TIMP2 peptide

Conditional deletion of Gata4 in Sertoli cells (SCs) of adult mice has been shown to increase permeability of the blood-testis barrier (BTB) and disrupt spermatogenesis. To gain insight into the molecular underpinnings of these phenotypic abnormalities, we assessed the impact of Gata4 gene silencing in cell culture models. Microarray hybridization identified genes dysregulated by siRNA-mediated inhibition of Gata4 in TM4 cells, an immortalized mouse SC line. Differentially expressed genes were validated by quantitative RT-PCR analysis of primary cultures of Gata4(flox/flox) mouse SCs that had been subjected to cre-mediated recombination in vitro. Depletion of GATA4 in TM4 cells and primary SCs was associated with altered expression of genes involved in key facets of BTB maintenance, including tight/adherens junction formation (Tjp1, Cldn12, Vcl, Tnc, Csk) and extracellular matrix reorganization (Lamc1, Col4a1, Col4a5, Mmp10, Mmp23, Timp2). Western blotting and immunocytochemistry demonstrated reduced levels of tight junction protein-1, a prototypical tight junction protein, in GATA4-depleted cells. These changes were accompanied by a loss of morphologically recognizable junctional complexes and a decline in epithelial membrane resistance. Furthermore, Gata4 gene silencing was associated with altered expression of Hk1, Gpi1, Pfkp, Pgam1, Gls2, Pdk3, Pkd4, and Ldhb, genes regulating the production of lactate, a key nutrient that SCs provide to developing germ cells. Comprehensive metabolomic profiling demonstrated impaired lactate production in GATA4-deficient SCs. We conclude that GATA4 plays a pivotal role in the regulation of BTB function and lactate metabolism in mouse SCs.

The present study was aimed at evaluating the seminal plasma proteins and sperm parameters of Curraleiro Pe-Duro bulls. Semen was collected from 10 bulls by electroejaculation, and sperm parameters were evaluated in fresh and frozen-thawed semen. Seminal plasma proteins were analyzed by 2-D SDS-PAGE and mass spectrophotometry. Tools in computational biology were used to generate bioinformatic knowledge and evaluate gene ontology, protein-protein interactions, phylogenetic trees and multiple sequence alignments. Sperm motility in fresh and frozen thawed semen was 78.8 +/- 1.8% and 21.2 +/- 1.6%, respectively. Pearson's correlations were evaluated (p < 0.05). Sperm motility and vigor in fresh semen were correlated with clusterin, TIMP2 and cathepsin S (r = 0.64-0.71) and sperm defects were related to inhibitor of carbonic anhydrase and BSP 5 (r = 0.78-0.80). Clusterin, BSP 5, alpha-enolase, creatine kinase M-type, glyceraldehyde-3-phosphate dehydrogenase, BSP 3, albumin, and 5'-nucleotidase and legumain were correlated with acrosome intact live sperm (r = 0.80-0.64). Associations were detected between sperm vigor and spermadhesin 1 (r = -0.89), and between sperm defects in fresh semen and spermadhesin 1 and clusterin (r = -0.81). Sperm motility in frozen-thawed semen was associated with BSP 1, spermadhesin 1, clusterin and spermadhesin Z13 (r = 0.64-0.85). The percent of motile sperm after freeze-thawing was negatively correlated (r = -0.64) with the amount of spermadhesin 1 in the seminal, plasma. Based on in silica analysis, TIMP2 interacted with BSP1, BSP3, BSP5 and metalloproteinases. Molecular functions of proteins associated with sperm parameters were binding, catalytic activity and enzymatic regulation. Amino acid sequences of spermadhesin 1 and BSP 1 from Bos taurus, and other domestic species were similar. Phylogenetic tree analysis demonstrated that clusterin from Bos taurus was related to Ovis aries and domains of clusterin, spermadhesin 1, BSP 1 and inhibitor of carbonic anhydrase were