Age-related histopathological and biochemical testicular damages were ameliorated by vitamin C administration / Los daños testiculares histopatológicos y bioquímicos relacionados con la edad mejoraron con la administración de vitamina C

Introduction and objectives: Aging is an irreversible process associated with decreased biological functions that can lead to the reduction of reproductive organs capacities in males and females. Paternal age is a significant predictor of offspring health and development. So, the aim of this study was to evaluate the effects of vitamin C on histopathological and biochemical testicular changes following aging process with a focus on stereological methods. Material and methods: For this study, 48 adult male NMRI mice were divided into two control and experimental groups. Mice in experimental group were supplemented with vitamin C (150mg/kg) including 24-h interval by oral gavage for 33 weeks. Same regime was performed for animals in control group except that vitamin C was replaced by water. Then, right testes were extracted for stereological and left testes were used for molecular analyses on weeks 8, 12, and 33. (AU)

Introducción y objetivos: El envejecimiento es un proceso irreversible asociado a una disminución de las funciones biológicas que puede conducir a la reducción de la capacidad de los órganos reproductivos en hombres y mujeres. La edad paterna es un predictor significativo de la salud y el desarrollo de la descendencia. Por lo tanto, el objetivo de este estudio fue evaluar los efectos de la vitamina C sobre los cambios testiculares histopatológicos y bioquímicos posteriores al proceso de envejecimiento con un enfoque en los métodos estereológicos.Material y métodos: Para este estudio, 48 ratones NMRI machos adultos se dividieron en dos grupos de control y experimentales. Los ratones del grupo experimental se suplementaron con vitamina C (150mg/kg), incluido un intervalo de 24 horas mediante sonda oral durante 33 semanas. Se realizó el mismo régimen para los animales del grupo de control, excepto que se reemplazó la vitamina C por agua. Luego, se extrajeron los testículos derechos para estereología y los testículos izquierdos se utilizaron para análisis moleculares en las semanas 8, 12 y 33. (AU)

[Clinicopathological characteristics of Klinefelter syndrome: a testicular biopsy analysis of 87 cases].

Objective: To investigate the clinicopathological characteristics of testicular biopsies from Klinefelter syndrome (KS) patients. Methods: The testicular biopsy specimens of 87 patients with KS (a total of 107 biopsy specimens) were collected from the Department of Pathology, Peking University Third Hospital, Beijing, China from January 2017 to July 2022. All patients were diagnosed as KS by peripheral blood karyotyping analysis. The testicular histopathologic features, testicular volume and hormone levels were evaluated retrospectively. The histopathologic analysis was used to assess the quantity and morphology of Leydig cells, the spermatogenic state of seminiferous tubules, the thickening of the basement membrane of seminiferous tubules and the changes of stroma. Results: Leydig cell proliferative nodules were seen in 95.3% (102/107) of KS testicular biopsy tissues. The eosinophilic inclusion bodies and lipofuscin in Leydig cells were found in 52.3% (56/107) and 57.9% (62/107) of specimens, respectively. The Sertoli cell only seminiferous tubules and the hyalinized tubules were found in 66.4% (71/107) and 76.6% (82/107) of the examined tissues, respectively. The tubules with complete spermatogenic arrest were found in 15.9% (17/107) of specimens, and 5.6% (6/107) of the specimens showed low spermatogenesis or incomplete spermatogenic arrest. In 85.0% (91/107) of the specimens, increased thick-walled small vessels with hyaline degeneration were identified. Conclusions: The most common features of KS testicular specimens are Leydig cell proliferative nodules, hyaline degeneration of seminiferous tubules and proliferation of thick-walled blood vessels. Testicular biopsy specimens of KS are rare. The pathologists can make a tentative diagnosis of KS based on the histological findings, combined with the ultrasound and laboratory results, which is helpful for further diagnosis and treatment of KS.

Age-related histopathological and biochemical testicular damages were ameliorated by vitamin C administration.

INTRODUCTION AND OBJECTIVES: Aging is an irreversible process associated with decreased biological functions that can lead to the reduction of reproductive organs capacities in males and females. Paternal age is a significant predictor of offspring health and development. So, the aim of this study was to evaluate the effects of vitamin C on histopathological and biochemical testicular changes following aging process with a focus on stereological methods. MATERIAL AND METHODS: For this study, 48 adult male NMRI mice were divided into two control and experimental groups. Mice in experimental group were supplemented with vitamin C (150mg/kg) including 24-h interval by oral gavage for 33 weeks. Same regime was performed for animals in control group except that vitamin C was replaced by water. Then, right testes were extracted for stereological and left testes were used for molecular analyses on weeks 8, 12, and 33. RESULTS: Our findings showed low semen quality, decreased level of serum Luteinizing hormone (LH), Follicle-stimulating hormone (FSH), and testosterone along with increased reactive oxygen species (ROS) production and higher apoptotic gene expression following aging. Stereological studies showed that the volume of testes, the length of seminiferous tubules, and the number of spermatogenic and none-spermatogenic cells decreased significantly during aging. Also, vitamin C consumption for 33 weeks significantly improved biochemical and histological indices. The impact of aging on male reproduction seems to be inevitable worldwide. Therefore, the use of protective and preventive remedies conserving male fecundity is very important and based on our results, vitamin C is a beneficial candidate for improving age-related testicular changes due to aging process.

Spectrum of Histologic Findings in Orchiectomy Specimens of Patients Seeking Male to Female Physical Adaptation.

Context. Hormonal therapy followed by orchiectomy is of the standard of care in management of gender identity disorder in patients seeking male to female transition. The orchiectomy specimens from these patients are routinely subjected to histopathologic evaluation. We discuss the spectrum of histopathologic findings, incidental findings, and cost analysis of processing these specimens. Design. Orchiectomy specimens from patients seeking male to female transition received at our institution from January 2019 to June 2021 were included in the study. Data including patient age, history of hormonal therapy, testicular weight, histopathologic findings, number of tissue sections, and processing cost were collected. Results. A total of 79 specimens were identified. Mean patient age was 36.7 ± 14.5 years. Mean testicular weight was 28.0 ± 8.3 g (right) and 27.8 ± 9.1 g (left). Histologic evaluation showed diminished or absent spermatogenesis in 100% and fibrosis of seminiferous tubules in 96% of specimens. Benign, incidental findings, none of which altered patient management were present in 6 specimens (8%). For most specimens, 3 sections per testis were submitted. This resulted in a mean of 5.8 ± 1.1 tissue sections submitted per specimen. Conclusions. Orchiectomy specimens from patients with gender dysphoria always demonstrate hormone-therapy effects albeit with varying degree. The chances of discovering any incidental finding of clinical significance are negligible. Diligent gross inspection and minimal tissue sampling with additional sampling reserved for gross abnormalities can adequately document the histologic findings in a cost-effective manner.

Clinicopathological characteristics of Klinefelter syndrome: a testicular biopsy analysis of 87 cases / 中华病理学杂志

Objective: To investigate the clinicopathological characteristics of testicular biopsies from Klinefelter syndrome (KS) patients. Methods: The testicular biopsy specimens of 87 patients with KS (a total of 107 biopsy specimens) were collected from the Department of Pathology, Peking University Third Hospital, Beijing, China from January 2017 to July 2022. All patients were diagnosed as KS by peripheral blood karyotyping analysis. The testicular histopathologic features, testicular volume and hormone levels were evaluated retrospectively. The histopathologic analysis was used to assess the quantity and morphology of Leydig cells, the spermatogenic state of seminiferous tubules, the thickening of the basement membrane of seminiferous tubules and the changes of stroma. Results: Leydig cell proliferative nodules were seen in 95.3% (102/107) of KS testicular biopsy tissues. The eosinophilic inclusion bodies and lipofuscin in Leydig cells were found in 52.3% (56/107) and 57.9% (62/107) of specimens, respectively. The Sertoli cell only seminiferous tubules and the hyalinized tubules were found in 66.4% (71/107) and 76.6% (82/107) of the examined tissues, respectively. The tubules with complete spermatogenic arrest were found in 15.9% (17/107) of specimens, and 5.6% (6/107) of the specimens showed low spermatogenesis or incomplete spermatogenic arrest. In 85.0% (91/107) of the specimens, increased thick-walled small vessels with hyaline degeneration were identified. Conclusions: The most common features of KS testicular specimens are Leydig cell proliferative nodules, hyaline degeneration of seminiferous tubules and proliferation of thick-walled blood vessels. Testicular biopsy specimens of KS are rare. The pathologists can make a tentative diagnosis of KS based on the histological findings, combined with the ultrasound and laboratory results, which is helpful for further diagnosis and treatment of KS.

Sertoli and Germ Cells Within Atrophic Seminiferous Tubules of Men With Non-Obstructive Azoospermia.

Background: Infertile men with non-obstructive azoospermia (NOA) have impaired spermatogenesis. Dilated and un-dilated atrophic seminiferous tubules are often present in the testes of these patients, with the highest likelihood of active spermatogenesis in the dilated tubules. Little is known about the un-dilated tubules, which in NOA patients constitute the majority. To advance therapeutic strategies for men with NOA who fail surgical sperm retrieval we aimed to characterize the spermatogonial stem cell microenvironment in atrophic un-dilated tubules. Methods: Testis biopsies approximately 3x3x3 mm3 were obtained from un-dilated areas from 34 patients. They were classified as hypospermatogenesis (HS) (n=5), maturation arrest (MA) (n=14), and Sertoli cell only (SCO) (n= 15). Testis samples from five fertile men were included as controls. Biopsies were used for histological analysis, RT-PCR analysis and immunofluorescence of germ and Sertoli cell markers. Results: Anti-Müllerian hormone mRNA and protein expression was increased in un-dilated tubules in all three NOA subtypes, compared to the control, showing an immature state of Sertoli cells (p<0.05). The GDNF mRNA expression was significantly increased in MA (P=0.0003). The BMP4 mRNA expression showed a significant increase in HS, MA, and SCO (P=0.02, P=0.0005, P=0.02, respectively). The thickness of the tubule wall was increased 2.2-fold in the SCO-NOA compared to the control (p<0.05). In germ cells, we found the DEAD-box helicase 4 (DDX4) and melanoma-associated antigen A4 (MAGE-A4) mRNA and protein expression reduced in NOA (MAGE-A: 46% decrease in HS, 53% decrease in MA, absent in SCO). In HS-NOA, the number of androgen receptor positive Sertoli cells was reduced 30% with a similar pattern in mRNA expression. The γH2AX expression was increased in SCO as compared to HS and MA. However, none of these differences reached statistical significance probably due to low number of samples. Conclusions: Sertoli cells were shown to be immature in un-dilated tubules of three NOA subtypes. The increased DNA damage in Sertoli cells and thicker tubule wall in SCO suggested a different mechanism for the absence of spermatogenesis from SCO to HS and MA. These results expand insight into the differences in un-dilated tubules from the different types of NOA patients.

Assessment of the architecture and integrity of frozen-thawed testicular tissue from (pre)pubertal boys with cancer.

BACKGROUND: Testicular tissue freezing is proposed for fertility preservation to (pre)pubertal boys with cancer before highly gonadotoxic treatment. Studies accurately comparing human (pre)pubertal testicular tissue quality before freezing and after thawing are exceptional. No study has reported this approach in a systematic manner and routine care. OBJECTIVES: To assess the impact of a control slow freezing protocol on testicular tissue architecture and integrity of (pre)pubertal boys after thawing. MATERIALS AND METHODS: (Pre)pubertal boys (n = 87) with cancer from 8 Reproductive Biology Laboratories of the French CECOS network benefited from testicular tissue freezing before hematopoietic stem cell transplantation. Seminiferous tubule cryodamage was determined histologically by scoring morphological alterations and by quantifying intratubular spermatogonia and the expression of DNA replication and repair marker in frozen-thawed testicular fragments. RESULTS: A significant increase in nuclear and epithelial score alterations was observed after thawing (p < 0.0001). The global lesional score remained lower than 1.5 and comparable to fresh testicular tissue. The number of intratubular spermatogonia and the expression of DNA replication and repair marker in spermatogonia and Sertoli cells did not vary significantly after thawing. These data showed the good preservation of the seminiferous tubule integrity and architecture after thawing, as previously reported in our studies performed in prepubertal mice and rats. DISCUSSION: The current study reports, for the first time, the development of a semi-quantitative analysis of cryodamage in human (pre)pubertal testicular tissue, using a rapid and useful tool that can be proposed in routine care to develop an internal and external quality control for testicular tissue freezing. This tool can also be used when changing one or several parameters of the freezing-thawing procedure. CONCLUSION: Control slow freezing protocol without seeding maintains the seminiferous tubule architecture and integrity, the concentration of spermatogonia and the expression of DNA replication and repair marker in spermatogonia and Sertoli cells after thawing.

Stereological properties of seminiferous tubules in infertile men with chromosomal and genetic abnormalities.

BACKGROUND: Male infertility is caused by genetic anomalies in 15-30% of cases. This study aimed to determine stereological properties of seminiferous tubules in infertile men with genetic anomalies, including Klinefelter Syndrome (KS), Y chromosome microdeletions (MYC) and CFTR gene mutations (CFTR); and to compare them to seminiferous tubules of men with obstructive azoospermia of non-genetic origin (control group). METHODS: The study was conducted on 28 human testis biopsy specimens obtained from 14 patients with MYC, 18 samples from 9 patients with KS, and 6 samples from 3 patients with CFTR. Whenever possible, a bilateral biopsy was included in the study. The control group had 33 samples from 18 patients (3 of them with a solitary testis). Qualitative and quantitative (stereological) analysis of seminiferous tubules (including the status of spermatogenesis, volume, surface area, length and number of tubules) were performed in all groups. RESULTS: Qualitative histological analysis revealed significant impairment of spermatogenesis in KS and MYC, whereas testicular parenchyma was fully maintained in CFTR and control groups. Spermatogenesis was most seriously impaired in KS. All stereological parameters were significantly lower in KS and MYC, compared to the CFTR and control groups. The total volume, surface and length of seminiferous tubules were significantly lower in KS compared with MYC. CONCLUSIONS: Stereological analysis is valuable in evaluating male infertility, whereas qualitative histological analysis can be helpful in assessing sperm presence in testicular tissue of patients with KS or MYK undergoing TESE.

Depletion of high-mobility group box 2 causes seminiferous tubule atrophy via aberrant expression of androgen and estrogen receptors in mouse testis†.

High-mobility group box 2, a chromatin-associated protein that interacts with deoxyribonucleic acid, is implicated in multiple biological processes, including gene transcription, replication, and repair. High-mobility group box 2 is expressed in several tissues, including the testis; however, its functional role is largely unknown. Here, we elucidated the role of high-mobility group box 2 in spermatogenesis. Paraffin-embedded testicular tissues were obtained from 8-week-old and 1-year-old wild-type and knock-out mice. Testis weight and number of seminiferous tubules were decreased, whereas atrophic tubules were increased in high-mobility group box 2-depleted mice. Immunohistochemistry revealed that atrophic tubules contained Sertoli cells, but not germ cells. Moreover, decreased cell proliferation and increased apoptosis were demonstrated in high-mobility group box 2-depleted mouse testis. To elucidate the cause of tubule atrophy, we examined the expression of androgen and estrogen receptors, and the results indicated aberrant expression of androgen receptor and estrogen receptor alpha in Sertoli and Leydig cells. Southwestern histochemistry detected decreased estrogen response element-binding sites in high-mobility group box 2-depleted mouse testis. High-mobility group box 1, which has highly similar structure and function as high-mobility group box 2, was examined by immunohistochemistry and western blotting, which indicated increased expression in testis. These findings indicate a compensatory increase in high-mobility group box 1 expression in high-mobility group box 2 knock-out mouse testis. In summary, depletion of high-mobility group box 2 induced aberrant expression of androgen receptor and estrogen receptor alpha, leading to decreased germ cell proliferation and increased apoptosis which resulted in focal seminiferous tubule atrophy.

Phycobiliproteins Ameliorate Gonadal Toxicity in Male Mice Treated with Cyclophosphamide.

Cyclophosphamide (CP)-which is used to treat autoimmune diseases and cancer-is related to gonadotoxicity attributed to oxidative stress. As phycobiliproteins (PBPs) are strong antioxidants that are unexplored as protective agents against male gonadotoxicity, our work aimed to investigate the effects of PBP crude extract on testicular damage and sperm parameter alterations caused by CP in mice. Three doses of PBP (50, 100, and 200 mg/kg) were tested in the experimental groups (n = 8 per group), administered concomitantly with 100 mg/kg CP. After 42 days receiving PBP daily and CP weekly, body and relative testicular weights, serum testosterone levels, testicular lipoperoxidation and antioxidant enzyme activity levels, and testicular histology and sperm parameter alterations were assessed. The results showed that PBP crude extract at 200 mg/kg prevented testosterone serum reduction, body weight loss, lipoperoxidation and enzyme activity increments, and sperm parameter alterations and partially ameliorated relative testicular weight reductions and histological damage in CP-treated mice. In conclusion, we showed that PBP crude extract (200 mg/kg) mitigated oxidative damage in the testes and ameliorated alterations in sperm parameters in mice treated with CP (100 mg/kg); therefore, PBP extract could be considered as a potential protective agent against CP toxicity.

CEP164 is essential for efferent duct multiciliogenesis and male fertility.

Cilia are evolutionarily conserved microtubule-based structures that perform diverse biological functions. Cilia are assembled on basal bodies and anchored to the plasma membrane via distal appendages. In the male reproductive tract, multicilia in efferent ducts (EDs) move in a whip-like motion to prevent sperm agglutination. Previously, we demonstrated that the distal appendage protein CEP164 recruits Chibby1 (Cby1) to basal bodies to facilitate basal body docking and ciliogenesis. Mice lacking CEP164 in multiciliated cells (MCCs) (FoxJ1-Cre;CEP164fl/fl) show a significant loss of multicilia in the trachea, oviduct, and ependyma. In addition, we observed male sterility; however, the precise role of CEP164 in male fertility remained unknown. Here, we report that the seminiferous tubules and rete testis of FoxJ1-Cre;CEP164fl/fl mice exhibit substantial dilation, indicative of dysfunctional multicilia in the EDs. We found that multicilia were hardly detectable in the EDs of FoxJ1-Cre;CEP164fl/fl mice although FoxJ1-positive immature cells were present. Sperm aggregation and agglutination were commonly noticeable in the lumen of the seminiferous tubules and EDs of FoxJ1-Cre;CEP164fl/fl mice. In FoxJ1-Cre;CEP164fl/fl mice, the apical localization of Cby1 and the transition zone marker NPHP1 was severely diminished, suggesting basal body docking defects. TEM analysis of EDs further confirmed basal body accumulation in the cytoplasm of MCCs. Collectively, we conclude that male infertility in FoxJ1-Cre;CEP164fl/fl mice is caused by sperm agglutination and obstruction of EDs due to loss of multicilia. Our study, therefore, unravels an essential role of the distal appendage protein CEP164 in male fertility.

A method for utilizing automated machine learning for histopathological classification of testis based on Johnsen scores.

We examined whether a tool for determining Johnsen scores automatically using artificial intelligence (AI) could be used in place of traditional Johnsen scoring to support pathologists' evaluations. Average precision, precision, and recall were assessed by the Google Cloud AutoML Vision platform. We obtained testicular tissues for 275 patients and were able to use haematoxylin and eosin (H&E)-stained glass microscope slides from 264 patients. In addition, we cut out of parts of the histopathology images (5.0 × 5.0 cm) for expansion of Johnsen's characteristic areas with seminiferous tubules. We defined four labels: Johnsen score 1-3, 4-5, 6-7, and 8-10 to distinguish Johnsen scores in clinical practice. All images were uploaded to the Google Cloud AutoML Vision platform. We obtained a dataset of 7155 images at magnification 400× and a dataset of 9822 expansion images for the 5.0 × 5.0 cm cutouts. For the 400× magnification image dataset, the average precision (positive predictive value) of the algorithm was 82.6%, precision was 80.31%, and recall was 60.96%. For the expansion image dataset (5.0 × 5.0 cm), the average precision was 99.5%, precision was 96.29%, and recall was 96.23%. This is the first report of an AI-based algorithm for predicting Johnsen scores.

Evaluation of multi-epitope recombinant protein as a candidate for a contraceptive vaccine.

Contraceptive vaccine (CV) is a valuable, non-invasive, and alternative method for purposeful contraception. Sperm antigens are useful targets for producing CVs due to their specialized expression in sperm. In this study, a recombinant protein containing three main sperm epitopes (IZUMO1, SACA3, and PH-20) was designed and evaluated as CV to control fertility in male mice. The chimeric recombinant protein was expressed and purified in E. coli. Male mice were immunized by 100 µg purified protein and sera were collected to assess IgG antibodies. Evaluating the reproductive performance, immunized male mice mated with normal-fertile female mice and mating rate and the number of newborns was studied. Immunized mice were sacrificed and necropsy and histopathology studies were conducted. The results revealed that the designed chimeric protein stimulated the immune system of the mice effectively. The level of IgG antibody was significantly higher in vaccinated mouse rather than control mouse. Eighty percent of the vaccinated mice became infertile and in the remaining ones, the number of children decreased to 4-6 offspring instead of 10-12 in normal mice. Histopathological studies showed that no organs including heart, brain, lung, liver, kidney and intestine were damaged. However, Normal spermatogenesis has been disrupted and necrotic spermatogonia cells were reported in Seminiferous tubules. We concluded that the designed chimeric protein containing IZUMO1, SACA3, and PH-20 epitopes can stimulate the immune system and cause male contraception without any side effects.

A novel glucokinase activator TMG-123 causes long-lasting hypoglycemia and impairs spermatogenesis irreversibly in rats.

The importance of glucose is well known as an energy source in testes. In order to evaluate the effects of long-lasting hypoglycemia on testes, a novel glucokinase activator, TMG-123, was dosed to rats at 5, 20 and 100 mg/kg for 13 weeks. As a result, plasma glucose levels decreased for several hours with increasing doses over the dose range of 5 to 100 mg/kg. No toxicological findings attributable to the test article were observed in clinical observation, measurements of body weight and food consumption, necropsy, and organ weight measurement. Histopathology showed scattered degeneration of seminiferous tubules in testes, and exfoliation of germ cells related to the degeneration of seminiferous tubules was observed in the lumen of both epididymides in the same animals at the end of the dosing period. Similar histopathological findings were noted at the end of the recovery period. In addition, a fertility study was conducted at the same doses for 13 weeks for males and 5 weeks for females. Sperm analysis showed decreases in the sperm concentration and the motility index and an increase in the incidences of sperm malformations. However, there were no abnormalities in the copulation or fertility rate. These results suggest that long-lasting hypoglycemia in rats is harmful to spermatogenesis and the testicular damage does not recover.

COVID-19 and male reproduction: Current research and unknown factors.

BACKGROUND: The testes are suspected target organs of SARS-CoV-2. However, the results of studies on the effect of COVID-19 on male reproduction are controversial. OBJECTIVE: To summarize current research on the effects of COVID-19 on male reproduction. METHODS: A systematic review of English literature was performed using PubMed and Ovid Embase up to 18 August 2020. Research articles on the presence of SARS-CoV-2 in semen, the effects of the virus on semen parameters and any pathological changes in the testes were evaluated. RESULTS: Fourteen studies were included in this review. Six of 176 survivors (3.4%) and 1 of 13 decedents (7.7%) in 2 of 12 studies were positive for viral RNA in semen and testicular tissue, respectively. After stratification of patient groups, we found that the virus was detected in the relatively early stage of infection, 6-16 days after disease onset, in semen from survivors. Two of 3 studies reported that some participants had substandard semen quality after COVID-19, and 1 study found that COVID-19 may impair semen quality in a severity-related manner. Pathological analyses showed that injuries to the seminiferous tubule occurred in all decedents (N = 11). Another study found that orchitic and testis fibrin microthrombi occurred in patients with fatal disease (100%, N = 2). Scrotal discomfort of orchiepididymitis or spermatic cord inflammation has also been reported in COVID-19 patients. CONCLUSION: Current studies suggest that semen is rarely considered a carrier of SARS-CoV-2 genetic material during the infection period but not in the semen of recovered patients. Fatal COVID-19 may cause testicular structure damage without the presence of virus.

Fetal and Postnatal Nicotine Exposure Modifies Maturation of Gonocytes to Spermatogonia in Mice.

Studies in laboratory animals have shown that male offspring from dams, exposed to nicotine during pregnancy and postnatal periods, show alterations in fertility, although the origin of this is still uncertain. In this study, we examined in a mouse model if the process of gonocyte maturation to spermatogonia was affected in male offspring from dams with nicotine administration during pregnancy and postnatal periods. BALB/C mice, with and without nicotine administrations in pregnancy and postnatal periods, were studied. The animals were euthanized at 3, 7, 10, 16, and 35 days postpartum (dpp). Testicular tissue samples were processed for histological, ultrastructural, and immunohistochemical studies; and testicular lipoperoxidation was determined. It was observed that in the nicotine-exposed animals, there was increased apoptosis and a reduction in the number of gonocytes that matured to spermatogonia. This gonocyte-spermatogonia maturation reduction was associated with a greater immunoreactivity to nicotinic acetylcholine receptors in the germ cells. Lipoperoxidation was similar in both groups until 16 dpp, with significant reduction at 35 dpp. Our findings suggest that nicotine intake during pregnancy and postnatal periods can affect the process of maturation of gonocytes to spermatogonia and the pool of available spermatogonia for spermatogenesis.

[Protective effect of taurine against formaldehyde-induced male reproductive toxicity in adult male rats].

OBJECTIVE: To study the effect of taurine on the reproductive toxicity damage induced by formaldehyde (FM) in adult male rats. METHODS: Forty-eight SD adult male rats were equally randomized into a normal control, an FM poisoning (FMP), a taurine intervention (TI), and an TI+FMP group. The control rats were given normal diet and gavage of saline, the rats of the FMP group treated intraperitoneally with FM at 10 mg/kg qd alt, those of the TI group intragastrically with taurine at 100 mg/kg qd, and those of the TI+FMP group with both FM and taurine at the above doses. After 30 days of treatment, the blood of the abdominal cardinal vein of the rats was extracted for measurement of the levels of serum hormones, the body weight, testis weight and testicular coefficient obtained, the testis tissue subjected to HE staining, and the expressions of Bcl-2 and Bax determined by Western blot. RESULTS: There were no statistically significant differences among the four groups of rats in the body weight, testis weight or testicular coefficient (P > 0.05). The rats in the FMP group showed obviously decreased testicular spermatogenic cells and disordered layers and loose structure of seminiferous tubules, which were basically restored to normal after taurine intervention. Compared with the normal controls, the animals of the TI group exhibited no significant abnormality, but those of the FMP group presented markedly reduced levels of serum T, LH and FSH (P < 0.05), and dramatically increased level of Gonadotropin-releasing hormone (GnRH) (P < 0.01). The levels of serum hormones were all significantly improved (P < 0.05) and that of the apoptotic protein Bax basically returned to normal (P < 0.05) after taurine intervention. CONCLUSIONS: Taurine has a certain protective effect against male reproductive toxicity caused by formaldehyde.

Deleterious effects of Pfaffia glomerata (Spreng.) Pedersen hydroalcoholic extract on the seminiferous epithelium of adult Balb/c mice.

Several plant species such as Pfaffia glomerata are widely used in traditional Brazilian medicine as stimulants and aphrodisiacs. In this regard, the aim of our study was to explore the effects of the long-term intake of the hydro-alcoholic root extract of P glomerata on the germ and somatic cells within the seminiferous tubules in adult Balb/c mice. The experimental groups were placed as: controls (water and DMSO), and treated with 300 and 400 mg/kg of the root extract. The number of germ and somatic cells, the proportion of pathological seminiferous tubules, and the germ cell apoptotic levels were evaluated. The volume and proportion of the seminiferous epithelium was decreased after the extract intake due to the increased germ cell apoptotic levels. Vacuolization of Sertoli cell cytoplasm was observed widely in pathological tubules, along with fully disorganized epithelia, showing multinucleated cells, which lead to decreased daily sperm production. Taken together, our results indicate that long-term intake of the P glomerata caused deleterious effects on spermatogenesis by inducing apoptosis and altering the seminiferous tubule's epithelial dynamics.

N-(3-Methozybenzyl)-(9Z,12Z,15Z)-octadecatrienamide from maca (Lepidium meyenii Walp.) ameliorates corticosterone-induced testicular toxicity in rats.

This study investigated the protective effects of maca ethanol extract (EEM) and N-(3-methozybenzyl)-(9Z,12Z,15Z)-octadecatrienamide (M 18:3) on corticosterone (CORT)-induced testicular toxicity. Male Wistar rats were divided into 5 groups. Except for the control group, CORT (40 mg per kg·bw) was injected subcutaneously for 21 consecutive days to induce testicular toxicity. 1 h before CORT injection, the rats were treated with EEM (400 mg per kg·bw) and M 18:3 (5 mg per kg·bw, 25 mg per kg·bw) by gavage, except for the control group and model group. Epididymal sperm and biochemical, and histological parameters were evaluated for the protective effects of the drugs. EEM (400 mg per kg·bw) and M 18:3 (5 mg per kg·bw, 25 mg per kg·bw) increased the sperm concentration and sperm motility, decreased the production of abnormal sperms, and increased the number of spermatogonia and primary spermatocytes in the seminiferous tubules of CORT-induced rats. Moreover, EEM and M 18:3 decreased the MDA levels and the positive expression rates of TUNEL, whereas they increased the activities of SOD, CAT, GSH-Px, and GST, and the contents of GSH in the testicles of CORT-induced rats. Furthermore, EEM and M 18:3 alleviated CORT-induced reduction in the positive expression rates of PCNA and Ki67 in the testicles of rats. Besides, EEM and M 18:3 reduced the expression levels of Keap-1 and increased the expression levels of Nrf2, HO-1, γ-GCS, and NQO1 in the testicles of CORT-induced rats. In summary, the protective effects of EEM and M 18:3 may be attributed to their anti-oxidative and anti-apoptotic properties.

Age-related changes of seminiferous tubule morphology, interstitial fibrosis and spermatogenesis in dogs.

There are age-related changes in testicular anatomy and physiology whereby there are modifications of sperm production and reproductive hormone functions. Effects of age on testicular microanatomy are well documented in humans, while there is limited understanding of these changes in dogs. The aim of this study was to evaluate age-related changes of seminiferous tubule morphology, interstitial fibrosis and spermatogenesis in dogs. Dogs (n = 32) were divided into four age groups: peripubertal (n = eight), relatively younger (n = seven), reproductively mature (n = seven) and relatively older (n = ten). Picrosirius Red stained sections were used for morphometrical analysis of testicular tissues, while the characteristics of seminiferous epithelium were assessed using a modified Johnsen scoring system for haematoxylin and eosin stained sections. Seminiferous epithelium and seminiferous tubule area increased from peripuberty to reproductive maturity, indicating there were changes during sexual maturation and subsequently there were decreases with further aging. There was a similar age-related trend for changes in seminiferous epithelium height with values being greatest in reproductively mature dogs; while there were no age-related differences in tubular diameter. Collagen content in the testicular interstitium gradually decreased from peripuberty to the age when dogs were reproductively mature and there were subsequent increases in relatively older dogs, thus, there was an association between the extent of testicular fibrosis and senescence. There was a decrease in spermatogenetic functions from relatively younger to older ages. Further investigations are warranted to establish mechanisms responsible for age-related changes of testicular morphology and related clinical implications.