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Expression and significance of Rap1A in testes of azoospermic subjects

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

1Department of Urology, Xijing Hospital, 2Department of Histology and Embryology, Fourth Military Medical University, Xi'an 710033, China

Asian J Androl 2004 Mar; 635-40


Keywords: Rap1A; azoospermia; cDNA microarray
Abstract

Aim: To evaluate the Rap1A mRNA expression and its significance in the testes of normal and azoospermic subjects. Methods: A cDNA microarray that contained Rap1A and some other genes such as RBM, EIF1AY was used to identify the differential gene expression profiles between the normal and azoospermic testes. cDNA probes were prepared by labeling mRNA from azoospermic and normal testicular tissues through reverse transcription with Cy5-dUTP and Cy3-dUTP, respectively. The mixed cDNA probes were then hybridized with cDNA microarray (each containing 4096 unique human cDNA sequences). The fluorescent signals were scanned and the values of Cy5-dUTP and Cy3-dUTP on each spot were analyzed and calculated. In situ hybridization was employed to detect the expression of Rap1A in the testes of 10 fertile and 39 azoospermic subjects. Results: One hundred and twenty-eight differentially expressed genes were found to be possibly related to azoospermia, of which 56 were up-regulated and 72, down-regulated genes. The mRNA expression of Rap1A in the spermatogenic cells of azoospermic was stronger than that in those of the fertile testes. Conclusion: Rap1A may play certain roles in the development of azoospermia.

1 Introduction

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.

2 Materials and methods

2.1 Subjects

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.

2.2 Instruments and reagents

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).

Table 1. Gene classification

Classification

Number

Proto-oncogene and tumor suppressor gene

1

Ionic channel and transport protein

2

Cyclins

3

Xeno-stress response protein

4

Cytoskeleton and movement

5

Apoptosis correlated protein

6

DNA synthesis, repair and rearrangement

7

DNA binding, transcription and transcription factor

8

Cellular receptor

9

Immune correlated

10

Cellular signal and transducin

11

Metabolism

12

Protein translation, synthesis

13

Development correlated

14

Other (maybe unclear)

15

2.3 Probe for cDNA microarray preparation

The total RNA was isolated using modified single-step extraction technique. Briefly, frozen tissues were crushed and homogenized in solution D and 1 % mercap-toethanol. The supernatant was extracted with phenol:chloroform (1:1), phenol:chloroform (5:1) and NaAc (pH 4.5) successively. The supernatant was precipitated in an equal volume of isopropanol at -20 for 1.5 hours and then precipitated in LiCl for purification. mRNAs were purified using the Oligotex mRNA Midi Kit (Quagen Inc., USA). Fluorescent cDNA probes were prepared by reverse transcription and then purification. The mRNA from normal testicular tissue was labeled with Cy3-dUTP and those from the azoospermic tissue, with Cy5-dUTP. Equal amounts of the two probes were mixed, precipitated by ethanol and resolved in 20 hybridization buffer (5SSC + 0.4 % SDS, 50 % formamide, 5Enhardt's solution).

2.4 Hybridization

Samples for cDNA hybridization were collected from one azoospermic and one normal testes. The samples were snap-frozen in liquid nitrogen within 15 min after biopsy and stored at -80 . Microarray slides were pre-hybridized in hybridization buffer containing 0.5 mg/mL denatured clupeine DNA at 42 for 6 hours. After denaturation at 95 for 5 min, the probe mixture was added onto the pre-hybridized slides and sealed with cover glass. After hybridizing in the HybChamber (United Gene Holdings Ltd., China) at 60 for 15 hours, the slides were washed in solutions of 2SSC + 0.2 % SDS, 0.1SSC + 0.2 % SDS and 0.1SSC successively for 10 min each and then dried at room temperature.

2.5 Scanning and analysis

The slides were scanned with a ScanArray 3000 laser scanner (General Scanning Inc., USA) at two wavelengths to obtain fluorescence intensities for both dyes (Cy3 and Cy5). The original value of each spot was normalized by the values of the housekeeping genes. The fluorescence intensities of Cy3 and Cy5 were analyzed and the ratio was calculated with ImaGene 3.0 software (BioDiscovery Inc.). The intensities of the two fluorescent signals represent the quantities of the two tagged probes. The ratio of Cy5:Cy3 in certain spots on the slide represents the mRNA abundance of this gene expressed in the azoospermic versus the normal tissues.

2.6 In situ hybridization

The probe for hybridization was designed and synthesized by the Boster Co. (Wuhan, China) using the Primer 3 software with respect to the sequence released in NCBI. The specific oligonucleotide sequence of the probe is 5'-TTTGTTCAGGGAATTTTTGTTGAAAAATAT-3', 5'-GTATATTCTATTACAGCTCAGTCC-ACGTTT-3' and 5' TGTGCCTTTTTAGAATCTTCT-GCAAAGTCA-3'. In situ hybridization was employed to detect the expression of Rap1A in the testes of 10 fertile and 39 azoospermic subjects according to the kit guideline. The probe was terminally labeled with digoxigenin-dUTP with a 1:75 working dilution. The sections were incubated with phosphatase-labeled anti-digoxigenin antibody (1:400) at 37 for 2 hours. To assess the specificity of labeling, control sections were co-incubated with a 100-fold excess of unlabeled probe and the corresponding DIG-labeled probe. No labeling above the background was detected in these sections. Positive staining shows a brown coloration appearing in the cytoplasm.

Hybridization was repeated at least for 3 times to eliminate false positive results. Only those genes with the Cy5-dUTP/Cy3-dUTP ratio more than 2.0 or less than 0.5 were selected.

2.7 Statistical analysis

The Chi-square test and SPSS 10.0 for Windows software were used to analyze the results.

3 Results

3.1 Gene expression

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.

Table 2. Up-regulated genes in azoospermic testes.

Genbank ID

Ratio

Gene Definition

Classification

NM_004681

3.248

Homo sapiens eukaryotic translation initiation factor 1A, Y chromosome (EIF1AY), mRNA

14

AL050152

3.436

cDNA DKFZp586K1220

15

NM_005917

3.448

malate dehydrogenase 1, NAD (soluble) (MDH1),

13

AL117407

3.485

cDNA DKFZp434D2050 (from clone DKFZp434D2050)

15

NM_001284

3.531

adaptor-related protein complex 3, sigma 1 subunit (AP3S1)

1, 12

NM_000699

3.583

amylase, alpha 2A; pancreatic (AMY2A)

5, 10, 12

NM_004369

3.605

collagen, type VI, alpha 3 (COL6A3), transcript variant 1

11

NM_004824

3.705

chromodomain protein, Y chromosome-like (CDYL)

8

NM_015216

3.733

KIAA0433 protein (KIAA0433)

15

AF338650

3.767

PDZ domain-containing protein AIPC (AIPC) mRNA

15

NM_003272

3.88

transmembrane 7 superfamily member 1 (upregulated in kidney) (TM7SF1)

15

NM_016090

3.902

RNA binding motif protein 7 (RBM7)

1

NM_016122

3.922

NY-REN-58 antigen (LOC51134)

10

AB020719

3.925

mRNA for KIAA0912 protein

15

NM_015550

3.93

oxysterol binding protein-like 3 (OSBPL3)

15

NM_007005

3.986

BCE-1 protein (BCE-1)

15

NM_014584

4.075

ERO1-like (S. cerevisiae) (ERO1L)

12

NM_013436

4.247

NCK-associated protein 1 (NCKAP1)

6

NM_023037

4.28

hypothetical protein CG003 (13CDNA73)

1, 15

NM_016353

4.58

rec (LOC51201)

1

D87684

4.754

mRNA for KIAA0242 protein

15

NM_021998

5.494

zinc finger protein 6 (CMPX1) (ZNF6)

13

NM_002069

5.516

guanine nucleotide binding protein (G protein), alpha inhibiting activity polypeptide

1, 11, 15

AL117234

5.66

Novel human gene mapping to chomosome X, isoform of dbl (proto-oncogene)

1

NM_002884

6.512

RAP1A, member of RAS oncogene family (RAP1A), mRNA

11

Table 3 Down-regulated genes in azoospermic testes

Genbank ID

Ratio

Gene Definition

Classification

NM_005916

0.109

MCM7 minichromosome maintenance deficient 7 (S. cerevisiae) (MCM7), mRNA

3 ,12

NM_006411

0.127

1-acylglycerol-3-phosphate O-acyltransferase 1

2

 

 

(lysophosphatidic acid acyltransferase, alpha) (AGPAT1)

 

NM_015112

0.127

KIAA0807 protein (MAST205), mRNA

15

AK023420

0.146

cDNA FLJ13358 fis, clone PLACE1000078

10, 15

NM_005345

0.147

heat shock 70kD protein 1A (HSPA1A), mRNA

15

NM_007098

0.149

clathrin, heavy polypeptide-like 1 (CLTCL1), transcript variant 2, mRNA

14

AL080218

0.165

mRNA; cDNA DKFZp586N1323 (from clone DKFZp586N1323)

15

NM_015909

0.169

neuroblastoma-amplified protein (LOC51594), mRNA

13, 15

NM_001257

0.17

cadherin 13, H-cadherin (heart) (CDH13), mRNA

15

AB051499

0.174

mRNA for KIAA1712 protein, partial cds

11

NM_004868

0.188

glycoprotein, synaptic 2 (GPSN2), mRNA

11,15

NM_001313

0.192

collapsin response mediator protein 1 (CRMP1), mRNA

14,11

NM_014762

0.193

24-dehydrocholesterol reductase (DHCR24), mRNA

6

NM_003682

0.197

MAP-kinase activating death domain (MADD), transcript variant 4, mRNA

15,1

NM_004814

0.198

U5 snRNP-specific 40 kDa protein (hPrp8-binding) (HPRP8BP), mRNA

3,13

NM_007317

0.201

kinesin-like 4 (KNSL4), mRNA

5, 8 ,13

NM_012145

0.201

deoxythymidylate kinase (thymidylate kinase) (DTYMK), mRNA

12

NM_005387

0.202

nucleoporin 98kD (NUP98), mRNA

5,13

NM_006819

0.202

stress-induced-phosphoprotein 1 (Hsp70/Hsp90-organizing protein) (STIP1), mRNA

13

NM_006341

0.202

MAD2 mitotic arrest deficient-like 2 (yeast) (MAD2L2), mRNA

11

NM_002714

0.209

protein phosphatase 1, regulatory subunit 10 (PPP1R10), mRNA

11

NM_000400

0.209

excision repair cross-complementing rodent repair deficiency,

7

 

 

complementation group 2 (xeroderma pigmentosum D) (ERCC2), mRNA

 

NM_000252

0.221

myotubular myopathy 1 (MTM1), mRNA

5

NM_002676

0.222

phosphomannomutase 1 (PMM1), mRNA

15,12

NM_005545

0.225

immunoglobulin superfamily containing leucine-rich repeat (ISLR), mRNA

10

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

Figure 2. Scattered dot image in cDNA microarray. Points between two inclined lines: genes not changed; Points out of these 2 lines: genes changed.

3.2 In situ hybridization

Rap1A mRNA positive staining was detected in the Leydig cells of both the azoospermic and normal testes. mRNA could also be seen in the spermatogenic cells in some sections of the azoospermic testes, but not in the normal testes. Rap1A mRNA expression was detected in spermatogenic cells of 29 of the 39 azoospermic testicular sections and only in 2 of the 10 normal testicular sections (Figure 3 and Table 4, P<0.01).

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.

 

Normal testes

Azoospermic testes

Negative

8

10

Weak positive

1

2

Positive

1

27

Total

10

39

Positive rate

20.00 %

74.36 %c

4 Discussion

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.

Acknowledgements

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.

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Correspondence to: Dr. Chen Shao, Department of Urology, Xijing Hospital, the Fourth Military Medical University, Xian 710033, China.
Tel: +86-29+337 5321, Fax: +86-29-337 5321
E-mail: drboyung@yahoo.com.cn
Received 2003-05-26 Accepted 2004-02-12

 

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