by Will W.M. Lee. / Thesis (M.Phil.) - Chinese University of Hong Kong. / Bibliography: leaves 101-110. / Chapter CHAPTER I --- GENERAL INTRODUCTION / Chapter A --- Spermatogenesis --- p.1 / Chapter B --- Sperm Maturation --- p.2 / Chapter C --- Ejaculation --- p.3 / Chapter D --- Sperms in Fertilization --- p.3 / Chapter E --- Aim of Spermatozoal Motility Studies --- p.7 / Chapter CHAPTER II --- AN ASSAY TO MEASURE THE SPERMATOZOAL PROGRESSIVE MOTION / INTRODUCTION --- p.8 / MATERIALS AND METHODS --- p.15 / Chapter A --- Sample Collection --- p.15 / Chapter B --- Cell Wash --- p.17 / Chapter C --- Media --- p.18 / Chapter D --- Motility Assay Chamber --- p.18 / RESULTS AND DISCUSSIONS --- p.22 / Chapter A --- Sperm Entry --- p.22 / Chapter B --- Sperm Motility --- p.32 / Chapter CHAPTER III --- EFFECT OF VARIOUS GROUPS OF CHEMICALS ON THE MOTILITY OF SPERMATOZOA / INTRODUCTION --- p.48 / MATERIALS AND METHODS --- p.55 / RESULTS AND DISCUSSIONS --- p.57 / Chapter A --- Energy Source --- p.57 / Chapter B --- "Phosphodiesterase Inhibitors and Cyclic 3',5'-Adenosine Monophosphate (cAMP)" --- p.59 / Chapter C --- p-Nitrophenyl Compounds --- p.64 / Chapter D --- Motility of Spermatozoa from Addicted Rat and Effect of Morphine on tozoa In VitroSperma- --- p.68 / Chapter E --- Metallic Ions and EDTA --- p.68 / Chapter CHAPTER IV --- EFFECT OF SEMINAL PLASMA ON SPERM MOTILITY --- p.77 / INTRODUCTION / MATERIALS AND METHODS --- p.79 / RESULTS --- p.82 / DISCUSSIONS --- p.98 / REFERENCES --- p.101 / Chapter APPENDIX I --- Spermatozoa Repellent as a Contraceptive --- p.111 / Chapter APPENDIX II --- Effect of p -Nitrophenylglycerol on Motility of Rat Epididymal Spermatozoa --- p.117

Apoptosis in testicular germ cells has been demonstrated in many species. Features of apoptosis reported in other species were used to confirm use of the TUNEL assay in stallion testes. Eight stallions with normal testicular size and semen quality were evaluated to determine the germ cell types and stages where apoptosis most commonly occurs. Mean numbers of TUNEL-positive germ cells per 100 Sertoli cell nuclei were highest in stages IV and V of the seminiferous epithelial cycle corresponding to meiotic divisions of primary spermatocytes and mitotic proliferation of B1 and B2 spermatogonia. Round and elongated spermatids were labeled less frequently by the TUNEL assay. To examine the relationships between germ cell apoptotic rate and spermatogenic efficiency, seminal traits were assessed to classify stallions into normal or reduced quality semen groups. Apoptotic rates were higher for stages IV-VI and stage VIII seminiferous tubules in stallions with reduced semen quality. Daily sperm production (DSP) per gram and per testis were lower for stallions with reduced semen quality. Regression analyses revealed negative linear relationships for germ cell apoptotic rate with DSP/g, DSP/testis, daily sperm output, progressively motile sperm and morphologically normal sperm in ejaculates. Mean circulating concentrations of inhibin were lower for stallions ejaculating reduced quality semen. Apoptotic rate was negatively correlated with concentrations of inhibin and estradiol-17b and positively correlated with concentrations of LH and FSH. To study germ cell apoptosis and formation of the Sertoli cell barrier during the initiation of spermatogenesis, tubule development was classified based on lumen score. Formation of a seminiferous tubule lumen was consistent with events leading to development of a Sertoli cell barrier. A primary wave of apoptosis removed early differentiating germ cell types prior to the formation of a tubule lumen facilitating both the formation of a tubule lumen and a Sertoli cell barrier. A second wave of apoptosis occurred after the formation of a lumen but before seminiferous tubule cross-sections contained a full complement of germ cells. In conclusion, apoptosis is an essential mechanism during normal spermatogenesis. Apoptosis also accounts for low numbers of normal sperm seen in the ejaculates of some stallions.

Thesis (Ph. D.)--Washington State University. / Includes bibliographical references.

Thesis (Ph. D.)--Bryn Mawr. / Recto of pl. i-viii contains letterpress descriptive of the plate following. Reprinted from the Journal of experimental zoology, vol. iv, no. 4. "Bibliography": p. 509-512.

Thesis (Ph. D.)--University of Michigan, 1927. / Descriptive letterpress on versos facing the plates. "Reprinted from the Journal of morphology and physiology, vol. 44, no. 3, December, 1927." Bibliography: p. 590-592.

Thesis (M. Phil.)--University of Hong Kong, 2010. / Includes bibliographical references (p. 161-190). Also available in print.

published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

Heat stress inhibits spermatogenesis partly by inducing apoptosis in the testicular germ cells. Using a cryptochid rat model, we identified a temperature-related ESTs 4 (TRS4) transcript from rat testis. Trs4 mRNA is specifically expressed in the mouse and rat testis from postnatal day 21 and 28 days onwards, respectively. Trs4 protein is located mainly in the elongating spermatids and mature spermatozoa at the acrosome and tail regions. Using a yeat-2-hybrid screening, Trs4 was found to bind Gstmu1, Rslh-2 and Ddc8 proteins. To further characterize the functional role of Trs4 in spermatogenesis, and study how Trs4 interacts its binding proteins for cellular functions, we aimed (1) to screen putative ES cells with Trs4 floxed allele for knockout mice generation, (2) to generate Trs4 deletion constructs and study the cellular localization of Trs4 and its putative binding partners in transfected spermatocyte GC-2spd(s) cell line, (3) to study how heat-treatment regulates the expression of Trs4 and apoptotic molecules. The Trs4 conditional targeting vector was constructed by flanking exons 4-6 with two LoxP sites and electroporated into ES cells. After screening of 480 clones, positive ES cell clones were identified by Southern blotting using 5’- and 3’- probes. Three putative positive clones were identified carrying the floxed allele. Trs4 protein contains putative ubiquitin-like motif (a.a. 119-224), IQ-calmodulin binding motif (a.a. 334-362) and a overlapping bipartite nuclear localization signal (BNL) (a.a. 346-362). Transfection of EGFP fused Trs4 truncated protein demonstrated that the IQ-calmodulin binding motif and BNL signal was important for localization of Trs4 protein in the cytoplasmic/Golgi regions; while the N-terminal contains ubiquitin-like motif and the C-terminal regions direct the expression of the EGFP-fusion protein mainly to the nucleus. The full-length sequence of Trs4 binding partners: Gstmu1, Rshl-2 and Ddc8 were cloned into the pDsRedmonomer-C1 vector, giving red fluorescence protein in the transfected cells. They were colocalized with EGFP-Trs4 in the cytoplasm of the cells, confirming that Trs4 and its interacting protein is likely interact with each other in vivo. As Trs4 colocalize with Gstmu1, a modulator of mitochondrial-dependent pathway in apoptosis, it is suggested that Trs4 is an upstream regulator of apoptosis under heat treatment in germ cells. The functional roles of Trs4 protein Trs4 and apoptotic molecules. The Trs4 conditional targeting vector was constructed by flanking exons 4-6 with two LoxP sites and electroporated into ES cells. After screening of 480 clones, positive ES cell clones were identified by Southern blotting using 5’- and 3’- probes. Three putative positive clones were identified carrying the floxed allele. Trs4 protein contains putative ubiquitin-like motif (a.a. 119-224), IQ-calmodulin binding motif (a.a. 334-362) and a overlapping bipartite nuclear localization signal (BNL) (a.a. 346-362). Transfection of EGFP fused Trs4 truncated protein demonstrated that the IQ-calmodulin binding motif and BNL signal was important for localization of Trs4 protein in the cytoplasmic/Golgi regions; while the N-terminal contains ubiquitin-like motif and the C-terminal regions direct the expression of the EGFP-fusion protein mainly to the nucleus. The full-length sequence of Trs4 binding partners: Gstmu1, Rshl-2 and Ddc8 were cloned into the pDsRedmonomer-C1 vector, giving red fluorescence protein in the transfected cells. They were colocalized with EGFP-Trs4 in the cytoplasm of the cells, confirming that Trs4 and its interacting protein is likely interact with each other in vivo. As Trs4 colocalize with Gstmu1, a modulator of mitochondrial-dependent pathway in apoptosis, it is suggested that Trs4 is an upstream regulator of apoptosis under heat treatment in germ cells. The functional roles of Trs4 protein / published_or_final_version / Obstetrics and Gynaecology / Master / Master of Philosophy

Spermatogenesis takes place in the seminiferous epithelium and it is a tightly regulated process that produces spermatozoa from spermatogonia. During spermatogenesis, germ cells have to traverse the seminiferous epithelium, from basal to adluminal compartment and finally reach the luminal edge of the seminiferous tubules at spermiation. During the transit of germ cells, they have to get across the blood-testis barrier (BTB), which is formed by adjacent Sertoli cells. Thus, although BTB is considered as one of the tightest blood-tissue barrier, the BTB undergoes cyclic restructuring to “open” transiently for the translocation of germ cells. However, the integrity of the BTB has to remain intact as the BTB is essential for the developing germ cells behind the barrier. For example, the BTB serves as an immunological barrier to “seal” developing germ cells from the systemic circulation. Since how the BTB restructuring is regulated remains elusive, the study herein aims to provide some information regarding to this events. The importance of the BTB to spermatogenesis was demonstrated by treating rats with 50 (lowdose) or 250 mg/kg b.w (high-dose) of adjudin. Although the BTB of rats was perturbed in both groups at week 6 post treatment, as shown by an in vivo BTB functional assay, the BTB of the low-dose group was found to have “resealed” at week 20 whereas the BTB of the high-dose group remained disrupted. Besides, despite almost all germ cells were depleted in both group of rats upon week 2 post treatment, spermatogonia were still present in the testis of rats no matter high- or low-dose of adjudin was used. However, spermatogenesis only recovered in low-dose treated group, which have an intact BTB. This suggests that after spermatogenesis is disrupted, its regeneration of spermatogenesis needs more than the existence of spermatogonia in which an intact BTB is required. After demonstrating the necessity of the BTB for spermatogenesis, the next question I addressed was how its restructuring was modulated. The involvement of mammalian target of rapamycin (mTOR) in manipulating the BTB was investigated. mTOR is able to form two distinct signaling complexes namely mTOR complex 1 (mTORC1) or mTORC2 by assembling with raptor or rictor, respectively. rpS6, which is a downstream molecule of mTORC1 was activated specifically during BTB restructuring and knockdown of rpS6 in cultured Sertoli cells was able to promote the TJ-barrier by inducing deposition of F-actin and BTB proteins at the cell-cell interface, suggesting the role of phosphorylated rpS6 is to “open” the BTB for the transit of spermatocytes. Moreover, the knockdown of rictor led to perturbation of TJ-barrier formed by cultured Sertoli cells via a PKC-α depending actin reorganization, causing internalization of BTB proteins. This indicates mTORC2 is necessary for the maintenance of the BTB and hence the two mTOR complexes work antagonistically to regulate the BTB in which mTORC1 is activated to promote the BTB restructuring while the expression of mTORC2 is essential to sustain the BTB integrity. / published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy