SUN-229 The Role of Testosterone in Spermatogenesis: Lessons from Proteome Profiling of Human Spermatozoa in Testosterone Deficiency

Testosterone is required during spermatogenesis to mantain the blood-testis barrier and for meiosis, Sertoli-spermatid adhesion and sperm release. Aim of the study was to better understand the effect of hypothalamic-pituitary-testicular axis on sperm protein composition and function.12 male patients with low testosterone and LH levels were consecutively enrolled for this study. Five normogonadic men were enrolled as control group in the protocol. If spermatozoa were detectable in seminal samples, the the ejaculates were washed and the sperm cells were selected using 50% PercollTM. The recovered sperm cells were then suspended in PBS and subjected to a residual leukocyte depletion using CD45 Dynabeads® magnetic cell sorting. The absence of leukocytes was further confirmed both by microscopic observation after staining and by performing a RT-PCR for CD45. Protein solubilization was independently performed on each sperm sample. A comparative proteomic analysis was performed comparing two pools: controls vs hypogonadic patients. The TMTduplexTM Isobaric Mass Tagging Kit was used for the peptide labeling. MS/MS analysis was performed using a LTQ Orbitrap Velos. Data were processed using Proteome Discoverer 1.4.1.14 software, using very stringent filtering criteria. Proteins identified by SEQUEST were analyzed using the PANTHER classification system. Proteomic results have been confirmed by Western Blot. The following primary antibody were used: anti-Lactoferrin polyclonal antibody and anti-PIP polyclonal antibodyWe included in our study 5 oligozoospermic hypogonadic patients with a sperm count >10 x 10(6)/ml and without leukocyte contamination. 42 proteins were under-expressed in the pool of hypogonadic patients; 12 of these 42 increased proteins have been previously reported to be related with spermatogenesis, sperm motility, sperm-oocyte interaction and acrosomal reaction. The distribution of proteins for molecular function revealed that the protein pattern impaired by low blood testosterone is involved in protein binding and hydrolase. Twelve proteins have been over-expressed in the pool of hypogonadic patients, including markers of defective spermatogenesis. Western-blot analysis confirmed proteomic data. This is the first application of high resolution mass spectrometry -based proteomics aimed to reveal an array of proteins in spermatozoa reflecting an impairment of spermatogenesis and sperm function in male secundary hypogonadism.