Carboxyfluorescein diacetate and propidium iodide were used as fluorescent stains to assess membrane integrity in sperm populations from ram and boar. The living spermatozoa were immobilized with low concentrations of formaldehyde so that individual stained cells could be observed in a suspension with the aid of a fluorescence microscope. Intracellular esterases liberated impermeant-free carboxyfluorescein from the permeant carboxyfluorescein diacetate and caused the product to accumulate and fluoresce green within the acrosome and the mitochondria as well as within the cytoplasm. Most of the spermatozoa (the intact ones) accumulated carboxyfluorescein in all compartments; however, a few cells (those with damaged plasma membranes) accumulated the stain only in the acrosome and/or the mitochondria, while others (all of whose membranes were damaged) remained entirely unstained. The impermeant propidium iodide did not stain any of the (intact) spermatozoa that accumulated carboxyfluorescein throughout their length, but stained all the others (the heads fluoresced red). The technique appeared to provide more reliable estimations of the percentage of functional cells than did motility estimations or assessments of acrosomal integrity (presence of normal apical ridge). The technique also demonstrated the sensitivity of the sperm plasma membrane to cold shock: virtually all cells rapidly became permeable to the stains after such stress. Assessments of boar sperm samples during preparative incubation for in-vitro fertilization indicated a considerable increase in the percentage of cells with damaged plasma membranes as incubation proceeded, in advance of the increase in the percentage of cells with discharged acrosomes.

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Use of fluorescent probes to assess membrane integrity in mammalian spermatozoa

Homozygous c-ros knockout male mice that lack prepubertal differentiation of the epididymal initial segment are healthy but sterile, despite normal sperm production and mating. Detailed computerized analysis of the motility of spermatozoa maturing in the epididymis revealed only minor defects. However, the majority of motile mature sperm released from the cauda epididymidis showed various extents of flagellar angulation that could not be corrected by raising extracellular osmolality. Measurement of the osmolality of cauda epididymal fluid showed no difference from the wild type. Studies in wild-type mice indicated a maturational change in the ability of motile sperm to maintain straight flagella during incubation, but angulation was induced in cauda sperm by the volume-sensitive ion channel blockers quinine, 5-nitro-2-(3-phenylpropylamino)-benzoic acid and BaCl(2), or by exposure to hypotonic media. Flagellar angulation, induced in the wild type or intrinsic to the knockout, was relieved upon demembranation by Triton X-100, confirming that it was a cell swelling phenomenon. A lack of response of immature wild-type sperm and mature knockout sperm to the channel blockers suggests that there is normally a development of the volume regulatory mechanisms upon maturation that is defective in sperm from the knockout animal. The resultant flagellar angulation may account for the reduction in sperm numbers in the oviduct of mated females and the failure to fertilize in vivo.

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Infertile spermatozoa of c-ros tyrosine kinase receptor knockout mice show flagellar angulation and maturational defects in cell volume regulatory mechanisms.

During incubation of ram spermatozoa at 1 x 10(7) cells mL-1 or less in a simple HEPES-buffered saline medium, high levels of cell death were detected using propidium iodide as a probe of viability (membrane integrity): some 70% of the cells died during 3 h incubation at 37 degrees C. Because the conditions of incubation were similar to those encountered during manipulations for in vitro fertilization, this phenomenon was investigated further. If ram spermatozoa were diluted in an equivalent sucrose-based medium, or if the saline medium was supplemented with 10% seminal plasma, survival was greatly improved (only 5-15% died during a 3-h incubation at 37 degrees C); the protective effect of seminal plasma resided in a 5-10 kDa fraction. Sperm death in the basal saline medium was strongly dependent on cell concentration below 5 x 10(7) spermatozoa mL-1 whereas little effect of concentration was seen in the sucrose medium or in the presence of seminal plasma. The presence of Ca2+ (2 mM), EGTA (1 mM) or mercaptoethanol (1 mM) enhanced sperm survival in saline medium, but no effect was gained by replacing NaCl with KCl, and neither BSA nor fetal calf serum were beneficial. However, when a combination of pyruvate (1 mM), lactate (21.7 mM), Mg2+ (0.4 mM), phosphate (0.3 mM) and Ca2+ (2 mM) was included in the saline medium (to render it similar to Tyrode's medium), cell survival was greatly improved (12% died during the 3-h incubation).(ABSTRACT TRUNCATED AT 250 WORDS)

Survival of ram spermatozoa at high dilution: protective effect of simple constituents of culture media as compared with seminal plasma

Effects of semen fractionation and dilution ratio on equine spermatozoal motility parameters.

In the cow, a specific glycoprotein-bovine oviduct-specific glycoprotein (BOGP)-is secreted by the epithelial cells of the oviduct at the follicular stage of the estrous cycle. In this study, we examined the effects of purified BOGP on the viability and motility of bovine spermatozoa in culture in vitro. Frozen-thawed bovine spermatozoa were incubated in modified Tyrode's solution (TALP) that contained purified BOGP (TALP-BOGP). In TALP-BOGP, both the viability and motility of bovine spermatozoa were more effectively maintained than in the control medium without any added protein. The increases in both the viability and motility of spermatozoa were dose-dependent. Spermatozoa were also incubated in TALP medium supplemented with bovine serum albumin, egg albumin, lactalbumin, or gastric mucin, and their viability and motility in these media were compared with that in TALP-BOGP. Both the viability and motility of spermatozoa were more effectively maintained in TALP-BOGP throughout a 12-hr incubation than in other media tested. An immunolabeling study demonstrated that a monoclonal antibody specific for BOGP reacted with the posterior region of the head, the middle portion, and the tail of spermatozoa that had been incubated with TALP-BOGP, suggesting that BOGP becomes specifically associated with particular regions of the spermatozoon. These results suggest that BOGP is a potent factor for maintenance of the viability and motility of sperm. On the basis of the present results, we also propose that BOGP may play an important role in sperm functions during the reproductive process. © 1995 wiley-Liss, Inc.

P.J. Bredderman

Papers

Factors stabilizing bull sperm cell volume and prolonging motility at high dilution

Alteration of cell volume in bull spermatozoa by factors known to affect active cation transport.