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Homologous genetic recombination is a central feature of meiosis in most sexually dividing organism. It leads to the establishment of new linkage relationships between genes and is crucial for the successful completion of meiosis. It is also necessary for a variety of important cellular events like immunoglobulin rearrangement, repair of chromosome damage, gene amplification, gene expression and sister chromatid exchange. Knowledge of the mechanisms of homologous recombination has come from extensive genetic studies on fungi. These studies have led to the formulation of various models which explain the genetic observations. Analysis of the fate of transfected DNA molecules into cells have revealed the existence of additional recombinational pathways in eukaryotes. The biochemistry of homologous recombination has mainly focused on the isolation and characterisation of the recombinase activity from various systems. The E. cold RecA protein remains the best characterised recombinase or pairing protein till date and its reactions in vitro, in particular the strand-transfer reaction, have laid the framework for isolating such proteins from eukaryotic systems. However, limited success has been achieved in the purification and characterisation of pairing activities from mammalian cells. The objective of the present study was to isolate and characterise a pairing activity from, a meiotic tissue of a mammalian system - the rat testis. Meiosis constitutes one of the important phases of the process of spermatogenesis. In rat, the various stages of spermatogenesis (the meiotic stages and spermiogenesis) can be broadly demarcated by the age of the rat. The pachytene stage of meiosis in rat is spread over nine days. Rat testicular nuclear extracts from animals, aged 38-42 days (the stages of spermiogenesis were absent and a significant proportion of the cells were in the pachytene stage during this period), were fractionated sequentially on phosphocellulose, heparin-agarose and ssDNA-cellulose columns. Fractions were tested for the ssDNA-dependent formation of slow-moving products on an agarose gel, using the strand-transfer away with M13mp19 RF III and ssDNA substrates. A partially purified fraction, which catalysed the formation of such products, was obtained from the ssDNA-cellulose column on eluting with 0.6 M KCI. In order to characterise the products formed by this partially purified fraction, they were analysed by electron microscopy. The majority of the observed structures represented multimers of the linear substrate. A significant proportion of the products resembled forked (Y-branched) structures and paired (duplex-duplex paired and 6s-duplex paired) structures. Length measurement of the multimers indicated the presence of a ligase activity in the partially purified fraction. The arms of the Y-branched structures were equal in length. The length of the molecule from the end of the stem to the end of either of the arms was the same as that of a monomer. The Y-branched structures were duplex in nature along their entire length. Exonucleases were not detected in the fraction; hence, these structures might actually represent paired structures. Typical r- and &-structures were not observed. The EM studies indicated that the slow-moving products, observed on the gel, might be ligated structures. The mobility of these products were found to be similar to those formed by a standard ligase - the T4 DNA ligase. Restriction digestion of the junctions of the ligated products revealed that the ends were intact and there was no nucleotide loss, as seen in the case of ligases involved in nonhomologous recombination. In order to characterise the type of ligase present, radiolabelled a-ATP was used to form the ligase-AMP adduct which was then separated by SDS-PAGE. Autoradiography revealed the presence of a major 100 kDa polypeptide (most probably DNA ligase I or 111), with a minor 65 kDa polypeptide (most probably DNA ligase 11). The latter, in turn, was found to be enriched in the ssDNA-cellulose fraction eluting with 0.8 M KC1. The formation of the ligated products was strictly dependent on the presence of ssDNA. Other characteristics of the ligase activity included the substitution of ATP by UTP. The ligase present in the 0.8 M fraction was found to be inhibited by ssDNA. Experiments to test the heat - stability of the products revealed the presence of a ssDNA-aggregating activity in the partially purified fraction. This aggregation was found to be specific for linear ssDNA. In the strand-transfer assay, the first step involves the transfer of the ssDNA circle on the linear duplex substrate accompanied by the partial displacement of the non-complementary strand of the duplex. It was reasoned that the linear end generated (partially-displaced strand) would be aggregated by the linear ssDNA-aggregating activity, thereby preventing the progression of the reaction and accounting for the absence of V - and a-structures under the EM. The formation of such aggregates was therefore monitored on the gel, using radiolabelled linear dsDNA. The formation of theme aggregates was found to be dependent on ssDNA and homology. ATP was essential for the reaction, though ATP hydrolysis was not necessary, since ATPW could substitute for ATP in the reaction. This suggested the presence of a pairing activity in the partially purified fraction. The aggregating activity was found to be enriched in the 0.5M KCl-fraction from the ssDNA-cellulose column. The molecular weight of the enriched polypeptide was found to be similar to that of histone Hl. Western blot analysis of the polypeptide with monospecific antibodies against histone Hl a and a comparison of the protein fingerprinting pattern of the polypeptide with that of histone Hla revealed the identity of the polypeptide to be histone Hla. Formation of stable protein-free aggregates of linear ssDNA was found to be a common property of histone H1 subtypes. This aggregation of linear ssDNA (non-complementary) did' not require ATP or Mg2+. Purified hietone H1 subtypes, however, could not cause homology-dependent aggregation of the linear duplex substrate. This study thus indicates the presence of a pairing activity and a ligase activity in a partially purified fraction from rat testicular nuclear extracts. Unlike other eukaryotic pairing activities characterised so far, this preparation is free from exonucleases. The homology-dependent duplex DNA aggregates, observed in this study, may therefore represent intermediates actually generated by the pairing activity.

Thesis (M. Phil.)--University of Hong Kong, 2001. / Includes bibliographical references (leaves.

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. / text

Spermatogenesis 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. / text

published_or_final_version / Zoology / Master / Master of Philosophy

published_or_final_version / Zoology / Master / Master of Philosophy

published_or_final_version / Zoology / Master / Master of Philosophy

Spring-born ram lambs were used to examine: (1) sexual maturational changes in LH, FSH and prolactin (PRL) secretion, testicular gonadotropin receptors, and testicular size and function; (2) predictability of yearling ram reproductive function from juvenile testicular size and reproductive hormone measurements. Despite continuous increases in testis size, serum LH-profile characteristics became greatest between 2 and 4 months and declined thereafter. However, LH-peak frequency increased by about 2-fold between 6 and 7 months; this was associated with marked increases in testosterone (T) secretion and spermatogenic function. Mean FSH and PRL levels were maximum at 2 months and 3 to 5 months, respectively, and decreased thereafter. Increases in steroidogenic and spermatogenic function were due partly to increases in testicular content of LH and FSH receptors. Yearling ram testis size and spermatogenic function were predictable from testis size at 5 to 6 months, neonatal (50 days) secretion of LH and T, and pubertal (150 days) secretion of T. However, combinations of testicular size and reproductive hormone measurements provided greater predictive power.

Thesis (M. Phil.)--University of Hong Kong, 1999. / Includes bibliographical references (leaves 67-76).