Effect of Active and Passive Immunization with Luteinizing Hormone-Releasing Hormone on Serum Luteinizing Hormone and Follicle-Stimulating Hormone Levels and the Ultrastructure of the Pituitary Gonadotrophs in Castrated Male Rats A. ARIMURA,* M. SHINO,** K. G. DE LA CRUZ,* E. G. RENNELS,** AND A. V. SCHALLY* Department of Medicine, Tulane University School of Medicine,* Veterans Administration Hospital, Neio Orleans, Louisiana,* and Department of Anatomy, the University of Texas Medical School at San Antonio, San Antonio, Texas** ABSTRACT. The effect of active and passive immunization with luteinizing hormone-releasing hormone (LHRH) on serum LH and follicle-stimulating hormone (FSH) levels and the ultrastructure of the pituitary gonadotrophs was investigated in castrated male rats. Two weeks after castration, the animals were immunized with Glu'-LHRH conjugated with human serum albumin (hSA), immunized with hSA only, or left uninjected. Immunogens were administered every 2 weeks. Four weeks after the initiation of immunization with hSA-Glu'-LHRH, 2 out of 4 rats showed parallel decreases in serum LH and FSH levels associated with a rise of serum antibody titer to LHRH. Serum LH and FSH levels remained suppressed throughout the experiment in these rats. On the other hand, both LH and FSH levels in hSAimniunized rats or non-immunized rats remained elevated, and typical castration cells containing large vacuoles were found in the pituitary. Although castration cells existed in the pituitary of rats which produced antibody to LHRH by active immunization, these cells were markedly degranulated, and secretory granules were scarce in the cytoplasm. In another experiment, rats were injected iv with one ml sheep anti-LHRH y-globulin (anti-LHRH)
or normal sheep y-globulin (NSG) eveiy 2 days for 3 weeks, starting one day after castration, when serum LH and FSH levels were already elevated. All the animals which received anti-LHRH showed a decrease in both serum LH and FSH levels, which remained low throughout the study, in a range comparable to those in intact normal male rats. On the other hand, in the animals which received NSG, both LH and FSH levels remained high or increased further throughout the experiment, and the pituitary contained numerous castration cells. Castration cells were completely absent from the pituitaries of rats treated with anti-LHRH, suggesting that castration cells are formed as a result of increased secretion of LHRH. Some FSH gonadotrophs in these castrated rats were atrophic. It was difficult to distinguish the LH gonadotrophs in rats which were either actively or passively immunized with LHRH; however, they seem not to have contributed significantly to the development of castration cells. In any case, antibody to the LHRH decapeptide drastically affected both LH and FSH cells, providing additional evidence for the concept that LHRH represents the physiological LHRH and FSHRH. (Endocrinology 99: 291, 1976)
T
HE ADMINISTRATION of anti-serum against synthetic luteinizing hormonereleasing hormone (LHRH) into proestrous rats prevented the preovulatory surge of both luteinizing hormone (LH) and folliclestimulating hormone (FSH) and blocked ovulation (1). This was most probably due to neutralization by the antiserum of endogenous LHRH, the secretion of which might Received September 4, 1975. This study was supported in part by USPH research grants HD-06555 (AA), AM-07467 (AVS), AM-12583 (EGR) and the Veterans Administration.
have increased on the afternoon of proestrus, resulting in the surge of LH and FSH secretion. The result also suggested that the LHRH decapeptide represents not only physiological LHRH but also FSHRH, or that physiological FSHRH is immunologically indistinguishable from LHRH (1). However, an argument could be made that, although the preovulatory surge of FSH as well as of LH is induced by an increased secretion of LHRH, tonic secretion of FSH could still be maintained by another FSHRH which is distinct from the LHRH decapeptide. The presence of such an FSHRH has
291 The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 10 July 2015. at 12:43 For personal use only. No other uses without permission. . All rights reserved.
292
ARIMURAETAL.
been postulated by some investigators, and a fraction of pig hypothalamic extracts was reported to possess much greater FSHRH activity than LHRH in an in vitro test (2,3). If there is another physiological FSHRH which is immunologically dissimilar to the LHRH decapeptide, the tonic secretion of FSH would not be suppressed by the administration of antiserum against LHRH. In such a case, if the effect of the antiserum to LHRH lasted a sufficiently long period, the pituitary LH cells would undergo a degenerative change, but the FSH cells would remain unaltered. Kurosumi (4) reported that rat LH cells can be clearly distinguished morphologically from FSH cells by electron microscopic examination. However, Rennels et al. (5) were unable to recognize two morphologically distinct populations of gonadotrophs after gonadectomy in rats, and immunocytochemical studies have thus far failed to separate clearly FSH and LH-containing gonadotrophs (6). Kurosumi et al. (7) have recently reported that the cellular changes which follow gonadectomy in the rat are mainly related to the cell they designated as the FSH gonadotroph. In the present study, the effects of active immunization with LHRH and the chronic administration of immuno y-globulin against LHRH on pituitary gonadotrophs in castrated male rats were examined by electron microscopy. Serum LH and FSH levels, and the serum levels of the antibody to LHRH in the experiment with active immunization, were also measured.
Endo • 1976 Vol 99 • No 1
group. Glu'-LHRH (Dr. D. H. Coy) was conjugated with human serum albumin (hSA), and 1 mg of hSA-Glu'-LHRH complex was homogenized in complete Freund's adjuvant (Calbiochem.). This immunogen preparation was injected into the rats of Group 1 in multiple sites as described elsewhere (8). Group 2 was immunized with hSA emulsified with complete Freund's adjuvant. Each animal received 8 immunizations at 2week intervals. Group 3 consisted of castrated control animals. One and a half ml of blood was collected between 0900 and 1000 h, before injecting the immunogen once a week, from the jugular vein of rats anesthetized with Surital (sodium thiamylal, Parke-Davis). Blood was allowed to clot at 4 C; the serum was separated by centrifugation and kept at — 20 C until assayed for LH and FSH and for anti-LHRH titer. Experiment 2 Group 1 rats, under ether anesthesia, received 1 ml of sheep anti-LHRH y-globulin into the jugular vein every 2 days starting on the day after castration. Group 2 received 1 ml of normal sheep y-globulin, similarly. Each animal received 6 iv injections and then 7 SC injections of sheep serum, because repeated injection into the jugular vein resulted in a considerable granulation of tissue, making further injections difficult. Subcutaneous injection of anti-LHRH y-globulin was found to be equally effective in neutralizing endogenous LHRH, as judged by the blockade of ovulation in cycling hamsters (9). Blood was collected from the jugular vein between 0900 and 1000 h before injecting antiLHRH or normal sheep y-globulin once a week, starting on the day following castration. Serum samples were separated and kept at - 2 0 C until assayed for LH and FSH. Determination of serum LH and FSH
Methods and Materials Male rats (Sprague-Dawley) weighing 300-350 g on arrival were kept in animal quarters equipped with air conditioning and controlled light (0500 to 1900 h), and fed Purina laboratory chow supplemented with fresh vegetables and water ad libitum. Experiment 1 Two weeks after castration, the rats were divided into 3 groups consisting of 3-5 rats per
Serum LH was determined by radioimmunoassay (RIA) as described by Niswenderef al. (10, 00-RAT LH assay), using rabbit anti-sheep LH serum, GDN no. 15, which was kindly supplied by Dr. Gordon D. Niswender. NIH-LH-S-17 was used as the reference standard preparation. Serum FSH was determined by radioimmunoassay, as described by Daane and Parlow (11), using the NIAMDD rat FSH RIA kit. All samples of one experiment were assayed in duplicate in the same RIA. The RIA data were analyzed by the program of Duddleson et al. (12) using an
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 10 July 2015. at 12:43 For personal use only. No other uses without permission. . All rights reserved.
LHRH AND PITUITARY GONADOTROPHS IBM-7044 computer. The coefficients of withinassay variation at 80% and 20% B/Bo were 9.6% (at 0.27 ng) and 4.8% (at 1.43 ng) for LH, and 8.5% (at 44.9 ng) and 4.3% (at 545.5 ng) for FSH, where B is cpm of the bound tracer for samples and Bo is cpm for the buffer control. The assay sensitivities derived from median variance ratios were 0.11 ng NIH-LH-S-17 and 7.6 ng NIAMDD-RAT-FSH-1, respectively. Determination serum
of anti-LHRH activity in the
The titer of antibody of LHRH in the serum was arbitrarily expressed as per cent of binding of the total radioactivity of [125I]iodoLHRH with test serum at 1:700 dilution. Binding took place in a system similar to that used in the radioimmunoassay for LHRH described elsewhere (13). Sheep anti-LHRH y-globulin Two sheep were immunized with hSA-Glu1LHRH conjugate. The conjugate, emulsified with complete Freund's adjuvant and corresponding to 2 mg Glu'-LHRH, was injected into each sheep at multiple sites every two weeks. At the first immunization, the complete Freund's adjuvant was reinforced by the addition of dried Mycobacterium tuberculosis, and 1 ml of pertussis vaccine was also injected into each animal as described by Vaitukaitis et al. (14). At the fifth immunization, 4 mg LHRH dissolved in 2 ml 40% polyvinylpyrrolidone (PVP) was injected iv. Three months after the initiation of immunization the serum of one of the sheep (no. 772) showed considerable binding with [125I]LHRH, being 99% at 1:70 dilution. It was possible to establish an excellent standard curve for the RIA of LHRH using antiserum no. 772 over a range of 16 to 4096 pg of synthetic LHRH. The antigenic determinant of this antiserum, as determined by cross reaction with various synthetic peptides corresponding to fragments of LHRH (8), resided between Leu7 and Gly10-NH2 of LHRH. The antibody-tracer binding was not affected by other hypothalamic and pituitary hormones, including TRH, somatostatin, rat LH, FSH, and prolactin. Sheep anti-LHRH y-globulin and normal sheep y-globulin were precipitated in 2 steps by using the ammonium sulfate method, as described elsewhere (15). After the mixture was dissolved in water, it was dialyzed against water
293
at 4 C for 48 h to remove the remaining ammonium sulfate and then lyophilized. Before injection, the lyophilized y-globulin was dissolved in 0.9% saline so that the concentration was the same as that in the original serum. Histological examination of the pituitary gland In experiment 1, all animals were sacrificed by decapitation, following withdrawal of blood under Surital anesthesia, 16 weeks after castration. In experiment 2, they were sacrificed one month after castration. The anterior pituitary glands were quickly exposed and bisected in the midline; one half was fixed in Bouin's solution for light microscopy, and the other in 1% osmium tetroxide in 0.1M cacodylate buffer solution (pH 7.4) for several hours. The fixation was followed by the procedure previously described by Shiino et al. (16) for electron microscopy.
Results Experiment 1 Serum LH and FSH levels of the castrated rats are depicted in Fig. 1. Two weeks after the castration, when the experiment was started, serum LH and FSH levels were greatly elevated, being in the range of from 16 to 56 ng/ml for LH and from 1.1 to 2.2 fig for FSH. The LH levels in normal adult rats were lower than 1 ng/ml, and the FSH levels were lower than 0.5 fig/ml. In 2 out of 5 rats (No. 2 and No. 5), which were immunized with hSA-Gli^-LHRH (group 1), both LH and FSH levels started decreasing 1 week after the second immunization, and, 8 weeks after thefirstimmunization, reached levels comparable with basal levels in intact rats. Then the LH levels rose slightly, but remained much lower than those in the other 2 animals (one other died during the experiment), in which LH levels fluctuated during the experiment, but never fell below 13 ng/ml. In those 2 rats whose LH decreased considerably, serum FSH levels also started to decrease 1 week after the second immunization and remained below 1 /xg/ml most of the time, though there were slight fluctuations. The other 2 animals showed considerable
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 10 July 2015. at 12:43 For personal use only. No other uses without permission. . All rights reserved.
294
Endo • 1976 Vol 99 • No 1
ARIMURAETAL. SERUM LH LEVELS
SERUM FSH LEVELS
Group 1
Group 1
FIG. 1. Semm LH and FSH levels in castrated rats. Group 1: immunized with hSA-Glu 1 LHRH. Group 2: immunized with hSA. Group 3: not immunized. Immunization was started 2 weeks after castration.
Note, 2 (rats No. 2 and No. 5) out of 4 rats of group 1 showed parallel decreases in serum LH and FSH levels 4 weeks after the initiation of immunization.
4
6
8 10 12 14
Weeks after Castration
16
2
4
6
8 10 12 14
16
Weeks after Castration
fluctuations of FSH levels; however, these never fell below 1.3 fig/ml. In group 2, which was immunized with hSA, and group 3, consisting of non-immunized control castrated rats, both LH and FSH levels remained high, although considerable fluctuations were present. It is interesting that synchronous fluctuations of FSH were observed in groups 2 and 3. Serum LH and FSH levels and the levels of antibody to LHRH in rats No. 2 and No. 5 in group 1 are illustrated in Fig. 2. It is clear that as the antibody titer started rising, both LH and FSH levels started falling. Antibody titers kept increasing or remained elevated, but LH levels started rising again, although to a small extent. No antibody to
LHRH could be detected in the sera of the other 2 animals in group 1 at any time during the study. In the electron microscopic examination, numerous castration cells1 were seen in the pituitary glands of both hSA-immunized (group 2) and non-immunized (group 3) castrated rats. The ultrastructure of castration cells observed in the anterior pituitary 1
The term "castration cell" is commonly used to denote gonadotrophic cells which become markedly enlarged and highly modified morphologically after gonadectomy in the rat. The cellular changes are progressive, and after a period of several weeks most of the modified cells assume the appearance of a signet ring due to the formation of a large vacuole which fills most of the cytoplasm.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 10 July 2015. at 12:43 For personal use only. No other uses without permission. . All rights reserved.
295
LHRH AND PITUITARY GONADOTROPHS
40-,
1 I J JI Group I
?
FIG, 2. Senim LH and FSH levels and titers of antibody to LHRH in castrated rats No. 2and No. 5, which generated antibody during immunization with hSAGlu'-LHRH. Arrows indicate the times of immunization. AntiLHRH titer is expressed by per cent of binding of [125I]LHRH with the serum at 1:700 dilution. Note that when the antibody titer started to rise, both LH and FSH levels started falling.
r-4.0
^ IOO
-3.0
-75
-2.0 =?
-50
Rat # 2
20-
-25
10-
rlOO
75 >
-50
-25 r
12
14
Weeks after Castration
glands of castrated rats which were immunized with hSA was quite similar to that of non-immunized castrated animals, and was characterized by dilated cisternae of the endoplasmic reticulum, large vacuoles, many immature granules in a well-developed Golgi zone, and abundant mature secretory granules in the cytoplasm (Fig. 3). These castration cells resembled the FSH gonadotrophs described by Kurosumi (4). LH-type gonadotrophs, as described by Kurosumi (4), were present, but this cell seemed not to be greatly altered by castration. The pituitary glands of only those rats in groups 1 (rats no. 2 and no. 5), which showed a marked lowering of serum FSH and LH, were examined by electron microscopy. Many castration cells were also present in the pituitaries of both animals.
However, they contained scarce secretory granules in the cytoplasm and in the Golgi zone. Consequently, the cytoplasm of this type of cell appeared as a network of dilated cisternae of the endoplasmic reticulum (Fig. 4). The LH-type gonadotroph described by Kurosumi (4) appeared not to be modified by castration, and these cells were only rarely seen in the glands from the castrated animals immunized with LHRH. Experiment 2 One day after castration, both LH and FSH levels were already elevated, compared with those in intact male rats (Fig. 5). Six days after the first injection of anti-LHRH yglobulin (group 1), both LH and FSH levels were decreased in these rats, and remained low thereafter, at levels similar to or slightly
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 10 July 2015. at 12:43 For personal use only. No other uses without permission. . All rights reserved.
296
ARIMURA ETAL.
Endo • 1976 Vol 99 • No 1
FIG. 3. A typical castration cell containing a large vacuole (v) in the anterior pituitary gland of a castrated rat (exp. 1, group 2) immunized with human serum albumin emulsified with complete Freund's adjuvant. (x8,100).
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 10 July 2015. at 12:43 For personal use only. No other uses without permission. . All rights reserved.
LHRH AND PITUITARY GONADOTROPHS
297
FIG. 4. An example of a markedly degranulated castration cell (rat No. 3), which was immunized with Glu'-LHRH conjugated with human serum albumin. (x8,100).
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 10 July 2015. at 12:43 For personal use only. No other uses without permission. . All rights reserved.
Endo • 1976 Vol 99 • No ]
ARIMURA ETAL.
298 Group I (onti-LHRH)
Group 1 (onti-LHRH)
LH
FSH
20
1
7
15
Group 2 (NSG)
22
28
LH
1
7
15
Group 2 (NSG)
22
28
FSH
, 30
FIG. 5. Serum LH and FSH levels of castrated male rats. Group 1: injected with antiLHRH y-globulin every 2 days. Group 2: injected with normal sheep y-globulin (NSG) every 2 days. Injection was started 1 day after castration. Both LH and FSH levels were already elevated on the day after castration. Note that both LH and FSH levels fell and remained low during
passive
immunization
with LHRH.
22 DAYS
28 AFTER
1
7
15
28
CASTRATION
higher than those found in normal male rats. On the other hand, both LH and FSH levels in rats in group 2, which received normal sheep y-globulin (NSG), remained high or continued increasing throughout the experiment, except for one rat in which FSH decreased 7 days after castration. However, the FSH level in this animal rose thereafter. Numerous castration cells were seen in the anterior pituitary glands of castrated rats which received normal sheep y-globulin (Fig. 6), but the occurrence of typical castration cells was completely prevented by treatment with antibody to LHRH y-globulin (Fig. 7). The castration cells in group 2 showed an enlargement of cell size, swollen cisternae of the endoplasmic reticulum, a large Golgi zone, and abundant secretory granules in the cytoplasm (Fig. 8). These castration cells seemed to originate from Kurosumi's FSH-gonadotrophs, but, in this kind of cell, there were very few of the large opaque granules usually seen in normal
FSH cells. Again, the LH cells of Kurosumi (4) seemed not to be involved in the development of castration cells. On the other hand, in the castrated rats given anti-LHRH y-globulin (group 1) some of the FSH cells were comparatively small in size, and contained scarce secretory granules and poor Golgi zones, indicating that these cells were morphologically atrophic (Fig. 9). However, most of the FSH cells were similar to the FSH cells of normal intact male rats, and typical castration cells were not present. The LH gonadotrophs of Kurosumi were rarely recognized in the passively immunized castrated rats. In view of this, it seems likely that these cells were markedly suppressed by the anti-LHRH y-globulin. Discussion The results of the present study indicate that either active or passive immunization of male rats with synthetic LHRH decapep-
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 10 July 2015. at 12:43 For personal use only. No other uses without permission. . All rights reserved.
LHRH AND PITUITARY GONADOTROPHS
\ '
299
v
® FIG. 6. Numerous castration cells (arrows) observed in the anterior pituitary gland of a castrated rat (exp. 2, group 2) which was injected with normal sheep y-globulin starting the day after castration. Hematoxylin and PAS stain. (x440).
tide greatly suppressed serum LH and FSH levels which were elevated by castration.
to LHRH. Therefore, antibody to LHRH can not only block the preovulatory surge of LH and FSH, but can also chronically supFurthermore, the suppression persisted throughout the experiment, which suggests press serum LH and FSH levels in castrated •that the elevation of LH and FSH secretion rats. Fraser et al. reported that active imresulting from castration is due to an in- munization of male rats resulted in a supcreased secretion of endogenous LHRH and pression of basal serum LH and FSH levels FSHRH, which can be neutralized by an (18). Our results together with those of antibody to the LHRH decapeptide. Fraser others (17-19) strongly suggest that the et al. recently reported that active im- physiological FSHRH, as well as LHRH, munization of ovariectomized rats with is immunologically indistinguishable from LHRH lowered elevated serum LH and LHRH decapeptide. This view is further FSH levels and that there was a reciprocal supported by the fact that similar findings correlation between the gonadotrophin were obtained by different investigators levels and the levels of antibody to LHRH who used different antibodies, which pos(17). They stated that long-term inhibition sess different antigenic determinants. The of LHRH could not be satisfactorily site of neutralization of endogenous LHRH achieved by the injection of antisera. How- by the antibody is unknown, but LHRH in ever, the present study shows that long-term the median eminence or in the hypophysial suppression of LHRH and FSHRH is also portal blood could be bound to antibody possible by chronic treatment with antibody and lose biological activity before it reaches
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 10 July 2015. at 12:43 For personal use only. No other uses without permission. . All rights reserved.
300
ARIMURA ETAL.
Endo • 1976 Vol 99 • No 1
FIG. 7. Anterior pituitary cells of a castrated rat (exp. 2, group 1) injected with anti-LHRH •y-globulin starting the day after castration. Hematoxylin and PAS stain. (x440).
the pituitary gland. In any case, the pituitary of rats which were actively or passively immunized with LHRH would be deprived of the influence of hypothalamic LHRH. In the first experiment, numerous castration cells were definitely observed in the pituitary glands of castrated rats given the control immunization and also in those actively immunized with LHRH. It is clear that castration cells would have been present before antibody to LHRH was formed, since the immunizations with LHRH were started two weeks after the castration. However, the finding of scarce immature and mature secretory granules in the castration cells of rats which generated antibody to LHRH following active immunization, probably due to the neutralization of endogenous LHRH by antibody to LHRH, indicates an interruption of normal stimulation by hypothalamic LHRH, which probably maintains synthesis and secretion
of gonadotrophins. This assumption is supported by assay results in which immunization with LHRH decreased both LH and FSH serum levels to those found in intact rats as early as eight weeks after the first immunization. Zambrano et al. (20) reported that the incubation of pituitary glands with LHRH in the culture medium for three and six hours led to formation of typical "signet ring" castration cells, and that the appearance of such signet ring cells was prevented by adding puromycin to the incubation medium together with LHRH. They interpreted these findings to mean that castration cells are stimulated to develop in the gland by an elevation in hypothalamic LHRH after castration. Consequently, the complete absence of castration cells after the injection of antibody against LHRH, starting on the day after castration, may be due to a neutralization of most of the
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 10 July 2015. at 12:43 For personal use only. No other uses without permission. . All rights reserved.
LHRH AND PITUITARY GONADOTROPHS
301
FIG. 8. An example of castration cells of a castrated rat (exp. 2., group 2) injected with normal sheep -/-globulin as a control. Dilated cistemae of the endoplasmic reticulum, a large Golgi area, and abundant secretory granules are seen. (x8,100).
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 10 July 2015. at 12:43 For personal use only. No other uses without permission. . All rights reserved.
302
ARIMURA ETAL.
Endo • L976 Vol 99 • Xo 1
FIG. 9. An example of an atrophic FSH-gonadotroph in the anterior pituitary gland of a castrated rat (esp. 2, group 1) injected with anti-LHRH y-globulin, beginning the day after castration. (x8,100).
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 10 July 2015. at 12:43 For personal use only. No other uses without permission. . All rights reserved.
LHRH AND PITUITARY GONADOTROPHS endogenous LHRH in these animals. Our assay results in the passively immunized castrated rats, in which both LH and FSH levels were decreased after the first injection of anti-LHRH y-globulin, and remained low thereafter, are in good agreement with the morphological observations. In the present study, the castration cells resembled, in ultrastructural aspects, the FSH cells described by Kurosumi (4). The LH gonadotrophs of Kurosumi were rarely recognized in both the actively and passively immunized rats. It is considered likely that the LH cells were suppressed by these immunization procedures. However, they seem not to have contributed significantly to the development of castration cells, in agreement with the recent findings of Kurosumi et al. (7). Acknowledgments The authors wish to thank Mr. P. Taylor, Mrs. Joan Gauthier, and Mrs. Deborah Pierson for their excellent technical help, Dr. A. J. Kastin and Ms. G. A. Farley for their help in preparing the manuscript, and Dr. G. D. Nisvvender and NIAMDD Rat Pituitary Hormone Program for the reagents used in radioimmunoassay for rat LH and FSH.
References 1. Arimura, A., L. Debeljuk, and A. V. Schally, Endocrinology 95: 323, 1974. 2. Gunner Johanson, K. N., B. L. Curries, K. Folkers, and C. Y. Bowers, Biochem Biophys Res Commun 50: 8, 1973.
303
3. Fawcett, C. P., A. E. Beezley, and J. E. Wheaton, Endocrinology 96: 1311, 1975. 4. Kurosumi, K.,Arch Histoljap 29: 329, 1968. 5. Rennels, E. G., E. M. Bogdanove, A. Arimura, M. Sato, and A. V. Schally,Endocrinology 88: 1318, 1971. 6. Moriarty, G. C., Endocrinology 97: 1215, 1975. 7. Kurosumi, K., Y. Kawarai, Y. Yukitake, and K. Inoue, Proc 13th Gun ma Symposium on Endocrinology, Biology of Reproduction and its Hormonal Control, 1975, Abstract no III-4. 8. Arimura, A., H. Sato, D. H. Coy, R. B. Worobec, A. V. Schally, N. Yanaihara, T. Hashimoto, C. Yanaihara, and N. Sukuta, Ada Endocrinol (Kbh) 2: 222, 1975. 9. de la Cruz, A., A. Arimura, K. G. de la Cruz, and A. V. Schally, Endocrinology 98: 490, 1976. 10. Niswender, G. D., A. R. Midgley, S. E. Monroe, and L. E. Reichert, Proc Soc Exp Biol Med 128: 807, 1968. 11. Daane, T. A., and A. F. Parlow, Endocrinology 88: 653, 1971. 12. Duddleson, W. G., A. R. Midgley, Jr., and G. D. Niswender, Comput Biomed Res 5: 205, 1972. 13. Arimura, A., H. Sato, T. Kumasaka, R. B. Worobec, L. Debeljuk, J. D. Dunn, and A. V. Schally, Endocrinology 93: 1092, 1973. 14. Vaitukaitis, J., J. B. Robbins, E. Nieschlag and G. T. Ross, 7 Clin Endocrinol Metab 33: 988, 1971. 15. Weir, D. M., Handbook of Experimental Biology, F. A. Davis Co., Philadelphia, 1967, p. 4. 16. Shiino, M., A. Arimura, A. V. Schally, and E. G. Rennels.Z ZellforschMikroskAnat 128:152,1972. 17. Fraser, H. M., S. L. Jeffcoate, A. Gunn, and D. T. Holland, J Endocrinol 64: 191, 1975. 18. Fraser, H. M., A. Gunn, S. L. Jeffcoate, and D. T. Holland,/ Endocrinol 63: 399, 1974. 19. Koch, Y., P. Chobsieng, U. Zor, M. Fridkin, and H. R. Lindner, Biochem Biophys Res Commun 55: 623, 1973. 20. Zambrano, D., S. Cuerdo-Rocha, and I. Bergmann, Cell Tiss Res 150: 179, 1974.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 10 July 2015. at 12:43 For personal use only. No other uses without permission. . All rights reserved.
or normal sheep y-globulin (NSG) eveiy 2 days for 3 weeks, starting one day after castration, when serum LH and FSH levels were already elevated. All the animals which received anti-LHRH showed a decrease in both serum LH and FSH levels, which remained low throughout the study, in a range comparable to those in intact normal male rats. On the other hand, in the animals which received NSG, both LH and FSH levels remained high or increased further throughout the experiment, and the pituitary contained numerous castration cells. Castration cells were completely absent from the pituitaries of rats treated with anti-LHRH, suggesting that castration cells are formed as a result of increased secretion of LHRH. Some FSH gonadotrophs in these castrated rats were atrophic. It was difficult to distinguish the LH gonadotrophs in rats which were either actively or passively immunized with LHRH; however, they seem not to have contributed significantly to the development of castration cells. In any case, antibody to the LHRH decapeptide drastically affected both LH and FSH cells, providing additional evidence for the concept that LHRH represents the physiological LHRH and FSHRH. (Endocrinology 99: 291, 1976)
T
HE ADMINISTRATION of anti-serum against synthetic luteinizing hormonereleasing hormone (LHRH) into proestrous rats prevented the preovulatory surge of both luteinizing hormone (LH) and folliclestimulating hormone (FSH) and blocked ovulation (1). This was most probably due to neutralization by the antiserum of endogenous LHRH, the secretion of which might Received September 4, 1975. This study was supported in part by USPH research grants HD-06555 (AA), AM-07467 (AVS), AM-12583 (EGR) and the Veterans Administration.
have increased on the afternoon of proestrus, resulting in the surge of LH and FSH secretion. The result also suggested that the LHRH decapeptide represents not only physiological LHRH but also FSHRH, or that physiological FSHRH is immunologically indistinguishable from LHRH (1). However, an argument could be made that, although the preovulatory surge of FSH as well as of LH is induced by an increased secretion of LHRH, tonic secretion of FSH could still be maintained by another FSHRH which is distinct from the LHRH decapeptide. The presence of such an FSHRH has
291 The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 10 July 2015. at 12:43 For personal use only. No other uses without permission. . All rights reserved.
292
ARIMURAETAL.
been postulated by some investigators, and a fraction of pig hypothalamic extracts was reported to possess much greater FSHRH activity than LHRH in an in vitro test (2,3). If there is another physiological FSHRH which is immunologically dissimilar to the LHRH decapeptide, the tonic secretion of FSH would not be suppressed by the administration of antiserum against LHRH. In such a case, if the effect of the antiserum to LHRH lasted a sufficiently long period, the pituitary LH cells would undergo a degenerative change, but the FSH cells would remain unaltered. Kurosumi (4) reported that rat LH cells can be clearly distinguished morphologically from FSH cells by electron microscopic examination. However, Rennels et al. (5) were unable to recognize two morphologically distinct populations of gonadotrophs after gonadectomy in rats, and immunocytochemical studies have thus far failed to separate clearly FSH and LH-containing gonadotrophs (6). Kurosumi et al. (7) have recently reported that the cellular changes which follow gonadectomy in the rat are mainly related to the cell they designated as the FSH gonadotroph. In the present study, the effects of active immunization with LHRH and the chronic administration of immuno y-globulin against LHRH on pituitary gonadotrophs in castrated male rats were examined by electron microscopy. Serum LH and FSH levels, and the serum levels of the antibody to LHRH in the experiment with active immunization, were also measured.
Endo • 1976 Vol 99 • No 1
group. Glu'-LHRH (Dr. D. H. Coy) was conjugated with human serum albumin (hSA), and 1 mg of hSA-Glu'-LHRH complex was homogenized in complete Freund's adjuvant (Calbiochem.). This immunogen preparation was injected into the rats of Group 1 in multiple sites as described elsewhere (8). Group 2 was immunized with hSA emulsified with complete Freund's adjuvant. Each animal received 8 immunizations at 2week intervals. Group 3 consisted of castrated control animals. One and a half ml of blood was collected between 0900 and 1000 h, before injecting the immunogen once a week, from the jugular vein of rats anesthetized with Surital (sodium thiamylal, Parke-Davis). Blood was allowed to clot at 4 C; the serum was separated by centrifugation and kept at — 20 C until assayed for LH and FSH and for anti-LHRH titer. Experiment 2 Group 1 rats, under ether anesthesia, received 1 ml of sheep anti-LHRH y-globulin into the jugular vein every 2 days starting on the day after castration. Group 2 received 1 ml of normal sheep y-globulin, similarly. Each animal received 6 iv injections and then 7 SC injections of sheep serum, because repeated injection into the jugular vein resulted in a considerable granulation of tissue, making further injections difficult. Subcutaneous injection of anti-LHRH y-globulin was found to be equally effective in neutralizing endogenous LHRH, as judged by the blockade of ovulation in cycling hamsters (9). Blood was collected from the jugular vein between 0900 and 1000 h before injecting antiLHRH or normal sheep y-globulin once a week, starting on the day following castration. Serum samples were separated and kept at - 2 0 C until assayed for LH and FSH. Determination of serum LH and FSH
Methods and Materials Male rats (Sprague-Dawley) weighing 300-350 g on arrival were kept in animal quarters equipped with air conditioning and controlled light (0500 to 1900 h), and fed Purina laboratory chow supplemented with fresh vegetables and water ad libitum. Experiment 1 Two weeks after castration, the rats were divided into 3 groups consisting of 3-5 rats per
Serum LH was determined by radioimmunoassay (RIA) as described by Niswenderef al. (10, 00-RAT LH assay), using rabbit anti-sheep LH serum, GDN no. 15, which was kindly supplied by Dr. Gordon D. Niswender. NIH-LH-S-17 was used as the reference standard preparation. Serum FSH was determined by radioimmunoassay, as described by Daane and Parlow (11), using the NIAMDD rat FSH RIA kit. All samples of one experiment were assayed in duplicate in the same RIA. The RIA data were analyzed by the program of Duddleson et al. (12) using an
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 10 July 2015. at 12:43 For personal use only. No other uses without permission. . All rights reserved.
LHRH AND PITUITARY GONADOTROPHS IBM-7044 computer. The coefficients of withinassay variation at 80% and 20% B/Bo were 9.6% (at 0.27 ng) and 4.8% (at 1.43 ng) for LH, and 8.5% (at 44.9 ng) and 4.3% (at 545.5 ng) for FSH, where B is cpm of the bound tracer for samples and Bo is cpm for the buffer control. The assay sensitivities derived from median variance ratios were 0.11 ng NIH-LH-S-17 and 7.6 ng NIAMDD-RAT-FSH-1, respectively. Determination serum
of anti-LHRH activity in the
The titer of antibody of LHRH in the serum was arbitrarily expressed as per cent of binding of the total radioactivity of [125I]iodoLHRH with test serum at 1:700 dilution. Binding took place in a system similar to that used in the radioimmunoassay for LHRH described elsewhere (13). Sheep anti-LHRH y-globulin Two sheep were immunized with hSA-Glu1LHRH conjugate. The conjugate, emulsified with complete Freund's adjuvant and corresponding to 2 mg Glu'-LHRH, was injected into each sheep at multiple sites every two weeks. At the first immunization, the complete Freund's adjuvant was reinforced by the addition of dried Mycobacterium tuberculosis, and 1 ml of pertussis vaccine was also injected into each animal as described by Vaitukaitis et al. (14). At the fifth immunization, 4 mg LHRH dissolved in 2 ml 40% polyvinylpyrrolidone (PVP) was injected iv. Three months after the initiation of immunization the serum of one of the sheep (no. 772) showed considerable binding with [125I]LHRH, being 99% at 1:70 dilution. It was possible to establish an excellent standard curve for the RIA of LHRH using antiserum no. 772 over a range of 16 to 4096 pg of synthetic LHRH. The antigenic determinant of this antiserum, as determined by cross reaction with various synthetic peptides corresponding to fragments of LHRH (8), resided between Leu7 and Gly10-NH2 of LHRH. The antibody-tracer binding was not affected by other hypothalamic and pituitary hormones, including TRH, somatostatin, rat LH, FSH, and prolactin. Sheep anti-LHRH y-globulin and normal sheep y-globulin were precipitated in 2 steps by using the ammonium sulfate method, as described elsewhere (15). After the mixture was dissolved in water, it was dialyzed against water
293
at 4 C for 48 h to remove the remaining ammonium sulfate and then lyophilized. Before injection, the lyophilized y-globulin was dissolved in 0.9% saline so that the concentration was the same as that in the original serum. Histological examination of the pituitary gland In experiment 1, all animals were sacrificed by decapitation, following withdrawal of blood under Surital anesthesia, 16 weeks after castration. In experiment 2, they were sacrificed one month after castration. The anterior pituitary glands were quickly exposed and bisected in the midline; one half was fixed in Bouin's solution for light microscopy, and the other in 1% osmium tetroxide in 0.1M cacodylate buffer solution (pH 7.4) for several hours. The fixation was followed by the procedure previously described by Shiino et al. (16) for electron microscopy.
Results Experiment 1 Serum LH and FSH levels of the castrated rats are depicted in Fig. 1. Two weeks after the castration, when the experiment was started, serum LH and FSH levels were greatly elevated, being in the range of from 16 to 56 ng/ml for LH and from 1.1 to 2.2 fig for FSH. The LH levels in normal adult rats were lower than 1 ng/ml, and the FSH levels were lower than 0.5 fig/ml. In 2 out of 5 rats (No. 2 and No. 5), which were immunized with hSA-Gli^-LHRH (group 1), both LH and FSH levels started decreasing 1 week after the second immunization, and, 8 weeks after thefirstimmunization, reached levels comparable with basal levels in intact rats. Then the LH levels rose slightly, but remained much lower than those in the other 2 animals (one other died during the experiment), in which LH levels fluctuated during the experiment, but never fell below 13 ng/ml. In those 2 rats whose LH decreased considerably, serum FSH levels also started to decrease 1 week after the second immunization and remained below 1 /xg/ml most of the time, though there were slight fluctuations. The other 2 animals showed considerable
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 10 July 2015. at 12:43 For personal use only. No other uses without permission. . All rights reserved.
294
Endo • 1976 Vol 99 • No 1
ARIMURAETAL. SERUM LH LEVELS
SERUM FSH LEVELS
Group 1
Group 1
FIG. 1. Semm LH and FSH levels in castrated rats. Group 1: immunized with hSA-Glu 1 LHRH. Group 2: immunized with hSA. Group 3: not immunized. Immunization was started 2 weeks after castration.
Note, 2 (rats No. 2 and No. 5) out of 4 rats of group 1 showed parallel decreases in serum LH and FSH levels 4 weeks after the initiation of immunization.
4
6
8 10 12 14
Weeks after Castration
16
2
4
6
8 10 12 14
16
Weeks after Castration
fluctuations of FSH levels; however, these never fell below 1.3 fig/ml. In group 2, which was immunized with hSA, and group 3, consisting of non-immunized control castrated rats, both LH and FSH levels remained high, although considerable fluctuations were present. It is interesting that synchronous fluctuations of FSH were observed in groups 2 and 3. Serum LH and FSH levels and the levels of antibody to LHRH in rats No. 2 and No. 5 in group 1 are illustrated in Fig. 2. It is clear that as the antibody titer started rising, both LH and FSH levels started falling. Antibody titers kept increasing or remained elevated, but LH levels started rising again, although to a small extent. No antibody to
LHRH could be detected in the sera of the other 2 animals in group 1 at any time during the study. In the electron microscopic examination, numerous castration cells1 were seen in the pituitary glands of both hSA-immunized (group 2) and non-immunized (group 3) castrated rats. The ultrastructure of castration cells observed in the anterior pituitary 1
The term "castration cell" is commonly used to denote gonadotrophic cells which become markedly enlarged and highly modified morphologically after gonadectomy in the rat. The cellular changes are progressive, and after a period of several weeks most of the modified cells assume the appearance of a signet ring due to the formation of a large vacuole which fills most of the cytoplasm.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 10 July 2015. at 12:43 For personal use only. No other uses without permission. . All rights reserved.
295
LHRH AND PITUITARY GONADOTROPHS
40-,
1 I J JI Group I
?
FIG, 2. Senim LH and FSH levels and titers of antibody to LHRH in castrated rats No. 2and No. 5, which generated antibody during immunization with hSAGlu'-LHRH. Arrows indicate the times of immunization. AntiLHRH titer is expressed by per cent of binding of [125I]LHRH with the serum at 1:700 dilution. Note that when the antibody titer started to rise, both LH and FSH levels started falling.
r-4.0
^ IOO
-3.0
-75
-2.0 =?
-50
Rat # 2
20-
-25
10-
rlOO
75 >
-50
-25 r
12
14
Weeks after Castration
glands of castrated rats which were immunized with hSA was quite similar to that of non-immunized castrated animals, and was characterized by dilated cisternae of the endoplasmic reticulum, large vacuoles, many immature granules in a well-developed Golgi zone, and abundant mature secretory granules in the cytoplasm (Fig. 3). These castration cells resembled the FSH gonadotrophs described by Kurosumi (4). LH-type gonadotrophs, as described by Kurosumi (4), were present, but this cell seemed not to be greatly altered by castration. The pituitary glands of only those rats in groups 1 (rats no. 2 and no. 5), which showed a marked lowering of serum FSH and LH, were examined by electron microscopy. Many castration cells were also present in the pituitaries of both animals.
However, they contained scarce secretory granules in the cytoplasm and in the Golgi zone. Consequently, the cytoplasm of this type of cell appeared as a network of dilated cisternae of the endoplasmic reticulum (Fig. 4). The LH-type gonadotroph described by Kurosumi (4) appeared not to be modified by castration, and these cells were only rarely seen in the glands from the castrated animals immunized with LHRH. Experiment 2 One day after castration, both LH and FSH levels were already elevated, compared with those in intact male rats (Fig. 5). Six days after the first injection of anti-LHRH yglobulin (group 1), both LH and FSH levels were decreased in these rats, and remained low thereafter, at levels similar to or slightly
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 10 July 2015. at 12:43 For personal use only. No other uses without permission. . All rights reserved.
296
ARIMURA ETAL.
Endo • 1976 Vol 99 • No 1
FIG. 3. A typical castration cell containing a large vacuole (v) in the anterior pituitary gland of a castrated rat (exp. 1, group 2) immunized with human serum albumin emulsified with complete Freund's adjuvant. (x8,100).
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 10 July 2015. at 12:43 For personal use only. No other uses without permission. . All rights reserved.
LHRH AND PITUITARY GONADOTROPHS
297
FIG. 4. An example of a markedly degranulated castration cell (rat No. 3), which was immunized with Glu'-LHRH conjugated with human serum albumin. (x8,100).
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 10 July 2015. at 12:43 For personal use only. No other uses without permission. . All rights reserved.
Endo • 1976 Vol 99 • No ]
ARIMURA ETAL.
298 Group I (onti-LHRH)
Group 1 (onti-LHRH)
LH
FSH
20
1
7
15
Group 2 (NSG)
22
28
LH
1
7
15
Group 2 (NSG)
22
28
FSH
, 30
FIG. 5. Serum LH and FSH levels of castrated male rats. Group 1: injected with antiLHRH y-globulin every 2 days. Group 2: injected with normal sheep y-globulin (NSG) every 2 days. Injection was started 1 day after castration. Both LH and FSH levels were already elevated on the day after castration. Note that both LH and FSH levels fell and remained low during
passive
immunization
with LHRH.
22 DAYS
28 AFTER
1
7
15
28
CASTRATION
higher than those found in normal male rats. On the other hand, both LH and FSH levels in rats in group 2, which received normal sheep y-globulin (NSG), remained high or continued increasing throughout the experiment, except for one rat in which FSH decreased 7 days after castration. However, the FSH level in this animal rose thereafter. Numerous castration cells were seen in the anterior pituitary glands of castrated rats which received normal sheep y-globulin (Fig. 6), but the occurrence of typical castration cells was completely prevented by treatment with antibody to LHRH y-globulin (Fig. 7). The castration cells in group 2 showed an enlargement of cell size, swollen cisternae of the endoplasmic reticulum, a large Golgi zone, and abundant secretory granules in the cytoplasm (Fig. 8). These castration cells seemed to originate from Kurosumi's FSH-gonadotrophs, but, in this kind of cell, there were very few of the large opaque granules usually seen in normal
FSH cells. Again, the LH cells of Kurosumi (4) seemed not to be involved in the development of castration cells. On the other hand, in the castrated rats given anti-LHRH y-globulin (group 1) some of the FSH cells were comparatively small in size, and contained scarce secretory granules and poor Golgi zones, indicating that these cells were morphologically atrophic (Fig. 9). However, most of the FSH cells were similar to the FSH cells of normal intact male rats, and typical castration cells were not present. The LH gonadotrophs of Kurosumi were rarely recognized in the passively immunized castrated rats. In view of this, it seems likely that these cells were markedly suppressed by the anti-LHRH y-globulin. Discussion The results of the present study indicate that either active or passive immunization of male rats with synthetic LHRH decapep-
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 10 July 2015. at 12:43 For personal use only. No other uses without permission. . All rights reserved.
LHRH AND PITUITARY GONADOTROPHS
\ '
299
v
® FIG. 6. Numerous castration cells (arrows) observed in the anterior pituitary gland of a castrated rat (exp. 2, group 2) which was injected with normal sheep y-globulin starting the day after castration. Hematoxylin and PAS stain. (x440).
tide greatly suppressed serum LH and FSH levels which were elevated by castration.
to LHRH. Therefore, antibody to LHRH can not only block the preovulatory surge of LH and FSH, but can also chronically supFurthermore, the suppression persisted throughout the experiment, which suggests press serum LH and FSH levels in castrated •that the elevation of LH and FSH secretion rats. Fraser et al. reported that active imresulting from castration is due to an in- munization of male rats resulted in a supcreased secretion of endogenous LHRH and pression of basal serum LH and FSH levels FSHRH, which can be neutralized by an (18). Our results together with those of antibody to the LHRH decapeptide. Fraser others (17-19) strongly suggest that the et al. recently reported that active im- physiological FSHRH, as well as LHRH, munization of ovariectomized rats with is immunologically indistinguishable from LHRH lowered elevated serum LH and LHRH decapeptide. This view is further FSH levels and that there was a reciprocal supported by the fact that similar findings correlation between the gonadotrophin were obtained by different investigators levels and the levels of antibody to LHRH who used different antibodies, which pos(17). They stated that long-term inhibition sess different antigenic determinants. The of LHRH could not be satisfactorily site of neutralization of endogenous LHRH achieved by the injection of antisera. How- by the antibody is unknown, but LHRH in ever, the present study shows that long-term the median eminence or in the hypophysial suppression of LHRH and FSHRH is also portal blood could be bound to antibody possible by chronic treatment with antibody and lose biological activity before it reaches
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 10 July 2015. at 12:43 For personal use only. No other uses without permission. . All rights reserved.
300
ARIMURA ETAL.
Endo • 1976 Vol 99 • No 1
FIG. 7. Anterior pituitary cells of a castrated rat (exp. 2, group 1) injected with anti-LHRH •y-globulin starting the day after castration. Hematoxylin and PAS stain. (x440).
the pituitary gland. In any case, the pituitary of rats which were actively or passively immunized with LHRH would be deprived of the influence of hypothalamic LHRH. In the first experiment, numerous castration cells were definitely observed in the pituitary glands of castrated rats given the control immunization and also in those actively immunized with LHRH. It is clear that castration cells would have been present before antibody to LHRH was formed, since the immunizations with LHRH were started two weeks after the castration. However, the finding of scarce immature and mature secretory granules in the castration cells of rats which generated antibody to LHRH following active immunization, probably due to the neutralization of endogenous LHRH by antibody to LHRH, indicates an interruption of normal stimulation by hypothalamic LHRH, which probably maintains synthesis and secretion
of gonadotrophins. This assumption is supported by assay results in which immunization with LHRH decreased both LH and FSH serum levels to those found in intact rats as early as eight weeks after the first immunization. Zambrano et al. (20) reported that the incubation of pituitary glands with LHRH in the culture medium for three and six hours led to formation of typical "signet ring" castration cells, and that the appearance of such signet ring cells was prevented by adding puromycin to the incubation medium together with LHRH. They interpreted these findings to mean that castration cells are stimulated to develop in the gland by an elevation in hypothalamic LHRH after castration. Consequently, the complete absence of castration cells after the injection of antibody against LHRH, starting on the day after castration, may be due to a neutralization of most of the
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 10 July 2015. at 12:43 For personal use only. No other uses without permission. . All rights reserved.
LHRH AND PITUITARY GONADOTROPHS
301
FIG. 8. An example of castration cells of a castrated rat (exp. 2., group 2) injected with normal sheep -/-globulin as a control. Dilated cistemae of the endoplasmic reticulum, a large Golgi area, and abundant secretory granules are seen. (x8,100).
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 10 July 2015. at 12:43 For personal use only. No other uses without permission. . All rights reserved.
302
ARIMURA ETAL.
Endo • L976 Vol 99 • Xo 1
FIG. 9. An example of an atrophic FSH-gonadotroph in the anterior pituitary gland of a castrated rat (esp. 2, group 1) injected with anti-LHRH y-globulin, beginning the day after castration. (x8,100).
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 10 July 2015. at 12:43 For personal use only. No other uses without permission. . All rights reserved.
LHRH AND PITUITARY GONADOTROPHS endogenous LHRH in these animals. Our assay results in the passively immunized castrated rats, in which both LH and FSH levels were decreased after the first injection of anti-LHRH y-globulin, and remained low thereafter, are in good agreement with the morphological observations. In the present study, the castration cells resembled, in ultrastructural aspects, the FSH cells described by Kurosumi (4). The LH gonadotrophs of Kurosumi were rarely recognized in both the actively and passively immunized rats. It is considered likely that the LH cells were suppressed by these immunization procedures. However, they seem not to have contributed significantly to the development of castration cells, in agreement with the recent findings of Kurosumi et al. (7). Acknowledgments The authors wish to thank Mr. P. Taylor, Mrs. Joan Gauthier, and Mrs. Deborah Pierson for their excellent technical help, Dr. A. J. Kastin and Ms. G. A. Farley for their help in preparing the manuscript, and Dr. G. D. Nisvvender and NIAMDD Rat Pituitary Hormone Program for the reagents used in radioimmunoassay for rat LH and FSH.
References 1. Arimura, A., L. Debeljuk, and A. V. Schally, Endocrinology 95: 323, 1974. 2. Gunner Johanson, K. N., B. L. Curries, K. Folkers, and C. Y. Bowers, Biochem Biophys Res Commun 50: 8, 1973.
303
3. Fawcett, C. P., A. E. Beezley, and J. E. Wheaton, Endocrinology 96: 1311, 1975. 4. Kurosumi, K.,Arch Histoljap 29: 329, 1968. 5. Rennels, E. G., E. M. Bogdanove, A. Arimura, M. Sato, and A. V. Schally,Endocrinology 88: 1318, 1971. 6. Moriarty, G. C., Endocrinology 97: 1215, 1975. 7. Kurosumi, K., Y. Kawarai, Y. Yukitake, and K. Inoue, Proc 13th Gun ma Symposium on Endocrinology, Biology of Reproduction and its Hormonal Control, 1975, Abstract no III-4. 8. Arimura, A., H. Sato, D. H. Coy, R. B. Worobec, A. V. Schally, N. Yanaihara, T. Hashimoto, C. Yanaihara, and N. Sukuta, Ada Endocrinol (Kbh) 2: 222, 1975. 9. de la Cruz, A., A. Arimura, K. G. de la Cruz, and A. V. Schally, Endocrinology 98: 490, 1976. 10. Niswender, G. D., A. R. Midgley, S. E. Monroe, and L. E. Reichert, Proc Soc Exp Biol Med 128: 807, 1968. 11. Daane, T. A., and A. F. Parlow, Endocrinology 88: 653, 1971. 12. Duddleson, W. G., A. R. Midgley, Jr., and G. D. Niswender, Comput Biomed Res 5: 205, 1972. 13. Arimura, A., H. Sato, T. Kumasaka, R. B. Worobec, L. Debeljuk, J. D. Dunn, and A. V. Schally, Endocrinology 93: 1092, 1973. 14. Vaitukaitis, J., J. B. Robbins, E. Nieschlag and G. T. Ross, 7 Clin Endocrinol Metab 33: 988, 1971. 15. Weir, D. M., Handbook of Experimental Biology, F. A. Davis Co., Philadelphia, 1967, p. 4. 16. Shiino, M., A. Arimura, A. V. Schally, and E. G. Rennels.Z ZellforschMikroskAnat 128:152,1972. 17. Fraser, H. M., S. L. Jeffcoate, A. Gunn, and D. T. Holland, J Endocrinol 64: 191, 1975. 18. Fraser, H. M., A. Gunn, S. L. Jeffcoate, and D. T. Holland,/ Endocrinol 63: 399, 1974. 19. Koch, Y., P. Chobsieng, U. Zor, M. Fridkin, and H. R. Lindner, Biochem Biophys Res Commun 55: 623, 1973. 20. Zambrano, D., S. Cuerdo-Rocha, and I. Bergmann, Cell Tiss Res 150: 179, 1974.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 10 July 2015. at 12:43 For personal use only. No other uses without permission. . All rights reserved.
