Assessment of the degree of contamination of rat germ cell preparations using specific cDNA probes

Abstract

Recent reports showing a decrease in sperm count in men have brought new concerns about male infertility. Animal models have been widely used to provide some relevant information about the human male gamete, and extrapolations are made to men and to the clinical context. The present study assesses one of the methods used for separation of germ cells of the adult rat testis, namely centrifugal elutriation followed by density gradients (Percoll ® ). This method was chosen since it presents the best results for cell purity in separating germ cells from the rat testis. A comparison between continuous and discontinuous Percoll ® gradients was performed in order to identify the best type of gradient to separate the cells. Maximal cell purity was obtained for spermatocytes (81 ± 8.2%, mean ± SEM) and spermatids (84 ± 2.6%) using centrifugal elutriation followed by continuous Percoll ® gradients. A significant difference in purity was observed between elongating spermatids harvested from continuous Percoll ® gradients and from discontinuous gradients. Molecular analysis was used to assess cell contamination by employing specific probes, namely transition protein 2 (TP2), mitochondrial cytochrome C oxidase II (COX II), and sulfated glycoprotein 1 (SGP1). Molecular analysis of the samples demonstrated that morphological criteria are efficient in characterizing the main composition of the cell suspension, but are not reliable for identifying minimal contamination from other cells. Reliable cell purity data should be established using molecular analysis.

Identification of differentially expressed genes of primary spermatocyte against round spermatid isolated from human testis using the laser capture microdissection technique

“…The COX II mRNA has been reported to be strongly expressed in pachytene spermatocytes of the testes of adult and prepubertal rats. The highest level of expression occurs in germ cells at stages VII-VIII of the spermatogenic cycle [21] and reflects a differential expression between spermatocyte and spermatid [22]. The functional significance of the high expression of COX II mRNA in pachytene spermatocytes and its stage-dependent changes is not clearly showed.…”