Spermatocyte Stage Research Articles

Defective spermatogenesis and testosterone levels in kinase suppressor of Ras1 (KSR1)-deficient mice.

The aim of this study was to clarify the role of the protein kinase suppressor of Ras1 (KSR1) in spermatogenesis. Spermatogenesis in ksr1 -/- mice was studied in testicular tissue and epididymal spermatozoa by light and transmission electron microscopy and by immunofluorescence using antibodies to ghrelin and 3β-hydroxysteroid dehydrogenase (3β-HSD). Blood testosterone levels were also assessed. ksr1 -/- mice showed reduced epididymal sperm concentration and motility as compared with wild- type (wt) mice. Testis tissue from ksr1 -/- mice revealed a prevalent spermatogenetic arrest at the spermatocyte stage; the interstitial tissue was hypertrophic and the cytoplasm of the Leydig cells was full of lipid droplets. Ghrelin signal was present in the seminiferous tubules and, particularly, in the interstitial tissue of wt mice; however, in ksr1 -/- mice ghrelin expression was very weak in both the interstitial tissue and tubules. On the contrary, the signal of 3β-HSD was weak in the interstitial tissue of wt and strong in ksr1 -/- mice. Testosterone levels were significantly increased in the blood of ksr1 -/- mice (P <0.05) as compared with wt. The results obtained reveal the importance of the KSR scaffold proteins in the spermatogenetic process. The study of the molecular mechanisms associated with spermatogenetic defects in a mouse model is essential to understand the factors involved in human spermatogenesis.

Atypical septate junctions maintain the somatic enclosure around maturing spermatids and prevent premature sperm release in Drosophila testis.

ABSTRACTTight junctions prevent paracellular flow and maintain cell polarity in an epithelium. These junctions are also required for maintaining the blood-testis barrier, which is essential for sperm differentiation. Septate junctions in insects are orthologous to the tight junctions. In Drosophila testis, major septate junction components co-localize at the interface of germline and somatic cells initially, and then condense between the two somatic cells in a cyst after germline meiosis. Their localization is extensively remodeled in subsequent stages. We find that characteristic septate junctions are formed between the somatic cyst cells at the elongated spermatid stage. Consistent with previous reports, knockdown of essential junctional components – Discs-large-1 and Neurexin-IV – during the early stages disrupted sperm differentiation beyond the spermatocyte stage. Knockdown of these proteins during the final stages of spermatid maturation caused premature release of spermatids inside the testes, resulting in partial loss of male fertility. These results indicate the importance of maintaining the integrity of the somatic enclosure during spermatid coiling and release in Drosophila testis. It also highlights the functional similarity with the tight junction proteins during mammalian spermatogenesis.This article has an associated First Person interview with the first author of the paper.

Защита сперматогенеза β-каротином при радиационных воздействиях. Сообщение 1: однократное острое внешнее γ-облучение. Краткосрочное назначение каротиноида

Purpose: Experimental evaluation of β-carotene correction of radiation induced by single acute γ-irradiation of spermatogenesis disorders in male (♂) F1 CBAxC97Bl mice and non-linear rats with short-term carotenoid prescription. Material and methods: Single external gamma irradiation ♂ was performed at the IGUR facility (137Cs, dose rate 0.029 Gy/s). The β-carotene suspension was administered ♂ orally 19 and 4 hours before, 4 and 24 hours after irradiation. The damage and effectiveness of protection of spermatogenesis of irradiated ♂ were judged by the state of their reproductive function. Results: The effects of correction of the short-term prescription of β-carotene radiation-induced by a single acute external gamma irradiation at doses of 3 and 5 Gy of spermatogenesis disorders in mice and rats at stages of mature spermatozoa, spermatids and spermatogonium have been established. β-carotene in ♂ mice reduced total embryonic mortality in 8–14 and 77–84 days after irradiation at a dose of 3 Gy from 46 to 36 and from 41 to 28 %, preimplantation – after 8–14 and 15–21 days – from 24 up to 13 and from 31 to 22 %, postimplantation – after 0–7 and 77–84 days – from 24 to 15 and from 25 to 7 %. In ♂ rats 0–7 days after irradiation at a dose of 5 Gy β-carotene reduced total, pre-, post- and induced postimplantation embryonic mortality from 62 to 41, from 34 to 17, from 41 to 30 and from 38 to 26 %, respectively. After 13–20 days – preimplantation mortality from 27 to 11 %. At the stage of spermatozoa after irradiation at a dose of 3 Gy increased the breeding efficiency from 70 to 100 %, fecundity – by 23–31 %; reduced total and preimplantation mortality from 49 to 39 % and from 41 to 27 %, respectively. At the stage of spermatids –sterilization was prevented and the efficiency of breeding was normalized, reduced post- and induced postimplantation mortality from 25 to 20 and from 17 to 12 %, respectively. The therapeutic and prophylactic effects of β-carotene at the spermatocyte stage are not revealed. In the spermatogonium stage, the carotenoid increased the total number and number of live embryos in pregnant female (♀) from 36 to 55 and from 20 to 50 %, reduced total and pre-implantation mortality from 81 to 56 and from 69 to 42 %, respectively. Conclusions: The prospects of β-carotene use for correction of gonadal radiation injuries in the risk groups of exposure to ionizing radiation and the inclusion of β-carotene in the scheme of drug prevention of acute radiation injuries of spermatogenesis are shown.

Characterization of inter-Sertoli cell tight and gap junctions in the testis of turtle: Protect the developing germ cells from an immune response

It is conceivable that early developing germ cells must across the basal to the luminal region of seminiferous tubules (STs) during spermatogenesis is associated with extensive restructuring of junctional complex. However, very limited information is documented about these junctional complexes in reptiles. In the present study we have determined the localization of inter-Sertoli cell tight junctions (TJ's), protein CLDN11 and gap junction protein Cx43 during spermatogenesis in the testis. In early spermatogenesis, weak immunoreactivity of CLDN11and focal localization of Cx43 was observed around the Sertoli cell in the luminal region, but completely delaminated from the basal compartment of STs. In late spermatogenesis, strong focal to linear localization of CLDN11and Cx43 was detected at the points of contact between two Sertoli cells and around the early stages of primary spermatocytes in the basal compartment of STs. In late spermatogenesis, localization of CLDN11and Cx43 was drastically reduced and seen only around Sertoli cells and spermatogonia near the basal lamina. However, transmission electron microscopy revealed that inter-Sertoli cell tight junctions were present within the basal compartment of STs, leaving the spermatogonia and early primary spermatocytes in the basal region during mid spermatogenesis. Gap junctions were observed between Sertoli cells, and Sertoli cells with spermatogonia and primary spermatocytes throughout spermatogenesis. Moreover, adherens and hemidesmosomes junctions were observed during spermatogenesis. The above findings collectively suggest that the intensity and localization of TJ's and gap junctions vary according to the spermatogenetic stages that might be protected the developing germ cells from own immune response.

Expression pattern and functional analysis of the two RING box protein RBX in spermatogenesis of Chinese mitten crab Eriocheir sinensis

Studies in E. sinensis have shown that ubiquitination mediated by Cullin-RING E3 ligases (CRLs) plays important roles in spermatogenesis. In other species, CRLs are also essential in cell cycle progression, DNA replication, signal transduction, gene transcription, and development. The catalytic RING component, the RING box protein, is an important part of CRLs. However, there have been few studies on CRLs in crustaceans. In this study, we cloned two RING box protein genes from the Chinese mitten crab, Eriocheir sinensis, termed Es-RBX1 and Es-RBX2 The full length Es-RBX1 cDNA comprises 741 nucleotides, and encodes a protein of 124 amino acid residues, whereas the Es-RBX2 cDNA comprises 1325 nucleotides, and encodes a protein of 110 amino acid residues. Bioinformatics analysis showed that the domains and structure of the RBX proteins have been highly conserved during evolution. Quantitative real-time polymerase chain reaction and western blotting showed that Es-RBX1 is highly expressed in the testis, particularly during the spermatocyte stage, whereas Es-RBX2 did not show specific expression in the male reproductive system. Furthermore, Es-RBX1 is mainly distributed in the nucleus, and changed its location with the development of the nucleus. Co-immunoprecipitation showed that Es-RBX1 could bind Cullin4. These results suggested that Es-RBX1 plays a key role in spermatogenesis of E. sinensis though forming a complex with Cullin4.

Radiation dosimetry and repair kinetics of DNA damage foci in mouse pachytene spermatocyte and round spermatid stages.

Accurate quantification of DNA double strand breaks (DSB) in testicular germ cells is difficult because of cellular heterogeneity and the presence of endogenous γH2AX. Here, we used confocal microscopy to quantify DNA damage and repair kinetics following γ-irradiation (0.5-4 Gy) in three major mouse male germ cell stages, early and late pachytene spermatocytes and round spermatids (RSs), following a defined post irradiation time course. Dose-response curves showing linear best fit validated γH2AX focus as a rapid biodosimetric tool in these substages in response to whole body in vivo exposure. Stage specific foci yield/dose and repair kinetics demonstrated differential radiosensitivity and repair efficiency: early pachytenes (EP) repaired most rapidly and completely followed by late pachytene (LP) and RSs. Repair kinetics for all three stages followed 'exponential decay' in response to each radiation dose. In pachytenes immediate colocalisation of γH2AX and 53BP1, which participates in non-homologous end-joining repair pathway, was followed by dissociation from the major focal area of γH2AX by 4 h demonstrating ongoing DSB repair. These results confirm the differential radiosensitivity and repair kinetics of DSBs in male germ cells at different stages. Taken together, our results provide a simple and accurate method for assessing DNA damage and repair kinetics during spermatogenesis.

Immunohistochemical analysis of RHAMM expression in normal and neoplastic human tissues: a cell cycle protein with distinctive expression in mitotic cells and testicular germ cells.

Expression of Receptor for Hyaluronic Acid Mediated Motility (RHAMM) increases cellular motility and RHAMM overexpression promotes invasive phenotype and metastasis of cancer cells. RHAMM has been suggested as a biomarker for poor prognosis in several tumor types, including lung, breast, colorectal, gastric, pancreatic ductal, and ovarian cancers. RNA studies showed restricted RHAMM expression in normal tissues, but its protein expression data in tissues were limited. In light of its potential as a prognostic marker and a therapeutic target, we performed immunohistochemical analysis to systematically characterize RHAMM expression in normal and neoplastic human tissues. Among 29 normal adult tissues, RHAMM protein showed restricted expression and was observed in the thymus, lymph node/tonsil, small intestine, colon, skin, bone marrow, placenta, and testis. The cellular distribution patterns of RHAMM in these normal tissues were consistent with RHAMM being a G2/M cell cycle protein, and this was further supported in comparison to the expression of cyclin B2, another G2/M protein. However, unlike the subcellular localization of cyclin B2, RHAMM decorated mitotic spindles in both anaphase and metaphase. RHAMM expression in tumor tissues is variable; and higher RHAMM protein expression is associated with histologically higher-grade tumors in general. Distinct from its expression in somatic tissues, RHAMM showed diffuse, strong, stage-specific expression in the spermatocyte stage of germ cells in adult testis. The neoplastic counterpart, spermatocytic tumor, also showed strong RHAMM expression. This unique expression in testis suggests that RHAMM may function during normal testicular germ cell maturation.

Species-specific expression of phosphoglycerate kinase 2 (PGK2) in the developing porcine testis

Whereas stage-specific markers for spermatogonial cells have been well investigated in mouse, the specific markers of germ cells in the testis of domestic animals have not been well defined. Phosphoglycerate kinase (PGK), an enzyme that converts 1,3-bisphosphoglycerate and adenosine diphosphate to 3-phosphoglycerate and adenosine triphosphate, has two isozymes: PGK1 and PGK2. In mouse, PGK1 exists only during the early stages of spermatogenesis, and PGK2 is then expressed during the pachytene spermatocyte stage. In this study, we investigated the localization of PGK2 in the developing porcine testis, and compared the similarities and differences in its expression with that of the PGK2 in mouse. The PGK2 protein was found to be exclusively expressed in spermatids of the adult mouse testis, whereas PGK2-positive cells were observed in the prepubertal and postpubertal testes of pigs. Based on this result, we examined the expression of PGK2 in in vitro-cultured porcine undifferentiated spermatogonia and found it to be maintained in the cultured cells. To verify this result and identify the spermatogonial stem cell-like potential in recipient testes, PKH26 dye-stained PGK2-positive cells were transplanted into the testes of busulfan-treated immunodeficient mouse that had been depleted of both testicular germ cells and somatic cells. The transplanted cells colonized the recipient testis at 8 weeks post transplantation, and fluorescence microscopy identified the cells in the basement membranes of the seminiferous tubules of the injected mouse. Taken together, our results suggest that PGK2 is expressed differently in the testes of mouse and pigs according to developmental stage. This finding should contribute to the study of spermatogenesis and the production of transgenic domestic animals through in vitro spermatogonial sperm cell culture.

Assessment of Polyscias fruticosa (L.) Harm (Araliaceae) leaf extract on male fertility in male Wistar rats

Background: Polyscias fruticosa is used widely as food, disease remedy and as an ornamental across Afro-Asian countries. For instance, P. fruticosa is used traditionally as an anti-asthma, anti-tussive, and a muco-suppressant herbal remedy for asthmatics in Ghana. Although many studies have investigated the pharmacological basis of the ethnobotanical uses of P. fruticosa, however, its effect on the reproductive system remains completely unknown. Aim of study: This study assessed effects of Polyscias fruticosa leaf extract (PFE) on male fertility and toxicity in adult male Wistar rats. Materials and methods: after crude preparation of PFE, it was subjected to qualitative phytochemical, thin layer chromatography (TLC) and gas chromatography mass spectrometry (GC-MS) analyses. Effect of PFE was assessed on male fertility and toxicity by using healthy adult male Wistar rats. Rats were randomly assigned to: normal saline (5 ml/kg po, n = 5), Clomiphene Citrate (50 mg/kg po; n = 5) and PFE (100, 200 and 500 mg/kg po; n = 5 respectively) groups and treated for 21 days. On day 22 rats were sacrificed and male fertility parameters (left testis weight, relative testis weight, caudal epididymal weight, caudal epididymal sperm count, sperm motility, sperm morphology and assessment of male sex hormones and testicular histology) were assessed. Results: There were no significant changes in bodyweight, weight of left testis, weights of right and left caudal epididymides between treatments groups (PFE and clomiphene citrate) and control. Caudal epididymal sperm count increased in PFE (100 and 500 mg/kg)-treated rats relative to control. Sperm motility relatively increased in PFE-treated rats compared to control. Sperm abnormality decreased in PFE-treated rats; especially in PFE (100 mg/kg) group compared to control. Serum testosterone levels decreased inversely with serum luteinizing hormone (LH) levels in PFE-treated rats compared to control. There were minimal-to-no-alterations in histological sections of testis, except vacuolations at primary spermatocyte stage. Glycosides, saponins, cyanogenic glycosides, sterols and alkaloids were detected in PFE. Conclusion: PFE improved caudal epididymal sperm count and may be useful as male fertility enhancer but exhaustive safety studies on key male sex organs needs to be established.

Spermatogonial deubiquitinase USP9X is essential for proper spermatogenesis in mice.

USP9X (ubiquitin-specific peptidase 9, X chromosome) is the mammalian orthologue of Drosophila deubiquitinase fat facets that was previously shown to regulate the maintenance of the germ cell lineage partially through stabilizing Vasa, one of the widely conserved factors crucial for gametogenesis. Here, we demonstrate that USP9X is expressed in the gonocytes and spermatogonia in mouse testes from newborn to adult stages. By using Vasa-Cre mice, germ cell-specific conditional deletion of Usp9x from the embryonic stage showed no abnormality in the developing testes by 1 week and no appreciable defects in the undifferentiated and differentiating spermatogonia at postnatal and adult stages. Interestingly, after 2 weeks, Usp9x-null spermatogenic cells underwent apoptotic cell death at the early spermatocyte stage, and then, caused subsequent aberrant spermiogenesis, which resulted in a complete infertility of Usp9x conditional knockout male mice. These data provide the first evidence of the crucial role of the spermatogonial USP9X during transition from the mitotic to meiotic phases and/or maintenance of early meiotic phase in Usp9x conditional knockout testes.

Subcellular distribution of calcium during spermatogenesis of zebrafish, Danio rerio.

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.

Somatic PI3K activity regulates transition to the spermatocyte stages in Drosophila testis.

Spermatogenesis, involving multiple transit amplification divisions and meiosis, occurs within an enclosure formed by two somatic cells. As the cohort of germline cells divide and grow, the surface areas of the somatic cells expand maintaining a tight encapsulation throughout the developmental period. Correlation between the somatic cell growth and germline development is unclear. Here, we report standardization of a quantitative assay developed for estimating the somatic roles of target molecules on germline division and differentiation in Drosophila testis. Using the assay, we studied the somatic roles of phosphatidylinositol-3-kinase (PI3K). It revealed that the expression of PI3KDN is likely to facilitate the early germline development at all stages, and an increase in the somatic PI3K activity during the early stages delays the transition to spermatocyte stage. Together, these results suggest that somatic cell growth plays an important role in regulating the rate of germline development.

The potential role of mesenchymal stem cells in a hypoxia model induced by sodium nitrite in testes of male albino rats

&#x0D; Introduction: The present work aims to examine the possible role of stem cells on biochemical markers and histopathological alterations of hypoxia caused by sodium nitrite (NaNO2) toxicity in testes of male rats.&#x0D; Methods: In this study, 96 adult male albino rats were divided into 6 groups (16 rats each). Group 1 (G1) was the control group and received distilled H2O. Group 2 (G2) received daily NaNO2 (35 m/kg bwt/ day) via subcutaneous injection for 3 weeks. Group 3 (G3) received NaNO2 for 2 weeks and were then injected once with 2*106 mesenchymal stem cells (MSCs) intravenously and sacrificed 4 weeks later. Group 4 (G4) received NaNO2 for 2 weeks and were then injected with 2*106 MSCs followed by daily NaNO2 injection for 1 week; rats in G4 were sacrificed 4 weeks from MSCs treatment. Group 5 (G5) rats were treated with NaNO2 for 2 weeks and then left to recover for 4 weeks. Finally, Group 6 (G6) rats were treated with NaNO2 for 3 weeks and left to recover for 3 weeks, after which point they were sacrificed.&#x0D; Results: The results showed that NaNO2 caused oxidative damage and histopathological alterations in the rat testes, as well as increased the levels of testes malondialdehyde (MDA), nitric oxide (NO) and DNA fragmentation percentage (DNA F %). Moreover, NaNO2 decreased the elevated activities of testes catalase (CAT) and total antioxidant activity (TAA), in comparison to the control group. The histological results illustrated different distortions, vacuolization and lipid accumulations in interlobular space as well as diminution of inter cellular germ cell layers, absence of Leydig cells, irregular basement membrane of tubule, and separation within spermatogenic cells. In addition, congestion and dilation of intertubular and peripheral blood capillaries were found. Nevertheless, the administration of stem cells reduced the danger actions of sodium nitrite by enhancing biochemical marker concentration.&#x0D; Conclusion: There was an improvement in the histology of the rat testes, including a relatively normal order in the different stages of spermatogonia and loss of different stages of spermatocytes. Regarding the recovery period, there was also a significant improvement in each of the biochemical parameters assessed and in the histological lesions.&#x0D; &#x0D; &#x0D; &#x0D;

Inhibitory effect of &lt;i&gt;Taraxacum officinale&lt;/i&gt; L (Compositae) aqueous root extract on spermatogenesis

Purpose: To investigate if T. officinale root aqueous extract has anti-spermatogenic activity similar to that of the whole plant which was shown previously to inhibit spermatogenesis. Methods: T. officinale aqueous extract was prepared by soaking 100 g of dried materials in 1 L distilled water for two days at 45 oC. Fifty adult male rats were divided into five groups and treated for 60 days. Four groups were gavaged with the whole plant or root aqueous extract in low or high doses. The male rat rats were allowed to mate with female rats. The control group received distilled water. Sperm count, motility and morphology as well as chromatin integrity were evaluated. Results: Serum testosterone level, sperm parameters, pregnancy rate and average number of fetuses per pregnant females decreased significantly in the treated groups compared to control and in the rootreceiving rats compared to the whole plant-receiving rats. Female rats which were mated with high dose root-receiving males did not deliver fetuses. Cross sections of seminiferous tubules of T. officinale treated rats showed lesions and disorganized germinal epithelium. Late spermatogenesis maturation arrest (spermatid stage) was observed in all of the treated groups except the high dose root-receiving group which showed early maturation arrest (spermatocyte stage). In addition, the mRNA level of two spermatogonial stem cell markers responsible for self-renewal and proliferation of spermatogonia increased in high dose-receiving rats. Conclusion: T. officinale root aqueous extract has inhibitory effects on spermatogenesis. Further studies are required to identify specific ingredient(s) in T. officinale that may be useful as male contraceptive(s). Keywords: Taraxacum officinale , Dandelion, GDNF family receptor alpha 1, Macrophage Colony- Stimulating Factor, Promyelocytic Leukaemia Zinc-Finger, Testosterone, Sperm count

A core component of the CUL4 ubiquitin ligase complexes, DDB1, regulates spermatogenesis in the Chinese mitten crab, Eriocheir sinensis

Studies in mammals have shown that damaged DNA-binding protein 1 (DDB1) is a multifunctional protein that recognizes UV-induced DNA lesions and activates nucleotide excision repair process, and could also be a linker protein for Cullin4 in ubiquitination to regulate cell cycle progression. However, there are few studies of DBB1 in crustaceans. In this study, a cDNA representing the DDB1 gene from Eriocheir sinensis (Es-DDB1) was cloned successfully. The full length Es-DDB1 cDNA comprises 4871 nucleotides, and encodes an open-reading frame (ORF) of 1137 amino acid residues. Bioinformatics' analysis showed that the domains and structure of Es-DDB1 have been highly conserved during evolution. Antibodies against Es-DDB1 and Es-Cul4 were raised using a prokaryotic expression system. Moreover, a co-immunoprecipitation assay showed that Es-DDB1 could bind Es-Cul4 in the testis of Eriocheir sinensis. Furthermore, quantitative real-time PCR and Western blotting showed high expression in the testis, particularly during the spermatocyte stage. Immunofluorescence assays showed that Es-DDB1 was mainly distributed in the cytoplasm in the early and middle developmental stages. These results indicated that Es-DDB1 might play a key role in spermatogenesis of E. sinensis.

Latent effect of gamma irradiation on reproductive potential and ultrastructure of males' testes of Culex pipiens (Diptera; Culicidae)

Laboratory male pupae of Culex pipiens were exposed to 23, 41, 74 and 128 Gy doses of gamma radiation according to the LD25, LD50, LD75 and LD90 calculation, respectively. The inherited deleterious effects of gamma radiation were observed in the F1, F2 and F3 generations. Levels of sterility index in the F1 and F2 were higher than those of untreated control but in the F3 generation there was a semi-sterility compared with the control. Ultrastructure of normal males' testes of C. pipiens was studied using transmission electron microscopy. Histopathological responses were observed in the irradiated testes of C. pipiens. Gamma radiation had greatly affected the testes, such as (i) rupture, necrosis, degeneration and small vacuoles were reported in the testicular wall (ii) an abnormal distribution of the developmental stages of spermatogonia and spermatocytes leading to a general decrease in the rate of spermatogenesis; and (iii) deformity of sperm inhibitting the movements and the fertility of the sperm led to the decrease in the reproductive potential of C. pipiens. Consequently, these radiation doses are consistent with those used in the already established Sterile Insect Technique (SIT) programmes against Culex pipiens.

Ultrastructural Localization of Intracellular Calcium During Spermatogenesis of Sterlet (Acipenser ruthenus).

Calcium regulates many intracellular events such as growth and differentiation during different stages of gamete development. The aim of this study was to localize and quantify the intracellular distribution of calcium during different developmental stages of spermatogenesis in sterlet, Acipenser ruthenus, using a combined oxalate-pyroantimonate technique. The distribution of calcium was described in spermatogonium, spermatocyte, spermatid, and spermatozoon stages. In the spermatogonium and spermatocyte, calcium deposits were mainly localized in the nucleus and cytoplasm. The spermatid had calcium in the nucleus, developing acrosomal vesicle, and cytoplasm. Intracellular calcium transformed from scattered deposits in spermatogonia and spermatocyte stages into an unbound form in spermatid and the spermatozoon. The proportion of area covered by calcium increased significantly (p<0.05) from early to late stages of spermatogenesis. The largest proportion of area covered by calcium was observed in the nucleus of the spermatozoon. In conclusion, although most of the intracellular calcium is deposited in limited areas of the spermatogonium and spermatocyte, it is present an unbound form in the larger area of spermatids and spermatozoa which probably reflects changes in its physiological function and homeostasis during the process of male gamete production in spermatogenesis.

Characterization of MAGEG2 with testis-specific expression in mice.

Male germ cell development is a well-defined process occurring in numerous seminiferous tubules of the testis. Uncovering testicular novel genes related to intrinsic regulation of spermatogenesis is essential for the understanding of spermatogenesis. In the present study, we investigated mouse Mageg2, which belongs to a group of melanoma-associated antigens (MAGEs). Mageg2 is transcribed in the testis specifically, and its expression level is increased at the pachytene spermatocyte stage, indicating that Mageg2 is expressed predominantly in germ cells. We generated an antibody against mouse MAGEG2 for further characterization at the protein level. Immunoblot analysis suggested that MAGEG2 has specific testicular expression and the expression primarily occurred in pachytene spermatocytes. Proteomic analyses demonstrated that mouse MAGEG2 binded to testicular germ cell-specific serine/threonine-protein kinase 31 (STK31) and heat shock protein 9 (HSPA9). Direct binding with both interaction partners was confirmed by co-immunoprecipitation. We found that STK31 and HSPA9 bind MAGEG2 directly but not with each other. Interestingly, MAGEG2 reduced the kinase activity of STK31. Our study suggests that mouse MAGEG2 has at least two functions, including chaperone activity related to HSPA9 and regulation of pachytene spermatocyte-specific kinase, STK31. Altogether, our results provide the first information about MAGEG2 at the transcript and protein levels and suggest its potential molecular functions.

Knockdown of Sucla2 decreases the viability of mouse spermatocytes by inducing apoptosis through injury of the mitochondrial function of cells

Sucla2, a β subunit of succinyl coenzyme A synthase, is located in the mitochondrial matrix. Sucla2 catalyzes the reversible synthesis of succinate and adenosine triphosphate (ATP) in the tricarboxylic acid cycle. Sucla2 expression was found to be correlated with the capacitation of boar spermatozoa. We have previously reported that Sucla2 was decreased in the testes of rats with spermatogenesis failure after exposure to endocrine disruptor BDE47. Yet, the expression model of Sucla2 in spermatogenesis and the function of Sucla2 in spermatogenic cells are still unclear. Our objective was to explore the localization of Sucla2 during mouse spermatogenesis and its function in the mouse spermatocyte line (GC2). The localization of Sucla2 in the mouse testis was explored through immunohistochemistry (IHC). Sucla2 was knocked down in GC2 cells and its expression was detected by Western blot (WB) to verify the efficiency of the siRNA transfection. Mitochondrial membrane potential (MMP), apoptosis and ROS of GC2 were detected by flow cytometry. ATP production was measured by the luminometric method and the presence of Bcl2 of GC2 was detected by WB. Sucla2 is highly expressed in all germ cells but not in interstitial cells. Coarse Sucla2 signals are found in spermatocytes in stages VII–XII of mouse spermatogenesis. In GC2 cells, knockdown of Sucla2 decreased cell viability and MMP, induced apoptosis of GC2 cells, decreased ATP production, and Bcl2 expression, and increased ROS levels. Sucla2 is related to the developmental stages of mouse spermatogenesis. Knockdown of Sucla2 decreases the viability of mouse spermatocytes by inducing apoptosis via decreased mitochondrial function of the cells

Molecular characterization and sub-cellular distribution of JNK and JIP4 protein kinases in spermatogenesis and acrosome reaction of the Chinese mitten crab Eriocheir sinensis H. Milne Edwards, 1853 (Crustacea: Brachyura: Varunidae)

The c-Jun N-terminal protein kinase mitogen-activated protein kinases (JNK MAPKs) are an evolutionarily conserved family of serine/threonine protein kinases involved in controlling cell growth, differentiation, and apoptosis in mammalian testis and germ cells. As a scaffold protein of the JNK pathway, JNK-interacting protein 4 (JIP4) is found in some mammalian spermatozoa and participates in certain male reproductive processes. Few have reported on the roles of JNK and JIP4 in the Chinese mitten crab Eriocheir sinensis H. Milne Edwards, 1853. Based on transcriptome sequences from the testis of E. sinensis , we successfully cloned full-length cDNAs of JNK and JIP4 (designated Es-JNK and Es-JIP4 , respectively). Western blotting showed relatively high expression of Es -JNK and Es -JIP4 proteins in the testis. Expression levels of total Es -JNK, phosphorylated Es -JNK ( Es -p-JNK), and Es -JIP4 proteins decreased gradually during spermatogenesis. Es-JNK and Es-JIP4 mRNAs were mainly localized in the nucleus from the spermatogonium stage to the secondary spermatocyte stage and were widely distributed in the nuclear cup, apical cap, acrosomal tubule, and acrosomal vesicle of spermatids. Es -JNK, Es -p-JNK, and Es -JIP4 proteins were nevertheless mainly dispersed in the cytoplasm and intercellular substance of spermatogonium and primary spermatocytes, in the nucleus of secondary spermatocytes, and in the apical cap, acrosomal tubule, acrosomal vesicle, and nuclear cup of spermatids. During the acrosome reaction induced by the calcium ionophore A23187, Es -JNK, Es -p-JNK, and Es -JIP4 proteins were found mainly expressed in the apical cap and acrosomal vesicle by immunofluorescence microscopy. The results implicate functions for Es -JNK and Es -JIP4 in spermatogenesis and the acrosome reaction in E. sinensis .