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 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 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 The functional role of the copper transporter 1 in spermatogenesis and in cisplatin-induced testicular injury(2019-05) Ghaffari, Rashin; Mukhopadhyay, Somshuvra; Richburg, John H.; Vokes, Steven A; Tucker, Haley O; Vasquez, Karen MCisplatin (cis-diamminedichloroplatinum(II) (cDDP)) is a highly effective chemotherapeutic drug used for the treatment of various cancers including ovarian, bladder, cervical, head and neck, small-cell and non-small-cell lung cancer. Unfortunately, cDDP is encumbered by its dose-limiting toxicity including prolonged azoospermia and, in some cases, permanent male infertility. Since patients that undergo cDDP based chemotherapy for these types of cancers are mostly young men in their prime reproductive age (ages 15-44), one of the major concerns after their recovery is infertility which would affect their quality of life. Therefore, it important to understand the mechanism of this reproductive toxicity in order to develop strategies to alleviate cDDP induced testicular injury that results after chemotherapy exposure. It has been well established that cDDP induces apoptosis in tumor and normal cell types, however, mechanisms leading to its long-lasting reproductive toxicity is less understood. Whether cDDP-induced impairment of spermatogenesis is a result of direct drug exposure to the germ cells (GCs) or results from a secondary effect to the somatic Sertoli cells (SCs) function remains unclear. An increasing body of evidence has implicated the major role of the high-affinity copper (Cu) transporter 1 (SLC31A1, CTR1), which functions in the uptake of Cu into mammalian cells, in cDDP sensitivity. The level of Ctr1 expression is recognized to be highly associated with cDDP accumulation and sensitivity in eukaryotic cells. The testis is one of the organs that express high Ctr1 mRNA and protein levels, which suggests that CTR1 protein may play a significant role in cDDP induced toxicity in testis. Moreover, given the essential role of CTR1 in Cu homeostasis, very little is known on the Cu transport mechanism and the physiological role of CTR1 protein in testis. Thus, this dissertation is focused on deciphering the role of CTR1 in testicular Cu homeostasis and its functional role in cDDP-induced testicular injury. In this thesis, two independent mouse models were generated with the conditional knockout of Ctr1 in either GCs (GC [superscript ΔCtr1]) or SCs (SC [superscript ΔCtr1]) using the Cre-Lox system. Loss of Ctr1 in GCs revealed the essential role of its expression in GCs to maintain functional spermatogenesis. GC [superscript ΔCtr1] mice exhibited a significant reduction of testicular mass by a severe progressive loss of GCs starting at postnatal day (PND) 28 leading to testis hypoplasia by adulthood. No spermatogenic recovery was observed in GC [superscript ΔCtr1] testis beyond PND 41, despite the presence of undifferentiated spermatogonial cells. On the other hand, SC [superscript ΔCtr1] mice were indistinguishable from their WT littermates with respect to fertility, spermatogenesis, growth, and development. Upon examination of testicular Cu level, SC [superscript ΔCtr1] mice exhibited significant reduced Cu and Cu-dependent cellular activities. The results from the two independent mouse models reveal, for the first time, the differential cell-specific role of CTR1 expression by testicular cell types (GCs and SCs) in maintaining functional spermatogenesis and Cu homeostasis. Finally, the role of CTR1 expression by SCs in cDDP-induced testicular injury was examined. Due to the testicular hypoplasia of the adult GC [superscript ΔCtr1] mouse testis, cDDP treatment of GC [superscript ΔCtr1] mice was not performed. Acute treatment of cDDP (5mg/kg) on SC [superscript ΔCtr1] mice was done and analyzed. Interestingly, SC [superscript ΔCtr1] mice testis exhibited higher resistance to cDDP-induced GC apoptosis than WT mice. Moreover, cDDP treated SC [superscript ΔCtr1] mice testis displayed significantly less platinum levels than their treated WT littermates. The results in this study provide the first evidence that loss of CTR1 expression by SCs enhances testis resistance to cDDP toxicity by reducing the accumulation of cDDP in testis. Taken together, the observations described within this thesis demonstrate the physiological role of CTR1 for functional spermatogenesis and Cu homeostasis in testis. Furthermore, loss of CTR1 expression in SCs revealed a newly identified pathway for cDDP uptake and toxicity. The mechanism(s) underlying the Cu transport system in testis and its effect on fertility will provide insights into the possible development of targeted therapies to improve fertility in individuals who are affected from diseases connected to an imbalance of Cu metabolism. Furthermore, the involvement of CTR1 in cDDP injury to the SCs sets the foundation to further understand and design strategies to protect or alleviate long-lasting reproductive injury following cDDP therapy