Cryopreservation Increases Coating of Bull Sperm by Binder of SPerm Proteins BSP3 and BSP5.

Abstract

Cryopreserved semen is used widely in the dairy industry to inseminate cows. This practice has allowed the affordable dissemination of semen from superior bulls throughout the world, but the fertility of cows has decreased since the 1950s, more so when artificial insemination with cryopreserved semen has been used. In order to address this problem, it is necessary to learn more about how sperm processing affects sperm quality and function, particularly at a molecular level. This study focused on the Binder of SPerm (BSP) proteins, which are secreted by the seminal vesicles and coat bull sperm. There is evidence that BSPs serve to bind sperm to the oviductal epithelium in order to hold the sperm in a storage reservoir in the oviduct, and that BSPs play a role in the capacitation of sperm. The sperm plasma membrane is modified by extension and cryopreservation of semen. Some of these modifications resemble those caused by capacitation. During incubation under capacitating conditions, we found that BSP5 is rapidly shed from sperm and BSP3 on sperm is quickly reduced in apparent molecular mass from 15 to 13 kDa. To determine if cryopreservation modifies the sperm BSP protein coat, we compared the amounts of 15 and 13 kDa BSP3 and BSP5 on frozen and fresh sperm. We also tested whether dilution of fresh semen in a commercially used milk extender modifies the BSP coat, by comparing the BSPs on fresh sperm diluted in milk extender with those diluted in TALP medium. Fresh semen (n = 6 bulls for BSP3, n = 7 bulls for BSP5) was diluted 1:5 in milk extender or TALP and incubated at 38.5 °C for 1 h. Then, seminal plasma, extender, and dead sperm were removed from live sperm by density gradient centrifugation through BoviPure (Nidacon). Frozen/thawed sperm from 3 straws from each of 4 bulls were isolated in the same manner and then pooled for use in all experiments. Proteins were extracted from 5 million sperm per sample, resolved by 18% SDS-PAGE and electroblotted to nitrocellulose. Seminal plasma was used as a positive control. Blots were labeled with custom-made antibodies against peptides in the N terminal domains of BSP3 and BSP5 (GenScript) and a commercially available β-tubulin antibody (abcam) to calibrate the loading of each lane. HRP-conjugated secondary antibodies and chemiluminescence were used to visualize the protein bands, which were recorded using the Bio-Rad ChemiDoc XRS+ imaging system. The intensity of each BSP band was normalized to the intensity of the β-tubulin band within the same lane. The amounts of 15 and 13 kDa BSP3 and of BSP5 detected in frozen sperm samples were greater (P < 0.03) than in fresh sperm diluted in either milk extender or TALP, but no significant differences were detected between the two dilution media. We concluded that the cryopreservation process enhances the binding of BSP5 and both forms of BSP3 to sperm. It is plausible that abnormally high binding of both BSP3 forms and BSP5 to cryopreserved sperm hinder the release of sperm from the oviductal epithelium, which is required for sperm to move out of the storage reservoir, and/or that excess binding of BSPs modifies the timing of capacitation, thereby reducing the chance that fertilization will occur at the optimal time after ovulation. This may explain the finding that the amount of BSP5 in semen samples is negatively correlated with bull fertility. Semen provided by Genex Cooperative, Inc; research supported by USDA NIFA NRICGP 2008-35203-19031.