Spermatogenesis is a complex process of male germ cell development in which the diploid spermatogonia undergo series of mitotic divisions and differentiation steps culminating into the preleptotene spermatocytes which then enter into the meiotic prophase following a single replication cycle. This phase is characterized by meiotic recombination and is followed by reduction division resulting in haploid round spermatids. These cells then undergo extensive morphological and nuclear changes to form a unique cell, spermatozoa. This entire germ cell differentiation process occurs in a unique environment present inside the seminiferous tubules which is created by the Sertoli cells, the somatic cells in the tubules by forming junctions with each other thus providing unique milieu to the developing germ cells. Within the tubule, the germ cells are also arranged in an orderly manner called stages of spermatogenesis indicating a complex mechanism of germ cell differentiation. This complex differentiation process is a consequence of developmentally and precisely regulated differential gene expression (Eddy, 2002). Unraveling the molecular mechanisms involved in the male germ cell development is an uphill task due to the complexity of the cyto-architecture existing in the tubules and further complicated by unavailability of established germ cell lines and lack of cell culture systems that facilitate the germ cell differentiation in vitro. Comparative gene expression analysis of spermatogenesis in nematodes, flies and rodents revealed highly conserved transcriptomes and have provided some insights into its regulation (Schlecht and Primig, 2003). However, these data fail to represent the genetic and biological complexity of human spermatogenesis. In the present study, an attempt has been made to identify the genes that are differentially expressed in human tetraploid and haploid germ cells and to investigate the mechanism of regulation of the genes expressed in the post-meiotic germ cells. To identify the cell type specific genes, expression profiling of the human tetraploid and haploid germ cells was carried out using cDNA microarray. These cells were purified by centrifugal elutriation (Meistrich et al., 1981; Shetty et al., 1996) from the human testicular tissues obtained from the patients undergoing orchidectomy as treatment for prostate cancer. Purity of the enriched population of the germ cells was ascertained by DNA flow cytometry and by RT-PCR analysis using the known cell-specific markers and ruling out contamination of the somatic cells such as the Sertoli cells and the Leydig cells. Microarray experiments were carried out with the RNA isolated from each cell type and labeling the cDNA with Cy3/Cy5-dUTP and hybridizing to the human 19K array chip (University Health Network, Toronto, Canada) containing 19,200 ESTs. Two independent hybridizations were carried out using the germ cells isolated from two individuals and the microarray data were analyzed using Avadis 3.1 software (Strand Life Sciences, India). Analysis of the microarray data following normalization revealed that 723 transcripts showed higher expression in the meiotic cells whereas 459 transcripts showed higher expression in the post-meiotic germ cells. Microarray data were validated further by RT-PCR analysis of some of the differentially regulated genes. The DAVID analysis (Database for Annotation, Visualization and Integrated Discovery; http://david.abcc.ncifcrf.gov/) of these genes revealed that many genes associated with diverse functions and pathways appeared to be differentially expressed in both cell types. It is known that many biological systems exhibit distinct temporal gene expression profiles during different processes related to cell cycle, stress response and differentiation. Similarly, there are sets of genes, which respond to specific stimuli, appear to be synchronized in their expression. Such ‘synexpressed’ genes have been shown to be regulated by common transcription regulatory processes and have similar upstream transcription factor binding sites (Niehrs and Pollet, 1999). And therefore, having identified genes that appeared to be differentially expressed in the haploid and the tetraploid germ cells, attempt was made to analyze transcription factor binding sites in the promoter of those genes. In silico promoter analysis of several genes showing higher post-meiotic expression was carried out in order to identify the common regulatory motifs. Analysis of the annotated promoters (available from Eukaryotic Promoter Database; http://www.epd.isb-sib.ch/) of about forty genes highly expressed in the post-meiotic germ cells using TFSEARCH program (http://www.cbrc.jp/ research/db/TFSEARCH.html) confirmed that many genes had common transcription factor binding sites. Interestingly, almost all of the analyzed genes harbored SRY (Sex determining Region in Y)/SOX (SRY-box containing) binding motifs. In addition, the promoters of genes such as Protamine 1 and 2, Transition protein 1 and 2, A kinase (PRKA) anchor protein 4 that are known to be expressed post-meiotically, also harbor SRY binding sites suggesting that SRY may be one of the key regulators of the post-meiotic gene expression. SRY is a HMG-box containing member of Sox-family of architectural transcription factors. SRY is encoded by the Y chromosome and was first discovered as the testis-determining factor in mammals (Koopman et al., 1991). SRY HMG-box is eighty amino acids conserved motif that binds to the minor groove of the DNA in a sequence-dependent manner resulting in its bending and thus regulating the gene expression. The RT-PCR analysis of the human haploid and tetraploid germ cells showed very high expression of SRY in the post-meiotic cells further suggesting key role of SRY in the post-meiotic gene regulation. Role of SRY in the post-meiotic gene expression was investigated by determining the effect of SRY on human Protamine 1 (PRM1) promoter, a gene known to be exclusively expressed in the round spermatids and as indicated above, harbors many SRY binding sites in its promoter. SRY cDNA was cloned into the mammalian expression vector, pcDNA3.1 and the PRM1 promoter was cloned into the promoter-less pGL3 Basic vector upstream of the Luciferase reporter gene. Co-transfection of both constructs led to up-regulation of PRM1 promoter activity in both HeLa cells and LNCaP cells in a dose-dependent manner clearly demonstrating the role of SRY in PRM1 gene expression. Sequential deletion of the SRY binding sites in the PRM1 promoter led to the identification of the critical SRY binding motif important for SRY-mediated upregulation of PRM1 gene expression. This was confirmed by demonstrating in vitro binding of SRY to its critical binding site in the PRM1 promoter by gel shift assay using the nuclear extract of the HeLa cells transfected with FLAG-tagged SRY. The human SRY is an atypical transcription factor that binds DNA through its HMG, but unlike the mouse Sry and other Sox proteins, lacks the trans-activation domain and therefore requires other factors for its actions. Recently, the glutamine-rich, zinc-finger containing transactivator, Specificity protein 1 (Sp1) has been identified as one such interacting partner (Wissmuller et al., 2006). RT-PCR analysis showed that human SP1 is highly expressed in the haploid germ cells and could up-regulate PRM1 expression which harbors two SP1 binding sites in its promoter. When co-transfected, SRY and SP1 up-regulated PRM1 promoter in co-operative manner suggesting that SP1 may act in coordination with SRY in regulating PRM1. All these data taken together clearly signifies a critical role of SRY in post-meiotic germ cell gene expression. Recent reports suggest that SRY is also expressed in the adult human brain and prostate. However, its role in these tissues is not clearly understood. The Y chromosome has been shown to be frequently lost in prostate cancer and has also been shown to suppress the tumorigenicity of the PC-3 prostate cancer cells suggesting that the Y chromosome encoded genes may be involved in tumor suppression. SRY can physically interact with the androgen receptor (AR) and thereby interfere in its downstream signaling (Yuan et al., 2001). Since the prostate tumors show initial androgen-dependency, it was interesting to look at the role of SRY in the prostate cancer. To decipher the effect of SRY on the androgen-responsive LNCaP cells, stable clones of LNCaP expressing human SRY were generated. These clones showed significant decrease in growth in response to 5α-dihydrotestosterone (DHT) compared to the vector transfected or the parental LNCaP cells. In the soft agar colony formation assay, the SRY expressing LNCaP formed smaller colonies as compared to the controls in presence of DHT. Preliminary experiments in male athymic nude mice demonstrated that one of the SRY expressing clones showed reduced tumor growth compared to control cells suggesting that SRY may play a role in prostate cancer progression by decreasing the sensitivity to DHT. To summarize, the present study has identified several genes differentially expressed in the human haploid and tetraploid germ cells and further showed that SRY may be one of the key regulators of the post-meiotic gene expression.

Call number: LD2668 .T4 1963 N48 / Master of Science

Call number: LD2668 .T4 1966 F245 / Master of Science

published_or_final_version / abstract / Zoology / Doctoral / Doctor of Philosophy

published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

No description available.

No description available.

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&lt / 0.05) were evident between the three groups. No significant differences (P&gt / 0.05) were found between the groups with respect to the APO-Direct assay. A significant difference (P&lt / 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.

Specification of a germ cell lineage and appropriate maturation are essential for the transfer of genetic information from one generation to the next. Germ cells form from pluripotent precursor cells that migrate into the gonadal ridge and undergo commitment to either the female or male lineage. In the fetal ovary, germ cells enter meiotic prophase I, then arrest at the diplotene stage; in the testis germ cells do not begin meiosis until puberty. Abnormal differentiation of germ cells can result in malignant transformation. Somatic cells play a key role in modulating the developmental fate of the germ cells. Research into germ cell development during fetal life has almost exclusively focused on studies in rodents, but we, and others, have reported several fundamental differences in the expression of germ cell specific markers in the human compared with the mouse. The studies described in this thesis have investigated germ cell-specific gene expression and the possible impact of the somatic cells during development. This was achieved by studying human fetal gonads obtained during the 1st and 2nd trimesters of pregnancy and through the use of both wild-type and mutant mouse ES cell lines. Studies on germ cells in the human fetal testis have extended the findings of others, and confirmed that germ cell populations at different stages of maturation co-exist in the human fetal testis, a situation that is in contrast to that in rodents. For example expression of M2A and AP2γ was restricted to the OCT4-positive gonocyte population, while VASA and NANOS1 were localised exclusively to the to the OCT4-negative prespermatogonia. DAZL was expressed in both populations. Analysis also revealed that both the gonocyte and prespermatogonial populations proliferate throughout the 2nd trimester. Recent studies have implicated retinoic acid (RA) in the control of meiotic entry in germ cells of the fetal mouse ovary. In this study we demonstrated for the first time that two genes implicated in the action of RA in mouse gonad, STRA8 and NANOS2, are also expressed in a similar sexspecific- manner in the human fetal gonads, and that the RA receptors are present in both somatic and germ cells suggesting that RA may regulate germ cell function in the human as well as the mouse. However, whilst the mesonephros appears to be the primary site of RA synthesis in the mouse our initial studies indicate that in the human the gonad itself may be a more likely site of RA biosynthesis. In the fetal mouse testis, RA is degraded by the enzyme Cyp26b1 present in the somatic cells and germ cells do not enter meiosis, our novel findings suggest that CYP26B1 is more abundant in the human fetal ovary than the testis, suggesting that meiotic entry may be controlled by an alternative signalling pathway in the human. One of the methods that can aid our understanding of somatic cell gene expression in the gonad is in vitro culture. To date, there have been no published reports of the successful in vitro culture of somatic cells from the human fetal testis. In the current study, populations of human somatic cells were dissociated and maintained in vitro and characterised. Analysis demonstrated that cells expressing mRNAs characteristic of Sertoli cells, Leydig cells and peritubular myoid (PTM) cells were present initially, but long-term culture resulted in downregulation in expression of mRNAs specific for Sertoli cells and Leydig cells, suggesting that these cells either failed to survive or underwent alterations to their phenotype. In contrast PTM/fibroblast cells proliferated in vitro and initially maintained androgen receptor expression. These cultures therefore hold promise for studies into the signalling or cell-cell interactions in testicular somatic cells especially those relevant to the PTM population. Several studies have claimed differentiation of putative germ cells from ES cells. In the current study, analysis of mouse ES cell lines has expanded on results showing that ES cells and early germ cells express a number of genes in common. Kit signalling was shown to be important for ES cell survival as they differentiate although expression of Kit was heterogeneous. We also demonstrated that ES cells that did not express Kit displayed a decreased expression of the early germ cell genes Blimp1, Fragilis and Stella, implicating Kit signalling in the control of germ cell-associated gene expression in ES cells. This may be important to future studies optimising germ cell derivation from ES cells. In conclusion, this study has demonstrated important differences in protein expression patterns in germ cells of the human fetal testis compared to the mouse, and has raised questions about whether the proposed mechanism controlling meiotic entry of germ cells in the mouse can be applied to the human. The establishment of a system for culturing human fetal gonadal somatic cells may lead to further understanding of gene expression and development in the human fetal testis, and data suggest that the Kit/Kitl signalling system may influence germ cell gene expression in mouse ES cells.