Andrology. 2019 May;7(3):373-381. doi: 10.1111/andr.12610. Epub 2019 Mar 28.

Detection of protamine 2 in bovine spermatozoa and testicles.

Hamilton TRS1, Simões R2, Mendes CM1, Goissis MD1, Nakajima E3, Martins EAL3, Visintin
JA1, Assumpção MEOA1.
Author information
1
Department of Animal Reproduction, School of Veterinary Medicine, University of
São Paulo, Avenida Professor Doutor Orlando Marques de Paiva, Sao Paulo,
Brazil.
2
Center for Natural and Human Sciences, Federal University of ABC, Santo André,
Brazil.
3
Process Development Laboratory, Inovation Division, Butantan Institute, São
Paulo, Brazil.
Abstract
BACKGROUND:
Sperm 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.

OBJECTIVES:
The 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 METHODS:

For 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.

RESULTS:
We 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.

CONCLUSION:
Our 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.

© 2019 American Society of Andrology and European Academy of Andrology.

KEYWORDS:
bull; immunoidentification; protamination; protamine 2; sperm chromatin

PMID:

30920782

DOI:

10.1111/andr.12610

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