Immotile bovine caput epididymal sperm contain levels of protein phosphatase activity twofold higher than do mature motile caudal sperm. Comparison of the inhibition profiles of endogenous phosphatase activities detected by okadaic acid (OA) and calyculin A (CA) revealed a pattern consistent with the predominance of a type 1 protein phosphatase (PP1). Immunoblot analysis identified PP1 gamma 2 (the testis-specific isoform of PP1) as the only PP1 isoform in sperm and showed little protein phosphatase 2A (PP2A). In addition, of the known PP1 inhibitors, i.e., DARPP-32, inhibitor 1 (I1), and inhibitor 2 (I2), only I2-like activity was detected in sperm. Inhibition of PP1 by the heat-stable I2-like activity purified from sperm could be reversed with purified glycogen synthase kinase-3 (GSK-3). Furthermore, sperm extracts contain an inactive complex of PP1 and I2 (termed PP1I) that could also be activated by purified GSK-3. The presence of GSK-3 in sperm was demonstrated by activation of purified PP1I, and quantitation revealed that immotile caput sperm contained sixfold higher GSK-3 activity than motile caudal sperm. Immunoblot analysis confirmed the expression of GSK-3 in sperm and revealed the occurrence of both the alpha and beta isoforms. Our findings suggest that the higher PP1 activity measured in immotile sperm, presumably due to higher GSK-3 activity, is responsible for holding motility in check. This conclusion was supported by the observation that the phosphatase inhibitors OA and CA, at micromolar and nanomolar levels, respectively, were able to induce motility in completely immotile bovine caput epididymal sperm and to stimulate the kinetic activity of mature caudal sperm. The intrasperm levels of cAMP, pH, and calcium were unaltered by treatment with these inhibitors. The results suggest a biochemical basis for the development and regulation of sperm motility and a possible physiological role for the PP1/I2/GSK-3 system.

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The presence of both cyclic 3':5'-adenosine monophosphate (cAMP) and the enzymes required for its synthesis, degradation, and apparent expression of action have been documented in mammalian spermatozoa. Several studies ) have shown that motility can be induced or maintained by cyclic nucleotides and phosphodiesterase inhibitors in bovine spermatozoa from the cauda epididymidis and that increased rates of respiration and fructolysis occur concomitantly. Advantage was taken in these studies of the fact that the motility of bovine spermatozoa from the cauda epididymidis can be substantially reduced by simple incubation at 37\s=deg\Cin isotonic buffers over a relatively short period of time (several hours). Since bovine spermatozoa from the caput epididymidis resemble quiescent caudal cells in that they are characterized by a virtual absence of motility (Igboeli & Foote, 1968) and low glycolytic rates (D. D. Hoskins, unpublished observations), the question arises as to whether the level of cAMP is a prime determinant in the epididymal development of the capacity for motility and increased metabolic rates. The same question can be asked about the cAMP-dependent protein kinases since these enzymes, present in large amounts in bovine spermatozoa from the cauda epididymidis (Hoskins, Casillas & Stephens, 1972;Garbers et al., 1973b), have been implicated in the physiological expression of all actions of cAMP (Kuo & Greengard, 1969a, b). In this communication, we report the levels of cAMP and cAMP-dependent and -independent protein kinases in bovine spermatozoa from the caput and cauda epididymidis.Cyclic AMP was determined by the method of Gilman (1970) except that commercial bovine heart cAMP-dependent protein kinase (Sigma Chemical Co.) was used as the cAMP-binding protein. Assays were performed on sper¬ matozoa from the epididymides of testes stored overnight at 4CC. A prewarmed (37°C) solution consisting of 42 mM-KCl, 88 mM-NaCl, 5 mM-MgS04, 10 mM-KH2P04 and 10 mM-tris, pH 7-2 was used to isolate, wash, and suspend the spermatozoa. Spermatozoa were obtained from the distal cauda epididymidis by the method of Henle & Zittle (1942) and from the distal portion of the caput, after excess blood vessels had been trimmed away, by gently shaking the 131

A computer-automated sperm motility assay (CASMA) system has been developed that provides a rapid and accurate analysis of multiple sperm movement parameters and a new measure of linearity, the linear deviation angle. CASMA provides objective, unbiased sampling and accurate quantitation of the movement characteristics of 200 sperm cells in 20 min. It consists of a microscope-mounted video camera, a high-resolution video disk recorder, a video digitizer/memory board mounted in an IBM 9000 microcomputer, and newly developed software. After manual recording, at 60 frames/s, of the video sequences (takes) of sperm suspensions, each take is automatically played back frame by frame, digitized, and stored in video memory. The software searches each frame, recognizes sperm cells, randomly selects a preset number for analysis, and traces each cell through the sequence to generate sperm "tracks" that are then stored in disk memory. This process is repeated for each take. Analysis of the stored tracks of each take yields the mean +/- SEM of the standard sperm motility parameters: percent motile (%M), curvilinear velocity (VC), net velocity (Vn), position-averaged velocity (Va), linear index (Vn/Va), progressiveness ration (Vn/Vc), and curvilinear progressiveness ratio (Va/Vc). Additionally, CASMA allows measurement of the linear deviation angle, a more direct measure of the linearity of sperm movement. For statistical comparisons, multiple takes can be considered either as replicates or separate experimental determinations. Finally, for more detailed analysis, each individually stored track, with its associated parameters, or histogram distributions of all sperm for each parameter can be displayed and printed. The performance of CASMA was evaluated by comparison of CASMA-determined movement parameters with manually determined values derived from the same sperm cells in the same video sequence and by comparison with published values determined using microcinematographic techniques. In each case, the CASMA values were essentially identical to those determined by manual measurements. Finally, CASMA accurately quantitates the linearity of sperm movement, a characteristic previously determined only by much more time-consuming methods. CASMA is a rapid and accurate system for measuring washed bull sperm motility and has reliably analyzed monkey and elephant sperm. The system has the potential to quantitate motility equally well with sperm from any species that have similar sperm head size.

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