| Tarim red deer (Cervus elaphus yarkandensis) is a unique deer subspecies of Xinjiang, and it is also the only red deer subspecies that inhabits in the desert landscape in the world, so it has important ecological position in the desert community. In recent years, because of the influence of the development and economic activities, their habitat is constantly shrinking, and population is declining more and more, species survival is facing a serious threat. So research on their protection reproductive biology has a positive effect on the continuation, recovery of their population.Sperm capacitation is critical to fertilization, and it is a very complex physiological phenomenon. There are a series of physiological and biochemical changes happening in the sperm capacitating process and the process is regulated by many factors. The main regulatory factors include cholesterol receptors, albumin, bicarbonate, calcium ion and protein tyrosine phosphorylation, and so on. Among them, protein tyrosine phosphorylation of sperm is an important regulatory factor in the various physiological and biochemical process, such as sperm capacitation, ultra-activate motility maintenance and acrosome reaction trigger. At present, the mechanism of sperm capacitation and affection of the main regulatory factors are not yet fully clear, and the efficient in vitro sperm capacitation system of deer has not been implemented. Though albumin, bicarbonate and calcium ions could be basic components of in vitro capacitation solution to study the initial incident in the sperm capacitation, the effections of these three components on the ability of sperm capacitation have species-specific characteristic, and even if for the same species, there are also differences between different reports. The rates of sperm penetration are all low in the deer in vitro fertilization (IVF) system reported so far.In this study, with the help of the techniques of several subject, such as molecular biology, biochemistry and reproductive physiology, Tarim wapiti sperm was used as experimental material, the in vitro sperm capacitation system was screened firstly, then in vitro sperm capacitation and the occurrence and mechanism of protein tyrosine phosphorylation of sperm, which was the key molecular incident in the process, were reseached. On these bases, the affection mechanism of several major affective or inducible factors on in vitro capacitation and protein tyrosine phosphorylation of Tarim wapiti sperm was analysesed.The results are shown as follows:1. Only three clear tyrosine phosphorylation protein can be detected before capacitation incubation of fresh sperm, and their molecular weight were 10 ku,40 ku and 47 ku, respectively, while the number of protein bands, which were phosphorylated in tyrosine. increased to 7 after capacitation incubation, their molecular weight were 10 ku,14 ku,25 ku, 32 ku,40 ku,47 ku and 55 ku, respectively (P< 0.05).2. Only six clear tyrosine phosphorylation protein can be detected before capacitation incubation of cryopreservated sperm, and their molecular weight were 10 ku,14 ku,25 ku,40 ku,47 ku and 55 ku, respectively, while many proteins were phosphorylated in tyrosine after capacitation incubation, their molecular weight were 10 ku,14 ku,25-32 ku,40 ku,47 ku,55 ku and 60-85 ku, respectively (P< 0.05). Furthermore, the phosphorylation levels of these proteins, especially the proteins with low molecular relatively increased along with the process of phosphorylation, and were relatively high in the period of 1 to 2 h after capacitation culture (P<0.05).3. Difference in the proteins, which were phosphorylated in tyrosine and detected from the freeze-thaw sperm membrane protiens of different male deer using the method of Western blot, were more large. The molecular weights of the proteins, which were phosphorylated in tyrosine, were concentrated in low molecular weight protein (molecular weight is between 10 to 85 ku), while protein with high molecular weight (molecular weight more than 85 ku) barely be phosphorylated in tyrosine, and a certain difference exists between the levels of sperm protein phosphorylation of different individual (P< 0.05).4. The conventional in vitro capacitating methods used in mammalian sperm can cause capacitation in vitro and protein tyrosine phosphorylation of the tarim red deer sperm. Among them, the rates of sperm capacitation and the levels of proteins phosphorylation in tyrosine in the IA group, the heparin group and caffeine group, were higher than those in the control group and Percool group (P<0.05), and the phosphorylation levels in tyrosine of the proteins with the molecular weight of 35 ku,40 ku and 55 ku were relatively more high, and especially the rates of sperm capacitation and the levels of proteins phosphorylation in tyrosine in the heparin group were higher than other groups (P< 0.05).5. The participation of BSA, HCO3- and Ca2+ were required in the in vitro capacitation of Tarim wapiti sperm. When the sperm in vitro capacitation system was short of BSA, HCO3-and Ca2+ simultaneously or separately, sperm protein tyrosine phosphorylation level is reduced, and this change can be detected specially along with the extension of incubation time. on the contrary, the level of sperm protein tyrosine phosphorylation is relatively high when the three ingredients exist at the same time. especially, the changes of the low molecular weight protein (10 ku,18 ku,25 to 32 ku) is more obvious. Nevertheless, the level of sperm protein tyrosine phosphorylation decreased significantly (P<0.05), when be lack of Ca2+ and HCO3 simultaneously. These results showed that HCO3- and Ca2- have important regulating roles in the capacitation and protein tyrosine phosphorylation process of tarim red deer, and BSA help strengthen the capacitation and protein tyrosine phosphorylation process.6. BSA is beneficial to the in vitro capacitation and protein tyrosine phosphorylation of tarim red deer sperm, and protein tyrosine phosphorylation, and when a can a certain concentration (6-10 mg/mL) of BSA was added to the capacitation liquid, the levels of in vitro capacitation and protein tyrosine phosphorylation increased, have significant difference compared with that of group with low concentration (3 mg/mL) (P<0.05). Especially, the expression level of the proteins tyrosine phosphorylation with molecular weight of 55 kn protein increased significantly (P<0.05), but there was no significant difference between the levels of 6 mg/mL and 10 mg/mL groups (P< 0.05), so the best usage of BSA was considered to be 6 mg/mL.7. A certain concentration of HCO3- was good to the in vitro capacitation and protein tyrosine phosphorylation of tarim red deer sperm. When HCO3- was short or in low density, the sperm vitality and the rate of sperm capacitation decreased (P<0.05); the rate of sperm capacitation decreased was highest when HCO3- was in concentration of 15～25 mM, but the rate of acrosome reaction increases and show the time-dependence when the concentration of HCO3- was up to 37 mM (P< 0.05). In addition, under the condition of low concentration of HCO3- the level of tyrosine phosphorylation protein expression was relatively low, while the phosphorylation level of protein with molecular weight of 40～56 ku expression was increased relatively when the concentration increased to 15 mM, and had a time-dependent trend (P< 0.05). So the best usage of HCO3- was considered to be 25 mM.8. A certain concentration of Ca2+ is beneficial to the in vitro capacitation and protein tyrosine phosphorylation of tarim red deer sperm, the sperm vitality in every group with a low concentration of Ca2+(0-2.2 mM) were basically similar to each other, but the high concentration (3.5-5.0 mM) of Ca2+ inhibited the sperm motility significantly (P<0.05), and promoted the early arising of sperm acrosome reaction(P<0.05); In addition, the concentration of Ca2+ was higher, the level of tyrosine phosphorylation protein expression was lower. The group with 1.1 mM was relatively better than other groups (P<0.05), So the best usage of Ca2+ was considered to be 1.1 mM.9. the adding serum to in vitro capacitation system of tarim red deer sperm was conducive to obtain a better capacitation effect, Among the results of adding estrus sheep serum (ESS), non-estrus sheep serum (NSS) and estrus deer serum (EDS), respectively, the percentage of capacitation sperm induced by ESS was highest, and the tyrosine phosphorylation level of protein with molecular weight of 55 ku increased significantly (P<0.05), these results suggested that the estrus sheep serum was more effective than estrus deer serum (P<0.05).10. BSA was not required to be added when the estrus sheep serum exist in capacitation system. The effect of combination of the estrus sheep serum with heparin was obviously better than that of combination of BSA with heparin. The combination of the estrus sheep serum with heparin not only obviously increased the phosphorylation levels of some proteins with low molecular weight (10±2 ku,14±2 ku,25±3 ku and 47±3 ku), but also improved the phosphorylation levels of some proteins with high molecular weight (70～110 ku) (P<0.05).11. The applicable in vitro capacitation incubation system of tarim red deer sperm screened was considered to be:using the adjusted TALP based liquid, adding 6 mg/mL of BSA, capacitating induced with ESS (20%) and heparin (20μg/mL) for 4 h after washing sperm, and collecting sperm with high vitality using the upstream method, the incubation conditions was 5% CO2,38.5℃ and saturated humidity. By comparison, application of the system could significantly improve the protein phosphorylation levels of more than 10 protiens such as those with molecular weight of 10 ku,14 ku,25～32 ku,40 ku,47～55 ku and 70～110 ku, respectively (P<0.05). |
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