0013-7227/79/1055-1162$02.00/0 Endocrinology Copyright © 1979 by The Endocrine Society
Vol. 105, No. 5 Printed in U.S.A.
Stimulation of Endogenous Follicle-Stimulating Hormone Release during Estrus by Exogenous Follicle-Stimulating Hormone or Luteinizing Hormone at Proestrus in the Phenobarbital-Blocked Rat* OLADAPO A. ASHIRU AND CHARLES A. BLAKEf Department of Anatomy, University of Nebraska Medical Center, Omaha, Nebraska 68105
FSH remained low during proestrus and estrus. After two iv injections of rat LH which caused plasma LH to rise more than 75-fold, plasma FSH rose during late proestrus and remained elevated during the morning of estrus. The stimulatory effects of plasma gonadotropins on FSH secretion were ovulation independent. The results indicate that in the phenobarbitalblocked rat, small selective elevations in plasma rat FSH or large selective elevations in plasma rat LH during proestrus, which are similar to FSH and LH levels observed in nonblocked rats, can stimulate pituitary FSH release during the time interval in which the second phase of FSH release would normally occur. The results also suggest that 1) the spontaneous LH surge and the associated first phase of increased plasma FSH are both involved in mediating the second phase of increased plasma FSH, and 2) the magnitude and/or duration of a rise in plasma gonadotropin during proestrus is important in effecting a threshold response which results in stimulating FSH release during estrus. (Endocrinology 105: 1162, 1979)
ABSTRACT. We previously reported that administration of LHRH on proestrus restores the two phases of increased plasma FSH during the periovulatory FSH surge in plasma in phenobarbital-blocked rats. In this study, we determined if the LHRHinduced restoration of the second phase of the FSH surge, which occurs during late proestrus and the morning of estrus, might be mediated by pituitary LH and/or FSH release during proestrus. In phenobarbital-blocked animals, rat FSH or LH was injected between 1500-1800 h on proestrus. Blood was collected by decapitation or through indwelling atrial cannulae during proestrus and estrus for RIA of FSH and LH. After sc or iv injection of FSH, plasma FSH but not LH was elevated during the late afternoon and evening of proestrus and during the morning of estrus. The FSH elevations during estrus reflect endogenous FSH release as hypophysectomy before the injection of FSH blocked the response. Subcutaneous or iv injection of rat LH did not elevate plasma FSH acutely. After sc injection of a dose of rat LH which caused plasma LH to rise about 5-fold, plasma
I
N THE cyclic rat, the periovulatory increases in serum FSH concentration can be divided into two phases. The first occurs during proestrus and is associated with the preovulatory increase in serum LH concentration. The second phase starts during late proestrus and continues into estrus. It is not associated with elevated serum LH (1-6). We have recently reported that administration of an ovulation-blocking dose of sodium phenobarbital during the early afternoon of proestrus completely blocks the proestrous LH surge and both phases of increased serum FSH (6). In the phenobarbital-blocked rat, administration of LHRH during proestrus not only restored the proestrous increases in serum LH and FSH (6-8) but Received December 15, 1978. * This work was supported by grants from the NIH (HD-11011 and HD-07097), the NSF (RIAS-SER 77-06922), and the Population Council (Biomedical Fellowship to O.A.A.). A portion of this study has been published in abstract form (Ashiru, O. A., and C. A. Blake, Physiologist 21: 3, 1978). f To whom all correspondence and requests for reprints should be addressed.
caused a virtually perfect simulation of the second phase of increased serum FSH as well (6). There is evidence to suggest that LHRH-induced restoration of the second phase of FSH release may be mediated by pituitary LH and/or FSH release during proestrus. Injection of an ovulation-blocking dose of pentobarbital at proestrus is highly effective in blocking the proestrous LH surge. However, it causes only a temporary and partial blockade of the first phase of FSH release. Serum FSH rises during the late afternoon to early evening of proestrus. Thereafter, the serum FSH rises to reach levels during estrus which approach values observed during the spontaneous increase in serum FSH during estrus (Blake, C. A., unpublished) (2, 9). These observations suggest that an increase in serum FSH concentration during proestrus may be involved in causing pituitary FSH release during estrus. Chappel and Barraclough (9) have demonstrated that the iv injection of highly purified rat LH during the afternoon of proestrus causes plasma FSH to rise within 3 h in the pentobarbital-treated rat. As pentobarbital was effective in blocking the first phase of increased plasma
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GONADOTROPIN STIMULATION OF FSH RELEASE
FSH during that 3-h period, their results clearly indicate that elevations in rat plasma LH during proestrus can cause the release of pituitary FSH during proestrus. It has yet to be demonstrated if elevations in plasma LH during proestrus in the absence of an increase in plasma FSH will cause a second phase of FSH release during estrus. In this study, we have used the phenobarbital-blocked proestrous rat as our model to determine if the second phase of increased FSH might be caused by pituitary LH and/or FSH release during proestrus. Materials and Methods Animals Virgin female Sprague-Dawley rats (Simonsen Lab., Gilroy, 7 CA) were kept in a room with the light on from 0500-1900 h daily for 26 or more days before daily vaginal lavages were taken. Rats which exhibited two or more consecutive 4-day • estrous cycles were used (208-295 g BW).
v
Effects of exogenous gonadotropins at proestrus on serum FSH and LH during proestrus and estrus in rats sacrificed by guillotine
Control rats were sacrificed by decapitation at 1400,1800, or * 2200 h on proestrus or at 0200, 0600, or 1000 h on estrus for collection of trunk blood and subsequent RIA of serum FSH and LH. Additional rats were injected ip with phenobarbital (100 mg/ kg BW) at 1200-1400 h on proestrus and divided into the - following treatment groups: 1) no additional treatment, 2) sc injection of 5 jug rat FSH (NIAMDD-rat FSH-I-3)1 at 1700 h on proestrus, and 3) sc injection of 50 /xg bovine LH (NIH-LH» BIO)2 at 1500 and 1600 h on proestrus. Hormones were dissolved in 0.4 ml saline; 0.2 ml was injected sc in each side in the ilio-inguinal region. Rats were decapitated for collection of trunk blood at 1800 or 2200 h on proestrus or * at 0200, 0600, or 1000 h on estrus. The Fallopian tubes were checked for the presence of ova in the rats sacrificed at 0600 or 1000 h on estrus. Effects of exogenous gonadotropins at proestrus on plasma FSH and LH during proestrus and estrus in serially bled rats Rats were injected ip with phenobarbital (100 mg/kg BW) at 1200-1300 h on proestrus. A single polyvinyl cannula was then inserted into the right atrium of the heart, as previously de* scribed (10), using ether as an anesthetic when needed. Rats were divided into the following treatment groups: 1) no additional treatment, 2) sc injection of 5 or 7.5 jug rat FSH at 1700 _> h on proestrus, 3) iv injection of 2.5, 5, or 7.5 jug rat FSH at 1700 h on proestrus, 4) sc injection of 50 jug bovine LH at 1500 and '
1
NIAMDD-rat FSH-I-3 was reported to have a LH biological potency of less than 0.002 X NIH-LH-Sl and a FSH biological potency of 1502 x NIH-FSH-Sl. NIH-LH-B10 was reported to have a LH biological potency of 1.06 X NIH-LH-Sl and a FSH biological potency of less than 0.05 x NIHFSH-Sl.
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1600 h on proestrus, 5) sc injection of 250 jug bovine LH at 1500 and 1600 h on proestrus, 6) sc injection of 5 jug rat LH (NIAMDD-rat LH-I-4)3 at 1500 h on proestrus, and 7) iv injection of 2 jug rat LH at both 1600 and 1800 h on proestrus. When given iv, hormones were dissolved in 0.2 ml saline and injected through the cannula. The cannula was then flushed with an additional 0.6 ml saline. Rats were bled (0.7 ml) through the cannulae just before injection and at 4-h intervals from 1800 h on proestrus until 1000 h on estrus. An additional sample was collected at 5 min after all iv injections of hormone. Additional blood samples were collected after sc injection of both LH preparations. After each bleeding, blood was centrifuged at 4 C and plasma was stored frozen at -20 C until subsequent RIA of FSH and LH. The packed red and white blood cells were resuspended in an equal volume of heparinized saline (10 U/ml) and injected into the rats through the cannulae within 20 min after each bleeding. The Fallopian tubes were checked for the presence of ova after the 1000 h bleeding on estrus. Effects of hypophysectomy on the plasma FSH response to injection of rat FSH on proestrus Phenobarbital was administered at 1200-1300 h on proestrus. Rats were hypophysectomized by the method of Gay (11) at 1600-1650 h under supplemental ether anesthesia. Rats were then given one of the two following treatments: 1) sc injection of 5 jug rat FSH at 1700 h on proestrus or 2) iv injection of 1, 2, or 5 /xg rat FSH at 1700 h on proestrus. Rats were bled through the cannulae as described above. After the 1000 h bleeding on estrus, the Fallopian tubes were checked for the presence of ova and the sella turcica was examined to determine if hypophysectomy was complete. RIAs Rat FSH was measured with the materials and instructions supplied with the FSH assay kit distributed by NIAMDD. The antiserum was S6. LH was measured by the method of Niswender et al. (12), as previously described (13). This RIA system measures both bovine and rat LH (14). FSH is expressed as nanograms per ml serum or plasma in terms of NIAMDD-rat FSH-RP-1, which has a biological potency equivalent to 2.1 X NIH-FSH-Sl. LH is expressed in terms of NIAMDD-rat LHRP-1, which has a biological potency equivalent to 0.03 x NIHLH-Sl. A serum pool (150 /xl) was assayed for FSH in triplicate in each of five assays. The mean (±SE) serum FSH concentration for the 15 determinations was 570 ± 14 ng/ml. The coefficient of variation within assays ranged from 2-11% and between assays was 10%. The serum pool (10 jul) was assayed for LH in triplicate in each of three assays. The mean (±SE) serum LH concentration for the nine determinations was 1389 ± 24 ng/ ml. The coefficient of variation within assays ranged from 2-6% and between assays was 5%. Statistics Statistical comparisons of data were performed by one-way 3
NIAMDD-rat LH-I-4 was reported to have a LH biological potency of 1.0 x NIH-LH-Sl and a FSH biological potency of less than 0.04 x NIH-FSH-Sl.
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Endo • 1979 Vol 105 • No 5
ASHIRU AND BLAKE
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analysis of variance (for repeated measures) for separate groups of rats to determine changes over time. Comparison between groups was made by two-way analysis of variance (treatment and time periods). Subsequent Newman-Keuls tests were performed. P values less than 0.05 were considered significant, and significance was tested at the 0.05 and 0.01 levels.
Results Mean serum LH and FSH concentrations for control rats decapitated between 1400 h on proestrus and 1000 h on estrus are shown in Fig. 1. In agreement with previous studies (1-6), serum LH was elevated only during proestrus. Serum FSH rose (P < 0.01) approximately 3-fold by 1800 h on proestrus, decreased (P < 0.05) by 2200 h on proestrus, rose {P < 0.05) again by 0200 h on estrus and remained elevated (P < 0.01) at 0600 and 1000 h on estrus above values observed at 1400 h on proestrus. The administration of phenobarbital between 12001400 h on proestrus blocked the LH surge (not illustrated; serum LH concentration ranged from 27-37 ng/ml) and the two phases of increased serum FSH (Fig. 1). Similarly, phenobarbital blocked the plasma LH (not illustrated; plasma LH concentration in individual rats ranged from 28-36 ng/ml) and FSH (Fig. 1) rises in rats bled serially through indwelling atrial catheters. In phenobarbital-blocked rats, sc or iv injection of
control phen
highly purified rat FSH did not elevate radioimmunoassayable serum/plasma LH (not illustrated; serum/ plasma LH concentration in individual rats remained low, 27-34 ng/ml). Sc injection of 5 /tg rat FSH at 1700 h on proestrus caused a small but significant (P < 0.05) increase in radioimmunoassayable serum/plasma FSH by 1800 h on proestrus (Fig. 2). The FSH concentrations rose further (P < 0.01) by 2200 h on proestrus and remained elevated through 1000 h on estrus. A similar plasma FSH response was observed in two rats administered 7.5 jug FSH (Fig. 2). The elevations in plasma FSH observed during estrus after sc injection of rat FSH must be due primarily to measurement of endogenous FSH rather than to measurement of exogenous FSH. When rats were hypophysectomized shortly before sc injection of FSH on proestrus, plasma FSH concentrations were low during estrus (Fig. 2). In one hypophysectomized rat, plasma FSH peaked at 1800 h on proestrus, 1 h after the injection, and failed to fall appreciably during the next 4 h. In the other hypophysectomized rat, plasma FSH peaked at 2200 h on proestrus, 5 h after the injection. These observations suggest that the injected FSH entered the plasma over a prolonged period. The increment and pattern of plasma FSH in the hypophysectomized rats were similar to those observed during the first phase of FSH release in intact rats (Fig. 2 vs. Ref. 8). The elevations in plasma FSH concentrations observed during estrus after the iv injection of 2.5-7.5 fig rat FSH at 1700 h on proestrus (Fig. 3) must also be due primarily to the measurement of endogenous FSH rather than to
1000r
800-
600 E X 400 TIME
FIG. 1. Mean (±SE) serum LH and FSH concentrations for control rats during proestrus (P) and estrus (E) are plotted. Indicated times encompass the interval from 1400 h on P to 1000 h on E. In this and subsequent figures: • - - • and A- -A, serum values in blood collected by decapitation; • — • and A—A, plasma values in blood collected serially through atrial catheters. The numbers to the right of the mean plasma FSH values at 1000 h on E represent the number of rats bled. Those to the right of the mean serum FSH values at 0600 or 1000 h represent the numbers or range of numbers of rats decapitated at each time period from 1400 h on P to 1000 h on E. FSH and LH were measured on the same samples. Any SE with a value less than 10 has not been plotted. The administration of phenobarbital (phen; 100 mg/ kg BW) at 1200-1400 h on P blocked the entire periovulatory FSH surge.
200
18
22
02
06
10
-H4 TIME
FIG. 2. Effects of sc injection of FSH (rat 1-3) at 1700 h on proestrus (P) in phenobarbital-blocked rats on serum/plasma FSH during P and estrus (E). Serum/plasma LH levels did not rise. Plasma FSH values for two individual rats administered 7.5 fig are plotted. Plasma FSH values for two individual rats administered 5 /ig and hypophysectomized at 1600-1650 h on P are designated HYP. The Xs at 0600 and 1000 h indicate that plasma FSH was below the sensitivity of the assay on the volume of plasma assayed.
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GONADOTROPIN STIMULATION OF FSH RELEASE 1800
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2500 1
2000 1600
-L\
•
SO»>g/ln|«cl|i
A 2S0M0/ln|*ctlon
g 1500 c
1400
. 1000 500
1200 400
1000
I Si 200
800
600
TIME
FIG. 4. Effects of sc injection of bovine LH at 1500 and 1600 h on proestrus (P) in phenobarbital-blocked rats on serum/plasma FSH and LH during P and estrus (E). Plasma FSH values at 1500, 1530, and 1600 h on P in rats injected with 50 or 250 jLtg LH at 1500 h were similar and have been combined.
400-
200
18
22
02
06
10
TIME
FIG. 3. Effects of iv injection of FSH (rat 1-3) at 1700 h on proestrus (P) in phenobarbital-blocked rats on plasma FSH during P and estrus (E). Plasma LH levels did not rise. Plasma FSH values for two individual rats administered 7.5 jig are plotted. Four rats were hypophysectomized (HYP) at 1600-1650 h on P before the injection of FSH. The Xs at 0200-1000 h indicate that plasma FSH was below the sensitivity of the assay for the volume of plasma assayed.
the measurement of exogenous FSH. Plasma FSH concentrations were low during estrus when rats were hypophysectomized shortly before iv injection of 1 or 2 jug (Fig. 3) or 5 jug (five rats not illustrated) exogenous FSH at proestrus. Two sc injections of 50 jug bovine LH did not elevate radioimmunoassayable serum FSH during proestrus or estrus (Fig. 4). It did cause a serum LH pattern, presumably in terms of assayable bovine LH, similar to that observed during the spontaneous LH surge (Fig. 4 vs. Ref. 10 and Fig. 1). When the dosage was increased 5fold to two 250-jng injections, the plasma FSH level rose during estrus to levels which approached but did not reach control values during estrus (Fig. 4 vs. Fig. 1). Subcutaneous and iv injections of highly purified rat LH on proestrus to phenobarbital-blocked rats caused an elevation in plasma LH but not in plasma FSH by 1800 h on proestrus (Fig. 5). The sc injection at 1500 h caused a 5-fold elevation in plasma LH by 1800 h. Plasma FSH did not rise in these rats during proestrus or estrus. Intravenous injection of a smaller dose caused plasma LH to rise over 30-fold within 5 min. It then declined to approach basal levels within 115 min. A second identical injection caused a similar LH response. Although the
FIG. 5. Effects of injection of rat LH in phenobarbital-blocked rats on plasma FSH and LH during proestrus (P) and estrus (E).
peak LH concentrations were in the upper range of values observed during the spontaneous LH surge, the elevations in plasma LH from 1500-2200 h on proestrus were in the physiological range observed in nonblocked rats (Fig. 5 vs. Ref. 10). In these LH-injected rats, plasma FSH rose (P < 0.05) by 2200 h on proestrus and reached values during estrus which were similar to values observed in control rats during estrus (Fig. 5 vs. Fig. 1). The effects of the FSH and LH preparations and their different routes of administration on ovulation are shown in Table 1. The occurrence of ovulation and the stimulation of endogenous FSH release during estrus were
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ASHIRU AND BLAKE
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TABLE 1. Effects of sc or iv injection of gonadotropins on ovulation Hormone
Dose (/xg)
Route
No. of rats
No. of ovulating rats
Rat FSH Rat FSH Rat FSH Rat FSH Rat FSH Bovine LH Bovine LH RatLH
5 7.5 2.5 5 7.5 100 500 5 4
sc sc iv iv iv sc sc sc iv
10 2 4 4 2 17 5 3 4
0 0 0 2 2 17 5 0 4
RatLH
No. of ova/ovulating rat
1,2 2,3 27 >7 >7
dissociated. Rats administered 100 /xg bovine LH ovulated fully (seven or more ova), but serum FSH was not elevated during estrus. Rats administered rat LH iv and those administered rat FSH sc had elevated plasma FSH during estrus, but only those given LH ovulated. Discussion The results of this study clearly indicate that elevations in plasma LH or plasma FSH during proestrus can stimulate the release of pituitary FSH during estrus in the phenobarbital-blocked rat. A small prolonged elevation in rat plasma FSH during proestrus, similar to that observed during the first phase of FSH release in nonblocked rats, is sufficient to stimulate pituitary FSH release during the time when the second phase of FSH release would normally occur. Similarly, a large elevation in rat plasma LH during proestrus, similar to that observed during the proestrous LH surge in nonblocked rats, will stimulate pituitary FSH release during the time that the second phase of FSH release would normally occur. These results suggest that both the proestrous LH surge and the associated first phase of increased plasma FSH are involved in mediating the second phase of increased plasma FSH in the cyclic rat. As small elevations in plasma rat LH during proestrus are ineffective in elevating plasma FSH, it would appear that the magnitude and/or duration of a rise in plasma gonadotropins during proestrus, at least in the case of plasma LH, are important in effecting a threshold response which ultimately results in a stimulation of pituitary FSH release during estrus. Although the effects of iv bovine LH on stimulating a rise in plasma FSH were not tested, bovine LH is probably less effective than rat LH in this regard, as indicated by the results obtained in rats given bovine LH sc. Only when plasma LH was elevated above proestrous LH surge levels for an extended period of time (presumably assayable in terms of bovine LH) did plasma FSH rise during estrus to levels which simulated a second phase of plasma FSH. In terms of the relatively small elevation in rat plasma
Endo • 1979 Vol 105 • No 5
FSH which is stimulatory to FSH release compared to the higher elevations in rat plasma LH which are needed to stimulate a rise in plasma FSH, it would appear that FSH is more effective than LH in stimulating FSH secretion. However, in physiological terms, a simulated proestrous LH surge caused a near-perfect simulation of the second phase of FSH release. The plasma FSH response observed during late proestrus and estrus after physiological elevations of plasma FSH during proestrus was greater than that observed in controls. This enhancement of FSH secretion may be due to the FSH dosages and routes of administration employed. However, phenobarbital-blocked rats may have more pituitary FSH available for release during late proestrus than do controls. Pituitary FSH concentration fell by more than half from 1400-2200 h on proestrus in controls but did not decrease during this period in phenobarbital-blocked rats (Ashiru, O. A., and C. A. Blake, unpublished). It may be that once threshold levels of plasma LH and FSH are reached during proestrus in the normal rat, additional proestrous increases in plasma LH and FSH enhance FSH release during late proestrus and estrus, but this additional release would be limited by the amount of pituitary FSH available for release. There is evidence to suggest that once plasma FSH has reached threshold levels during proestrus to effect a stimulatory response on its own secretion during estrus, additional increases in plasma gonadotropins during proestrus will stimulate additional pituitary FSH release during estrus. In rats in which the proestrous increase in plasma FSH was partially suppressed with pentobarbital, plasma FSH at 1000 h on estrus was higher after sc injection of ovine LH or FSH on proestrus than after sc injection of saline (15). Thus, increases in ovine LH during proestrus superimposed on a lower than normal rise in plasma FSH during proestrus can apparently stimulate additional pituitary FSH release by 1000 h on estrus. Similarly, the increase in plasma FSH at 1000 h on estrus observed after the injection of ovine FSH on proestrus could be due to the additive stimulatory effects of exogenous and endogenous FSH during proestrus. Alternatively, the increased plasma FSH on estrus could be due to the measurement of ovine FSH which has entered the plasma compartment over a prolonged period, as rat FSH did in the present study. There is a substantial latency in the time of onset of pituitary FSH release in response to an increase in plasma LH or FSH during the afternoon of proestrus. In the pentobarbital-blocked rat, Chappel and Barraclough (9) observed an increase in plasma FSH at 3 but not at 2 h after iv injection of rat LH on proestrus. Our results are in agreement with theirs. An increase in plasma FSH was observed between 2-6 h after iv injection of rat LH. The difference in plasma FSH patterns after sc injection
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GONADOTROPIN STIMULATION OF FSH RELEASE of 5 /xg rat FSH in intact and hypophysectomized proestrous rats suggests that the latency of the FSH-positive feedback on its own secretion is between 1-5 h. The mechanism by which increases in plasma gonadotropins exert their stimulatory effect on FSH secretion is clearly independent of ovulation. In all cases in which exogenous LH stimulated FSH release during estrus, rats ovulated fully (seven or more ova). However, rats given 100 jug bovine LH ovulated fully, and serum FSH did not rise during estrus. Therefore, the LH threshold to stimulate pituitary FSH release would appear to be higher than that needed to induce ovulation. In contrast, increases in plasma FSH can stimulate pituitary FSH release without causing ovulation. It is not clear if FSH itself or if FSH and LH contamination in the FSH preparation caused partial ovulation in some of the rats given the higher doses of FSH iv. As FSH injection did not elevate radioimmunoassayable plasma rat LH and since the sc injection of rat LH elevated plasma LH 5fold but did not cause ovulation, FSH must be at least partially responsible for causing the partial ovulation observed in some of the rats given the higher doses of FSH iv. In the nonblocked rat and hamster, the proestrous increases in serum gonadotropins are associated with a decline in serum estrogen concentration (3, 16). Chappel et al. (17) have reported implantation of silastic capsules containing 17/J-estradiol at 1800 h on proestrus to suppress the second phase of increased serum FSH in the hamster. They suggested that the initiation of the second phase of FSH release requires, at least in part, the decline in serum 17/8-estradiol. We have conducted similar experiments in the rat (Ashiru, 0 . A., and C. A. Blake, unpublished). The preliminary data suggest that the implantation of silastic capsules containing 17/6-estradiol at 1300 h on proestrus partially suppresses both phases of increased plasma FSH. However, the suppressions are relatively slight and it is our contention that a decrease in 17/?-estradiol titers alone is not the mechanism by which increases in proestrous plasma gonadotropin levels initiate and stimulate pituitary FSH release during estrus.
Acknowledgments The authors thank Dr. L. E. Reichert, Jr., Dr. G. D. Niswender, Dr. A. F. Parlow, and the NIAMDD (Pituitary Hormone Distribution
1167
Program) for their gifts of materials used for RIA and the biological studies.
References 1. Gay, V. L., A. R. Midgley, Jr., and G. D. Niswender, Patterns of gonadotrophin secretion associated with ovulation, Fed Proc 29: 1880, 1970. 2. Daane, T. A., and A. F. Parlow, Periovulatory patterns of rat serum follicle stimulating hormone and luteinizing hormone during the normal estrous cycle: effects of pentobarbital, Endocrinology 88: 653, 1971. 3. Butcher, R. L., W. E. Collins, and N. W. Fugo, Plasma concentrations of LH, FSH, prolactin, progesterone and estradiol-17/? throughout the 4-day estrous cycle of the rat, Endocrinology 94: 1704, 1974. 4. Gay, V. L., and R. L. Tomacari, Follicle-stimulating hormone secretion in the female rat: cyclic release is dependent on circulating androgen, Science 184: 75, 1974. 5. Smith, M. S., M. E. Freeman, and J. D. Neill, The control of progesterone secretion during the estrous cycle and early pseudopregnancy in the rat: prolactin gonadotropin and steroid levels associated with rescue of the corpus luteum of pseudopregnancy, Endocrinology 96: 219, 1975. 6. Ashiru, O. A., and C. A. Blake, Restoration of the periovulatory follicle-stimulating hormone surges in sera by luteinizing hormone releasing hormone in phenobarbital-blocked rats, Life Sci 23: 1507, 1978. 7. Blake, C. A., Simulation of the proestrous luteinizing hormone (LH) surge after infusion of LH-releasing hormone in phenobarbital-blocked rats, Endocrinology 98: 451, 1976. 8. Blake, C. A., Simulation of the early phase of the proestrous folliclestimulating hormone rise after infusion of luteinizing hormonereleasing hormone in phenobarbital-blocked rats, Endocrinology 98: 461, 1976. 9. Chappel, S. C, and C. A. Barraclough, Further studies on the regulation of FSH secretion, Endocrinology 101: 24, 1977. 10. Blake, C. A., A detailed characterization of the proestrous luteinizing hormone surge, Endocrinology 98: 445, 1976. 11. Gay, V. L., A stereotaxic approach to transauricular hypophysectomy, Endocrinology 81: 1177, 1967. 12. Niswender, G. D., A. R. Midgley, Jr., S. E. Monroe, and L. E. Reichert, Jr., Radioimmunoassay for rat luteinizing hormone with anti-ovine LH serum and ovine LH-I131, Proc Soc Exp Biol Med 128: 807, 1968. 13. Blake, C. A., R. L. Norman, and C. H. Sawyer, Validation of an ovine-ovine LH radioimmunoassay for use in the hamster, Biol Reprod 8: 299, 1973. 14. Niswender, G. D., A. R. Midgley, Jr., and L. E. Reichert, Jr., Radioimmunologic studies with murine, bovine, ovine, and porcine luteinizing hormone, In Rosenberg, E. (ed.), Gonadotropins, GeronX, Inc., Los Altos, 1968, p. 299. 15. Schwartz, N. B., and W. L. Talley, Effects of exogenous LH or FSH on endogenous FSH, progesterone and estradiol secretion, Biol Reprod 17: 820, 1978. 16. Baranczuk, R., and G. S. Greenwald, Peripheral levels of estrogen in the cyclic hamster, Endocrinology 92: 805, 1973. 17. Chappel, S. C, R. L. Norman, and H. G. Spies, Effects of estradiol on serum and pituitary gonadotropin concentrations during selective elevations of follicle stimulating hormone, Biol Reprod 19: 159, 1978.
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Vol. 105, No. 5 Printed in U.S.A.
Stimulation of Endogenous Follicle-Stimulating Hormone Release during Estrus by Exogenous Follicle-Stimulating Hormone or Luteinizing Hormone at Proestrus in the Phenobarbital-Blocked Rat* OLADAPO A. ASHIRU AND CHARLES A. BLAKEf Department of Anatomy, University of Nebraska Medical Center, Omaha, Nebraska 68105
FSH remained low during proestrus and estrus. After two iv injections of rat LH which caused plasma LH to rise more than 75-fold, plasma FSH rose during late proestrus and remained elevated during the morning of estrus. The stimulatory effects of plasma gonadotropins on FSH secretion were ovulation independent. The results indicate that in the phenobarbitalblocked rat, small selective elevations in plasma rat FSH or large selective elevations in plasma rat LH during proestrus, which are similar to FSH and LH levels observed in nonblocked rats, can stimulate pituitary FSH release during the time interval in which the second phase of FSH release would normally occur. The results also suggest that 1) the spontaneous LH surge and the associated first phase of increased plasma FSH are both involved in mediating the second phase of increased plasma FSH, and 2) the magnitude and/or duration of a rise in plasma gonadotropin during proestrus is important in effecting a threshold response which results in stimulating FSH release during estrus. (Endocrinology 105: 1162, 1979)
ABSTRACT. We previously reported that administration of LHRH on proestrus restores the two phases of increased plasma FSH during the periovulatory FSH surge in plasma in phenobarbital-blocked rats. In this study, we determined if the LHRHinduced restoration of the second phase of the FSH surge, which occurs during late proestrus and the morning of estrus, might be mediated by pituitary LH and/or FSH release during proestrus. In phenobarbital-blocked animals, rat FSH or LH was injected between 1500-1800 h on proestrus. Blood was collected by decapitation or through indwelling atrial cannulae during proestrus and estrus for RIA of FSH and LH. After sc or iv injection of FSH, plasma FSH but not LH was elevated during the late afternoon and evening of proestrus and during the morning of estrus. The FSH elevations during estrus reflect endogenous FSH release as hypophysectomy before the injection of FSH blocked the response. Subcutaneous or iv injection of rat LH did not elevate plasma FSH acutely. After sc injection of a dose of rat LH which caused plasma LH to rise about 5-fold, plasma
I
N THE cyclic rat, the periovulatory increases in serum FSH concentration can be divided into two phases. The first occurs during proestrus and is associated with the preovulatory increase in serum LH concentration. The second phase starts during late proestrus and continues into estrus. It is not associated with elevated serum LH (1-6). We have recently reported that administration of an ovulation-blocking dose of sodium phenobarbital during the early afternoon of proestrus completely blocks the proestrous LH surge and both phases of increased serum FSH (6). In the phenobarbital-blocked rat, administration of LHRH during proestrus not only restored the proestrous increases in serum LH and FSH (6-8) but Received December 15, 1978. * This work was supported by grants from the NIH (HD-11011 and HD-07097), the NSF (RIAS-SER 77-06922), and the Population Council (Biomedical Fellowship to O.A.A.). A portion of this study has been published in abstract form (Ashiru, O. A., and C. A. Blake, Physiologist 21: 3, 1978). f To whom all correspondence and requests for reprints should be addressed.
caused a virtually perfect simulation of the second phase of increased serum FSH as well (6). There is evidence to suggest that LHRH-induced restoration of the second phase of FSH release may be mediated by pituitary LH and/or FSH release during proestrus. Injection of an ovulation-blocking dose of pentobarbital at proestrus is highly effective in blocking the proestrous LH surge. However, it causes only a temporary and partial blockade of the first phase of FSH release. Serum FSH rises during the late afternoon to early evening of proestrus. Thereafter, the serum FSH rises to reach levels during estrus which approach values observed during the spontaneous increase in serum FSH during estrus (Blake, C. A., unpublished) (2, 9). These observations suggest that an increase in serum FSH concentration during proestrus may be involved in causing pituitary FSH release during estrus. Chappel and Barraclough (9) have demonstrated that the iv injection of highly purified rat LH during the afternoon of proestrus causes plasma FSH to rise within 3 h in the pentobarbital-treated rat. As pentobarbital was effective in blocking the first phase of increased plasma
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GONADOTROPIN STIMULATION OF FSH RELEASE
FSH during that 3-h period, their results clearly indicate that elevations in rat plasma LH during proestrus can cause the release of pituitary FSH during proestrus. It has yet to be demonstrated if elevations in plasma LH during proestrus in the absence of an increase in plasma FSH will cause a second phase of FSH release during estrus. In this study, we have used the phenobarbital-blocked proestrous rat as our model to determine if the second phase of increased FSH might be caused by pituitary LH and/or FSH release during proestrus. Materials and Methods Animals Virgin female Sprague-Dawley rats (Simonsen Lab., Gilroy, 7 CA) were kept in a room with the light on from 0500-1900 h daily for 26 or more days before daily vaginal lavages were taken. Rats which exhibited two or more consecutive 4-day • estrous cycles were used (208-295 g BW).
v
Effects of exogenous gonadotropins at proestrus on serum FSH and LH during proestrus and estrus in rats sacrificed by guillotine
Control rats were sacrificed by decapitation at 1400,1800, or * 2200 h on proestrus or at 0200, 0600, or 1000 h on estrus for collection of trunk blood and subsequent RIA of serum FSH and LH. Additional rats were injected ip with phenobarbital (100 mg/ kg BW) at 1200-1400 h on proestrus and divided into the - following treatment groups: 1) no additional treatment, 2) sc injection of 5 jug rat FSH (NIAMDD-rat FSH-I-3)1 at 1700 h on proestrus, and 3) sc injection of 50 /xg bovine LH (NIH-LH» BIO)2 at 1500 and 1600 h on proestrus. Hormones were dissolved in 0.4 ml saline; 0.2 ml was injected sc in each side in the ilio-inguinal region. Rats were decapitated for collection of trunk blood at 1800 or 2200 h on proestrus or * at 0200, 0600, or 1000 h on estrus. The Fallopian tubes were checked for the presence of ova in the rats sacrificed at 0600 or 1000 h on estrus. Effects of exogenous gonadotropins at proestrus on plasma FSH and LH during proestrus and estrus in serially bled rats Rats were injected ip with phenobarbital (100 mg/kg BW) at 1200-1300 h on proestrus. A single polyvinyl cannula was then inserted into the right atrium of the heart, as previously de* scribed (10), using ether as an anesthetic when needed. Rats were divided into the following treatment groups: 1) no additional treatment, 2) sc injection of 5 or 7.5 jug rat FSH at 1700 _> h on proestrus, 3) iv injection of 2.5, 5, or 7.5 jug rat FSH at 1700 h on proestrus, 4) sc injection of 50 jug bovine LH at 1500 and '
1
NIAMDD-rat FSH-I-3 was reported to have a LH biological potency of less than 0.002 X NIH-LH-Sl and a FSH biological potency of 1502 x NIH-FSH-Sl. NIH-LH-B10 was reported to have a LH biological potency of 1.06 X NIH-LH-Sl and a FSH biological potency of less than 0.05 x NIHFSH-Sl.
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1600 h on proestrus, 5) sc injection of 250 jug bovine LH at 1500 and 1600 h on proestrus, 6) sc injection of 5 jug rat LH (NIAMDD-rat LH-I-4)3 at 1500 h on proestrus, and 7) iv injection of 2 jug rat LH at both 1600 and 1800 h on proestrus. When given iv, hormones were dissolved in 0.2 ml saline and injected through the cannula. The cannula was then flushed with an additional 0.6 ml saline. Rats were bled (0.7 ml) through the cannulae just before injection and at 4-h intervals from 1800 h on proestrus until 1000 h on estrus. An additional sample was collected at 5 min after all iv injections of hormone. Additional blood samples were collected after sc injection of both LH preparations. After each bleeding, blood was centrifuged at 4 C and plasma was stored frozen at -20 C until subsequent RIA of FSH and LH. The packed red and white blood cells were resuspended in an equal volume of heparinized saline (10 U/ml) and injected into the rats through the cannulae within 20 min after each bleeding. The Fallopian tubes were checked for the presence of ova after the 1000 h bleeding on estrus. Effects of hypophysectomy on the plasma FSH response to injection of rat FSH on proestrus Phenobarbital was administered at 1200-1300 h on proestrus. Rats were hypophysectomized by the method of Gay (11) at 1600-1650 h under supplemental ether anesthesia. Rats were then given one of the two following treatments: 1) sc injection of 5 jug rat FSH at 1700 h on proestrus or 2) iv injection of 1, 2, or 5 /xg rat FSH at 1700 h on proestrus. Rats were bled through the cannulae as described above. After the 1000 h bleeding on estrus, the Fallopian tubes were checked for the presence of ova and the sella turcica was examined to determine if hypophysectomy was complete. RIAs Rat FSH was measured with the materials and instructions supplied with the FSH assay kit distributed by NIAMDD. The antiserum was S6. LH was measured by the method of Niswender et al. (12), as previously described (13). This RIA system measures both bovine and rat LH (14). FSH is expressed as nanograms per ml serum or plasma in terms of NIAMDD-rat FSH-RP-1, which has a biological potency equivalent to 2.1 X NIH-FSH-Sl. LH is expressed in terms of NIAMDD-rat LHRP-1, which has a biological potency equivalent to 0.03 x NIHLH-Sl. A serum pool (150 /xl) was assayed for FSH in triplicate in each of five assays. The mean (±SE) serum FSH concentration for the 15 determinations was 570 ± 14 ng/ml. The coefficient of variation within assays ranged from 2-11% and between assays was 10%. The serum pool (10 jul) was assayed for LH in triplicate in each of three assays. The mean (±SE) serum LH concentration for the nine determinations was 1389 ± 24 ng/ ml. The coefficient of variation within assays ranged from 2-6% and between assays was 5%. Statistics Statistical comparisons of data were performed by one-way 3
NIAMDD-rat LH-I-4 was reported to have a LH biological potency of 1.0 x NIH-LH-Sl and a FSH biological potency of less than 0.04 x NIH-FSH-Sl.
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Endo • 1979 Vol 105 • No 5
ASHIRU AND BLAKE
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analysis of variance (for repeated measures) for separate groups of rats to determine changes over time. Comparison between groups was made by two-way analysis of variance (treatment and time periods). Subsequent Newman-Keuls tests were performed. P values less than 0.05 were considered significant, and significance was tested at the 0.05 and 0.01 levels.
Results Mean serum LH and FSH concentrations for control rats decapitated between 1400 h on proestrus and 1000 h on estrus are shown in Fig. 1. In agreement with previous studies (1-6), serum LH was elevated only during proestrus. Serum FSH rose (P < 0.01) approximately 3-fold by 1800 h on proestrus, decreased (P < 0.05) by 2200 h on proestrus, rose {P < 0.05) again by 0200 h on estrus and remained elevated (P < 0.01) at 0600 and 1000 h on estrus above values observed at 1400 h on proestrus. The administration of phenobarbital between 12001400 h on proestrus blocked the LH surge (not illustrated; serum LH concentration ranged from 27-37 ng/ml) and the two phases of increased serum FSH (Fig. 1). Similarly, phenobarbital blocked the plasma LH (not illustrated; plasma LH concentration in individual rats ranged from 28-36 ng/ml) and FSH (Fig. 1) rises in rats bled serially through indwelling atrial catheters. In phenobarbital-blocked rats, sc or iv injection of
control phen
highly purified rat FSH did not elevate radioimmunoassayable serum/plasma LH (not illustrated; serum/ plasma LH concentration in individual rats remained low, 27-34 ng/ml). Sc injection of 5 /tg rat FSH at 1700 h on proestrus caused a small but significant (P < 0.05) increase in radioimmunoassayable serum/plasma FSH by 1800 h on proestrus (Fig. 2). The FSH concentrations rose further (P < 0.01) by 2200 h on proestrus and remained elevated through 1000 h on estrus. A similar plasma FSH response was observed in two rats administered 7.5 jug FSH (Fig. 2). The elevations in plasma FSH observed during estrus after sc injection of rat FSH must be due primarily to measurement of endogenous FSH rather than to measurement of exogenous FSH. When rats were hypophysectomized shortly before sc injection of FSH on proestrus, plasma FSH concentrations were low during estrus (Fig. 2). In one hypophysectomized rat, plasma FSH peaked at 1800 h on proestrus, 1 h after the injection, and failed to fall appreciably during the next 4 h. In the other hypophysectomized rat, plasma FSH peaked at 2200 h on proestrus, 5 h after the injection. These observations suggest that the injected FSH entered the plasma over a prolonged period. The increment and pattern of plasma FSH in the hypophysectomized rats were similar to those observed during the first phase of FSH release in intact rats (Fig. 2 vs. Ref. 8). The elevations in plasma FSH concentrations observed during estrus after the iv injection of 2.5-7.5 fig rat FSH at 1700 h on proestrus (Fig. 3) must also be due primarily to the measurement of endogenous FSH rather than to
1000r
800-
600 E X 400 TIME
FIG. 1. Mean (±SE) serum LH and FSH concentrations for control rats during proestrus (P) and estrus (E) are plotted. Indicated times encompass the interval from 1400 h on P to 1000 h on E. In this and subsequent figures: • - - • and A- -A, serum values in blood collected by decapitation; • — • and A—A, plasma values in blood collected serially through atrial catheters. The numbers to the right of the mean plasma FSH values at 1000 h on E represent the number of rats bled. Those to the right of the mean serum FSH values at 0600 or 1000 h represent the numbers or range of numbers of rats decapitated at each time period from 1400 h on P to 1000 h on E. FSH and LH were measured on the same samples. Any SE with a value less than 10 has not been plotted. The administration of phenobarbital (phen; 100 mg/ kg BW) at 1200-1400 h on P blocked the entire periovulatory FSH surge.
200
18
22
02
06
10
-H4 TIME
FIG. 2. Effects of sc injection of FSH (rat 1-3) at 1700 h on proestrus (P) in phenobarbital-blocked rats on serum/plasma FSH during P and estrus (E). Serum/plasma LH levels did not rise. Plasma FSH values for two individual rats administered 7.5 fig are plotted. Plasma FSH values for two individual rats administered 5 /ig and hypophysectomized at 1600-1650 h on P are designated HYP. The Xs at 0600 and 1000 h indicate that plasma FSH was below the sensitivity of the assay on the volume of plasma assayed.
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GONADOTROPIN STIMULATION OF FSH RELEASE 1800
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2500 1
2000 1600
-L\
•
SO»>g/ln|«cl|i
A 2S0M0/ln|*ctlon
g 1500 c
1400
. 1000 500
1200 400
1000
I Si 200
800
600
TIME
FIG. 4. Effects of sc injection of bovine LH at 1500 and 1600 h on proestrus (P) in phenobarbital-blocked rats on serum/plasma FSH and LH during P and estrus (E). Plasma FSH values at 1500, 1530, and 1600 h on P in rats injected with 50 or 250 jLtg LH at 1500 h were similar and have been combined.
400-
200
18
22
02
06
10
TIME
FIG. 3. Effects of iv injection of FSH (rat 1-3) at 1700 h on proestrus (P) in phenobarbital-blocked rats on plasma FSH during P and estrus (E). Plasma LH levels did not rise. Plasma FSH values for two individual rats administered 7.5 jig are plotted. Four rats were hypophysectomized (HYP) at 1600-1650 h on P before the injection of FSH. The Xs at 0200-1000 h indicate that plasma FSH was below the sensitivity of the assay for the volume of plasma assayed.
the measurement of exogenous FSH. Plasma FSH concentrations were low during estrus when rats were hypophysectomized shortly before iv injection of 1 or 2 jug (Fig. 3) or 5 jug (five rats not illustrated) exogenous FSH at proestrus. Two sc injections of 50 jug bovine LH did not elevate radioimmunoassayable serum FSH during proestrus or estrus (Fig. 4). It did cause a serum LH pattern, presumably in terms of assayable bovine LH, similar to that observed during the spontaneous LH surge (Fig. 4 vs. Ref. 10 and Fig. 1). When the dosage was increased 5fold to two 250-jng injections, the plasma FSH level rose during estrus to levels which approached but did not reach control values during estrus (Fig. 4 vs. Fig. 1). Subcutaneous and iv injections of highly purified rat LH on proestrus to phenobarbital-blocked rats caused an elevation in plasma LH but not in plasma FSH by 1800 h on proestrus (Fig. 5). The sc injection at 1500 h caused a 5-fold elevation in plasma LH by 1800 h. Plasma FSH did not rise in these rats during proestrus or estrus. Intravenous injection of a smaller dose caused plasma LH to rise over 30-fold within 5 min. It then declined to approach basal levels within 115 min. A second identical injection caused a similar LH response. Although the
FIG. 5. Effects of injection of rat LH in phenobarbital-blocked rats on plasma FSH and LH during proestrus (P) and estrus (E).
peak LH concentrations were in the upper range of values observed during the spontaneous LH surge, the elevations in plasma LH from 1500-2200 h on proestrus were in the physiological range observed in nonblocked rats (Fig. 5 vs. Ref. 10). In these LH-injected rats, plasma FSH rose (P < 0.05) by 2200 h on proestrus and reached values during estrus which were similar to values observed in control rats during estrus (Fig. 5 vs. Fig. 1). The effects of the FSH and LH preparations and their different routes of administration on ovulation are shown in Table 1. The occurrence of ovulation and the stimulation of endogenous FSH release during estrus were
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ASHIRU AND BLAKE
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TABLE 1. Effects of sc or iv injection of gonadotropins on ovulation Hormone
Dose (/xg)
Route
No. of rats
No. of ovulating rats
Rat FSH Rat FSH Rat FSH Rat FSH Rat FSH Bovine LH Bovine LH RatLH
5 7.5 2.5 5 7.5 100 500 5 4
sc sc iv iv iv sc sc sc iv
10 2 4 4 2 17 5 3 4
0 0 0 2 2 17 5 0 4
RatLH
No. of ova/ovulating rat
1,2 2,3 27 >7 >7
dissociated. Rats administered 100 /xg bovine LH ovulated fully (seven or more ova), but serum FSH was not elevated during estrus. Rats administered rat LH iv and those administered rat FSH sc had elevated plasma FSH during estrus, but only those given LH ovulated. Discussion The results of this study clearly indicate that elevations in plasma LH or plasma FSH during proestrus can stimulate the release of pituitary FSH during estrus in the phenobarbital-blocked rat. A small prolonged elevation in rat plasma FSH during proestrus, similar to that observed during the first phase of FSH release in nonblocked rats, is sufficient to stimulate pituitary FSH release during the time when the second phase of FSH release would normally occur. Similarly, a large elevation in rat plasma LH during proestrus, similar to that observed during the proestrous LH surge in nonblocked rats, will stimulate pituitary FSH release during the time that the second phase of FSH release would normally occur. These results suggest that both the proestrous LH surge and the associated first phase of increased plasma FSH are involved in mediating the second phase of increased plasma FSH in the cyclic rat. As small elevations in plasma rat LH during proestrus are ineffective in elevating plasma FSH, it would appear that the magnitude and/or duration of a rise in plasma gonadotropins during proestrus, at least in the case of plasma LH, are important in effecting a threshold response which ultimately results in a stimulation of pituitary FSH release during estrus. Although the effects of iv bovine LH on stimulating a rise in plasma FSH were not tested, bovine LH is probably less effective than rat LH in this regard, as indicated by the results obtained in rats given bovine LH sc. Only when plasma LH was elevated above proestrous LH surge levels for an extended period of time (presumably assayable in terms of bovine LH) did plasma FSH rise during estrus to levels which simulated a second phase of plasma FSH. In terms of the relatively small elevation in rat plasma
Endo • 1979 Vol 105 • No 5
FSH which is stimulatory to FSH release compared to the higher elevations in rat plasma LH which are needed to stimulate a rise in plasma FSH, it would appear that FSH is more effective than LH in stimulating FSH secretion. However, in physiological terms, a simulated proestrous LH surge caused a near-perfect simulation of the second phase of FSH release. The plasma FSH response observed during late proestrus and estrus after physiological elevations of plasma FSH during proestrus was greater than that observed in controls. This enhancement of FSH secretion may be due to the FSH dosages and routes of administration employed. However, phenobarbital-blocked rats may have more pituitary FSH available for release during late proestrus than do controls. Pituitary FSH concentration fell by more than half from 1400-2200 h on proestrus in controls but did not decrease during this period in phenobarbital-blocked rats (Ashiru, O. A., and C. A. Blake, unpublished). It may be that once threshold levels of plasma LH and FSH are reached during proestrus in the normal rat, additional proestrous increases in plasma LH and FSH enhance FSH release during late proestrus and estrus, but this additional release would be limited by the amount of pituitary FSH available for release. There is evidence to suggest that once plasma FSH has reached threshold levels during proestrus to effect a stimulatory response on its own secretion during estrus, additional increases in plasma gonadotropins during proestrus will stimulate additional pituitary FSH release during estrus. In rats in which the proestrous increase in plasma FSH was partially suppressed with pentobarbital, plasma FSH at 1000 h on estrus was higher after sc injection of ovine LH or FSH on proestrus than after sc injection of saline (15). Thus, increases in ovine LH during proestrus superimposed on a lower than normal rise in plasma FSH during proestrus can apparently stimulate additional pituitary FSH release by 1000 h on estrus. Similarly, the increase in plasma FSH at 1000 h on estrus observed after the injection of ovine FSH on proestrus could be due to the additive stimulatory effects of exogenous and endogenous FSH during proestrus. Alternatively, the increased plasma FSH on estrus could be due to the measurement of ovine FSH which has entered the plasma compartment over a prolonged period, as rat FSH did in the present study. There is a substantial latency in the time of onset of pituitary FSH release in response to an increase in plasma LH or FSH during the afternoon of proestrus. In the pentobarbital-blocked rat, Chappel and Barraclough (9) observed an increase in plasma FSH at 3 but not at 2 h after iv injection of rat LH on proestrus. Our results are in agreement with theirs. An increase in plasma FSH was observed between 2-6 h after iv injection of rat LH. The difference in plasma FSH patterns after sc injection
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GONADOTROPIN STIMULATION OF FSH RELEASE of 5 /xg rat FSH in intact and hypophysectomized proestrous rats suggests that the latency of the FSH-positive feedback on its own secretion is between 1-5 h. The mechanism by which increases in plasma gonadotropins exert their stimulatory effect on FSH secretion is clearly independent of ovulation. In all cases in which exogenous LH stimulated FSH release during estrus, rats ovulated fully (seven or more ova). However, rats given 100 jug bovine LH ovulated fully, and serum FSH did not rise during estrus. Therefore, the LH threshold to stimulate pituitary FSH release would appear to be higher than that needed to induce ovulation. In contrast, increases in plasma FSH can stimulate pituitary FSH release without causing ovulation. It is not clear if FSH itself or if FSH and LH contamination in the FSH preparation caused partial ovulation in some of the rats given the higher doses of FSH iv. As FSH injection did not elevate radioimmunoassayable plasma rat LH and since the sc injection of rat LH elevated plasma LH 5fold but did not cause ovulation, FSH must be at least partially responsible for causing the partial ovulation observed in some of the rats given the higher doses of FSH iv. In the nonblocked rat and hamster, the proestrous increases in serum gonadotropins are associated with a decline in serum estrogen concentration (3, 16). Chappel et al. (17) have reported implantation of silastic capsules containing 17/J-estradiol at 1800 h on proestrus to suppress the second phase of increased serum FSH in the hamster. They suggested that the initiation of the second phase of FSH release requires, at least in part, the decline in serum 17/8-estradiol. We have conducted similar experiments in the rat (Ashiru, 0 . A., and C. A. Blake, unpublished). The preliminary data suggest that the implantation of silastic capsules containing 17/6-estradiol at 1300 h on proestrus partially suppresses both phases of increased plasma FSH. However, the suppressions are relatively slight and it is our contention that a decrease in 17/?-estradiol titers alone is not the mechanism by which increases in proestrous plasma gonadotropin levels initiate and stimulate pituitary FSH release during estrus.
Acknowledgments The authors thank Dr. L. E. Reichert, Jr., Dr. G. D. Niswender, Dr. A. F. Parlow, and the NIAMDD (Pituitary Hormone Distribution
1167
Program) for their gifts of materials used for RIA and the biological studies.
References 1. Gay, V. L., A. R. Midgley, Jr., and G. D. Niswender, Patterns of gonadotrophin secretion associated with ovulation, Fed Proc 29: 1880, 1970. 2. Daane, T. A., and A. F. Parlow, Periovulatory patterns of rat serum follicle stimulating hormone and luteinizing hormone during the normal estrous cycle: effects of pentobarbital, Endocrinology 88: 653, 1971. 3. Butcher, R. L., W. E. Collins, and N. W. Fugo, Plasma concentrations of LH, FSH, prolactin, progesterone and estradiol-17/? throughout the 4-day estrous cycle of the rat, Endocrinology 94: 1704, 1974. 4. Gay, V. L., and R. L. Tomacari, Follicle-stimulating hormone secretion in the female rat: cyclic release is dependent on circulating androgen, Science 184: 75, 1974. 5. Smith, M. S., M. E. Freeman, and J. D. Neill, The control of progesterone secretion during the estrous cycle and early pseudopregnancy in the rat: prolactin gonadotropin and steroid levels associated with rescue of the corpus luteum of pseudopregnancy, Endocrinology 96: 219, 1975. 6. Ashiru, O. A., and C. A. Blake, Restoration of the periovulatory follicle-stimulating hormone surges in sera by luteinizing hormone releasing hormone in phenobarbital-blocked rats, Life Sci 23: 1507, 1978. 7. Blake, C. A., Simulation of the proestrous luteinizing hormone (LH) surge after infusion of LH-releasing hormone in phenobarbital-blocked rats, Endocrinology 98: 451, 1976. 8. Blake, C. A., Simulation of the early phase of the proestrous folliclestimulating hormone rise after infusion of luteinizing hormonereleasing hormone in phenobarbital-blocked rats, Endocrinology 98: 461, 1976. 9. Chappel, S. C, and C. A. Barraclough, Further studies on the regulation of FSH secretion, Endocrinology 101: 24, 1977. 10. Blake, C. A., A detailed characterization of the proestrous luteinizing hormone surge, Endocrinology 98: 445, 1976. 11. Gay, V. L., A stereotaxic approach to transauricular hypophysectomy, Endocrinology 81: 1177, 1967. 12. Niswender, G. D., A. R. Midgley, Jr., S. E. Monroe, and L. E. Reichert, Jr., Radioimmunoassay for rat luteinizing hormone with anti-ovine LH serum and ovine LH-I131, Proc Soc Exp Biol Med 128: 807, 1968. 13. Blake, C. A., R. L. Norman, and C. H. Sawyer, Validation of an ovine-ovine LH radioimmunoassay for use in the hamster, Biol Reprod 8: 299, 1973. 14. Niswender, G. D., A. R. Midgley, Jr., and L. E. Reichert, Jr., Radioimmunologic studies with murine, bovine, ovine, and porcine luteinizing hormone, In Rosenberg, E. (ed.), Gonadotropins, GeronX, Inc., Los Altos, 1968, p. 299. 15. Schwartz, N. B., and W. L. Talley, Effects of exogenous LH or FSH on endogenous FSH, progesterone and estradiol secretion, Biol Reprod 17: 820, 1978. 16. Baranczuk, R., and G. S. Greenwald, Peripheral levels of estrogen in the cyclic hamster, Endocrinology 92: 805, 1973. 17. Chappel, S. C, R. L. Norman, and H. G. Spies, Effects of estradiol on serum and pituitary gonadotropin concentrations during selective elevations of follicle stimulating hormone, Biol Reprod 19: 159, 1978.
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