Identification of novel genes expressed during spermatogenesis in stage-synchronized rat testes by differential display

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Abstract

The molecular mechanism regulating spermatogenesis at different developmental stages remains largely unknown. In a vitamin A-deficiency (VAD) rat model, five distinct histologically defined, stage-synchronized testes: (i) resting spermatogonia and preleptotene spermatocytes at Day 0 of post-vitamin A treatment (PVA); (ii) early pachytene spermatocytes at Day 7 PVA; (iii) late pachytene at Day 15 PVA; (iv) round spermatids at Day 25 PVA; and (v) elongated spermatids at Day 35 PVA were used to study gene expression profiles by mRNA differential display. Twenty-four differentially expressed cDNA fragments were identified and cloned; oligonucleotide sequence analyses indicated that there are 12 novel gene sequences, half of which share no apparent match in current GenBank/EMBL databases. Other 12 VAD clones share sequence homology to membrane channel and transport, transcription and translation, cell cycle and morphogenesis, inducer and transducer, surface or secreted glycoproteins or enzymes, and other miscellaneous molecules. Semi-quantitative RT-PCR analyses against different stages of VAD testes demonstrated: (i) restricted expression of VAD1.2 and 1.3 (novel) on Day 25 PVA when round spermatids form; (ii) escalating pattern of VAD12 (Cx43) in Sertoli cells; and (iii) relative constant levels of VAD4 (A5D3), VAD26.1 (ribonuclease), and VAD27 (GRP8) in spermatogenesis.

Section snippets

Materials and methods

Preparation of synchronized rat testes. Male, 20-day-old, Sprague–Dawley rats were fed with a diet deficient in vitamin A (ICN, Cleveland, OH) (Groups A–D, n=4) or normal rat chow (Group E, n=4) for 60–70 days. All animals were held in Sealsafe IVC individually ventilated cages (Tecniplast, Buguggiate, Italy). The ethical guidelines set forth by the University’s Committee on Using Live Animals for Teaching and Research were adhered to strictly. After histological verification of spermatogenic

Synchronization of VAD testes

After treating 20-day-old SD rats with a VAD diet for 2 months, the animals showed signs of vitamin A deficiency, which included huffed fur, hairlessness, weight loss or cessation of growth, and discharges from the eyes. The representative histology of the testes from VAD rats after designated PVA (retinol) treatment is shown in Fig. 1. At Day 0 PVA, the rat testis contained mainly type A spermatogonia and few preleptotene spermatocytes just before retinol replacement (Fig. 1A). This indicated

Discussion

To elucidate the molecular mechanism regulating the developmental process of male gametes, we employed the mRNA differential display [4] to dissect the molecular mechanisms governing spermatogenesis. This approach is a well-established method that is supplementary to the conventional subtractive hybridization technique and has been used to study the gene expression during spermatogenesis in mice [12], [13], rat [14], [15], [16], and male infertility in human [17]. The present study utilizes the

Acknowledgements

This research was supported by a public-funded grant (HKU7272/01M) to J.M.L. from the Research Grants Council of Hong Kong. We thank Miss Pauline Leung, Mr. Andy Leung, and Eric Leung for technical assistance and Mr. Cheuk Hung Lee for editorial comments.

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    Present address: Microbiology and Tumor Biology Centre, MTC Box 280, Karolinska Institute, Stockholm S-171 77, Sweden.

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