Sertoli Cell Vacuolation Research Articles

Assessment of Dichlorvos, Cypermethrin, Synthetic Camphor, Pinenes, and Kerosene: Impact on Testosterone Levels, Glutathione Concentration, and Testicular Histology in Male Wistar Rats

Pesticides are integral to modern agriculture and public health but pose significant risks to human and environmental health. This study investigates the impact of selected pesticides on male reproductive health via inhalation in an improvised chamber. Male Wistar rats were assigned to groups and exposed to individual pesticides and a combined mixture to determine lethal doses. Tissue samples from 54 rats were analyzed histologically and biochemically for effects on testes and epididymides, with measured serum testosterone and GSH levels. Sniper consistently showed the highest significant decrease in GSH levels across all exposure levels (p < 0.05). A significant dose-dependent relationship was observed in the recovery groups from 25% to 75% exposure (p < 0.05). Pesticide exposure led to a substantial decrease in serum testosterone levels in male Wistar rats at 25% (all pesticides, p < 0.05), 50% (DD Force, Sniper, Kerosene, Combined, p < 0.05), and 75% (DD Force, Industrial Camphor, Edible Camphor, Kerosene, p < 0.01) concentrations. Escalating damage in testicular and epididymal structures, with a more pronounced severity at higher exposure levels, including epithelial degeneration, Sertoli cell vacuolation, seminiferous tubule derangement, and ductal atrophy. Increased exposure to high doses of pesticides induces oxidative stress in male Wistar rats which could potentiate decreased testosterone levels and adversely impact the histological architecture of the testes and epididymides. Pesticide detoxifications are somewhat evident following withdrawal from the environmental insults. Significant and persistent damage occurred at higher levels, suggestive of substantial reproductive toxicity following exposure.

The effects of vitamin E supplementation on sperm parameters, chromatin integrity, and gene expression before and after freezing in aged mice.

Some age-related testicular changes, such as Sertoli cell vacuolization and blood-testis barrier breakdown, reduce total sperm production and male fertility. Therefore, this study investigated the effect of vitamin E on restoring testicular function in aged mice. Sperm cryo-resistance was also assessed. Twenty-eight 48-week-old male Naval Medical Research Institute mice were divided into four groups for a daily gavage of vitamin E: the control group received distilled water, while the three treatment groups were administered 100, 200, and 400 mg/kg, respectively, for 4 weeks. Subsequently, semen analyses, DNA fragmentation index (DFI), and protamine deficiency tests were conducted. Testicular histology, tissue antioxidant enzyme activity, and gene expression levels were also assessed. The two higher dosages of vitamin E were associated with a higher sperm count, greater progressive motility, and improved sperm morphology (p<0.05). These benefits were also evident after sperm freezing (p<0.05). Although chromatin abnormalities increased following vitrification, the treatment groups showed better outcomes (p<0.05). The tubular diameter, epithelium height, and luminal diameters remained unchanged with age. The tissue antioxidant capacity was greater in the groups receiving the high doses of vitamin E. Additionally, significant increases in inhibitor of DNA binding protein-4 (Id4) and GDNF family receptor alpha-1 (Gfra1) expression were observed in the higher vitamin E dosage groups, and promyelocytic leukemia zinc finger protein (Plzf) expression was notably present in the 400 mg/kg treatment group compared to the control group (p<0.05). Antioxidant supplementation might enhance reproductive outcomes in aging males. The observed effects included improved sperm cryo-resistance, which is advantageous for future applications such as sperm freezing or fertility preservation.

Evaluation of chemical castration using intra-testicular injection of zinc gluconate into the testis of the male donkey versus surgical castration: antimullerian hormone as an endpoint marker

BackgroundChemical castration of male animals is an alternative to surgical castration for inducing azoospermia, consequent sterility. Intra-testicular injection of zinc gluconate has been used for chemical castration in several animal species. However, its application to equine species, such as donkeys, has yet to be reported. This study aimed to evaluate the use of zinc gluconate for the chemical castration of male donkeys and to compare its effectiveness relative to routine surgical castration. For this purpose, investigations of serum testosterone and anti-Müllerian hormone levels, testicular ultrasonographic echogenicity, and histopathological findings were performed.MethodsFourteen clinically healthy adult male donkeys were randomly and equally divided into two groups. The donkeys in group I (n = 7) underwent surgical castration. The donkeys in group II (n = 7) received intra-testicular zinc gluconate injections. The donkeys were kept under close clinical observation for 60 days. Abnormalities in donkey behavior and gross alterations in the external genitalia were recorded daily. Serum testosterone and anti-Müllerian hormone (AMH) levels were measured 15 days before the start of the treatment and 15, 30, 45, and 60 days after treatment. The testicles of group II donkeys were evaluated ultrasonographically. At the end of the study, the testes were removed and histologically examined.ResultsSerum testosterone levels significantly declined compared to pre-castration levels in surgically castrated donkeys (group I), but donkeys exposed to chemical castration (group II) showed a non-significant reduction in testosterone levels. Donkeys in the surgical group had considerably lower serum AMH levels. In contrast, there was a non-significant (p > 0.05) increase in AMH levels in the chemical group compared with the pre-sterilization level. In addition, ultrasonographic examination revealed that the testicular echo-density had changed, as observed by a few scattered hyperechoic regions throughout the entire testis parenchyma. The histopathological investigation confirmed the presence of necrosis of the spermatogenic epithelium, increased thickness of the basement membrane of the seminiferous tubules, marked interstitial fibrosis, and shrinkage of the seminiferous tubules. Furthermore, syncytial giant cells were present in the lumen of seminiferous tubules and were associated with Sertoli cell vacuolation. Donkeys subjected to chemical castration (group II) had orchitis, as confirmed histopathologically.ConclusionIntra-testicular injection of zinc gluconate resulted in histopathological and ultrasonographic testicular changes in adult male donkeys, which may affect their reproductive potential. However, it did not significantly alter serum testosterone or AMH levels, indicating that it cannot be used as a substitute for surgical castration in male donkeys.

Supplementation of feedlot lambs with cactus cladodes as strategy to improve testicular function

The aim of this study was to evaluate the effects of diets containing cactus cladodes genotypes on plasma testosterone levels, testicular histopathological and histomorphometric parameters, and oxidative stress markers in lambs. Thirty-six male, intact Santa Inês lambs (22.0 ± 2.9 kg initial body weight), were to feedlot for 86 days. A completely randomized design was used with three dietary treatments (control diet with Tifton-85 hay as the only roughage; and two more diets with Miúda or OEM cactus cladodes partially replacing hay) and twelve replicates. There was no influence of the diets on the testicular weight (P = 0.414) and gonadosomatic index (P = 0.384) of lambs. The testosterone serum concentrations were almost twice as higher in lambs fed Miúda cactus cladodes compared to control treatment. There was greater incidence and severity of lesions in the testicular parenchyma of animals that received control diet: loosening of germ cell epithelium, germ cell desquamation and vacuolization of Sertoli cells. The seminiferous tubule diameter and height of the seminiferous epithelium were higher in lambs fed OEM cactus cladodes (P = 0.003). The tubular volume and Leydig cells volume were higher in animals fed with cactus cladodes (P < 0.05). The levels of malondialdehyde were higher in the lambs of control group compared to OEM group (P = 0.039) and the testicular concentration of nitric oxide was higher in control group (P = 0.009). The diet containing OEM cactus cladodes increased the levels of superoxide dismutase. Our results indicate that diets containing cactus cladodes promote antioxidant protection to the testicular parenchyma and preserve the spermatogenic process of lambs.

Exposure to China dust exacerbates testicular toxicity induced by cyclophosphamide in mice.

This study investigated the potential effects of China dust (CD) exposure on cyclophosphamide (CP)-induced testicular toxicity in mice, focusing on spermatogenesis and oxidative damage. CP treatment reduced testicular and epididymal weight and sperm motility and enhanced sperm abnormality. Histopathological examination presented various morphological alterations in the testis, including increased exfoliation of spermatogenic cells, degeneration of early spermatogenic cells, vacuolation of Sertoli cells, a decreased number of spermatogonia/spermatocytes/spermatids, along with a high number of apoptotic cells. In addition, the testis exhibited reduced glutathione (GSH) levels and glutathione reductase (GR) activity and enhanced malondialdehyde (MDA) concentration. Meanwhile, CD exposure exacerbated testicular histopathological alterations induced by CP. CD exposure also aggravated oxidative damage by increasing the lipid peroxidative product MDA and decreasing GSH levels and antioxidant enzyme activities in the testis. These results suggest that CD exposure exacerbates CP-induced testicular toxicity in mice, which might be attributed to the induction of lipid peroxidation and reduced antioxidant activity.

Effects of Dietary Crude Glycerin Concentration on Testicular Morphology and Oxidative Stress Markers and on Plasma Testosterone Concentrations

We evaluated the effects of feeding 6%, 12% or 18% crude glycerin, containing 80.5% glycerol, on testicular histomorphometry and markers of oxidative stress and on plasma testosterone concentrations in lambs. Body weight, testicular biometric measurements, gonadosomatic index and net weight of the testicles were higher for the treated groups (P<0.05) compared with a control group that did not receive dietary glycerin. The mean total length of seminiferous tubules was higher in the 6% group (P<0.05), while the mean total tubular and seminiferous epithelium volumes increased in all treated groups (P<0.05). The volume of Leydig cells increased in the 12% group, while their number per gram of testicle decreased (P<0.05). There was a decrease in mean nuclear diameter and mean volume of Leydig cells, and an increase in the mean number of these cells per gram of testicle, in the 18% group (P<0.05). Plasma testosterone concentrations were unaffected. There was desquamation of seminiferous epithelium and vacuolation of Sertoli cells in the treated groups. Variable degrees of spermatocyte necrosis and the presence of giant cells were seen in all groups and there was intense vacuolation of Sertoli cells in the 12% and 18% groups. Superoxide dismutase and catalase production increased most in the 12% and 18% groups (P<0.05), while glutathione production was higher in the 18% group (P<0.05). Mean nitric oxide concentration decreased in all treated groups (P<0.05), while malondialdehyde production was higher in the 18% group than in the control and 6% groups (P<0.05). We conclude that the inclusion of 6% glycerin in the diet of lambs results in changes in testicular morphology that have been previously associated with improved reproductive function, but without evidence of oxidative stress.

Histoprotective effect of rutin against cisplatin-induced toxicities in tumor-bearing mice: Rutin lessens cisplatin-induced toxicities.

Cisplatin is an effective anticancer drug used against a variety of cancers. The full therapeutic potential of cisplatin is often hampered due to concurrent development of various side effects in the hosts. Rutin, a naturally occurring bioflavonoid shows several pharmacological activities. It has been earlier reported by us that rutin and cisplatin in combination show better antitumor activity against murine ascites Dalton's lymphoma. As cisplatin is given to cancer-bearing hosts only, the present study was undertaken to explore the histoprotective effect of rutin against some toxicities induced by cisplatin in tumor-bearing mice. Cisplatin treatment caused severe damages in tissue architecture such as degenerated hepatocytes with nuclear condensation and sinusoidal dilatation in the liver, glomerular deterioration, infiltration of cells, and tubular congestion in the kidney, and vacuolization of Sertoli cells or dense granules in the cytoplasm and damaged seminiferous tubules in the testes. In the rutin plus cisplatin combination-treated mice, all the abnormal tissue architectural features were decreased. Further, as compared to cisplatin treatment, combination treatment did not show any significant elevation in the liver functional biomarkers (serum aspartate aminotransferase, alanine aminotransferase and alkaline phosphatase) and renal functional biomarkers (serum urea and creatinine levels). The combination treatment reduced the sperm abnormalities also as compared to the cisplatin alone treatment. The in vitro hemolysis assay of red blood cells and scanning electron microscopy revealed that combination treatment lessened the cisplatin-induced hemolysis and abnormalities in RBCs. Thus, the present findings demonstrate that rutin has histoprotective ability against cisplatin-induced toxicities in tumor-bearing mice.

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.

Exploration of small RNA biomarkers for testicular injury in the serum exosomes of rats

Testicular injury is often observed in drug development. Serum hormones are usually used as noninvasive biomarkers for testicular injury; however, their sensitivities are low. Therefore, it is difficult to monitor testicular injury in drug development. In recent years, molecules in body fluid exosomes have attracted attention as biomarkers for diseases. In this study, small RNAs in serum exosomes were analyzed to identify noninvasive biomarkers of testicular injury in rats, which are often used in preclinical drug development. The rat models of testicular injury were prepared by a single oral administration of 2000 mg/kg ethylene glycol monomethyl ether, in which spermatocyte degeneration and Sertoli cell vacuolation were observed, or 400 mg/kg carbendazim, in which Sertoli cell vacuolation and seminiferous tubule dilation were observed. Serum exosomal small RNA-seq analysis of these models was performed. The analysis identified 3 small RNAs that fluctuated in common between the models, and miR-423-5p and miR-128-3p were selected as candidate markers. For evaluating these candidate markers in other testicular injury models, the models were prepared by a single oral administration of 60 mg/kg 1,3-dinitrobenzene or 500 mg/kg nitrofurazone, and spermatocyte degeneration and Sertoli cell vacuolation were observed. In qPCR analysis, these exosomal miRNAs were upregulated in all models except for the 1,3-dinitrobenzene model, in which severe hemolysis was observed. By contrast, these miRNAs in whole serum extracts did not significantly change in any of the models. In conclusion, we identified miR-423-5p and miR-128-3p in serum exosomes as noninvasive biomarkers for testicular injury in rats.

Global deletion of the RNA decapping enzyme Dcp2 postnatally in male mice results in infertility

The post-transcriptional regulation of gene expression plays an important role in many essential biological processes. The RNA decapping enzyme Dcp2 is a crucial enzyme involved in RNA degradation. Dcp2 proteins are highly expressed in the testis and brain in adult mice. This study aimed to investigate the in vivo functions of Dcp2. An inducible Dcp2 knockout mouse model was established. No obvious health abnormalities were observed after postnatal global deletion of Dcp2 in male mice. However, Dcp2-deleted male mice were infertile and showed Sertoli cell vacuolization and germ cell degeneration. Dcp2 deletion resulted in testicular atrophy, reduced number of epididymal sperm, and increased apoptosis in seminiferous tubules. However, spermatocyte-specific deletion of Dcp2 did not compromise the fertility. The findings of this study indicated that Dcp2 was important for spermatogenesis and male fertility.

ZnO Nanoparticles Induced Male Reproductive Toxicity Based on the Effects on the Endoplasmic Reticulum Stress Signaling Pathway.

PurposeThe aim of this study was to evaluate the adverse effects of ZnO NPs on male reproductive system and explore the possible mechanism.MethodsIn this study, the effect of oral administration of 50, 150 and 450 mg/kg zinc oxide nanoparticles (ZnO NPs) in adult male mice was studied over a 14-day period.ResultsThe results showed that the number of sperms in the epididymis and the concentration of testosterone in serum were decreased with an increased dose of ZnO NPs. Testicular histopathological lesions like detachment, atrophy and vacuolization of germ cells were observed. The results showed that increased dosage of ZnO NPs correspondingly up-regulated the IRE1α, XBP1s, BIP, and CHOP (P<0.05) which are genes related to ER stress. These observations indicated that ZnO NPs had adverse effects on the male reproductive system in a dose-dependent manner possibly through ER stress. The expression of caspase-3 was significantly increased in all the treated groups (P<0.001), which reflected the possible activation of apoptosis. Additionally, there was significant down-regulation of the gene StAR (P<0.05), a key player in testosterone synthesis. When an ER-stress inhibitor salubrinal was administered to the 450 mg/kg ZnO NPs treatment group, the damages to the seminiferous tube and vacuolization of Sertoli and Leydig cells were not observed. Furthermore, the testosterone levels in the serum were similar to the control group after the subsequent salubrinal treatment.ConclusionIt may be inferred that the ZnO NP's reproductive toxicity in male mice occurred via apoptosis and ER-stress signaling pathway.

Ipomoea brasiliana poisoning on buck reproduction

ABSTRACT: Swainsonine-containing plants comprise a group of important poisonous plants in Brazil. This research aimed to characterize both the behavioral changes related to reproduction and appearance of lesions in the reproductive system of bucks poisoned by Ipomoea brasiliana. I. brasiliana plants were collected and administered at a dose of 4g/kg (800µg swainsonine/kg) to two groups of bucks for 45 days. Goats from Group I were euthanized on the 46th day of the experiment, and goats from Group II were euthanized on the 120th day. Group III was composed of goats that did not receive I. brasiliana and were euthanized on the 120th day of the experiment. Reproductive behavioral changes were observed starting on day 20 and were characterized by an absence of courtship behavior, and Flehmen reflex, decrease or loss of libido and inability to perform mating. After 120 days, Group II goats showed no regression of the changes in their reproductive behavior or improvement of their seminal parameters. The main defects observed in the sperm of goats that consumed I. brasiliana were cytoplasmatic droplets, bent tails and detached tails. The main histopathological findings were reported in tests, with cytoplasmic vacuolization of germline and Sertoli cells, generalized impairment of spermatogonia maturation with exfoliation of degenerative cells, cell fragments, rare abnormal spermatocytes in the seminiferous lumen and disappearance of Leydig cells. Results of this study confirmed the hypothesis that I. brasiliana causes testicular degeneration in male goats.

Characterization of the Testicular Toxicity of 3-Nitro-1,2,4-Triazol-5-One and 2,4-Dinitroanisole in Rats ( Rattus norvegicus).

Nitrotriazolone (3-nitro-1,2,4-triazol-5-one; NTO) and dinitroanisole (2,4-dinitroanisole; DNAN), insensitive energetic materials used in explosive formulations, have induced testicular toxicity and oligospermia in repeated-dose oral toxicity tests. To identify the target site of testicular toxicity of NTO and DNAN, Sprague Dawley rats were orally dosed with NTO (500 mg/kg/d) or DNAN (50 or 100 mg/kg/d) in corn oil for 1, 3, 7, or 14 days. Degeneration of germinal epithelium occurred in multiple tubule stages on days 7 and 14 in treated rats. Degeneration increased in severity with time and was characterized by degeneration/apoptosis of pachytene spermatocytes and round and elongating spermatids, depletion of step 19 spermatids, luminal spermatogenic cell sloughing, multinucleate cells, and pronounced Sertoli cell vacuolation. Serum luteinizing hormone and follicle-stimulating hormone did not differ between NTO- and DNAN-treated and control rats on any sampling day. Serum testosterone levels reduced only in rats given 50 mg/kg/d DNAN for 7 days. These results suggest that the initial site of testicular injury for both NTO and DNAN is the Sertoli cell.

Exposure to Concentrated Ambient PM2.5 Compromises Spermatogenesis in a Mouse Model: Role of Suppression of Hypothalamus-Pituitary-Gonads Axis.

Epidemiological studies link ambient fine particulate matter (PM2.5) pollution to abnormalities in the male reproductive system. However, few toxicological studies have investigated this potentially important adverse effect of PM2.5 pollution. Therefore, in the present study, we analyzed the effects of PM2.5 exposure on spermatogenesis and hypothalamic-pituitary-gonadal (HPG) axis in a murine model. Fourteen male C57BL/6J mice were subjected to a 4-month exposure to filtered air or concentrated ambient PM2.5 (CAP). Their sperm count, testicular histology, spermatogenic parameters, and the major components of HPG axis were assessed. Exposure to CAP significantly reduced sperm count in the epididymis. This was accompanied by Sertoli cell vacuolization, immature germ cell dislocation, and decreases in pachytene spermatocytes and round spermatids of stage VII seminiferous tubules, suggesting a marked impairment of spermatogenesis in these mice. This impairment of spermatogenesis appeared to be attributable to a suppression of HPG axis subsequent to CAP exposure-induced hypothalamic inflammation, as exposure to CAP significantly increased TNFα and IL1b mRNA levels and meanwhile decreased gonadotropin-releasing hormone mRNA expression in the hypothalamus. Moreover, CAP exposure significantly reduced circulating testosterone and follicle-stimulating hormone, testicular testosterone and mRNA expression of follicle-stimulating hormone target gene SHBG and luteinizing hormone target genes P450scc, 17βHSD, and StAR. The present data demonstrate that exposure to ambient PM2.5 impairs spermatogenesis in murine model, raising the concern over effects of ambient PM2.5 pollution on the male reproductive function.

Chemical sterilisation of Bos indicus bull calves following intratesticular injection of zinc acetate: Effects on semen quality and testicular changes

The aim of this study was to determine the effects in Bos indicus bull calves of intratesticular administration of 1mL of either saline (n=9) or one of the two doses of zinc acetate (ZA1, 57.75mg, n=10 or ZA2, 71.75mg, n=10) on semen quality and testicular changes. Semen was collected by electroejaculation on Days 343, 524 and 783 and animals were slaughtered on Day 860. Treatment reduced median maximum number of progressively motile and morphologically normal sperm collected (P=0.001) and the percentage of animals in which sperm were recovered (saline: 100%, 9/9; ZA1: 44.9%, 4/9 and ZA2: 40.0%, 4/10; P=0.013). Compared to saline treated controls, treatment with ZA reduced the mean diameter of the testes after Day 34 of treatment (treatment×time, P=0.013) and total testicular weight at slaughter (treatment: mean±SEM; saline: 569.4±59.0g, ZA1: 249.3±72.9g, ZA2: 247.5±68.1g; P=0.004). Histological changes in testes of bulls treated with ZA were characterized by germ cell depletion, vacuolation of Sertoli cells, interstitial fibrosis, epididymal duct atrophy with variable remnants of testicular tissue and degeneration. We conclude that intratesticular administration of two doses of ZA in B. indicus calves is able to severely impair spermatogenesis and cause varying degrees of testicular degeneration and a reduction in testicular diameter and mass. Further investigation is required to determine ways of obtaining more consistent results from treatment.

Effects of chronic tramadol administration on testicular tissue in rats: an experimental study.

In a prospective experimental study, the effects of chronic tramadol administration on gonadotrophic and sex hormones, histopathological and morphometrical alterations in rat testicular tissue were investigated in a laboratory setting. Tramadol was given alone to adult male albino rats. Gonadotrophic and serum sex hormone levels were measured and testicular pathological and morphometric changes were observed in treated vs. After 30 days of treatment, tramadol induced a decrease in LH, FSH and testosterone serum levels. Histologically, degenerative changes in the seminiferous tubules were observed. They showed shrinkage, separation of tubular basement membrane, disorganisation and vacuolisation of spermatogenic layers. Morphometric analysis revealed significant decrease in the mean values of the tubular diameter and epithelial height. Ultrastructural abnormalities were detected in all cells of spermatogenic lineage in addition to the appearance of apoptotic cells. Sertoli cell vacuolation, huge lipid droplets and disrupted Sertoli cell junctions were observed. Leydig cells showed euchromatic nuclei and dilated smooth endoplasmic reticulum. In view of these findings, it is concluded that tramadol induces alterations in sex hormonal levels in conjunction with disruption of the normal histological structure of rat testis. This might lead to the risk of male infertility. Therefore, tramadol should be used with caution with appropriate dose monitoring.

Inhibin B as a Marker of Sertoli Cell Damage and Spermatogenic Disturbance in the Rat

This study was designed to determine the effects of Compound A on spermatogenesis including assessment of inhibin B levels and on fertility in the male rat over a 15 to 19 weeks treatment and a 19 weeks treatment-free period in control and 30, 60, and 180 mg/kg dose groups (n = 22/group). Compound A in a dose-dependent manner induced various degrees of spermatogenic alterations compatible with Sertoli cells being the primary target, for example, inter- and intracellular Sertoli cell vacuolization and altered cellular morphology followed by germ cell degeneration and marked reduction of epididymidal sperm numbers. Blood-testis barrier remained intact (electron microscopy and hyperosmotic fixation test) until germ cells disappeared. Mating behavior and weights of androgen-dependent prostate and seminal vesicles remained unaffected. Inhibin B levels correlated only with moderate to severe spermatogenic alterations. Ten animals with inhibin B levels below detection limit were encountered and five of these animals were fertile in week 19 but following 19 weeks without treatment, another five animals were rendered infertile and inhibin B levels remained undetectable. In the rat, inhibin B only reflects major spermatogenic alterations and markedly reduced inhibin B levels might indicate irreversibility of these alterations and even infertility.

The Inhibin B (InhB) Response to the Testicular Toxicants Mono‐2‐Ethylhexyl Phthalate (MEHP), 1,3 Dinitrobenzene (DNB), or Carbendazim (CBZ) Following Short‐term Repeat Dosing in the Male Rat

The objective of this study was to evaluate the utility of plasma Inhibin B (InhB) as a biomarker of testicular injury in adult rats following short-term exposure to the known Sertoli cell toxicants mono-2-ethylhexyl phthalate (MEHP), 1,3 dinitrobenzene (DNB), or carbendazim (CBZ). Following oral gavage administration of the compounds for 2 or 7 days, the rats were evaluated for clinical signs, body weight, food consumption, organ weights, plasma hormone levels, and gross and microscopic pathology. MEHP, DNB, and CBZ produced a range of testicular toxicity characterized by minimal exfoliation of germ cells as demonstrated by increased cellular debris in the epididymis (MEHP) to more severe and dose/duration responsive Sertoli cell vacuolation, germ cell degeneration, and multinucleated giant cells of germ cell origin (DNB and CBZ). The slight to moderate Sertoli and germinal cell injuries did not correlate with significant changes in plasma InhB levels following 2- or 7-day exposures. However, more severe injury to germinal epithelium following up to 7 days of exposure, but not after 2 days of exposure, correlated with decreased plasma InhB levels and less consistently with increases in plasma follicle stimulating hormone. In conclusion, under the conditions of these studies, changes in InhB were not an effective early onset marker of testicular toxicity or an effective marker for slight to moderate levels of acute injury, and only reflected more severe disruption of spermatogenesis. Changes in plasma InhB and follicle stimulating hormone were poorly correlated except in some instances of moderate to marked testicular toxicity.

Reproductive and developmental toxicity screening test of 3-cyanopyridine in rats

Crl:CD(SD)rats were given 3-cyanopyridine by gavage at 0, 5, 30 or 180mg/kg/day. Males were dosed for 42 days beginning 14 days before mating, and females for 40–53 days beginning 14 days before mating to day 3 of lactation, including throughout the mating and gestation periods. General toxicity, mainly liver damage, was observed in males at ≥30mg/kg/day and in females at ≥5mg/kg/day. Sertoli cell vacuolation was observed at 180mg/kg/day, and spermatocyte damages were observed at ≥30mg/kg/day. Effects on estrous cycles, corpora lutea and implantations, and unsuccessfully mated females, despite additional mating, were observed at 180mg/kg/day. Delayed initiation of delivery, dystocia, and deaths or moribundities of pregnant females were observed at 180mg/kg/day, and only two pregnant rats delivered live pups at that dose. The NOAEL for reproductive/developmental toxicity was concluded to be 30mg/kg/day.

Enhanced mitophagy in Sertoli cells of ethanol-treated rats: morphological evidence and clinical relevance

Although chronic ethanol consumption results in Sertoli cell vacuolization and augmented testicular germ cell apoptosis via death receptor and mitochondrial pathways, Sertoli cells are resistant to apoptosis. The aim of this study was to examine whether the activation of autophagy in the Sertoli cells of ethanol-treated rats (ETR) may have a role in their survival. Adult Wistar rats were fed either 5% ethanol in Lieber-DeCarli liquid diet or an isocaloric control diet for 12weeks. The TUNEL method demonstrated that Sertoli cells were always TUNEL-negative despite the presence of many apoptotic germ cells in ETR, supporting our previous studies. Electron microscopy revealed the presence of large numbers of autophagic vacuoles (AVs) in Sertoli cells of ETR compared to few AVs in control testes. Most of the AVs in Sertoli cells of ETR enveloped and sequestered damaged and abnormally shaped mitochondria, without cytoplasm, indicating mitochondrial autophagy (mitophagy). Immuno-electron microscopy showed the localization of LC3, a specific marker of early AVs (autophagosomes), around AVs sequestering mitochondria in Sertoli cells of ETR. Immunohistochemical staining of LC3 demonstrated a punctate pattern in Sertoli cells of ETR, confirming the formation of autophagosomes, while LC3 puncta were almost absent in control testes. Moreover, increased immunoreactivity of LAMP-2, a lysosomal membrane protein and marker of late AVs (autolysosomes), was mainly observed in Sertoli cells of ETR, with weaker expression in control testes. Via the deletion of pro-apoptotic damaged mitochondria, enhanced Sertoli cell mitophagy in ETR may be an anti-apoptotic mechanism that is essential for spermatogenesis.