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Germplasm preservation of livestock or endangered animals and expansion of germline stem cells are important. The purpose of this study is to investigate whether supplementation of trehalose to the freezing medium (FM) reduces tissular damage and improves the quality of testicular cells in the cryopreserved bovine testicular tissues. We herein established an optimized protocol for the cryopreservation of bovine testicular tissues, and the isolation as well as culture of bovine germ cells containing spermatogonial stem cells (SSCs) from these tissues. The results showed that FM containing 10% dimethyl sulfoxide (Me2SO/DMSO), 10% knockout serum replacement (KSR) and 20% trehalose (FM5) combined with the uncontrolled slow freezing (USF) procedures has the optimized cryoprotective effect on bovine testicular tissues. The FM5 + USF protocol reduced the cell apoptosis, maintained high cell viability, supported the structural integrity and seminiferous epithelial cohesion similar to that in the fresh tissues. Viable germ cells containing SSCs were effectively isolated from these tissues and they maintained germline marker expressions in the co-testicular cells and co-mouse embryonic fibroblasts (MEF) feeder culture systems respectively, during the short-term culture. Additionally, upregulated transcriptions of spermatogenic differentiation marker C-KIT and meiotic marker SYCP3 were detected in these cells after retinoic acid-induced differentiation. Together, FM5 + USF is suitable for the cryopreservation of bovine testicular tissues, with benefits of reducing the apoptosis, maintaining the cell viability, supporting the testicular structure integrity, and sustaining the survival and differentiation potential of bovine germ cells containing SSCs.

Introduction: Diabetes is still a world health problem that can cause many complications. Male infertility is one of the diabetic complications. This condition is caused by oxidative stress in diabetic patients. African bitter leaf is believed to contain an antioxidant compound that can repair male infertility. Purpose: This study aims to determine the effect of administering African bitter leaf extract on spermatogenic cell count in hyperglycemic Wistar rats. Method: This study use Rattus norvegicus as an animal model, which was administrated with alloxan to induce hyperglycemic. P0 group were given Na CMC 0,1%, Control group were given glibenclamide 0,63/kg bodyweight for 14 days. 100mg/kg bodyweight (P1), 200mg/kg (P2), and 400mg/kg (P3) of African bitter leaf extract were administrated for 14 days. In the end, the animals were sacrificed, and testicle histopathologic sections were made. Results: Significant result (P

Spermatogenesis is a multifaceted and meticulously orchestrated process involving meiosis, chromatin build up, transcriptional and translational hushing, and spermiogenesis. Male germ cell lines GC-1spg (GC-1) and GC-2(spd)ts (GC-2) provide a useful resource to comprehend the molecular events occurring during such a tightly regulated process. Using cDNA microarray, expression profiling of GC-1 and GC-2 cell lines was done to precisely understand their characteristics and uniqueness. Our observations indicate that whilst both the cell lines are indeed of testicular origin, GC-2 is not haploid as was originally thought. Data analysis of the 23,351 transcripts detected in GC-1 and 20,992 in GC-2 cell lines demonstrates an 80% transcript overlap between GC-1 and GC-2 cells and ~ 40% similarity of both with the primary spermatocyte transcriptome. 3152 and 793 transcripts exclusive to GC-1 and GC-2, respectively, were identified. The presence of transcripts for 36 genes was validated in these cell lines including those showing testis-specific expression, as well as genes not reported previously. Overall, this study provides the transcriptome database of GC-1 and GC-2 cells. Analysis of the data demonstrates the transcriptomic transitions between GC-1 and GC-2 thus providing a glimpse to the process of germ cell differentiation from type B spermatogonium into preleptotene spermatocyte.

Purpose: To investigate the comparative effects of Withania coagolans extract and morphine on spermatogenesis in rats Methods: W. coagolans was collected from Sistan and Baluchestan, Iran and 50 and 100 mg/kg body weight doses of methanol extract and 5, 10 and 15 mg/kg body weight doses of morphine were administered parenterally to the rats which were divided into groups. Blood samples were collected and the levels of luteinizing hormone (LH), follicle stimulating hormone (FSH), and testosterone were assayed. The testicular tissue was isolated for histopathological examination. Results: No significant changes were observed in levels of LH, FSH and testosterone in treated groups (p < 0.05). However, there was significant difference between the treated groups for extract plus morphine groups, in terms of the number of spermatogonium, spermatocytes and spermatide variation. Moreover, the results indicate tissue disorders in all groups relative to control. The extract caused more disturbances in spermatogenesis compared to morphine, and appears to improve parameters related to spermatogenesis. Conclusion: The results show that the higher dose of Withania coagolans extract (100 mg/kg) exerts varying effects on reproductive parameters. Moreover, the lower dose of Withania coagolans extract (50 mg/kg) enhanced spermatogenesis while also protecting against the damaging effects of morphine.

In the present study, we describe the spermatogenesis of Allenbatrachus grunniens, a benthic toadfish from Estuarine Pranburi River, Thailand, at the ultrastructural level. According to the nuclear and cytoplasmic characteristics, A. grunniens spermatogenic cells are classified into five stages. The spermatogonium contains the prominent nucleus and locates in the periphery of the seminiferous lobule. The spermatogonium develops into the primary spermatocyte and then into secondary spermatocyte, which was characterized by the dramatical increase in heterochromatin. The subsequent spermatids exhibit the centriole formation, chromatin condensation, mitochondrial redistribution, and reduced cytoplasm. After the final morphological differentiation, the spermatozoon shows an oval head with a high degree of chromatin condensation, a small midpiece consisted of prominent basal bodies surrounded by eleven mitochondria, and the biflagellate tail. Our observation demonstrates the ultrastructure of Batrachoididae spermatozoa, which can help to understand their reproductive biology.

Despite studies on the effects of medicinal plants on reproductive performance, the effect of extracts broccoli and caraway on serum and testicular oxidative biomarkers, testicular structure and function and sperm quality before and after cryopreservation has not been studied. Sixty-three male mice were divided into nine controlled and treated groups as follow: control, broccoli (200 mg/kg), broccoli (300 mg/kg), caraway (200 mg/kg), caraway (300 mg/kg), broccoli -caraway (200 mg/kg), broccoli (300 mg/kg)-caraway (200 mg/kg), broccoli (200 mg/kg)-caraway (300 mg/kg), broccoli-caraway (300 mg/kg). After 42 days of treatment, the animals were sacrificed and blood sample and testicular tissue were collected for biochemical and histological measurements. Sperm quality was also measured before and after cryopreservation. The results showed that the diameter and number of spermatogonium, primary spermatocytes, spermatids and sperm count were significantly increased by broccoli (300 mg/kg), while level of them were significantly decreased by caraway (300 mg/kg) compared to other groups (p 0.01). Sperm viability and motility after thawing significantly improved by broccoli (300 mg/kg) compared to control. Testosterone levels significantly increased by broccoli (300 mg/kg) compared to control and caraway (300 mg/kg). The serum and testicular SOD and CAT activity significantly increased by broccoli (300 mg/kg) compared to other groups (p 0.05). MDA and DNA fragmentation levels significantly increased by caraway (200 and 300 mg/kg) compared to others (p 0.01). It can be concluded that broccoli extract in a dose-dependent manner than caraway extract could improve serum and testes oxidative biomarkers, testicular structure and function, and sperm quality before and after cryopreservation.

A complete hydatidiform mole (CHM) is a conceptus with only sperm-derived chromosomes. Here, we report on a CHM with genomic DNA identical to that of the paternal somatic cells. The CHM developed in a woman who had undergone intrauterine implantation of a blastocyst obtained through in vitro injection of a presumed round spermatid into one of her oocytes. The CHM was genetically identical to peripheral white cells of her husband and contained no maternally derived nuclear DNA. We hypothesize that a spermatogonium, rather than a round spermatid, was inadvertently selected for the procedure. The CHM developed into a gestational trophoblastic neoplasia, which resolved after chemotherapy. (Funded by the Japan Society for the Promotion of Science.).

The Jumonji family (Jmj family) is a large family of proteins that contain the JmjC domain. Various members of this family are lysine (K)-specific demethylases, which remove methylation from the lysines of histones, and thus, are known as Jmj-KDMs. In this study, all Jmj-KDMs were identified in the orange-spotted grouper Epinephelus coioides and compared with those from Homo sapiens, Danio rerio, Takifugu rubripes, Oreochromis niloticus, and Xenopus tropicalis. The comparison revealed the teleost-specific loss of jmjd1a and the teleost-specific expansion of kdm5b, jmjd3, jmjd2a, and kdm6a in E. coioides. Analysis of the temporal expression pattern of Jmj-KDM genes in E. coioides enabled their division into different types according to their sex-related expression patterns. The kdm6 subfamily genes kdm6aa and jmjd3 (kdm6bb), which are also teleost specific, were selected for further analysis. Quantitative PCR analysis revealed the significantly higher expression of kdm6bb in bisexual-phase gonads and testes compared with ovary. Histological expression pattern analysis based on in situ hybridization (ISH) showed the high expression of kdm6aa in primary-growth stage oocytes, cortical-alveolus stage oocytes and granulosa cells, whereas kdm6aa and jmjd3 were highly expressed in Sertoli cells and spermatogonium. These results suggest that Jmj-KDMs, especially the kdm6 subfamily genes kdm6aa and jmjd3, are involved in sex-related processes in E. coioides.

Background: Acesulfame potassium is a low calorie sweetener. Considering its growing rate of consumption, investigation of its potential impacts on health is necessary. In the current study, the effect of intraperitoneal injection of this substance was investigated during pregnancy and after breast-feeding on the spermatogenesis of adult male rats. Materials and Methods: In this experimental research, acesulfame potassium (50, 100, 200 and 400 mg/kg body weight) was injected intraperitoneally into pregnant (Wistar) rats and then into the male infants after breast-feeding until maturity. After anaesthesia and blood sampling from the adult males, the serum levels of testosterone, FSH and LH were measured using the ELISA method. Subsequently, some of the rats were anatomized from each group, and histological sections were prepared from their testicles using hematoxylin-eosin staining. The histological sections were examined in terms of morphology and spermatogonium, spermatocyte and spermatid cell counts by optical microscopy. Also, the weight changes in the mothers and infants were examined during the trial period. The data were analysed by the ANOVA method. Results: The average weight of the infants treated at dosages of 50 and 200 mg/kg body weight increased significantly compared to that of the other groups. No significant difference was observed in the weight ratio of testes to the body and LH value in any of the groups. While the FSH value showed significant reduction in the infants receiving dosages of 50 and 100 mg/kg body weight (P < 0.01), testosterone at dosages of 50, 200, and 400 mg/kg body weight increased significantly (P < 0.001). Histological studies did not show significant disruption in the spermatogenesis process and the average number of cells counted. Conclusion: It seems that consumption of acesulfame potassium during pregnancy and infancy probably does not have a negative impact on testicular development and spermatogenesis after puberty.

Background/Aim Cryptorchidism, or undescended testis (UDT) occurs in 1%–4% of newborn males and leads to a risk of infertility and testicular malignancy. Recent research suggests that infertility and malignancy in UDT may be caused by abnormal development of the neonatal germ cells, or gonocytes, which normally transform into spermatogonial stem cells (SSC) or undergo apoptosis during minipuberty at 2–6 months in humans (2–6 days in mice). We aimed to identify the current knowledge on how UDT is linked to infertility and malignancy. Methods Here we review the literature from 1995 to the present to assess the possible causes of infertility and malignancy in UDT, from both human studies and animal models. Results Both the morphological steps and many of the genes involved in germ cell development are now characterized, but the factors involved in gonocyte transformation and apoptosis in both normal and cryptorchid testes are not fully identified. During minipuberty there is evidence for the hypothalamic–pituitary axis stimulating gonocyte transformation, but without known direct control by LH and androgen, although FSH may have a role. An arrested gonocyte maybe the origin of later malignancy at least in syndromic cryptorchid testes in humans, which is consistent with the recent finding that gonocytes are normally absent in a rodent model of congenital cryptorchidism, where malignancy has not been reported. Conclusion The results of this review strengthen the view that malignancy and infertility in men with previous UDT may be caused by abnormalities in germ cell development during minipuberty. Type of study Systematic review (secondary, filtered) Level of evidence Level I.

Background and aim: Aging is one of the causes of male infertility, and abnormal global DNA methylation and imprinting defects have been characterized in testis during biological aging. One of the important emerging approaches aims to take advantage of the healing properties of young blood plasma to limit the progression of aging in various organs in the body. We aimed to show whether blood plasma transfer has an effect on DNA methylation and spermatogenetic cell development. In addition, we aimed to show whether the young plasma transfer to old mice has an effect on the rejuvenation of the old and whether the impaired DNA methylation and PCNA expression in old age can be restored. Methods: Groups were (i) young control, (ii) young plasma transfer to aged, (iii) aged control, (iv) aged plasma transfer to young. We utilized IHC and WB in protein level of Dnmts. For the global DNA methylation level, we used 5-methylcytosine staining. We also analyzed PCNA protein expressions in all groups by IHC. Results: We found that transfusion of young plasma into the old animal restored DNA methylation and PCNA expression as it did in the young animal. Most importantly, we observed an increase in spermatogonia and spermatid counts in older animals after young blood plasma transfer. Conclusions: Our findings show that young plasma transfer can restore epigenetic disorders that occur with aging and solve infertility problems by increasing the sperm count that decreases. It needs to be supported by different studies, especially human studies. © 2022 The Authors We would like to thank all authors who provided published data.

Stage- and cell type-specific biomarkers are important for understanding spermatogenesis in mammalian testis. The present study identified several testicular cell marker proteins in 6- and 24-month old bovine testes. In 6-month old bovine testes, spermatogonia and spermatocytes were detected but complete spermatogenesis occurred in 24-month old testes. The diameters of the seminiferous tubules increased significantly in the 24-month old testes compared with those in the 6-month old testes. Protein Gene Product 9.5 (PGP9.5), also known as the undifferentiated spermatogonium marker, and GATA4 (GATA binding protein 4), vimentin, and SOX9 (SRY-Box Transcription Factor 9) were detected in the basement membrane region. Interestingly, ID4 (inhibitor of DNA binding protein 4; previously known as the undifferentiated cell marker) proteins were located in the basement membrane region but their expression patterns were different from those of PGP9.5. Co-immunohistochemistry results showed that ID4 was detected in the Sertoli cells expressing vimentin and SOX9 in 6- and 24-month old bovine testes. This result indicated that ID4 is a putative biomarker of Sertoli cell in the bovine system, which is different from the rodent models. Thus, these results will contribute in understanding the process of spermatogenesis that is different in bovines compared to other species.

We assessed the effect of chondroitin sulfate in dose of 60 mg/kg during the period of spermatogenesis (the time between the first division of the spermatogonium and the sperm ejaculation from the testes), including the term of ripening in the epididymis — 70 days. Prolonged administration of chondroitin sulfate has no negative effect on the processes of spermatogenesis, especially on young cells of the spermatogenic epithelium — spermatogonia, and does not increase the number of tubules on the 12 stages of meiosis, that is, it does not reduce the reserve of spermatogenesis. Our findings testify, that the administration of chondroitin sulfate has no negative effect on the level of androgenic saturation and the functioning of the reproductive system of male rats.

This study aimed to determine honey's potential in increasing the number of spermatogenic cells (spermatogonium, spermatocyte and spermatid) of rats (Rattus norvegicus) with nutritional deficiencies. Twenty four male rats (Rattus norvegicus) were randomly divided into four groups. Negative control (K-) mice was not fasted nor given honey. Positive control (K+) mice was fasted for 5 days without the administration of honey. The treatment group rats were fasted for 5 days followed by administration of honey 30% (P1) and 50% (P2) in drinking water for 10 days. On the 76th day all rats were sacrificed and their testes were collected for histological slides using Hematoxylin-Eosin staining. The results showed that fasting treatment for five days (group K +) caused a decrease (p

Sex-ratio is considered as an end point during endocrine disrupting chemicals (EDCs) evaluation. Many fish species including Japanese medaka have XX/XY sex determination mechanism, however, sex reversal (SR) can be induced by external and genetic factors. SR imposed an imbalance in natural sex ratio of a population living in any ecosystem. Considering SR as an end point, we aimed to investigate the potential EDC effects of graphene oxide (GO), a nanocarbon, using Japanese medaka as a model. One-day post-hatch (dph) medaka fries were exposed to GO (2.5, 5.0, 10.0 and 20 mg/L) for 96 h without food, followed by 6 weeks depuration in a GO-free environment with feeding. Phenotypic sex was determined by gonad histology; genotypic sex by genotyping Y-chromosome-specific male sex determining gene, dmy. Our data indicated testes in both XY and XX genotypes, while ovaries were only in XX females. Histopathology of XY and XX testis showed isogenic spermatocysts with active spermatogenesis. Distribution of spermatocytes (SPTs), not the spermatogonium (SPGs), showed enhancement in XY than XX testis. Female phenotypes had single ovary, either in stage 0 or 1. Ovo-testis/testis-ova were absent in XX or XY gonads. GO (2.5–20 mg/L) had inconsistent concentration-dependent effect in both SPGs and SPTs; however, no effect on ovarian follicles. Despite genotypic differences (XY/XX), in the histopathology/histochemistry of liver and kidneys GO effects was found to be minimum. Taken together, present study showed spontaneous induction of SR in some XX genotypes; however, exposure of fasting fries to GO had no apparent EDC effects.

Transmission electron microscopy of the spermatozoon of six species of intertidal sea urchins has shown that each species has a sperm with unique dimensions, indicating that spermatozoon morphology could be used as a taxonomic character. However, although the structure of the sperm is species-specific, all sperm have a similar appearance which is typical of the Echinoidea. The sperm head is composed of a conical nucleus capped by a small, spheroidal acrosome which is undifferentiated internally. The mid-piece consists of a single ring-shaped mitochondrion which surrounds the centrioles. An examination of spermatogenesis reveals that each spermatogonium and spermatocyte develops a flagellum, the flagellum being resorbed prior to cell division. In addition a few small vesicles which resemble proacrosomal vesicles form in primary spermatocytes, a feature which has not been reported for echinoids, although such vesicles are a common feature of pre-spermiogenic cells of ophiuroids and holothurians. Spermiogenesis in all species is similar to descriptions published by other workers.

Linija zarodnih celic vključuje vse razvojne stadije celic, ki vodijo v nastanek spermijev in jajčec. Od somatskih celic se izvorne zarodne celice ločijo že v zgodnjem embrionalnem razvoju, nato migrirajo do mesta gonad in tam diferencirajo v spermatogonije in oogonije, ki predstavljajo zarodne matične celice. Zaradi zmožnosti samoobnavljanja in diferenciacije v gamete so spermatogoniji in oogoniji primerni za transplantacijo ali krioprezervacijo. Za povečanje uspešnosti transplantacije in krioprezervacije je potrebno željene celice predhodno namnožiti. Cilj našega dela je bil vzpostaviti celično kulturo spermatogonijev in oogonijev potočne postrvi. Celice smo po izolaciji iz gonad očistili z gradientnim centrifugiranjem ter jih nasadili v osnovno in bogato gojišče. Dodatno stopnjo čistosti smo dosegli z diferencialnim presajanjem. Po šestnajstih dneh gojenja smo delitve celic preverili z detekcijo z EdU. Z merjenjem fluorescence smo dokazali, da so se spermatogoniji in oogoniji v celični kulturi delili. Na podlagi rezultatov lahko zaključimo, da sta gradientno centrifugiranje in diferencialno presajanje učinkoviti metodi za čiščenje spermatogonijev in oogonijev. Prav tako pa obogatitev osnovnega gojišča ugodno vpliva na rast celic. Z vzpostavitvijo celične kulture smo uspešno namnožili spermatogonije in oogonije ter jih tako pripravili za nadaljnjo uporabo. The germ cell line includes all cell types that lead to the development of eggs and sperm. The somatic cells already separate from the primordial germs cells in the early stage of embryonic development. Afterwards they migrate to the gonads where they differentiate into spermatogonia and oogonia which represent germ stem cells. The ability of self-renewal and differentiation into gametes gives the spermatogonia and oogonia potential use for transplantation and cryopreservation. To improve the success of transplantation and cryopreservation isolated cells need to be multiplied beforehand. The objective of our study was to establish spermatogonial and oogonial cell culture in brown trout. After isolating the cells from gonads we enriched these cells with gradient centrifugation and then seeded them in a basic and enriched medium. Additional enrichment of spermatogonia and oogonia was achieved by differential plating. After sixteen days of cultivation we used EdU detection to determine cell proliferation. The observed fluorescence shows evidence of spermatogonia and oogonia proliferation. Based on our results we can conclude that gradient centrifugation and differential plating are efficient methods for cell enrichment. We also concluded that a positive effect on cell growth is shown in cells seeded in the enriched medium. With cell culturing we achieved the proliferation of spermatogonia and oogonia and prepared them for further use.

This study investigated the effects of rutin against reproductive damage caused by toxic mercury in male rats. Thirty-five Sprague Dawley rats were used. Control group was injected with saline for 7 days. The rutin-100 group received 100 mg/kg/b.w. rutin for 7 days. Mercuric chloride (HgCl2 ) group received 1.23 mg/kg/b.w. of HgCl2 for 7 days. Mercury chloride + rutin-50 group received 50 mg/kg/b.w. rutin and HgCl2 1.23 mg/kg/b.w. for 7 days. HgCl2 + rutin-100 group received 100 mg/kg/b.w. rutin and HgCl2 1.23 mg/kg/b.w. for 7 days. It was detected that HgCl2 treatment increased malondialdehyde (MDA) levels, tumour necrosis factor-α (TNF-α) and cyclooxygenase-2 (COX-2) expressions, necrosis and degeneration of spermatogonium, dead and abnormal sperm percentages; tubular walls thinning; and decreased antioxidant enzyme activities and sperm motility. It was determined that rutin application reduced testicular damage caused by HgCl2 . In conclusion, rutin administration may treat HgCl2 toxicity in testes.

The objective was to evaluate the effect of replacing soybean meal with the detoxified castor bean cake on testicular morphometry and spermatogenesis of sheep. Were used 24 uncastrated, 9-month old sheep weighing 29±0.8 kg they were randomly distributed among three treatments: T1 = 0%, T2 = 50%, and T3 = 100% substitution of soybean meal with detoxified castor bean cake. The animals were fed with Aruana grass pastage (Panicum maximum ‘Aruana’) and a ration for 90 days. After slaughtering, the testicles were collected and histological slides were prepared with tissue fragments. The data were evaluated for normality using the Shapiro-Wilk test, and analysis of variance was carried out at 5% level of significance. Substitution of soybean meal with detoxified castor bean cake had no effect on any of the assessed variables at the tested levels (P >0.05). The mean yield of spermatogenesis was 72.91 rounded spermatids per spermatogonium; the mean of total number of germ cells held by a Sertoli cell was 12.09; the mean of the testicular spermatic reserve was 31.82×109 and that per testicular gram was 238.28×106; the mean of daily spermatic production was 3.03×109 and that per testicular gram was 22.69×106; and the total number of Sertoli cells was 4.15×109 and that per testicular gram was 34.51×106. The results show that it is possible to replace 100% of the soybean meal with detoxified castor bean cake in sheep diet without any effects on spermatogenesis; however, it is important to perform seminal evaluations in future studies. RESUMO O objetivo com este estudo foi avaliar o efeito da substituição do farelo de soja pela torta de mamona destoxificada sobre a espermatogênese em ovinos. Foram utilizados 24 ovinos, com peso médio de 29±0,8kg e 9 meses de idade, distribuídos aleatoriamente em três tratamentos: T1= 0%, T2= 50% e T3= 100% de substituição do farelo de soja pela torta de mamona destoxificada. Os animais foram alimentados com pastagem de capim Aruana (Panicum maximum cv. Aruana) e ração durante 90 dias. Por ocasião do abate, coletou-se os testículos para mensurações e avaliação da espermatogênese. Os dados foram avaliados quanto à normalidade por meio do teste de Shapiro-Wilk e utilizou-se a Análise de Variância com 5% de significância. Não houve efeito da substituição do farelo de soja pela torta de mamona destoxificada nos níveis testados (P>0,05) para nenhuma das variáveis avaliadas. O rendimento médio da espermatogênese foi de 72,91 espermátides arredondadas produzidas a partir de uma espermatogônia; a média do número total de células germinativas sustentadas por uma de célula de Sertoli foi 12,09; a média da reserva espermática testicular total foi de 31,82x109 e de 238,28x106 por grama de testículo; a média da produção espermática diária foi de 3,03x109 e de 22,69x106 por grama de testículo e o número total de células de Sertoli foi de 4,15x109 e de 34,51x106 por grama de testículo. Os resultados demonstram que é possível substituir 100% do farelo de soja pela torta de mamona destoxificada na dieta de ovinos sem prejuízos no processo de espermatogênese.

The defective human survival motor neuron 1 (SMN1) gene leads to spinal muscular atrophy (SMA), the most common genetic cause of infant mortality. We previously reported that loss of SMN results in rapid differentiation of Drosophila germline stem cells and mouse embryonic stem cells (ESCs), indicating that SMN also plays important roles in germ cell development and stem cell biology. Here, we show that in healthy mice, SMN is highly expressed in the gonadal tissues, prepubertal spermatogonia, and adult spermatocytes, whereas low SMN expression is found in differentiated spermatid and sperm. In SMA-like mice, the growth of testis tissues is retarded, accompanied with gamete development abnormalities and loss of the spermatogonia-specific marker. Consistently, knockdown of Smn1 in spermatogonial stem cells (SSCs) leads to a compromised regeneration capacity in vitro and in vivo in transplantation experiments. In SMA-like mice, apoptosis and accumulation of the R-loop structure were significantly elevated, indicating that SMN plays a critical role in the survival of male germ cells. The present work demonstrates that SMN, in addition to its critical roles in neuronal development, participates in mouse germ cell and spermatogonium maintenance.

Introduction: Phototherapy is the most common treatment for neonatal jaundice. This study sought to determine ultrastructural changes in testis, at different time-points, after 48 hours of conventional phototherapy was given to newborn rats. Methods: Newborn male Wistar rats (n = 36) were divided into two groups as follows – group 1 (G1), control (without phototherapy) and group 2 (G2), exposure to conventional phototherapy for 48 h. Six animals from each group were sacrificed on postnatal days (PND) 70, 100 and 130. The testes were dissected out and processed for Transmission Electron Microscopy (TEM). Results: TEM showed that G2 on PND 70 and 100 showed damaged organelles, including nuclei, mitochondria, endoplasmic reticulum, vacuoles and electron dense bodies in the testes. Seminiferous Tubule on PND130 showed lesser damage. On PND70 ST wall thickness (STWT) of G2 was significantly higher (P < 0.001) than G1 STWT of G2 was significantly lower than G1 on PND100 (P = 0.047) and on PND130 (P < 0.001). Mitochondrial diameter in spermatogonia was significantly higher in G2 on PND70 (P = 0.001), PND100 (P = 0.031) and PND130 (P = 0.028). Primary spermatocytes in G2 also had larger mitochondria on PND70 (P < 0.001), PND100 (P = 0.007) and PND130 (P = 0.008). Further, spermatids had larger mitochondria in G2 on PND70 (P < 0.001), PND100 (P = 0.044) and PND130 (P < 0.001). Conclusion: Phototherapy causes degenerative changes in rat testis on PND70 and 100 that partially recover by PND 130.

Summary Mammalian male germ-cell development consists of three distinct phases: primordial germ cell (PGC) development, male germ-cell specification for spermatogonium development, and ensuing spermatogenesis. Here, we show an in vitro reconstitution of whole male germ-cell development by pluripotent stem cells (PSCs). Mouse embryonic stem cells (mESCs) are induced into PGC-like cells (mPGCLCs), which are expanded for epigenetic reprogramming. In reconstituted testes under an optimized condition, such mPGCLCs differentiate into spermatogonium-like cells with proper developmental transitions, gene expression, and cell-cycle dynamics and are expanded robustly as germline stem cell-like cells (GSCLCs) with an appropriate androgenetic epigenome. Importantly, GSCLCs show vigorous spermatogenesis, not only upon transplantation into testes in vivo but also under an in vitro culture of testis transplants, and the resultant spermatids contribute to fertile offspring. By uniting faithful recapitulations of the three phases of male germ-cell development, our study creates a paradigm for the in vitro male gametogenesis by PSCs.

Information on the reproductive biology of toadfish remains limited. In this study, we examined the structure and development of gonads in the grunting toadfish Allenbatrachus grunniens (Linnaeus, 1758) using morphological, histological and ultrastructural methods. The fish were collected from the Pranburi River Estuary, Thailand, during the annual reproductive period for this species (January to December 2018). The ovary of this species was paired and had elongated sac-like structures parallel to the kidneys and the digestive tract. In females, we identified 4 oocyte differentiation phases in the ovary including oogonia proliferation phase, a primary growth phase that was further classified into 2 steps (perinucleolar and oil droplets-cortical alveolar steps), and a secondary growth phase that also contained 2 steps (secondary growth and full-grown oocyte steps) and post-ovulatory phases, indicating an asynchronous pattern in ovarian development for this species. Transmission electron microscopy showed the 4 layers including the zona pellucida, basement membrane, granulosa cells and theca cells, all of which initially appeared in the oil droplets-cortical alveolar stage. The zona pellucida and the granulosa cells were highly developed during the secondary growth stage. The granulosa cells contained abundant smooth endoplasmic reticulum near the mitochondria. In males, the spermatogenesis was classified into spermatogonium to spermatozoa. Finally, we associated the morphological gonadal developments (stage I - IV) and the gonadosomatic index (GSI value) with the cellular developmental processes in both sexes. These results help integrate various levels of reproductive observations, which will be applied to understanding the reproductive cycle and development for aquaculture.

Stembark of Pakan Banyu ( Croton argyratus Blume ) empirically used as traditional contraception. The stembark contained active substances as steroids, saponins and alkaloids tannins alleged antifertillity for men. The purpose of this study was to demonstrate the potential of Pakan Banyu stembark ethanol extract to the total number of spermatogonium cells, primary spermatocytes, spermatids and spermatozoa in male rats. This study was an experimental research design posttest only control group design using 20 male rats into 4 groups of 5 mice. The control rats were used in treatment group, the rats were given the pakan banyu stembark extract at a dose of 100, 200 and 500 mg / kg with 0.5% CMC Na suspending agent. The treatment was given orally for 15 days at the 16 th days surgery was done to the testicles organs, then microanatomy preparations were made using paraffin method and using HE colour. Analysis of data using One-way ANOVA and Kruskal Wallis with 95% confidence level. The results showed that there is no effect of Pakan Banyu stembark ethanol extract against spermatogonium cells, primary spermatocytes, spermatids and spermatozoa.

Background Our understanding of the molecular mechanisms implementing pubertal maturation of the testis in vertebrates is incomplete. This topic is relevant in Atlantic salmon aquaculture, since precocious male puberty negatively impacts animal welfare and growth. We hypothesize that certain miRNAs modulate mRNAs relevant for the initiation of puberty. To explore which miRNAs regulate mRNAs during initiation of puberty in salmon, we performed an integrated transcriptome analysis (miRNA and mRNA-seq) of salmon testis at three stages of development: an immature, long-term quiescent stage, a prepubertal stage just before, and a pubertal stage just after the onset of single cell proliferation activity in the testis. Results Differentially expressed miRNAs clustered into 5 distinct expression profiles related to the immature, prepubertal and pubertal salmon testis. Potential mRNA targets of these miRNAs were predicted with miRmap and filtered for mRNAs displaying negatively correlated expression patterns. In summary, this analysis revealed miRNAs previously known to be regulated in immature vertebrate testis (miR-101, miR-137, miR-92b, miR-18a, miR-20a), but also miRNAs first reported here as regulated in the testis (miR-new289, miR-30c, miR-724, miR-26b, miR-new271, miR-217, miR-216a, miR-135a, miR-new194 and the novel predicted n268). By KEGG enrichment analysis, progesterone signaling and cell cycle pathway genes were found regulated by these differentially expressed miRNAs. During the transition into puberty we found differential expression of miRNAs previously associated (let7a/b/c), or newly associated (miR-15c, miR-2184, miR-145 and the novel predicted n7a and b) with this stage. KEGG enrichment analysis revealed that mRNAs of the Wnt, Hedgehog and Apelin signaling pathways were potential regulated targets during the transition into puberty. Likewise, several regulated miRNAs in the pubertal stage had earlier been associated (miR-20a, miR-25, miR-181a, miR-202, let7c/d/a, miR-125b, miR-222a/b, miR-190a) or have now been found connected (miR-2188, miR-144, miR-731, miR-8157 and the novel n2) to the initiation of puberty. Conclusions This study has - for the first time - linked testis maturation to specific miRNAs and their inversely correlated expressed targets in Atlantic salmon. The study indicates a broad functional conservation of already known miRNAs and associated pathways involved in the transition into puberty in vertebrates. The analysis also reveals miRNAs not previously associated with testis tissue or its maturation, which calls for further functional studies in the testis. Electronic supplementary material The online version of this article (10.1186/s12864-017-4205-5) contains supplementary material, which is available to authorized users.

Calcium plays a variety of vital regulatory functions in many physiological and biochemical events in the cell. The aim of this study was to describe the ultrastructural distribution of calcium during different developmental stages of spermatogenesis in a model organism, the zebrafish (Danio rerio), using a combined oxalate–pyroantimonate technique. Samples were treated by potassium oxalate and potassium pyroantimonate during two fixation stages and examined using transmission electron microscopy to detect electron dense intracellular calcium. The subcellular distribution of intracellular calcium was characterized in spermatogonium, spermatocyte, spermatid, and spermatozoon stages. The area which is covered by intracellular calcium in different stages was quantified and compared using software. Isolated calcium deposits were mainly detectable in the cytoplasm and the nucleus of the spermatogonium and spermatocyte. In the spermatid, calcium was partially localized in the cytoplasm as isolated deposits. However, most calcium was transformed from isolated deposits into an unbound pool (free calcium) within the nucleus of the spermatid and the spermatozoon. Interestingly, in the spermatozoon, calcium was mainly localized in a form of an unbound pool which was detectable as an electron-dense mass within the nucleus. Also, sporadic calcium deposits were scattered in the midpiece and flagellum. The proportional area which was covered by intracellular calcium increased significantly from early to late stages of spermatogenesis. The extent of the area which was covered by intracellular calcium in the spermatozoon was the highest compared to earlier stages. Calcium deposits were also observed in the somatic cells (Sertoli, myoid, Leydig) of zebrafish testis. The notable changes in the distribution of intracellular calcium of germ cells during different developmental stages of zebrafish spermatogenesis suggest its different homeostasis and physiological functions during the process of male gamete development.

In all teleosts the testes are mainly paired organs composed of seminiferous tubules or lobules entering the testicular main ducts and the spermatic duct forming an unpaired structure opening at the genital papilla. The interstitial tissue and the germinal epithelium consist of germinal cells (spermatogonia, spermatocytes, spermatids, spermatozoa) supported by somatic (=Sertoli) cells. During spermatogenesis the germinal and somatic cells form spermatocysts, which are the functional unit providing a blood testis barrier. This cyst starts to develop when Sertoli cells processes enclose a single spermatogonium comprising an isogenic clone of secondary spermatogonia and all germinal cells at further stages of spermato- and spermiogenesis. The testes in more basal taxa are tubular with anastomosing tubules. The tubules in the testes of most externally fertilizing species show a similar structure alongside the entire testis, but in species practicing insemination, different morphological adaptations coupled with dividing testis into a spermatogenic and a aspermatogenic part occur. The spermatogenic part is a place filled with cysts containing developing spermatozoa, while the aspermatogenic part is a storage organ for spermatozoa as well as sperm bundles called spermatozeugmata or spermatophores. In more derived taxa the testes are lobular. Additionally, according to the distribution of spermatogonia, the lobular testis are classified as: a) unrestricted spermatogonial testis type, where spermatogonia are situated along the lobules (e.g. in Percidae), and b) restricted spermatogonial testis type, where spermatogonia are situated only at the distal end of the lobule just beneath the tunica albuginea as in Atherinomorpha. Independent of testis type the process of spermatogenesis finally leads to the development of species-specific spermatozoa, which in all teleosts are anacrosomal. The spermatozoal ultrastructure shows a great diversity in shape and size of the head (mainly the nucleus), location and number of mitochondria, number of flagella or their lack, mutual arrangement of centrioles and the intercentriolar apparatus as well as the presence of additional structures such as pseudoflagella, striated rootlet, accessory microtubules, lateral fins, cytoplasmic sleeve, fenestrated membrane, dense fibers etc. According to the mode of fertilization and the presence or lack of the flagellum, the spermatozoa are classified as aquasperm (aflagellate, uniflagellate and biflagellate) or introsperm with a simple or complex structure.

Insecticide chlorpyrifos (CPF) with increased oxidative stress, structural destruction, and hemostasis of testicular tissue leads to male infertility. The present study investigated the protective effect of exercise (Exe) and eugenol supplementation (Sup) on CPF-induced testicular spermatogenic disorders in male rats. In this experimental study, 21 adult male albino rats were divided into seven groups, control (Co: 6 weeks), CPF (6 weeks), Co + Oil (2 weeks healthy food and 4 weeks oil), Co + Dimethylsulfoxide (DMSO: 6 weeks), CPF + Sup (2 weeks CPF and 4 weeks CPF + Sup), CPF + Exe (2 weeks CPF and 4 weeks CPF + Exe), and CPF + Exe + Sup (2 weeks CPF and 4 weeks CPF + Exe + Sup) group. All treatments were done intraperitoneally (5 days a week). Exe groups were subjected to run at moderate exercise intensity for 5 days per week over 6 weeks. DMSO groups were administered to the equal volume of vehicle for 6 consecutive weeks. Finally, the animals were sacrificed with Co2 gas and then alterations in testicular histology and sperm parameters were evaluated. Protein expression of PLZF and IGFα in the CPF group showed a significant decrease compared with the control group (p ˂ 0.001 for both). It was shown that CPF + Exe + Sup (p ˂ 0.001) and CPF + Sup (p ˂ 0.01) groups had a significant increase in protein expression of PLZF, but the protein expression of IGFα showed a significant increase just in the CPF + Exe + Sup group (p ˂ 0.001). Also, CPF caused a significant decrease in Leydig counts, Sertoli cell count, spermatogonium counts, spermatocyte cell count, spermatid cell count, and tunica thickness as well as a significant increase in testicle diameter (p ˂ 0.01) and ducts diameter compared with the control group. It seems that aerobic exercise with eugenol supplementation suppresses the disruption effects of CPF on testicular tissue (cellular and structural) by increasing the antioxidant capacity and improving the secretion of sex hormones. Therefore, the aerobic exercise with supplement of the eugenol has potential therapeutic targets for male infertility that need further study.