Thesis (M. Phil.)--University of Hong Kong, 2000. / Includes bibliographical references (leaves 65-75).

Abstract Carbonic anhydrases (CAs) are a group of zinc-containing metalloenzymes that catalyze the interconversion of carbon dioxide and bicarbonate (CO2 + H2O ⇔ HCO3- + H+). They are present in almost all organs and are implicated in various biological functions, the most important of which is participation in the regulation of ion, water, and acid-base balance. Recently, some members of the CA gene family have been suggested to promote cell proliferation and to act as trophic growth factors. The present study was undertaken to examine the distribution of CA isoenzymes in the reproductive system, to attain a more detailed view on their linkage to the reproductive processes and to neonatal development. The expression of membrane-bound CA IX and CA XII was studied in the female and male reproductive tracts by immunohistochemistry and western blotting. CA XII was found to be expressed in the basolateral plasma membrane of luminal and glandular epithelia in human uterus. In human efferent ducts, it was located in the basolateral plasma membrane of luminal epithelium, where it coexpressed with Aquaporin-1. In epididymal duct, CA XII was only expressed in occasional epithelial cells. These cells coexpressed CA II, suggesting that they represent apical mitochondria-rich cells (AMRC). CA IX was also expressed in the basolateral plasma membrane of luminal epithelium in human efferent ducts, but its expression was not uniform among the tubules. These findings suggest that basolateral plasma membrane-associated CA IX and CA XII contribute, along with CA II and CA IV, to the regulation of acid-base balance and water transport in the reproductive tract. Western blotting of rat Leydig tumor cells and testis for CA II revealed an unidentified 66-kDa polypeptide band. The polypeptide was successfully purified from several rat tissues using CA inhibitor affinity chromatography. The amino acid sequence of the polypeptide showed it to be identical to NonO/p54nrb, a non-POU domain-containing octamer-binding protein previously implicated in transcriptional regulation. The recombinant NonO/p54nrb was shown to display CA activity, and the antibody to it predominantly immunostained the nuclei in lymphocytes, where CA activity was also detected histochemically. Accordingly, the nuclear Leydig cell CA immunoreactivity represents NonO/p54nrb. It is classified as a novel, nonclassical CA, and it may participate in pH-related events in the nucleus. Human and rat milk was found to contain CA VI by immunohistochemistry and western blotting. The enzyme purified from human milk by CA inhibitor affinity chromatography was confirmed by PNGase F digestion and amino acid sequence as CA VI. The CA VI concentrations in human colostral milk were approximately eight times higher than those in mature milk (34.7 mg/l vs. 4.5 mg/l). Secretion of CA VI into milk is suggested by its localization in the alveolar epithelium of the rat mammary gland. The structural and functional stability of CA VI in an acidic milieu, its suggested growth-supporting function in taste bud stem cells, and its high concentration in colostrum suggest that it is an essential factor for the growth and development of the newborn alimentary canal.

M.Tech. (Homoeopathy) / Acne vulgaris is a common skin condition, affecting mostly adolescents. This study attempts to demonstrate the effect of the homoeopathically prepared remedy Testis compositum in the treatment of acne vulgaris. Thirty participants were selected for the study, but only 28 completed the study. The study was conducted over a period of 8 weeks. All the participants formed the control group during the first two weeks of the study, and then formed the experimental group for the next six weeks. During the control period, the participants received placebo medication. At the start of the control period, and at two week intervals through the duration of the study, the participants were assessed by counting the acne lesions – only facial Acne vulgaris was assessed during the trial. At the start of the control period, the start of the experimental period, and after completion of all treatment, frontal and bilateral facial photographs were taken to enable visualisation of the changes that occurred during the study. The results were statistically analysed using the t-test, the Wilcoxon test and descriptive statistics. The results show that treatment with Testis compositum had a significant effect in improving acne vulgaris.

Philosophiae Doctor - PhD / The role of male germ cell death in spermatogenesis is an important one as it removes dysfunctional or genetically damaged germ cells and is necessary to maintain an optimal germ cell to Sertoli cell ratio. The formation of the bloodtestis barrier requires the elimination of excessive germ cells and a surge of germ cell apoptosis occurs prior to puberty regulating the ratio of germ cells to Sertoli cells. The aim of this study was to evaluate the presence of four apoptotic markers on sperm from patients with various grades of fertility using flow cytometry. Furthermore, any correlations between the apoptotic marker assays and the standard semen analysis results were identified. This study compares early and late parameters of apoptosis with morphological features in spermatozoa in the same samples. The three sample groups were identified as: teratozoospermic [G-pattern] (n=26), teratozoospermic [P-pattern] (n=98) and oligoteratozoospermic [Ppattern] (n=36). Standard semen analysis was conducted on the semen samples according to the WHO guidelines. Four apoptotic marker assays using flow cytometry was applied in this study to examine the apoptotic alterations in ejaculate sperm. These assays included the Annexin-V staining for the determination of phosphatidylserine exposure, APO-Direct to identify DNA fragmentation, caspase-3 to detect expression of this active protease during early apoptosis and Fas expression. For the Annexin-V and caspase-3 assays, statistically significant differences (P<0.05) were evident between the three groups. No significant differences (P>0.05) were found between the groups with respect to the APO-Direct assay. A significant difference (P<0.05) was found when comparing the teratozoospermic [G-pattern] group and the oligoteratozoospermic [P-pattern] group for the Fas assay. A strong positive correlation was evident between the Fas and the caspase-3 assays in the teratozoospermic [G-pattern] group. For the teratozoospermic [P-pattern group] the following positive correlations existed between the APO-Direct and the Fas assays, APO-Direct and caspase-3 assays and between caspase-3 and Fas assays. The only strong positive correlation was between the caspase-3 and APO-Direct assays in the oligoteratozoospermic [P-pattern] group. The presence of spermatozoa showing microscopic features resembling apoptosis has been identified in ten human ejaculate samples per sample group. Electron microscopy was used to identify morphological features of apoptosis in these human sperm samples. Classical apoptosis as observed in diploid cells could be identified in sperm and these included: loose fibrillarmicrogranular chromatin network, presence of vacuoles in the nuclear chromatin, membranous bodies within the vacuoles of the chromatin, partially disrupted nuclear membranes, plasma membrane protuberances and apoptotic bodies containing cytoplasmic vacuoles and dense masses. This study has confirmed that semen samples with abnormal semen parameters exhibit the presence of apoptotic markers in sperm. The identification of apoptotic markers on the sperm suggests that abnormalities occur during their developmental process, however, the exact mechanism thereof remains unclear. These findings may suggest that certain apoptotic markers may be an indicator of abnormal sperm function and possibly indicative of male infertility. / South Africa

Bisphenol (BP) A is a common manufacturing chemical in polycarbonate plastics and has been widely used in plastics, epoxy resin liners of canned foods, dental materials, and thermal receipts. Human exposure to BPA is associated with a negative impact on human health including the development and function of the reproductive system due to its action as an endocrine-disrupting chemical (EDC). Numerous experimental studies have demonstrated that BPA impairs both male and female reproductive function, despite the variation in study paradigms such as dose, exposure route, timing, and outcomes measured. Due to the toxicological effects of BPA, BPA analogues such as BPS have been used as alternatives for BPA. However, recent evidence has suggested these BPA analogues can induce similar or even more severe toxic effects as BPA, and health risks of exposure to replacement bisphenols need to be considered. Therefore, my study was designed to examine whether prenatal exposure to BPE and BPS negatively impacts on male and female reproductive function in mice. Pregnant females were orally administrated corn oil (control), BPA, BPE, and BPS (0.5, 20, or 50mg/kg/day) from gestational day 11 (the presence of vaginal plug=1) to birth, and reproductive tissues in F1 mice were collected and analyzed in both neonatal and adult mice. In males, I observed reduced sperm counts and quality, disrupted stages of spermatogenesis in adults and increased germ cell apoptosis in neonatal testis following prenatal BPA, BPE or BPS exposure. Particularly, I found the expression of methyltransferases for DNA methylation and histone modification was also affected by prenatal exposure to BPA, BPE, or BPS in neonatal testis, suggesting a potential of epigenetic alterations in F1 males. In females, prenatal exposure to BPE and BPS accelerated the onset of puberty, disrupted estrous cyclicity, and caused several fertility problems especially in aged mice. In the neonatal ovaries, I also observed that BPE and BPS inhibit germ cell nest breakdown comparable to BPA. These results suggest that prenatal exposure to BPE and BPS with physiologically relevant doses affects male and female reproductive function probably due to germ cell development defects in the developing gonads. Finally, to understand their complete impact on male and female fertility, a study of transgenerational effects of BPE and BPS is performed to examine the transgenerational effects of prenatal exposure to BPA, BPE and BPS on reproductive function in F3 offspring. To be called transgenerational, expression of the specific phenotype will be continued at least across three generations. As described in previous studies, the direct exposure of a pregnant female (F0 generation) results in the exposure of the embryos (F1 generation) and the germline that will generate the next generation (F2 generation). Thus, I orally exposed to control treatment (corn oil), BPA, BPE or BPS (0.5 or 50 μg/kg/day) from gestational day 7 to birth in pregnant females (F0). Mice from F1 and F2 offspring were used to generate the F3 generation. In F3 males, prenatal exposure to BPA, BPE, and BPS induces persistence and even more severe phenotypes in sperm counts and motility in the F3 generation than in the F1 offspring. The expression of DNA and histone methyltransferases were transgenerationally increased by BPA, BPE and BPS exposure in both neonatal and adult testis. In F3 females, prenatal exposure to BPA, BPE, and BPS accelerated the onset of puberty and exhibited abnormal estrous cyclicity, and those females exhibited similar fertility problems as those in the F1 generation. However, BPA, BPE and BPS exposure did not affect neonatal follicular development such as germ cell nest breakdown or follicle numbers in the ovary on postnatal day 4. Taken together, our results suggest that prenatal exposure to BPA analogues, BPE and BPS, have transgenerational effects on male and female reproductive function in mice. Our findings suggest the hypothesis that transgenerational epigenetic alterations in germ cells may lead to reproductive disorders/dysfunction in the F3 generation.

No description available.

The Testis Specific Serine Kinases (Tssks) are a family of proteins that show testis and sperm-specific expression. Members of this family are most conserved among mammals, however there are homologs in vertebrates like birds and amphibians, chordates, and other invertebrates like insects and cnidarians. This specific expression suggests that these kinases are highly regulated. Analysis of murine and human Tssk1, Tssk2, and Tssk6 sequences show that these genes are comprised of one exon each, suggesting they are retrotransposons. The expression of these genes shows their importance, since many retrotransposons are silenced due to the foreign nature of the DNA, and knock-out mouse models have shown that these kinases are required for fertility. Understanding the properties of these kinases not only expands our scientific knowledge, but also lends itself to understanding fertility issues in men as well as being a contraceptive target. We looked at an epigenetic regulation factor, DNA methylation at CpG dinucleotides, to see if this caused the testis-specific gene expression we saw. Tssk2 and preliminary results from Tssk1 showed that there is no differential methylation at CpG dinucleotides or between tissues. Preliminary results for Tssk6 did show one site that may be differentially methylated, thus the tissue specific expression. We then started looking further into biochemically characterizing TSSK1 and TSSK2 to determine functionally relevant sites and new substrates. Understanding how these kinases function in sperm is relevant in our understanding in the fertility field and poses new targets for developing contraceptives.

This document only includes an excerpt of the corresponding thesis or dissertation. To request a digital scan of the full text, please contact the Ruth Lilly Medical Library's Interlibrary Loan Department (rlmlill@iu.edu).

Further investigation of possible regulatory pathway of NYD-SP24 demonstrated that the cell cycle of NYD-SP24 overexpressing cells was incompletely blocked at G2/M phase, and one of cell cycle-related genes, p21, shown to be an inducer of differentiation in different types of cell system, was found upregulated in a p53-independent manner, consistent with a role of NYD-SP24 in differentiation. (Abstract shortened by UMI.) / Spermatogenesis is a unique cell differentiation process consisting of three main phrases, namely mitosis, meiosis and postmeiosis. The differentiation of germ cells in the process involves distinct transcriptional programs, each under control of different transcription factor network. Many testis-specific transcription factors have been reported previously, however, few detailed studies have been done. This thesis describes the characterization and functional studies of a newly discovered testis-specific transcription factor, NYD-SP24, and the investigation of the possible regulatory pathway of NYD-SP24 in spermatogenesis. / The results demonstrated that in normal human tissues, NYD-SP24 was specifically and highly expressed in the testis but not in other somatic tissues, indicating its possible role in spermatogenesis. In the mouse model, mRNA and protein of NYD-SP24 mouse homolog gene (mNYD-SP24) were increased in the first wave of spermatogenesis. During the differentiation of germ cells, mNYD-SP24 mRNA and protein were confined to spermatocyte, round spermatid and spermatozoa. In hyperthermic mouse model, expression of mNYD-SP24 was decreased following the damage to germ cells by heat shock, but returned to normal level upon recovery of spermatogenesis. These results suggest the involvement of NYD-SP24 in spermatogenesis. / To identify the possible downstream targets of NYD-SP24, microarray and two-dimension gel technologies were performed in NYD-SP24 overexpressing cells and control cells. The results of microarray showed that 16 genes were upregulated and 4 genes were downregulated in NYD-SP24 overexpressing cells. In the two-dimension gel analysis, 12 protein spots were found to be altered significantly, with 8 increased and 4 decreased. Among these proteins, 3 were successfully identified by mass spectrometry. The nuclei localization in germ cells and the ability of NYD-SP24 to alter gene expression profile suggest that NYD-SP24 may be a testis-specific transcription factor, involved in gene regulation in spermatogenesis. / Zhu Hu. / "June 2005." / Adviser: Chan Hsiao Chang. / Source: Dissertation Abstracts International, Volume: 67-07, Section: B, page: 3607. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (p. 136-146). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract in English and Chinese. / School code: 1307.

Die Transkriptionsfaktoren der p53 Familie besitzen diverse Aufgaben sowohl während der Tumorentstehung als auch während der Entwicklung. In seiner ursprünglichen, evolutionär konservierten Rolle hat p63, ein Mitglied der p53 Familie, die Aufgabe die genetische Stabilität von Keimzellen zu gewährleisten und beeinflusst zudem die Keimbahnentwicklung. Mit Hilfe von „knockout“ (KO) Mausmodellen wurde bereits der Einfluss der 3 Transkriptionsfaktoren p53, p63 und p73 auf die weibliche und männliche Keimbahn untersucht. Während p53KO und p63KO Mäuse fruchtbar sind, führt der gemeinsame Verlust aller p73 Isoformen oder der Verlust der transkriptionell aktiven Isoform TAp73 hingegen zur Infertilität der Tiere. Weibliche TAp73KO Tiere sind deshalb infertil, da es bei ihnen zu einer Inhibition der Ovulation kommt, die Blastozystenentwicklung gestört ist und Spindeldefekte in den Oozyten auftreten. Eine Erklärung für die Infertilität von männlichen TAp73KO Mäusen wurde hingegen noch nicht gegeben. In der vorliegenden wissenschaftlichen Studie beschreiben wir eine bisher unbekannte Funktion des Transkriptionsfaktors TAp73: seinen Einfluss auf die Entwicklung und den Erhalt der adulten männlichen Keimbahn. Untersuchungen an Mausmodellen zeigen, dass „total“ p73KO und Isoform-spezifische TAp73KO Mäuse im Alter von 6 Wochen oder älter einen starken Verlust von sich entwickelnden Keimzellen in den testikulären Tubuli aufweisen. ΔNp73KO Tiere zeigen diesen Phänotyp nicht und ihre Testis Morphologie entspricht der der Wildtyp (WT) Tiere. Der Phänotyp der p73KO und TAp73KO Testes lässt sich durch eine starke Reduktion in der Zahl der späten Stadien der Spermatozyten und Spermatiden charakterisieren, wohingegen die Zahl der basalen Spermatogonien und pachytänen Spermatozyten des Keimepithels nicht betroffen ist (reguläre Zellzahl und Proliferation). Die Depletion von p73 oder TAp73 führt zu einer Anhäufung von apoptotischen und unreifen Keimzellen im Lumen des Epididymis, was auf eine atypische, verfrühte Ablösung der Keimzellen aus dem Keimepithel schließen lässt. Diese Beobachtung wird dadurch bestätigt, dass die Sertoli-Zellen, welche die Keimzellen stützen und ernähren, im Elektronenmikroskop verkürzte cytoplasmatische Arme sowie eine verstärkte Vakuolisierung aufweisen. Außerdem zeigt das Keimepithel große strukturelle Veränderungen, die dadurch gekennzeichnet sind, dass die Keimzellen nicht mehr dicht zu einem Epithel gepackt sind und die Sertoli-Keimzell-Verbindung gestört ist. Der Verlust der Epithelstruktur begleitet von der Reduktion der Keimzellen scheint das Ergebnis eines Defekts der Blut-Testis-Schranke (BTS) zu sein. Dies wird durch einen in vivo BTS Permeabilitätstest und die gestörte Morphologie der Sertoli-Sertoli „tight junctions“ deutlich. Der funktionelle Verlust der BTS führt zur Zerstörung der epithelialen Polarität und der Umgebung der sich entwickelnden Keimzellen. Somit wird auch die basal-luminal gerichtete Migration der Keimzellen entlang der Keimzelltaschen der Sertoli-Zellen verhindert. Der molekulare Hintergrund für das Ungleichgewicht in der Reorganisation der Zell-Zell Verbindungen wurde mit Hilfe der quantitativen Expressionsanalyse des Gesamtgenoms untersucht. Dabei wurde Testis Gewebe von TAp73KO mit WT Mäusen verglichen, um TAp73-Zielgene zu finden. Die Depletion von TAp73 führt zur Induktion von Genen, die an der Adhäsion und Zellmigration beteiligt sind; Integrine und Proteaseinhibitoren wie Timp1 und die Serpine zeigen eine Hochregulation in der Expression. Es ist bekannt, dass diese Proteine bei der Reorganisation von Zell-Zell Verbindungen im Testis involviert sind. Die Studie zeigt, dass TAp73 vorwiegend in den Keimzellen exprimiert wird und dass diese parakrin auf die Sertoli-Zellen zu wirken scheinen, da die Überexpression von TAp73 Zielgenen in isolierten Sertoli-Zellen in der Langzeitkultur zurückgeht. Zusammenfassend konnte zum ersten Mal gezeigt werden, dass die Funktion von TAp73 für die adulte Spermatogenese unerlässlich ist. TAp73 reguliert im Testis ein Transkriptionsprogramm von Genen, die an Adhäsion und Migration beteiligt sind, sichert den Zusammenhalt des Keimepithels und verhindert den frühzeitigen Verlust von Keimzellen. Somit wird die ungestörte Keimzellentwicklung ermöglicht. Umgekehrt führt die Depletion von TAp73 zu starken Defekten in Zell-Zell Verbindungen von sich entwickelnden Keimzellen im Keimepithel, was die Infertilität von p73KO und TAp73KO Mäusen erklärt.