Browsing by Subject "Spermatogenesis"
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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 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 Effects of Strenuous Exercise on Stallion Sperm Quality(2012-10-19) Rosenberg, Jennifer L.Some stallions are expected to perform athletically and breed contemporarily. Athletic activity has the potential, especially during the summer months, to induce thermal stress to the testes, resulting in reduced reproductive capability due to decreased sperm quality and libido. There is concern in the horse industry about what level of exercise, if any, affects the reproductive capability of a stallion. Thermal stress associated with training and exercise may impact sperm quality and the future reproductive capability of the stallion. The goal of this study was to determine the effect of strenuous exercise on stallion sperm quality. The objectives were to measure changes in body and scrotal temperatures following strenuous exercise and sperm quality following strenuous exercise. Miniature Horse stallions (n = 7), implanted with subdermal thermosensory devices in the subcutaneous neck and scrotal tissue, were assigned to treatment group based on age and semen quality. Exercising stallions (EX; n = 3) were exercised 4 d/wk for 90 min for 12 wk, while non-exercising stallions (CN) were tied in the shade. Semen was collected from stallions for 5 consecutive days every 4 wk to evaluate semen quality (raw, 24 h and 48 h cooled). Subcutaneous scrotal (SQST), rectal (RT) and neck (NT) temperatures were recorded along with heart rate. Spermatozoa data were normally distributed; therefore, they were subjected to parametric analysis by repeated measures (wk) using the PROC MIXED procedure (SAS v 9.1; SAS Inst. Inc., Cary, NC). Model included treatment (CN or EX), time (wk 0, 4, 8, or 12), and stallion as the subject of the repeated measures. Compared to the CN group, EX stallions had elevated temperatures (avg RT 39.27 vs 37.07 degrees, NT 39.77 vs 37.44 degrees C, and SQST 34.90 vs 33.40 degrees C; P < 0.0001). There was no difference in sperm quality between treatment groups (P > 0.05). In this study, strenuous exercise in Miniature Horse stallions, did not affect sperm quality. This suggests that anecdotal reports of reduced sperm quality in stallions in training may have other causes other than elevated scrotal and body temperature. While previous studies have illustrated that prolonged insulation of the testes reduces semen quality, strenuously exercising stallions for up to 90 min under hot and humid ambient conditions may not be harmful to spermatogenesis.Item Foxo Transcription Factors Control Spermatogonial Stem Cell Self-Renewal And The Initiation Of Spermatogenesis(2013-01-17) Goertz, Meredith Johanna; Castrillon, Diego H., M.D., Ph.D.Spermatogonial stem cells (SSCs), as the foundation for spermatogenesis, must maintain a balance of both self-renewal and differentiation. Although several factors important for these processes have been identified, the fundamental mechanisms regulating SSC self-renewal and differentiation remain essentially unknown. The work presented here describes the discovery of a role for the Foxo forkhead transcription factors in mouse spermatogenesis and SSCs. Foxo1 was found to specifically mark mouse gonocytes, and its cytoplasmic-to-nuclear translocation delineated the gonocyte-to-SSC transition in neonatal testes. In adults, Foxo1 is specifically expressed within a subset of undifferentiated spermatogonia with stem cell potential. Genetic analyses showed that Foxo1 was required for both SSC maintenance as well as the initiation of spermatogenesis, with limited contributions from Foxo3 and Foxo4. Conditional inactivation of PI3K/Akt pathway components in the male germ line confirmed that PI3K signaling regulates Foxo1 stability and subcellular localization, revealing that the Foxos are crucial effectors of PI3K/Akt signaling in SSCs. The nuclear localization of Foxo1, indicating functional activation, was found to correlate with Gfralpha1 expression and thus stem cell potential. Subsequent gene expression analyses identified a complex network of Foxo gene targets that rationalized both the maintenance of SSCs and initiation of differentiation by the Foxos. Taken together, these findings demonstrate that the Foxos, particularly Foxo1, are essential in maintaining the spermatogonial stem cell population, and regulation of the Foxos through the PI3K/Akt signaling pathway is a critical process underlying SSC self-renewal versus differentiation.Item Germ cell apoptosis and death receptor response in the rodent testis after acute mono-(2-ethylhexyl) phthalate and cisplatin exposure(2002-12) Giammona, Charles John; Richburg, John H.Item Human spermatogenesis: a study of the spermatogonial population and ultrastructural aspects of spermatozoal development(Texas Tech University, 1976-12) Smith, Fannie ENot availableItem Identification and characterization of cystatin-related epididymal and spermatogenic (CRES) expression in anterior pituitary gonadotropes(Texas Tech University, 2001-08) Sutton-Walsh, Heather GayleMale factor infertility contributes to half of all infertility cases, yet current knowledge remains unable to diagnose all but gross abnormalities in male reproductive physiology. In an effort to identify proteins involved in initiating epididymal sperm maturation, our lab identified the cystatin-related epididymal spermatogenic (CRES) gene based on its preferential expression in the initial epididymal segment. Cres represents a unique subgroup of the cystatin family of cysteine protease inhibitors in that its expression is restricted to reproductive tissues and its sequence predicts specificity distinct from classic cystatins. CRES protein is localized to the caput epididymal lumen and the sperm acrosome. Both compartments contain active proteases responsible for protein processing events that are required for sperm function; however, the regulatory mechanisms controlling these processing events are poorly understood. These observations led to the proposal that CRES protein regulates protein processing in these tissues. We hypothesized that CRES might also regulate proteolysis in the pituitary gland for two reasons: (1) the pituitary gland expresses a number of proteases involved in preprotein processing; and (2) the pituitary gland is functionally connected with the gonad via the hypothalamic-pituitary-gonadal axis. Thus, the goal of these studies was to determine whether CRES is expressed in the pituitary gland and if so, to examine its regulation by hypothalamic and/or gonadal factors. These studies demonstrate that CRES is expressed in the anterior pituitary gland and is co-localized intracellularly with leutinizing hormone (LH) in gonadotrope secretory granules. To examine the regulation of Cres mRNA, we developed a semi-quantitative RT-PCR assay and showed that, unlike steroid hormones, GnRH is the primary physiologic regulator of Cres gene expression. Specifically, GnRH administration causes a rapid decrease in Cres mRNA levels, an effect opposite from its potent stimulation of LHp mRNA expression. In contrast, intracellular CRES and LHP protein levels were regulated similarly by steroid hormones, consistent with their being packaged within the same subset of secretory granules. Taken together, these studies indicate that CRES function is influenced by subtle alterations in the hypothalamic-pituitary-gonadal axis, suggesting it may regulate processing events important at specific times for gonadotrope secretory functions.Item The modulation of apoptosis in testicular germ cells following toxicant-induced cellular stress(2007-12) McKee, Chad Marcus, 1975-; Richburg, John H.Di-(2-ethylhexyl) phthalate (DEHP) is a ubiquitous environmental toxicant. The active metabolite of DEHP, mono-(2-ethylhexyl) phthalate (MEHP), is ultimately responsible for disrupting the process of spermatogenesis and promoting germ cell death. In addition, this toxicant has been positively correlated with developmental problems such as cryptorchidism, a derangement of the seminiferous tubules, and a syndrome called testicular dysgenesis, leading to reduced sperm number. The potential impact of MEHP on human fertility justifies a detailed investigation into the mechanisms by which this agent causes germ cell death. MEHP is known to directly target and damage the Sertoli cell, a testicular cell whose main function is to support the development of the principle germ cell types from the earliest stem cell to the most mature spermatozoa. This dissertation examines the downstream effect of Sertoli cell damage on germ cell homeostasis and the proteins that modulate the sensitivity of germ cells to undergo apoptotic elimination. Specifically, the stabilization of the p53 protein is proposed to be an important upstream determinant of Fas-mediated apoptosis in germ cells following MEHP exposure. Furthermore, that the resulting cell death is the result of increased death receptor expression and c-FLIPL ubiquitinylation. The mechanism is speculated to reside in the spermatocyte sub-population of germ cells, which appears to be most responsive to changes in apoptosis. Exposures of wild type mice to MEHP caused an increased p53 stability and elevated protein levels of the membrane-bound death receptors Fas and DR5 in testicular spermatocytes. The expression of these proteins occur coincident with increases in spermatocyte apoptosis and are driven by p53 activity. To further assess the mechanisms responsible for the sensitivity of germ cells to undergo p53-mediated apoptosis, we used the germ cell line GC-2spd(ts) (a p53 temperature sensitive spermatocyte-like cell line that allows for p53 nuclear localization at 32°C but not 37°C). Induction of the p53 protein led to higher levels of the death receptors DR5 and Fas, activation of caspase-8, and decreases in c-FLIPL. Addition of TRAIL (the cognate ligand for DR5) and the agonistic DR5 agonistic antibody MD5-1, triggered a robust synergistic increase of apoptosis in GC-2 cells maintained at the p53 permissive temperature (32°C). DR5 levels on the germ cell plasma membrane were considerably enhanced following these treatments. Immunoprecipitation of c-FLIPL suggests that the protein is ubiquitinylated after cellular stress and concomitant with p53 activity. Experiments also reveal that c-FLIPL levels may be influenced by Itch, a regulatory protein able to label targets for the proteasomal degradation using a ubiquitinylating E3 ligase. Immunohistochemical detection in adult wild type mouse testis show robust increases in Itch protein levels upon MEHP treatment (1g/kg) and subsequently localization to the cytoplasm of meiotic spermatocyte germ cells. Western blot analysis of testis from MEHP treated mice also show a correlation between the reduction of c-FLIPL and an increase in Itch threonine-222 phosphorylation, a necessary modification for its E3 ligase function. These results provide a possible model in which the removal of Sertoli cell support promotes germ cell death through the extrinsic pathway, ultimately leading to disruption of spermatogenesis and testicular dysgenesis in mammals. However, removal of Itch also show increases in apoptosis and Itch protein deficient mice demonstrate defects in meiosis. Thus, Itch may also play a novel role in the cell cycle.Item Spermatogenesis of Anolis carolinensis(Texas Tech University, 1959-06) Pruitt, Celia RalstinA cytological study of the spermatogenesis of the American chameleon lizard, Anelis carolinensis, of the family Iguanidas, was made. Testicular tissue, taken from animals collected around Schriever, Louisiana (elevation 15 feet), was fixed in a modification of the Bouin-Allen fluid (corrected for altitude). Slides were prepared in the usual manner and stained in iron hemotoxylin. The diploid number of chromosomes was found to be thirty-four and the haploid number, seventeen. The prophase stages, as well as the metamorphosis of the spermatozoa, followed a general reptilian trend in development. Morphological evidence of sex-chromosomes was encountered. These chromosomes were most obvious in the side view of the primary spermatocyte cells. The sex-chromosome is bipartite and has spindle fiber attachments at one pole of the cell only.Item Item Item Item Item Item Item Spermatogenesis of Bufo woodhousei: a thesis in zoology(Texas Tech University, 1955-01) McIlroy, Anne SalmanPor a number of years the spermatogenesis of amphibians has been of interest to zoologists because (1) these are the first vertebrates to have emerged from the water during the process of evolution; (2) the sex chromosome relationship of reptiles has not been definitely determined, and research in this field has been disappointing; hope has been maintained that the amphibia may furnish some information leading to the solution of the problem introduced in the field of cytology by the heterozygosity of the male in mammals and the heterozygosity of females in birds. The purpose of this work is to see if a species of toad, Bufo woodhousei, will throw a light on the problem.Item Spermatogenesis of Bufo woodhousei: a thesis in zoology(Texas Tech University, 1955-01) McIlroy, Anne SalmanNot availableItem Spermatogenesis of Phrynosoma cornutum(Texas Tech University, 1951-01) Cavazos, Larry F.Not available