Energy Metabolism of Spermatozoa. IV. Effect of Calcium on Respiration of Mature Epididymal Sperm of the Rabbit

BIOLOGY OF REPRODUCTION 13, 1-9 Energy Effect (1975) Metabolism of Spermatozoa. of Calcium on Respiration Epididymal Sperm BAYARD of the Rabbit T. STOREY of Reproductive of Obstetrics Department of Biophysics of Physiology and Physical Biology, Gynecology, and and Department Biochemistry, of Pennsylvania School Philadelphia, Pennsylvania of Medicine, 19174 ABSTRACT Epididymal sperm flushed from the caudae of mature rabbits with Tris-saline buffer containing EDTA are motile. The endogenous rate of respiration is stimulated by glucose or pyruvate. There is no further stimulation of the respiration rate by added Ca+2, and this rate is sensitive to inhibition by oligomycin which inhibits mitochondrial ATP synthesis and slows respiration, but which does not inhibit mitochondrial Ca+2 uptake with attendant increase in respiration rate. The sperm cell membrane is not permeable to Ca2, as shown by the observation that addition of the ionophore A23187, which makes cell membranes selectively permeable to Ca+2, results in a marked stimulation of respiration by added Ca+2. The stimulated rate is insensitive to inhibition by oligomycin, showing that the Ca+2 has direct access to the mitochondria which take up this cation in an energy-linked reaction. The presence of 45 mM l-1C03 in the medium stimulates the rate with pyruvate, but not with glucose, and does not affect the response to added Ca+2. Cyclic AMP and dibutyl cyclic AMP, with or without added Ca+2, also do not stimulate respiration with glucose or pyruvate as substrate. Mature rabbit sperm from the cauda epididymis are apparently intrinsically motile, needing only freedom from restraint and oxygen; a trigger in the form of Ca+2 is not needed. INTRODUCTION The in divalent many control cell cation physiological of many by its volved involvement ta in and was ster energy Chang the 1967; have maintain incubated the motility of spermatozoa was this ion. Morton (Chance, 1970). reported the that motility at 37 C, of but AMP they inhibitor diesterase synthesis. found of cyclic (Rail hydrolysis Sutherland, by 1958; sperm been February September has et al., 1971a, 1973; Rogers Ca2 inhibitors on hamthat (Drevius, 1971; Mann, and Chang, 1973; evident have profound are effects that the produced are Casillas Ca2 and at 5 synthesis Butcher this than 23, 1975. 25, 1974. rabbit 1 mM in report, Ca+2 the on as yet unclear. on which spermatozoa might can and also effects AMP Outside the of effects species Effects be that cyclic from is lacking. is AMP reported concerning spermatozoa It these spermatozoa. hamster and 1973). cyclic inhibited information motility HaesungMorton al., (1971) slightly as has spermatozoa, by Hoskins al., 1972; 1973;Tash 1973b; that means monkey et 1973; et reports of and et al., Schoenfeld to discussed 1973a), metabolism these 10 cyclic phosphodiesterase Chulavanatol, from evident et a!., of Garbers -induced after recently b; Hicks and Morton, effect sperm Ca2 decay of intracellular and maintenance Garbers 1973; stimulatory charern of Accepted Received motility the phospho- to role and hamof restored begun 30 mm. The possible AMP in the initiation Mori- In support of this suggesthat 10 mM caffeine, an AMP and had 1965; guinea pig, rat, and rabbit maintained in the absence of et al. (1974) have reported Ca2 activates motility in quiescent epididymal sperm through activation cyclic tion, in- 1962) which (Garbers Hoskins, of those Lehninger, (1970) to of 1969), regulation particularly metabolism et al., required spermatozoa that ster in its functions Manery, Sutherland, motility role through and 1968; enzymes, Lehninger a central processes (Bianchi, intracellular plays properties membranes and and Ca+2 expected, of other Ca2 on since Division Department University IV. of Mature STORE rabbit as oviduct fluid originally anni (1965). in demonstrated and has The effect of to in have shown of Ca on tion of spermatozoa on these the never been indirect in up with of mature an of in containing of quent regard no other of and of ymis which effects therefore, Spermatozoa mitoin is taken results RESULTS a sperm respiration effects of fluid. laboratory spermatozoa the but the 1974) The are, reports has this rabbit motility, processes paper in as Ca2 rabbit of in regard process. intact 1964; 1968) HC03 Keyhani, is increased stimulation This 2 and energy-linked metabolic this been Williams, Brackett, to Ca2 (Storey respiration Ca+2 1967; rabbit has interest. investiga- epididymal flushed a mature 0.1 washing apparent cells 70 motility, rabbit. AND Spermatozoa were flushed from the excised epididymides of mature male White New Zealand rabbits and washed once with a medium composed of 75 mM NaCI, 75 mM KCI, 0.2 mM EDTA, and 20 mM Tris, pH 7.4, followed by centrifugation at 800g. The sedimented sperm twice, in a medium mM Tris, pH 7.45, cells were composed resuspended of 150mM and washed NaCI and 20 by centrifugation at 800 g. After the second cells were resuspended in a minimal volume medium, Hypotonically treated spermatozoa were prepared by resuspending the sedimented cells from the first wash in a solution composed of 10 mM NaPi and 2 mM EDTA, containing 0.4 percent bovine serum albumin (BSA). The cells were sedimented at 800 g, then resuspended and washed twice in a medium composed of 113 mM KCI, 3 mM MgCl2, 5 mM KPi, and 20 mM Tris, pH 7.4, and then resuspended in a minimal volume of this medium. The respiratory activity of both intact and hypotonically treated spermatozoa was determined on cell suspensions at 25 C using the miniaturized oxygen electrode assembly described previously (Keyhani and Storey, NaCI-Tris 1973a). medium, followed wash, the of this Intact and cells were hypotonically assayed treated in the cells in did ADP rises the fully active 3 by of resuspended glucose as glucose gives fold followed greater which than is not respiration by synthesis, respiration tory (Chance, rate is uncoupler Fig. with 1B, the Reed and Mg+2 and brane demonstrated in which compound in a probe of to in Reed the of to been shown treated by as can plasma (1972a) this an particular Ca+2 Lardy Ca2 act in and of are to presence, be memhave ionophore permeability. respiration plasma cells the the experiment prior b) use the 187 across A23187-treated rate of sperm cations, its membrane the the Ca2 respira- 1799. the has divalent detail ATP addition to (1972a, cells. The inhibition maximal in the transported of 1A), Ca2. Ca2 A23 Ca2: rapidly 1.6 (Fig. mitochondrial on of is for (...truncated)