Indian Journal of Animal Research

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Research Article

Indian Journal of Animal Research, volume 55 issue 4 (april 2021) : 394-400

Proteins and Heparin Binding Proteins of Spermatozoa and Seminal Plasma in Beetal Bucks: Purification, Characterization and Role in In vivo Fertility

Navjot S. Dhillon1, Ranjna S. Cheema1,*, Sumit Singhal2
1Department of Veterinary Gynecology and Obstetrics, College of Veterinary Sciences, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana-141 004, Punjab, india.
2Directorate Livestock Farms, College of Veterinary Sciences, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana-141 004, Punjab, india.
Cite article:- Dhillon S. Navjot, Cheema S. Ranjna, Singhal Sumit (2020). Proteins and Heparin Binding Proteins of Spermatozoa and Seminal Plasma in Beetal Bucks: Purification, Characterization and Role in In vivo Fertility . Indian Journal of Animal Research. 55(4): 394-400. doi: 10.18805/ijar.B-3974.

ABSTRACT

Background: The proteome analysis of seminal plasma and spermatozoa is of special relevance in livestock. Heparin binding proteins (HBPs) found in the seminal plasma of several mammals are shown to bind to sperm membrane and affect a series of events that contribute to normal fertility, such as sperm capacitation, formation of the oviduct reservoir and binding to the oocyte. Profiles of HBPs from seminal plasma and sperm membranes have been associated with sperm fertility. Although, HBPs present in the SP are described in several species, but little is known about HBPs in buck. 

Methods: Seminal plasma (SP) and sperm extracts (SE) of 13 bucks were subjected to heparin-sepharose affinity chromatography. Sperm extract, seminal plasma and purified HBPs and Non-HBPs were fractionated by SDS-PAGE. Total 78 females (6 per buck) were mated with 13 bucks. Bucks were divided into two groups, G-I (high fertile, 83.3-100% FR) and G-II (low fertile, 50-66.7% FR). Relationship between HBPs and fertility rate was observed. 

Result: SDS – PAGE of SP and SE resulted in resolution of 22 (10-240 kDa) and 21 (10-270 kDa) bands, respectively. Based on fertility rate 15 and 13 kDa proteins were absent in SP of higher number of GI-compared to G-II bucks. Fourteen bands ranging from 10 – 180 kDa and 10 – 150 kDa were separated from SP-NHBP and SP-HBP. SP-HBPs of 75, 35, 30, 28, 25 and 13 kDa were present in higher (28.6%, 42.5%, 26.2%, 40.5%, 14.3% and 36.2%) number of high fertile than low fertile bucks. NHBP and HBP purified from SE resolved into 11 bands ranging from 10 – 135 kDa and 10 – 120 kDa, respectively. SE-HBP of 53 kDa, 50/45 kDa and 25 kDa were present in higher percentage of high fertile than low fertile bucks. 

INTRODUCTION

The proteome analysis of seminal plasma (SP) and spermatozoa is of special relevance in livestock. It does not only contribute towards our basic understanding of reproductive biology, but also contribute in identifying the factors related to infertility reduction, promotion of fertilizing ability and semen preservation techniques (Duncan and Thompson, 2007). Seminal plasma proteins (SPP) modulate sperm maturation, protection against micro-organisms, complement induced attacks and oxidative stress (Moura et al., 2007). Thus, SP-HBP play a vital role in sperm survival and the overall fertilization process and any alteration in these proteins can be directly related to infertility. HBP have been identiûed in various species, e.g. bovine (Cheema et al., 2016), equine (Calvete et al., 1997), buffalo (Singh et al., 2016a, Gupta et al., 2018) and canine (de Souza et al., 2006). HBP found in the SP of several mammals were shown to bind to sperm membrane and affect a series of events that contribute to normal fertility, such as sperm capacitation, formation of the oviduct reservoir and binding to the oocyte. HBP from sperm membranes have been associated with bull fertility (Bellin et al., 1994).
 
Male fertility is an important factor since numerous goats are to be bred by a single buck. Traditional available methods to detect male fertility in mammals include sperm morphology, motility, cervical mucus penetration assay, acrosome reaction, sperm zona pellucida penetration. Clinical assessments of the values for these variables and association with fertility are still not widely accepted. HBP have been indicated as a biochemical marker to predict the fertility potential of bulls. Sperm from high-fertility bulls have a greater frequency of acrosome reactions in response to heparin-like material (Lenz et al., 1988) and have a greater binding affinity for heparin (Marks and Ax, 1985) than sperm from less-fertile bulls. There is hardly any information available on the HBP as a valuable diagnostic tool to predict the fertility of Beetal buck spermatozoa. Therefore, present study was conducted to isolate / characterize HBP from buck SP / sperm extracts (SE), identify their relationship with fertility.

MATERIAS AND METHODS

Experimental animals and management
 
All the procedures were approved by Institutional ethical committee (GADVASU/2018/IAEC/46/06). The experiments were conducted in Reproductive Biology Lab, Department of Veterinary Gynecology and Obstetrics, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana Punjab, India from August 2018 to April 2019. All the experimental bucks were kept under loose housing system throughout the study period at Goat Research Farm of the university.
 
Experimental design
 
Thirteen sexually mature Beetal bucks were randomly selected. Semen was collected twice a week using goat artificial vagina. SP was separated by centrifugation of semen at 3000 rpm for 5 min. SE and SP were subjected to affinity chromatography for purification of HBP. Sperm extract proteins (SEP), SPP, SE-HBP and SP-HBP and unbound protein fractions (NHBP) were characterized by SDS-PAGE.
 
Fertility trial and pregnancy diagnosis
 
Total 78 females (6 per buck) were mated with 13 bucks to assess the fertilizing potential and to establish a link between fertility and SP-HBP / SE-HBP. Goats showing natural heat symptoms were mated in isolation with the prior selected buck according to mating history and pedigree record in order bucks to avoid inbreeding depression. Proper record was maintained regarding breeding of each buck. Conception rate was confirmed ultra-sonographically after fifty days of mating. Bucks were divided into two groups, G-I and G-II exhibiting 83.35-100% and 50-66.66% FR, respectively. Therefore, SEP, SPP, HBP and NHBP were compared based on two fertility rates i.e. high and low.
 
Preparation of sperm extracts (SE) for SDS -PAGE and affinity chromatography
 
Fresh semen was washed twice with PBS, pH 7.4. Sperm pellet (500 x 106) was suspended in 1.0 ml of 2% SDS in 62.5 mM Tris-HCl (pH 6.8) containing 50 mM EDTA, 25% glycerol and protease inhibitors (Cocktail, SERVA). Sperm suspensions were sonicated at 20 Watts for 3 x 20 secs, centrifuged at 10000 rpm for 15 min. SE were stored in aliquots at -20°C till further use. Total protein was measured in SE and SP by the method of Lowry et al., (1951).
 
Purification of HBP by affinity chromatography
 
HBP from SP and SE were purified by affinity chromatography (Manaskova et al., 2002) using cyanogen bromide activated heparin bound sepharose (Farooqui, 1980). About 0.5 ml of SP or SE was loaded and circulated through the column for 15 min for absorption of HBP to the heparin bound resins. NHBP and HBP were eluted with 10 mM Tris HCl and 1M - 2.0 M NaCl at a flow rate of 1 ml/min in the tubes racked in a fraction collector, respectively. The recovered HBP and NHBP fractions were pooled in agreement with the observed curve, obtained from optical density (280 nm) detected by UV monitor attached to the fraction collector. The pooled fractions of HBP and NHBP were concentrated by spinning through protein concentrators (Millipore, 3 kDa) and analyzed for protein content. Area of each peak was calculated using Microsoft excel. The molecular weight of HBP and NHBP were confirmed by SDS-PAGE on 10% acrylamide gels.
 
Sodium dodecyl sulphate-Polyacrylamide gel electrophoresis (SDS-PAGE, Laemmli, 1970)
 
SDS-PAGE of SE, SP, SP-HBP/NHBP and SE-HBP/NHBP was performed under reduced-denaturing conditions in a vertical system using 10% separating gel concentration. A Protein molecular weight marker (10-180 kDa, SMO-BIO), was also run along the samples. Gels were stained with commassie brilliant blue stain and destained till clear background is obtained against dark blue bands. The gel images were captured using Syngene gel documentation and analysis-system (Syngene Gel-Doc, Model-Alpha Imager 1220, Alpha Innotech Corporation, USA) and analyzed by Gene Snap Image Acquisition software (Syngene). The molecular weights were assigned to different proteins on comparing with the standard.
 
Statistical analysis
 
The mean and standard error were calculated using Microsoft excel program. Significant differences in HBP, NHBP among the groups were tested by ANOVA using SPSS16 program (Student version for windows, SPSS Inc.233 South Wacker Drive, 11th floor Chicago, IL 60606-6412). Normality of the data was assessed using the Shapiro-Wilk test and homogeneity of variances was evaluated using the Levene test.

RESULTS AND DISCUSSSION

Affinity chromatography of seminal plasma and sperm extracts
 
Representative graphic images of NHBP and HBP, purified from SP and SE are shown in Fig 1. HBP could be eluted from SP of 5, 3 and 5 bucks with 1M, 1.5M and 2.0M NaCl, respectively. In SE, it could be possible to elute HBP with 1.0M, 1.5M and 2.0M NaCl from 4, 5 and 4 bucks, respectively. Average area of NHBP / HBP peak was non-significantly (p>0.05) / significantly (p<0.05) higher in SP of G-II than G-I bucks (Table 1). However, an average HBP concentration was non-significantly high (23.8 vs. 20.6%) in SP of G-I compared to G-II, but the reverse was the case for NHBP. Average area of NHBP peak in SE was with mol. wt. of 22/30/38 kDa, 28/45/47 kDa, 24/34/55/59/70 kDa and 50/62/75 kDa showed a frequency distribution (FD) of 87.5%, 75%, 25% and 37.5% in sperm membrane proteins of Assam hill goats (Deori et al., 2018).
 

Fig 1: Representative graphic images of heparin-sepharose affinity chromatography of seminal plasma (A, B) and sperm extracts (C, D) of bucks.


 

Table 1: Area (OD x Vol) and percentage of total protein in Peak I and II of seminal plasma and sperm extracts of bucks based on fertility rate.


 
Observations on area and protein concentration of heparin HBP and NHBP peaks indicated that area of peak was not proportional to the protein concentration in SP as well as SE. It can be predicted from these observations that quantity of SP-HBP and SE-HBP is related to SE-HBP. Variable fertility of bucks, which was more prominent in SE-HBP variable concentration of HBP was reported in SP of cross-bred cattle (Srivastava et al., 2012, Cheema et al., 2016), buffalo (Singh et al., 2014). Gupta et al., (2018) also reported an average HBP content of 2.04±0.098 mg/109 spermatozoa in buffalo bulls. Variations in HBP content of SP and SE was observed among the bucks during the present study as also reported in bulls (Nauc and Manjunath, 2000 and Gupta et al., 2018).
 
Characterization of seminal plasma and sperm proteins by SDS - PAG
 
SDS - PAGE of SP and SE resulted in resolution of 21 (10-240 kDa) and 21 (10-270 kDa) bands, respectively (Fig 2). Seventeen and sixteen protein bands were consistently present in SE and SP of Beetal bucks. However, Deori et al., (2018) could separate 20 different proteins (10-75 kDa) with consistent pattern of only 6 proteins in spermatozoa of 8 Assam hill bucks. Sixteen protein bands ranging from 14-97 kDa were reported in SP of Anglo - Nubian goats (Teixeria et al., 2006). Protein bands of 13, 15, 18 and 28 kDa in SE showed a distribution frequency of 68.5%, 31.8%, 92.8% and 69.2% in 13 Beetal bucks. Distribution frequency of 240, 90, 38, 28, 25 and 12 kDa peptides in SP was 76.9%, 53.9%, 53.9%, 84.6%, 84.6% and 23.1%, respectively. The proteins with mol. wt. of 22/30/38 kDa, 28/45/47 kDa, 24/34/55/59/70 kDa and 50/62/75 kDa showed a frequency distribution (FD) of 87.5%, 75%, 25% and 37.5% in sperm membrane proteins of Assam hill goats (Deori et al., 2018).
 

Fig 2: SDS-PAGE pattern of sperm extracts (A) and seminal plasma (B) in beetle goat buck. About 100 µg protein was loaded on 12% acrylamide gel. Separated proteins were stained with Coomassie brilliant Blue R-250. Std (Marker), Lanes 1-14: Buck numbers.


 
Characterization of non - heparin (peak 1) and heparin (peak 2) bound proteins in seminal plasma by SDS - PAGE
 
Out of 14 bands detected in NHBP (10-180 kDa) and HBP (10-150 kDa), only ten and five bands were consistently observed in 13 bucks (Fig 3). FD of NHBPs of 35, 25 and 44 kDa was 53.8%, 46.1% and 7.7%, respectively (Table 2). HBPs of 25, 100/75 kDa, 35/10, 30, 28, 40 and 10 kDa showed a FD of 92.3%, 84.6%, 76.9%, 69.2%, 61.5%, 53.8% and 46.1% among the 13 bucks (Table 2). It indicated buck’s effect on SP- NHBP and HBP. La Falci et al., (2002) identified HAP with mol wts of 73-104 kDa, 119 kDa and 178 kDa from goat SP. Villemure et al., (2003) indicated the presence of GSP-14 kDa, GSP-15 kDa, GSP -20 kDa and GSP-22 kDa in goat SP by gelatin agarose chromatography but only GSP-20 and 22 kDa had affinity for heparin. But, in the present study, proteins of 13, 17, 20, 25 showed an affinity for heparin. Electrophoretic analysis of affinity purified HBP fraction from SP of Nellore bulls (Fernandes et al., 2009), rams (Martin et al., 2013), buffalo bulls (Singh et al., 2014) and cross bred bulls (Cheema et al., 2016) revealed the presence of 8 (15 - 63 kDa), 13 (10 - 232 kDa), 8/6/9 (13 - 71 kDa/14 - 61 kDa/10 - 170 kDa) and 14 (14-150 kDa) bands, respectively. Therefore, variation in number of HBP bands was also evident in other species.
 

Fig 3: SDS-PAGE pattern of heparin sepharose eluted peak I (A, Non-heparin binding proteins) and peak II (Heparin binding proteins) from sperm extracts. About 100 µg protein was loaded on 12% acrylamide gel. Separated proteins were stained with Coomassie brilliant Blue R-250. Std (Marker), Lanes 1-13: Buck numbers.


 

Table 2: Frequency distribution (%) and molecular weights (kDa) of non - heparin and heparin bound proteins in seminal plasma and sperm extracts.


 
Characterization of non-heparin binding and heparin binding proteins in sperm extracts by SDS-PAGE
 
NHBP and HBP purified from SE resolved into 11 bands ranging from 10-135 kDa and 10-120 kDa, respectively (Fig 4). Four and one band of NHBP and HBP were consistently detected in all bucks, respectively (Table 2). FD of 44/18 kDa, 85/50/25 kDa and 15 kDa non-HBP was 92.3%, 84.6% and 61.5%, respectively (Table 2). HBP’s of 120/20 kDa, 18 kDa, 50/45/37 kDa, 25/15 kDa and 31kDa showed a FD of 92.3%, 84.6%, 69.2% and 53.8% and 46.1%, respectively. Therefore, buck factor was also evident for variation in number of sperm NHBP and HBP.
 

Fig 4: SDS-PAGE pattern of heparin sepharose peak I (A, Non-heparin binding proteins) and peak II (Heparin binding proteins) from seminal plasma. About 100 µg protein was loaded on 12% acrylamide gel. Separated proteins were stained with Coomassie brilliant Blue R-250. Std (Marker), Lanes 1-14: Buck numbers.


 
Relationship of proteins and HBP in SP and SE with in vivo fertility
 
It was found that 20 kDa SP protein was present in all bucks except one exhibiting high FR and those of 15/13 kDa were absent in higher number (4/6 and 3/6) of G-1 compared to G-II bucks (3/7 and1/7). Another protein of 28 kDa was absent in 1/6 and 2/7 bucks of G-I and G-II, respectively. SE proteins of 2/6, 2/6, 4/6, 1/6, 1/6 and 3/6 bucks exhibiting high FR were devoid of 240, 90, 38, 28, 25 and 12 kDa proteins. These proteins were also not detected in 1/7, 4/7, 2/7, 1/7, 1/7 and 7/7 bucks, exhibiting low FR. Detection of 240 and 38 kDa proteins in higher number of G-I compared to G-II bucks indicated their role in fertility. Conversely, presence of 90 and 12 kDa proteins in higher number of G-II than G-I may be related to sub-fertility of bucks. Nandi et al., (2012) did partial characterization of 14 kDa protein detected in goat spermatozoa and opined that it may play a crucial role in the acrosomal membrane fusion.

There was no difference in distribution of SP-NHBP among the two groups based on FR. However, difference in distribution of SP-HBP was observed among the bucks based on FR (Table 3). Number of SP-HBP bands constantly present in G-I and G-II bucks was eight and five, respectively. However, frequency distribution of 100/30/28 kDa and 40/13 kDa SP-HBP in G-I was 83.3% and 66.7%, respectively. A FD of 85.7%, 71.4%, 57.1%, 42.8% and 28.57% for 100/25 kDa, 75/40 kDa, 35/30 kDa, 26 kDa and 13 kDa HBP was noticed in SP-HBP of G-II bucks. It indicated that 75, 35, 30, 28, 25 and 13 kDa SP-HBP were present in higher (28.6%, 42.9%, 26.2%, 40.5%, 14.3% and 38.2%) number of G-I as compared to G-II bucks.
 

Table 3: Frequency distribution of Heparin bound proteins in seminal plasma and sperm extracts based on fertility rate of bucks.


 
Two bands of SE-HBP were detected in all bucks of G-I (120/10 kDa) and G-II (18/10 kDa). FD for 53/50/45/20/18 kDa, 37/25 kDa and 31/15 kDa SE-HBP was 83.3%, 66.7% and 50% in G-I bucks (Table 3). However, FD of 120/20 kDa, 37 kDa, 50/45/31/15 kDa, 53/25 kDa in G-II was 85.71%, 71.4%, 57.1% and 42.8% respectively. It revealed that SE-HBP of 53 kDa, 50/45 kDa and 25 kDa were present in higher percentage (40.4%, 26.1% and 23.9%) of G-I than G-II bucks. SPP of bucks having affinity for heparin may be homologous to BSP proteins. Villemure et al., (2003) also revealed that sequence of GSP-20 and 22 was homologous to BSP.
 
It may be predicted that SP-HBP and SE-HBP of 75, 35, 30, 28, 25, 13 kDa and 53, 50, 45 and 25 kDa are associated with higher fertility of Beetal bucks. Mor et al., (2006) suggested that heparin binding moiety of sperm membrane protein may be required for modulation of sperm motility. Number of motile and viable spermatozoa were significantly (p<0.05) higher and MDA concentration was non-significantly low in GI bucks compared to G-II (Dhillon et al., 2019). Therefore, SE-HBP of 53, 50, 45 and 25 kDa may have contributed to high sperm attributes vis a vis fertility by reducing oxidative stress in G-I bucks. There is no supporting data regarding relationship of HBP with fertility in bucks, but HBP have been related to fertility of bulls (Bellin et al., 1994, 1998 and Sprott et al., 2000).

CONCLUSION

This is the first report in goats that reveals role of SP-HBP and SE-HBP in fertility. SP-HBP and SE-HBP of 75, 35, 30, 28, 25, 13 kDa and 53, 50, 45 and 25 kDa may be established as fertility associated proteins in goat, but before that validity of these proteins in higher number of animals and further analysis by MALDI-TOF or LC-MS analysis is essential.

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