Temperature-related sequence 4 (Trs4) is a transcript identified from the testis of a cryptorchid rat model. TRS4 is firstly detected in round spermatids on postnatal day 21 mice, and is localized on the acrosome and tails of elongating spermatids and mature spermatozoa, suggesting a role in spermatogenesis. The present study aimed to investigate the regulatory and functional roles of Trs4 in spermatogenesis. To investigate the hormonal regulation of Trs4 in vitro, mouse germ cell were co-cultured with Sertoli (TM4) cell-line for 24 hours with 1μM or 10nM testosterone and the expression of Trs4 was quantitated. It was found that testosterone treatment at physiological level (10nM) significantly down-regulated Trs4 mRNA expression. However, TRS4 protein level did not change significantly. To investigate the functional roles of Trs4 and its interacting proteins (Rshl2, Gstmu1 and Ddc8) in spermatogenesis, the scrotal heat-treated rat model was used. The expression of Trs4, Rshl2, Gstmu1 and Ddc8 transcript were investigated on day 1, 9, 25, 37, 46, 56 and 79 post-heat treatment. Trs4, Rshl2 and Ddc8 transcripts were down-regulated on day 9 after heat-treatment, and recovered from day 37 onward. Immunohistochemical staining of TRS4 and RSHL-2 proteins showed they specifically localized in the cytoplasm of elongating spermatids in rat seminiferous tubules, suggesting that they might interact in regulating the development of elongating spermatids. Ten sessions of electroacupuncture (EA) was applied to the rats on day 9-36 to study the effect of EA in spermatogenesis. It was found that EA did not have any effect on the expression of Trs4, Rshl-2 and Ddc8 during recovery from hyperthermia when compared to the heat-treated only group. However, qPCR analysis on Gstmu1 mRNA expression showed a significant decrease in expression on day 46, and consistent with our TUNEL assay indicating a decrease in apoptosis in the EA group. Therefore, it is possible that Trs4 interacts with Gstmu1 and acts as anti-apoptotic factor for cell survival. To understand functional roles of Trs4 in mouse spermatogenesis, five Trs4 chimeric mice were generated in our laboratory and germline transmissible 〖Trs4〗^(flox/+) mice were identified. Sycp1-Cre mice were used to breed with Trs4 floxed mice to generate Trs4 floxed mice expressing Cre recombinase in spermatocytes (〖Trs4〗^(flox/+);〖Sycp-Cre〗^(+/0)) and aiming to generate Trs4 heterozygous KO mouse. However, after screening 74 offspring, no Trs4 heterozygous KO mouse was identified in this study and was likely to be caused by the low efficiency of recombination in Sycp1-Cre mice. Then, Zp3-Cre mice that expressing Cre-recombinase in oocyte were employed. Crossing of 〖Trs4〗^(flox/+):Zp3-〖Cre〗^(+/0) mice and Trs4 floxed mice could not produce heterozygous or homozygous Trs4 KO mouse, but suggested an aberrant recombination process happened in vivo. PCR assay confirmed DNA rearrangement occurred in regions flanking the exon 4 to the second LoxP site in Trs4 floxed mice. Furthermore, the absence of 〖Trs4〗^(flox/flox) homozygous transgene suggested a possibility of insertional mutagenesis in the transgene. Future study is needed to confirm and characterize the insertion site of the floxed allele in order to generate Trs4 knockout mice for fertility study. / published_or_final_version / Obstetrics and Gynaecology / Master / Master of Philosophy

No description available.

Thesis (Ph. D.)--Columbia University. / Vita. Reprinted from Journal of morphology, supplement to vol. xv. Bibliography: p. 267-269.

Thesis (Ph. D.)--University of Texas at Austin, 2002. / Vita. Includes bibliographical references. Available also from UMI Company.

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.

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Thesis (Ph. D.)--University of Texas at Austin, 2007. / Vita. Includes bibliographical references.

Thesis (Ph. D.)--University of Chicago, 1900. / Includes bibliographical references (p. 57-59).

Thesis (Ph. D.)--Yale University, 1920. / "Author's abstract of this paper issued by the [Wistar Institute] Bibliographic Service, March 28." "Reprinted from Journal of morphology, vol. 35, no. 2, June, 1921." Bibliography: p. 409-410.

Thesis (Ph. D.)--Columbia University. / Vita. Reprinted from Journal of morphology, supplement to vol. xv. Bibliography: p. 267-269.