Since 1976 many studies have been reported on the occurrence and functional significance of ecto-protein kinases in a variety of cell types although their precise biochemical identity is largely unknown. This study reports for the first time purification to apparent homogeneity of an ecto-protein kinase (ecto-CIK) and some of its characteristics using caprine sperm as the cell model. The ecto-CIK is a unique membrane-specific serine/threonine protein kinase. It is a strongly basic 115 kDa protein made up of two subunits: 63 and 55 kDa. The ecto-kinase undergoes a remarkable lateral movement on the outer cell surface culminating in capping on the sperm acrosomal tip. MPS, its major protein substrate is also located on the acrosomal tip. Both ecto CIK and MPS serve as potential regulators of flagellar motility. This novel enzyme appears to be major kinase responsible for the reported regulation of mammalian cellular functions by modulating phosphorylation of the membrane-bound proteins.

We have demonstrated the location of a cyclic AMP independent serine/threonine protein kinase (ecto-CIK) on the outer surface of mature goat spermatozoa. We purified and characterized the major physiological protein substrate (MPS) of ecto-CIK. 32P-labeled membrane proteins phosphorylated by endogenous ecto-CIK of intact cauda-epididymal spermatozoa was solubilized with 1% Triton X-100 and then fractionated by following several chromatographic techniques like Sephacryl S-300 molecular sieve chromatography, DEAE-cellulose ion-exchange chromatography and chromatofocussing. The MPS of ecto-CIK has been purified to apparent homogeneity and it was found to be a monomeric protein of 100 kDa. Three isoforms of MPS have been found with pI of 6.37, 6.05, and 5.14 and all these isoforms served as the specific substrate of ecto-CIK. The ecto-kinase has nearly 30 times greater affinity for MPS as compared to casein the most potent exogenous protein substrate. Addition of MPS (pI 5.14) antibody caused head-to-head sperm agglutination. The Fv/Fab fragment of anti-MPS caused significant inhibition of sperm motility. The data show that MPS is an ecto-protein localized on the sperm head. MPS may thus play an important role for the regulation of sperm-egg interaction.

Previously we have purified and characterized a unique plasma membrane-specific cyclic AMP-independent ecto-protein kinase (ecto-CIK) as well as its ecto-phosphoprotein substrate (MPS) using caprine sperm model. This study reports for the first time the role of the sperm external surface protein phosphorylation system on sperm acrosome reaction, which is essential for fertilization. Calcium ionophore A23187 has been used to trigger the sperm acrosome reaction in vitro. Treatment of sperm cells with CIK antibody (dil: 1:500) causes a significant decrease (approx. 50%) in percentage of acrosome reacted sperm. Onset of the acrosome reaction causes a remarkable increase in the rate of acrosin release from the cells in the medium. However, CIK antibody inhibits significantly (approx. 50%) the acrosin release. The level of membrane-bound MPS as estimated by ELISA and the degree of its phosphorylation catalyzed by the endogenous ecto-CIK, increase significantly with the progress of the acrosome reaction. Both the parameters increase by approximately 100% during the 20 min of the reaction. MPS antibody (1:100 dilution) markedly decreases (approx. 75%) percentage of acrosome-reacted sperm. MPS antibody as well shows high efficacy to inhibit acrosin release from spermatozoa. The results demonstrate that the cell-surface protein kinase and its protein substrate are essential for membrane fusion component of acrosome reaction. The data are consistent with the view that MPS regulates acrosomal membrane fusion with the overlying plasma membrane by the mechanism of its phosphorylation and dephosphorylation.

Previous studies from our laboratory have identified MPS, a 100-kDa protein, as the major phosphoprotein substrate of caprine sperm ecto-cyclic AMP independent protein kinase. In this study the isolated (32)P-labelled MPS has been incorporated into mature caprine (Capra indicus) cauda-epididymal spermatozoa with the help of cell electroporation technique to investigate the effect of MPS on sperm flagellar motility. The optimum conditions for electroporation of sperm cells consisted of exposure of 0.2 ml of sperm cells (2 x 10(8)/ml) to external electric field of intensity 1.5 kV/cm and capacitation of 25 microF at 4 degrees C and post-pulse incubation at 37 degrees C for 1 hr. when nearly 50% of the cells lost motility. Scanning electron micrographs (SEM) demonstrate the formation of micro-pores and local osmotic swelling in the electroporated spermatozoa. MPS incorporation was maximal when its concentration was 30 microg/ml (300 pmol) in the medium and when the post-pulse incubation time was 60 min. At maximum (75%) MPS incorporation, total and forward motility increments were also maximum: 34% (P < 0.01) and 32% (P < 0.01), respectively. The subcellular fractionation data show that major portion of the introduced MPS was bound to the plasma-membrane of spermatozoa. The 32P-labelled electrophoresed intact spermatozoa lost radioactivity due to the action of the endogenous ecto-phosphoprotein phosphatase. Therefore MPS is primarily localised on the sperm external surface leaving its phosphate group(s) oriented in the extracellular medium. The data provided further evidence to strengthen the view that MPS is an ecto-phosphoprotein and that it plays an important role in the regulation of sperm flagellar motility.

Sperm forward motility is an essential parameter in mammalian fertilization. Studies from our laboratory have identified and characterized a few unique sperm motility regulatory proteins/glycoproteins from the male reproductive fluids and mammalian blood serum. The purified sperm motility-initiating protein MIP from caprine epididymal plasma as well as the forward motility-stimulating factor FMSF and motility-stimulating protein MSP from buffalo and goat serum, respectively, have high efficacy to initiate or increase motility in nonmotile or less motile sperm. "ntibody of sperm motility inhibitory factor MIF-II has the high potential to enhance sperm vertical velocity and forward motility by increasing intracellular cyclic adenosine monophosphate c"MP level. The appearance and disappearance of D-galactose-specific lectin and its receptor along the epididymis has been reported to be involved in motility regulation in spermatozoa. " novel synthetic cryopreservation method and role of lipid to protect membrane damage during cryopreservation have been demonstrated. Motility-promoting proteins may be extremely useful for improving cattle breeding and breeding of endangered species, thereby helping in enhanced production of animal products as well as in the conservation of animals. Isolated proteins and developed cryopreservation technology may also be beneficial in human infertility clinics to increase the chance of fertilization.Keywords: Spermatozoa, Epididymis, Motility regulatory proteins, Cryopreservation, Reproduction © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. . IntroductionLivestock is a very important subsector of Indian agricultural production system. The overall contribution of the livestock is almost . %. India ranks first in milk production in the world . million tons , which is mostly contributed by cattle and buffalo. The bull concentrates in itself a high economic value and thus need to be maintained on proper nutrition and management to obtain optimum performance in terms of semen production [ ]. The demand for the best males has increased considerably due to a shortage in the number of proven bulls having better semen characteristics for sustaining a successful breeding program [ ].Reproductive techniques facilitate the breeding of farm animals, which ultimately improve milk production and growth in dairy industry. "rtificial insemination "I is a first-generation reproductive biotechnology that profoundly contributed to genetic improvement, particularly in dairy cattle. Such impact would not have been possible without successfully freezing bull semen. Quality control of frozen sperm is of utmost importance for the sperm to be used in "I [ ]. Sperm cryopreservation allows prolonged preservation of semen and a wider use of a male gamete [ ]. Thi...