Reduced cytochrome oxidase activity and increased protein tyrosine phosphorylation of mitochondria-rich fractions of buffalo (Bubalus bubalis) spermatozoa after a cycle of freezing and thawing.

The motility and fertility of mammalian spermatozoa are compromised when they are cryopreserved. Sperm mitochondrial proteins play a vital role in conferring motility. However, the effects of cryopreservation on mitochondria-specific proteins remain primarily unexplored in domestic animals, including buffaloes, so the present study aimed to evaluate this issue. Mitochondria were isolated from both non-cryopreserved and cryopreserved buffalo spermatozoa by sonication followed by sucrose density gradient ultracentrifugation. The purity of the mitochondrial preparation was assessed by cytochrome oxidase assay and electron microscopy. Mitochondria separated from cryopreserved buffalo spermatozoa were associated with significantly lower (P ≤ 0.05) cytochrome oxidase activity as compared with non-cryopreserved spermatozoa. The intensities of two low-molecular-mass mitochondrial proteins (30.1 kDa and 26.1 kDa) were significantly reduced as compared with the non-cryopreserved group. In addition, in cryopreserved buffalo sperm mitochondria, the intensities of three tyrosine phosphorylated proteins (126.6, 106.7 and 26 kDa) increased significantly compared with the non-cryopreserved group. Of these, tyrosine phosphorylation of the 26-kDa mitochondrial protein of cryopreserved sperm was very intense and unique because it could not be detected in the mitochondria of non-cryopreserved sperm. Thus, the study confirmed that both cytochrome oxidase activity and the proteins of buffalo sperm mitochondria undergo significant cryogenic changes in terms of quantity and quality after a cycle of freezing and thawing and this may be one of the important causes of reduced post-thaw motility and fertility of cryopreserved buffalo spermatozoa.

Sperm antioxidant defences decrease during epididymal transit from caput to cauda in parallel with increases in epididymal fluid in the goat (Capra hircus).

The status of antioxidant defences of both spermatozoa and their associated fluids during epididymal transit from the caput to cauda have not been studied so far in any species. Herein we report for the first time that sperm antioxidant defences, namely Cu,Zn-superoxide dismutase (Cu,Zn-SOD) and catalase activity, decrease significantly (P<0.05) from the caput to cauda during epididymal transit in parallel with increases in Cu,Zn-SOD, total SOD and total glutathione peroxidase (GPx) activity in the luminal fluid of the respective segments. However, levels of GPX1 and GPX3 in epididymal fluid did not change significantly from the caput to cauda. Catalase was detected for the first time in goat spermatozoa. A significantly higher total antioxidant capacity of caudal fluid than of the caput suggests a requirement for a rich antioxidant environment for the storage of spermatozoa. The retention of cytoplasmic droplets in most of the caudal spermatozoa confirmed that these droplets do not contribute to the increased antioxidant defences of cauda epididymidal fluid. Thus, the antioxidant defences of the spermatozoa and their associated epididymal fluid are modulated from the caput to cauda in a region-specific manner. This may be one of the compensatory mechanisms of epididymal fluid to scavenge any excess reactive oxygen species produced in the microenvironment of spermatozoa.

Loss of heat shock protein 70 from apical region of buffalo (Bubalus bubalis) sperm head after freezing and thawing.

The post-thaw fertility of frozen-thawed mammalian spermatozoa is substantially low as compared with that of fresh sperm. Furthermore, the post-thaw fertility of the cryopreserved buffalo sperm has been reported to be poor as compared with that of cattle sperm. Recently, heat shock protein 70 (HSP70) has been found to play a critical role in mammalian fertilization and early embryonic development in boar and cattle. However, the presence of such fertility-related HSP70 in buffalo sperm and its status after cryopreservation has not been reported so far. Thus, a study was conducted to determine the effect of cryopreservation on the level and distribution pattern of HSP70 molecule in buffalo sperm after cryopreservation. Buffalo semen samples, after dilution in semen extender, were aliquoted in straws and divided into two groups. One group was not cryopreserved, and the other group was cryopreserved for 60 days. Sperm proteins were extracted from both non-cryopreserved (NC) and cryopreserved (C) sperm and subjected to Western blot analysis for detection of HSP70 using a monoclonal anti-HSP70 antibody. The distribution pattern of these proteins in buffalo sperm was also monitored before and after cryopreservation using indirect immunofluorescence technique. A prominent 70-kDa protein band of HSP70 protein was detected in protein extracts of both NC and C buffalo sperm. Densitometry analysis revealed that the intensity of 70-kDa HSP70 protein band of cryopreserved sperm decreased significantly (P < 0.05) compared with that of NC sperm. However, the level of HSP70 in cryopreserved extended seminal plasma (ESP) did not change as compared with that of NC samples indicating a possible degradation of HSP70 in the spermatozoa itself rather than leakage of the protein into the ESP. Furthermore, Western blot also confirmed that several HSP70 immunoreactive protein bands detected in the ESP were contributed by the egg yolk that was added to the extender. Immunocytochemistry revealed that HSP70 proteins were distributed over the apical region of sperm head and/or acrosome, post-acrosomal, and middle piece regions of NC buffalo spermatozoa. However, the fluorescence signal of apical region of sperm head was lost significantly (P < 0.05) after a cycle of freezing and thawing. Thus, the present study confirmed that there was loss of HSP70 from buffalo sperm head after freezing and thawing of buffalo spermatozoa, and this may be one of the causes of the reduced post-thaw fertility of sperm in this species.

Temporal leakage of Cu,Zn superoxide dismutase and loss of two low-molecular-weight forms of glutathione peroxidase-1 from buffalo (Bubalus bubalis) sperm after freezing and thawing.

The postthaw motility and fertility of frozen-thawed buffalo spermatozoa are substantially low as compared with those of cattle sperm. The sperm motility and fertility have been positively correlated with the antioxidant enzyme activities of human and canine sperm. However, the extent of antioxidant enzyme loss during cryopreservation, although reported for human and cattle sperm, is still not clear for buffalo sperm. Thus, in the present study, an attempt was made to determine the activities of various antioxidant enzymes in buffalo spermatozoa cryopreserved for various durations (0, 30, and 60 days) and the mechanism of antioxidant enzyme loss, if any, during the process. Total superoxide dismutase (SOD) activity of cryopreserved sperm decreased and that of extended seminal plasma increased progressively with the increase in duration of cryopreservation indicating the possible time-dependent leakage of these enzymes from cryopreserved sperm into the extended seminal plasmas. The catalase and glutathione peroxidase (GPx) enzyme activities could not be detected in buffalo sperm but could be detected in fresh and extended seminal plasmas. Total GPx activities of extended seminal plasma decreased progressively with the increase in duration of cryopreservation. To confirm the presence of these enzymes at protein levels, specific antioxidant enzymes such as Cu,Zn SOD of 16 kDa and three molecular weight forms (57.7, 40.9, and 26.05 kDa) of GPx-1 were detected in buffalo sperm by Western blot. Furthermore, the intensities of 16-kDa Cu,Zn SOD in 60-day cryopreserved sperm and those of two low-molecular-weight forms of GPx-1 (40.9 and 26.05 kDa) in 30-day cryopreserved sperm decreased significantly (P < 0.05) as compared with those of noncryopreserved (0-day cryopreserved) sperm indicating selective and temporal leakage of only low-molecular-weight antioxidant proteins in the initial phase. However, all the mentioned GPx-1 forms disappeared in 60-day-old cryopreserved sperm. Immunocytochemistry experiment also revealed that Cu,Zn SOD proteins are distributed over the acrosomal region of noncryopreserved buffalo spermatozoa, and the fluorescence signal decreased substantially in 60-day cryopreserved sperm. Thus, the present study reported that there is temporal leakage of Cu,Zn SOD and loss of two low-molecular-weight forms of GPx-1 from the cryopreserved buffalo spermatozoa after freezing and thawing.

Cryogenic changes in proteases and antiprotease activities of buffalo (Bubalus bubalis) and cattle (Bos taurus) semen.

The postthaw motility and fertility of buffalo and cattle semen is reduced when they are cryopreserved for artificial insemination. In the present study, an attempt was made to characterize the cryogenic changes in proteases and antiprotease activities (APA) of buffalo and cattle semen because these proteolysis regulators have been reported to be associated with sperm motility and fertility. Buffalo sperm demonstrated at least two major proteases of 45 and 42 kDa and three minor proteases of 95, 52, and 33 kDa. Similarly, cattle sperm demonstrated three major proteases of 62, 45, and 42 kDa and two minor proteases of 85 and 78 kDa. Buffalo seminal plasma demonstrated at least three major proteases of 78, 68, and 62 kDa and one minor protease of 98 kDa and cattle seminal plasma demonstrated one major protease of 68 kDa and two minor proteases of 78 and 75 kDa. Except for the 45 kDa protease, most of the previously mentioned proteases were found to be metalloproteinases. Compared with fresh sperm, cryopreserved buffalo and cattle sperm demonstrated a major protease band of 52/49 kDa and the activity of this protease reduced progressively with the duration of cryopreservation. On the contrary, compared with the fresh seminal plasma, cryopreserved buffalo and cattle semen extenders displayed the presence of a new protease band of 45 kDa and demonstrated that this protease activity was leaked from buffalo and cattle cryopreserved spermatozoa. Buffalo and cattle seminal plasmas displayed at least two major APA of 86 and 26 kDa. Compared with buffalo, cattle seminal plasma demonstrated significantly greater APA. Thus, the present study demonstrated the presence of an array of proteases and APA in buffalo and cattle semen and the activities of which changed during cryopreservation. The leakage of the specific protease activity and changes in the proteases and APA might be attributed to reduced motility and fertility of cryopreserved semen in these species.