Percentage Of Seminiferous Tubules Research Articles

Alcoholic beer consumption permutes P21 and cyclin D1 expression, oxidative stress factors, and histomorphometric parameters in rat testis

BackgroundEthanol consumption is increasingly prevalent in communities and has several side effects for humans. Chronic alcohol consumption is often associated with decreased libido and infertility. This study aimed to evaluate the impact of beer on spermatogenesis, proteins, and gene expression of P21 and cyclin D1 in testicular tissue. MethodTwenty-four male mice were assigned to 4 groups. The control group received normal saline, and the experimental groups received alcoholic beer (3 g/kg BW as 20% v/v). After 7, 15, and 35 days, mice were sacrificed, and a part of testicular tissues was stored at −70 °C to measure the protein and gene expression of P21 and cyclin D1 by Western blot and real-time PCR. Moreover, this assay measured the levels of malondialdehyde (MDA), superoxide dismutase (SOD), and catalase (CAT). Histomorphometry and histopathology were done. ResultsThe consumption of alcoholic beer led to increased protein and gene expression of P21 and decreased expression of cyclin D1, as well as reduced levels of SOD and CAT, increased levels of MDA, and subsequent tissue damage in the testicular tissue in a time-dependent manner. Also, the diameter of seminiferous tubules and the thickness of the germinal epithelium were reduced in a time-dependent manner, and the percentage of seminiferous tubules with tubular differentiation, replacement, and negative spermiogenesis coefficients increased significantly. ConclusionAlcoholic beer disrupted the process of cell division in the testes by reducing the expression of cyclin D1, increasing p21 expression, and inducing oxidative stress, which in turn reduced sperm production and quality.

No effects of the antiandrogens cyproterone acetate (CPA), flutamide and p,p'-DDE on early sexual differentiation but CPA-induced retardation of embryonic development in the domestic fowl (Gallus gallus domesticus).

Because a wide range of environmental contaminants are known to cause endocrine disorders in humans and animals, in vivo tests are needed to identify such endocrine disrupting chemicals (EDCs) and to assess their biological effects. Despite the lack of a standardized guideline, the avian embryo has been shown to be a promising model system which responds sensitively to EDCs. After previous studies on the effects of estrogenic, antiestrogenic and androgenic substances, the present work focuses on the effects of in ovo exposure to p,p'-DDE, flutamide and cyproterone acetate (CPA) as antiandrogenic model compounds regarding gonadal sex differentiation and embryonic development of the domestic fowl (Gallus gallus domesticus). The substances were injected into the yolk of fertilized eggs on embryonic day one. On embryonic day 19 sex genotype and phenotype were determined, followed by gross morphological and histological examination of the gonads. Treatment with flutamide (0.5, 5, 50 µg/g egg), p,p'-DDE (0.5, 5, 50 µg/g egg) or CPA (0.2, 2, 20 µg/g egg) did not affect male or female gonad development, assessed by gonad surface area and cortex thickness in both sexes and by the percentage of seminiferous tubules in males as endpoints. This leads to the conclusion that antiandrogens do not affect sexual differentiation during embryonic development of G. gallus domesticus, reflecting that gonads are not target organs for androgens in birds. In ovo exposure to 2 and 20 µg CPA/g egg, however, resulted in significantly smaller embryos as displayed by shortened lengths of skull, ulna and tarsometatarsus. Although gonadal endpoints were not affected by antiandrogens, the embryo of G. gallus domesticus is shown to be a suitable test system for the identification of substance-related mortality and developmental delays.

O-210 Cluster analysis reveals two hidden subgroups among patients with cryptozoospermia

Abstract Study question Can cryptozoospermic patients be sub classified based on clinical parameters? Summary answer Cryptozoospermic patients can be subdivided in two subgroups based on the histological phenotype of their testicular tissues, testicular volume and FSH levels. What is known already Cryptozoospermia is a severe form of oligozoospermia in which patients present with a sperm concentration of ≤ 0.1 million per milliliter. Due to such low sperm concentration, cryptozoospermic patients depend on the surgical procedure of testicular sperm extraction (TESE) to retrieve sperm for intracytoplasmic sperm injection (ICSI). The general etiology underlying cryptozoospermia remains hitherto unknown, likely due to the high heterogeneity within this patient cohort regarding histological and endocrine parameters. Study design, size, duration We retrospectively selected 132 cryptozoospermic patients (Crypto) who underwent TESE during their fertility treatment. Exclusion parameters were genetic diseases, a history of cryptorchidism or tumors and hormonal treatment. As controls, we selected 160 patients with obstructive azoospermia and normal spermatogenesis. All 292 patients were used for principal component analysis (PCA) followed by hierarchical clustering on principal components (HCPC). Participants/materials, setting, methods PCA and hierarchical clustering were performed considering age, testicular volume, hormonal values (FSH, LH, testosterone and free testosterone), ejaculate parameters (Ejaculate pH and volume, sperm concentration and total sperm count) and histological information. Histological analyses were performed on two PAS-stained sections from two independent biopsies per testis and patient. The percentage of seminiferous tubules containing elongated spermatids, round spermatids, spermatocytes or spermatogonia, as well as Sertoli cell only and hyalinized tubules was assessed. Main results and the role of chance The PCA and hierarchical clustering subdivided the patient cohort into 3 different clusters. Cluster 1 included all the controls (n = 160) and 2 Crypto patients. Remaining Crypto patients were equally subdivided between cluster 2 (n = 65) and cluster 3 (n = 65). The control patients in cluster 1 were characterized by the highest percentages of tubules with elongated spermatids (>80%), normal testicular volume and hormonal values. Characteristic of Crypto patients in cluster 2 was the arrest of germ cell differentiation at the level of spermatocytes in the majority of seminiferous tubules. In contrast, the majority of seminiferous tubules in Crypto patients in cluster 3 showed a Sertoli cell only phenotype or even tubular shadows. Interestingly, the more severe histological phenotype of the cluster 3 patients was accompanied by higher FSH and LH levels as well as lower testicular volume compared to cluster 2 patients. Despite this, the percentage of tubules with full spermatogenesis was similar in the two Crypto groups and no difference was found in the sperm retrieval rate after TESE in the two patient groups. Limitations, reasons for caution Uncovering the existence of subgroups within the cohort of Crypto-patients is the prerequisite for unveiling potentially distinct etiologies. Comprehensive genetic and scRNA-seq analyses of these Crypto-subgroups remain to be performed to validate the findings. Wider implications of the findings Overall, this study offers novel approaches towards the understanding of the etiology underlying the reduced sperm formation in Crypto patients and the division into two subgroups will facilitate the strategic search for underlying causes. Moreover, this approach could be applied to any infertility type in order to identify hidden subgroups. Trial registration number not applicable

Protective effects of Equisetum arvense methanolic extract on testicular tissue disorders in streptozotocin-induced diabetic murine model.

Diabetes in a long period can damage the testicular tissue and impair the male fertility potential. Recently, different herbal treatments have been used for the prevention of type I diabetes and its pathological effects. Methanolic extract of Equisetum arvense has anti-oxidant and hypoglycemic properties. Thus, the current study aimed to evaluate the protective effects of Equisetum arvense methanolic extract (EE) on diabetes-induced detrimental effects in mice testicular tissue. Thirty-two adult male mice were randomly divided into four groups including control-sham, diabetic (induced by streptozotocin, 50.00 mg kg-1 for five days), diabetic + EE 250 (250 mg kg-1) and diabetic + EE 500 (500 mg kg-1). After 45 days, all animals were euthanized and their testicles were dissected out and undergone histological analyses. Moreover, the serum level of testosterone was evaluated. Analyses showed that seminiferous tubules diameter, Leydig cells number per mm2 of the connective tissue, Sertoli cells number per tubule, serum level of testosterone and percentage of seminiferous tubules with positive tubular differentiation, repopulation and spermiogenesis indices were significantly decreased in the diabetic group in comparison with control-sham group. The administration of EE in test groups significantly decreased the adverse effects of diabetes (especially 500 mg kg-1). The results of this study revealed that diabetes disturbs spermatogenesis and spermiogenesis processes in mice. Meanwhile, the EE prevents diabetes-induced damages in mice testicular tissue, which may be associated with its hypoglycemic and antioxidative activities.

Impact of chronic vitamin A excess on sperm morphogenesis in mice.

The increasing availability of fortified foods and supplements has caused an overconsumption of vitamin A (VA), above the recommended level. To date, the effects of chronic VA excess (VAE) on spermatogenesis remain unclear. This study aims to investigate the long-term excessive intake of VA effects on spermatogenesis in mice. Dams were initially fed a control diet (4IU/g) or a VAE diet (250IU/g), 4weeks prior to mating and during pregnancy. Dams and their male pups continued this diet regimen until the offspring reached 12weeks of age. At 12weeks of age, epididymis caudal spermatozoa and testes were collected. For histological analysis, sections were stained with periodic acid-Schiff-hematoxylin, and quantitative PCR was used to detect changes in gene expression in the testes of the VAE mice. Sperm motility and morphology were evaluated to detect the endpoint of VAE toxicity. Body weights were not significantly different between the control and VAE groups. Testicular cross-sections from the control and VAE mice contained a normal array of germ cells, and the daily sperm production was similar between the two groups. However, the percentage of seminiferous tubules in stages VII and VIII was significantly lower in the VAE mice than in the control. In addition, significant changes in the expression of genes involved in retinoid metabolism, spermatogenesis, and spermiogenesis were detected in the testes of the VAE mice. Consistently, sperm motility and head morphology were significantly impaired in the VAE mice. Our findings suggest that long-term dietary intake of VAE was able to influence both pre- and post-meiotic spermatogenesis. As a result of testicular toxicity, we demonstrated, to the best of our knowledge, for the first time that long-term VAE caused sperm-head abnormalities.

Testis structure, duration of spermatogenesis and daily sperm production in four wild cricetid rodent species (A. cursor, A. montensis, N. lasiurus, and O. nigripes).

Although rodents represent approximately 40% of all living mammalian species, our knowledge regarding their reproductive biology is still scarce. Due to their high vulnerability to environmental changes, wild rodents have become beneficial models for ecological studies. Thus, we aimed to comparatively investigate key functional testis parameters in four sexually mature wild rodent species (A. cursor, A. montensis, N. lasiurus, and O. nigripes). These species belong to the Cricetidae family, which is the most diverse family of rodents in South America, with a total of ~120 species in Brazil. The results found for the gonadosomatic index and the sickled sperm head shape observed strongly suggest that the species here evaluated are promiscuous, prolific, and short-lived. The duration of spermatogenesis was relatively short and varied from ~35–40 days. Both the percentage of seminiferous tubules (ST) in the testis parenchyma (~95–97%) and the number of Sertoli cells (SC) (~48–70 million) per testis gram were very high, whereas a fairly good SC efficiency (~8–13 round spermatids per SC) was observed. In comparison to other mammalian species studied, particularly the rodents of the suborder Myomorpha (i.e. hamsters, rats and mice), the rodents herein investigated exhibited very high (~62–80 million) daily sperm production per testis gram. This impressive spermatogenic efficiency resulted mainly from the short duration of spermatogenesis and quite high values found for the ST percentage in the testis and the SC number per testis gram. We expect that the knowledge here obtained will help conservation programs and the proper management of wildlife.

Human amnion mesenchymal stem cells restore spermatogenesis in mice with busulfan-induced testis toxicity by inhibiting apoptosis and oxidative stress

BackgroundBefore starting gonadotoxic therapies, cryopreservation of mature sperm has been proposed worldwide as a method for male fertility preservation and for enabling the conception of a healthy baby with assisted reproductive technology (ART); however, these technologies are not feasible for prepubertal boys and men with spermatogenic failure. Transplantation of mesenchymal stem cells has exhibited successful therapeutic benefits in restoring spermatogenesis via gonadal graft angiogenesis, transplanted cell clonogenesis, and disordered somatic compartment recovery. This study aimed to elucidate the fertility protective effects and the underlying mechanisms of human amnion mesenchymal stem cells (hAMSCs) against busulfan-induced testis toxicity.MethodsAn in vivo busulfan-induced testis toxicity mouse model and an in vitro busulfan-administered mouse Sertoli cell line were employed to evaluate the efficacy and mechanisms of hAMSC transplantation on male fertility preservation. The process of spermatogenesis was evaluated histologically, and the percentage of seminiferous tubules with vacuoles was evaluated by HE staining. Semen parameters were calculated by computer-assisted semen analysis. ELISA was employed to test the testosterone concentration and the levels of oxidative- and antioxidative-associated substances LDH, MDA, GR, SOD, GPx, and CAT. The rates of proliferation (Ki67), apoptosis (Annexin V), and ROS were measured by FACS. The fluorescence intensity of a marker of apoptosis (TUNEL) and a meiosis gene in spermatogenesis (SCP3) were detected by immunofluorescence assay. The expression of mRNA in germ cell-specific (GCS) genes (Dazl, Ddx4, and Miwi) and meiosis genes (Scp3, Cyclin A1, and Stra8) was tested by qPCR. The expression of antiapoptotic proteins (SURVIVIN and BCL2), apoptotic proteins (CASPASE3 and CASPASE9), GCS proteins (Dazl, Ddx4, and Miwi), and meiosis proteins (Scp3, Cyclin A1, and Stra8) was tested by western blotting.ResultshAMSC transplantation following disruption by busulfan-induced testis toxicity restored spermatogenesis, elevating testosterone levels and enhancing testicular weight, size, and semen parameters in vivo. In addition, hAMSCs clearly ameliorated cell apoptosis, enhanced cell proliferation, repressed oxidative damage, and augmented oxidative defense in vivo and in vitro. Moreover, hAMSCs distinctly increased the expression of the GCS genes Dazl, Ddx4, and Miwi and the meiosis genes Scp3, Cyclin A1, and Stra8 in vivo.ConclusionshAMSCs might represent a promising tool for the use in regenerative medicine, as these cells can restore spermatogenesis in a busulfan-induced testis toxicity mouse model and facilitate activity in a busulfan-administered mouse Sertoli cell line by resisting apoptosis and oxidative stress.

Paternal cadmium exposure increases the susceptibility to diet-induced testicular injury and spermatogenic disorders in mouse offspring.

The impairments of gestational cadmium (Cd) exposure on testicular development and male fertility in offspring have been reported. Here, we investigated the effect of paternal low-concentration cadmium exposure on testicular development and spermatogenesis in offspring. Five-week-old male mice were exposed to cadmium chloride (100mg/L) in drinking water for 20 weeks. Results presented that Cd did not affect the testicular histology and sperm count in mice. After mating with untreated females, pregnant mice and pups were then evaluated. No significant difference in the rate for successful pregnancy and the body weight of pups was observed in Cd-exposed mice compared to the controls. Male offspring were given with a chow and high-fat diet from postnatal day (PND) 35 to PND70. Our data indicated that high-fat diet obviously decreased No. of sperm in epididymides of adult offspring due to paternal Cd exposure. Testicular histology revealed that the percentage of seminiferous tubules in stages IX-XII and the atypical residual bodies positive tubules in CdH (paternal cadmium exposure and pubertal high-fat diet) group were higher than these in CdC (paternal cadmium exposure and pubertal chow diet) group. Further analysis demonstrated that high-fat diet markedly accelerated testicular apoptosis, as determined by TUNEL assay and immunostaining for cleaved caspase-3, in male offspring due to paternal Cd exposure. Collectively, high-fat diet exacerbates the damage of testicular development and spermatogenesis in offspring due to paternal cadmium exposure.

Metabolomics-based pathway changes in testis fragments treated with ethinylestradiol in vitro.

There is a need to develop in vitro models to test drugs and chemicals that induce toxicity in the male reproductive system. We have evaluated an in vitro mouse testis organ culture model capable of producing viable, fertilization-proven sperm as a possible toxicity test model. Although this in vitro model was limited to round spermatid differentiation, histopathology observations could still be performed. Liquid chromatography/mass spectrometry analysis (LC/MS)-based metabolomics was used to measure metabolome changes of chemically treated in vitro testis fragments. On Postnatal Day 5, C57BL/6J mouse testes were divided into four fragments, which were placed onto a 1.5% agarose gel cube and cultured in α-MEM including 0.4% AlbuMAX I (Day 0). On Day 1 of culture, testis fragments were treated with 0 (control), 0.01, or 1 nM ethinylestradiol (EE). On Day 20 of culture, the testis fragments were collected for LC/MS and histology analysis. Several metabolites involved in glycogen metabolism and glycolysis pathways (uridine diphosphate-glucose, glucose phosphate, and pyruvate), in the tricarboxylic acid cycle pathway (oxaloacetate and aspartate), and in the arginine and proline metabolism (arginine and spermine) were significantly altered in the 1 nM EE treated group compared to the control group. The metabolite changes were associated with an increase in percentage of seminiferous tubules with round spermatids as well as dose-dependent dead cells. These findings suggest that EE treatment may cause testicular toxicity by affecting glycogen metabolism and energy pathways. To confirm these findings, further experiments will be necessary using other testicular toxicants.

Effects of varicocele on 4-aminobutyrate aminotransferase expression and its potential role in surgically varicocele-induced rat testes

Objective To investigate the expression of 4-aminotransferase (ABAT) and its effect on spermatogenesis in experimental varicocele rat model. Methods A total of 36 specific pathogen free (SPF) male SD rats were randomly divided into varicocele group and sham operation group, with 18 rats in each group. The experimental rat varicocele was induced by partially occluding left renal vein and completely ligating the branches of the left spermatic vein (LSV). After 4 weeks, the using hematoxylin and eosin (HE) staining, Masson and glycogen staining were used to evaluate the two groups of kidney and testis histology change, evaluation of varicocele rat model. After the model was constructed, the localization of ABAT protein in rat testis was further analyzed by immunofluorescence, and the expression of ABAT protein in varicocele was detected by Western blotting, and the possible role was analyzed. Results 4, 6, 8 weeks after the operation, the varicocele group in the kidney tissue pathological changes were not found, and the left spermatic vein diameter was greater than 1 mm [(1.52±0.23) mm vs. (0.26±0.03) mm], the weight of the left testis [(1.61±0.08) g vs. (1.93±0.11) g] and the percentage of seminiferous tubules (58% vs. 18%) were lower than the control group, which indicates that the successful construction of varicocele animal model. Immunofluorescence analysis showed that ABAT protein was mainly expressed in the cytoplasm of spermatogenic cells. Compared with the control group, with the prolonged duration of varicocele, Western blotting showed that the expression of ABAT protein was down regulated in the varicocele group, which is positive with the degree of spermatogenesis damage. Conclusion In experimental rat varicocele model, varicocele affects the expression of ABAT protein, which may be related to varicocele damage spermatogenesis. Key words: 4-transaminase; Varicocele; Rat testis

Evaluation of an in vitro mouse testis organ culture system for assessing male reproductive toxicity.

Development of an in vitro system capable of producing mature sperm remains a challenging goal, with only few successes reported. Such a system, could be used to test agents for potential toxicity to the male reproductive system; to explore this, we exposed immature mouse testis fragments in culture to ethinylestradiol (EE), a well-known testicular toxicant in vivo. Testis fragments from postnatal day 5 mice were cultured in Albumax I medium. After 24 hr of culture, fragments were treated with 0.01, 0.1 or 1 nM EE, then harvested after 20 days in culture and examined for histology or gene expression measures by quantitative PCR. There was substantial variability between fragments in the degree of spermatogenesis observed. The percentage of seminiferous tubules containing any dead germ cells increased as a result of EE exposure in a dose dependent fashion. This was accompanied with a decreased percentage of tubules with round spermatids. Expression of estrogen receptor 1, cytochrome P450, family 11, subfamily a, and polypeptide 1 also was reduced, depending on the dose. These gene expression changes in the testis fragments are similar to those seen after animals have been exposed to EE. Gene expression changes in testis fragments are encouraging, but the variability across samples will need to be reduced for this in vitro system to become a generally applicable method for assessing testicular toxicants.

L-Cysteine Partially Protects Against Acrylamide-Induced Testicular Toxicity.

Background:Acrylamide is a widespread substance with many areas of utilization. Acrylamide also forms a part of high-temperature-processed starchy foods. To date, numerous in vivo and in vitro studies have documented that acrylamide poses toxic effects on various organ systems.Aims:To determine the potential protective effect of L-cysteine on acrylamide-induced testicular toxicity.Study Design:Animal experimentation.Methods:We randomly divided 28 rats into four groups: control (0.9% saline), L-cysteine (150 mg/kg), acrylamide (40 mg/kg), and acrylamide+L-cysteine. After a 10-day intraperitoneal injection period, we euthanized the animals, recorded their body and testis weights, collected blood samples for serum testosterone measurement, and excised the testes for histopathological and morphometric evaluation. Immunohistochemical scoring of proliferating cell nuclear antigen and bax proteins was performed.Results:Acrylamide reduced body (p<0.01) and testis weights (p<0.05), seminiferous tubule diameter (p<0.001), and proliferating cell nuclear antigen expression (p<0.05) but increased bax protein expression (p<0.01) and the percentage of seminiferous tubules containing multinucleated giant cells (p<0.001). However, no significant change was observed in serum testosterone level of the experimental groups when compared with that of controls. L-cysteine administered with acrylamide decreased multinucleated giant cell number (p<0.001) and reversed the reduced proliferating cell nuclear antigen positivity (p<0.001) but showed no effect in restoring other parameters compared with the group treated with acrylamide alone.Conclusion:Considering the dose and duration employed, the present study concluded that L-cysteine partially protects testis against acrylamide-induced toxic effects.

Pubertal chlorocholine chloride exposure inhibits testicular testosterone synthesis by down-regulating steroidogenic enzymes in adult rats

Chlorocholine chloride (CCC) is widely used to regulate plant growth. Considerable attention has been focused on its reproductive and developmental toxicities. In order to investigate the effects of pubertal CCC exposure on testicular testosterone (T) synthesis, male SD rats were exposed to CCC by oral gavage at doses of 0, 75, 150 and 300 mg/kg bw/day from postnatal day 23 to 70. We observed that pubertal CCC exposure lowered the body weight and the mean Johnsen’s score. The percentage of seminiferous tubules with deciduous spermatogenic cells was increased in the 75 and 150 mg/kg bw/day groups. In addition, pubertal CCC exposure reduced the testicular absolute weights in the 75 and 300 mg/kg bw/day groups as well as the sperm motility in epididymides in the 150 mg/kg bw/day group. A significant decrease of testicular T was observed while levels of hypothalamic gonadotropin-releasing-hormone (GnRH) and serum luteinizing hormone (LH) were increased. Protein levels of steroidogenic acute regulatory (StAR), cholesterol side-chain cleavage enzyme (P450scc) and 3β-hydroxysteroid dehydrogenase (3β-HSD) were decreased. Taken together, these results indicate that pubertal CCC exposure in rats might decrease testicular T synthesis by suppressing the expression of steroidogenic enzymes, which partially lead to an impairment on spermatogenesis.

Implications of body condition and seasonality on morphological and functional parameters of testes of Myotis nigricans (Chiroptera: Vespertilionidae).

The insectivorous bat Myotis nigricans is widely distributed throughout the Neotropics, including Brazil, and has a reproductive biology that is affected by climate and food availability. To evaluate the reproductive capacity of this species, morphofunctional parameters of the testes were correlated with environmental variables and the body condition of individuals captured. After bats had been killed, their testes were removed, fixed in Karnovsky's fluid for 24h and embedded in resin for evaluation by light microscopy. The mean annual tubulosomatic index (0.58%) and the percentage of seminiferous tubules in the testes (88.96%) were the highest ever recorded for the Order Chiroptera. The percentage of Leydig cells and volume of the cytoplasm of Leydig cells were higher in the rainy than dry season (80.62±3.19% and 573.57±166.95μm, respectively; mean±s.d.). Conversely, the percentage of nuclei of the Leydig cells in the dry season (26.17±3.70%; mean±s.d.) and the total number of Leydig cells (6.38±1.84×109; mean±s.d.) were higher in the dry season. The results of the present study could help in future conservation of these bats because they provide a better understanding of the bats' reproductive strategies and how the species can adapt to changes.

Ghrelin modulates testicular damage in a cryptorchid mouse model.

Cryptorchidism or undescended testis (UDT) is a common congenital abnormality associated with increased risk for developing male infertility and testicular cancer. This study elucidated the effects of endogenous ghrelin or growth hormone secretagogue receptor (GHSR) deletion on mouse reproductive performance and evaluated the ability of ghrelin to prevent testicular damage in a surgical cryptorchid mouse model. Reciprocal matings with heterozygous/homozygous ghrelin and GHSR knockout mice were performed. Litter size and germ cell apoptosis were recorded and testicular histological evaluations were performed. Wild type and GHSR knockout adult mice were subjected to creation of unilateral surgical cryptorchidism that is a model of heat-induced germ cell death. All mice were randomly separated into two groups: treatment with ghrelin or with saline. To assess testicular damage, the following endpoints were evaluated: testis weight, seminiferous tubule diameter, percentage of seminiferous tubules with spermatids and with multinucleated giant cells. Our findings indicated that endogenous ghrelin deletion altered male fertility. Moreover, ghrelin treatment ameliorated the testicular weight changes caused by surgically induced cryptorchidism. Testicular histopathology revealed a significant preservation of spermatogenesis and seminiferous tubule diameter in the ghrelin-treated cryptorchid testes of GHSR KO mice, suggesting that this protective effect of ghrelin was mediated by an unknown mechanism. In conclusion, ghrelin therapy could be useful to suppress testicular damage induced by hyperthermia, and future investigations will focus on the underlying mechanisms by which ghrelin mitigates testicular damage.

Evaluation of Culture Time and Media in an In Vitro Testis Organ Culture System

The complexity of spermatogenesis makes development of appropriate in vitro testis models challenging. A novel in vitro mouse testis culture system has been reported but not yet evaluated as an alternative model for male reproductive toxicity testing. We assessed the effects of media composition on sperm differentiation and testis morphology of cultured mouse testis fragments. Testes from postnatal day 5 B6:CBA-Tg(Acrv1-EGFP)2727Redd/J male mice were cultured in knockout serum replacement (KSR) or Albumax I (Albumax) medium. Enhanced green fluorescent protein (EGFP) expression was examined on days 35, 42, 45, and 49 of culture. Histology and flow cytometry were performed for testis morphology and spermatid differentiation. EGFP signals were first observed in round spermatids on day 22 of culture (corresponding to postnatal day 27) and were observed until the end of culture, indicating testis-specific protein expression. A-kinase anchor protein 4 expression, a marker of elongated spermatid (step 15-16) occurred earlier in explants cultured in KSR than Albumax medium (typically day 35 and after day 42 of culture, respectively). The percentage of seminiferous tubules with elongated spermatid was higher in Albumax than KSR medium from days 45 to 49 of culture. Albumax medium may facilitate or support better morphology and spermatid production than KSR medium. Further studies need to improve spermatid production and refinement of this in vitro testis culture system that may be useful as a supplement to current male reproductive toxicity testing or an alternative model in cases where in vivo testing may be unfeasible. Birth Defects Research 109:465-474, 2017. © 2017 Wiley Periodicals, Inc.

Irradiation affects germ and somatic cells in prepubertal monkey testis xenografts.

Does irradiation evoke adverse effects in germ and somatic cells in testis xenografts from prepubertal monkeys? In addition to the expected depletion of germ cells, a dose-dependent effect of irradiation was observed at the mRNA and protein level in Sertoli and peritubular myoid cells. Testicular irradiation studies in monkeys have focused on the dose-dependent effects on germ cells. Previous studies using intact animals or xenografts reported that germ cells are highly sensitive to irradiation. Their depletion was demonstrated by morphometric and histological analyses. The effect of irradiation on expression of Sertoli and peritubular myoid cell markers, however, has not yet been described. The testes of two prepubertal macaques (Macaca fascicularis) were dissected into testicular fragments. Fragments were randomly exposed in vitro to one of the following three doses of irradiation: 0 Gy, n = 60; 1 Gy, n = 54; 4 Gy, n = 72. Non-irradiated control fragments (0 Gy) were placed into the Faxitron for 6.6 min without irradiation. For 1 Gy and 4 Gy irradiation was applied for 1.7 and 6.6 min, respectively. Grafts were then either immediately analyzed or subcutaneously implanted under the back skin of 39 nude mice and analyzed after 6.5 months. Post grafting, 133 testicular xenografts were retrieved. The body weight, serum testosterone level and seminal vesical weight of the host mice as well as the number and weight of retrieved grafts were determined. Larger grafts were used to evaluate both mRNA expression profiles and protein expression patterns. In total, 71 testicular fragments were used for morphometric and histological analysis while 68 fragments were analyzed for gene expression. For PCR arrays, M. fascicularis-specific primer sequences were employed. Irradiation-induced changes in the transcript levels of 34 marker genes were determined for each testicular graft. The effects of irradiation on peritubular myoid cells and Sertoli cells were confirmed by immunohistochemical analysis of chemokine (C-X-C motif) ligand type 11 (CXCL11), alpha smooth muscle actin (SMA) and chemokine (C-X-C motif) ligand type 12 (CXCL12). The four testes gave rise to 106 xenografts, which were individually analyzed, limiting the role of chance despite using only two monkeys in the study. Prior to grafting, the two donors displayed spermatogonia as the most advanced germ cell type in 95% and 70% of seminiferous tubules, respectively, while remaining tubules contained SCO. No spermatocytes were encountered prior to grafting in either monkey. After 6.5 months, non-irradiated grafts displayed spermatocytes in 15.4% and 1.8% of seminiferous tubules indicating an induction of meiosis. Irradiation resulted in a complete absence of spermatocytes. The percentage of seminiferous tubules containing spermatogonia declined in a dose-dependent manner. In non-irradiated xenografts, ~40% of tubules contained spermatogonia. This proportion was reduced to 3.4% and 4.3% in the 1 Gy treated group and to 1.3% and 0.2% in 4 Gy irradiated grafts. A dose-dependent decline in mRNA levels of selected germ cell marker genes supported the morphologically detected loss of germ cells. Irradiation had no effect on CXCL12 transcript levels. At the protein level, CXCL12-positive Sertoli cells were most abundant in the 1 Gy group compared to the 4 Gy group (P < 0.05), indicating a potential role of CXCL12 during recovery of primate spermatogenesis. The most prominent radiation-evoked changes were for CXCL11, which was localized to smooth muscle cells of blood vessels and seminiferous tubules. Transcript levels declined in a dose-dependent manner in grafts from both monkeys (MM687: P < 0.01 (0 Gy versus 4 Gy), MM627: P < 0.05 (0 Gy versus 4 Gy), P < 0.001 (1 Gy versus 4 Gy)). CXCL11 patterns of protein expression revealed irradiation-dependent changes as well. That peritubular cells are affected by X-irradiation was substantiated by changes at the transcript level between 1 and 4 Gy exposed groups (P < 0.01) and at the protein level of SMA (P < 0.05, 0 Gy versus 4 Gy). n/a. The spermatogonial stem cell system in primates is remarkably different from rodents. Therefore, data from a non-human primate may be more relevant to man. However, species-specific differences amongst primates cannot be fully excluded and the use of only two donors may raise concerns toward the generalization of the findings. There may also be important differences across the prepubertal period (e.g. infancy, early childhood) that are not represented by the ages included in the present study. This study is the first to indicate relevant testicular somatic cell responses following irradiation of prepubertal primate tissue. In addition to the well-known depletion of germ cells, the changes in Sertoli, and in particular peritubular myoid, cells may have important consequences for spermatogenic recovery. These novel findings should be taken into consideration when irradiation effects are assessed in tumor survivors. Interdisciplinary Center for Clinical Research (IZKF) Münster (Schl2/001/13) and the Excellence Cluster 'Cells in Motion' at the University Münster. There are no conflicts of interest to declare.

Follicle-stimulating hormone enhances recovery from low-dose doxorubicin-induced spermatogenic disorders in mice.

We aimed to investigate the effects of FSH for promoting spermatogenesis in mice with low-dose doxorubicin-induced spermatogenesis impairment. Eight-wk-old male imprinting control region mice were divided into three groups. Groups D and F received 0.5mg/kg of doxorubicin twice weekly for 5weeks. Group C received saline instead of doxorubicin. After inducing spermatogenesis impairment, group D was treated daily with saline for 4weeks. Group F was given 1IU of recombinant human FSH daily for 4weeks. Spermatogenesis recovery was evaluated based on the testis weight, sperm count, histological assessment, and mating. The percentage of sperm with unfragmented deoxyribonucleic acid (DNA) was analyzed by single-cell pulsed-field gel electrophoresis, and the serum FSH levels were measured. The elevation of serum FSH advanced slowly. The testis weight, sperm count, percentage of seminiferous tubules with spermatogenesis, percentage of sperm with unfragmented DNA and pregnancy rate were significantly increased by the administration of FSH. Our study findings indicated that the immediate administration of exogenous FSH can promote the recovery from impaired spermatogenesis induced by low-dose doxorubicin before endogenous FSH increases to the maximum level.

Ultrasonographic caput epididymis diameter is reduced in non-obstructive azoospermia compared with normozoospermia but is not predictive for successful sperm retrieval after TESE.

Is the ultrasonographic determination of the caput epididymis diameter predictive for sperm retrieval after testicular sperm extraction (TESE) in non-obstructive azoospermia (NOA)? Ultrasonographic determination of the caput epididymis diameter did not give any relevant clinical information in NOA and was not predictive for positive sperm retrieval after TESE. The diameter of the caput epididymis in ultrasonography (US) has a diagnostic relevance in azoospermic men to correctly identify obstructive azoospermia; however, its clinical value in NOA is not yet determined. We performed a retrospective study of 100 azoospermic and 160 normozoospermic men attending a university infertility clinic. Participants were submitted to scrotal US to determine the mean value of bilateral testicular volumes (ml), the bilateral longitudinal caput diameter (mm) and the antero-posterior diameter of the corpus (mm) epididymis. The number of spermatozoa retrieved after TESE and the testicular histology of azoospermic men was obtained and the percentage of seminiferous tubules with elongated spermatids (%T) was used to classify cases with normal spermatogenesis (obstructive azoospermia) (OA) (n = 20; %T ≥ 80) or with NOA (n = 80; %T < 70). The US testes volumes and caput diameters were reduced (P < 0.05) in NOA compared with OA and with normozoospermia, but the corpus values were not different. The caput diameter in the side submitted to biopsy was significantly reduced when germinal epithelium was absent (Sertoli cell only) (P < 0.05) and the lowest value of caput diameter was observed when the seminiferous epithelium and tubule lumen were absent (testicular hyalinosis). On the contrary, a total arrest of spermatogenesis at the first meiosis level, or a defect of spermiogenesis resulting in scattered elongated spermatids in each tubule, did not show a reduced diameter of caput epididymis compared with normozoospermia. The caput diameter did not show any difference between NOA patients with or without successful sperm retrieval at TESE. On the contrary testicular volume was significantly reduced in NOA patients with no sperm retrieval (P = 0.0037). The caput diameter was not correlated with the number of retrieved sperm, the serum level of follicle stimulating hormone, or with the percentage of tubules with elongated spermatids at histological analysis. The aetiology of NOA was not included in the statistical analysis due to the low rate of cases with a specific aetiology for a testicular failure. Larger studies should exclude the possibility that besides testicular histology, aetiology of NOA might influence the diameter of caput epididymis. Moreover, whether a reduced diameter of caput epididymis is only a result of a testicular pathologic phenotype or whether it may underscore a primitive dysfunction influencing the number of ejaculated spermatozoa is not yet determined. We reported that US diameter of the caput epididymis is reduced in cases of NOA but, in contrast with the testicular volume, it is independent of the completion of spermatogenesis and subsequent presence of spermatozoa in the epididymis. Therefore ultrasonographic determination of caput epididymis diameter is not predictive for positive sperm retrieval after TESE in cases of a primitive testicular failure. Our novel findings may help to define which reproducible parameters of scrotal US should be assessed in the work-up of male infertility. This work was supported by the Ministero dell'Università e Ricerca (I) PRIN 2009. The authors declare no competing interest.

Silymarin protects from varicocele-induced damages in testis and improves sperm quality: evidence for E2f1 involvement

This study was designed to evaluate the protective effect of silymarin (SMN) on varicocele-induced damage in testis and its effects on sperm parameters and on antioxidant status. Wistar rats were divided into three groups: control-sham, varicocele-induced, and SMN-treated varicocelized (50mg/kg, orally) rats. The sperm count, DNA integrity, and histone-protamine transition was evaluated after 42 days. The antioxidant status was analyzed by determining testicular malondialdehyde (MDA) and total thiol molecules (TTM). The endocrine status of the testicular tissue was estimated by counting the normal Leydig cell distribution/mm2 and by determination of serum testosterone. The expression of E2f1 mRNA was analyzed using RT-PCR. Carbohydrate depletion and lipid foci replacement in germinal cells were examined by histochemical analyses. Silymarin rehabilitated the varicocele-induced Leydig cell degeneration and testosterone reduction. In addition, SMN recovered the varicocele-induced reduction of TTM and lowered significantly (P < 0.05) the varicocele-elevated content of MDA. The SMN treatment resulted in a significant (P < 0.05) down-regulation of the VCL-up-regulated E2f1 mRNA. Silymarin-treated animals were protected from varicocele-induced testicular atrophy and these animals showed a significant (P < 0.05) increase in the percentage of seminiferous tubules with positive tubular differentiation, repopulation, and spermiogenesis indices. Furthermore, SMN improved the varicocele-induced carbohydrate reduction in germinal cells. Our data suggest that in addition to oxidative stress, alteration in the testicular endocrine function plays a crucial role in the pathogenesis of varicocele. Moreover, the protective effects of SMN on varicocele-induced damage may reflect its antioxidant property, which may be mediated via the E2f1 transcription factor.