Detection of protamine 2 in bovine spermatozoa and testicles

Abstract

BackgroundSperm DNA integrity is crucial for transmission of genetic information to future generations and DNA damage can occur during chromatin packaging. Chromatin packaging involves the replacement of somatic nucleosomal histones by nuclear proteins called protamines. Protamine 1 (PRM1) is transcribed and translated in spermatids of all mammals; however, protamine 2 (PRM2) is transcribed in low levels in spermatids and it is not yet described in bull mature spermatozoa.ObjectivesThe aim of this study was to assess gene and protein expression of PRM2 and corroborate gene and protein expression of PRM1 in bull spermatozoa and testis.Materials and methodsFor this purpose, absolute q‐RT‐PCR was performed to calculate the number of copies of PRM1 and PRM2 mRNAs in bovine epididymal spermatozoa and testicular tissue. Western blot and mass spectrometry were performed to identify PRM1 and PRM2 in samples of bovine epididymal spermatozoa. Samples of bovine testicular tissue were collected to identify PRM1 and PRM2 by immunohistochemistry.ResultsWe evaluated that the number of PRM1 mRNA copies was about hundred times higher than PRM2 mRNA copies in sperm and testicular samples (p < 0.0001). In addition, we estimated the PRM1: PRM2 ratio based on mRNA number of copies. In spermatozoa, the ratio was 1: 0.014, and in testicle, the ratio was 1: 0.009. We also evaluated the immunolocalization for PRM1 and PRM2 in bovine testis, and both proteins were detected in spermatids. Western blot and mass spectrometry in bovine epididymal spermatozoa confirmed these results.ConclusionOur work identifies, for the first time, PRM2 in bovine epididymal spermatozoa and in testis. Further studies are still needed to understand the role of PRM2 on the chromatin of the spermatozoa and to verify how possible changes in PRM2 levels may influence the bull fertility.

Sperm DNA fragmentation: causes and identification

“…In mice, DNA integrity is altered when the ratio between protamines 1 and 2 is disrupted (Suganuma et al, 2005). In bovine, a recent study from our research group identified, for the first time, PRM2 in bovine epididymal spermatozoa and in testis (Hamilton et al, 2019). This finding could suggest that the ratio PRM1:PRM2 could be important in bovine fertility also.…”

“…Protamine types 1 and 2 (PRM1, PRM2) are found in mammalian sperm. PRM1 is expressed in all mammals (Carrell et al, 2007), whereas PRM2 is described in human, primate, murine and equine sperm (Maier et al, 1990;Queralt et al, 1995) and recently in bovine (Hamilton et al, 2019). Protamine type 3 is described as a PRM2 isoform and is reported in rat (Schluter and Engel, 1995), human, ape and Old World monkeys (reviewed by Steger and Balhorn, 2018).…”

“…After that, cysteines must be reduced and protamines separated by electrophoresis. SDS polyacrylamide acetic acid-urea gel electrophorese is indicated (Seligman et al, 1994;Hamilton et al, 2019). Scanning the gel previously stained with Coomassie blue allows the identification of protamines.…”

An improved acetic acid‐urea polyacrylamide electrophoresis method to evaluate bovine sperm protamines

“…To isolate bovine sperm nuclear proteins, we used a protocol described by Dogan et al (2015) and adapted by Hamilton et al (2019).…”

“…The additional amino acids could lead to an altered protein conformation and function; however, it remains unclear whether both transcripts for bull protamine 2 are translated and have different binding activities to DNA. Recently, the gene and protein expression of protamine 2 was described in bovine sperm (Hamilton et al., 2019). More recently, it has been shown that sperm cells with protamine deficiency may form epigenetically altered pronuclei and are associated with infertility and reduced embryogenesis capability (Carrell, 2019).…”