Characterization of membrane proteins in fresh and frozen spermatozoa in Assam hil HILL GOAT

Abstract

Thirty six pooled ejaculates from nine Assam Hill Goat bucks aged 2 to 2.5 years collected by artificial vagina method were used to study the fresh and frozen semen characteristics, characterization of seminal plasma proteins, characterization of sperm membrane proteins in fresh and frozen semen and to study the effect of supplementation of three membrane stabilizers, each at two different concentrations viz. 50 and 80 mM sucrose, 50 and 100 mM trehalose, and 100 and 150ng/ml IGF-1 to tris-citric acid fructose egg yolk glycerol extender (TCFEYG) on post-thaw semen characteristics and on sperm membrane proteins of frozen semen. Characterization of two fertility related membrane proteins viz. ADAM1 and ADAM2 were also done in fresh and frozen spermatozoa of Assam Hill goat by western blotting and immunolocalization using anti ADAM1 and anti ADAM2 antibodies raised in rabbit respectively. The mean per cent progressive sperm motility, HOST-reacted sperm and intact acrosome was significantly (plt0.01) higher in fresh semen than in semen frozen in different extenders. The mean per cent post-thaw progressive motility, HOST-reacted sperm and intact acrosome differed significantly (plt0.01) between the different extenders. However, no significant difference was observed in the mean per cent HOST-reacted sperm between TCFEYG supplemented with 100mM trehalose and TCFEYG supplemented with 100ng/ml IGF-1 and no significant difference was observed in the mean per cent intact acrosome between TCFEYG supplemented with 100mM trehalose and TCFEYG supplemented with 100ng/ml IGF-1 and between TCFEYG supplemented with 50mM sucrose and TCFEYG supplemented with 50mM trehalose. The mean per cent post-thaw progressive sperm motility, HOST-reacted sperm and intact acrosome in frozen semen was found to be the highest in TCFEYG supplemented with 150ng/ml IGF-1. newlineSDS- PAGE of seminal plasma and sperm membrane extract of fresh semen revealed the presence of 20 and 24 protein bands respectively with molecular weights ranging from10 kDa to 240 kDa. The SDS-PAGE electrophoretogram of sperm membrane proteins of semen frozen using TCFEYG and TCFEYG supplemented with 50mM sucrose (TCFEYG + 50mM S) and, 80mM sucrose (TCFEYG + 80mM S) revealed 21 protein bands with molecular weights ranging from 10 kDa to 240 kDa. The 21 protein bands were same with that observed in the sperm membrane of fresh spermatozoa, except three protein bands. These three proteins of molecular weights 23 kDa (~Phosphatidyl-ethanolamine-binding protein), 29 kDa (~Proacrosin binding protein) and 42 kDa (~tyrosine- phosphorylated SPACA1) were absent in TCFEYG and TCFEYG supplemented with 50mM, and 80mM sucrose. The SDS-PAGE electrophoretogram of sperm membrane proteins of semen frozen using TCFEYG supplemented with 50mM trehalose (TCFEYG + 50mM T), and 100mM trehalose (TCFEYG + 100mM T) revealed 22 protein bands with molecular weights ranging from 10 kDa to 240 kDa. The 22 protein bands were same with that observed in the sperm membrane of fresh spermatozoa, except two protein bands. These two proteins of molecular weights 29 kDa (~Proacrosin binding protein) and 42 kDa (~tyrosine- phosphorylated SPACA1) were absent in TCFEYG supplemented with 50mM, and 100mM trehalose. The supplementation of trehalose to the basic TCFEYG extender at 50mM and 100mM concentrations, however, had a protective effect on the sperm membrane protein of 23kDa when compared to the basic TCFEYG extender and TCFEYG supplemented with 50 and 80mM sucrose. The SDS-PAGE electrophoretogram of sperm membrane proteins of semen frozen using TCFEYG newlinesupplemented with 100ng/ml IGF-1 (TCFEYG + 100ng/ml IGF-1), and 150ng/ml IGF-1 (TCFEYG + 150ng/ml IGF-1) revealed 21 protein bands with molecular weights ranging from 10 kDa to 240 kDa. The 21 protein bands were same with that observed in the sperm membrane of fresh spermatozoa, except three protein bands. These three proteins of molecular weights 29 kDa (~Proacrosin binding protein), 130 kDa (~ lipoprotein binding protein) and 240 kDa (~golgi-associated retrograde protein) were absent in TCFEYG supplemented with 100ng/ml, and 150ng/ml IGF-1. Supplementation of the basic tris extender with IGF1 at concentrations of 100ng/ml and 150ng/ml resulted in protection of sperm membrane proteins of molecular weights 23kDa (~Phosphatidyl-ethanolamine-binding protein) and 42 kDa (~tyrosine- phosphorylated SPACA1) during the freeze-thaw process when compared to the basic tris extender and tris extender supplemented with sucrose. The 23 kDa protein was however, also found to be protected in the tris extender supplemented with trehalose. newlineADAM1 was detected as three bands of ~ 25kDa, 66kDa and~ 90kDa in the sperm membrane extract of fresh sperm. However, in the sperm membrane extract of semen frozen using TCFEYG, TCFEYG + 80mM S, TCFEYG + 50mM S, TCFEYG + 50mM T, TCFEYG + 100mM T, TCFEYG + 100ng/ml IGF-1 and TCFEYG + 150ng/ml IGF-1 extenders it was detected as ~25kDa, 66kDa and ~90kDa; ~25kDa, 66kDa and ~90kDa; ~25kDa, ~49kDa,~66kDa, ~90kDa and ~110kDa; ~25kDa,66kDa and ~90kDa; ~25kDa, 66kDa and ~90kDa; ~25kDa, ~49kDa,~66kDa, ~90kDa and ~110kDa ; and ~25kDa, ~49kDa,~66kDa, ~90kDa and ~110kDa respectively. In the present study, freeze-thaw process and supplementation of tris extender used for freezing of semen with membrane stabilizers such as sucrose, trehalose and IGF-1 has been found to result in certain variations in the molecular weight of ADAM1. However, reactive protein bands of 25kDa, 66kDa and 90kDa were found to be consistently present in the fresh sperm as well as sperm frozen in different extenders. ADAM2 was detected as two bands of ~ 80kDa and~ 130kDa in the sperm membrane extract of fresh sperm. However, in the sperm membrane extract of sperm frozen using TCFEYG, TCFEYG + 80mM S, TCFEYG + 50mM S, TCFEYG + 50mM T, TCFEYG + 100mM T, TCFEYG + 100ng/ml IGF-1 and TCFEYG + 150ng/ml IGF-1 extenders it was detected as ~70kDa, ~80kDa, ~100kDa and ~130kDa; ~70kDa, 80 kDa and ~130kDa; ~70kDa, ~80kDa,~90kDa, ~100kDa and ~130kDa; ~70kDa, ~80kDa, and ~100kDa; ~70kDa, ~80kDa, and ~100kDa; ~70kDa, ~80kDa, and ~100kDa; and ~70kDa, ~80kDa, and ~100kDa respectively. In the present study, freeze-thaw process and supplementation of tris extender used for freezing of semen with membrane stabilizers such as sucrose, trehalose and IGF1 has been found to result in certain variations in the molecular weight of ADAM2. However, a protein band of 80 kDa was found to be consistently present in the fresh sperm as well as sperm frozen in different extenders. Immunolocalization of the ADAM1 and ADAM2 proteins revealed the presence of the proteins in the acrosomal region of sperm cells in both fresh and frozen semen. Present study revealed no change in the localization of ADAM1 and ADAM2 post freezing thereby indicating that there is no effect of freezing on the distribution of these two proteins. newlineIt was concluded that cryopreservation of Assam Hill Goat semen resulted in alterations in sperm membrane proteins, however, supplementation of membrane stabilizers exerted protective effects. Based on post-thaw semen characteristics and study on membrane proteins it was found that IGF-1 at 150ng/ml was superior to other membrane stabilizers in maintaining post-thaw semen quality.