R C Strickler — Page 5

PubMedDate not supplied

The effect of tumor necrosis factor on human sperm motility in vitro.

Tumor necrosis factor (TNF alpha) is present in elevated levels in peritoneal fluid from infertile women with endometriosis. The effect of TNF alpha on human sperm motility in vitro was evaluated utilizing peritoneal fluid from infertile women with minimal endometriosis containing 0, 100, 400, or 800 U of TNF alpha/ml as well as similar concentrations of recombinant human TNF alpha. No reduction in progressive and total motility was found at recombinant TNF alpha concentrations of 100 U ml. However, 500 and 1000 U of recombinant TNF alpha/ml caused a significant reduction in progressive and total sperm motility after 4 and 21 hours of incubation when compared with controls. Similarly, peritoneal fluid containing 100 U of TNF alpha/ml did not significantly reduce progressive and total sperm motility after either 4 or 21 hours of incubation; but peritoneal fluid containing 400 U of TNF alpha/ml reduced progressive sperm motility after 4 and 21 hours and total sperm motility after 21 hours of incubation. Peritoneal fluid with a TNF alpha concentration of 800 U/ml caused a significant reduction in both progressive and total sperm motility after 4 and 21 hours when compared with controls of TNF alpha-negative peritoneal fluid. The addition of polyclonal rabbit anti-TNF alpha antibody or 30-min heat inactivation at 56 C of TNF alpha-positive peritoneal fluid reversed the inhibitory effect on sperm motility. The ability of TNF alpha to cause a significant reduction of sperm motility in vitro suggests that this may be a mechanism for the infertility observed in women with minimal endometriosis.

Ascitic Fluid↗

PubMedDate not supplied

Affinity labeling in the presence of the reduced diphosphopyridine nucleotide NADH identifies peptides associated with the activities of human placental 3 beta-hydroxy-delta 5-steroid dehydrogenase/isomerase.

OBJECTIVE: We sought to identify peptides associated with activity in the primary structure of human placental 3 beta-hydroxy-delta 5-steroid dehydrogenase/isomerase (3 beta-HSD/isomerase). METHODS: Purified human placental 3 beta-HSD/isomerase was affinity-radioalkylated by 2 alpha-bromo [2'-14C]acetoxyprogesterone (2 alpha-[14C]BAP) in the presence or absence of the reduced diphosphopyridine nucleotide, NADH. NADH protected both 3 beta-HSD and isomerase from inactivation by 2 alpha-[14C]BAP. Tryptic peptides of unprotected and NADH-protected radioalkylated enzyme were purified by high-pressure liquid chromatography. The amino acid sequence of each radiolabeled peptide was determined and localized within the cDNA-derived primary structure of the enzyme. RESULTS: According to the sequence analyses, NADH shifted radioalkylation by 2 alpha-[14C]BAP away from the Arg-250 peptide (251GQFYYISDDTPHQSYDNLNYTLSK274) and toward the Lys-135 tryptic peptide (136EIIQNGHEEEPLENTWPAPYPHSK159). Based on amino acid analysis to quantitate radioactivity incorporated per nmol peptide, NADH decreased the radiolabeling of His262 in the Arg-250 peptide by 8.2-fold. His142 in the Lys-135 peptide was radiolabeled by 2 alpha-[14C]BAP only in the presence of NADH. CONCLUSIONS: We have previously reported that the substrate pregnenolone blocks the inactivation of 3 beta-HSD by 2 alpha-[14C]BAP through the protection of His262 in the Arg-250 peptide. Protection by NADH against the inactivation of isomerase as well as 3 beta-HSD is evidence that 2 alpha-[14C]BAP binds at the active sites of both enzyme activities. Because the same Arg-250 peptide has been affinity-alkylated in studies that targeted each of the two activities, we propose that the 3 beta-HSD and isomerase reactions are catalyzed in this region of the enzyme protein.

Amino Acid Sequence↗