Response of epididymal duct to the temporary depletion of spermatozoa induced by testicular irradiation in mice.код для вставкиСкачать
THE ANATOMICAL RECORD 207:17-24 (1983) Response of Epididymal Duct to the Temporary Depletion of Spermatozoa Induced by Testicular Irradiation in Mice KAZUHIRO ABE, HIROKO TAKANO, A N D 'I'AKASHI I T 0 Dc~part t n c w t of A natomy, Hok ka d o Utr LOCV ally Sc h 001 of M d r L I 11 c, Sapporo 060, Japan ABSTRACT The mouse epididymal duct can be histologically divided into five segments (I-V), and the principal cells in segment I1 appear to secrete periodic acid-Schiff (PAS)-positive material into the lumen. In this study, male dd-mice received one, two, or four 800-R doses of radiation beginning a t age 50 days. Mice receiving multiple doses were irradiated a t 1-week intervals. After irradiation, marked depletion of spermatozoa, or aspermia, occurred in the epididymal duct for 2 to 16 weeks after a latency period of 3 to 4 weeks according to the times of irradiations. During oligospermia or aspermia, PASpositive inclusions appeared in the principal cells in segment IV. The inclusions occupied a supranuclear position and appeared as round granules and globules measuring 2-15 pm in diameter, and increased in number, size, and staining intensity with time. They disappeared after reappearance of spermatozoa. The findings suggest that PAS-positive material may bind to spermatozoa and, if not bound, is reabsorbed by the principal cells in segment IV and deposited as intracellular inclusions, and the principal cells in segment IV are capable of digesting the accumulated PAS-positive material. The mouse epididymal duct is divided into five segments (I-V) according to the morphological differences of the principal cells (Takano, 1980; Abe et al., 1982a, 1983a). The principal cells in segment I1 have PAS-positive cytoplasm and appear to secrete PASpositive material into the lumen (Abe et al., 1982a). Such PAS-positive material may contain the specific epididymal glycoprotein which binds to spermatozoa and renders spermatozoa capable of fertilization (Lea et al., 1978; Olson and Hamilton, 1978; Faye et al., 1980; Moore, 1980).The principal cells in segment IV acquire PAS-positive cytoplasmic inclusions after ligation of the efferent duct or epididymal duct proximal to segment 11(Abe et al., 1982a). These findings suggest that the PAS-positive inclusions in segment IV appear under the following conditions: 1) release of PAS-positive material into segment IV; and 2) interruption of flow of spermatozoa from the testis into segment IV. Thus we assumed that, unless bound to spermatozoa, the specific epididymal glycoprotein is absorbed by the principal cells in segment IV. Ligation at the efferent duct or epididymal duct proximal to segment I1 stops the flow 0 1983 ALAN R. LISS, INC not only of spermatozoa but also of secretions from the testis into segment Tv. In this study, to understand more clearly effects of the presence of spermatozoa on the function of the principal cells in segments I1 and IV, we attempted to eliminate spermatozoa temporarily by testicular irradiation. In addition, to demonstrate the association of the intraepithelial PAS-positive inclusions in segment IV with the intraluminal PAS-positive material from segment 11, the epididymal duct a t the junction between segments I11 and IV was ligated after irradiation and before disappearance of spermatozoa in the epididymal duct. The changes induced in the epididymal duct were analyzed by qualitative and quantitative morphology. MATERIALS AND METHODS In this experiment involving 115 male ddmice, 102 mice received one, two, or four 800R doses of radiation beginning at age 50 days. Mice receiving multiple doses were irradiated at 1-week intervals, Five mice received two radiation doses and underwent unilatKrct,ivrd September 20, 1982; iicceptcd April 20, 1983 18 K. ABE, H. TAKANO, AND T. IT0 era1 ligation of the epididymal duct between segments I11 and IV, according to the previous procedure (Abe et al., 1982b), 2 weeks after the second irradiation. Eight mice, 60 to 90 days old, nonirradiated, served as controls. During irradiation, the mice were anesthetized with pentobarbital sodium (Nenibutal) injected intraperitoneally, placed supine, and covered with wet cotton and a 6-mm thick plastic board in order to achieve the optimum effective depth (see “build up” in the textbook by Jones and Cunningham, 1971). The wet cotton filled the space between the plastic board and the lower body of the mouse. Cephalad to the umbilical level, the body was shielded by placing a 5-cm thick lead slab on the plastic board. From a 6oC0 source, 800 R was administrated at a dose rate of 125 R per minute to the lower half of the body. After irradiation, the mice were killed a t various intervals and the epididymides and testes were removed. They were fixed in Bouin’s solution for 3 hours and then embedded in paraffin. Serial 6-pm longitudinal sections of the epididymis and testis were cut, stained with periodic acid-Schiff (PAS) and hematoxylin, and examined by light microscopy. For quantitative observations, in one section among the serial sections from each epididymis, the one ductal cross section showing the most marked appearance of the intraepithelial PAS-positive inclusions plus the three neighboring sections were selected. The minimum and maximum inner diameters of these ductal profiles and the number and size of the PAS-positive inclusions were rneasured. The ductal circumferences were calculated using the minimum and maximum diameters. The appearance of the intraepithelial PAS-positive inclusions in each epididymis was graded using the equation C d, 2 C d + + iL x 100, where d+ = diameters of inclusions staining more weakly than the luminal material, d, + = diameters of the inclusions staining more intensely than the luminal material, and L = the total circumferential length of the ducts. spermatocytes were regenerated. Spermatozoa disappeared a t 5 weeks and reappeared at 7 weeks postirradiation. Two of four 800-R irradiations depressed the number of type A spermatogonia and accelerated the sequential disappearance of the other spermatogenetic cells. Recovery of spermatogenesis began 3 weeks after the last irradiation but was more protracted. Thus, two and four irradiations resulted in disappearance of spermatozoa from the seminiferous epithelium from 4 to 10 weeks and 4 to 16 weeks after the first irradiation, respectively, Epididymal Duct In the normal epididyniis, the epididyinal duct in all segments except for segment I contains spermatozoa and PAS-positive material (Figs. 1, 2a, 3a). In segments IV and V, the lumen is extremely distended with abundant spermatozoa. The principal cells in segment IV were 15-20 pm high and about 8 pin ’I ,111 II’ -lV + RESULTS Testis The seminiferous tubule epithelium showed disappearance of type B spermatogonia and leptotene and zygotene spermatocytes 1week after a single 800-R irradiation. Cells in all stages of spermatogenesis disappeared and reappeared sequentially. At 3 weeks postirradiation, spermatogonia and Fig. 1. Longitudinal section of the testis and epididyinis of the mouse. The epididymal duct is divided into five segments (1-V). Scgmcnt I1 appears dark because the epithelial cells are stained with periodic acid-Schiff (PAS).The duct in segments IV and V contains abundant spermatozoa (black content of the lumen). PAS-hematoxylin. x7. EPIDIDYMAL PAS-POSITIVE MATERIAL AND SPERMATOZOA Fig. 2. Segment IV of the mouse epididymal duct. a) Normal. The duct contains numerous spermatozoa and PAS-positive material. b) Segmciit IV 10 weeks after the first of the two irradiations. The duct contains strongly PAS-positive material and little spermatozoa. PAS-positive inclusions (arrows) are seen in the cpithelial cells. c) 19 Segment IV in the duct which h a d been ligated between segments 111 and IV 2 weeks after the second irradiation, is observed 4 weeks after ligation. The duct is decreased in diaimtcr, and shows no spermatozoa and little PASpositive matcrial in the lumen and no inclusions i n the epithelium. PAS-hematoxylin. x 170. wide and had round nuclei, about 6 pm in appeared in the epididymal duct (Fig. 3b). These cells disappeared soon after spermatodiameter (Fig. 3a). After irradiation, aspermia or marked oli- zoic regeneration. The luminal material was gospermia occurred in the epididymal duct more strongly PAS-positive in the spermatofor a time of duration after a latency period, zoa-depleted ducts when compared with the as shown in Fig. 4. Spermatozoa disappeared controls (Figs. 2b, 3b,c). During aspermia, PAS-positive inclusions sequentially from the proximal to distal segments of the duct. In segment IV, after one appeared in the principal cells in segment irradiation, spermatozoa were normal in IV, especially in the proximal region (Figs. number until 4 weeks, disappeared at 5 2b, 3b,c). They usually occupied a supranuweeks, began to reappear a t 6 weeks, and clear position and appeared as round granbecame normal in number again a t 7 weeks ules and globules measuring 2-15 p m in postirradiation. After two and four irradia- diameter (Figs. 3b,c). The inclusions intions, spermatozoa in segment IV were nor- creased in number, size, and staining intenmal in number until 3 weeks but disappeared sity with time. Figs. 5 and 6 show the relationship bea t 4 weeks, and aspermia persisted until 10 weeks and 16 weeks after the first irradia- tween the disappearance of spermatozoa from tion, respectively. Spermatozoa in segment the epididymal duct and the appearance of IV became nearly normal in number a t 15 the PAS-positive inclusions in the principal weeks and recovered incompletely at 20 cells in segment IV. After one irradiation, weeks after the first irradiation of two and the epithelium in segment IV was normal for the initial 4 weeks while spermatozoa were four irradiations, respectively. As spermatozoa disappeared, spermatoge- present. Then, as spermatozoa were depleted netic cells, mainly consisting of spermatids, from the duct, the PAS-positive inclusions 20 K. ABE, H. TAKANO, AND T. IT0 Fig. 3 . Segment IV of the epididymal duct. a ) Normal. The lumen has many spa-matozoa and PAS positive material. The epithelial cells have no inclusions. b, c) Ten weeks after the first of four irradiations. The lumen contains strongly PAS-positive material and has spermatids (ST) and few spermatozoa. The principal cells contain PAS-positive inclusions varying in size and staining intensity (arrows). PAS-hematoxylin. ~ 6 5 0 . Fig. 4. Changes in number of spermatozoa in the cpididymal duct of segment IV after one (1 x ), two ('2 x I, and four (4 x 1 irradiations (arrows).The normal range (standard deviation) is shown by parallel lines. Bars represent standard deviations. The initial changes after two irradiations are almost t h c hiltlie as those after four irradiations. 21 EPIDIDYMAL PAS-POSITIVE MATERIAL AND SPERMATOZOA . !X 'X . 0 . . . - . . .* : -.*. t we. 6 -&e- a -.- .* rn r-10 12-15 7-9 * 20 Weeks Fig. 5. Intraepithelial PAS-positive inclusions in segmcnt IV various weeks after onc (1x1 irradiation and after the first of two ( 2 X ) and four (4X ) irradiations. Each dot represents the grade of appearance of the inclusions in each epididymis. Dots in (-) exhibit epididymides which had no inclusions (0%in grade). Bars among dots represent average values. Black belts show the duration in which the duct contains many spermatozoa. Note that PAS-positive inclusions appeared and increased in grade with time during aspermia and that epididymides had little or no inclusions after reappearance of spermatozoa. IOpm : Diameter 25 : Number/mm 30% : Grade t t 4 1 4 . 4 Irradiation 0 Spermatozoa ( - ) 4 8 Weeks Fig. 6. Possible changes, summarized from the results, in diameter, number, or grade of PAS-positive inclusions in segment IV after one (1 X), two (2 X), and four (4x1 irradiations (arrows). Thick lines represent the periods of aspermia. The diameter and number of the inclu- 12 16 20 sions showed similar changes to that in the grade. During the phase of the most significant changes, the averaged inclusions were about 10 pm in diameter, numbered about 25 per mm of the lateral aspect of the epithelium, and 30% in grade. 22 K. ABE, H. TAKANO. AND T. IT0 appeared a t 5 weeks and became more significant 6 weeks after the irradiation. The inclusions disappeared after reappearance of spermatozoa. After four irradiations, the PAS-positive inclusions were not observed for initial 3 weeks after the first irradiation while spermatozoa were still present. Following disappearance of spermatozoa from the duct, the inclusions appeared 4 weeks after the first irradiation, increased in grade until about 10 weeks, and began to decrease in grade at 16 weeks (Figs. 5 , 6). During the phase of the most significant changes, the averaged PAS-positive inclusions were about 10 pm in diameter, numbered about 25 per mm of the lateral aspect of the epithelium, and about 30% in grade (Fig. 6). In the group irradiated twice, the radiation-induced changes were of intermediate severity between those after one to four irradiations. The inclusions were still present in the duct, which showed incomplete recovery of spermatozoa 15 weeks and 20 weeks after the first of two and four irradiations, respectively (Fig. 7). Segments I, 11, 111, and V showed no distinctive postirradiation changes. In the mice with ducts ligated 2 weeks after the second irradiation, the diameters of the 50- 40- -< c 30( 5 " '' 0 C o o -5 20-0 "C ._ --2+ n 0 .= 10- 0 0 'OD 0 0 100 moob o 800 . . .* 200 300 Spermatozoa : Number/lO>ml Fig. 7. Relationship between the number of spermatozoa a n d the PAS-positive inclusions during recovery of spolmntozoa. Dots a n d circles represent thc valucs 12 to 15 weeks a n d 20 weeks after t h e first of two a n d four irradiations, respectively. duct and the lumen in segment IV were decreased, and neither luminal PAS-positive material nor intraepithelial PAS-positive inclusions were seen on the ligated side 4 weeks after the operation (Fig. 2c). The contralatera1 nonligated epididymis showed the PASpositive inclusions. DISCUSSION Radiation induced disappearance of spermatozoa from the epididymal duct after a latency period. This may be related to the sensitivity of spermatogenic cells to irradiation and the duration of the spermatogenetic cycle in the seminiferous epithelium. Oakberg (1956) observed the mouse testis histologically after a 100-R radiation dose and estimated the duration of spermatogenesis from type A spermatogonia to spermatozoa is 34.5 days. The present study showed that spermatozoa in the seminiferous epithelium disappeared 5 weeks after 800-R irradiation. Thus, the interval from irradiation to aspermia in the testis corresponds to the duration of spermatogenesis and indicates that only the earliest cell types in the spermatogenic series were destroyed when irradiated with the larger 800-R dose, as is the case with the 100 R used by Oakberg (1956). However, the destructive effect on the young spermatogenic cells seems to be exaggerated by multiple irradiations, because spermatozoa disappeared earlier and showed a more retarded recovery after four irradiations than after one irradiation. In contrast to the radiosensitivity of spermatogenic cells, the interstitial cells and Sertoli cells showed morphologically no detectable damage. It is also indicated that the interstitial cell androgen production and Sertoli cell function were almost unaffected by radiation. Because the segmental differentiation of the epididymal epithelial cells is dependent upon androgen, the segmental differences of the epididymal duct disappear after gonadectomy (Cavazos, 1958; Alexander, 1972), but after irradiation the segmental differences of the epididymal duct are maintained. In addition, because differentiation of the initial segment also requires the intraluminal androgen-binding protein produced by Sertoli cells, the initial segment shows decrease of epithelial height-dedifferentiation-after efferent duct ligation (Fawcett and Hoffer, 19791, but shows no such changes after irradiation. Thus, the present study suggests that flow of the testicular fluid into the epididymal epithelial cells retain EPIDIDYMAL PAS-POSITIVE MATERIAL AND SPERMATOZOA their functions under the influence of androgen after irradiation. It has been suggested that the biochemical response of the epididymal epithelial cells is affected by irradiation (Ito, 1966; Gupta and Bawa, 1971). Morphologically, however, despite damage of spermatogenic cells after irradiation, the epididymal duct did not show any changes during the initial postirradiation latency period. In addition, because changes in segment IV after the latency period were prevented by ligation between segments 111 and IV and similar changes could be induced by ligation of the efferent duct without irradiation (Abe et al., 1982a), the changes in segment IV after radiation are definitely considered due to no radiation damage and to be concerned with a functional activity of this segment. Thus, it is considered that irradiation induces a temporary disappearance of spermatozoa from the epididymal duct, leaving the ductal epithelium functionally active and the flow of the testicular fluid uninterrupted. During the period of ductal aspermia, the intraepithelial PAS-positive inclusions, such as shown in the ligation experiment (Abe et al., 1982a1, depended upon the release of the luminal material from the segment 11. In segment 11, the cytoplasm of the principal cells stains with PAS, the apical portions of the cells are strongly positive, and the stereocilia are embedded within strongly PASpositive material; and the lumen from segment I1 distally contains PAS-positive material together with spermatozoa (Takano, 1980). These findings suggest that the luminal PAS-positive material is secreted by the principal cells in segment 11. The ligation between segments I11 and IV obstructs the flow of the luminal material to segment IV and prevents the appearance of the PAS-positive inclusions (Abe et al., 1982a). Thus, the PAS-positive inclusions in the principal cells in segment IV appear when the duct in segment IV received the PAS-positive material from segment I1 but no spermatozoa. Duration of the appearance of the PAS-positive inclusions clearly depends on the duration of ductal aspermia. Recent studies in the rat, rabbit, and hamster demonstrated that the principal cells in the epididymal duct distal to the initial segment secrete specific epididymal glycoproteins or proteins which bind to the spermatozoa membrane (Lea et al., 1978; Olson and Hamilton, 1978; Faye et al., 1980; Moore, 1980). In the mouse, Vernon et al. (1982), 23 using a monoclonal antibody, demonstrated immunohistologically that a n antigen binding to sperm tails is secreted from the principal cells in a short segment of the distal epididymal head. Such substances have been hypothesized to be responsible for rendering spermatozoa capable of fertilization (see reviews by Bedford, 1975, and Hamilton, 1975). Because of the stainability and the site of secretion, the luminal PAS-positive material in the mouse may contain such a specific epididymal glycoprotein. The epididymal principal cells are known to be absorptive as well as secretory (Moniem and Glover, 1972; Moore and Bedford, 1979; Fain-Maurel et al., 1981). It is therefore likely that the specific epididymal glycoprotein is reabsorbed by the principal cells in segment IV and deposited as intracellular inclusions if not bound to spermatozoa. The present study showed that the inclusions appeared 1 week after the disappearance of spermatozoa in the epididymal duct. In the previous study, we observed that PASpositive inclusions develop in segment IV beginning 1 week after efferent duct ligation (Abe et al., 1982a). Vernon et al. (1982) also examined the sperm tail antigen of the mouse epididymal duct 10 days after efferent duct ligation, but found no labeling of the luminal material and no intracellular deposits in the principal cells corresponding to segment IV. Thus, the site of the antigen secretion seems to correspond to segment 111, but the sperm tail antigen demonstrated by Vernon et al., (1982)may be different from the PAS-positive material demonstrated by our group. The PAS-positive inclusions disappeared soon after the reappearance of spermatozoa. This suggests that the principal cells are capable of digesting the accumulated PAS-positive material. The principal cells contain dense bodies and multivesicular bodies which contain acid-phosphatase and are lysosomal in character (Friend, 1969; Moniem and Glover, 1972). We have observed that the principal cells in segment IV in normal mice have specific dense bodies and peculiar inclusions, both of which are considered to represent various stages of digestion of accumulated materials (Abe et al., 1983b). The particular inclusions which contain a bundle of tubules occur only in segment IV, and they may be concerned with a specific function such as absorption of the glycoprotein in this segment. Thus, even under normal conditions, the principal cells may absorb and digest the glycoprotein which is un- 24 K. ABE, H . TAKANO, AND T. I T 0 bound to spermatozoa. Although the sperm coating antigen secreted from the proximal epididymis has been shown only on the stereocilia of the principal cells without cytoplasmic labeling in the distal epididymis, absorption of the antigen has been also suggested from the distinctive cytoplasmic labeling of the clear cells in the distal epididymis (Vernon et al., 1982). As demonstrated above, the principal cells in segment IV can absorb the luminal PASpositive material coming from segment 11. The intracellular PAS-positive inclusions are formed by the absorbed luminal material and depend directly on the absence of spermatozoa from the lumen. The luminal PAS-positive material could be bound to or used by spermatozoa in the lumen. In this study, temporary depletion of spermatozoa in the epididymal duct induced by testicular irradiation clearly demonstrated the relationship between spermatozoa, PAS-positive material secreted in segment 11, and the function of the principal cells in segment IV. The luminal PAS-positive material also may play a role in the differentiation of the principal cells in segment IV, as shown previously (Abe et al., 1982b). ACKNOWLEDGMENTS We wish to thank Professor Goro Irie, Department of Radiology, Hokkaido University School of Medicine, for advice on the radiation method and Libby Cone, S.B., University of Massachusetts Medical School, for correction of English in the manuscript. The irradiation was performed at Central Institute of Radioisotope Science, Hokkaido University. This study was supported by a grant from the Ministry of Education, 1981 (5670002). LITERATURE CITED Abe, K., H. Takano, and T. Ito (1982a) Response of the epididymal duct in the corpus epididymidis to efferent or epididymal duct ligation i n the mouse. J. Reprod. Fertil., 64:69-72. Abe, K., H. Takano, and T. Ito (198213) Appearance of peculiar epithelial cells in the epididymal duct of the mouse ligated epididymis. Biol. Reprod., 26:501-509. Ahe, K., H. Takano, and T. Ito (1983a)Ultrastructure of the mouse epididymal duct with special reference to the regional differences of the principal cells. Arch. Histol. Jpn., 46151-68. Abe, K., H. Takano, a n d T. Ito (198313)Tubule-containing inclusions in the epithelial cells of the mouse epididyma1 duct. Arch. Histol. Jpn., 46: (in press). Alexander, N. J. (1972)Prenatal development of the ductus epididymidis in the rhesus monkey. The effects of fetal castration. Am. J . Anat., 135:119-134. Bedford, J. M. (1975) Maturation, transport and fate of spermatozoa i n the epididymis. In: Handbook of Physiology. Male Reproductive System, Sect. 7., Vol. 5 , D. W. Hamilton and R. 0. Greep, eds. American Physiological Society, Bethcsda, pp. 303-317. Cavazos, L. F. (1958) Effects of testosterone propionate on histochemical reactions of epithelium of r a t ductus epididymidis. Anat. Rec., 132:209-227. Fain-Maurel, M.A., J. P. Dadoune, and M. F. Alfonsi (1981) High-resolution autoradiography of newly formed proteins in t h e epididymis after incorporation of tritiated amino acids. Arch. Androl., 6:249-266. Fawcett, D. W., and A. P. Hoffer (1979) Failure of exogenous androgen to prevent regression of the initial segments of the r a t epididymis after efferent duct ligation or orchidectomy Biol. Reprod., 20:162-181. Faye, J.C., L. Duguet, M. Mazzuca, and F. Bayerd (1980) Purification, radioimmunoassay, and immunohistochemical localization of a glycoprotein produced by the r a t epididymis. Biol. Reprod., 23:423-432. Friend, D. S. (1969) Cytochemical staining of multivesicular body and Golgi vesicles. J. Cell Biol., 41:269-279. Gupta, G. S., and S. R. Bawa (1971) Phosphatases in testes and epididymides of albino rats after partial body y-irradiation. J. Reprod. Fertil., 27:451-454. Hamilton, D. W. (1975) Structure and function of the epithelium lining the ductuli effercntes, ductus epididymidis and ductus deferens in the r a t . In: Handbook of Physiology. Male Reproductive System, Sect. 7., Vol. 5 , D. W. Hamilton and R. 0. Greep, eds. American Physiological Society, Bcthesda, pp. 259-301. Ito, H. (1966) Histochemical observations of oxidative enzymes in irradiated testis and epididymis. Rad. Res., 28.266-277. Jones, H . E., and J. R. Cunningham (1971) The Physics of Radiology, 3rd ed., Charles C. Thomas, Springfield. Lea, 0. A,, P. Petrusz, and F. S. French (1978) Purification and localization of acidic epididymal glycoprotein (AEG): A sperm coating protein secreted by the r a t epididymis. Int. J . Androl., ISuppl.], 2t592-607. Moniem, K. A., and T. U. Glover (1972) Comparative histochemical localization of lysosomal enzymes in mammalian epididymides. J. Anat., 111:437-452. Moore, H. D. M., and J . M. Bedford (1979) The differential absorptive activity of epithelial cells of the r a t epididymis before and after castration. Anat. Rec., 193t313-328. Moore, H . D. M. (19801 Localization of specific glycoproteins secreted by the rabbit and hamster epididymis. Biol. Reprod., 22705-718. Oakberg, E. F. (1956)Duration of spermatogenesis in the mouse and timing of stages o f t h e cycle of the seminiferous epithelium. Am. J. Anat., 99:507-516. Olson, G. E., and D. W. Hamilton (1978) Characterization of the surface glycoproteins of r a t spermatozoa. B i d . Reprod., 19126-35. Takano H. (1980) Qualitative and quantitative histology and histogenesis of t h e mouse epididymis, with special emphasis on t h e regional difference. Acta Anat. Nippon, 551573-587, (In Japanese with English Abstract.) Vernon, R. B., C. H. Muller, J. C. Herr, F. A. Feucher, and E. M. Eddy (1982)Epididymal secretion of a mouse sperm surface component recognized by monoclonal antibody. Biol. Reprod., 26:523-535.