External ionic conditions, internal pH and motility of ram and boar spermatozoa.

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.

Water insoluble fraction of egg yolk maintains porcine sperm motility by activating adenylate cyclase.

The decrease in motility of porcine cauda epididymal sperm was less than that of caput epididymal sperm in the medium containing bicarbonate. This may be due to the difference of sensitivity of adenylate cyclase to bicarbonate between mature and immature sperm; activation of mature sperm enzyme by bicarbonate was higher than that of immature sperm. Nondialysable fraction of egg yolk prevented the decrease in motility of immature sperm in the presence of bicarbonate, but it was not effective for the motility of mature sperm under the same condition, because only bicarbonate is sufficient for the maintenance of its motility. In the absence of bicarbonate, both mature and immature sperm required egg yolk to maintain motility. The favorable effect of egg yolk on the motility is ascribed to the enhancement of intracellular cAMP level. Partial fractionation of egg yolk showed that water-insoluble lipoprotein fraction contains factor(s) which activates adenylate cyclase in sperm plasma membrane. This is the first report in which high molecular weight activator of the sperm enzyme was demonstrated.

Effects of freezing-thawing on the spermatozoon nucleus: a comparative chromatin cytophotometric study in the porcine and human species.

Freezing-thawing effects on the nuclei of porcine and human spermatozoa were studied by determining native DNA percentage from fluorescence after acridine orange (AO) staining and by analyzing chromatin structure by a quantitative microspectrophotometric study of Feulgen-DNA complexes before and after freezing. The study of boar spermatozoa revealed no alteration in native DNA percentage after freezing. However, native DNA percentage decreased significantly in human spermatozoa. Feulgen-DNA content and sperm nuclear surface area decreased in both species after freezing. These results prompted us to hypothesize an overcondensation of sperm chromatin after freezing-thawing. This overcondensation may be related to the lower conception rates obtained with human and porcine semen after cryostorage via defective decondensation.

Preliminary study of water and some element contents in boar spermatozoa, before, during and after freezing.

Boar semen was analysed by electron microscopy coupled to image analysis and X-ray energy dispersive spectroscopy, during the usual process for freezing and thawing in field conditions. Freeze-substitution and freeze-quenching permitted recording of real or potential intracellular ice before, during, and after freezing. Heads and flagella displayed two different osmotic properties before freezing. Heads were dehydrated progressively before and during freezing, while flagella were hydrated before freezing and were only dehydrated during freezing. All parts of the thawed cells were rehydrated. Ice crystal damage was mostly present in frozen mitochondria and axonemes and the acrosomes were strongly affected by thawing. The total amounts of Na, Cl, Ca, K, Mg, and Zn per cell were only elevated in frozen and thawed midpieces while the heads were permeable both to water and elements at that time.

Changes in sperm surface membrane and luminal protein fluid content during epididymal transit in the boar.

The surface membrane protein of boar sperm and the proteins in the fluid surrounding the gametes were analyzed during epididymal transit. The present study demonstrated that sequential dramatic changes occur in protein composition of the sperm membrane and epididymal fluid during epididymal transit. The maturation process of the boar sperm surface was characterized by a complex sequential evolution of the composition and orientation of macromolecules in the sperm membrane. Epididymal maturation resulted in the progressive disappearance of most of the surface testicular compounds, which were either renewed or masked by new permanent or transient low molecular weight polypeptides on the boar sperm surface membrane. In the fluid surrounding the spermatozoa, composition of the luminal proteins was altered throughout the epididymal transit and several new compounds were characterized. Very few proteins were correlated either with blood plasma or sperm surface compounds.

Nucleus of the boar spermatozoon: structure and modifications in frozen, frozen-thawed, or sodium dodecyl sulfate-treated cells.

After cryosubstitution and Epon embedding, or after Nanoplast embedding and very thin sectioning, the chromatin of ejaculated or diluted boar spermatozoa appears to be formed of DNA fibers embedded in a quite homogeneous matrix. After sodium dodecyl sulfate (SDS) treatment, and to a lesser extent after freeze-thawing, the DNA fibers are present mostly between cords, probably proteinaceous in nature. The quantity of free sulfhydryl (SH) groups, as calculated from staining by DACM and flow fluorometry, is increased in thawed or SDS-treated cells. The quantity of NH2 groups, calculated from electron microscopy image analysis of alcoholic phosphotungstic acid-stained cells, is decreased in thawed nuclei. The DNA is more accessible to the fluorochrome ethidium bromide after freeze-thawing, and its sensitivity to HCl hydrolysis is modified, during the Feulgen-like staining procedure using acriflavine. The X-ray energy dispersive analysis of cryosections of nuclei indicates that the slight separation of DNA and nucleoproteins in freeze-thawed spermatozoa could result from a dramatic modification of the nuclear ionic environment during thawing.

[Flagellar motility amd movement of boar spermatozoa during epididymal transit]. / Analyse de la motilité et du mouvement flagellaire des spermatozoïdes de verrat au cours du transit épididymaire.

The motility of boar spermatozoa during epididymal transit was analysed in vitro using various techniques. From the head to the cauda there was an increase of the percentage of motile and progressive spermatozoa. During maturation there was a progressive reduction of flagellar bend curvature while flagellar beat frequencies increased. A three dimensional pattern of flagellar beating responsible for cell rotation and straight line progression of spermatozoa was observed only in caudal epididymis. The addition of epididymal fluid protein to the media could increase the number of motile cells at the various levels but had no influence on the characteristics of flagellar bending.

[Acetylcarnitine and spermatozoa: relationship with epididymal maturation and motility in the boar and man ]. / Acétylcarnitine et spermatozoïdes: relation avec la maturation épididymaire et la mobilité chez le verrat et l'homme.

The level of carnitine and acetylcarnitine in spermatozoa of boar epididymal origin and of human ejaculates was demonstrated. In the epididymal fluid of boars, the concentration of carnitine (nmol/mg protein) began to increase from 20 in the distal caput to rise progressively to 700 in the distal cauda. By contrast, the carnitine content of spermatozoa only started to increase in the proximal cauda where the concentration of carnitine in the fluid was 200-300 nmol/mg protein, then gradually increased in spermatozoa from more distal sites. The increase in the acetylcarnitine content of spermatozoa paralleled that of the carnitine amount, represented 50% of total carnitine (carnitine + acetylcarnitine) and coincided with the acquisition of progressive motility. In two populations of human seminal spermatozoa selected by migration and characterised by a very large difference in their percentage of progressively motile cells, higher carnitine and acetylcarnitine contents (40%) were found in migrated spermatozoa compared to the residual population. These results suggest that accumulation of carnitine and its metabolite may be an important factor in the acquisition and the maintenance of progressive motility. Measurement of acetylcarnitine content of human seminal spermatozoa could be used as a marker of epididymal maturation.

The distribution of carnitine and acetylcarnitine in the epididymis and epididymal spermatozoa of the boar.

In the epididymal fluid of boars, the concentration of carnitine (nmol/mg protein) began to increase from 20 in the distal caput, then rose progressively to 700 in the distal cauda. By contrast, the carnitine content of spermatozoa only started to increase in the proximal cauda where the concentration of carnitine in the fluid was 200-300 nmol/mg protein then gradually increased in spermatozoa from more distal sites. The increase in the acetylcarnitine content of spermatozoa paralleled that of the carnitine amount and represented 50% of the total carnitine (carnitine + acetylcarnitine). We conclude that the acetylcarnitine content of epididymal spermatozoa may be used as a marker of maturation.

Relations between the fertilizing ability, motility and metabolism of epididymal spermatozoa.

This paper compares the principal modifications taking place in spermatozoa during epididymal transit in several species. The relations between changes in the epididymal medium and modifications in the metabolism, motility and/or fertility of spermatozoa are reviewed with particular reference to spermatozoon forward motility. The fertilizing ability of motile testicular and epididymal spermatozoa has been described. It is suggested that the maturation process may induce a series of spermatozoal changes in cyclic AMP content, external membrane composition and nuclear structure throughout the genital tract; these changes would be under the control of the epididymal epithelium. The origins of these phenomena are practically unknown.

Metabolism of boar spermatozoa before, during preparation for, and after storage in liquid nitrogen.

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 degrees C. Glycerol was added at 15 degrees C and during further cooling to 5 degrees 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 degrees C and from 15 to 5 degrees C. With essentially anaerobic conditions, although more lactate was accumulated this occurred only while the cells were cooling from 30 to 15 degrees C, and no further accumulation could be detected during cooling from 15 to 5 degrees C. When boar spermatozoa were incubated at 37 degrees 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.

Effects of osmolality, bicarbonate and buffer on the metabolism and motility of testicular, epididymal and ejaculated spermatozoa of boars.

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.