NEW EVIDENCE DEMONSTRATING THE MULTIVALENT NATURE OF HUMAN CHORIONIC GONADOTROPIN*

FERTILITY AND STERR.ITY Copyright 1971 by The Williams & Wilkins Co. Vol. 22, No.1, January 1971 Printed in U.S.A. NEW EVIDENCE DEMONSTRATING THE MULTIVALENT NATURE OF HUMAN CHORIONIC GONADOTROPIN* S. HAMASHIGE, M.D., J. D. ALEXANDER, B.S., E. V. ABRAVANEL, AND M. A. ASTOR, B.A. Department of Pathology, School of Medicine, University of California, Irvine, California, and the Orange County Medical Center, Orange, California Commercial preparations of human chorionic gonadotropin (HCG) with a potency of 2000 to 3000 I.U'/mg. contain at least 2 pregnancy-specific antigenic moieties which show partial identity by immunoelectrophoretic analysis. l 4 These HCG preparations can be chromatographically fractionated to yield at least 3 pregnancy-specific components (Fr I-I, Fr I-II, and Fr ID, each possessing distinct electrophoretic characteristics. 5 Varying degrees of the immunologic activities of HCG and spermiation in frogs indicated the presence of a gonadotropic principle in each of these 3 HCG components. However, Fr I-I did not cause gonadal responses in the intact male rat and in the intact female rabbit. Recently, dissociation in the responses of the sex organs of hypophysectomized male rats, inability to cause a gonadal response in hypophysectomized female rats, and histologically distinct spermatogenesis in the testes of hypophysectomized rats were presented as evidence that the gonadotropic principle in Fr I-I was distinct from those of Fr I-II and Fr 11.6 This report presents studies of the neutralization of the gonadotropic activities of homologous and heterologous antigens by Fr I-I and Fr II antisera as further evidence for the distinctiveness of Fr I-I. This impression is supported further by results of steroidogenic and spermatogenic tests in hypophysectomized rats. Differences in * This research was aided by Grant 1'-409 from the American Cancer Society and by a research grant from the California College of Medicine, University of California, Irvine, Calif. A preliminary report of this study was presented at the 51st meeting of the Endocrine Society, New York, June 1969. 26 steroidogenesis and spermatogenesis suggest that Fr I-II, Fr II, and HCG are also dissimilar to each other. These experimental results are the basis for a proposal that HCG is a multivalent hormone complex. MATERIALS AND METHODS HCG containing 2000 to 3000 I.U'/mg., purchased commercially (Vitamerican Corporation, Little Falls, N. J.), was used to prepare chromatographic fractions Fr I I, Fr I-II, and Fr 11.5 The characteristics of these preparations and their antisera have been described. 5 Hypophysectomized Wi star rats weighing about 100 gm. were purchased (Simonsen Laboratories, Gilroy, Calif.) and rested for 14 days prior to use in gonadotropic, steroidogenic, and spermatogenic tests. A gonadotropic test which is dependent upon test material containing sufficient HCG activity to cause a significant (p = O. 0001) increase in the mean weight of the sex organs of hypophysectomized rats has been described. 6 When neutralization of gonadotropic activity was studied, the previously described procedure was used, except that antisera were used as diluents in place of saline. Gonadotropic activity of the test material was considered to have been neutralized by antisera when the mean weights of the sex organs were not significantly greater than those of control rats injected with saline. A steroidogenic test was dependent upon HCG to repair the steroid biosynthetic capabilities of testes after a 14-day posthypophysectomy regression period. HCG activity was considered present when the

January 1971 MULTIVALENCE OF CHORIONIC GONADOTROPIN 27 postinjection testes contained steroids not present in the preinjection testes. Hypophysectomized, hemigonadectomized rats (5/test group) were given 2 0.5-ml. subcutaneous injections of test material at 24 hr. intervals and the testes were excised 48 hr. after the second injection. Steroids were extracted from homogenized testes by the method of McKerns and Nordstrand. 7 Steroid content extracts of testes were analyzed as trimethylsilyl derivatives in a gas chromatograph (GC-4, Beckman Instruments, Fullerton, Calif.) with a hydrogen flame ionization detector. SE-30 (5%) on Chromosorb WHP (100-120 mesh) at 260 0 C., OV-225 (3%) on Chromosorb WHP (100-120 mesh) at 230 0 C., and OV- 17 (5%) on Chromosorb WHP (100-120 mesh) at 260 0 C. were the columns (Beckman Instruments) and column temperatures used in the analyses. Identification of steroids based on retention time and quantitation by absolute calibration were preformed on two different columns. Steroid standards, chromatographic grade, used in the calibration were purchased commercially (Calbiochem, Los Angeles, Calif.). A spermatogenic test was dependent upon HCG to cause histologically discernible changes in the seminiferous epithelium of testes. Histologic changes attributed to HCG were those features present in the postinjection testes which were not present in the preinjection testes surgically excised on the last day of the regression period. Hypophysectomized, hemigonadectomized rats (5/test group) were given 6 0.5-ml. subcutaneous injections of test material at 24-hr. intervals. The postinjection testes, excised 24 hr. after the sixth injection, and the preinjection testes were processed in the following manner. Immediately after excision, all testes were sliced into small pieces and fixed in a 2.5% gluteraldehyde solution, postfixed in a 1 % osmic acid solution, and embedded in Epon. Thin sections (0.5 p.) were cut with an ultramicrotome, stained with toluidine blue, and photographed using phase contrast. In these tests, rats were considered not to be hypophysectomized and were discarded when discernible growth occurred during the regression period, when the preinjection testis exceeded 150 mg. in weight, or when microscopic sections of the testes failed to show histologically discernible hypophysectomy-induced atrophy. RESULTS Responses in the Mean Weights of the Sex Organs of Hypophysectomized Rats Injected with HCG Preparations. An increase in the mean weight of the testes and a change in the ratio of testes weight to the weight of the prostate and seminal vesicles were employed as indicators of gonadotropic activity. The validity and relative sensitivity of the test system were based on the finding that a significant (p = 0.0001) increase in the mean weight of the testes and a significant (p = 0.01) increase in the sex organ ratio, as compared with those of control rats injected with saline, were caused by injections of 10 p.g of HCG (Table 1). Mean testes weights of TABLE 1. Weights of Testes, Prostate, and Seminal Vesicles of Hypophysectomized* Wistar Rats Injected with Fr I-I, Fr I-II, Fr II, and HCG Test antigen Saline HCG FrI-I FrI-II FrII i'l!. 10 150 10 1 Gonadotropic indexes Mean weight oftestest mg./l00gm. body weight Ratio of testes/ prostate and seminal vesicles 240 2.8 339 3.2 312 3.3 308 2.7 363 2.8 * The mean weight of testes of intact rats of comparable age and size was 1081 mg./100 gm. of body weight and the ratio of testes/prostate and seminal vesicles was 5.5. t The mean weight of testes from a minimum of 10 rats which were 18 days posthypophysectomized. Average S.D. = 51 mg./100 gm. of body weight.

28 HAMASHIGE ET AL. Vol. 22 312, 308, and 363 mg./l00 gm. of body weight, which were about 30% greater than those of control rats, demonstrated that Fr I-I, Fr I-II, and Fr II contained gonadotropic principles of varying potencies. However, the sex organ ratio of 3.3 for Fr I-I rats was significantly (p = 0.01) greater than that of control rats. The sex organ ratios of 2.7 and 2.8 for Fr I-II and Fr II rats were identical (p = 0.92) with those of the control rats. Hyperemia and hypertrophy of the ovaries were used as indexes of HCG activity in hypophysectomized female rats (Table 2). A significant (p = 0.0001) increase in the mean weight and hyperemia of the ovaries in 9 to 10 rats injected with HCG (25 ~g.), not present in rats injected with saline, indicated the validity and relative sensitivity of this test. Ovarian weights of 35.0 and 39.3 mg./l00 gm. of body weight and grossly discernible hypermia of ovaries in at least 8 to 10 rats demonstrated that Fr I-II (25 ~g.) and Fr II (5 ~g.) contained gonadotropic activity. However, as much as 1500 ~g. of Fr I-I failed to produce discernible gonadotropic TABLE 2. Weights of Ovaries of Hypophysectomized" Wistar Rats Injected with Fr I-I, Fr I-II, Fr II, and HeG Test antigen Saline HCG Fr I-I FrI-II Fr II "g. 25 1500 25 5 Gonadotropic indexes Mean weight of ovariest mg./l00 gm. body weight Hyperemia ofovaries:j; 22.3 0/10 35.2 9/10 20.5 0/10 35.0 8/10 39.3 9/10 * The mean weight of ovaries of intact rats of comparable age and size was 42.0 mg./l00 gm. of body weight. t The mean weights of ovaries from a minimum of 10 rats which were 18 days posthypophysectomized. Average S.D. = 6.2 mg./l00 gm. of body weight. * Ratios indicate number of rats with hyperemia of ovaries/number of rats tested. effects in female rats, whereas only onetenth as much of the same preparation produced hypertrophy of the testes. The specific biologic activity of the Fr I-I in female rats was not determined because of the prohibitively large amount of material that may be required. Neutralization studies employing antisera to block the gonadotropic activity of homologous and heterologous antigens were used to reconfirm the sex-dependent expression of the biologic activity of Fr I-I. Mean testes weights of 248, 271, 269, and 256 mg./100 gm. of body weight, not significantly (p = >0.10) greater than those of control rats, showed that the gonadotropic activity of 150 ~g. of Fr I-I was neutralized by antisera containing about 500-1000 hemagglutination units (H.U.) of antibody activity, irrespective of whether the antisera were from rabbits (RB 34 and RB 35) immunized with Fr I-lor rabbits (RB 46 and RB 52) immunized with Fr II (Table 3). Mean weights of 259, 235, 258, and 280 mg./100 gm. of body weight, not significantly (p = >0.10 greater than those of control rats, showed that the gonadotropic activity of 1.0 ~g of Fr II was neutralized by homologous (RB 46 and RB 52) or heterologous antisera (RB 34 and RB TABLE 3. The Neutralizing Ability of Fr I-I and Fr II Antisera on the Gonadotropic Effect of Fr I-I on the Testes of Hypophysectomized Wistar Rats Test antigen FrI-I Fr I-I Fr I-I FrI-! 150 150 150 150 Antiserum * (specific activity) Anti-Fr I-I RB35 (640) RB 34(1280) Anti-Fr II RB 46 (1280) RB 52 (640) Gonadotropic index (weight oftestest) mg./1oogm. body weight 248 271 269 256 * Numbers indicate hemagglutination units that are reciprocals of titers. t Mean weights of testes from a minimum of 10 rats which were 18 days posthypophysectomized. Average S.D. = 26 mg./loo gm. of body weight.

January 1971 MULTIVALENCE OF CHORIONIC GONADOTROPIN 29 35) containing about 1000 H.D., reconfirming the similarity of Fr I-I and Fr II antisera (Table 4). However, when aliquots of these same antisera were used to neutralize the gonadotropic activity of 5.0 p.g. of Fr II in female rats, a distinct difference was noted (Table 5). Ovaries with mean weights of 24.8 and 24.7 mg./l00 gm. of body ~eight, not significantly greater than TABLE 4. The Neutralizing Ability of Fr II and Fr I-I Antisera on the Gonadotropic Effect of Fr II on the Testes of Hypophysectomized Wistar Rats -- Gonadotropic Test Antiserum'" index antigen (specific (weight activity) oftestest) "g. mg./loogm. body weight Anti-Fr II FrII 1 RB46 (640) 259 FrII 1 RB52 (320) 235 Anti-Fr I-I Fr II 1 RB34 (2560) 258 Fr II 1 RB 35 (640) 280 * Numbers indicate hemagglutination units that are reciprocals of titers. t Mean weights of testes from a minimum of 10 rats which were 18 days posthypophysectomized. Average S.D. = 41 mg./100 gm. of body weight. TABLE 5. The Neutralizing Ability of Fr II and Fr I-I Antisera on the Gonadotropic Effect of Fr II on the Ovaries of Hypophysectomized Wistar Rats Test antigen Fr II FrII FrII FrII "g. 5 5 5 5 Antiserum'" (specific activity) Anti-Fr II RB 52 (320) RB46(640) Anti-Fr I-I RB 34 (12,800) RB 35 (6,400) Gonadotropic indexes Weight of ovariest mg./loogm. body weight Hyperemia of ovaries:t: 24.8 1/10 24.7 1/10 33.3 8/10 38.0 9/10 * Numbers indicate hemagglutination units that are reciprocals of titers. t The mean weights of ovaries from a minimum of 10 rats which were 18 days posthypophysectomized. Average S.D. = 7 mg./100 gm. of body weight. :J: Ratios indicate number of rats with hyperemia of ovaries/number of rats tested. those of control rats, and the absence of ovarian hyperemia in 18 of 20 rats signified that neutralization had been achieved by homologous antisera containing less than 1000 H.D. In contrast, mean ovarian weights of 33.3 and 38.0 mg./l00 gm. of body weight, significantly (p = 0.0001) greater than those of control rats given saline, and hyperemia of ovaries of 17 of 20 rats indicated that more than 5000 H.D. of Fr I-I antisera were unable to neutralize the gonadotropic activity of Fr II in female rats, suggesting a distinct sex-dependent dissociation of biologic activities. Steroid Content of Testes of Hypophysectomized, Hemigonadectomized Rats Injected with HCG Preparations. The steroid content of postinjection testes of hypophysectomized and hemigonadectomized rats was used as an indicator of HCG activity. Postinjection testes of rats given HCG (10 p.g.) contained about 1 p.g. of testosterone and 7 p.g. of estradiol/100 gm. of testes, whereas the preinjection testes of these rats did not contain measurable amounts of either steroid (Table 6). The preinjection and postinjection testes of control rats given saline did not contain detectable amounts of either steroid. The steroid contents of postinjection testes of rats given Fr I-I, Fr I-II, and Fr II were different, although the preinjection control testes of all test groups were similar and TABLE 6. Testosterone and Estradiol Content of Testes of Hypophysectomized* and Hemigonadectomized rats Injected with Fr I-I, Fr I-II, Fr II, and HCG Antigen Testosterone Estradiol "g. "g./loogm. testes Saline 0 0 RCG 10 1 7 Fr I-I 150 36 8 Fr I-II 10 5 7 Fr lit 1 0 0 * Preinjection control testes of each test group did not contain testosterone or estradiol. t Chromatogram showed a large, unidentified peak distinct from those of testosterone and estradiol.

30 HAMASHIGE ET AL. Vol. 22 did not contain measurable amounts of steroids. Testosterone (36 Itg.) and estradiol (8 Itg.)/IOO gm. of testes indicated that an androgen was the principle steroid produced by injections of Fr I-I, whereas 5 Itg. of testosterone and 7 Itg. of estradiol/i 00 gm. of testes showed that biosynthe.sis of similar amounts of androgenic and estrogenic steroids was induced by Fr I-II. However, no measurable amount of either steroid was present in the postinjection testes of rats given Fr II. Instead, the chromatograms of testicular extracts of Fr II rats showed a large unknown peak with a retention time distinct from that of testosterone, estradiol, dehydroepiandrosterone, androstenedione, estrone, or estriol. Spermatogenesis in Hypophysectomized, Hemigonadectomized Rats Injected with HCG Preparations. A spermatogenic test was dependent upon HCG to induce histologically discernible differences in the seminiferous epithelium. Histologic changes attributed to HCG were those features present in the postinjection testes of rats with surgically excised preinjection testes showing marked posthypophysectomy-induced atrophy evident in gross and microscopic examination. Three histologically distinct types of seminiferous tubules were observed in hypophysectomized and hemigonadectomized rats given 6 daily injections of 5 Itg. of HCG (Fig. 1). Seminiferous tubules showing varying numbers of medium-sized spermatogenic cells having a round, vesicular nucleus with a single chromatin clump (Fig. 1, C and D), tentatively identified as spermatids, predominated. A second histologic type of seminiferous tubule, present in lesser numbers, was characterized by an apparent increase in the number of small spermatogenic cells with a round nucleus composed of beaded chromatin strands and a small amount of clear cytoplasm (Fig. la), currently designated as spermatogonia. The third histologic type of seminiferous tubule present in HCG-injected testes showed proliferation of spermatogonia accompanied by clusters of large spermatogenic cells (pachytene spermatocytes?) with a round to oval nucleus showing irregular aggregation of chromatin and a moderate amount of granular cytoplasm (Fig. IB). The testes of rats injected with Fr I-I, Fr I-II, or Fr II were characterized by the presence of only one of the three histologic variants of seminiferous tubules. (Figs. 2, 3, and 4). Proliferation of spermatogonia similar to that of HCG-injected testes was induced (Fig. 2, Band C) when 300 Itg. of Fr I-I were used as the test antigen. Furthermore, a few spermatogenic cells, distinguishable from spermatogonia by their larger size, coarser nuclear chromatin, and a more opaque, granular cytoplasm, tentatively designated as leptotene spermatocytes (Fig. 2, Band D), while not present in HCG-injected testes (Fig. 1), were present in Fr I-I testes. Proliferation of spermatogonia with a central cluster of pachytene spermatocytes, more pronounced than those seen in HCG testes (Fig. IB), characterized the testes of rats injected with 15 Itg. of Fr I-II (Fig. 3, B, C, and D). Seminiferous tubules with varying numbers of spermatids without spermatogonia, leptotene spermatocytes, or pachytene spermatocytes (Fig. 4, B, C, and D) were present in testes of rats injected with 2.0 Itg. of Fr II. Further proliferation and/or maturation of spermatogenic cells was not induced when the amount of Fr I-I, Fr I-II, or Fr II injected was increased 2-fold. These testes showed a decrease in the population of the seminiferous epithelium, with further dilatation of the tubules forming large, histologically empty lumina with variable amounts of cellular debris (results not shown). Smaller increments of Fr I-I, Fr I-II, or Fr II injection produced histologic changes similar to those shown in Figs. 1 ~4,

January 1971 MULTIVALENCE OF CHORIONIC GONADOTROPIN 31 FIG. 1. Photomicrographs showing postinjection testis of a hypophysectomized rat injected with 5,.g of ReG. A (left upper quadrant), a tubule showing proliferation of spermatogonia. B (right upper quadrant), a tubule showing proliferation of spermatogonia with a central cluster of a few pachytene spermatocytes. C (left lower quadrant), a tubule showing spermatids. D (right lower quadrant), a tubule showing spermatids and gaps in the basal germ cell layer. All four photomicrographs show interstitial cell hypertrophy and hyperplasia. X 415.

FIG. 2. Photomicrographs showing the preinjection and the postinjection testes of a hypophysectomized rat injected with 300 "g. of Fr I-I. A (left upper quadrant), tubules of the preinjection testis showing hypophysectomy-induced atrophy. B (right upper quadrant), a tubule of the postinjection testis showing proliferation of spermatogonia and a few leptotene spermatocytes. C (left lower quadrant), a tubule of the postinjection testis showing proliferation of spermatogonia. D (right lower quadrant), a tubule of the postinjection testis showing mitosis of basal germ cells, proliferation of spermatogonia, and leptotene spermatocytes. X 415. Note: The postinjection testis also shows intersititial cell hypertrophy and hyperplasia not present in the preinjection testis. 32

January 1971 MULTIVALENCE OF CHORIONIC GONADOTROPIN 33 FIG. 3. Photomicrograph showing the preinjection and postinjections testes of a hypophysectomized rat injected with 15 JLg of Fr I-II. A (left upper quadrant), tubules of the preinjection testis showing hypophysectomy-induced atrophy. B (right upper quadrant), a tubule of the postinjection testis showing proliferation of spermatogonia and pachytene spermatocytes. C (left lower quadrant), a tubule of the postinjection testis showing proliferation of spermatogonia and pachytene spermatocytes. D (right lower quadrant), a tubule showing pachytene spermatocytes without proliferation of spermatogonia. X 415. Note: The postinjection testis also shows interstitial cell hypertrophy and hyperplasia not present in the preinjection testis.

FIG. 4. Photomicrograph showing the preinjection arid postinjection testes of a hypophysectomized rat injected with 2'llg of Fr II. A (left upper quadrant), tubules of the preinjection testis showing hypophysectomy-induced atrophy. B (right upper quadrant), a tubule of the postinjection testis showing large numbers of spermatids. C (left lower quadrant), a tubule of the postinjection testis showing degenerating spermatids, a thinned epithelium, and gaps in the basal germ cell layer. D (right lower quadrant), a tubule of the postinjection testis showing a few spermatids, a thinned epithelium, and gaps in the basal germ cell layer. X 415. Note: The postinjection testis also shows interstitial cell hypertrophy and hyperplasia not present in the pre injection testis. 34

January 1971 MULTIVALENCE OF CHORIONIC GONADOTROPIN 35 but of a markedly lesser degree (results not shown). Despite histologically discernible differences in spermatogenesis, similarities were also observed. All HeG preparations caused an apparent increase in the interstitial cell population, and hypertrophy was suggested by an increased nuclear size and an increased amount of granular cytoplasm in the Leydig's cells. (Figs. 1-4); however, mitoses of interstitial cells were not observed. Postinjection testes of all three test groups showed diminution of osmophilic bodies as compared with the preinjection testes. No changes in the Sertoli's cells were discernible histologically. COMMENTS Four preparations, Fr I-I, Fr I-II, Fr II, and HeG, have been compared with each other using gonadal responses, steroidogenesis, and spermatogenesis in hypophysectomized rats as indexes. Divergences and dissociations in the results and an absence of parallelism between international units of HeG activity and test responses were the basis for a proposal that HeG is a hormone complex. Fr I-I caused hypertrophy of the testes, but did not produce an increase in the weight of accessory sex organs (Table 1) and did not induce gonadotropic activity in female rats (Table 2). Furthermore, Fr I-I antisera with over 5000 H.U. were unable to neutralize the hypertrophy and hyperemia of ovaries caused by Fr II (Table 5), whereas only 1000 H.U. of Fr I-I antisera neutralized the hypertrophy of testes, prostate, and seminal vesicles caused by Fr II (Table 3). These results suggest a sex-dependent dissociation of action which appeared to be peculiar to Fr I-I. Also, the testes of Fr I-I rats contained 4.5 j.tg. of testosterone/j.tg. of estradiol-a steroid ratio unlike those of other test groups (Table 6). Proliferation of primitive germ cells in the seminiferous tubules which characterized testes of Fr I-I rats in a previous experiment 6 was also characteristic of testes of Fr I-I rats in this experiment, despite larger dosages administered over a longer period (Fig. 2). The leptotene spermatocytes present in the testes of Fr I-I rats (Fig. 2), but not observed in Fr I-I testes in previous experiments, may be the results of increased dosages or the longer course of injection. A positive gonadal response in all hypophysectomized rats (Tables 1 and 2) indicated not only an absence of a sex-dependent dissociation of activities, but that Fr I-II was dissimilar to Fr I-I. The presence of testosterone and estrogen in the testes of Fr I-II rats in about equal amounts was another indicator of dissimilarity between Fr I-I and Fr I-II (Table 6). Pachytene spermatocytes, which differentiated testes of Fr I-II rats from Fr I-I rats, persisted, despite increased dosages administered over a longer period (Fig. 2). Proliferation of spermatogonia, not present in the testes of rats given only 2 injections of Fr I_II 6, was a new finding (Fig. 2). Nevertheless, this test group offers the best demonstration that further maturation of spermatogenic cells was not promoted by increased increments of test antigen. Fr II was indistinguishable from Fr I-II in gonadal response tests except for differences in specific biologic activities (Tables 1 and 2). However, testosterone and estradiol, present in measurable amounts in extracts of Fr I-II testes, were not present in extracts of Fr II testes (Table 6). The possibility that steroidogenesis had occurred in Fr II testes was suggested by a large, unidentified peak not present in chromatograms of other extracts of testes and a marked diminution of the cholesterol peak. The relative retention time of this unidentified peak was within the limits of that for progesterone, and confirmatory studies are in progress. The unique-

36 HAMASHIGE ET AL. Vol. 22 ness of Fr II was supported further by spermatogenic studies. Spermatogenic cells in Fr II testes, previously suspected of being different from those present in Fr I-II testes,6 were discernibly different in this study (Fig. 4). HCG was also tested and failed to show complete parallelism of activity with any of its 3 chromatographic fractions. A significant (p = 0.003) increase in the weight of the testes with only partial hypertrophy of the accessory sex organs suggests that HCG was qualitatively similar to Fr I-I (Table 1). However, the absence of a sex-dependent dissociation suggested that HCG was similar to Fr I-II and Fr II in hypophysectomized female rats (Table 2). The steroid content of HCG testes was unlike that of other testes, showing a 7: 1 predominance of estrogen to testosterone (Table 6). Finally, in the spermatogenic tests, HCG testes (Fig. 1) showed three histologically distinct types of seminiferous tubules not unlike those present in other testes (Figs. 2-4), suggesting partial identity with Fr I-I, Fr I-II, and Fr II. Incomplete hypophysectomy as a cause of these divergent results is not indicated. The mean weight of the testes of rats used in the gonadal response tests was less than 400 mg./loo gm. of body weight with a testes to accessory sex organ weight ratio of less than 3 (Table 1). Furthermore, these testes showed histologically discernible atrophy and an absence of spermatogenic cell associations. Comparable intact rats had a mean testicular weight of 1081 mg./loo gm. of body weight, a sex organ ratio of 5.6, and the spermatogenesis was histologically distinct. All rats were preselected for the steroidogenic and spermatogenic tests, and only those rats with preinjection testes weighing less than 150 mg./gm. of body weight and showing marked atrophy by histologic examination were used. Consequently, although it is impossible to be absolutely certain that all rats were completely hypophysectomized, it is unlikely that pituitary gonadotropin stimulation in these rats was a major factor. Cross-contamination of fractions was not demonstrated in previous characterizations of Fr I-I, Fr I-II, and Fr II by immunoelectrophoresis, disc electrophoresis, hemagglutination inhibition, and bioassays in intact animals.5 The sex-dependent dissociation of activities indicates that the maximum contamination of Fr I-I by Fr I II and Fr II was less than 2% (Tables 1 and 2). The inability of more than 5000 H.U. of Fr I-I antisera to neutralize the gonadal effect of heterologous antisera in female rats (Table 5) signifies the absence of Fr I-II and Fr II antibodies in Fr I-I antisera, therefore, that Fr I-I was not cross-contaminated. The varied pattern of steroid content in the testes does not suggest the distribution of a single, biologically active principle in all 3 fractions (Table 6). Distinct and characteristic differences in spermatogenesis in the testes of the various test groups suggested that Fr I-I, Fr I-II, and Fr II were discrete preparations (Figs. 1-4). Consequently, it appears reasonable to regard the variance in activities as intrinsic and inherent to the various HCG preparations. Differences in dosages cannot be implicated as the cause of variance in gonadotropic activities, for there is no constant relationship between international units of HCG activity and positive gonadal responses. Fr I-I (150 JIg.) was unable to cause a significant increase in the mean weight of the prostate gland and seminal vesicles of intact, immature rats, indicating an absence of international units of HCG activity.5 However, 150 JIg. of Fr I-I readily produced spermiation in frogs, 5 caused hypertrophy of the testes in hypophysectomized rats (Table 1), induced steroidogenesis (Table 6), and initiated proliferation of spermatogenic cells (Fig.

January 1971 MULTIVALENCE OF CHORIONIC GONADOTROPIN 37 2). These findings indicate a complete dissociation between positive responses in several biologic test systems and international units of HCG activity in a standard bioassay. The presence of a pregnancyspecific substance containing few or no international units of HCG activity in standard bioassays but possessing HCG activity by immunoassays has also been detected. 8 Further discrepancies between international units of HCG activity and test responses were observed in steroidogenic and spermatogenic tests. Although comparable amounts of HCG (26-30 LU.), Fr I-II (20-40 LD.), and Fr II (6-8 LU.) were administered, the differences in the steroid content of the testes appear to be more of a qualitative, rather than of a quantitative, nature (Table 6). The maximum degree of maturation of spermatogenic cells was observed in the testes of rats injected with 12-16 LU. of activity in Fr II (Fig. 4), whereas the maximum proliferation of germ cells was observed in the testes of rats injected with 300 Jlg. of Fr I-I containing no international units of HCG activity (Fig. 2). Furthermore, the maximum number of histologic variants of seminiferous tubules was observed in the testes of rats injected with 13-16 1. U. of HCG (Fig. 1), whereas the testes of rats injected with 30-80 LU. of activity in Fr I-II demonstrated a homogeneity of histologic changes (Fig. 3). Consequently, it appears that the indexes employed in this study were dependent upon biologic responses dissimilar to those of a standard bioassay. The divergences and dissociations in test responses are best explained on the basis that HCG contains a mosaic of determinants for biologic responses and that the complements of biologic determinants present in Fr I-I, Fr I-II, and Fr II were dissimilar. The absence of parallelism of activities between HCG and any one of its components supports a previous proposal that the gonadotropic capabilities of HCG were a summation of complementary and anticomplementary activities. Therefore, HCG appears to be multivalent and may be composed of several gonadotropic hormones. The experimental findings presented may be of particular relevance to structure-function studies of HCG. Gonadal hypertrophy, steroidogenesis, and spermatogenesis were not elicited by neuraminidase-treated Fr I-I, Fr I-II, Fr II, or HCG, suggesting that the carbohydrate moiety was an intrinsic portion of the determinant group for biologic activities (results not shown). Homogeneity of the HCG protein does not ensure homogeneity of carbohydrate content. 9 Additional indicators of carbohydrate moieties appear desirable, and meaningful structure-function relationships may not be possible until homogeneity of gonadotropic activity can be defined. The dissociation of androgenic from estrogenic steroid biosynthesis, separation of proliferation from maturation of spermatogenic cells, and the synchrony in the maturation of spermatogenic cells are interesting endocrinologic observations. SUMMARY Human chorionic gonadotropin (HCG) is composed of at least three electrophoretically distinct pregnancy-specific components possessing varying immunologic characteristics. These pregnancy-specific components have been compared with each other and with HCG, using gonadal responses, steroidogenesis, and spermatogenesis in hypophysectomized rats as indexes. Divergences and dissociations in results not attributable to incomplete hypophysectomy of test animals, cross-contamination of preparations, or variations in dosages were observed. These findings are best explained on the basis that HCG contains a multiplicity of sites for biologic

38 HAMASHIGE ET AL. Vol. 22 activity and is, therefore, a multivalent hormone complex. REFERENCES 1. HAMASHIGE, S., AND ARQUILLA, E. R hnmunologic and biologic study of human chorionic gonadotropin. J Clin Invest 43:1163, 1964. 2. WaDE, C. E., AND BAGSHAWE, K. D. The purification of chorionic gonadotropin. In Ciba Foundation Study Group No. 22, Little, Boston, 1965. 3. ROBYN, CLAUDE. Contribution a la caracterisation immunologique des gonadotropines urinaires humaines. Rev Belg Path 31 :334, 1965. 4. YODER, J. M., ADAMS, E. C., JR., CHAMBLISS, K. W., AND LOUGHMAN, B. E. Antigens of human chorionic gonadotropin preparations. fractionation of pregnancy urine human chorionic gonadotropic and isolation of one of its specific antigens. J Clin Endocr 27:509, 1967. 5. HAMASHIGE, S., AsTOR, M. A., ARQUILLA, E. R, AND VAN THIEL, D. H. Human chorionic gonadotropin: A hormone complex. J Clin Endocr 27: 1690,1967. 6. HAMASHIGE, S., AND AsTOR, M. A. Newobservations on tile gonadotropic action of human chorionic gonadotropin derived by study of chromatographic fractions. Fertil Steril 20:1029, 1969. 7. McKERNS, K. W., AND NORDSTRAND, E. Stimulation of the rat ovary by gonadotropins and separation of steroids by gas chromatography. Biochim Biophys Acta 104:237, 1965. 8. WIDE, L., AND HOBSON, B. M. New method for the diagnosis of hydatidiform mole. Lancet 2:699, 1964. 9. BELL, J. J., CANFIELD, R F., AND SCIARRA, J. J. Purification and characterization of human chorionic gonadotropin. Endocrinology 84:298, 1969.