Evidence for an Epididymal Origin of Bovine Sperm Forward Motility Protein,2

Transcription

1 BIOLOGy OF REPRODUCTION 19, (1978) Evidence for an Epididymal Origin of Bovine Sperm Forward Motility Protein,2 HOWARD BRANDT, TED S. ACOTT, DEBORAH J. JOHNSON and DALE D. HOSKINS3 Oregon Regional Primate Research Center 505 N.W. 185th Avenue Beaverton, Oregon ABSTRACT Forward mobility protein (FMP) is a glycoprotein found in bovine seminal plasma that can induce forward motion in immotile caput spermatozoa under conditions of elevated cyclic AMP levels. This paper presents evidence that this seminal plasma protein is of epididymal origin. FMP activity is present in decreasing amounts in caudal epididymal fluid, seminal plasma and rete testis fluid. The FMP of caudal epididymal fluid displays properties identical to those of seminal plasma FMP after heat, freeze-thaw, trypsin or $3-glactosidase treatment. Further physical evidence for the similar character of epididymal and seminal plasma FMP is shown by the fact that the complex Sepharose 6B elution profiles of the forward motility inducing activity in the two fluids are nearly identical. Since rhesus monkey seminal plasma induces forward motion in bovine caput sperm, we were able to use the seminal plasma of vasectomized monkeys to assess the contribution of the accessory glands to FMP production. The typical motility inducing profile seen in the semen of the normal monkey was not observed in the ejaculate of the vasectomized animal. This indicates that FMP is not produced by the accessory glands. These data indicate an epididymal origin of FMP and suggest a physiological role for this protein in the acquisition of sperm motility. INTRODUCTION Although it has long been known that mammalian sperm acquire the capacity for forward motility as they traverse the epididymis (Tournade, 1913), the biochemical basis for this acquisition has remained obscure. Recent studies have shown that ligation of the distal corpus epididymis of the rabbit produces, in days, caput spermatozoa with high motility. This implies that the acquisition of the capacity for motility in this species does not require exposure to more distal portions of the epididymis and that the pattern of the Accepted April 26, 1978 Received February 9, 1978 Publication No. 981 of the Oregon Regional Primate Research Center. 2This investigation was supported by General Research Support Grant RR00163, Grant for the Operation of the Oregon Regional Primate Research Center, from the Animal Resources Branch, Division of Research Resources, National Institutes of Health, as well as Program Project Grant HD05969 from the Center for Population Research, National Institute of Child Health and Human Development, National Institutes of Health. reprint requests to Dale D. Hoskins. onset of motility can be changed by simple retardation of sperm in the proximal portion of this organ (Bedford, 1975). Burgos and Tovar (1974) have carried out similar ligation studies in the rat and have shown that immature sperm, retarded for up to 10 days in the caput epididymidis, display the same pattern of motility acquisition as caput sperm fron the nonligated epididymis, that is, they move in circles with a marked stiffness in the neck and midpiece. These authors postulated that the acquisition of sperm motility in the rat is due to a factor or factors produced by the epididymis and secreted into the lumen. The nature of these hypothetical factors or their epididymal site of synthesis, however, has not been determined. Calvin and Bedford (1971) have suggested that the mechanism by which sperm become motile involves an increased rigidity of the flagellum, due to the formation of disulfide bonds. In 1974, we suggested that the intrasperm content of camp may also be required for motility initiation in developing bovine sperm since submotile mature (caudal) bovine sperm are stimulated by camp phosphodiesterase inhibitors and the absolute intrasperm levels of camp nearly double during epididymal 830

2 EPIDIDYMAL ORIGIN OF FMP 831 transit (Hoskins et al., 1974). However, we found that the addition of high levels of camp phosphodiesterase inhibitors causes immature (caput) sperm only to twitch and that further addition of seminal plasma is required for induction of forward progression (Hoskin et al., 1978). Recently, we have partially purified the factor in seminal plasma responsibbe for the initiation of motility synergistically with theophylline and have shown that it is a glycoprotein (Acott and Hoskins, 1976, 1978). The obvious question is whether this seminal plasma protein which we have called forward motility protein (FMP) is involved in the acquisition of motility during development. In this report, we provide evidence that seminal FMP is of epididymal origin. MATERIALS AND METHODS Freshly ejaculated bovine semen was supplied by All West Breeders, Inc., Burlington, WA. Sperm was sedimented and washed as previously described (Hoskins et al., 1975). Contents of the cauda epididymidis were obtained from freshly slaughtered bulls by the method of Henle and Zittle (1942) and epididymal fluid was obtained after removal of sperm by centrifugation at 2,500 X g for 10 mm (20#{176}C). Rete testis fluid was obtained from the testes of freshly slaughtered bulls by dissection of the caput epididymidis away from the testis (for exposure of the ductus efferentes, creation of a small incision into the ducts and collection of the exuded fluid with a pipette. Testicular sperm were removed by centrifugation at 2,500 X g for 10 mm (20#{176}C). All reproductive tract fluids were routinely heated at 95#{176}C for 10 mm, centrifuged at 100,000 X g for 60 mm (4#{176}C) and dialyzed against assay buffer (10 mm Tris HCI, 42 mm KCI, 103 mm NaCI, 5 mm MgSO4. 10 mm KH2PO4. ph 7.4) for 16 h (4#{176}C).Seminal plasma and caudal fluids were stored at -20#{176}C.In contrast, rete testis fluid was stored at 4#{176}Cand used within 24 h of collection. Sperm motility was quantified by the method of Acott and Hoskins (1978) except that the reaction volume was reduced to 300 MI. Briefly, the forward motility assay used bovine sperm from a minced section of the distal caput portion of the epididymis. The sperm were extracted into assay buffer and layered over 5 volumes of an 8% Ficoll solution in the same buffer and centrifuged for 5 mm at 500 X g and then for 5 mm at 1,150 X g. The sperm pellet was resuspended in assay buffer containing 5mg/mi BSA. All of the above operations were conducted at room temperature. Aliquots chosen to give a final concentration of 2.5 X 10 sperm/mi were added to assay buffer containing 33 mm theophylline, 10 mm glucose, 10 mg/mb BSA and the material to be tested. After a 20 mm incubation at 37#{176}C,the sperm were placed on a prewarmed (37#{176}C)hemocytometer (0.1 mm in depth) and a 5 second exposure photograph was taken under dark field illumination. The quantity of sperm tracks represents the number of moving sperm and the lengths of the tracks represent their velocity. The forward motility index (FMI) is defined as the product of the percentage of motile sperm and the mean velocity expressed in Mm/second. Seminal plasma from 6 vasectomized rhesus monkeys was pooled. These samples were kindly supplied by Dr. Nancy Alexander. The normal monkey seminal plasma was a pool of 3 different animals. Both poois were heattreated (95#{176}C, 10 mm), centri fuged (2,500 X g. 10 mm) and dialyzed against assay buffer. Protein concentration was determined by the method of Lowrey et al. (1951) with bovine serum albumin as the standard.,<200 U / 10 RESULTS AND DISCUSSION Since the FM! assay measures a physiological parameter, i.e., induction of motion, rather than a chemical entity, we sought in the studies described here to provide preliminary data on the origin of seminal plasma FMP by comparing the chemical and physical properties of epididymal and seminal plasma factors. Figure 1 shows that concentration-dependence curves for both fluids are similar in i.. D 3 do 60? ijg PROTEIN FIG. 1. Concentration dependence of the ability to initiate forward motility in bovine caput sperm with bovine seminal plasma (A) and bovine epididymab - caudal fluid (B). i F-...

3 832 BRANDT ET AL. TABLE 1. Concentration of forward motility protein in bovine reproductive tract fluids. Fluid FMI/Ml FMI/uga Maximum observed FMI (mean ± SEM) Caudal epididymal fluid Seminal plasma Retetestisfluid abased on protein value before heat treatment. The values for FMI/ul and FMI4ig were derived from linear portions of the concentration curves obtained for each fluid. that saturation of forward-motion-inducing activity with each occurs between 500 and 600 FM! units. Clearly, the specific activity of epididymal fluid is approximately 5 times greater than that of seminal pbasma (Table 1). These data support an epididymal origin of FMP since, of the the body fluids tested thus far, only seminal plasma and epididymal fluid induce such high maximab FMI values (Hoskins et ab., 1978). In addition, similarity between the factors from these two fluids is shown by the observations that both are stable to heat and freeze-thaw treatment, labile to proteolytic digestion by trypsin and partially (50%) inactivated by preincubation for 2 h with j3-galactosidase (Table 2). Incubation with the latter enzyme for an additional 2 h does not increase inactivation of either fluid factor. Bovine reproductive tract fluids contain, as measured by the induction of forward motion in bovine caput spermatozoa, the highest levels of FMP in any of the biobogical fluids studied to date. The specific forward motility index (FMI/pg protein) of fluid from the bovine cauda epididymidis is nearly 500 times that of blood serum (Hoskins et al., 1978). Table 1 shows that, among reproductive tract fluids, the highest activity is found in fluid from the caudal epididymidis and the lowest in rete testis fluid. The larger standard error shown for rete testis fluids stems from the poor precision of the photographic assay at low FM! values. At first glance, it may seem somewhat paradoxical that rete testis fluid and seminal plasma are of equal specific activity when FMI vabues are expressed on a pg protein basis. It must be recalled, however, that seminal plasma protein bevels are as much as 20 times greater than those found in rete fluid (Mann, 1975). Thus, when expressed on a volume basis, seminal plasma contains much more FMP than rete testicular fluid (Table 1). An additional approach to determining whether FMP is of epididymal origin is to assess whether the amount present in the fluid contributed by the epididymis is sufficient to account for all the FMP found in seminal plasma. This question can be approached indirectly by using the amount of glycerylphosphorylcholine (which is solely of epididymal origin) found in seminal plasma to assess the proportion of fluid contributed by the epididymis to a typical ejaculate (Mann, 1975). Used in conjunction TABLE 2. The effects of various degradative procedures on the stability of the forward-motilityinducing factor(s) in seminal plasma and caudal epididymal fluid. Procedurea Seminal plasma Heat (90#{176}C,10 mm) Freeze-thaw Trypsin 0 16 ji-galactosidase Caudal epididymal fluid Values are expressed as a percentage of activity remaining in untreated controls. Fluids were frozen (-20#{176}C) and thawed (3 7#{176}C) 10 times. Enzyme treatments were as follows: One mg of trypsin/ml (specific activity 200) was incubated at ph 7.2 with seminal plasma and caudal epididymab fluid for 2 h (25#{176}C). The reaction was stopped with 1 mg/mi of soybean trypsin inhibitor (specific activity 280). Activity lost was expressed as a percentage of a sample that had soybean trypsin inhibitor added before the 2 h incubation period. One mg of 3-galactosidase/ml (specific activity 360) was incubated at ph 7.5 for 2 and 4 h (25#{176}C)with seminal plasma and caudal fluid. The treated fluids were assayed immediately after incubation. Controls containing -galactosidase at the same dilution as the treated fluids showed this enzyme had no effect on motility assay results.

4 EPIDIDYMAL ORIGIN OF FMP 833 with data on the levels of protein found in bovine epididymal fluid and seminal plasma (Mann, 1975), it is possible to first estimate the contribution of epididymal protein to the total protein in the ejaculate and then to estimate what the relative FM! specific activities in the two fluids should be. We infer from these rough calculations that epididymal fluid should have a specific activity 3.5 times higher than seminal plasma if it is the sole source of FMP. The data shown in Table 2 indicate that the specific activity of epididymal fluid is in fact 4.5 times greater than that in seminal plasma. Thus, the amount of FMP in epididymal fluid could easily account for all the activity found in seminal plasma. Probably the best piece of physical evidence that the two fluid factors from seminal plasma and caudal epididymal fluid are identical is that both show the same activity profile when passed through a column of Sepharose 6B (Fig. 2). At first glance, this 4-peak pattern indicates that there are actually four factors, not one. However, when the fractions from any one of the peaks are pooled, concentrated and rechromatographed on the same column, the original 4-peak pattern is regenerated (Hoskins et al., 1978). We have interpreted this result as a tendency for FMP to exist in a state of multiple aggregate equilibria. Although this condition prevents the assignment of a unique molecular weight by gel chromatography, we can use this unusual 4-peak profile as a fingerprint for the detection of FMP. The fact that C >. -p elution peak heights are dissimilar is not disturbing since they represent only the relative amount of one molecular size form present. When it is recognized that Sepharose 6B fractionates proteins over a large molecular weight range (2.5 X X 106 daltons) the elution profiles shown are remarkably similar. a Although these observations do not provide proof that the two factors are the same, they do suggest that they are quite similar. Proof of identity demands purification and characterization of FMP from both fluids. The photographic motility assay is not particularly well suited to protein purifications since it is expensive and somewhat laborious. Efforts are currently under way to circumvent these problems by development of a radioummunoassay from seminal plasma FMP. The final evidence for the epididymal origin of seminal plasma FMP is based on studies which showed that FMP is widely distributed in the seminal plasma of laboratory and domestic animals and that the forward motility proteins present in the seminal plasma of these species lack specificity with regard to activation of bovine caput spermatozoa (Hoskins et al., 1978). Thus, rhesus monkey (Macaca mulatta) seminal pbasma, as well as bovine seminal plasma, induces forward motion in bovine caput sperm. Therefore, it was reasoned that the seminal plasma of vasectomized monkeys should not contain FMP if this glycoprotein is produced in the epididymis. The data shown in Fig. 3 confirm that this is true. 400 Although a reproducible but bow activity was detected in the seminal plasma from ui: a FIG. 2. Chromatographic profile of seminal plasma (A) and epididymal caudal fluid (B) on Sepharose 6B. Vo iaq PROTEIN FIG. 3. Concentration dependence of the ability to initiate forward motility in bovine sperm with normal (.) monkey and vasectomized (0) monkey seminal plasma.

5 834 BRANDT ET AL. vasectomized animals (Fig. it is important to note that this activity cannot be explained by production of a low amount of FMP by male reproductive tract accessory glands. If such production did occur, an FM! value in the range found in the seminal plasma of normal monkeys (Fig. 3) would have been observed when saturating levels of vasectomized monkey seminal plasma were used. The data in Fig. 3 clearly show that this is not the case since the concentration-dependence curve leveled off at an FM! value of approximately 300. The low activity detected in vasectomized monkey seminal plasma can probably best be explained as an action of a nonspecific protein promoting a low level of motility. Such a bow level was detected in bovine serum and in many of the homogenates of bovine organs that we tested (Brandt et al., 1977) provided that high levels of proteins were used. The specific activity, relative to bovine seminal plasma (100%), observed in vasectomized monkey seminal plasma (14.8%) is, in fact, close to that observed for bovine vitreous humor (17.9%) (Brandt et al., 1977). Through our experiments with vasectomized monkeys, we have ruled out the possibility that FMP is produced by the accessory glands. These data, however, have not eliminated the testis as the ultimate source of FMP. As noted above, we have used both the specific activity (FMI/pg protein or p1 fluid) and the maximum obtainable FMI (600) as determinants of whether a given fluid contains FMP. First, note from Table 2 that the specific activity of caudal epididymab fluid is nearly 5 times greater than that of rete fluid. If FMP were produced in the testis, these data would demand that 80% of all non-fmp proteins in rete fluid be absorbed by the epididymal epithelium and that no new proteins of epididymal origin be added into the epididymal bumen. We consider this unlikely. Second, using the criterion for the presence of FMP as the ability to induce an FMI of 600, rete testis fluid does not contain FMP (Table 1). These data do not, however, preclude a mechanism whereby the testis produces a partially active, precursor form of FMP (pro- FMP) which is then activated in the epididymis. In fact, this idea was supported by preliminary evidence which showed that catalytic amounts of epididymab fluid increase the FMI of rete testis fluid (Brandt et a!., 1977). Subsequently, however, we have been unable to induce a 600 FMI with this activated rete fluid. We thus consider the possibility that the testis produces a pro-fmp as remote and suggest instead that FMP is produced by neither the accessory glands nor the testis but by the epididymis. We suggest that this protein instills in bovine sperm the capacity to become progressively motile when admixed with accessory gland secretions. The transition of sperm from an immotile state in situ to a highly motile state in semen probably involves a dramatic increase in the intrasperm content of camp (Morton et al., 1974; Cascieri et al., 1976). It should be stressed that the motility state of bovine caput sperm activated by phosphodiesterase inhibitors approximates but does not duplicate the motility state of ejaculated bovine sperm. Suspensions of ejaculated sperm have a higher percentage of cells showing forward progression and display a characteristic swirling motion not shown by activated caput sperm. We recognize that there is probably much more to motility induction than just the elevation of camp bevels and addition of FMP. Nonetheless, we are encouraged by the fact that we can induce forward motion in 40-50% of bovine caput sperm and that their average velocity is between 70 and 80 pm/second, a value approximately two-thirds of that reported for ejaculated bovine sperm (Nelson, 1975). The data presented here suggest that the epididymis produces a protein, FMP, which is involved in the physiological initiation of motility. RE FE RENCES Acott, T. S. and Hoskins, D. D. (1976). Characterization of factors which initiate forward motility in sperm from the bovine caput epididymis. Fed. Proc. 35, Acott, T. S. and Hoskins, D. D. (1978). J. Biol. Chem. In Press. Bedford, J. M. (1975). Maturation, transport, and fate of spermatozoa in the epididymis. In: Handbook of Physiology. (R. 0. Greep and E. B. Estwood, eds.). Sect. 7. Vol. 5. American Physiological Society, Washington, D.C. pp Brandt, H., Acott, T. S., Johnson, D. J. and Hoskins, D. D. (1977). The initiation of forward motility in bovine caput spermatozoa by rete testicular and epididymal fluid protein(s). 10th Annual Meeting of the Society for the Study of Reprod. p. 29 (Abstr.). Burgos, M. H. and Tovar, E. S. (1974). Sperm motility in the rat epididymis. Fertil. Steril. 25, Calvin, H. and Bedford, M. S. (1971). Formation of disulfide bonds in the nucleus and accessory structures of mammalian spermatozoa during maturation in the epididymis. J. Reprod. Feet. (Supple.) 13,

6 EPIDIDYMAL ORIGIN OF FMP 835 Cascieri, M., Amann, R. P. and Hammerstadt, R. H. (1976). Adenine nucleotide changes at initiation of bull sperm motility. J. Biob. Chem. 251, Henle, G. and Zittle, L. A. (1942). Studies of the metabolism of bovine epididymal spermatozoa. Am. J. Phys. 136, Hoskins, D. D., Stephens, D. T. and Hall, M. J. (1974). Cyclic adenosine 3 :5 monophosphace and protein kinase levels in developing bovine spermatozoa.j. Reprod. Fert. 37, Hoskins, D. D., Brandt, H. and Acott, T. S. (1978). Initiation of sperm motility in the mammalian epididymis. Fed. Proc. as symposium presented at 1977 FASEB Meeting. In Press. Lowry, 0. H., Rosebrough, N. J., Fart, A. L. and Randall, R. J. (1951). Protein measurement with the folin phenol reagent. J. Biol. Chem. 193, Mann, T. (1975). Biochemistry of semen. In: Handbook of Physiology. (R. 0. Greep and E. B. Estwood, eds.). Sect. 7. Vol. 5. American Physiological Society, Washington, D. C. pp Morton, B., Lum, J. H., Albagbi, L. and Joos, T. (1974). The activation of motility in quiescent hamster sperm from the epididymis by calcium and cyclic nucleotides. Biochem. Biophys. Rca. Comm. 56, Nelson, L. (1975). Spermatozoa motility. In: Handbook of Physiology. (R. 0. Greep and E. B. Estwood, eds.). Sect. 7. Vol. 5. American Physiological Society, Washington, D.C. pp Tournade, A. (1913). Difference de motilite des spermatozoids prelenes dans les diners segments de l epididyme. C. R. Soc. Biol. (Paris) 74,