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.
Highlights
Many hypotheses for the various processes leading to conception in vivo are extrapolated from studies on other eutherian mammals
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
Transitional protein 2 (TP2), mitochondrial cytochrome C oxidase II (COX II), and sulfated glycoprotein 1 (SGP1) are cDNA probes which recognize mRNA encoding specific proteins expressed in different germ cells
Summary
Many hypotheses for the various processes leading to conception in vivo are extrapolated from studies on other eutherian mammals. The objective of the present study was to investigate the accuracy of identifying morphological cell contamination of rat germ cells separated by a combination of methods (mechanical and enzymatic digestion, followed by centrifugal elutriation and a further step of Percoll® purification). COX II is present in mitochondria of all germ cells and Sertoli cells, but its highest expression occurs in pachytene spermatocytes [4]. These cDNA probes were employed to determine the purity of samples stained with Periodic Acid Schiff and hematoxylin (PAS + H), and viewed under the light microscope. Labeling of extracted mRNA with a 32P cDNA probe was detected using a PhosphorImager and the data were analyzed using computer software, reducing subjective bias
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Schedule a callMore From: Brazilian journal of medical and biological research = Revista brasileira de pesquisas medicas e biologicas
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