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A. Depeiges and J. L. Dacheux

Summary. Lizard spermatozoa, which are non-motile in the testis, develop the ability to swim as they pass along the excurrent duct. The addition of caffeine, a phosphodiesterase inhibitor, induced forward motility in spermatozoa from the caput epididymidis and increased the velocity of spermatozoa from the distal part of the epididymis. Caffeine had no effect on the motility of testicular spermatozoa. This suggests that sperm motility in this species is cyclic AMP-dependent but this factor alone is not sufficient to induce testicular sperm motility. In samples from the distal region of the epididymis, sperm motility was maximal in April just after the breeding season and then decreased significantly during the following months. A parallel can be drawn between these data and the levels of testosterone in the plasma. In the lizard, as in mammals, the epididymis may play an important role in the maturation of spermatozoa.

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J. L. Dacheux, M. Paquignon, and Y. Combarnous

Summary. Spermatozoa from the corpus epididymidis of boars and rams showed head-to-head agglutination when diluted. The occurrence of agglutination coincided with the appearance of motility but preceded the ability to bind to zona-free hamster eggs. Head-to-head agglutination was inhibited by the addition of caudal epididymal plasma. A protein acting as an antagglutinin on spermatozoa from the corpus epididymidis was extracted from cauda epididymal plasma and partly purified.

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J-L. Gatti, C. Chevrier, M. Paquignon, and J-L. Dacheux

Internal pH and motility of testicular, epididymal and ejaculated ram and boar spermatozoa were studied as a function of external ionic composition. Internal pH was estimated by the amine distribution method and motility was characterized by percentage of cells that were motile and flagellar beat frequency. Upon dilution in media at different external pH values, internal pH of boar and ram spermatozoa changed rapidly towards the external pH. High external concentrations of Na+ or K+ had no effect on the rate of equilibration and only a slight effect on the final internal pH value, ruling out a role of Na+–H+ or K+–H+ exchange mechanisms in this process. In both species, a linear relationship was observed between internal and external pH but equilibration was incomplete suggesting that there is a complex regulatory mechanism. This result was unaffected by epididymal maturation and ejaculation. Ram and boar testicular spermatozoa showed no increase in movement after dilution, suggesting that simple changes in internal pH are not a sufficient trigger for motility. At high external pH, internal pH increased and motility of epididymal boar spermatozoa was initiated. Motility of ejaculated boar spermatozoa, and epididymal and ejaculated ram spermatozoa was less dependent upon external pH and affected only very slightly by the internal pH changes. K+ or Na+ had almost no effect on motility just after dilution. After 1 h of incubation, movement decreased. Maintenance of motility in sodium or potassium showed a sharp external pH optimum. Media without Na+ and K+ allowed a better conservation of motility at external pH > 8 for ram epididymal and ejaculated spermatozoa and at external pH > 6 for boar ejaculated spermatozoa.

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C. Jeulin, J. L. Dacheux, and J. C. Soufir

In the male reproductive tract, very high concentrations (mmol l−1) of free l-carnitine and acetyl-l-carnitine are found in the epididymides, seminal plasma and spermatozoa. It has been reported that the uptake of free l-carnitine by spermatozoa might be related to the epididymal maturation of the sperm membrane, since a greater uptake was found by caput than by cauda spermatozoa in vitro. However, the free l-carnitine concentrations estimated inside the gametes were never greater than those of the surrounding medium. In this study, we investigated the mechanism of transport of free l-carnitine and its ester acetyl-l-carnitine, through the plasma membrane of mature and immature epididymal boar spermatozoa. In vitro, we found a passive diffusion of both compounds to the spermatozoa, whatever the maturation stage. The spermatozoa might progress in the epididymal lumen and accumulate high amounts of free l-carnitine. The active uptake of free l-carnitine occurs only across epididymal mucosa. These results are in agreement with those reported on cells of other organs that exchange pharmacological free l-carnitine concentrations (mmol l−1) by a passive mechanism through the plasma membrane. The acetylation of high amounts of free l-carnitine inside the spermatozoa was found only in caudal spermatozoa. This result suggests that oxidative metabolism (producing acetyl CoA) might be more active in mature cells. The acetyl-l-carnitine added to the incubation medium of boar spermatozoa was hydrolysed. Enzymatic activity of the sperm membrane is low and this may partially explain the low concentrations of acetyl-l-carnitine found in the caudal epididymal plasma.

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Nathalie Josso, J. Y. Picard, J. L. Dacheux, and M. Courot

Summary. Boar rete testis fluid was tested for its capacity to induce Müllerian regression in 14·5-day-old rat Müllerian ducts. Weak activity was present in crude RTF, but after gel filtration and 5-fold concentration, greater activity was detected in 1 out of 7 pools of the eluted fractions. The biologically active fraction (mol. wt 160 000–310 000) coincided with the elution of authentic labelled anti-Müllerian hormone, obtained from bovine fetal testes. These results indicate that a small amount of anti-Müllerian hormone is still synthesized in post-natal life.

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M. Jean, J-L. Dacheux, F. Dacheux, P. Sagot, P. Lopes, and P. Barriere

The involvement of proteins extracted from spermatozoa of fertile semen in sperm–zona binding was examined under hemizona assay conditions. One droplet of a suspension of spermatozoa was exposed to sperm proteins and then tested for zona binding, while a parallel semen suspension droplet incubated with culture medium served as a control. The reliability of the test was increased by relating the number of spermatozoa bound to each inseminated hemizona to the surface area of the hemizona and expressed as the binding index. For spermatozoa incubated with extracted proteins, the binding index was greater than (P = 0.001) that of controls (125.2±45.1 versus 63.6±29.2, respectively). As a first control, two other protein sources (fetal calf serum and human follicular fluid) were tested in the hemizona assay. No significant differences were found in zona binding for other protein-exposed spermatozoa compared with controls. As a second and reverse control, exposure of one hemizona to sperm proteins before insemination with untreated spermatozoa induced a marked decrease (P = 0.0003) in sperm binding, compared with that of the matched hemizona not exposed to sperm proteins (control) (3.4 ±1.4 versus 74.5±6.8, respectively). Taken together, these findings confirm the involvement of extracted sperm proteins in sperm–zona interactions. Therefore, in the cases in which fertilization in vitro fails because of a lack of sperm–zona binding, incubation of deficient spermatozoa with proteins extracted from spermatozoa of fertile ejaculates should restore their ability to interact with the oocyte and, thus, should enhance the prognosis for in vitro fertilization.

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T. O'Shea, J.-L. Dacheux, and M. Paquignon

Summary. Boar spermatozoa incorporated more [14C]glycerol into lipid when incubated with 200 mm- than with 25 mm-glycerol. Measurements were made of the metabolism of spermatozoa while they were being prepared for frozen storage. [14C]Glucose was converted to CO2 and lipid while the cells were cooling to 15°C. Glycerol was added at 15°C and during further cooling to 5°C glucose metabolism was greatly reduced but [14C]glycerol was converted to CO2 and lipid. Under aerobic conditions spermatozoa accumulated lactate while cooling from 30 to 15°C and from 15 to 5°C. With essentially anaerobic conditions, although more lactate was accumulated this occurred only while the cells were cooling from 30 to 15°C, and no further accumulation could be detected during cooling from 15 to 5°C. When boar spermatozoa were incubated at 37°C after storage in liquid nitrogen, metabolism of glycerol was greater than metabolism of glucose. It is suggested that this preferential use of glycerol during cooling and after storage may be one facet of its cryoprotective function. After storage, boar spermatozoa incorporated relatively less [14C]stearic and [14C]palmitic acids into phospholipids (especially phosphatidyl choline) than did freshly collected cells. Caffeine stimulated the oxygen uptake of freshly collected and thawed cells.

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Elizabeth M. Edwards, J.-L. Dacheux, and G. M. H. Waites

Summary. Spermatozoa were collected from the rete testis and vas deferens of conscious rams. The endogenous oxygen uptake of the spermatozoa was unaffected by α-chlorohydrin added in vitro, although this compound abolished the stimulation of oxygen uptake caused by the addition of glycerol. The metabolism of [14C]glycerol by testicular and epididymal spermatozoa was markedly reduced by α-chlorohydrin, CO2 production and lactate accumulation being almost totally inhibited. These effects were dependent upon a period of preincubation of the spermatozoa with α-chlorohydrin alone, since the presence of glycerol protected the spermatozoa from its action. Longer exposure and a higher concentration of α-chlorohydrin were needed with testicular than with epididymal spermatozoa to achieve a maximal effect. The metabolism of [14C]glucose by both sperm types was also inhibited by α-chlorohydrin. Spermatozoa of the ram are therefore susceptible to the action of α-chlorohydrin throughout the epididymis, although more mature spermatozoa are more affected. It is suggested that α-chlorohydrin is converted to an intermediate which is the agent responsible for the inhibition of glycolysis in spermatozoa.

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J. L. Dacheux, T. O'Shea, and M. Paquignon

Summary. Spermatozoa were collected from the rete testis of conscious boars, from the cauda epididymidis by retro-flushing, and by ejaculation. Testicular spermatozoa showed no progressive motility, and that of ejaculated was greater than that of epididymal spermatozoa. Glycolysis and respiration of testicular spermatozoa, while lower than that of the more mature cells, were only slightly affected by the incubation conditions. Epididymal spermatozoa converted 83% of the glucose they utilized to CO2 or lactate, but testicular cells converted only 35% to these metabolites. Synthesis of lipid was greatest by testicular spermatozoa. With the more mature cells hyperosmolar conditions depressed CO2 production, but increased lactate production, and these changes were greater for ejaculated than for epididymal spermatozoa. Glycolysis plus respiration of these cells was related to their motility. These results were interpreted as showing increasing motility, glycolysis and respiration with maturation, but also decreased synthetic capacity and increased sensitivity to the environment.

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C. C. Pariset, J. M. F. Feinberg, J. L. Dacheux, and S. J. Weinman

Summary. Calmodulin concentration and cAMP-dependent protein kinase activity were simultaneously determined on ram spermatozoa collected by cannulation of successive segments of the epididymal tubule. Epididymal transit was characterized on one hand by an overall decrease in the calmodulin level and on the other by a dramatic rise in the cAMP-dependent protein kinase activity. In contrast to the calmodulin level, the cAMP-dependent protein kinase activity was correlated with the acquisition of flagellar beat. No further alterations in the level of these two proteins could be detected as spermatozoa acquired progressive motility.