Analysis of the different molecular markers expression in bovine spermatogonial stem cells

• Grant number: 12/01998-2
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: April 01, 2012
• End date: December 31, 2012
• Field of knowledge: Agronomical Sciences - Veterinary Medicine - Animal Reproduction
• Principal Investigator: José Antonio Visintin
• Grantee: Alexandre Hinkelmann Bruno
• Host Institution: Faculdade de Medicina Veterinária e Zootecnia (FMVZ). Universidade de São Paulo (USP). São Paulo , SP, Brazil

Abstract

Spermatogonial Stem Cells (SSCs) are undifferentiated cells that can undergo both self-renewal and differentiation, giving rise to spermatozoa through spermatogenesis. There are many biotechnological applications for these cells, as: fertility reestablishment of an infertile animal, production of transgenic organisms, immortalization of a high value genetics of an animal by cryopreservation or in vitro culture of SSCs. Specific genes that are expressed in the SSCs can be used as molecular markers, increasing the efficiency of isolating a pure or highly enriched stem cell population and allowing the successful identification of these cells, as described in different animal species. The synthetized proteins from the expression of molecular markers are easily recognized by specific antibodies. The DBA is a lectine used as specific marker to the only precursor of bovine SSCs. The ± 6 integrine is found in the surface of this type of cells. The SSEA-4, until this moment, has only been used as a molecular marker for primates and humans SSCs. To reach better results in the process involving the SSCs, is extremely important to develop better methods for isolation, purification and identification of these cells. The purification by flow cytometer sorting is an efficient and quickly way to obtain the SSCs. However, this technique is limited because of the lack of knowledge about bovine SSCs surface markers. The quantity of SSCs also differs, decreasing according to animal aging. This way, some markers have the expression increased or decreased depending of the animal age.For this purpose, this study has as an objective to evaluate and compare the expression of 3 bovine SSC's molecular markers using testicles from pre-pubertal (n=10) and adults (age between 3 and 4 years old, n=10). The ± 6 integrine and SSEA4's expressions, and the affinity for the DBA lectine of the SSCs will be evaluate by flow cytometer and immunohistochemistry. It is expected that the cells provided by pre-pubertal animals have a higher expression of these markers than the adults. These results will be complementary and very important for our research group, increasing the comprehension of a new molecular marker expression (SSEA-4), only described in many species, but bovine, and helping to elucidate mechanisms underlying the process. (AU)