We use spermatogenesis as a model to study the genetic regulation of germ cell development and differentiation. We also study the molecular genetics of aberrant sexual development in human caused by mutations in the luteinizing hormone receptor

Genetic Regulation of Spermatogenesis
Wu, Pang, Baxendale, Rennert, Chan in collaboration with Chen,a Li,b Dym,c Sud
Spermatogenesis is a tightly regulated process; it is characterized by spermatogonial mitotic expansion and differentiation of stem cells, meiosis of spermatocytes, and transformation of spermatids to spermatozoa. Specific changes occur in the cell population, with each step resulting in a more differentiated state. Knowledge of the factors that result in the differentiation of germ cells will be helpful in enhancing our understanding of the biological changes that occur during human differentiation. One of the goals of this research is to delineate the genes that regulate spermatogenesis by using expression gene profiling techniques, including DNA microarrays and serial analysis of gene expression (SAGE).

We profiled the expressed genes of three germ cell populations, namely, type A spermatogonia (spermatogonial stem cell), pachytene spermatocytes, and round spermatids. Type A spermatogonial cells were isolated from the testes of six-day-old mice while spermatocytes and spermatids were isolated from the testes of 60-day-old mice by using the STAPUT technique. Highly purified total RNA isolated from the germ cells was reverse-transcribed and labeled with 33P-dCTP. Labeled cDNA was hybridized to mouse GeneFilters from ResGen with 5,148 cDNA elements. Duplicate microarray hybridizations were performed on two different preparations of germ cells at the same stage of spermatogenesis. Only genes that gave comparable signals between duplicate microarrays were considered to be truly expressed. This procedure identified 79 cDNAs that can be clustered into 12 changing patterns among the cells at the three stages of spermatogenesis studied. Among these cDNAs, only 12 are known mouse genes, 16 are expressed sequence tags (ESTs) with similarity to known genes, and the rest undefined ESTs. The 10 most abundant cDNAs in type A spermatogonia are ESTs. The large number of ESTs underscores the fact that little is known about the genetic regulation of spermatogenesis and suggests leads for further investigation. Germ cell RNA was also used to generate SAGE libraries. So far, we have performed SAGE with pachytene spermatocytes. Preliminary analysis of 18,500 tags yielded classes of transcripts with distinctly different abundance. Among the 23 most abundant transcripts (abundance of each gene was >0.2 percent of transcriptome) (Table 1), there were three tags with no match in the mouse SAGEmap database, again suggesting that our knowledge of genes expressed in germ cells is very limited.

Table 1: The most abundant tags (>0.2% of transcriptome)

We will continue to analyze germ cell transcriptomes at different stages of spermatogenesis and study the change of the transcriptome of germ cells under different experimental conditions that affect spermatogenesis. We will also investigate the effect of abrogation of some of the selected genes on spermatogenesis. In addition, we plan within the coming year to generate a SAGE database for mouse germ cell genes.

Physiological and Genetic Effects of Constitutively Activated Luteinizing Hormone Receptor
Zhang, Pang, Wu, Rennert, and Chan in collaboration with Leschek,e Martin, and Sud
Constitutively activating mutations of the human luteinizing hormone/chorionic gonadotropin hormone receptor (LHR) causes familial male-limited precocious puberty (FMPP). We have identified two FMPP patients who developed testicular neoplasia. To study the impact of constitutive activation of the LH/hCG signaling pathway on spermatogenesis and sexual development and the potential tumorigenic effect of a constitutively activated LHR, we have generated an in vitro cell model and are in the process of generating a transgenic animal model. The mouse Leydig cells, MA-10, were transfected with an LHR carrying an activating mutation. The profile of expressed genes in cells expressing the mutated LHR was compared with that of control cells. Preliminary studies showed several genes associated with cell proliferation, e.g., CDC23, phosphatidylinositol transfer protein alpha, and a gene known to be located in the multiple endocrine neoplasia type 1 site were up-regulated, and two cell differentiation genes, namely, the Slit2 gene and the inducible GTPase Ral-B gene, were down-regulated in MA-10 cells expressing a constitutively activated hLHR (Table 2). Interestingly, two genes known to be involved in spermatogenesis, namely, Vav-T gene and tpr1, were also down-regulated in cells expressing the mutated LHR. We are now repeating this study with LHR carrying different activating mutations and by using a more extensive mouse cDNA microarray. In addition to the in vitro study, we are generating constructs of LHR carrying activating mutations for introduction into mouse ES cells to generate a transgenic animal model of FMPP for the study of the impact of constitutively activated LHR gene on spermatogenesis and sexual development.

Table 2: Genes affected by hLHR-Asp578Tyr

Up-Regulated Genes CDC23 Phospholipase C b3 neighboring gene located in the MEN1 site Phosphatidylinositol transfer protein alpha Down-Regulated Genes Neurogenic extracellular slit protein (Slit2) Inducible GTPase Ral-B mRNA Vav-T gene