Viability of ram testicular spermatozoa following cryopreservation in rete testis fluid

Mammalian spermatozoa have been shown to possess cAMP-dependent protein kinase (A-PK) and endogenous substrate proteins for this enzyme. A study of the kinase system was undertaken to determine changes that may be associated with sperm maturation by comparing immature testicular with mature cauda epididymal and ejaculated spermatozoa. Absolute activity levels of A-PK, stimulated over a concentration range of 10(-9) to 10(-5) M, was significantly greater in testicular than ejaculated spermatozoa. At an optimal cAMP concentration (10(-6) M), testicular spermatozoa had significantly greater amounts of cAMP-dependent protein kinase activity than did cauda or ejaculated spermatozoa. Electrophoretic analysis and autoradiography of NP-40-soluble protein extracts revealed the presence of two substrate proteins (Mr = 62,000 and 44,000) in all three types of spermatozoa. In addition, a phosphoprotein (Mr = 20,000) was detected in mature cauda and ejaculated but not immature testicular spermatozoa. The phosphorylation of these substrate proteins was both dose and time dependent. Examination of cyclic AMP phosphodiesterase activity revealed significantly higher levels in testicular than ejaculated spermatozoa. These results indicate marked alterations in cAMP-modulated protein phosphorylation and dephosphorylation systems in ram spermatozoa during epididymal maturation.

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Characterization of cAMP‐dependent protein kinase and its endogenous substrate proteins in ram testicular, cauda epididymal, and ejaculated spermatozoa

Wooten

Voglmayr

Wrenn

1987

Gamete Research

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Susceptibility of epididymal boar sperm to cold shock and protective action of phosphatidylcholine

1987

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Rapidly cooling (cold shocking) washed cauda boar sperm irreversibly reduced motility and respiration and greatly increased the uptake of 45Ca2+; the plasma membranes were removed and the acrosomes detached from nuclei. The motility, respiration, and calcium uptake of the less mature caput sperm were largely unaffected; and there was little damage to the ultrastructure. This indicates that boar sperm becomes less resistant to cold shock as they mature in the epididymis. The oxygen uptake, glucose breakdown, and lactic acid production of control caput sperm was less than that of cauda sperm. This suggest that the maturation of sperm in the epididymis of the boar involves an increase in both the glycolytic and oxidative phases of glucose metabolism. The presence of 2.0 mg/ml phosphatidylcholine (lecithin) in the medium prevented ultrastructural damage to cauda sperm on cold shock, and motility and respiration were maintained at levels similar to those of control sperm. Although the presence of phospholipid reduced the large calcium influx following cold shock, it was still greater that that of control sperm. The "protective" effect against cold shock was not maintained after rewashing the sperm free of phosphatidylcholine prior to cold shock, indicating a fairly "loose" interaction of the phospholipid with boar sperm membranes that was easily disrupted.

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Characteristics and in vitro fertilizing ability of giant panda (Ailuropoda melanoleuca) frozen‐thawed epididymal spermatozoa obtained 4 hours postmortem: A case report

Pérez-Garnelo¹,

Garde

Pintado³

et al. 2004

Zoo Biology

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When Chu-Lin, a male giant panda (studbook #249), died at Madrid Zoo, his reproductive tract was removed 4 hr postmortem and the epididymal spermatozoa were collected. Extended sperm were kept at 51C for 4 hr, loaded into straws, and frozen for 7 min in liquid nitrogen vapor before the straws were plunged into liquid nitrogen. Two straws were thawed and evaluated. Sperm motility was assessed in fresh, refrigerated, and thawed spermatozoa (75%, 60%, 35%, respectively). Sperm viability and acrosome status were estimated using a triplestain technique (TST). The results showed 33% live sperm with intact acrosomes after thawing. A hypoosmotic swelling (HOS) test demonstrated the retention of membrane integrity in 72% of thawed sperm. To evaluate the in vitro fertilizing ability of thawed sperm, a sperm penetration assay (SPA) was performed. The values obtained for the percentage of penetration and the penetration index were 62% and 1.78 sperm/oocyte, respectively. The results obtained demonstrate that epididymal sperm recovered from a giant panda postmortem can be successfully cryopreserved. The sperm fertilizing ability demonstrated in vitro after thawing may provide a final opportunity for this male to contribute to the currently small germplasm reserves of this endangered species, and to reproduce in the future through assisted reproductive technology.

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Viability of ram testicular spermatozoa following cryopreservation in rete testis fluid

Cited by 7 publications

References 43 publications

Characterization of cAMP‐dependent protein kinase and its endogenous substrate proteins in ram testicular, cauda epididymal, and ejaculated spermatozoa

Characterization of cAMP‐dependent protein kinase and its endogenous substrate proteins in ram testicular, cauda epididymal, and ejaculated spermatozoa

Susceptibility of epididymal boar sperm to cold shock and protective action of phosphatidylcholine

Characteristics and in vitro fertilizing ability of giant panda (Ailuropoda melanoleuca) frozen‐thawed epididymal spermatozoa obtained 4 hours postmortem: A case report

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