Bo Yang¹, He Wang¹, Xiao-Kang Gao¹, Bao-Qi Chen¹, Yuan-Qiang Zhang², He-Liang Liu¹, Yong Wang¹, Wei-Jun Qin¹, Rong-Liang Qin¹, Guo-Xing Shao¹, Chen Shao¹

In the recent fifty years, the occurrence of azoospermia has an upward trend all over the world [1]. Generally speaking, the treatment of azoospermia is still a serious challenge due to insufficient understanding of the mechanism of its genesis. Increasing attention has been paid to the research on the molecular pathology of azoospermia and genes related to it. Spermatogenesis is a very complicated process and it is reasonable to assume that many genes may be potentially involved. Rap1A is a member of the Ras superfamily, that plays an important role in cellular regulation, including cell growth, survival, differentiation, cytokine production, chemotaxis and vesicle trafficking [2]. In this study, the Rap1A mRNA expression and its significance in the testes of normal and azoospermic subjects were evaluated.

From September to December 2002, 39 azoospermic males with an average age of 26.8 years and marital age of 2.2 years visited this Hospital. They had no obvious manifestations of gonad or pituitary hypofunction. Azoospermia was confirmed by at least 3 successive semen analyses. Upon testicular biopsy, 22 cases showed hypospermatogenesis and 17, germ cell arrest. Control tissues were collected from 10 young fertile males died of accidents with a normal testicular biopsy.

cDNA microarray slides with 4096 spots were purchased from the United Gene Technique Ltd. (China); it contains 16 genes as the negative, 16 genes as the blank and 96 housekeeping genes as the positive control. The classification of the genes is indicated in Table 1. Cy3-dUTP and Cy5-dUTP were purchased from the Amer-sham Phamacia Biotech Inc. (USA), Oligotex mRNA Midi Kit from the Quagen Inc. (USA), ScanArray 3000 scanner from the General Scanning Inc. (USA) and ImaGene 3.0 software from the BioDiscovery Inc. (USA).

A total of 128 genes was found to be differentially expressed in the azoospermic, but not in the normal testes. There were 56 up-regulated and 72 down-regulated genes (Tables 2 and 3). Of particular interest was the over-expression of Rap1A mRNA in the azospermic testis, being 6.5 fold greater than that in the normal testis. Figure 1 is one image of the hybridization arrays and Figure 2, the distribution of genes in the hybridization.

Figure 1. Results of cDNA microarray. Red points: up-regulated genes; blue points: down-regulated genes; yellow points: genes not changed.

Figure 3. Results of in situ hybridization. 3A (azoospermic testis): Strong hybridization signals found in both spermatogenic (big arrows) and Leydig cells (small arrows) (40). 3B (normal testis): Strong hybridization signal found only in Leydig cells (40).

Table 4. In situ hybridization results of spermatogenic cells. ^cP<0.01.

Male infertility is a major medical problem since it accounts for 30 % to 50 % of all conception failures [3]. Genetic alterations are likely to be involved in a significant percentage of severe infertility [4-6]. Nearly 7100 genes are expressed in human testis but few have been thoroughly investigated [7]. The relationship between the majority of those genes and the development of azoospermia has not been fully clarified [8]. Rap1A and its interaction partner Krit1 play important roles during mouse embryogenesis [9]. A high level of Rap1A expression has been detected in the spermatogenic cells of rat and is believed to participate in spermatogenesis [10]. Rap1A is a downstream target gene and is an integral component in the cAMP-Epac-Rap1 signaling pathway [11]. Rap1A may down-regulate the cell activation induced by cAMP-elevating agents [12]. The cAMP responsive element modulator protein (CREM) is a master-switch in the transcriptional response to cAMP [13]. In patients with predominant round spermatid maturation arrest, CREM expression was significantly reduced [14]. The cAMP response element-binding protein (CREB) also plays a physiological role in the Sertoli and Leydig cells [15]. The present paper is the first study reporting the up-regulated expression of Rap1A mRNA in the spermatogenic cells in human azoospermic testis. We assume that the direct or indirect effect of Rap1A on CREM or CREB may be one of the mechanisms contributing to hypo-spermatogenesis or germ cell arrest. It might inhibit spermatogenesis by its small GTPase pathway role.

Other differentially expressed genes identified in this study may also be involved in the regulation of spermato-genesis. For example, the Collapsin response mediator protein 1 (Crmp1) gene, a development-correlated and testis-specific gene, was down-regulated in the study. Crmp1 is expressed during spermatogenesis and is presumably involved in testicular germ cell differentiation and sperm formation in mice [16]. Another down-regulated gene identified in this study, Phosphomanno-mutase 1 (PMM1), is a metabolism-related gene. The PMM1 protein blocks sperm maturation probably by inhibiting the spermatogenic metabolism.

The insufficiency of the present study may include the relatively small sample number and the possibility of missing low abundance mRNAs.

The study was supported by the Population and Family Planning Special Grants (No.200102), Shaanxi, China. The authors are grateful to Dr. Xin-Hai Zhang for his assistance.