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Spermatogenic Cells (spermatogenic + cell)
Selected AbstractsThe significance of feeding for reproduction in a male Metastriata tick, Haemaphysalis longicornis (Acari: Ixodidae)ACTA ZOOLOGICA, Issue 1 2000Tomohide Matsuo In Haemaphysalis longicornis, secretions of the male accessory genital glands were regenerated by re-feeding for 3 or 4 days, although the secretions were almost completely released during the first copulation. It was also shown that spermatogenesis continued during re-feeding, since prospermia (elongated spermatids) were deposited in the seminal vesicle. A potent male seeks a receptive female on the host for copulation. The movement of males to different attachment sites occurred between the third and fourth day of re-feeding, and completely re-fed males (for 4 days) were able to copulate successfully. Spermatogenic cells, ranging from spermatogonia at the anterior end to prospermia at the posterior end, were found in fed males. Degeneration of spermatocytes at the great growth phase and developing spermatids prior to final development of prospermia were seen in virgin males without re-feeding after the first meal. Fully elongated spermatids (prospermia) appeared morphologically normal up to 10 days after the first feeding. Degeneration of spermatocytes and developing spermatids occurred from the second day and was almost complete by the fourth day. The degenerating cells shrank, became electron-dense, and finally died. A reduction in secretions of the four lobes of the accessory glands occurred during the 10 days after feeding. [source] The expression of glutathione reductase in the male reproductive system of rats supports the enzymatic basis of glutathione function in spermatogenesisFEBS JOURNAL, Issue 5 2002Tomoko Kaneko Glutathione reductase (GR) recycles oxidized glutathione (GSSG) by converting it to the reduced form (GSH) using an NADPH as the electron source. The function of GR in the male genital tract of the rat was examined by measuring its enzymatic activity and examining the gene expression and localization of the protein. Levels of GR activity, the protein, and the corresponding mRNA were the highest in epididymis among testes, vas deferens, seminal vesicle, and prostate gland. The localization of GR, as evidenced by immunohistochemical techniques, reveals that it exists at high levels in the epithelia of the genital tract. In testis, GR is mainly localized in Sertoli cells. The enzymatic activity and protein expression of GR in primary cultured testicular cells confirmed its predominant expression in Sertoli cells. Intracellular GSH levels, expressed as mol per mg protein, was higher in spermatogenic cells than in Sertoli cells. As a result of these findings, the effects of buthionine sulfoximine (BSO), an inhibitor for GSH synthesis, and 1,3-bis(2-chlorethyl)-1-nitrosourea (BCNU), an inhibitor for GR, on cultured testicular cells were examined. Sertoli cells were prone to die as the result of BCNU, but not BSO treatment, although intracellular levels of GSH declined more severely with BSO treatment. Spermatogenic cells were less sensitive to these agents than Sertoli cells, which indicates that the contribution of these enzymes is less significant in spermatogenic cells. The results herein suggest that the GR system in Sertoli cells is involved in the supplementation of GSH to spermatogenic cells in which high levels of cysteine are required for protamine synthesis. In turn, the genital tract, the epithelia of which are rich in GR, functions in an antioxidative manner to protect sulfhydryl groups and unsaturated fatty acids in spermatozoa from oxidation during the maturation process and storage. [source] p19Ink4d and p18Ink4c cyclin-dependent kinase inhibitors in the male reproductive axisMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 8 2007Gregory M. Buchold Abstract The loss of the cyclin-dependent kinase inhibitors (CKIs) p18Ink4c and p19Ink4d leads to male reproductive defects (Franklin et al., 1998. Genes Dev 12: 2899,2911; Zindy et al., 2000. Mol Cell Biol 20: 372,378; Zindy et al., 2001. Mol Cell Biol 21: 3244,3255). In order to assess whether these inhibitors directly or indirectly affect male germ cell differentiation, we examined the expression of p18Ink4c and p19Ink4d in spermatogenic and supporting cells in the testis and in pituitary gonadotropes. Both p18Ink4c and p19Ink4d are most abundant in the testis after 18 days of age and are expressed in purified populations of spermatogenic and testicular somatic cells. Different p18Ink4c mRNAs are expressed in isolated spermatogenic and Leydig cells. Spermatogenic cells also express a novel p19Ink4d transcript that is distinct from the smaller transcript expressed in Sertoli cells, Leydig cells and in other tissues. Immunohistochemistry detected significant levels of p19Ink4d in preleptotene spermatocytes, pachytene spermatocytes, condensing spermatids, and Sertoli cells. Immunoprecipitation-Western analysis detected both CKI proteins in isolated pachytene spermatocytes and round spermatids. CDK4/6-CKI complexes were detected in germ cells by co-immunoprecipitation, although the composition differed by cell type. p19Ink4d was also identified in FSH+ gonadotrophs, suggesting that this CKI may be independently required in the pituitary. Possible cell autonomous and paracrine mechanisms for the spermatogenic defects in mice lacking p18Ink4c or p19Ink4d are supported by expression of these CKIs in spermatogenic cells and in somatic cells of the testis and pituitary. Mol. Reprod. Dev. 74: 997,1007, 2007. © 2007 Wiley-Liss, Inc. [source] Use of a highly sensitive quantitative telomerase assay in intracytoplasmic sperm injection programmes for the treatment of 47,XXY non-mosaic Klinefelter men,ANDROLOGIA, Issue 4 2002Y. Yamamoto Summary. We evaluated the role of the sensitive quantitative telomerase assay (SQTA) in the management of men with non-mosaic Klinefelter's syndrome (KS). Diagnostic testicular biopsy (DTB) was performed in 24 men with KS. A part of the DTB was stained and the remaining fragment was processed for the SQTA. After 3,18 months, a therapeutic testicular biopsy (TTB) was performed in the same testicle and the recovered specimens were processed to identify spermatozoa. Men with a SQTA outcome equal to 0.00 Units ,g,1 protein (n=7) demonstrated therapeutic testicular biopsy material that was negative for spermatogenic cells. In five men with a SQTA outcome of 8.11,38.03 Units ,g,1, the most advanced germ cell was the spermatogonium/primary spermatocyte. In the remaining 12 men, the most advanced spermatogenic cell in the TTB was the spermatozoon. In these men, the SQTA outcome was equal to 25.76,92.68 Units ,g,1 protein. Using 39.00 Units ,g,1 protein as a cut-off value, the accuracy of the SQTA in identifying men positive for spermatozoa was 91.6%. It appears that the SQTA has a role for identifying non-mosaic KS men who have testicular spermatozoa. [source] The expression of glutathione reductase in the male reproductive system of rats supports the enzymatic basis of glutathione function in spermatogenesisFEBS JOURNAL, Issue 5 2002Tomoko Kaneko Glutathione reductase (GR) recycles oxidized glutathione (GSSG) by converting it to the reduced form (GSH) using an NADPH as the electron source. The function of GR in the male genital tract of the rat was examined by measuring its enzymatic activity and examining the gene expression and localization of the protein. Levels of GR activity, the protein, and the corresponding mRNA were the highest in epididymis among testes, vas deferens, seminal vesicle, and prostate gland. The localization of GR, as evidenced by immunohistochemical techniques, reveals that it exists at high levels in the epithelia of the genital tract. In testis, GR is mainly localized in Sertoli cells. The enzymatic activity and protein expression of GR in primary cultured testicular cells confirmed its predominant expression in Sertoli cells. Intracellular GSH levels, expressed as mol per mg protein, was higher in spermatogenic cells than in Sertoli cells. As a result of these findings, the effects of buthionine sulfoximine (BSO), an inhibitor for GSH synthesis, and 1,3-bis(2-chlorethyl)-1-nitrosourea (BCNU), an inhibitor for GR, on cultured testicular cells were examined. Sertoli cells were prone to die as the result of BCNU, but not BSO treatment, although intracellular levels of GSH declined more severely with BSO treatment. Spermatogenic cells were less sensitive to these agents than Sertoli cells, which indicates that the contribution of these enzymes is less significant in spermatogenic cells. The results herein suggest that the GR system in Sertoli cells is involved in the supplementation of GSH to spermatogenic cells in which high levels of cysteine are required for protamine synthesis. In turn, the genital tract, the epithelia of which are rich in GR, functions in an antioxidative manner to protect sulfhydryl groups and unsaturated fatty acids in spermatozoa from oxidation during the maturation process and storage. [source] Inducible 70 kDa heat shock protein does not protect spermatogenic cells from damage induced by cryptorchidismINTERNATIONAL JOURNAL OF ANDROLOGY, Issue 2 2007Wieslawa Widlak Abstract Accumulation of inducible heat shock proteins (e.g. Hsp70i) during cellular stress confers thermotolerance, reduces the consequences of damage and facilitates cellular recovery, while abrogation of Hsp70i expression renders sensitivity to apoptosis. Testis translocation into abdominal cavity, which results in temperature elevation, does not induce expression of the Hsp70i proteins. Despite constitutive expression of testis-specific Hsp70 proteins, spermatocytes are very sensitive to damage at elevated temperatures. To test whether Hsp70i protein could protect testes from heat-induced damage, we have engineered transgenic mice that over-express this protein selectively in spermatocytes and spermatids. We demonstrate that the testes of cryptorchid transgenic mice, like those of wild-type mice, exhibit reduced weight and smaller sizes of their seminiferous tubules, disorganization of their germinal epithelium structures, appearance of multinucleated giant cells, and reduced populations of germ cells. The data show that constitutive expression of Hsp70i does not protect the seminiferous epithelium against cryptorchidism-induced damage. [source] Effects of lithium carbonate on rat seminiferous tubules: an ultrastructural studyINTERNATIONAL JOURNAL OF ANDROLOGY, Issue 6 2006O. Zarnescu Summary Lithium salts are commonly used for treatment of bipolar disorder but prolonged treatment with therapeutic doses induces substantial toxic effects. In the present study we examined the effects of lithium carbonate on the ultrastructure of rat seminiferous tubules. Rats were exposed to lithium carbonate at doses of 35 mg/kg/day for 21 days. After lithium treatment, the tunica propria widened and folded together with convolutions of the basement membrane, myoid cells and lymphatic endothelium. In the seminiferous epithelium loss of germ cell attachment and appearance of expanded intercellular spaces between spermatogenic cells were observed. Early stages of spermatogenic cells showed nuclear protrusions or swellings because of an extensive enlargement of the outer nuclear membrane. Round spermatids exhibited abnormally shaped acrosomes and dilation of the subacrosomal space. Many abnormal, degenerated late spermatids with random orientation were seen towards the basal and adluminal compartments of the seminiferous epithelium. In addition, spermatids exhibited alteration in F-actin bundle ectoplasmic specialization and contained many mitochondria-associated granular bodies. [source] Methyl tert -butyl ether (MTBE)-induced cytotoxicity and oxidative stress in isolated rat spermatogenic cellsJOURNAL OF APPLIED TOXICOLOGY, Issue 1 2007Dongmei Li Abstract Methyl tert -butyl ether (MTBE) is a class of synthetic organic chemical. In the USA, MTBE pollution is regarded as a serious environmental problem. The objective of the present study was to investigate the cytotoxic effects and oxidative stress induced by MTBE in isolated rat spermatogenic cells. In cytotoxic experiments, spermatogenic cells isolated from the testes of adult Sprague-Dawley rats by a mechanical procedure without the use of trypsin were incubated with medium alone (control), 0.5, 5, 50 mm MTBE, respectively, for 6, 12 and 18 h. MTT assay, staining with fluorescein diacetate (FDA) and propidium iodide (PI) and flow cytometric analyses were used. In oxidative stress experiments, the spermatogenic cells were incubated with medium alone (control) and with 0.5, 50 ,m, 5 mm MTBE. For 1, 2, 6, 12, 18 h incubation, ROS production was tested using a 2,,7,-dichlorofluorescein diacetate (DCHF-DA) probe; for 1, 3, 6, 12, 18 h incubation, cytosolic superoxide dismutase (SOD) and extracellular SOD (SODEX) activity was assessed; and for 18 h incubation, lipid peroxidation was assessed. The results showed that MTBE at high doses significantly decreased the spermatogenic cell viability and increased plasma membrane damage and the ratio of necrotic cells compared with the control. Assessment of the MTBE-induced oxidative stress revealed that MTBE increased the production of reactive oxygen species (ROS) and enhanced lipid peroxidation. In addition, although SODEX activity increased at a high dose level, cytosolic SOD activity decreased. These results suggest that an increase of MTBE-induced ROS production and an enhancement of membrane lipid peroxidation may play an important role in its cytotoxicity in isolated rat spermatogenic cells. Copyright © 2006 John Wiley & Sons, Ltd. [source] Novel identification of peripheral dopaminergic D2 receptor in male germ cells,JOURNAL OF CELLULAR BIOCHEMISTRY, Issue 1 2007Carola Otth Abstract Dopamine is a recognized modulator in the central nervous system (CNS) and peripheral organ functions. The presence of peripheral dopamine receptors outside the CNS has suggested an intriguing interaction between the nervous system and other functional systems, such as the reproductive system. In the present study we analyzed the expression of D2R receptors in rat testis, rat spermatogenic cells and spermatozoa, in different mammals. The RT-PCR analysis of rat testis mRNA showed specific bands corresponding to the two dopamine receptor D2R (L and S) isoforms previously described in the brain. Using Western blot analysis, we confirmed that the protein is present in rat testis, isolated spermatogenic cells and also in spermatozoa of a range of different mammals, such as rat, mouse, bull, and human. The immunohistochemistry analysis of rat adult testis showed that the receptor was expressed in all germ cells (pre- and post-meiotic phase) of the tubule with staining predominant in spermatogonia. Confocal analysis by indirect immunofluorescence revealed that in non-capacitated spermatozoa of rat, mouse, bull, and human, D2R is mainly localized in the flagellum, and is also observed in the acrosomal region of the sperm head (except in human spermatozoa). Our findings demonstrate that the two D2 receptor isoforms are expressed in rat testis and that the receptor protein is present in different mammalian spermatozoa. The presence of D2R receptors in male germ cells implies new and unsuspected roles for dopamine signaling in testicular and sperm physiology. J. Cell. Biochem. 100: 141,150, 2007. © 2006 Wiley-Liss, Inc. [source] Aberrant distribution of junctional complex components in retinoic acid receptor alpha-deficient miceMICROSCOPY RESEARCH AND TECHNIQUE, Issue 6 2010Sanny S.W. Chung Abstract Retinoic acid receptor alpha (RAR,)-deficient mice are sterile, with abnormalities in the progression of spermatogenesis and spermiogenesis. In this study, we investigated whether defective retinoid signaling involved at least in part, disrupted cell,cell interactions. Hypertonic fixation approaches revealed defects in the integrity of the Sertoli-cell barrier in the tubules of RAR,-deficient testes. Dye transfer experiments further revealed that coupling between cells from the basal to adluminal compartments was aberrant. There were also differences in the expression of several known retinoic acid (RA)-responsive genes encoding structural components of tight junctions and gap junctions. Immunostaining demonstrated a delay in the incorporation of zonula occludens (ZO-1), a peripheral component protein of tight junctions, into the Sertoli cell tight junctions. Markedly reduced expression of connexin-40 in mutant pachytene spermatocytes and round spermatids was found by in situ hybridization. An ectopic distribution of vimentin and disrupted cyclic expression of vimentin, which is usually tightly regulated during spermiogenesis, was found in RAR,-deficient testes at all ages examined. Thus, the specific defects in spermiogenesis in RAR,-deficient testes may correlate with a disrupted cyclic expression of RA-responsive structural components, including vimentin, a downregulation of connexin-40 in spermatogenic cells, and delayed assembly of ZO-1 into Sertoli cell tight junctions. Interestingly, bioinformatic analysis revealed that many genes that are components of tight junctions and gap junctions contained potential retinoic acid response element binding sites. Microsc. Res. Tech., 2010. © 2009 Wiley-Liss, Inc. [source] Localizations of intracellular calcium and Ca2+ -ATPase in hamster spermatogenic cells and spermatozoaMICROSCOPY RESEARCH AND TECHNIQUE, Issue 8 2006H.L. Feng Abstract Calcium plays a predominant role regulating many functional processes of spermatogenesis and fertilization. The purpose of the present study is to define the exact location of calcium as well as examine the role it plays during spermatogenesis and sperm capacitation. Testes and epididymides were obtained from adult healthy male hamsters. Spermatozoa were incubated with modified Tyrode's medium up to 4 h at 37°C for sperm capacitation in vitro. Samples of the testes and sperm cells were analyzed by cytochemical techniques to determine the location of calcium and Ca2+ -ATPase and the percentage of acrosome reactions under light and electron microscopy. The data showed that (1) Sertoli cells exhibited numerous calcium precipitates as large, round, electron-dense bodies distributed throughout the cytoplasm and the mitochondrial matrix. Fine calcium precipitates existed in fewer numbers in the intracellular storage sites of spermatogonia and primary spermatocytes, in sharp distinction to secondary spermatocyte and spermatids, which showed an abundance of large and round calcium precipitates, especially in the mitochondrial matrix of spermatids. More calcium deposits were distributed in the plasma membrane (PM), acrosome membrane, and matrices of the acrosome and mitochondria following capacitation; (2) Ca2+ -ATPase was found in the endoplasmic reticulum system and PM of noncapacitated spermatozoa as well as Sertoli cells. Capacitated spermatozoa showed a weak signal. These results suggest that the presence of calcium in spermatogenic cells might play a role in cell growth and differentiation during spermatogenesis. The Ca2+ -ATPase function may be inhibited during capacitation, leading to an increase in acrosomal calcium level and triggering of acrosomal exocytosis. Microsc. Res. Tech., 2006. © 2006 Wiley-Liss, Inc. [source] Expression of zinc finger protein 105 in the testis and its role in male fertilityMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 6 2010Huaxin Zhou Using an in silico approach, we identified a putative zinc finger domain-containing transcription factor (zinc finger protein 105, ZFP105) enriched in the adult mouse testis. RT-PCR analyses showed that Zfp105 was indeed highly expressed in adult mouse testis and that its expression was regulated during postnatal development. To further characterize Zfp105 expression, we generated a Zfp105:,-galactosidase (LacZ) knock-in reporter mouse line (Zfp105LacZ/+) in which a Zfp105:LacZ fusion gene was expressed. Whole-mount LacZ analyses of adult Zfp105LacZ/+ tissues showed robust LacZ staining in the testis, very weak staining in the ovary, and no staining in the spleen, liver, kidney, heart, lung, thymus, adrenal gland, uterus, or oviduct. Sectional LacZ staining showed that ZFP105 was highly expressed in pachytene spermatocytes. ZNF35, the human ortholog of Zfp105, was also highly expressed in human testis. Immunofluorescence analysis showed that ZNF35 was located primarily in the cytoplasm of male germ cells. More importantly, reduced male fertility was observed in adult Zfp105LacZ/LacZ mice. Histological studies showed the presence of undifferentiated spermatogenic cells in the lumen of seminiferous tubules at stage VII and in the epididymal lumen of adult Zfp105LacZ/LacZ mice. Taken together, our results suggest that ZFP105 is a male germ-cell factor and plays a role in male reproduction. Mol. Reprod. Dev. 77: 511,520, 2010. © 2010 Wiley-Liss, Inc. [source] Transgenic sperm produced by electrotransfection and allogeneic transplantation of chicken fetal spermatogonial stem cellsMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 4 2010Fei Yu To study self-renewal, genetic modification, and differentiation of avian spermatogonial stem cells (SSCs), we isolated chicken SSCs from fetal testes on the 16th hatching day via enzyme digestion, and then cultured the SSCs over 2 months after purification in vitro. SSCs were identified by alkaline phosphatase staining and SSEA-1 fluorescence. The EGFP gene was transfected into SSCs by three different methods: electroporation, liposome transfer and calcium acid phosphate precipitation. The transfection rate and cell survival rate using electroporation were higher than when using liposomes or calcium acid phosphate (20.52% vs. 9.75% and 5.61%; 69.86% vs. 65.00% and 51.16%, respectively). After selection with G418 for 8 days, the transgenic SSCs were transplanted into the testes of cocks treated with busulfan. Twenty-five days after transplantation, the recipients' semen was light ivory in color, and the density of spermatozoa was 3.87 (×107/ml), with 4.25% expressing EGFP. By 85 days after transplantation, the number of spermatozoa increased to 32.7 (×107/ml) and the rate of EGFP expression was 16.25%. Frozen sections of the recipients' testes showed that transgenic SSCs were located on the basal membrane of the seminiferous tubules and differentiated into spermatogenic cells at different stages. The EGFP gene was successfully amplified from the DNA of all recipients' semen samples. Mol. Reprod. Dev. 77: 340,347, 2010. © 2010 Wiley-Liss, Inc. [source] Functional assessment of centrosomes of spermatozoa and spermatids microinjected into rabbit oocytes,MOLECULAR REPRODUCTION & DEVELOPMENT, Issue 3 2009Masahito Tachibana Abstract Although intracytoplasmic sperm injection (ICSI) is a widely used assisted reproductive technique, the fertilization rates and pregnancy rates of immature spermatids especially in round spermatid injection (ROSI) remain very low. During mammalian fertilization, the sperm typically introduces its own centrosome which then acts as a microtubule organizing center (MTOC) and is essential for the male and female genome union. In order to evaluate the function of immature germ cell centrosomes, we used the rabbit gamete model because rabbit fertilization follows paternal pattern of centrosome inheritance. First, rabbit spermatids and spermatozoa were injected into oocytes using a piezo-micromanipulator. Next, the centrosomal function to form a sperm aster was determined. Furthermore, two functional centrosome proteins (,-tubulin and centrin) of the rabbit spermatogenic cells were examined. Our results show that the oocyte activation rates by spermatozoa, elongated spermatids, and round spermatids were 86% (30/35), 30% (11/36), and 5% (1/22), respectively. Sperm aster formation rates after spermatozoa, elongated spermatids, and round spermatids injections were 47% (14/30), 27% (3/11), and 0% (0/1), respectively. The aster formation rate of the injected elongating/elongated spermatids was significantly lower than that of the mature spermatozoa (P,=,0.0242). Moreover, sperm asters were not observed in round spermatid injection even after artificial activation. These data suggest that poor centrosomal function, as measured by diminished aster formation rates, is related to the poor fertilization rates when immature spermatogenic cells are injected. Mol. Reprod. Dev. 76: 270,277, 2009. © 2008 Wiley-Liss, Inc. [source] Intratesticular localization of the organic solute carrier protein, OSCP1, in spermatogenic cells in miceMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 10 2008Kazuyuki Hiratsuka Abstract Organic solute carrier protein 1 (OSCP1) is a recently described human gene that facilitates the transport of various organic solutes into the cell, when expressed in frog eggs. In this study, we cloned a mouse ortholog of OSCP1 encoding 379 amino acid protein, with 94% homology to the human counterpart. The mouse OSCP1 mRNA was predominantly expressed in the testis, in which it was attributed to the spermatogenic cells, except the spermatogonia. Immunohistochemistry confirmed that OSCP1 protein is continuously expressed during spermatogenesis in a stage- and cell type-specific manner, in the leptotene spermatocytes at stage IX through step 15 spermatids. Subcellular fractionation of mouse testis homogenates indicated that OSCP1 is a 45-kDa cytosolic protein. Moreover, when green fluorescent protein-OSCP1 fusion constructs were transfected into cultured cells, the fluorescence localized evenly in the cytoplasm. These results suggest that mouse testis OSCP1 may indirectly mediate substrate uptake into meiotic and spermiogenic germ cells, within the cytosol. Mol. Reprod. Dev. 75: 1495,1504 © 2008 Wiley-Liss, Inc. [source] p19Ink4d and p18Ink4c cyclin-dependent kinase inhibitors in the male reproductive axisMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 8 2007Gregory M. Buchold Abstract The loss of the cyclin-dependent kinase inhibitors (CKIs) p18Ink4c and p19Ink4d leads to male reproductive defects (Franklin et al., 1998. Genes Dev 12: 2899,2911; Zindy et al., 2000. Mol Cell Biol 20: 372,378; Zindy et al., 2001. Mol Cell Biol 21: 3244,3255). In order to assess whether these inhibitors directly or indirectly affect male germ cell differentiation, we examined the expression of p18Ink4c and p19Ink4d in spermatogenic and supporting cells in the testis and in pituitary gonadotropes. Both p18Ink4c and p19Ink4d are most abundant in the testis after 18 days of age and are expressed in purified populations of spermatogenic and testicular somatic cells. Different p18Ink4c mRNAs are expressed in isolated spermatogenic and Leydig cells. Spermatogenic cells also express a novel p19Ink4d transcript that is distinct from the smaller transcript expressed in Sertoli cells, Leydig cells and in other tissues. Immunohistochemistry detected significant levels of p19Ink4d in preleptotene spermatocytes, pachytene spermatocytes, condensing spermatids, and Sertoli cells. Immunoprecipitation-Western analysis detected both CKI proteins in isolated pachytene spermatocytes and round spermatids. CDK4/6-CKI complexes were detected in germ cells by co-immunoprecipitation, although the composition differed by cell type. p19Ink4d was also identified in FSH+ gonadotrophs, suggesting that this CKI may be independently required in the pituitary. Possible cell autonomous and paracrine mechanisms for the spermatogenic defects in mice lacking p18Ink4c or p19Ink4d are supported by expression of these CKIs in spermatogenic cells and in somatic cells of the testis and pituitary. Mol. Reprod. Dev. 74: 997,1007, 2007. © 2007 Wiley-Liss, Inc. [source] Identification of a heat-shock protein Hsp40, DjB1, as an acrosome- and a tail-associated component in rodent spermatozoaMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 2 2007Masamichi Doiguchi Abstract Iba1 is a 17-kDa EF-hand protein highly expressed in the cytoplasm of elongating spermatids in testis. Using Iba1 as a bait, we performed yeast Two-hybrid screening and isolated a heat-shock protein Hsp40, DjB1, from cDNA library of mouse testis. To characterize DjB1 that is encoded by Dnajb1 gene, we carried out immunoblot analyses, in situ hybridization, and immunohistochemistry. Immunoblot analyses showed that DjB1was constitutively expressed in mouse testis and that its expression level was not changed by heat shock. Dnajb1 mRNA was exclusively expressed in spermatocytes and round spermatids in mouse testis, and Dnajb1 protein DjB1 was predominantly expressed in the cytoplasm of spermatocytes, round spermatids, and elongating spermatids. In mature mouse spermatozoa, DjB1 was localized in the middle and the end pieces of flagella as well as in association with the head (acrosomal region). Association of DjB1 with the acrosomal region in sperm head was also observed in rat spermatozoa. These data suggested that DjB1, which was constitutively expressed in postmeiotic spermatogenic cells in testis, was integrated into spermatozoa as at least two components, that is, sperm head and tail of rodent spermatozoa. Mol. Reprod. Dev. © 2006 Wiley-Liss, Inc. [source] Molecular cloning and characterization of SRG-L, a novel mouse gene developmentally expressed in spermatogenic cellsMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 9 2006Quanhong Ma Abstract Full-length cDNA of a novel mouse gene upregulated in late stages of spermatogenic cells was cloned from mouse testis using overlapping RT-PCR and RACE. The mRNA of the gene was expressed mainly in diplotene/pachytene spermatocytes, round and elongating spermatids. We named this gene as SRG-L (Spermatogenesis Related Gene expressed in late stages of spermatogenic cells, GenBank Accession No. AY352586). The tissue-specific analysis showed a higher expression level in testis and spleen. The gene is mapped on chromosome 8q33.1 and contains 18 exons. The full-length of cDNA is 2,843 bp with an open reading frame (ORF) of 2,625 bp that encodes a 104 kDa protein (874 amino acids) with a putative transmembrane region. The bioinformatics analysis revealed that the SRG-L has two conserved regions, transglutaminase-like homologues domain and D -serine dehydratase domain, rich phosphorylation sites and methylation sites. The SRG-L protein was detected in diplotene/pachytene spermatocytes and spermatids by immunohistochemical staining and Western blot. The results suggest that SRG-L may play definite roles regulating differentiation of germ cells during spermatogenesis, particularly during meiosis and spermiogenesis. Mol. Reprod. Dev. 1075,1083, 2006. © 2006 Wiley-Liss, Inc. [source] Cell-cycle regulation and mammalian gametogenesis: A lesson from the unexpectedMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 8 2006Abraham L. Kierszenbaum Abstract The progression of mammalian gametogenesis requires a precise balance between cell-cycle activities and elimination of defective gametogenic cells to ensure the perpetuation of species. Both spermatogonia and oogonia are stem cell populations committed to meiosis with the aim of generating haploid gametes for fertilization. At puberty, mitotically dividing spermatogonial cell cohorts maintain the ability of cell renewal and occupy niches in the seminiferous tubule. In contrast, mitotically dividing oogonial cell cohorts produced in the fetal ovary, are exclusively committed to meiosis and produce primordial follicles housing a primary oocyte surrounded by somatic follicular cells. A consistent physiological event during mammalian gametogenesis is the disposal of spermatogenic cells by apoptosis and ovarian follicles by atresia. Cyclin-dependent kinases (Cdks) and their cyclin partners coordinate the activities of the cell cycle. An additional cell-cycle regulatory component is the centrosome. The centrosome harbors regulatory proteins controlling the normal progression of the cell cycle. Changes in individual centrosome proteins can lead to cell-cycle arrest and a decrease in the genomic protective function of p53 that promotes apoptosis. Disruption of cyclin A1, Cdk2, and Cdk4 expression in transgenic mice results in infertility and gonadal atrophy. Cdk,cyclin complexes interact with regulatory proteins, which may fine-tune the activities of the complex. One of the many regulatory proteins is p12, a 115 amino acid growth suppressor polypeptide designated p12CDK2AP1, partner of Cdk2 and with binding affinity to DNA polymerase ,/primase. Overexpression of p12 is associated with testicular and ovarian atrophy without affecting fertility. Ectopic expression of p12 was driven by the keratin 14 promoter. Keratin 14 is the pairing partner of keratin 5 and both keratins are expressed in testis. The efficiency of keratin promoters in driving ectopic gonadal gene expression, the association of gonadal atrophy with the ectopic expression of a Cdk2 regulatory protein and the centrosome, as a reservoir of cell-cycle regulatory proteins, open new experimental opportunities to address still lingering questions concerning cell differentiation and division during mammalian gametogenesis. Mol. Reprod. Dev. 939,942, 2006. © 2006 Wiley-Liss, Inc. [source] Calpain 11 is unique to mouse spermatogenic cells,MOLECULAR REPRODUCTION & DEVELOPMENT, Issue 6 2006Irit Ben-Aharon Abstract The calpains are a family of calcium-dependent thiol proteases involved in intracellular processing of proteins. They occur as heterodimers containing one of various large subunits and a common small subunit. Some of the large subunits are expressed ubiquitously and others are expressed in a restricted set of tissues. We have cloned the cDNA for mouse calpain 11 and demonstrated that it is expressed specifically in the mouse testis. The mRNA begins to accumulate in the testis between days 14 and 16 after birth, corresponding to the period of pachytene spermatocyte development. The protein is detected by day 18 after birth, during mid to late pachytene spermatocyte development, and is present in the acrosomal region of spermatozoa from the cauda epididymis. The expression of calpain 11 during spermatogenesis and its localization in spermatozoa suggest that it is involved in regulating calcium-dependent signal transduction events during meiosis and sperm functional processes. Mol. Reprod. Dev. Published 2006 Wiley-Liss, Inc. [source] Stage-dependent Dishevelled-1 expression during mouse spermatogenesis suggests a role in regulating spermatid morphological changesMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 6 2006Pengpeng Ma Abstract Dishevelled (Dsh in Drosophila or DVL in mice) is a member of the highly conserved Wg/Wnt signaling pathway, which regulates important processes such as cell proliferation, polarity, and specification of cell fate. Three orthologous genes of Dishevelled (Dvl-1, Dvl-2, and Dvl-3) have been found in both humans and mice. They play pivotal roles in regulating cell morphology and a variety of changes in cell behaviors. In the present study, we show that the expression of Dvl-1 is stage-dependent during mouse spermatogenesis, although Dvl-2 and Dvl-3 show relative consistent expression. The expression of Dvl-1 mRNA first appears in pachytene spermatocytes, increases in round and elongating spermatids, and then turns to an undetectable level in mature sperm cells. Analyses of immunohistochemistry and immunofluorescence staining show that DVL-1 is present diffusely in the cytoplasm of pachytene spermatocytes and exhibits mainly a vesicular pattern and perinuclear distribution and a weak diffusely cytoplasmic signal in round and elongating spermatids. The vesicular pattern of DVL-1 has been observed by previous studies in somatic cells, and suggested to play roles in signal transduction. Immunoprecipitation experiments show that DVL-1 coimmunprecipitates with spermatogenic cells ,-actin rather than ,-tubulin. These results indicate that DVL-1 may be involved in spermatid morphological changes during mouse spermiogenesis through mediating signal transduction and/or regulating actin cytoskeleton organization. Mol. Reprod. Dev. © 2006 Wiley-Liss, Inc. [source] Tyrosine protein kinases and spermatogenesis: truncation mattersMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 4 2006Abraham L. Kierszenbaum Abstract Protein phosphorylation on serine/threonine or tyrosine residues represents a significant regulatory mechanism in signal transduction during spermatogenesis, oogenesis, and fertilization. There are several families of tyrosine protein kinases operating during spermatogenesis: the Src family of tyrosine protein kinases; the Fujinami poultry sarcoma/feline sarcoma (Fps/Fes) and Fes-related protein (Fer) subfamily of non-receptor proteins; and c-kit, the transmembrane tyrosine kinase receptor that belongs to the family of the PDGF receptor. A remarkable characteristic is the coexistence of full-length and truncated tyrosine kinases in testis. Most of the truncated forms are present during spermiogenesis. Examples include the truncated forms of Src tyrosine kinase hematopoietic cell kinase (Hck), FerT, and tr-kit. A feature of FerT and tr-kit is the kinase domain that ensures the functional properties of the truncated protein. FerT, a regulator of actin assembly/disassembly mediated by cortactin phosphorylation, is present in the acroplaxome, a cytoskeletal plate containing an F-actin network and linking the acrosome to the spermatid nuclear envelope. This finding suggests that Fer kinase represents one of the tyrosine protein kinases that may contribute to spermatid head shaping. The c-kit ligand, stem cell factor (SCF), which induces c-kit dimerization and autophosphorylation, exists as both membrane-associated and soluble. Although tyrosine protein kinases are prominent in spermatogenesis, a remarkable observation is the paucity of phenotypic alterations in spermatogenic cells in male mice targeted with Fer kinase-inactivating mutation. It is possible that the redundant functions of the tyrosine protein kinase pool present during spermatogenesis may explain the limited phenotypes of single mutant mice. The production of compound and viable mutant mice, lacking the expression of two or more tyrosine kinases, may shed light on this intriguing issue. Mol. Reprod. Dev. © 2006 Wiley-Liss, Inc. [source] Reciprocal regulation of the mouse protamine genes by the orphan nuclear receptor germ cell nuclear factor and CREM,MOLECULAR REPRODUCTION & DEVELOPMENT, Issue 4 2004Geoffrey C. Hummelke Abstract Germ cell nuclear factor (GCNF) is a member of the nuclear receptor superfamily, which is expressed in the adult predominantly in the male and female germ cells. In the male, GCNF is expressed in spermatogenic cells. GCNF binds as a homodimer to direct repeat response elements of the consensus half-site sequence, AGGTCA, with 0 bp spacing (DR0). Using this information, a search of genomic databases was performed to identify candidate GCNF responsive, spermatogenic-specific, genes that contain DR0 sequences. The mouse protamine genes are the strongest candidates identified to date, as they are post-meiotically expressed in round spermatids and contain DR0 elements in their proximal promoters. Previous work has shown that both recombinant and endogenous GCNF bind to DR0 elements in the mouse protamine 1 and 2 (Prm 1 and Prm 2) promoters with high affinity and specificity. The present work shows that in transient transfection assays in GC-1 and JEG-3 cells, co-transfection of a GCNF-VP16 expression plasmid with reporter plasmids containing either the wild type Prm 1 or Prm 2 promoter established that GCNF-VP16 is able to regulate transcription from both promoters in a DR0-dependent manner. Wild type GCNF, in contrast, acts as a repressor of basal transcription on both the Prm 1 and Prm 2 promoters in a DR0-dependent manner. Furthermore, CREM, activation of these promoters is also repressed by wild-type GCNF, indicating that GCNF also acts as a repressor of activated transcription. GCNF therefore defines a novel nuclear receptor-signaling pathway that may regulate a subset of genes involved in the terminal differentiation process of spermatogenesis, exemplified by the protamines. Mol. Reprod. Dev. 68: 394,407, 2004. © 2004 Wiley-Liss, Inc. [source] Di(n -butyl) Phthalate Induces Vimentin Filaments Disruption in Rat Sertoli Cells: A Possible Relation with Spermatogenic Cell ApoptosisANATOMIA, HISTOLOGIA, EMBRYOLOGIA, Issue 3 2010M. S. Alam With 3 figures Summary Phthalate esters have been extensively used as a plasticizer of synthetic polymers. Previous studies have revealed that some phthalate esters including di(n -butyl) phthalate (DBP) induce spermatogenic cell apoptosis, although its mechanism is not yet clear. The present study describes that disruption of Sertoli cell vimentin filaments by DBP administration may relate to spermatogenic cell apoptosis. The present histopathological study revealed that a single oral administration of 500 mg/kg DBP caused progressive detachment and displacement of spermatogenic cells away from the seminiferous epithelium and sloughing of them into the lumen. Degenerative spermatogenic cells characterized by chromatin condensation were frequently observed in DBP-treated rats. Ultrastructurally, the degenerative spermatogenic cells were separated from their neighbours, and a collapse of Sertoli cell vimentin filaments was recognized in DBP-treated rats. Sertoli cell cultures showed the increased number and size of vacuoles in their cytoplasm. In agreement with the in vivo experiment, vimentin filaments clearly showed a gradual collapse in DBP-exposed Sertoli cells in vitro. These in vivo and in vitro experiments indicate that DBP-induced collapse of Sertoli cell vimentin filaments may lead to detachment of spermatogenic cells, and then detached cells may undergo apoptosis because of loss of the support and nurture provided by Sertoli cells. [source] Distribution of Lectin-Bindings in the Testis of the Lesser Mouse Deer, Tragulus javanicusANATOMIA, HISTOLOGIA, EMBRYOLOGIA, Issue 3 2009S. Agungpriyono Summary The distribution of lectin bindings in the testis of the smallest ruminant, lesser mouse deer (Tragulus javanicus), was studied using 12 biotinylated lectins specific for d -galactose (peanut agglutinin PNA, Ricinus communis agglutinin RCA I), N -acetyl- d -galactosamine (Dolichos biflorus agglutinin DBA, Vicia villosa agglutinin VVA, Soybean agglutinin SBA), N -acetyl- d -glucosamine and sialic acid (wheat germ agglutinin WGA, s-WGA), d -mannose and d -glucose (Lens culinaris agglutinin LCA, Pisum sativum agglutinin PSA, Concanavalin A Con A), l -fucose (Ulex europaeus agglutinin UEA I), and oligosaccharide (Phaseolus vulgaris agglutinin PHA-E) sugar residues. In Golgi-, cap-, and acrosome-phase spermatids, lectin-bindings were found in the acrosome (PNA, RCA I, VVA, SBA, WGA and s-WGA), and in the cytoplasm (PNA, RCA I, VVA, SBA, WGA, LCA, PSA, Con A and PHA-E). s-WGA binding was confined to the spermatid acrosome, but other lectins were also observed in spermatocytes. In spermatogonia, VVA, WGA, Con A, and PHA-E bindings were observed. Sertoli cells were intensely stained with DBA and Con A, and weakly with PHA-E. In interstitial Leydig cells, RCA I, DBA, VVA, Con A, PSA, LCA, WGA and PHA-E were positive. UEA I was negative in all cell types including spermatogenic cells. Unusual distribution of lectin-bindings noted in the testis of lesser mouse deer included the limited distribution of s-WGA only in the spermatid acrosome, the distribution of DBA in Sertoli cells, Leydig cells and lamina propria, and the absence of UEA I in all type cells. The present results were discussed in comparison with those of other animals and their possible functional implications. [source] Quercetin protects hamster spermatogenic cells from oxidative damage induced by diethylstilboestrolANDROLOGIA, Issue 5 2010G. Li Summary Quercetin has been reported to be an efficient antioxidant which protects chicken spermatogonial cells from oxidative damage through increasing intracellular antioxidants and decreasing lipid peroxidation. Exposure to diethylstilboestrol (DES) could cause reproductive damage in males, which is associated with oxidative stress. This study was conducted to investigate the protective effects of quercetin on DES-induced oxidative damage in cultured hamster spermatogenic cells. The cells were treated with different concentrations of DES, and their growth status was observed under inverted microscope. The viability of spermatogenic cells was detected by 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT). The contents of superoxide dismutase (SOD) in supernatants and glutathione peroxidase (GSH-Px) in cells were detected with spectrophotography. The results showed that quercetin significantly inhibited the DES-induced damage on spermatogenic cells, with the exception of the low-dose group in which no significant difference was observed. The cell survival rate increased significantly in the middle- and high-dose groups. The contents of SOD and GSH-Px were significantly elevated after medication with quercetin (P < 0.01). It can be concluded that quercetin protects spermatogenic cells against DES-induced oxidative damage through increasing intracellular antioxidants and decreasing lipid peroxidation. Quercetin plays a very important role in ameliorating reproductive toxicity induced by environmental oestrogens. [source] Immunohistochemical distribution of S-100 protein and subunits (S100-, and S100-,) in the swamp-type water buffalo (Bubalus bubalis) testisANDROLOGIA, Issue 3 2003M. B. C. Cruzana Summary. The distribution and localization of S-100 protein (S-100) and its subunits (S100-, and S100-,) in the testis of swamp-type water buffalo were investigated using immunohistochemistry. S-100 was detected in the Sertoli cells in the convoluted seminiferous tubules, modified Sertoli cells lining the terminal segment of the seminiferous tubules and in the intratesticular excurrent ducts (straight tubules and rete testis). S100-, showed the same distribution and localization with that of S-100. However, the cytoplasmic extension of the Sertoli cells in S100-, staining showed less staining intensity compared with that of S-100. S100-, showed a positive staining only in the modified Sertoli cells of the terminal segment of the seminiferous tubule. Endothelial cells of blood vessels were also positive with the proteins while the Leydig and spermatogenic cells showed a negative reaction. The localization of S-100 in the testis of the water buffalo was in parallel with that of other artiodactyls which supports the hypothesis that this protein is a multifunctional protein. S100-, in the Sertoli cells suggests that this protein is involved in establishing blood,testis barrier. Its presence in the modified Sertoli cells and in the epithelium of the excurrent ducts suggest secretory and absorptive function, respectively. Meanwhile, S100-,, which was detected only in the modified Sertoli cells, is involved in the secretory activity of these cells that are related to exocrine function. [source] Use of a highly sensitive quantitative telomerase assay in intracytoplasmic sperm injection programmes for the treatment of 47,XXY non-mosaic Klinefelter men,ANDROLOGIA, Issue 4 2002Y. Yamamoto Summary. We evaluated the role of the sensitive quantitative telomerase assay (SQTA) in the management of men with non-mosaic Klinefelter's syndrome (KS). Diagnostic testicular biopsy (DTB) was performed in 24 men with KS. A part of the DTB was stained and the remaining fragment was processed for the SQTA. After 3,18 months, a therapeutic testicular biopsy (TTB) was performed in the same testicle and the recovered specimens were processed to identify spermatozoa. Men with a SQTA outcome equal to 0.00 Units ,g,1 protein (n=7) demonstrated therapeutic testicular biopsy material that was negative for spermatogenic cells. In five men with a SQTA outcome of 8.11,38.03 Units ,g,1, the most advanced germ cell was the spermatogonium/primary spermatocyte. In the remaining 12 men, the most advanced spermatogenic cell in the TTB was the spermatozoon. In these men, the SQTA outcome was equal to 25.76,92.68 Units ,g,1 protein. Using 39.00 Units ,g,1 protein as a cut-off value, the accuracy of the SQTA in identifying men positive for spermatozoa was 91.6%. It appears that the SQTA has a role for identifying non-mosaic KS men who have testicular spermatozoa. [source] Effects of 60 Hz 14 µT magnetic field on the apoptosis of testicular germ cell in miceBIOELECTROMAGNETICS, Issue 1 2009Yoon-Won Kim Abstract We recently reported that continuous exposure, for 8 weeks, of extremely low frequency (ELF) magnetic field (MF) of 0.1 or 0.5 mT might induce testicular germ cell apoptosis in BALB/c mice. In that report, the ELF MF exposure did not significantly affect the body weight or testicular weight, but significantly increased the incidence of testicular germ cell death. In the present study, we aimed to further characterize the effect of a 16-week continuous exposure to ELF MF of 14 or 200 µT on testicular germ cell apoptosis in mice. There were no significant effects of MF on body weight and testosterone levels in mice. In TUNEL staining (In situ terminal deoxynucleotidyl transferase-mediated deoxy-UTP nick end labeling), germ cells showed a significantly higher apoptotic rate in exposed mice than in sham controls (P,<,0.001). TUNEL-positive cells were mainly spermatogonia. In an electron microscopic study, degenerating spermatogonia showed condensation of nuclear chromatin similar to apoptosis. These results indicate that apoptosis may be induced in spermatogenic cells in mice by continuous exposure to 60 Hz MF of 14 µT. Bioelectromagnetics 30:66,72, 2009. © 2008 Wiley-Liss, Inc. [source] | ||||||||||||||||