Human Spermatogenesis : Differential Gene Expression And Regulation
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.