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

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Thesis (Ph. D.)--Washington State University, May 2009. / Title from PDF title page (viewed on June 19, 2009). "School of Molecular Biosciences." Includes bibliographical references.

Thesis (MMedVet (Equine Surgery))--University of Pretoria, 2006. / Includes bibliographical references. Also available in print format.

Thesis (MSc (Veterinary Science)) - University of Pretoria, 2002. / Includes bibliographical references.

Luteinizing hormone (LH), one of the two gonadotropin hormones released from the pituitary gland, binds its receptor (LHR) in the gonads to initiate steroid hormone production, as well as gametogenesis and ovulation. Mutations of amino acid sequence within the receptor can render it either inactive or constitutively active. All activating mutations result in male-limited precocious puberty. Males afflicted with this condition undergo puberty around 4 years of age, with elevated testosterone levels and premature skeletal development. In order to better understand how chronic ligand-mediated activation of the LHR affects gonadal development and function, a mouse model expressing a yoked hormone-receptor (YHR) complex, engineered by covalently linking the hormone human chorionic gonadotropin to the rat LHR, has been studied. YHR+ males have prepubertally elevated testosterone and decreased gonadotropin levels. Histological evaluation of the testes of these animals show significantly smaller seminiferous tubules and Leydig cell clusters. Finally, testis gene expression analysis revealed a significant decrease in the relative mRNA expression of three Leydig cell specific genes. Based on these results, it was hypothesized that premature activation of the LHR impairs postnatal Leydig cell development. In the testis there are two morphologically and developmentally distinct populations of Leydig cells, the fetal and the adult. The first objective of this study was to quantify the populations of cells in the adult Leydig cell lineage in both the YHR+ and the WT controls. Real-time RT-PCR, for markers of the immature and adult Leydig cell populations, as well as Leydig cell quantification, suggested a delay in adult Leydig cell development. Interestingly, there was a significant increase in the fetal Leydig cell population in the YHR+ mice. The second objective was to determine if the decrease in the adult population is due to either a decrease in proliferation or an increase in apoptosis in the YHR+ animal. There was not a difference in apoptosis between the WT and the YHR+ at any age examined, however, there was a decrease in progenitor Leydig cell proliferation in the YHR+ animals at 2 weeks of age. The final objective was to determine if elevated neonatal testosterone levels impairs the development of the adult Leydig cell population. Seven-day old WT pups were subjected to testosterone supplementation via subdermal implant. Quantification of the total Leydig cell population revealed a significant decrease in the number of adult Leydig cells in the testosterone-treated group similar to that seen in the YHR+ animal. Taken together, these data suggest that elevated neonatal testosterone levels resulting from premature LHR activation inhibits the proliferation of progenitor Leydig cells, resulting in fewer adult Leydig cells in the YHR+ animals.

The Reproductive Homeobox X-linked, Rhox, genes encode transcription factors that are expressed exclusively in the testis, epididymis, placenta, and ovary. While there are 33 Rhox genes in mice, only Rhox5 and Rhox8 are expressed in Sertoli cells, suggesting that they alone regulate the expression of somatic-cell gene products crucial for testicular metabolism and germ-cell development. Targeted deletion of Rhox5, the founding member of the Rhox gene cluster, in mice, results in decreased expression of the insulin-2 gene (Ins2) and other metabolic genes, male subfertility via reduced sperm number, increased germ-cell apoptosis and a reduced proportion of sperm with normal motility. Davis et al. developed a Rhox8 siRNA knockdown transgenic model to study possible functional similarities between Rhox5 and Rhox8 and reveal compensatory actions via the breeding of Rhox5/Rhox8 double knockout mice. They observed that loss of Rhox8 results in downregulation of the sex-determining region Y gene (Sox9). Further analysis of the role of Rhox5 in testicular metabolism regulation was completed by development of mutant constructs encoding combinations of Rhox5 functional domains and subsequent analysis via qRT-PCR, luciferase assay and immunohistochemistry in cell lines transfected with expression plasmids containing these mutants. Our results indicated direct interaction of RHOX5 with the Ins2 promoter. The homeodomain and amino-terminal domain construct being sufficient for promoter activation albeit at a lower level than the full-length RHOX5 construct. MacLean et al. conducted qRT-PCR analysis of cells transfected with plasmids encoding the other Rhox genes revealed that Rhox8 and Rhox11 were also capable of upregulating Ins2 expression at a lower level than Rhox5. Our analysis of metabolic gene expression in the Rhox8 knockdown model also revealed decreased expression of Ins2 as well as insulin receptor-1 (InsR1). Continued analysis of the Rhox8-KD model and Rhox5/Rhox8 double knockout mice in young and aged (~12 months) male mice revealed a subfertility phenotype characterized by reduced litter frequency and size, reduced total spermatozoa and reduced sperm forward motility. This was reflected by a decrease in RHOX8 and SOX9 protein expression maintained in the aged mice that was more severe in the double knockout animals. qRT-PCR analysis of altered gene expression in the Rhox8 single knockdown male mice revealed significant expression downregulation of fellow Rhox genes Rhox5 and Rhox10 and expression upregulation of the growth differentiation factor-9 (Gdf9) gene normally expressed in the somatic cells of the ovary. Based on the regulation of sex specific genes Sox9 and Gdf9 in the Rhox8 knockdown model as well as data published by Daggag et al. revealing that Rhox8 is the only Rhox member expressed in the somatic cells of the embryonic testis, we sought to elucidate the possible role of Rhox8 in sex determination and testicular differentiation/development. Due to the limitations of our current knockdown model whereby siRNA production is initiated postnatally due to its androgen-dependent promoter, we elected to develop a new Rhox8 knockdown model to characterize the embryonic functions of Rhox8. Rhox8 siRNA were cloned into a Cre-recombinase activated expression vector in which a highly active U6 promoter drives expression of the shRNA. The constitutively active form of this vector exhibited knockdown of Rhox8 in stable Rhox8 overexpressing cell lines developed for this purpose. This developed vector is being used to develop a new transgenic line. While waiting for the development of the new Rhox8 knockdown transgenic mice, we chose to characterize the mRNA and protein expression of Rhox8 in the embryonic testis. Using qRT-PCR and immunohistochemistry, expression of Rhox8 was confirmed and exhibited continually increased expression in the Sertoli cells over the course of embryonic day ~13.5-18.5 (E13.5-E18.5). To examine possible functions of Rhox8 in the embryonic testis prior to receipt of the new knockdown model, we adapted a protocol to transfect the new constitutively active Rhox8 knockdown into embryonic gonads and cultured them up to 72 hours after electroporation. This protocol yielded successful expression of GFP from a GFP-expression plasmid and this was maintained for up to 72 hours in E13.5 gonads. qRT-PCR analysis of gonads transfected with the Rhox8 knockdown expression plasmid revealed significant knockdown of Rhox8 and Sox9 mRNA expression at the 48-hour and 72-hour time points.

The annual testicular cycle of a teleost the lake chub (Couesius plumbeus) from a north temperate latitude (51°N) has been studied by histological and histochemical methods with a planimetric evaluation of the different spermatogenetic stages. The annual cycle is divided into five different stages and the cyclical testicular changes have been correlated with changing environmental conditions. Lobule boundary cells, considered to be homologue of the Leydig cells of higher vertebrates, have been identified, and the changes in their secretory activity have been reported. Temperature is the major environmental factor controlling the testicular cycle. The pituitary gland, through its gonadotropin(s), mediates between environmental changes and developments in the testes. Higher temperatures (16°-22°C) promote spermiogenesis and spermiation, whereas low temperatures (5°-12°C) are more conducive to gonial proliferation and the initial phase of spermatogenesis. Hypophysectomy affects the mitotic ability of the spermatogonia, completely blocks their transformation into spermatocytes and suppresses the secretory activity of the lobule boundary cells. Replacement therapy with fish gonadotropin and mammalian LH restores spermatogenesis to a large extent and maximum response is elicited with whole fish pituitary extract. It is proposed that the fish pituitary gonadotropin is similar to mammalian LH and in this species mammalian FSH is physiologically inactive in the restoration of testicular activity. Since whole fish pituitary produces a maximum response it is suggested that factors such as TSH and STH have a probable synergistic role in the testicular maturation in Couesius plumbeus. Evidence is presented that a weak endogenous rhythm of activity is partly responsible for the timing of various testicular changes. / Science, Faculty of / Zoology, Department of / Graduate

Testis-specific protein (TSP) variability has been examined in the three-spined stickleback, Gasterosteus aculeatus and related species including Gasterosteus wheatlandi, Punqitius pungitius, and Aulorhynchus flavidus, in order to determine if such proteins can act as molecular markers for different species of Gasterosteiformes and for different populations of a single species. Cytochemistry of the sperm histones of Gasterosteus aculeatus revealed that these basic proteins can be classified as intermediate sperm histones according to Bloch's (1969 and 1976) categories. Electron microscopy indicates that the chromatin in the nucleus condenses in a granular pattern as the somatic histones of the spermatogonia are replaced by the sperm histones in the spermatid during spermiogenesis in this teleost. The testis specific proteins (TSP's) of G. aculeatus and related species were characterized by electrophoresis on polyacrylamide gels and by hydrolysis of the amidoblack stained bands from the gel and subsequent amino acid analysis. The compositional analysis revealed that all the TSP's of the fish in the order Gasterosteiformes were intermediate type containing histidine, lysine, and arginine amino acid residues. However, the TSP's of different species could be distinguished by their electrophoretic mobilities on polyacrylamide gels and by differences in the amino acid composition. Apparently TSP's can act as molecular markers to distinguish these particular teleosts. To establish the electrophoretic pattern for the TSP's of mature, breeding G. aculeatus, the developmental profile was investigated over the course of a season for a population of these fish in Jericho Pond, Vancouver, B.C. As the testis matures, somatic histones are replaced gradually by one or several rapidly moving TSP's. Some protein bands that are present in fish with immature testes are removed by the time the electrophoretic pattern typical for mature males occurs during the breeding season. This pattern shows only rapidly moving TSP's and very low levels of somatic histones. Experiments using alkaline phosphatase indicated that the electrophoretic profile of the multiplicity of TSP'.s of mature fish was not due to differences in charge because of phosphorylation of serine side chains in TSP bands. In addition to acting as molecular markers for different species, the TSP's showed an electrophoretic profile in anadromous G. aculeatus that differed from the profile in freshwater fish with respect to band morphology and also the number of bands seen on long gels. Here, too, the similarity of the freshwater and anadromous TSP profiles was reinforced by similar digestion patterns with cyanogen bromide, confirming the presence of methionine in these proteins. However, different populations of anadromous G. aculeatus either from British Columbia or from Quebec were indistinguishable by electrophoretic analysis. Moreover the electrophoretic and amino acid analysis could not distinguish between G. aculeatus from different freshwater populations. There was no discernable trend for the multiplicity of TSP's from such populations. However, in the fish from two freshwater lakes there was an indication that the benthic forms (bottom dwellers) might have reached sexual maturity later than the limnetic forms (top dwellers) as the limnetic animals lost their somatic histones before the benthics did. From these analyses, TSP's apparently can act as molecular markers between different species of Gasterosteiformes, and to a lesser extent between anadromous and freshwater forms of G. aculeatus. This agrees with the findings of Mann et. al. ( 1982) that the spermatid/sperm-specific proteins of the frog genus Xenopus can distinguish between different species of the genus and somewhat between different subspecies of Xenopus laevis. Both the Gasterosteiformes and anuran TSP's are of the intermediate type. In several instances, particular species of fish gave anomalous results. For example, an anadromous G. aculeatus showed the presence of a band typical for the TSP of G. wheatlandi on polyacrylamide gel electrophoresis. Perhaps this is due to hybridization between Gasterosteidae of different species. Finally, incubation of TSP preparations at 37 °C indicated the presence of endogenous protease at neutral pH. Such a protease was not active at acid pH and therefore did not interfere with the electrophoretic analysis. / Science, Faculty of / Zoology, Department of / Graduate

During spermatogenesis in rainbow trout, a synchronous development of stem cells → spermatocytes → spermatids → mature sperm occurs, with replacement of the histones in chromatin by protamines. PART A: Histone methylation. Along with histone acetylation and phosphorylation, methylation of specific lysyl residues of histones H3 and H4 can be observed. This histone methylation occurs predominantly in the large diploid stem cells and primary spermatocytes, which actively synthesize DNA and histones. In spermatids, histone methylation is minimal and so probably has no role in the replacement of histones by protamine. Other labelling experiments suggest that histone H4 methylation is a late event in the cell cycle, occurring after the synthesis, acetylation and deacetylation of histone H4. This methylation may be necessary for histone phosphorylation or chromatin condensation prior to cell division. PART B: Chromatin subunit structure. When a sample of trout testis nuclei is digested with micrococcal nuclease, the DNA is cleaved almost entirely to discrete fragments approximately 200 base pairs long and multiples thereof. The same DNA fragments can be obtained when isolated chromatin, as opposed to intact nuclei, is nuclease digested. These DNA fragments can also be found in discrete chromatin "subunits" isolated from nuclease-digested nuclei. Sedimentation through sucrose gradients, or velocity sedimentation in an analytical ultracentrifuge separates these chromatin subunits into 11S (monomer), 16S (dimer), 22S (trimer) etc. species. Subunits can also be fractionated on a Sepharose 2B column equilibrated and run in low salt. High salt (>40 mM NaCl) or divalent cations (≃5 mM) cause subunit precipitation. Chromatin subunits have a protein:DNA ratio of approximately 1.2 and contain all the histones, including the trout-specific histone H6. There are however no detectable nonhistone chromosomal proteins. Mg⁺⁺ precipitates of the 11S chromatin monomers, when pelleted, are thin and clear, while oligomer Mg⁺⁺ pellets are thick and white. This could reflect a more symmetrical or ordered packing of 11S monomers, which are deficient in histone Hi. This histone may crosslink the larger oligomers, resulting in a disordered Mg⁺⁺ complex. These results are consistent with the subunit model of chromatin structure, based on 200 base pair long regions of DNA associated with histones. These subunits would be separated by nuclease-sensitive DNA spacer regions, and crosslinked by histone Hi. Testis consisting predominantly of early spermatids (meiotic tissue, containing mainly nucleohistone) gives similar yields of DNA fragments and 11S subunits. Later stage testis (protamine has replaced the histones) however, gives no DNA fragments or 11S subunits. This presumably reflects large differences in structure between nucleoprotamine and nucleohistone. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate