Elsevier

Reproductive Biology

Volume 17, Issue 3, September 2017, Pages 252-258
Reproductive Biology

Original article
Serine-like proteolytic enzymes from common carp Cyprinus carpio L. seminal plasma are able to degrade sperm proteins

https://doi.org/10.1016/j.repbio.2017.05.012Get rights and content

Abstract

The application of zymography, with sperm proteins as a substrate, allowed for the first time the visualisation of two serine proteinases with a molecular weight of 76 and 163 kDa from common carp Cyprinus carpio L. seminal plasma. Twenty four hours of incubation in a development solution with a pH of 7.5 and incubation at 37 °C were the best conditions for the visualisation of serine proteinase; however, proteolysis was also observed at 4 °C. Our results indicate that serine proteinase from common carp seminal plasma with a molecular weight of 76 and 163 kDa may be involved in the degradative mechanism of sperm proteins. This mechanism may be responsible for the removal of damaged sperms by the digestion of native sperm proteins.

Introduction

The seminal plasma of fish is a unique system responsible for many biological processes connected with the maturation, storage and/or aging of sperm. Firstly, seminal plasma proteins are related to regulating spermatogenesis in the testes and sperm motility [1], [2]. Secondly, species-specific proteins of seminal plasma protect sperm motility and its fertilisation ability during storage [3]. Lastly, proteins present in seminal plasma take part in remodelling the testes during and after seasonal breeding. Proteinases and their inhibitors play a crucial role in the mentioned processes [4], [5], [6].

Metalloproteinases and serine proteinases are the main proteinases whose activities were detected in the seminal plasma of teleosts using gelatine and casein as a substrate [7], [8], [9]. The activities of these enzymes were also detected in common carp Cyprinus carpio L. seminal plasma and testes fluid [10], [11]. In freshwater fish, the physiological functions of seminal plasma and/or testes fluid proteinases have been postulated to be related to the regulation of spermatogenesis, sperm motility activation and aging processes (removal of immature or damaged sperm) [12], [13].

Recently the trypsin-like proteinase and proteasome subunits were identified in common carp seminal plasma [14]. It is known that trypsin could be involved in various steps of spermatogenesis in the testis [15]. Similarly to the proteasome-ubiquitin complex [16], serine protease could be responsible for cell apoptosis and tissue turnover. However, there are no data proving the potential to degrade sperm protein by proteinases present in common carp semen.

Analysis of the proteolytic activity in biological material together with their inhibitors is usually impossible in one step. In fish, protease inhibitors are dominant proteins in seminal plasma [6]. For that reason, it is impossible to measure any proteolytic activity using simple kinetic methods. However, using the substrate polyacrylamide gel, it is possible to visualise and evaluate proteolytic activities in simple one step [7], [8], [10]. Therefore, the use of substrate electrophoresis is the most effective option for evaluation of the fish seminal plasma proteolytic enzymes.

In order to identify proteolytic activities in the seminal plasma of common carp, different substrates (gelatine, casein, albumin and haemoglobin) have been used [11]. It has been shown that gelatine is a suitable substrate for the identification of metalloproteinases and serine proteinases; however, when using casein, only serine proteinases were detected in common carp seminal plasma. On the other hand, other substrates, like sperm proteins, have never been used for the detection of enzymatic activity in seminal plasma. Incorporating the sperm proteins into polyacrylamide gel as a substrate may help to answer the question regarding the involvement of seminal plasma proteinases in sperm degradation.

The objective of this study was to examine whether seminal plasma proteinases are able to degrade sperm proteins. Moreover, the activity of proteolytic enzymes with regard to degrading sperm proteins was measured in different in vitro conditions such as temperature and pH. This allows us to discuss their potential physiological role in common carp reproductive tracts.

Section snippets

Semen collection and sperm extraction

Semen samples were collected through gentle abdominal massage of adult males of common carp (n = 10) originating from the Knieja Fishery Farm (50°49′31.6″N 19°29′34.6″E) in May. Before semen collection, the males were anaesthetised with 2-phenoxyethanol (Sigma-Aldrich, St. Louis, MO, USA) given at a dose of 0.5 mL L−1. Special care was taken during semen collection to avoid contamination with urine, faeces, blood or mucus.

The seminal plasma was separated from the sperms by centrifugation at 10,000 g

The effects of incubation time, pH of the development solution and temperature on the proteolytic activity of common carp seminal plasma using sperm extract as a substrate

The length of time of incubation did not affect the intensity of the detected bands of 76 and 163 kDa (Fig. 1a–c). Differences in the ROD of proteinase were observed in a wide range of pH (Fig. 2a). A high ROD in proteinases of 76 kDa was observed at a pH of 7.5, although these differences were not significant (Fig. 2b). On the other hand, the ROD intensity of proteinases of 163 kDa increased together with increasing pH (Fig. 2c). An incubation temperature of 4 °C was enough to develop the activity

Discussion

The application of zymography with sperm extract proteins as a substrate allowed us for the first time to visualise two serine proteinases with a molecular weight of 76 and 163 kDa from common carp seminal plasma. Twenty four hours of incubation in a developer solution with a pH of 7.5 and incubation at 37 °C were the best conditions for the visualisation of serine proteinase. Four fractions of sperm proteins obtained after GF were digested by serine proteinases from common carp seminal plasma.

Conflict of interest

The authors declare no conflict of interest.

Ethical approval

Approval from the Animal Experiments Committee in Olsztyn, Poland was obtained before starting any experiments.

Author contributions

Beata I. Cejko (lead author, designed the study, wrote the manuscript), Mariola Słowińska (performed research, edited the manuscript), Sylwia Judycka (performed research), Radosław K. Kowalski (designed the study, interpreted the data).

Acknowledgements

The presented study was supported by National Science Centre Grant No. N 311 351239 and funds appropriated to the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences, Olsztyn, Poland.

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