Effects of Cervical Stimulation and Anti-LH Releasing Hormone Serum on LH Releasing Hormone Content in the Hypothalamus MICHIO TAKAHASHI, J. JOE FORD, KOJ1 YOSHINAGA, AND ROY O. GREEP Laboratory of Human Reproduction and Reproductive Biology and Department of Anatomy, Harvard Medical School, Boston, Massachusetts 02115 abolished by iv injection of 2 ml LHRH antiserum at the time of cervical stimulation, but pseudopregnancy was still induced and diestrus prolonged 4 days longer than in the control group. Progestin concentrations in ovarian vein blood were comparable between anti-LHRH-treated and control groups. These results suggest that the LH released by cervical stimulation is dispensable for the induction of pseudopregnancy. The marked elevation of LHRH in the hypothalamus observed immediately after cervical stimulation must be related to the regulatory mechanism of the characteristic secretory pattern of LH during the period of pseudopregnancy. (Endocrinology 96: 453, 1975)
ABSTRACT. Hypothalamic content of LHRH was radioimmunoassayed after cervical stimulation on the morning of day of estrus. LHRH content rose 3-fold by 120 min after the stimulation, reaching a level equivalent to that of day 5 of pseudopregnancy. The increase was not steady as there were two early periods when the content did not increase. Measurement of pituitary LH content revealed a marked increase followed by a sudden decrease which corresponded respectively to the two periods when LHRH levels failed to rise. These changes in pituitary LH level suggest that both the synthesis and release of LH in the pituitary are promoted by the action of LHRH released from the hypothalamus. LH changes in the pituitary were
P
SEUDOPREGNANCY is one of the most remarkable phenomena in the reproductive activities of female rats. After induction of pseudopregnancy, rats secrete a level of progesterone (1,2) comparable to that of animals which have a functional luteal phase in the estrous cycle. Analysis of the mechanism of the induction of pseudopregancy is expected to provide information important to understanding the basic difference between the estrous cycle of rats and those animals with active corpora lutea which are more common among mammalian species. Hormonal changes following cervical stimulation have been intensively analyzed (3-6) since this exteroceptive stimulus is translated eventually into the hormonal maintenance of the pseudopregnant state. Utilizing synthetic LHRH (7,8) we have shown that specific antibodies generated Received June 17, 1974. For reprints: Koji Yoshinaga, Ph.D., LHRRB, Harvard Medical School, 45 Shattuck Street, Boston, Mass. 02115.
against LHRH can be used to neutralize endogenous LHRH, and to measure LHRH by means of radioimmunoassay (9). In the present study, we examined the effect of anti-LHRH serum on the induction of pseudopregnancy, and also investigated changes in hypothalamic LHRH content after cervical stimulation. Materials and Methods Animals and collection of samples. Adult female Charles River CD rats kept in 14 hr light (0500 to 1900 hr) and 10 hours darkness were used. Vaginal smears were examined daily for at least two cycles and those rats showing regular 4-day cycles were used in this experiment. The animals were injected intravenously with 2 ml of either normal rabbit serum (35 rats, the control group) or anti-LHRH serum (39 rats, the anti-LHRH treated group) at 0955 hr on day of estrus immediately before cervical stimulation. The uterine cervix was stimulated for 2 min with a glass rod at 1000 hr of estrous day. Five, 15, 45 and 120 min after cervical stimulation the rats were sacrificed by decapitation and hypothalami and pituitaries were collected from 5 rats both in the control and anti-LHRH treated
453
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454
TAKAHASHI, FORD, YOSHINAGA AND GREEP
groups. The control values before the treatment (0 min) were obtained from 5 intact estrous rats at 1000 hr. Five days after cervical stimulation, ovarian vein blood was collected from 5 rats of both control and anti-LHRH-treated groups. Blood was collected for 10 minutes under Nembutal (Abbott Lab., 40 mg/kg body weight, ip) anesthesia and heparin (sodium heparin (1 U/g body weight, iv) was used as an anticoagulant (10). After the collection of ovarian vein blood, the rat was decapitated and the hypothalamus and pituitary were collected. Smears were taken from 10 other rats of the control and 8 rats of the anti-LHRH treated group after cervical stimulation, and the length of diestrous smears was recorded. The generation of anti-LHRH serum and the measurement of LHRH. The anti-LHRH sera used for the radioimmunoassay and passive immunization were generated in rabbits in our laboratory using LHRH (TAP-023, Takeda Chemical Industries, Japan) conjugated with bovine serum albumin as antigen. The amide terminal of LHRH was hydrolyzed before conjugation. Two ml of this anti-LHRH serum blocked ovulation in cycling rats when administered around the critical period of proestrus. The characterization of the serum was previously reported in detail (9). The radioimmunoassay of LHRH was carried out according to the method described earlier (9). The hypothalamic area was dissected out immediately after decapitation of the animal and homogenized in 1 ml 0.2M ice chilled acetic acid. The homogenate was stored for 24 hr at 4 C and centrifuged at 20,000 x g for 1 hr. DupliFIG. 1. Hypothalamic content of LHRH after cervical stimulation. Two ml normal rabbit serum or antiLHRH serum was given immediately before the stimulation applied at 1000 0 5 IS 45 120 min 5 day hr on estrous day. estrus 10 30 60 1000 hr The value at 0 min stimulation time after stimulation was obtained from 5 intact estrous rats at 1000 hr. Each circle signifies the mean of samples taken from 5 rats and vertical line represents standard error of the mean.
LHRH ng/hypolhalamus
Endo • 1975 Vol 96 • No 2
cate aliquots of 200 /xl supernatant were neutralized with 0.2M NH4OH and incubated with the antisera and 125I-LHRH for 4 hours at 23 C and subsequent 44 hr at 4 C. After incubation antigen-antibody complex was separated by precipitation of unreacted labelled LHRH with Dextran T70 (Pharmacia Fine Chemicals, Sweden)-coated charcoal. The measurement of LH in the pituitary and progestin in ovarian vein blood. Pituitary samples were homogenized in neutral phosphate buffered saline and prepared according to the method described by Bast and Melampy (11) for radioimmunoassay of LH. LH was analyzed using NIAMD kit and LH level was expressed as concentration (/ig/mg anterior pituitary wet weight) in terms of NIAMD-LH-RP-1 standard. Progestin concentration in ovarian vein blood was measured by radioimmunoassay (12). Onetenth ml of plasma diluted with 0.9 ml water was extracted with 5 ml petroleum ether and the extract subjected to a Sephadex LH-20 chromatography. The fractions of progesterone and 20a-hydroxypregn-4-en-3-one (20a-OH-P) after conversion to progesterone by chromic acid oxidation, served for the assay.
Results
Hypothalamic content of LHRH (Fig. 1). In the control group, immunoassayable LHRH had increased by 300% 2 hr after cervical stimulation. The level observed at this time was comparable to that at day 5 after the stimulation. First, however, the content decreased significantly (p < 0.05) from 0 to 5 min and showed little change between 15 and 45 min. Similar trends were observed in the anti-LHRH-treated group. Thus, cervical stimulation was shown to cause a rapid and remarkable elevation in hypothalamic content of LHRH and treatment with anti-LHRH did not alter this response. Pituitary LH levels (Fig. 2). In the control group, immunoassayable LH concentration in the pituitary increased to a remarkably high level at 5 min and decreased sharply at 15 min after cervical stimulation. It then
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LHRH AFTER CERVICAL STIMULATION FIG. 2. Pituitary LH levels atter cervical NRS treated 30 stimulation. LH values are presented anti-LHRH treated in terms of NIAMD1 LH-RP-1. Two ml 20 normal rabbit serum or anti-LHRH serum was given immediately before the 10 stimulation applied at lOOO.hr on estrous day. The value at 0 min was obtained 15 45 120 0 from 5 intact estrous stim. estrus rats. Each bar sigtime in minutes tooohr nifies the mean of samples taken from 5 rats and vertical line at end of each bar represents standard error of mean.
gradually increased again until 120 min after the stimulation. In the anti-LHRHtreated group, however, no significant changes were observed in LH concentration. Induction of pseudopregnancy (Table 1) and progestin level (Table 2). All of the rats receiving cervical stimulation showed a long diestrus regardless of their subsequent treatment. The length of diestrus in the anti-LHRH treated group was significantly longer than that of the control group by approximately 4 days. The treatment of normally cycling rats with antiLHRH on the day of estrus extended diestrous length by one day but never induced pseudopregnancy (Table 1). Both progesterone and 20a-OH-P levels in ovarian vein blood in the anti-LHRH treated rats were exactly the same as those in the control pseudopregnant rats (Table 2). Discussion It is well documented that cervical stimulation evokes a significant elevation of peripheral LH level (4-6,13,14). According to Wuttke's report (6), the earliest rise in peripheral LH is detected 10 min after stimulation and elevated LH concentrations are found during the next 2 hr with a
455
TABLE 1. Influence of anti-LHRH serum on duration of diestrus after cervical stimulation Treatment
No. of rats
Length of diestrus (day, mean ± SE)
Normal rabbit serum + cervical stim.
10
12.3 ± 0.4**
Anti-LHRH serum + cervical stim.
8
16.2 ± 0.5**
Anti-LHRH serum .
6
3.0 ± 0
* Two ml normal rabbit serum or anti-LHRH serum was given iv at 0955 hr and the cervical stimulation was applied at 1000 hr on estrus. ** Significantly different, p < 0.01.
peak value around 30 to 60 min after stimulation. The sharp decrease in pituitary LH content observed from 5 to 15 min in the control group in our study is considered to be the result of LH release. Thus, the two periods (0 to 5 and 15 to 45 min) during which LHRH content did not increase, appear to reflect the release of LHRH and correspond well with the sharp rise of pituitary and peripheral LH levels. Although the anti-LHRH treatment did not alter the pattern of hypothalamic LHRH, changes in content of pituitary LH were completely absent. This absence is attributed to the neutralization of the released LHRH by antibody while LHRH was passing through the portal vessels. At 5 min after the cervical stimulation, hypothalamic LHRH content in the animals passively immunized with anti-LHRH serum was higher than that in the control animals, TABLE 2. Progestin concentration in ovarian vein blood 5 days after cervical stimulation Progestin (/xg/ml plasma)
Treatment*
No. of rats
Progesterone
20aHydroxypregn4-en-3-one
Normal rabbit serum
5
1.54 ± 0.24**
0.78 ± 0.13
Anti-LHRH serum
5
1.60 ± 0.24
0.77 ± 0.05
* Two ml normal rabbit serum or anti-LHRH serum was given iv at 0955 hr on estrus. ** Mean ± SE.
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456
TAKAHASHI, FORD, YOSHINAGA AND GREEP
though the difference is not significant. Therefore, there was not enough antiLHRH taken up in the hypothalamus to interfere with the assay, since contamination of anti-LHRH in the hypothalamic extract would cause underestimation of the real LHRH value. Successful induction of pseudopregnancy and activation of corpora lutea to secrete progesterone in the anti-LHRH treated animals by cervical stimulation indicates that the LH release ocurring immediately after cervical stimulation is dispensable for the induction of pseudopregnancy. This is in accord with the view of Spies and Niswender (4) who deny the role of LH for the induction of pseudopregnancy. They demonstrated that induction of pseudopregnancy failed in pelvic neurectomized rats in which an intact pattern of LH rise was still induced after cervical stimulation. McLean and Nikitovitch-Winer (15) reported that antiLH treatment at the time of cervical stimulation prevented induction of pseudopregnancy and that the estrous cycle is resumed within 4-5 days. The main difference between anti-LH and -LHRH treatments is that the former is assumed to preclude all the circulating LH for a certain period of time, while the latter prevents only the secretion of LH leaving the circulating LH intact. Therefore, it can be said that the LH released in response to cervical stimulation is dispensable, but LH at basal levels is still indispensable during the course of the induction of pseudopregnancy. More work is required before elucidation of the physiological significance of this reflexly released LH, but extension of the duration of pseudopregnancy in the anti-LHRHtreated rats in our study apparently signifies that this LH has some role in fixing the duration of pseudopregnancy. One possible explanation is that this LH together with simultaneously released FSH may cause growth of the following generation of follicles from the initial stage of pseudo-
Endo i 1975 Vol 96 . No 2
pregnancy. Therefore, the exclusion of this release of LH and FSH results in the delay of onset of follicular growth and affects the initiation of the cycle which follows pseudopregnancy. Recently, Neill's group (16-18) found that cervical stimulation elicits daily surges of prolactin and that these surges are induced and maintained in ovariectomized rats. These findings introduced the concept of "hypothalamic pseudopregnancy" wherein daily surges of prolactin proceed without the support of ovarian steroids. During pseudopregnancy LH is secreted at basal levels since anti-LH treatment suppresses decidual reaction (15,19) and progestin secretion (15). This view is endorsed by the results of measuring peripheral LH by radioimmunoassay during the period of pseudopregnancy (11). This characteristic secretory pattern of LH is also considered to be regulated by the CNS independently of ovarian steroids, since anti-LH treatment, except at the time of cervical stimulation, apparently depresses progesterone secretion but never induces the recurrence of the estrous cycle (15). The remarkable elevation of immunoassayable LHRH in the hypothalamus observed after cervical stimulation can thus be viewed as one of the manifestations of "hypothalamic pseudopregnancy." Acknowledgments The authors wish to thank Dr. M. Fujino of Takeda Chemical Industries, Japan, for the generous gift of synthetic LHRH. We are indebted to the Rat Pituitary Hormone Distributing Program, NIAMD, NIH for the LH radioimmunoassay kit. This work was supported by the Ford Foundation and NIH Grants HD-06047, HD-03736 and HD53973.
References 1. Fajer, A. B., and C. A. Barraclough, Endocrinology 81: 617, 1967. 2. Hashimoto, I., D. M. Henricks, L. L. Anderson, and R. M. Melampy, Endocrinology 82: 333, 1968. 3. Wuttke, W., and J. Meites, Proc Soc Exp Biol Med 125: 648, 1970.
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LHRH AFTER CERVICAL STIMULATION 4. Spies, H. G., and G. D. Niswender, Endocrinology 88: 937, 1971. 5. Bishop, W., R. Orias, C. P. Fawcett, L. Krulich, and S. M. McCann, Proc Soc Exp Biol Med 137: 1411, 1971. 6. Wuttke, W., Endocrinology 92: 1280, 1973. 7. Matsuo, H., A. Arimura, R. M. G. Nair, and A. V. Schally, Biochem Biophys Res Commun 45: 822, 1971. 8. Geiger, R., W. Konig, H. Wissman, K. Geisen, and F. Enzmann, Biochem Biophys Res Commun 45: 767, 1971. 9. Makino, T., M. Takahashi, K. Yoshinaga, and R. O. Greep, Contraception 8: 133, 1973. 10. Takahashi, M., and Y. Suzuki, Endocrinol Japon 18: 5, 1971. 11. Bast, J. D., and R. M. Melampy, Endocrinology 86: 506, 1970.
457
12. Abraham, G. E., K. Hopper, D. Tulchinsky, R. S. Swerdloff, and W. D. Odell, Anal Lett 4: 325, 1971. 13. Taleisnik, S., S. Caligaris, and J. J. Astrada, Endocrinology 79: 49, 1966. 14. Blake, C. A., and C. H. Sawyer, Neuroendocrinology 10: 358, 1970. 15. McLean, B. K., and M. B, Nikitovitch-Winer, Endocrinology 93: 316, 1973. 16. Freeman, M. E., and J. D. Neill, Endocrinology 90: 1292, 1972. 17. , M. S. Smith, S. J. Nazian, and J. D. Neill, Endocrinology 94: 875, 1974. 18. Smith, M. S., and J. D. Neill, Fed Proc 33: 238, 1974. 19. Chang, C. C , S. Badawy, and K. A. Laurence, Fertil Steril 22: 663, 1971.
Second Annual NICHD Workshop on the Testis The workshop will be held at the University of North Carolina, Chapel Hill, North Carolina on April 2-4, 1975. The topics this year will be "Steroid Synthesis and Metabolism in the Tubule"; Hormonal Regulation of Seminiferous Tubular Function." Presentation of papers and discussion will be confined generally to the invited participants, but there will be room for 50 observers. If you are interested in attending, please indicate your research activities in the field. For information, write to Dr. Richard J. Sherins, National Institute of Child Health and Human Development, Building 10, Room 12N-208, Bethesda, Maryland 20014.
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ABSTRACT. Hypothalamic content of LHRH was radioimmunoassayed after cervical stimulation on the morning of day of estrus. LHRH content rose 3-fold by 120 min after the stimulation, reaching a level equivalent to that of day 5 of pseudopregnancy. The increase was not steady as there were two early periods when the content did not increase. Measurement of pituitary LH content revealed a marked increase followed by a sudden decrease which corresponded respectively to the two periods when LHRH levels failed to rise. These changes in pituitary LH level suggest that both the synthesis and release of LH in the pituitary are promoted by the action of LHRH released from the hypothalamus. LH changes in the pituitary were
P
SEUDOPREGNANCY is one of the most remarkable phenomena in the reproductive activities of female rats. After induction of pseudopregnancy, rats secrete a level of progesterone (1,2) comparable to that of animals which have a functional luteal phase in the estrous cycle. Analysis of the mechanism of the induction of pseudopregancy is expected to provide information important to understanding the basic difference between the estrous cycle of rats and those animals with active corpora lutea which are more common among mammalian species. Hormonal changes following cervical stimulation have been intensively analyzed (3-6) since this exteroceptive stimulus is translated eventually into the hormonal maintenance of the pseudopregnant state. Utilizing synthetic LHRH (7,8) we have shown that specific antibodies generated Received June 17, 1974. For reprints: Koji Yoshinaga, Ph.D., LHRRB, Harvard Medical School, 45 Shattuck Street, Boston, Mass. 02115.
against LHRH can be used to neutralize endogenous LHRH, and to measure LHRH by means of radioimmunoassay (9). In the present study, we examined the effect of anti-LHRH serum on the induction of pseudopregnancy, and also investigated changes in hypothalamic LHRH content after cervical stimulation. Materials and Methods Animals and collection of samples. Adult female Charles River CD rats kept in 14 hr light (0500 to 1900 hr) and 10 hours darkness were used. Vaginal smears were examined daily for at least two cycles and those rats showing regular 4-day cycles were used in this experiment. The animals were injected intravenously with 2 ml of either normal rabbit serum (35 rats, the control group) or anti-LHRH serum (39 rats, the anti-LHRH treated group) at 0955 hr on day of estrus immediately before cervical stimulation. The uterine cervix was stimulated for 2 min with a glass rod at 1000 hr of estrous day. Five, 15, 45 and 120 min after cervical stimulation the rats were sacrificed by decapitation and hypothalami and pituitaries were collected from 5 rats both in the control and anti-LHRH treated
453
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454
TAKAHASHI, FORD, YOSHINAGA AND GREEP
groups. The control values before the treatment (0 min) were obtained from 5 intact estrous rats at 1000 hr. Five days after cervical stimulation, ovarian vein blood was collected from 5 rats of both control and anti-LHRH-treated groups. Blood was collected for 10 minutes under Nembutal (Abbott Lab., 40 mg/kg body weight, ip) anesthesia and heparin (sodium heparin (1 U/g body weight, iv) was used as an anticoagulant (10). After the collection of ovarian vein blood, the rat was decapitated and the hypothalamus and pituitary were collected. Smears were taken from 10 other rats of the control and 8 rats of the anti-LHRH treated group after cervical stimulation, and the length of diestrous smears was recorded. The generation of anti-LHRH serum and the measurement of LHRH. The anti-LHRH sera used for the radioimmunoassay and passive immunization were generated in rabbits in our laboratory using LHRH (TAP-023, Takeda Chemical Industries, Japan) conjugated with bovine serum albumin as antigen. The amide terminal of LHRH was hydrolyzed before conjugation. Two ml of this anti-LHRH serum blocked ovulation in cycling rats when administered around the critical period of proestrus. The characterization of the serum was previously reported in detail (9). The radioimmunoassay of LHRH was carried out according to the method described earlier (9). The hypothalamic area was dissected out immediately after decapitation of the animal and homogenized in 1 ml 0.2M ice chilled acetic acid. The homogenate was stored for 24 hr at 4 C and centrifuged at 20,000 x g for 1 hr. DupliFIG. 1. Hypothalamic content of LHRH after cervical stimulation. Two ml normal rabbit serum or antiLHRH serum was given immediately before the stimulation applied at 1000 0 5 IS 45 120 min 5 day hr on estrous day. estrus 10 30 60 1000 hr The value at 0 min stimulation time after stimulation was obtained from 5 intact estrous rats at 1000 hr. Each circle signifies the mean of samples taken from 5 rats and vertical line represents standard error of the mean.
LHRH ng/hypolhalamus
Endo • 1975 Vol 96 • No 2
cate aliquots of 200 /xl supernatant were neutralized with 0.2M NH4OH and incubated with the antisera and 125I-LHRH for 4 hours at 23 C and subsequent 44 hr at 4 C. After incubation antigen-antibody complex was separated by precipitation of unreacted labelled LHRH with Dextran T70 (Pharmacia Fine Chemicals, Sweden)-coated charcoal. The measurement of LH in the pituitary and progestin in ovarian vein blood. Pituitary samples were homogenized in neutral phosphate buffered saline and prepared according to the method described by Bast and Melampy (11) for radioimmunoassay of LH. LH was analyzed using NIAMD kit and LH level was expressed as concentration (/ig/mg anterior pituitary wet weight) in terms of NIAMD-LH-RP-1 standard. Progestin concentration in ovarian vein blood was measured by radioimmunoassay (12). Onetenth ml of plasma diluted with 0.9 ml water was extracted with 5 ml petroleum ether and the extract subjected to a Sephadex LH-20 chromatography. The fractions of progesterone and 20a-hydroxypregn-4-en-3-one (20a-OH-P) after conversion to progesterone by chromic acid oxidation, served for the assay.
Results
Hypothalamic content of LHRH (Fig. 1). In the control group, immunoassayable LHRH had increased by 300% 2 hr after cervical stimulation. The level observed at this time was comparable to that at day 5 after the stimulation. First, however, the content decreased significantly (p < 0.05) from 0 to 5 min and showed little change between 15 and 45 min. Similar trends were observed in the anti-LHRH-treated group. Thus, cervical stimulation was shown to cause a rapid and remarkable elevation in hypothalamic content of LHRH and treatment with anti-LHRH did not alter this response. Pituitary LH levels (Fig. 2). In the control group, immunoassayable LH concentration in the pituitary increased to a remarkably high level at 5 min and decreased sharply at 15 min after cervical stimulation. It then
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LHRH AFTER CERVICAL STIMULATION FIG. 2. Pituitary LH levels atter cervical NRS treated 30 stimulation. LH values are presented anti-LHRH treated in terms of NIAMD1 LH-RP-1. Two ml 20 normal rabbit serum or anti-LHRH serum was given immediately before the 10 stimulation applied at lOOO.hr on estrous day. The value at 0 min was obtained 15 45 120 0 from 5 intact estrous stim. estrus rats. Each bar sigtime in minutes tooohr nifies the mean of samples taken from 5 rats and vertical line at end of each bar represents standard error of mean.
gradually increased again until 120 min after the stimulation. In the anti-LHRHtreated group, however, no significant changes were observed in LH concentration. Induction of pseudopregnancy (Table 1) and progestin level (Table 2). All of the rats receiving cervical stimulation showed a long diestrus regardless of their subsequent treatment. The length of diestrus in the anti-LHRH treated group was significantly longer than that of the control group by approximately 4 days. The treatment of normally cycling rats with antiLHRH on the day of estrus extended diestrous length by one day but never induced pseudopregnancy (Table 1). Both progesterone and 20a-OH-P levels in ovarian vein blood in the anti-LHRH treated rats were exactly the same as those in the control pseudopregnant rats (Table 2). Discussion It is well documented that cervical stimulation evokes a significant elevation of peripheral LH level (4-6,13,14). According to Wuttke's report (6), the earliest rise in peripheral LH is detected 10 min after stimulation and elevated LH concentrations are found during the next 2 hr with a
455
TABLE 1. Influence of anti-LHRH serum on duration of diestrus after cervical stimulation Treatment
No. of rats
Length of diestrus (day, mean ± SE)
Normal rabbit serum + cervical stim.
10
12.3 ± 0.4**
Anti-LHRH serum + cervical stim.
8
16.2 ± 0.5**
Anti-LHRH serum .
6
3.0 ± 0
* Two ml normal rabbit serum or anti-LHRH serum was given iv at 0955 hr and the cervical stimulation was applied at 1000 hr on estrus. ** Significantly different, p < 0.01.
peak value around 30 to 60 min after stimulation. The sharp decrease in pituitary LH content observed from 5 to 15 min in the control group in our study is considered to be the result of LH release. Thus, the two periods (0 to 5 and 15 to 45 min) during which LHRH content did not increase, appear to reflect the release of LHRH and correspond well with the sharp rise of pituitary and peripheral LH levels. Although the anti-LHRH treatment did not alter the pattern of hypothalamic LHRH, changes in content of pituitary LH were completely absent. This absence is attributed to the neutralization of the released LHRH by antibody while LHRH was passing through the portal vessels. At 5 min after the cervical stimulation, hypothalamic LHRH content in the animals passively immunized with anti-LHRH serum was higher than that in the control animals, TABLE 2. Progestin concentration in ovarian vein blood 5 days after cervical stimulation Progestin (/xg/ml plasma)
Treatment*
No. of rats
Progesterone
20aHydroxypregn4-en-3-one
Normal rabbit serum
5
1.54 ± 0.24**
0.78 ± 0.13
Anti-LHRH serum
5
1.60 ± 0.24
0.77 ± 0.05
* Two ml normal rabbit serum or anti-LHRH serum was given iv at 0955 hr on estrus. ** Mean ± SE.
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456
TAKAHASHI, FORD, YOSHINAGA AND GREEP
though the difference is not significant. Therefore, there was not enough antiLHRH taken up in the hypothalamus to interfere with the assay, since contamination of anti-LHRH in the hypothalamic extract would cause underestimation of the real LHRH value. Successful induction of pseudopregnancy and activation of corpora lutea to secrete progesterone in the anti-LHRH treated animals by cervical stimulation indicates that the LH release ocurring immediately after cervical stimulation is dispensable for the induction of pseudopregnancy. This is in accord with the view of Spies and Niswender (4) who deny the role of LH for the induction of pseudopregnancy. They demonstrated that induction of pseudopregnancy failed in pelvic neurectomized rats in which an intact pattern of LH rise was still induced after cervical stimulation. McLean and Nikitovitch-Winer (15) reported that antiLH treatment at the time of cervical stimulation prevented induction of pseudopregnancy and that the estrous cycle is resumed within 4-5 days. The main difference between anti-LH and -LHRH treatments is that the former is assumed to preclude all the circulating LH for a certain period of time, while the latter prevents only the secretion of LH leaving the circulating LH intact. Therefore, it can be said that the LH released in response to cervical stimulation is dispensable, but LH at basal levels is still indispensable during the course of the induction of pseudopregnancy. More work is required before elucidation of the physiological significance of this reflexly released LH, but extension of the duration of pseudopregnancy in the anti-LHRHtreated rats in our study apparently signifies that this LH has some role in fixing the duration of pseudopregnancy. One possible explanation is that this LH together with simultaneously released FSH may cause growth of the following generation of follicles from the initial stage of pseudo-
Endo i 1975 Vol 96 . No 2
pregnancy. Therefore, the exclusion of this release of LH and FSH results in the delay of onset of follicular growth and affects the initiation of the cycle which follows pseudopregnancy. Recently, Neill's group (16-18) found that cervical stimulation elicits daily surges of prolactin and that these surges are induced and maintained in ovariectomized rats. These findings introduced the concept of "hypothalamic pseudopregnancy" wherein daily surges of prolactin proceed without the support of ovarian steroids. During pseudopregnancy LH is secreted at basal levels since anti-LH treatment suppresses decidual reaction (15,19) and progestin secretion (15). This view is endorsed by the results of measuring peripheral LH by radioimmunoassay during the period of pseudopregnancy (11). This characteristic secretory pattern of LH is also considered to be regulated by the CNS independently of ovarian steroids, since anti-LH treatment, except at the time of cervical stimulation, apparently depresses progesterone secretion but never induces the recurrence of the estrous cycle (15). The remarkable elevation of immunoassayable LHRH in the hypothalamus observed after cervical stimulation can thus be viewed as one of the manifestations of "hypothalamic pseudopregnancy." Acknowledgments The authors wish to thank Dr. M. Fujino of Takeda Chemical Industries, Japan, for the generous gift of synthetic LHRH. We are indebted to the Rat Pituitary Hormone Distributing Program, NIAMD, NIH for the LH radioimmunoassay kit. This work was supported by the Ford Foundation and NIH Grants HD-06047, HD-03736 and HD53973.
References 1. Fajer, A. B., and C. A. Barraclough, Endocrinology 81: 617, 1967. 2. Hashimoto, I., D. M. Henricks, L. L. Anderson, and R. M. Melampy, Endocrinology 82: 333, 1968. 3. Wuttke, W., and J. Meites, Proc Soc Exp Biol Med 125: 648, 1970.
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LHRH AFTER CERVICAL STIMULATION 4. Spies, H. G., and G. D. Niswender, Endocrinology 88: 937, 1971. 5. Bishop, W., R. Orias, C. P. Fawcett, L. Krulich, and S. M. McCann, Proc Soc Exp Biol Med 137: 1411, 1971. 6. Wuttke, W., Endocrinology 92: 1280, 1973. 7. Matsuo, H., A. Arimura, R. M. G. Nair, and A. V. Schally, Biochem Biophys Res Commun 45: 822, 1971. 8. Geiger, R., W. Konig, H. Wissman, K. Geisen, and F. Enzmann, Biochem Biophys Res Commun 45: 767, 1971. 9. Makino, T., M. Takahashi, K. Yoshinaga, and R. O. Greep, Contraception 8: 133, 1973. 10. Takahashi, M., and Y. Suzuki, Endocrinol Japon 18: 5, 1971. 11. Bast, J. D., and R. M. Melampy, Endocrinology 86: 506, 1970.
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12. Abraham, G. E., K. Hopper, D. Tulchinsky, R. S. Swerdloff, and W. D. Odell, Anal Lett 4: 325, 1971. 13. Taleisnik, S., S. Caligaris, and J. J. Astrada, Endocrinology 79: 49, 1966. 14. Blake, C. A., and C. H. Sawyer, Neuroendocrinology 10: 358, 1970. 15. McLean, B. K., and M. B, Nikitovitch-Winer, Endocrinology 93: 316, 1973. 16. Freeman, M. E., and J. D. Neill, Endocrinology 90: 1292, 1972. 17. , M. S. Smith, S. J. Nazian, and J. D. Neill, Endocrinology 94: 875, 1974. 18. Smith, M. S., and J. D. Neill, Fed Proc 33: 238, 1974. 19. Chang, C. C , S. Badawy, and K. A. Laurence, Fertil Steril 22: 663, 1971.
Second Annual NICHD Workshop on the Testis The workshop will be held at the University of North Carolina, Chapel Hill, North Carolina on April 2-4, 1975. The topics this year will be "Steroid Synthesis and Metabolism in the Tubule"; Hormonal Regulation of Seminiferous Tubular Function." Presentation of papers and discussion will be confined generally to the invited participants, but there will be room for 50 observers. If you are interested in attending, please indicate your research activities in the field. For information, write to Dr. Richard J. Sherins, National Institute of Child Health and Human Development, Building 10, Room 12N-208, Bethesda, Maryland 20014.
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