Leydig Cell Number Research Articles

Comparative testis structure and function in three representative mice strains.

Mice are widely used as experimental models due to several positive characteristics and in particular their suitability for studies involving molecular biology and transgenesis. Despite the large number of mice strains currently available, the literature regarding their basic reproductive biology is still relatively scarce. Herein, we comparatively evaluated several important and correlated parameters related to testis structure and function in sexually mature male mice of inbred (C57BL/6, n = 19; BALB/c, n = 17) and outbred (Swiss, n = 17) strains, frequently utilized in research. Swiss mice presented significant variation for many parameters evaluated, including higher sperm production, mainly when compared to the C57BL/6 strain. However, some key parameters such as the duration of spermatogenesis, the Sertoli cell number per testis, and the spermatogenic efficiency were similar among the different strains. Although presenting significantly higher Leydig cell (LC) proportion and numbers per testis gram and per testis, the anogenital index was smaller in Swiss mice. Estradiol levels were lower in C57BL/6, whereas testosterone levels and 3β-HSD expression were similar among strains. Regarding the LC/macrophages relationship, in comparison to the literature, we reported a much higher contribution of macrophages to the mouse intertubule. Thus, we estimated that there are around 1.6 macrophages per LC in BALB/c mice and this intriguing finding could be relevant to testis function in overall and spermatogonial biology in particular. Taken together, our results highlight the importance of knowing more accurately the testis structure and function in the different mice strains available for research, particularly when a specific testis parameter is being investigated.

Features of gonadal dysgenesis and Leydig cell impairment in testes with Sertoli cell-only syndrome.

There is evidence that disturbed spermatogenesis is associated with impaired Leydig cell function and that it may be the result of testicular dysgenesis during fetal/infant development. Sertoli cell-only syndrome (SCOS) is defined by complete lack of germ cells in the seminiferous epithelium. The pathogenesis of SCOS is still not well understood. The aim of the study is to evaluate testes with SCOS focusing on morphometric signs of testicular dysgenesis and markers of Leydig cell (LC) function in relation to hormonal status of studied infertile men. Forty-nine testicular biopsies of patients with SCOS and 15 controls with normal spermatogenesis (NOR) were studied. In each biopsy the seminiferous tubule diameter (STD), thickness of tubular membrane (TM), area fraction of intertubular space (AFIS) were measured and semi-quantitative assessment of the LC number was performed (LC-score). The results of histological examination were correlated with serum levels of FSH, LH, testosterone (T) and T/LH ratio. In SCOS group testicular volume (median [M]: 16.0 vs. 29.5; p < 0.001) and STD (M: 141.7 vs. 190.2; p < 0.001) were lower, while TM (M: 9.8 vs. 6.4; p < 0.001) and AFIS (M: 47.6 vs. 27.6; p < 0.001) were significantly higher in comparison to NOR group. LC-score was higher in SCOS than in NOR group (M: 2.2 vs. 1.1; p < 0.001). Abnormal AFIS and STD were present in 43% of SCOS biopsies and among them in 81% the increased LC-score was found. In SCOS group, the subjects had significantly higher levels of both gonadotropins (FSH, M: 19.9 vs. 3.4; p < 0.001; LH, M: 7.1 vs. 4.2; p < 0.001). Total serum testosterone level did not differ between studied groups; however, T/LH ratio was significantly lower in SCOS group (M: 2.3 vs. 3.8; p < 0.001). Negative correlation between LC-score and STD was observed in SCOS group (r = -0.48; p < 0.001). AFIS correlated positively with serum FSH level in NOR (r = 0.53; p < 0.05) and SCOS (r = 0.41; p < 0.05) group, while with LH, and negatively with T/LH ratio, only in SCOS (LH, r = 0.37; p < 0.05; T/LH, r = -0.36; p < 0.05) group. We have shown that substantial number of testes from subjects with SCOS presented abnormal morphometric features, which are recognized as the signs of testicular dysgenesis. Additionally, an increased number of Leydig cells simultaneously with abnormal T/LH ratio were found, which suggests an impaired function of these cells. Increased serum levels of LH and also FSH, may reflect dysfunction of Leydig cells. It seems that reproductive hormones levels reflect also the condition of testicular structure, and that FSH may be related to the changes in intertubular space area independently of impaired Leydig cell function.

Effects of acute hCG stimulation on serum INSL3 and 25-OH vitamin D in Klinefelter syndrome.

It has recently been suggested that the hypergonadotropic hypogonadism characterizing Klinefelter syndrome (KS) might not be due to a steroidogenic dysfunction per se, but mainly to an altered testosterone (T) secretion into the bloodstream. However, the Leydig cell functionality remains incompletely studied in KS, and new markers should be considered. Previous data indicated that chronic hCG stimulation influences the production of both insulin-like peptide 3 (INSL3) and 25-hydroxy-vitamin D (25-VD) in eugonadal men. To evaluate INSL3 and 25-VD serum levels, as markers of Leydig cell functionality, in association with sex steroids, after an acute hCG test in a group of KS patients and healthy volunteers. A retrospective analysis of a prospective case-control clinical trial was carried out. KS patients (n=11) and age-matched healthy controls (n=11) provided a basal blood sample (V0) immediately followed by a single intramuscular injection of hCG 5000IU. Blood samples were taken in the following five days (V1-V5). At baseline, INSL3 was lower in KS patients compared with controls (P=.007). When adjusted for INSL3 levels, the production of steroids was similar between KS patients and controls. 25-VD was in the insufficient range both in KS patients and in controls and was not different (P=.064). Acute hCG stimulation increased neither INSL3 nor 25-VD in both KS patients and controls. In controls, an inverse correlation was detected between INSL3 levels and body mass index (P=.020) and waist circumference (P=.020). INSL3 secretion is independent from steroidogenesis, and its production is mostly not influenced by acute hCG stimulation both in KS men and in controls. INSL3 serum levels should be considered as a marker of Leydig cell differentiation and numbers rather than steroidogenesis. 25-VD serum levels are also not increased by a single acute hCG administration, which was not able to restore the normal concentrations of 25-VD.

Lack of retinoid acid receptor-related orphan receptor alpha accelerates and melatonin supplementation prevents testicular aging.

The role of retinoid acid receptor-related orphan receptor alpha (RORα) on male reproductive functions during aging is unclear. Here, we analyze the morphological changes in the testis of both young and aged RORα-deficient mice, with and without melatonin supplementation. Young mutants showed vacuolation, degeneration and pyknosis of spermatogenic epithelium and Sertoli cells. Aged mutants showed atrophy of the seminiferous tubules and absence of mitotic spermatogenic cells. Absence of sperms in many tubules, loss of acrosomal cap, vacuolation and hypertrophy of Sertoli cells were detected in aged mice, with a significant reduction in the number of seminiferous tubules and a significant increase in the number of Leydig cells and telocytes. Repair in seminiferous tubules and interstitial tissues with enhancement of spermatogenesis was observed in melatonin-treated aged mice. Young mutants overexpressed VEGF that was weaker in aged animals and observed only in the spermatocytes, while melatonin increased VEGF expression in spermatocytes and spermatids. Caspase 3 increased in both young and aged mutant mice in all seminiferous tubules and interstitium; caspase 3 immunostaining in seminiferous tubules, however, showed a normal pattern of apoptosis with melatonin supplementation. The present study reports that age-dependent testicular changes in RORα mutant mice were recovered by melatonin treatment.

Curcumin Effectivity on Hepar and Reproductive Organ Recovery Male Mice (Mus musculus L) after Methoxychlor Exposure

Methoxychlor (MXC) is an insecticide (DDT derivates) that has the potential for bioaccumulation in mammal and causes a disruptive effect on the hepar and reproductive system. This study was done to find out the benefits of curcumin as a natural ingredient to overcome the negative impact of Methoxychlor (MXC) on hepar and male reproductive organ of Balb’C mice (Mus musculus L). The study was carried out in a Completely Randomized Design (CRD) Posttest Only Control Group Design used four treatments and six replications. The curcumin treatment after administration of MXC was carried out by gavage with curcumin doses: 0.05; 0,1; and 0.2 mg/g body weight, every day for two weeks, respectively. Histological observations of the liver, and testis was performed using the paraffin method and Hematoxylin Eosin stained. The results showed that MXC exposure caused liver disruption by increasing the number of pycnotic necrotic hepatocytes and hydrophic degeneration hepatocytes. On the male reproductive organ, MXC caused testis impairment by reducing the number of Sertoli cells and Leydig cells, spermatogenic cell counts, and the diameter of seminiferous tubules. The administration of curcumin at doses of 0.1 mg/g bw in mice exposed to methoxychlor can reduce the number of hydrophic degeneration hepatocytes and tend to reduce the number of pycnotic hepatocytes; and also increase the number of Sertoli cells, the number of spermatogenic cells, and the diameter of the seminiferous tubules, and tend to reduce the amount of Leydig cells. Curcumin treatment tends to recover hepar dan testis disruption of mice that were exposed by MXC.

The potential therapeutic effects of Lactobacillus plantarum and inulin on serum and testicular reproductive markers in diabetic male rats

BackgroundIt is well established that diminished reproductive health is one of the notable long-term outcomes of type 2 diabetes mellitus (T2DM), especially among males. Due to the global increasing rate of T2DM and infertility, we aimed to investigate the impact of Lactobacillus plantarum (L. plantarum), inulin, and their combinatory supplementation on fertility markers as well as testicular kisspeptin and androgen receptor (AR)’s expression in diabetic male rats.MethodsThirty-five Male Wistar rats with Streptozotocin-induced T2DM were supplemented with L. plantarum, inulin, or their combination for 8 weeks. At the end-point, the animals were sacrificed and serum, testicular, and seminal parameters were studied.ResultsAdministration of L. plantarum and inulin in diabetic male rats improved sperm motility and viability (P < 0.001, both) as well as testicular tissue development via increasing leydig cell number, testicular spermatid count, and diameter of seminiferous tubules (P < 0.001, all). Testicular expression of Kisspeptin was elevated by inulin supplementation (P = 0.01). L. plantarum administration increased testicular AR expression (P = 0.01). The expression of Kisspeptin showed a remarkable correlation with fertility markers (P < 0.001).ConclusionSupplementation with either L. plantarum, inulin, or their combination can prevent infertility caused by T2DM in male rats via improving testicular kisspeptin and AR expression, leydig cell count, and effectively increasing epididymal sperm motility and viability.

Pulmonary Emphysema Impairs Male Reproductive Physiology Due To Testosterone and Oxidative Stress Imbalance in Mesocricetus auratus.

This study evaluated whether pulmonary emphysema affects sperm quality, male reproductive organs, and testosterone levels in adult male hamsters. Mesocricetus auratus males (130-150g) were subdivided into a control group (C group) and an emphysema group (E group). The C group received an intratracheal instillation of saline solution (0.3mL/100g of body weight), and the E group received papain (40mg/100g of body weight). After 60days, the biometric, pulmonary, and reproductive parameters of each group were evaluated. The E group developed pulmonary emphysema, which decreased body weight and sperm quality compared to the C group. In oxidative stress-related assays, lipid peroxidation was increased in the testis and epididymis (caput and cauda) in the E group compared with the C group. However, only the caput epididymis showed a reduction in glutathione levels. Pulmonary emphysema also affected the testicle by inducing an increase in abnormal seminiferous tubules, accompanied by a decrease in seminiferous epithelium height. Spermatogenesis kinetics were also modified by pulmonary emphysema. The number of Leydig and Sertoli cells decreased in the E group, accompanied by an increase in the nuclear volume of Leydig cells. Testosterone concentration was increased in the E group. Similarly, pulmonary emphysema altered epididymal components in all regions. In conclusion, pulmonary emphysema affected the reproductive system in this experimental model, as shown by testicular and epididymal morphophysiology changes, hormonal alteration, and oxidative stress imbalance, inducing the loss of correct function in the male reproductive system.

Pathological Findings in the Testes of COVID-19 Patients: Clinical Implications

BackgroundCoronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), involves multiple organs. Testicular involvement is largely unknown. ObjectiveTo determine the pathological changes and whether SARS-CoV-2 can be detected in the testes of deceased COVID-19 patients. Design, setting, and participantsPostmortem examination of the testes from 12 COVID-19 patients was performed using light and electron microscopy, and immunohistochemistry for lymphocytic and histiocytic markers. Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the virus in testicular tissue. Outcome measurements and statistical analysisSeminiferous tubular injury was assessed as none, mild, moderate, or severe according to the extent of tubular damage. Leydig cells in the interstitium were counted in ten 400× microscopy fields. Results and limitationsMicroscopically, Sertoli cells showed swelling, vacuolation and cytoplasmic rarefaction, detachment from tubular basement membranes, and loss and sloughing into lumens of the intratubular cell mass. Two, five, and four of 11 cases showed mild, moderate, and severe injury, respectively. The mean number of Leydig cells in COVID-19 testes was significantly lower than in the control group (2.2 vs 7.8, p < 0.001). In the interstitium there was edema and mild inflammatory infiltrates composed of T lymphocytes and histiocytes. Transmission EM did not identify viral particles in three cases. RT-PCR detected the virus in one of 12 cases. ConclusionsTestes from COVID-19 patients exhibited significant seminiferous tubular injury, reduced Leydig cells, and mild lymphocytic inflammation. We found no evidence of SARS-CoV-2 virus in the testes in the majority (90%) of the cases by RT-PCR, and in none by electron microscopy. These findings can provide evidence-based guidance for sperm donation and inform management strategies to mitigate the risk of testicular injury during the COVID-19 disease course. Patient summaryWe examined the testes of deceased COVID-19 patients. We found significant damage to the testicular parenchyma. However, virus was not detected in testes in the majority of cases.

Exposure to low doses of malathion during juvenile and peripubertal periods impairs testicular and sperm parameters in rats: Role of oxidative stress and testosterone

Malathion is an organophosphate insecticide used in agriculture and for controlling vector-borne diseases such as Zika. Humans can be exposed to malathion by means of ingestion of contaminated food. The juvenile and peripubertal periods are a large window of vulnerability to the action of toxic agents. The aim of the present study was to evaluate the effects of low doses of malathion during the development of testes in the juvenile and peripubertal periods in rats. For this purpose, 45 male Wistar rats (postnatal day (PND) 25) were assigned to 3 experimental groups and treated for 40 days. The animals were exposed daily to malathion 10 mg/kg (M10 group) or 50 mg/kg (M50 group) diluted in 0.9 % saline via gavage. The control group received only the vehicle. On the 40th experimental day, the rats were anaesthetized and euthanized. The blood was collected for determination of testosterone concentration. The testes were removed and weighed. Spermatozoa from the vas deferens were used for sperm morphological analysis. The testes were used for evaluation of sperm count and oxidative stress status to determine the inflammatory profile and analysis of tissue constitution. The results showed that both malathion doses reduced the sperm count and increased the number of abnormal sperms. Furthermore, both doses altered the spermatogenetic process, delayed spermiogenesis, reduced the Leydig and Sertoli cell number and increased the thickness of tunica albuginea. The M10 group presented increased IL-10 levels and reduced GSH levels. These parameters did not change in the M50 group. However, the M50 group showed an increase in the number of abnormal seminiferous tubules, a decrease in plasma testosterone concentration and an increase in lipid peroxidation in the testes. In conclusion, the exposure to low doses of malathion during juvenile and peripubertal development resulted in testicular toxicity and compromised the testicular morphology and function.

In utero cadmium and dibutyl phthalate combination exposure worsens the defects of fetal testis in rats

Testicular dysgenesis syndrome might be due to the fetal testis defects caused by endocrine disruptors. Here, we report the combined effects of in utero exposure to cadmium (CdCl2, Cd) and di-n-butyl phthalate (DBP) on fetal testis development in rats. Pregnant Sprague-Dawley rats were randomly divided into four groups: control, Cd, DBP (250 mg/kg/day), and Cd + DBP. Cd (0.25 mg/kg/once) was intraperitoneally injected to the dam on gestational day 12 and DBP (250 mg/kg) was daily gavaged to the dam on gestational day 12 for 10 days. Cd, DBP, and Cd + DBP lowered serum testosterone levels in male fetuses. Cd and DBP did not alter fetal Leydig cell (FLC) number, but the combined exposure led to decreased FLC number. Cd did not affect FLC aggregation while DBP caused FLC aggregation and the combined exposure worsened FLC aggregation. Cd lowered FLC mRNA (Lhcgr, Star, Cyp11a1, and Insl3) levels and DBP lowered Lhcgr, Star, Insl3, and Nr5a1 levels. DBP up-regulated Scarb1 expression without affecting Cyp11a1 while the combined exposure antagonized DBP. These two chemicals and its combination did not affect Sertoli cell number and gene (Amh, Fshr, and Sox9) expression at current doses. In conclusion, the combined exposure of Cd and DBP exerts synergically antiandrogenic effects via targeting FLC development.

Effects of in utero exposure to diisodecyl phthalate on fetal testicular cells in rats

Diisodecyl phthalate (DIDP) is one of synthetic phthalate plasticizers. It is widely used in plastic products and is a potential endocrine disruptor. However, the effects of DIDP on fetal testicular cell development remain unclear. The objective of the present study was to determine the effects of DIDP on fetal testis development in rats after in utero exposure. Sprague Dawley dams were randomly divided into 5 groups and were daily gavaged with DIDP (0, 10, 100, 500, and 1000 mg/kg body weight) from gestational day 14–21. Serum testosterone levels, fetal Leydig cell number and distribution, testicular gene and protein expression in male pups were examined. DIDP decreased serum testosterone levels at 1000 mg/kg (1.37 ± 0.40 ng/mL, mean ± SE) when compared to the control level (3.14 ± 0.60 ng/mL). DIDP did not affect numbers of Leydig and Sertoli cells. DIDP significantly induced abnormal aggregation of fetal Leydig cells and increased the incidence of multinucleated gonocytes at 1000 mg/kg. Furthermore, DIDP down-regulated expression of Star, Cyp11a1, Hsd17b3, and Insl3 in fetal Leydig cells at 1000 mg/kg and Sox9 in Sertoli cells at 1000 mg/kg. In conclusion, the current study indicates that in utero exposure to high-dose DIDP disrupts the development of fetal testicular cells, thus affecting the male reproductive system.

Improvement of Rat Sperm Chromatin Integrity and Spermatogenesis with Omega 3 following Bleomycin, Etoposide and Cisplatin Treatment

The objective of this study is to examine the effects of omega 3 treatment on rat sperm chromatin condensation, DNA damage and spermatogenesis after bleomycin, etoposide and cisplatin (BEP) treatment. In this experimental study, 40 male rats were divided into four groups: Control, BEP, Omega 3 and BEP + Omega 3. Sperm chromatin condensation and DNA damage were assessed using aniline blue and acridine orange staining, respectively. Results show that the mean percentage of sperms with excessive histone and DNA damage was significantly increased in the BEP group after 9 weeks as compared to control group (p˂0.001). While, in the BEP + Omega 3 group, the mean percentage of sperm with excessive histone and DNA damage was decreased significantly compared with BEP group (p˂0.001). The testicular histomorphometric analysis indicated that omega 3 has a significant effect on the mean number of spermatogonia, primary spermatocytes, leydig cells and testicular histology properties following BEP treatment. The mean count of aforementioned cells significantly increased after omega 3 treatment compared with the BEP group (p˂0.001). Our data indicated omega 3 may be had beneficial effect for improving chromatin condensation, DNA damage during spermatogenesis and testicular histomorphic properties following BEP treatment.

PENGARUH PEMBERIAN MADU TERHADAP STAGING SPERMATOGENESIS DAN SEL LEYDIG PADA MENCIT (Mus musculus) YANG DIINFEKSI Toxoplasma gondii

The aim of this research is to know about the staging of spermatogenesis and leydig cells in mice that injected by Toxoplasma gondii after giving honey as a preventive substance. This research used 25 mice in age 2-4 month with 20-30 weight. Mice were divided into 5 groups (P0, P1, P2, P3 dan P4). P0 as a control teratment were given aquades in peroral, P1 was a group given honey in doses I (0.08ml/0.2ml/day) during 14 days in peroral, P2 was a group that given infection by T.gondii in doses 103 in peritonial, P3 was given honey in doses I 0.08ml/0.2ml/day) then injected by T.gondii, and P4 was given honey in doses II (0.12ml/0.2ml/day) then injected by T.gondii. This research conducted in 18 days, in day 14th mice were given honey and the day 15th–18th mice injected by T.gondii doses 103. The data result was analyzed with Analysis of Variant (ANOVA) and cotinued with Duncan. Comparision statistical showed with SPSS statistic 20 for Windows. The result of analysis, P0 and P1 were significantly different (p&lt;0.05), with the increase number of spermatogenic and leydig cells. P0 and P2 also significantly different, with the decrease number of spermatogenic and leydig cells. In P2 and P3 group were not significantly different (P&gt;0.05), also in P4 group were not significantly different with P0 and P1. The results of this research showed that honey in doses II can maintain the number of sprematogenic and leydig cells as a preventive substance against T.gondii.

EFEK PENEMBAKAN LASERPUNKTUR PADA TITIK OVA BEBEK MOJOSARI JANTAN TERHADAP JUMLAH SEL SERTOLI DAN LEYDIG

Demand for eggs and duck meat lately increased along with the increasing interest of the community to consume eggs and duck meat. Due to that reason researcher try to find the best way to enhance the reproductive ability of livestock., which is in this research by laserpuncture shot. This research used 3 treatments: the control group, first treatment with laserpuncture shot dose of 0.2 J, second treatment with laserpuncture shot dose of 0.4 J. Data was analyzed with ANOVA and further test by Duncan Multiple Range Test. Research result proved that Laserpuncture shot with dose of 0.2 J and 0.4 J decrease the number of sertoli and leydig cells.

Specificity and application of SOX2 antibody

Sox2 is known to play an important role in maintaining the totipotency and self-renewal of embryonic stem cells. The purpose of this study was to prepare an anti-chicken Sox2 polyclonal antibody using prokaryotic expression techniques, to evaluate its specificity and to use it to investigate the expression and distribution of Sox2 in the chicken brain and lungs. The chicken Sox2 gene was amplified and subcloned to a pET-30a vector to construct a prokaryotic expression vector, pET-Sox2. A His-Sox2 fusion protein was expressed, purified, and used to prepare an antichicken Sox2 polyclonal antibody. Western blotting revealed that the antichicken Sox2 antibody could specifically bind not only to the purified His-Sox2 fusion protein but also to the endogenous Sox2 protein in the testes of chicken, showing a distinct dose-dependent relationship between antigen and Sox2 antibody. Indirect immunofluorescent staining of Sox2-overexpressing cells showed strong nuclear and diffuse cytoplasmic immunoreactivity for Sox2 in the antichicken Sox2 antibody-staining cells. A CRISPR/Cas9 effector system-mediated Sox2 knockdown assay indicated that Sox2 expression in HEK 293T cells was downregulated in the presence of doxycycline but upregulated in the absence of doxycycline. In addition, cryosectioning and immunohistochemical staining illustrated that most spermatogonia in the seminiferous tubules, and a small number of Sertoli and Leydig cells, were positive for Sox2. The antichicken Sox2 antibody was also successfully used to investigate the expression and distribution of Sox2 in the chicken cerebellar cortex, optic tectum, cerebral cortex, and lungs. The results of this study confirmed the specificity of the antichicken Sox2 polyclonal antibody, which will be available for the study of biological functions of the chicken Sox2 gene and the self-renewal mechanisms of chicken pluripotent stem cells.

Maternal exposure to zearalenone in masculinization window affects the fetal Leydig cell development in rat male fetus

Zearalenone is a phenolic Fusarium mycotoxin, which is ubiquitous in human and animal feedstuff and often co-occurs with other mycotoxins. ZEA has been reported to disturb Leydig cell function and even cause the apoptosis to the Leydig cells. However, the effects of gestational exposure to zearalenone on fetal Leydig cells and the underlying mechanism remain unknown. Sprague Dawley dams were daily gavaged with 0, 2.5, 5, 10, and 20 mg/kg body weight ZEA from gestational day 14–21. On gestational day 21, rats were euthanized and serum testosterone levels were measured, and testes were collected for further evaluation of Leydig cell number, cell size, gene, and protein expression. Zearalenone significantly decreased anogenital distance and its index of male fetus, serum testosterone levels, Leydig cell proteins (SCARB1, STAR, CYP11A1, CYP17A1, and INSL3), and fetal Leydig cell number at 10 and/or 20 mg/kg by delaying the commitment of stem Leydig cells into the Leydig cell lineage and proliferation. Further study found that Notch signaling (RFNG, PSEN1, NOTCH1, and NOTCH3) was up-regulated by zearalenone. In conclusion, gestational exposure to high doses of zearalenone (10 and 20 mg/kg) blocks fetal Leydig cell development, thus possibly causing the anomalies of the male reproductive tract.

Long-term 1-nitropyrene exposure induces endoplasmic reticulum stress and inhibits steroidogenesis in mice testes

1-Nitropyrene (1-NP) is a representative nitro-polycyclic aromatic hydrocarbon from diesel exhaust. Recently, we found that maternal 1-NP exposure caused fetal growth retardation and disturbed cognitive development in adolescent female offspring. To investigate long-term 1-NP exposure on spermatogenesis and steroidogenesis, male mice were exposed to 1-NP (1.0 mg/kg/day) by gavage for 70 days. There was no significant difference on relative testicular weight, number of testicular apoptotic cells and epididymal sperm count between 1-NP-exposed mice and controls. Although long-term 1-NP exposure did not influence number of Leydig cells, steroidogenic genes and enzymes, including STAR, P450scc, P45017α and 17β-HD, were downregulated in 1-NP-expoed mouse testes. Correspondingly, serum and testicular testosterone (T) levels were reduced in 1-NP-exposed mice. Additional experiment showed that testicular GRP78 mRNA and protein were upregulated by 1-NP. Testicular phospho-IRE1α and sliced xbp-1 mRNA, a downstream molecule of IRE1α, were elevated in 1-NP-exposed mice. Testicular phospho-PERK and phospho-eIF2α, a downstream molecule of PERK pathway, were increased in 1-NP-exposed mice. Testicular NOX4, a subunit of NAPDH oxidase, and HO-1, MDA, two oxidative stress markers, were increased in 1-NP-exposed mice. Testicular GSH and GSH/GSSG were decreased in 1-NP-exposed mice. These results suggest that long-term 1-NP exposure induces reactive oxygen species-evoked ER stress and disrupts steroidogenesis in mouse testes.

Effects of gestational Perfluorooctane Sulfonate exposure on the developments of fetal and adult Leydig cells in F1 males

Studies have showed that some of the most common male reproductive disorders present in adult life might have a fetal origin. Perfluorooctane sulfonic (PFOS) is one of the major environmental pollutants that may affect the development of male reproductive system if exposed during fetal or pubertal periods. However, whether PFOS exposure during fetal period affects testicular functions in the adult is still unclear. Herein, we investigated the effects of a brief gestational exposure to PFOS on the development of adult Leydig- and Sertoli-cells in the male offspring. Eighteen pregnant Sprague-Dawley rats were randomly divided into three groups and each received 0, 1 or 5 mg/kg/day PFOS from gestational day 5–20. The testicular functions of F1 males were evaluated on day 1, 35 and 90 after birth. PFOS treatment significantly decreased serum testosterone levels of animals by all three ages examined. The expression level of multiple mRNAs and proteins of Leydig (Scarb1, Cyp11a1, Cyp17a1 and Hsd17b3) and Sertoli (Dhh and Sox9) cells were also down-regulated by day 1 and 90. PFOS exposure might also inhibit Leydig cell proliferation since the number of PCNA-positive Leydig cells were significantly reduced by postnatal day 35. Accompanied by changes in Leydig cell proliferation and differentiation, PFOS also significantly reduced phosphorylation of glycogen synthase kinase-3β while increased phosphorylation of β-catenin. In conclusion, gestational PFOS exposure may have significant long-term effects on adult testicular functions of the F1 offspring. Changes in Wnt signaling may play a role in the process.

An exploration of the role of Sertoli cells on fetal testis development using cell ablation strategy.

Sertoli cells (SCs) are presumed to be the center of testis differentiation because they provide both structural support and biological regulation for spermatogenesis. Previous studies suggest that SCs control germ cell (GC) count and Leydig cell (LC) development in mouse testes. However, the regulatory role of SCs on peritubular myoid (PTM) cell fate in fetal testis has not been clearly reported. Here, we employed Amh-Cre; diphtheria toxin fragment A (DTA) mouse model to selectively ablate SCs from embryonic day (E) 14.5. Results found that SC ablation in the fetal stage caused the disruption of testis cords and the massive loss of GCs. Furthermore, the number of α-smooth muscle actin-labeled PTM cells was gradually decreased from E14.5 and almost lost at E18.5 in SC ablation testis. Interestingly, some Ki67 and 3β-HSD double-positive fetal LCs could be observed in Amh-Cre; DTA testes at E16.5 and E18.5. Consistent with this phenomenon, the messenger RNA levels of Hsd3b1, Cyp11a1, Lhr, Star and the protein levels of 3β-HSD and P450Scc were significantly elevated by SC ablation. SC ablation appears to induce ectopic proliferation of fetal LCs although the total LC number appeared reduced. Together, these findings bring us a better understanding of SCs' central role in fetal testis development.

Характеристика жизнеспособности сперматозоидов у потомства самок крыс с экспериментальным сахарным диабетом 1 типа при действии иммобилизационного стресса

One of the main factors underlying the development of prenatal stress is maternal type 1 diabetes. Its influence on the formation of the male reproductive system of the offspring has not been well studied. Therefore, this study was aimed at the analysis of the spermatozoa motor activity characteristics in mature offspring of the mothers with experimental type 1 diabetes mellitus, subjected to the immobilization stress. The study was performed on white laboratory Wistar rats (females), in which the type 1 diabetes mellitus was inducted, and their mature offspring. Spermatozoa were obtained from the epididymis; their motility was taken into account when determining their content in a unit volume. In order to study the antistress resistance of sperm, the the experimental animals were subjected to a model of immobilization stress. We demonstrate that the sperm concentration is decreased in the offspring of female rats with experimental diabetes. This decrease also occurs in animals subjected to the immobilization stress, in both intact and experimental groups. The fertile to non-fertile germ cells ratio is also affected by the immobilization stress. We show that the number of Leydig cells and their activity index were reduced in experimental animals. Based on these results, we conclude that the fertility and antistress resistance of spermatogenesis are reduced in the offspring of the female rats with experimental diabetes mellitus. The hypothalamic-pituitary-gonadal system provides control of the reproductive system, primarily through testosterone, which is produced by the Leydig cells and is involved in the regulation of the spermatogenesis. The activation of the hypothalamic-pituitary-adrenal system under hypoglycemia leads to the suppression of the hypothalamic–pituitary–gonadal system activity. We propose that the immobilization stress raises the corticosteroid levels, which further inhibits the secretory activity of the Leydig cells, leading to the disruption of the spermatogenesis and the decrease in sperm concentration and motor activity.