucun ARNm TSAG10 détectable; l'analyse par transfert Western n'a pas non plus réussi à détecter la protéine TSAG10 dans les échantillons de sperme des patients, tandis que l'expression de TSGA10 a été clairement détectée dans les échantillons du témoin. De manière cohérente, l'analyse par immunofluorescence a démontré la présence du signal TSGA10 dans la partie médiane des spermatozoïdes du témoin, mais une absence totale de signal TSGA10 chez ceux des patients. CONCLUSION: Dans l'ensemble, notre étude identifie un nouveau variant pathogène de TSGA10 comme cause du syndrome des spermatozoïdes acéphaliques dans cette famille et fournit des informations concernant les manifestations cliniques associées aux variants de TSGA10 chez l'homme. MOTS-CLéS: Infertilité, TSGA10, Spermatozoïdes acéphaliques, Variations faux-sens.

Bisphenol S stimulates Leydig cell proliferation but inhibits differentiation in pubertal male rats through multiple mechanisms.

Bisphenol S (BPS) is a novel bisphenol A (BPA) analogue, a ubiquitous environmental pollutant that disrupts male reproductive system. Whether BPS affects Leydig cell maturation in male puberty remains unclear. Male Sprague-Dawley rats (age of 35 days) were daily gavaged to 0, 1, 10, 100, and 200 mg/kg/day from postnatal days 35-56. BPS at 1-10 mg/kg/day and higher doses markedly reduced serum testosterone and progesterone levels but it at 200 mg/kg/day significantly increased estradiol level. BPS at 100 and 200 mg/kg/day significantly elevated serum luteinizing hormone (LH) levels. BPS at 1-10 mg/kg/day and higher doses significantly reduced inhibin A and inhibin B levels. BPS at 100 and 200 mg/kg/day markedly increased CYP11A1+ Leydig cell number, but did not affect HSD11B1+ (a mature Leydig cell marker) cell number. BPS at 10 mg/kg/day and higher doses significantly downregulated the expression of Cyp11a1 and at 100 and 200 mg/kg/d significantly lowered Cyp17a1, Hsd11b1, and Nr5a1 in the testes. BPS at 100 and/or 200 mg/kg/day significantly elevated Lhb in the pituitary. BPS at 100 and 200 mg/kg/day significantly increased the phosphorylation of AKT1, AKT2, and CREB without affecting total AKT1, AKT2, and CREB levels. BPS at 1-100 µM significantly suppressed testosterone production and induced proliferation of primary immature Leydig cells after 24 h of treatment and these actions were reversed by estrogen receptor α antagonist, ICI 182780, and partially reversed by vitamin E. BPS at 0.1-10 µM significantly increased oxidative stress of Leydig cells in vitro. BPS also directly inhibited 17ß-hydroxysteroid dehydrogenase 3 activity at 10-100 µM. In conclusion, BPS causes hypergonadotropic androgen deficiency in male rats during pubertal exposure via activating ESR1 and inducing ROS in immature Leydig cells and directly inhibiting 17ß-hydroxysteroid dehydrogenase 3 activity.

Human Umbilical Cord Mesenchymal Stem Cells Encapsulated with Pluronic F-127 Enhance the Regeneration and Angiogenesis of Thin Endometrium in Rat via Local IL-1ß Stimulation.

Thin endometrium (< 7 mm) could cause low clinical pregnancy, reduced live birth, increased spontaneous abortion, and decreased birth weight. However, the treatments for thin endometrium have not been well developed. In this study, we aim to determine the role of Pluronic F-127 (PF-127) encapsulation of human umbilical cord mesenchymal stem cells (hUC-MSCs) in the regeneration of thin endometrium and its underlying mechanism. Thin endometrium rat model was created by infusion of 95% ethanol. Thin endometrium modeled rat uterus were treated with saline, hUC-MSCs, PF-127, or hUC-MSCs plus PF-127 separately. Regenerated rat uterus was measured for gene expression levels of angiogenesis factors and histological morphology. Angiogenesis capacity of interleukin-1 beta (IL-1ß)-primed hUC-MSCs was monitored via quantitative polymerase chain reaction (q-PCR), Luminex assay, and tube formation assay. Decreased endometrium thickness and gland number and increased inflammatory factor IL-1ß were achieved in the thin endometrium rat model. Embedding of hUC-MSCs with PF-127 could prolong the hUC-MSCs retaining, which could further enhance endometrium thickness and gland number in the thin endometrium rat model via increasing angiogenesis capacity. Conditional medium derived from IL-1ß-primed hUC-MSCs increased the concentration of angiogenesis factors (basic fibroblast growth factor (bFGF), vascular endothelial growth factors (VEGF), and hepatocyte growth factor (HGF)). Improvement in the thickness, number of glands, and newly generated blood vessels could be achieved by uterus endometrium treatment with PF-127 and hUC-MSCs transplantation. Local IL-1ß stimulation-primed hUC-MSCs promoted the release of angiogenesis factors and may play a vital role on thin endometrium regeneration.

Stem Leydig cells: Current research and future prospects of regenerative medicine of male reproductive health.

Stem cells are specialized cells that can renew themselves through cell division and can differentiate into multi-lineage cells. Mesenchymal stem cells are adult stem cells that exist in animal and human tissues. Mesenchymal stem cells have the ability to differentiate into mesodermal lineages, such as Leydig cells, adipocytes, osteocytes, and chondrocytes. Mesenchymal stem cells express cell surface markers, such as cluster of differentiation (CD) 29, CD44, CD73, CD90, CD105, and lack the expression of CD14, CD34, CD45 and HLA (human leukocyte antigen)-DR. Stem Leydig cells are one kind of mesenchymal stem cells, which are present in the interstitial compartment of testis. Stem Leydig cells are multipotent and can differentiate into Leydig cells, adipocytes, osteocytes, and chondrocytes. Stem Leydig cells have been isolated from rodent and human testes. Stem Leydig cells may have potential therapeutic values in several clinical applications, such as the treatment of male hypogonadism and infertility. In this review, we focus on the latest research on stem Leydig cells of both rodents and human, the expression of cell surface markers, culture, differentiation potential, and their applications.

Short-term exposure to perfluorotetradecanoic acid affects the late-stage regeneration of Leydig cells in adult male rats.

Perfluorotetradecanoic acid (PFTeDA) is one of perfluoroalkyl substances widely found in the environment. PFTeDA may cause the dysfunction of male reproductive system. However, whether PFTeDA affects the regeneration of Leydig cells remains unclear. The objective of this study was to examine the effects of short-term exposure of PFTeDA on the late-stage maturation of Leydig cells. Fifty-four adult Sprague-Dawley male rats were daily gavaged with PFTeDA (0, 10, or 20 mg/kg body weight) for 10 days, and then were injected intraperitoneally with ethylene dimethane sulfonate (EDS, 75 mg/kg body weight/once) to ablate Leydig cells to induce their regeneration. On day 21 (early stage) and 56 (late stage) after EDS, hormone levels, gene expression, and protein levels were measured. PFTeDA did not affect the early stage of Leydig cell regeneration, because it had no effect on serum testosterone, luteinizing hormone, and follicle-stimulating hormone levels, Leydig cell number, and its gene and protein expression. PFTeDA significantly reduced serum testosterone level and down-regulated the expression of Leydig cell genes (Cyp11a1, Hsd3b1, Cyp17a1, Hsd17b3, Hsd11b1, and Insl3) and their proteins (CYP11A1, HSD3B1, CYP17A1, HSD17B3, and INSL3), decreased the phosphorylation of AKT1 and ERK1/2, as well as lowered sperm count in the epididymis at 20 mg/kg. In conclusion, short-term exposure to PFTeDA blocks the late-stage maturation of Leydig cells.

Perfluorotridecanoic Acid Inhibits Leydig Cell Maturation in Male Rats in Late Puberty via Changing Testicular Lipid Component.

Perfluorotridecanoic acid (PFTrDA) is a long-chain (C13) perfluoroalkyl carboxylic acid. Here, we report the influence of PFTrDA exposure on the maturation of rat Leydig cells in late puberty in vivo. Male Sprague-Dawley rats were administered PFTrDA by gavage of 0, 1, 5, and 10 mg/kg/day from 35 days to 56 days postpartum. PFTrDA had no effect on body weight, testis weight, and epididymis weight. It significantly decreased the serum testosterone level after 5 and 10 mg/kg exposure, while it did not alter the serum estradiol level. The serum luteinizing hormone level was markedly reduced after 10 mg/kg PFTrDA exposure, while the follicle-stimulating hormone level was unchanged. Star, Cyp11a1, Cyp17a1, Hsd3b1, and Insl3 transcript levels in the testis were markedly lowered in the 1-5 mg/kg PFTrDA group and the Lhb transcript level in the pituitary in the 10 mg/kg group. CYP11A1 and HSD11B1-positive Leydig cell numbers were markedly reduced after 10 mg/kg PFTrDA exposure. Testicular triglyceride and free fatty acid (palmitic acid, oleic acid, and linoleic acid) levels were significantly reduced by PFTrDA, while Mgll (up-regulation) and Scarb1 and Elovl5 (down-regulation) expression were altered. AKT1 and AMPK phosphorylation was stimulated after 10 PFTrDA mg/kg exposure. In conclusion, PFTrDA delays the maturation of Leydig cells in late puberty mainly by altering the free fatty acid profile.

Inhibition of human sperm motility and capacitation by ziram is mediated by decreasing tyrosine protein kinase.

Many endocrine disruptors may interfere with sperm motility, hyperactivation, and capacitation, thereby leading to male infertility. In the current study, we screened 14 endocrine disruptors, including plant ingredients, cigarette ingredients, minerals, insecticides and fungicides, plastics, and plasticizers, to inhibit human sperm motility and forward motility. Only ziram, a dithiocarbamate fungicide, can effectively inhibit sperm motility, forward motility, hyperactivation, capacitation, and spontaneous acrosome reaction of normal human spermatozoa. Its half maximum inhibitory concentration (IC50) values were less than 4 µM. Ziram also inhibited sperm motility and forward motility of asthenozoospermia spermatozoa and IC50 values were about 6-8 µM. In addition, ziram inhibited normal sperm motility, calcium influx, reactive oxygen species, and mitochondrial membrane potential at 2.5 and/or 5 µM, with IC50 values ​​exceeding 100 µM, although it did not affect sperm DNA fragmentation up to 5 µM. Ziram-mediated inhibition of sperm motility and forward motility was irreversible. Forskolin, 8Br-cAMP, pentoxifylline, progesterone, vitamin E, and A23187 cannot prevent ziram-mediated inhibition of sperm motility and forward motility. Further studies have shown that ziram inhibited the level of tyrosine protein kinase with an IC50 value of about 10 µM, without affecting p21-activated kinase 4, and it caused damage to the mitochondrial structure of normal spermatozoa at 2.5 and 5 µM. In conclusion, ziram irreversibly inhibits human sperm motility, forward motility, and capacitation by reducing the level of tyrosine protein kinase and damaging the ultrastructure of mitochondria.

Androgen and Luteinizing Hormone Stimulate the Function of Rat Immature Leydig Cells Through Different Transcription Signals.

The function of immature Leydig cells is regulated by hormones, such as androgen and luteinizing hormone (LH). However, the regulation of this process is still unclear. The objective of this study was to determine whether luteinizing hormone (LH) or androgens contribute to this process. Immature Leydig cells were purified from 35-day-old male Sprague Dawley rats and cultured with LH (1 ng/ml) or androgen (7α-methyl-19- nortestosterone, MENT, 100 nM) for 2 days. LH or MENT treatment significantly increased the androgens produced by immature Leydig cells in rats. Microarray and qPCR and enzymatic tests showed that LH up-regulated the expression of Scarb1, Cyp11a1, Cyp17a1, and Srd5a1 while down-regulated the expression of Sult2a1 and Akr1c14. On the contrary, the expression of Cyp17a1 was up-regulated by MENT. LH and MENT regulate Leydig cell function through different sets of transcription factors. We conclude that LH and androgens participate in the regulation of rat immature Leydig cell function through different transcriptional pathways.

The Production of Testosterone and Gene Expression in Neonatal Testes of Rats Exposed to Diisoheptyl Phthalate During Pregnancy is Inhibited.

Background: Diisoheptyl phthalate (DIHP) is a phthalate plasticizer, which is a branched phthalate. Here, we reported the effects of gestational exposure to DIHP on testis development in male rats. Methods: Pregnant Sprague-Dawley rats were orally fed with vehicle (corn oil, control) or DIHP (10, 100, 500, and 1,000 mg/kg) from gestational day (GD) 12-21. At GD21, serum testosterone levels, the number and distribution of fetal Leydig cells, and testicular mRNA and protein levels, the incidence of multinucleated gonocytes, and focal testicular hypoplasia in the neonatal testis were measured. Results: DIHP increased the fetal Leydig cell cluster size and decreased the fetal Leydig cell size with LOAEL of 10 mg/kg. DIHP did not affect the fetal Leydig cell number. DIHP significantly lowered serum testosterone levels, down-regulated the expression of steroidogenesis-related genes (Lhcgr, Star, Cyp11a1, Hsd3b1, Cyp17a1, and Hsd17b3) and testis descent-related gene (Insl3) as well as protein levels of cholesterol side-chain cleavage enzyme (CYP11A1) and insulin-like 3 (INSL3). DIHP dose-dependently increased the percentage of multinucleated gonocytes with the low observed adverse-effect level (LOAEL) of 100 mg/kg. DIHP induced focal testicular hypoplasia. Conclusion: Gestational exposure to DIHP causes testis dysgenesis in rats.

Perfluorododecanoic acid delays Leydig cell regeneration from stem cells in adult rats.

Perfluorododecanoic acid (PFDoA) is an endocrine-damaging compound in contaminated food and water. However, the potential role and underlying mechanism of PFDoA in Leydig cell regeneration from stem Leydig cells remain unclear. The current study aims to investigate the effect of PFDoA on the regeneration of Leydig cells in the testis of rats treated with ethylene dimethane sulfonate (EDS). PFDoA (0, 5 or 10 mg/kg/day) was gavaged to adult Sprague-Dawley male rats for 8 days, and 75 mg/kg EDS was intraperitoneally injected to eliminate Leydig cells to initiate its regeneration from day 21-56 after EDS. The serum testosterone levels in the 5 and 10 mg/kg/day PFDoA groups were significantly reduced at day 21 after EDS and the levels of serum luteinizing hormone and follicle-stimulating hormone were significantly decreased in the 10 mg/kg/day PFDoA groups at day 56 after EDS. PFDoA significantly reduced Leydig cell number and proliferation at a dose of 10 mg/kg at days 21 and 56 after EDS. PFDoA significantly down-regulated the expression of Leydig cell-specific genes (Lhcgr, Scarb1, Star, Cyp11a1, Hsd3b1 and Cyp17a1) and their proteins at both doses at days 21 and 56 after EDS. PFDoA significantly down-regulated the gene expression of Sertoli cells (Fshr, Dhh, and Sox9) at 5 mg/kg or higher at days 21 and 56 after EDS. In addition, we found that PFDoA significantly inhibited EdU incorporation into putative stem Leydig cells and their differentiation into the Leydig cell lineage in vitro. In conclusion, short-term PFDoA exposure in adulthood delayed the regeneration of Leydig cells by preventing Leydig cells from stem cells via multiple mechanisms.

Perfluorotridecanoic acid inhibits fetal Leydig cell differentiation after in utero exposure in rats via increasing oxidative stress and autophagy.

Perfluorotridecanoic acid (PFTrDA) is a long-chain perfluoroalkyl substance, and its effect on the differentiation of fetal Leydig cells remains unclear. The objective of this study is to explore the effect of in utero PFTrDA exposure on the differentiation of fetal Leydig cells and investigate its underlying mechanisms. Pregnant Sprague-Dawley female rats were daily administered by gavage of PFTrDA at doses of 0, 1, 5, and 10 mg/kg from gestational day 14 to 21. PFTrDA had no effect on the body weight of dams, but significantly reduced the body weight and anogenital distance of male pups at birth at a dose of 10 mg/kg. PFTrDA significantly decreased serum testosterone levels as low as 1 mg/kg. PFTrDA did not affect fetal Leydig cell number, but promoted abnormal aggregation of fetal Leydig cells at doses of 5 and 10 mg/kg. PFTrDA down-regulated the expression of Insl3, Lhcgr, Scarb1, Star, Hsd3b1, Cyp17a1, Nr5a1, and Dhh as well as their proteins. PFTrDA lowered the levels of antioxidants (SOD1, CAT, and GPX1), induced autophagy as shown by increased levels of LC3II and beclin1, and reduced the phosphorylation of mTOR. In conclusion, PFTrDA inhibits the differentiation of fetal Leydig cells in male pups after in utero exposure mainly through increasing oxidative stress and inducing autophagy.

Short-term perfluorooctane sulfonate exposure impairs Leydig cell regeneration in the adult rat testis via targeting hedgehog signaling.

Perfluorooctane sulfonate is related to male reproductive dysfunction in rats and humans. However, the underlying mechanism remains unknown. Here, we reported the effects of short-term exposure to perfluorooctane sulfonate on the regeneration of Leydig cells in vivo and investigated possible mechanisms in vitro. After adult male Sprague-Dawley rats were gavaged perfluorooctane sulfonate (0, 5 or 10 mg/kg/day) for 7 days and then injected intraperitoneally ethane dimethane sulfonate next day to eliminate Leydig cells, the Leydig cell regeneration process was monitored. Perfluorooctane sulfonate significantly lowered serum testosterone levels, reduced the number of regenerated Leydig cells, down-regulated the expression of Lhcgr, Scarb1, Star, Cyp11a1, Hsd3b1, Cyp17a1, and Dhh) and their proteins at doses of 5 and 10 mg/kg 35 and 56 days after ethane dimethane sulfonate. Using a 3D seminiferous tubule culture system to study the development of stem Leydig cells, we found that perfluorooctane sulfonate inhibited stem Leydig cell proliferation and differentiation and hedgehog signaling pathway. In conclusion, a short-term exposure to perfluorooctane sulfonate can inhibit the development of stem Leydig cells into the Leydig cell lineage via direct suppression of hedgehog signaling pathway and indirect inhibition of desert hedgehog section by Sertoli cells.

Xylene delays the development of Leydig cells in pubertal rats by inducing reactive oxidative species.

Xylene is a cyclic hydrocarbon, which is commonly used as a solvent in dyes, paints, polishes, and industrial solutions. It is a potential environmental pollutant. Here, we report the effect of xylene exposure on Leydig cell development in male rats during puberty. Xylene (0, 150, 750, and 1500 mg/kg) was gavaged to 35-day-old male Sprague Dawley rats for 21 days. Xylene significantly reduced serum testosterone levels at 750 and 1500 mg/kg without affecting serum luteinizing hormone and follicle-stimulating hormone levels. Xylene reduced the number of HSD11B1-positive Leydig cells at the advanced stage at 1500 mg/kg. At 750 and 1500 mg/kg, xylene also reduced the cell size and cytoplasm size. It down-regulated the expression of Leydig cell-specific genes (Lhcgr, Scarb1, Star, Cyp11a1, Hsd3b1, Cyp17a1, and Hsd11b1) and proteins. In addition, xylene significantly reduced the ratio of phosphorus-GSK-3ß (pGSK-3ß/GSK-3ß), phosphorus-ERK1/2 (pERK)/ERK1/2, and phosphorus-AKT1 (pAKT1)/AKT1, and SIRT1 levels in the testes. In vitro Leydig cell culture showed that xylene induced oxidative stress by increasing the production of reactive oxygen species and lowing antioxidant (Sod2), and inhibited the production of testosterone, and down-regulated the expression of genes related to steroidogenesis, while vitamin E reversed the xylene-mediated effect as an antioxidant. In conclusion, xylene exposure may disrupt the development of pubertal Leydig cells by increasing reactive oxygen species production and reducing the expression of GSK-3ß, ERK1/2, AKT1, and SIRT1.

Perfluoroheptanoic acid induces Leydig cell hyperplasia but inhibits spermatogenesis in rats after pubertal exposure.

Perfluoroheptanoic acid (PFHpA) is a short-chain alternative to long-chain perfluoroalkyl substances, which have been reported to possess reproductive toxicity. However, it is unclear whether PFHpA affects Leydig cell development during puberty. The 35-day-old Sprague Dawley male rats were exposed to PFHpA by gavage with 0 (corn oil), 10, 50, and 100 mg/kg/day for 21 days. PFHpA did not affect the body weight of rats, but it reduced testis weight, relative testis weight, and epididymis weight at 100 mg/kg. It significantly increased serum testosterone, luteinizing hormone, and follicle-stimulating hormone levels at a dose of 100 mg/kg without affecting serum estradiol levels. PFHpA suppressed sperm production at a dose of 100 mg/kg. PFHpA induced Leydig cell hyperplasia (increased number of CYP11A1-positive Leydig cells) at a dose of 100 mg/kg, but down-regulated the expression of Cyp11a1, Hsd3b1, and Cyp17a1 in individual Leydig cell pe se and up-regulated the expression of Fshr in the Sertoli cell pe se. PFHpA did not affect the number of HSD11B1 (a biomarker for more mature Leydig cells) positive Leydig cells and SOX9 positive Sertoli cells. PFHpA increased BCL2, and the phosphorylation of AKT1, AKT2, ERK1/2, and JNK, but decreased BAX levels. However, it had no effect on SIRT1 and PGC-1α levels. In conclusion, PFHpA induces Leydig cell hyperplasia due to the increase in the secretion of luteinizing hormone through negative feedback after down-regulating the expression of steroidogenic enzymes and inhibiting testosterone production in individual Leydig cells. This proliferation may be mediated by increasing BCL2 and phosphorylation of AKT, ERK1/2, and JNK, and decreasing BAX level.

[Semen quality of infertile men carrying hepatitis B virus].

OBJECTIVE: To study the semen parameters of infertile men carrying hepatitis B virus (HBV) and the influence of HBV infection on semen quality. METHODS: We collected the semen samples from 782 infertility males aged 25－35 years old. According to the results of serological examinations, we divided the patients into groups A (HBsAg, HBeAb and HBcAb positive, n = 286), B (HBsAg, HBeAg and HBcAb positive, n = 230) and C (non-HBV control, n = 266), and comparatively analyzed the routine semen parameters, sperm acrosin activity, sperm DNA fragmentation index (DFI) and high DNA stainability (HDS) among the three groups of patients. RESULTS: Compared with the patients of groups B and C, those of group A showed markedly decreased sperm concentration (ï¼»88.20 ± 82.62ï¼½ and ï¼»89.29 ± 53.80ï¼½ vs ï¼»71.49 ± 60.03ï¼½ ×106/ml, P<0.05), progressively motile sperm (PMS) (ï¼»34.88 ± 15.60ï¼½% and ï¼»37.82 ± 13.63ï¼½% vs ï¼»30.70 ± 14.79ï¼½%, P<0.05), sperm motility (ï¼»45.77 ± 16.58ï¼½% and ï¼»48.16 ± 14.03ï¼½% vs ï¼»42.67 ± 17.23ï¼½%, P<0.05), sperm viability (ï¼»82.55 ± 7.55ï¼½% and ï¼»85.26 ± 6.39ï¼½% vs ï¼»81.07 ± 10.19ï¼½%, P>0.05) and morphologically normal sperm (MNS) (ï¼»6.93 ± 4.45ï¼½% and ï¼»7.27 ± 4.43ï¼½% vs ï¼»5.72 ± 3.47ï¼½%, P<0.05), with sperm concentration, PMS, sperm motility, sperm viability and MNS remarkably lower in group B than in C. Sperm acrosin activity was significantly reduced in group A in comparison with groups B and C (ï¼»57.07 ± 26.38ï¼½ vs ï¼»63.03 ± 28.75ï¼½ and ï¼»78.00 ± 33.49ï¼½ µIU/106, P<0.01), remarkably lower in group B than in C (P<0.01). The sperm DFI and HDS, however, were markedly higher in group A than in B (ï¼»14.79 ± 9.46ï¼½% vs ï¼»12.95 ± 7.29ï¼½% and ï¼»11.60 ± 5.98ï¼½%, P<0.05; ï¼»9.62 ± 6.20ï¼½% vs ï¼»8.43 ± 4.72ï¼½% and ï¼»8.41 ± 4.59ï¼½%, P<0.05), and both higher in group B than in C. CONCLUSIONS: Semen quality is lower in infertile men carrying HBV and therefore HBV infection is one of the causes of male infertility.

Bisphenols and Leydig Cell Development and Function.

Bisphenol A (BPA) is a ubiquitous environmental pollutant, mainly from the production and use of plastics and the degradation of wastes related to industrial plastics. Evidence from laboratory animal and human studies supports the view that BPA has an endocrine disrupting effect on Leydig cell development and function. To better understand the adverse effects of BPA, we reviewed its role and mechanism by analyzing rodent data in vivo and in vitro and human epidemiological evidence. BPA has estrogen and anti-androgen effects, thereby destroying the development and function of Leydig cells and causing related reproductive diseases such as testicular dysgenesis syndrome, delayed puberty, and subfertility/infertility. Due to the limitation of BPA production, the increased use of BPA analogs has also attracted attention to these new chemicals. They may share actions and mechanisms similar to or different from BPA.

Perfluoroalkyl substances cause Leydig cell dysfunction as endocrine disruptors.

Perfluoroalkyl substances (PFASs) are a group of man-made organic substances. Some of PFASs have been classified as persistent organic pollutants and endocrine disruptors. They might interfere with the male sex endocrine system, causing the abnormal development of the male reproductive tract and failure of pubertal onset and infertility. The present review discusses the development and function of two generations of Leydig cells in rodents and the effects of PFASs on Leydig cell development after their exposure in gestational and postnatal periods. We also discuss human epidemiological data for the effects of PFASs on male sex hormone levels. The structure-activity relationship of PFASs on Leydig cell steroidogenesis and enzyme activities are also discussed.

Oncostatin M inhibits differentiation of rat stem Leydig cells in vivo and in vitro.

Oncostatin M (OSM) is a pleiotropic cytokine within the interleukin six family of cytokines, which regulate cell growth and differentiation in a wide variety of biological systems. However, its action and underlying mechanisms on stem Leydig cell development are unclear. The objective of the present study was to investigate whether OSM affects the proliferation and differentiation of rat stem Leydig cells. We used a Leydig cell regeneration model in rat testis and a unique seminiferous tubule culture system after ethane dimethane sulfonate (EDS) treatment to assess the ability of OSM in the regulation of proliferation and differentiation of rat stem Leydig cells. Intratesticular injection of OSM (10 and 100 ng/testis) from post-EDS day 14 to 28 blocked the regeneration of Leydig cells by reducing serum testosterone levels without affecting serum luteinizing hormone and follicle-stimulating hormone levels. It also decreased the levels of Leydig cell-specific mRNAs (Lhcgr, Star, Cyp11a1, Hsd3b1, Cyp17a1 and Hsd11b1) and their proteins by the RNA-Seq and Western blotting analysis. OSM had no effect on the proliferative capacity of Leydig cells in vivo. In the seminiferous tubule culture system, OSM (0.1, 1, 10 and 100 ng/mL) inhibited the differentiation of stem Leydig cells by reducing medium testosterone levels and downregulating the expression of Leydig cell-specific genes (Lhcgr, Star, Cyp11a1, Hsd3b1, Cyp17a1 and Hsd11b1) and their proteins. OSM-mediated action was reversed by S3I-201 (a STAT3 antagonist) or filgotinib (a JAK1 inhibitor). These data suggest that OSM is an inhibitory factor of rat stem Leydig cell development.

Changes in human umbilical vein endothelial cells induced by endothelial nitric oxide synthase traffic inducer.

This study investigated the changes in human umbilical vein endothelial cells (HUVECs) induced by overexpression of endothelial nitric oxide synthase traffic inducer (NOSTRIN) and its role in cellular injury. Recombinant NOSTRIN-expressing and empty vectors were transfected into cultured HUVECs, and factor VIII-related antigen was examined by using immunohistochemical analysis. Growth curves were generated for both transfected and untransfected cells and these indicated that the proliferative ability of cells overexpressing NOSTRIN was significantly decreased. The expression of NOSTRIN and eNOS proteins was detected by using Western blot analysis, endothelial NOS (eNOS) activity was assayed by using spectrophotometry, and NO2 (-)/NO3 (-) levels were measured using nitrate reductase. Immunohistochemical analysis demonstrated that all groups expressed NOSTRIN in the plasma membrane and cytoplasm, and Western blot analysis confirmed that NOSTRIN levels were significantly higher in cells transfected with the NOSTRIN plasmid (P<0.01). The activity of eNOS and the levels of NO2 (-)/NO3 (-) were significantly decreased in NOSTRIN overexpressing cells as compared with empty vector and untransfected cells (P<0.01 and P<0.01, respectively). Morphological and ultrastructural changes were observed under light and electron microscopy, and it was found that NOSTRIN-overexpressing cells were elongated with deformities of the karyotheca, injury to the plasma membrane, increased lipids in the cytoplasm, and shortened microvilli. This study showed that overexpression of NOSTRIN had a significant effect on eNOS activity in HUVECs and resulted in significant cellular damage.