Browsing by Subject "Testis"
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Item Age-dependent alterations in spermatogenesis in itchy mice(2012-12) Dwyer, Jessica Leigh; Richburg, John H.; Mills, Edward; DiGiovanni, John; Huibregtse, Jon; Sanders, BobSpermatogenesis is an intricate process that strongly depends on the rapid turnover of short-lived proteins, both in the differentiating germ cells and in the supportive Sertoli cells. Recent evidence has demonstrated the importance of the ubiquitin-proteasome system for this turnover, with the final enzymatic E3 ligase providing the target specificity. One E3 ligase, Itch, has been well characterized in the immune system, but its role during spermatogenesis is not yet well understood. Mice lacking functional Itch protein display a late onset autoimmune disease characterized by severe inflammation, infiltration of immune cells into various organs, and most apparently chronic dermatitis, ultimately dying from pulmonary inflammation at 6 to 9 months of age. The work presented here evaluates the testes of itchy mice at two developmental time points, during the peri-pubertal period at postnatal day (PND) 28 and at adulthood, PND 56. Itchy mice are smaller in size and have lower spermatid head counts, most likely resulting from an increase in germ cell apoptosis rather than a decrease in Sertoli cell number. Litter sizes are reduced in the homozygous itchy colonies, with data suggesting a defect during fetal development and not in gamete production, although survival rates tend to be similar to that of wild type. At PND 28, itchy mice show a delay in spermatogenesis and an increase in meiotic figures, while PND 56 mice show alterations in germ cell layers, spermatid head formation, and irregular cell division. Examination of the previously identified targets of Itch revealed no significant increases in the testis, but led to discovery of immunoglobulin (IgG) deposits within the interstitial space. Changes in protein expression outside of the seminiferous epithelium suggest that cells of the immune system may be influencing proper development and functional spermatogenesis in the testis. While the previous studies using the itchy mice focused primarily on the late onset autoimmune dysfunction in these animals, increased spleen weights and changes in testicular protein are observed as early as PND 28, indicating that the loss of Itch impacts these animals much earlier during development. Taken together, these data indicate that Itch is required for functional spermatogenesis and that it may play different cellular roles depending on the developmental age of the animal. Future work is targeted at identifying the possible testis-specific targets of Itch and deciphering whether the observed phenotypes are the result of the primary loss of Itch or are a secondary effect from the overactive immune system.Item Altered spermatogenesis of death ligand gene deficient mice and the influence of phthalates in germ cell apoptosis and enhanced testicular cancer progression(2012-05) Lin, Yichen; Richburg, John H.; Bratton, Shawn B.; Mills, Edward M.; Sanders, Bob G.; Wright, Casey W.Testicular germ cell apoptosis is a process that begins in early development and continues in the adult testis. It is important during spermatogenesis for maintaining homeostasis of different types of germ cells. The number of sperm produced depends on the supportive capacity of surrounding Sertoli cells, which provide nutrition and an adaptive environment for growth and development of the germ cells. There are two major pathways that regulate germ cell apoptosis: extrinsic and intrinsic. We hypothesize that Sertoli cells use the extrinsic pathway to eliminate germ cells when exposed to phthalates, a common Sertoli cell toxicant. Death ligands, which are involved in the extrinsic pathway, were used in this research to test this hypothesis. Here, we demonstrate that: 1) the loss of FasL and TRAIL protein expression results in decreased production of mature spermatids in the adult testis, likely as a result of alterations in germ cell homeostatsis during the first wave of spermatogenesis. 2) The high baseline incidence of germ cell apoptosis in peripubertal FasL-/- and TRAIL-/- mice is correlated with increases in levels of TRAIL and FasL, respectively. 3) The decline in germ cell apoptosis observed after MEHP treatment in FasL-/- mice closely corresponds to the occurrence of increased levels of c-FLIP. 4) A more predominant role of FasL occurs in controlling the proper number of germ cells during the first wave of spermatogenesis in peri-pubertal mice. TRAIL is more critical for maintaining long-term homeostasis of the germ cell population in adult testis as well as in the reproductive function. 5) Several possible genes are involved in the altered spermatogenesis and development in the testis of gene-deficient mice. 6) Findings described in Chapter 6 indicate cellular mechanisms triggered by MEHP exposure that act to enhance tumor progression/metastasis in testicular embryonal carcinoma cells (NT2/D1). Taken together, these novel findings provide important mechanistic insights into the functional roles of FasL in the testis at distinct developmental periods and further indicate that FasL itself is required for the regulation of c-FLIP levels in the testis. Additionally, exposure to environmental toxicants, such as the phthalates, can enhance testicular cancer metastasis and invasion.Item An electron microscopic cytochemical study of the effects of testosterone propionate on human testes(Texas Tech University, 1972-05) Barham, Steven SNot availableItem An electron microscopic cytochemical study of the effects of x-irradiation on inosine diphosphatase and thiamine pyrophosphatase in the human testis(Texas Tech University, 1971-08) Brackeen, Randolph B.Not availableItem An electron microsopic, cytochemical study of the effects of x-irradiation on mammalian testes(Texas Tech University, 1970-12) Vollet, John JNot availableItem Characterization and function of cks2-a cyclin dependent kinase regulatory subunit-during spermatogenesis(2005-05) Attaya, Ebtesam Nabil; MacDonald, Clinton C.; Williams, Simon C.; Hutson, James C.; Schnieder, Brandt; Ravnik, StuartCell division is controlled by cyclin dependent kinases (CDKs) that are activated to phosphorylate various downstream targets. My goal was to study the CDKs and interacting proteins that control meiosis. CDK2beta, the alternatively spliced isoform of cyclin dependent kinase 2 (CDK2), may be a key meiotic regulator due to its expression at prophase of meiosis I in spermatogenesis. Therefore, we used the yeast two-hybrid system to identify binding partners of CDK2beta. The mouse homolog of CKS2 (CDC28 Kinase Subunit 2) was identified as a CDK2beta interactor after screening an adult mouse testis cDNA library. CKS2 was confirmed to bind to CDK2beta. Cks2 was more abundant in day 17 after birth and adult mice, and was only weakly detected in mutants that lack germ cells. Further, Cks2 mRNA levels were significantly higher in pachytene spermatocytes than early and late spermatids, while Cks1 was nearly undetectable. CKS2 protein was detected with both CDK2beta and cyclin A1 in germ cells during the first meiotic division. The Cks2 gene has three exons. Cks2-/- mice were generated by another group by deletion of exon 1. Cks2-/- mice are sterile due to a block in spermatogenesis which arrests at metaphase I of meiosis. I show here that the major biochemical effect in the absence of Cks2 was an increase of CCNA1 and CDK2-associated kinase activity. Surprisingly, reverse transcription-PCR analysis showed that a mutant form of Cks2 was being transcribed in testes of these mice. We also found that Cks2-/- mice produced a protein that was immunoreactive with the CKS antibody, suggesting that a translatable mRNA was present in testes of Cks2-/- mice. Cloning of the mutant Cks2 cDNA indicated that it consisted of a portion of the Cks2 coding sequence (exons 2 and 3) fused to portions of introns 1 from the Cks2 gene and the plasmid used to generate the Cks2-/- ES cells. We postulate that this cDNA is derived from a mRNA transcribed from a cryptic promoter active in the testes of Cks2-/- mice. Interestingly, the aberrant protein did not possess all of the function attributed to the wild type protein, as Cks2-/- mice were sterile. However, these data indicate that the interpretation that this phenotype is due to complete loss of Cks2 must be interpreted with care. While not definitive, these data suggest that a truncated form of Cks2 was being expressed in testes of these mice, and suggest a more complicated explanation for the observed infertility phenotype. These data indicate that CKS2 appears to act as a CDK2beta/CCNA1 repressor and that Cks2-/- mice generate a Cks2 mutant that is being transcribed and possibly translated in the testis.Item Characterization and function of cks2-a cyclin dependent kinase regulatory subunit-during spermatogenesis(Texas Tech University, 2005-05) Attaya, Ebtesam Nabil; MacDonald, Clinton C.; Williams, Simon C.; Hutson, James C.; Schnieder, Brandt; Ravnik, StuartCell division is controlled by cyclin dependent kinases (CDKs) that are activated to phosphorylate various downstream targets. My goal was to study the CDKs and interacting proteins that control meiosis. CDK2beta, the alternatively spliced isoform of cyclin dependent kinase 2 (CDK2), may be a key meiotic regulator due to its expression at prophase of meiosis I in spermatogenesis. Therefore, we used the yeast two-hybrid system to identify binding partners of CDK2beta. The mouse homolog of CKS2 (CDC28 Kinase Subunit 2) was identified as a CDK2beta interactor after screening an adult mouse testis cDNA library. CKS2 was confirmed to bind to CDK2beta. Cks2 was more abundant in day 17 after birth and adult mice, and was only weakly detected in mutants that lack germ cells. Further, Cks2 mRNA levels were significantly higher in pachytene spermatocytes than early and late spermatids, while Cks1 was nearly undetectable. CKS2 protein was detected with both CDK2beta and cyclin A1 in germ cells during the first meiotic division. The Cks2 gene has three exons. Cks2-/- mice were generated by another group by deletion of exon 1. Cks2-/- mice are sterile due to a block in spermatogenesis which arrests at metaphase I of meiosis. I show here that the major biochemical effect in the absence of Cks2 was an increase of CCNA1 and CDK2-associated kinase activity. Surprisingly, reverse transcription-PCR analysis showed that a mutant form of Cks2 was being transcribed in testes of these mice. We also found that Cks2-/- mice produced a protein that was immunoreactive with the CKS antibody, suggesting that a translatable mRNA was present in testes of Cks2-/- mice. Cloning of the mutant Cks2 cDNA indicated that it consisted of a portion of the Cks2 coding sequence (exons 2 and 3) fused to portions of introns 1 from the Cks2 gene and the plasmid used to generate the Cks2-/- ES cells. We postulate that this cDNA is derived from a mRNA transcribed from a cryptic promoter active in the testes of Cks2-/- mice. Interestingly, the aberrant protein did not possess all of the function attributed to the wild type protein, as Cks2-/- mice were sterile. However, these data indicate that the interpretation that this phenotype is due to complete loss of Cks2 must be interpreted with care. While not definitive, these data suggest that a truncated form of Cks2 was being expressed in testes of these mice, and suggest a more complicated explanation for the observed infertility phenotype. These data indicate that CKS2 appears to act as a CDK2beta/CCNA1 repressor and that Cks2-/- mice generate a Cks2 mutant that is being transcribed and possibly translated in the testis.Item Function and regulation of CCAAT/enhancer binding protein beta in Leydig cell development and steroidogenesis(Texas Tech University, 2002-08) Nalbant, DemetPituitary luteinizing hormone (LH) is required for the development of multiple cell types in both the male and female reproductive systems. In particular, LH promotes differentiation of testicular Leydig cells and stimulates steroid production by Leydig cells in the testis, theca, granulosa and luteal cells in the ovary. We have been interested in identifying nuclear transcription factors that are targets of LH signaling pathways in Leydig cells and concentrated on CCAAT/enhancer binding protein beta (C/EBPp). Our initial studies showed that C/EBPp is expressed in a differentiation specific pattern whose expression is stimulated by LH/human chorionic gonadotropin (hCG) and cyclic AMP (cAMP) in Leydig cells. We hypothesized that C/EBPp plays an important role in LH regulated Leydig cell development and steroidogenic function. To assess the specific roles of C/EBPp in Leydig cell function we have analyzed the steroidogenic capacity of Leydig cells from C/EBPp-deficient mice generated by gene targeting. This study revealed that testosterone production in male C/EBPp deficient mice is severally compromised suggesting that C/EBPp is essential for complete functional differentiation of Leydig cells. In order to understand how LH effects on Leydig cell differentiation and/or function may be mediated through C/EBPp, we attempted to identify genetic control elements that control C/EBPp transcription in steroidogenic and non-steroidogenic cells. We identified an evolutionarily conserved, steroidogenic cell-specific, distal enhancer element located in the C/EBPp 5'-flanking region. Our studies suggest that the activity of the enhancer may be, at least in part, controlled by as yet uncharacterized nuclear factors specifically detected in nuclear extracts of steroidogenic cells. These studies set the stage for elucidation of the molecular mechanisms controlling C/EBPp expression in steroidogenic cells in general, and may aid in uncovering alternative LH-dependent signaling pathways critical for functional maturation of Leydig cellsItem Hypothyroidism induced with iopanoic acid and propylthiouracil fails to disrupt testicular function as determined by serum testosterone, spermatogenesis, and seminiferous tubule morphology(Texas Tech University, 1987-05) Weiss, Stephen RobertThe role of the thyroid hormones, triiodothyronine and thyroxine, in the hormonal control of mammalian testicular function has not been clearly delineated. Previous data regarding the effects of hypothyroidism and hyperthyroidism on testicular function, specifically spermatogenesis, has provided only conflicting results; and it is equivocal whether hypothyroidism can effectively disrupt qualitative and quantitative spermatogenesis. To examine the effect of acute hypothyroidism on spermatogenesis, adult male rats were injected with either iopanoic acid (lOP) or propylthiouracil (PTU), two goitrogens with specific mechanisms of action, for four weeks. Controls were injected with propylene glycol. Weekly blood samples were analyzed for triiodothyronine, thyroxine and testosterone. At the end of the experimental period (5 weeks), testicular tissue was excised and seminiferous tubules were prepared for histological analysis. Serum assays of the thyroid hormones revealed that the treated animals had become hypothyroid. Analysis of serum testosterone concentrations revealed no differences among the groups. Light microscopy revealed no morphological changes of the seminiferous tubules or disruption of spermatogenesis in the lOP and PTU treated animals based on germ cell populations. These data suggest that acute hypothyroidism does not interfere with normal testicular function in the rat as determined by normal testosterone levels, tissue morphology and unchanged germ cell populations in the treated animals when compared to the controls.Item Immune-privileged Sertoli cells survive allotransplantation by inhibiting adaptive immune response(2012-12) Kaur, Gurvinder; Dufour, Jannette M.; Webster, Daniel R.; Huston, James C.; Bright, Robert K.Immune privileged Sertoli cells (SC) survive long-term and protect co-grafted cells when transplanted as allo- or xeno-grafts. However, their survival/protection mechanism remains unclear. The objective of this study was to investigate the immune privilege mechanism of SC. MSC-1 cells (a mouse Sertoli cell line), which lack some of the immunoprotective abilities associated with SC, as they are unable to survive in naïve immune competent animals, served as controls. SC and MSC-1 cells were transplanted as allografts into naïve BALB/c mice. SC survived throughout the study, whereas, very few MSC-1 cells were detected by day 11 and MSC-1 cell grafts were completely rejected within 20 days. To identify genes that are differentially regulated between SC and MSC-1 cells, microarray analysis was performed. 2369 genes with a ± 4-fold or higher level in SC compared to MSC-1 cells were obtained. Interestingly, genes involved in inhibiting adaptive immune response were upregulated in SC. Therefore, we hypothesized that SC survive as allografts by inhibiting humoral and/or cellular immune response. Analysis of SC or MSC-1 cell grafts and serum from transplanted animals for activation of humoral immune response revealed that antibody mediated cell death was not observed in either sets of grafts. Cell-mediated death (apoptosis) measured by TUNEL assay showed a significant increase in apoptotic cells at days 2, 5 and 8 in MSC-1 cell grafts, while very few apoptotic cells were observed in SC grafts. Immune cell infiltrate was examined and macrophages and CD4 T cells were detected in both sets of grafts. In contrast, SC grafts contained CD8 T cells, while little to no CD8 T cells were detected in MSC-1 cell grafts. Furthermore, early anti-inflammatory environment and T regulatory cells (CD4+Foxp3+ and CD8+Foxp3+ Tregs), associated with graft tolerance, were detected in SC grafts. MSC-1 cell grafts contained pro-inflammatory milieu and Tregs were either absent or very few were detected in rejected MSC-1 cell grafts. Overall, this led to the conclusion that SC have created a tolerogenic environment at the graft site by inhibiting humoral immune response, apoptosis and increasing the number of Tregs, which could be responsible for their long-term survival.Item Influence of vitamin E hypovitaminosis and E hypervitaminosis on 3 beta-hydroxysteroid dehydrogenase activity in mouse testes(Texas Tech University, 1972-12) Eastland, Kathryn SueNot availableItem Leydig cell development in boars(Texas Tech University, 1997-12) Geiger, Tammy LynnThree waves of Leydig cell development are found in the pig testes Clearly defined periods of cell proliferation and differentiation occur during fetal, perinatal and pre-pubertal periods. Luteinizing hormone (LH) regulates Leydig cell proliferation via growth factors in the rat. However, it is not known how LH affects Leydig cell development in the neonatal pig testis. The purpose of the study was to identify growth factors involved in Leydig cell proliferation in neonatal pigs In this study we investigated the effects of hCG and the following growth factors; EGF (TGFa), SEP, IL-lp, IGF-1 and TGFP in vitro on DNA synthesis in neonatal Leydig cells. Leydig cells were isolated from 3- to 5-day-old pigs using coUagenase dispersion and a Percoll® gradient centrifligation. The cells were then cultured for 48 hours in serum free media, washed and treated with growth factors and or hCG for 18 hours. Tritiated thymidine incorporation into DNA was assessed over a subsequent four hour incubation period. Incorporation of ['^Hj-thymidine was stimulated by hCG treatment with a 2.3-fold increase over control cultures. SIP also induced a significant increase ( p <.0001) in the incorporation of ["'HJ-thymidine into Leydig cell DNA. Similarly, EGF and IGF-1 also increased DNA synthesis in porcine Leydig cells. When IGF-1 and SIP were added together, they had an additive effect on Leydig cell DNA synthesis. TGFp had very little, if any, etTect on DN.A synthesis when added alone, but inhibited the stimulatory effects of other growth tactor and mitogens (SIP, hCG, EGF, and IGF-1) on DN.A. synthesis Our results indicate thai these growth factors may play a role in proliferation of Leydig cells during the perinatal period of development.Item Regulation of steriodogenesis in rodent leydig cells by corticotropin-releasing hormone (CRH)(Texas Tech University, 1995-08) Huang, Bu-Miin; Norman, Reid L.; Stocco, Douglas M.; Beale, Elmus G.; Droms, Kurt A.; Hutson, James C.; Weitlauf, Harry M.Not availableItem Testicular macrophages: isolation, characterization, and hormonal responsiveness(Texas Tech University, 1984-08) Yee, James BNot available