Histone H3 protein levels regulated by ubiquitylation-dependent proteolysis in sperm from infertile men

Abstract

The human sperm still retained 15% histone at most in its condensed nucleus after the replacement from histone to protamine in the nulear protein during spermiogenesis. Based on the important roles of sperm proteins in early embryo development, we proposed that altered expression level of sperm histone protein may lead to male infertility. In order to test this hypothesis and explore the related mechanism, semen samples from 115 male subjects between 30-40 years old attempting ICSI/IVF were collected and then assayed by semen analysis according to 2010 WHO standards. Sperm chromatin structure assay was also taken by flow cytometry after acridine orange staining. And the localization of histone H3 protein in sperm was detected by immunofluorescence while its expression level was analyzed using SDS-PAGE and Western blot. Lysine 48-linkage specific polyubiquitylated proteins and enzyme of 20S proteasome were also examined by SDS-PAGE and Western blot while the chymotrypsin-like activity of the proteasome was assayed using the fluorogenic substrate Suc-LLVY-AMC. The results showed that histone H3 protein expression was significantly increased in sperm from infertile men suffering from asthenospermia (A) or oligoasthenospermia (OA) compared with those from subjects with normospermia (N), even though there was no significant change in sperm chromatin structure. Lysine 48-linkage specific polyubiquitylated proteins were increased whereas the chymotrypsin-like activity of proteasome significantly decreased in sperm from A or OA infertile men. We conclude that histone H3 protein may be related to sperm abnormalities, which are causes of male infertility and that this increase may be caused by the decrease in the chymotrypsin-like activity of the proteasome to result in an increase in Lysine 48-linkage specific polyubiquitylated protein. (This project was supported by grants of the National Natural Science Foundation of China, No. 31071053).