Effects of Exogenous LH or FSH on Endogenous FSH, Progesterone and Estradiol Secretion1

Transcription

1 BIOLOGY OF REPRODUCTION 17, (1978) Effects of Exogenous LH or FH on Endogenous FH, Progesterone and Estradiol ecretion1 NEENA B. CHWARTZ and WILLIAM L. TALLEY Department of Biological ciences, Northwestern University, Evanston, Illinois ABTRACT In one set of experiments, an ovulatory dose of ovine LH or FH was injected, on the afternoon of proestrus, into rats which had received pentobarbital (PB). erum FH was elevated over noninjected controls at 1 h estrus. The adrenal glands were not necessary for this response. Ovariectomy, performed shortly before the gonadotrophin injection, caused an elevation in serum FH the following morning, which was not further increased by the injection of LH or FH. erum LH was not elevated nor was pituitary LH content decreased at 1 h estrus in any treatment group. In a second set of experiments, the time course of LU, FH, estradiol (E2) and progesterone (P4) was investigated, between proestrous afternoon (163 h) and estrous morning (1 h), in 3 treatment groups: a) saline controls (no PB), b) PB controls (no LH), c) PB + LH (8 Mg IV at 153 h). a) aline controls showed a maximum LU and FU primary surge at 183 h; LH returned to baseline by 23 h, while FH fell slowly through the night. P4 was elevated at 183 hand 23 h, then fell to baseline; E2 fell abruptly between 163 h and 183 h. b) In PB controls, serum LU and P were continuously low, but a small rise in FH occurred at 23 h and 4 h (estrus). E2 fell significantly also, but not as low as in the saline controls. c) LH, superimposed on PB, raised LH at 163 h, but no further rises occurred, while FH rose gradually to peak at 4 h and then fell part way to saline control levels by 1 h estrus. Thus, FH appeared to be released independently of LU. The injected LU also induced a rise in P4 at 163 h and 183 h, with E2 falling, as in saline controls, between 163 h and 183 h. In a third experiment, P4 injection was tested, superimposed on PB blockade. P4 failed to increase LH or FH (in a manner resembling LH injection), but did cause E2 to fall. It is concluded that the primary LH (and/or FH) surge, as well as exogenous LH or FH superimposed on PB blockade, can induce a release of FH independently of changes in measured steroids (serum P4, E2) and in the absence of concomitant LH release. The induction of FH release could not be detected in the absence of the ovary, but ovariectomy by itself increased FH release without LU release. The data suggest that gonadotrophins, as well as ovariectomy, may induce FH release by altering a nonsteroidal feedback from the ovaries. INTRODUCTION During an investigation of the effects of ovine LB and FH on ovulation (chwartz et al., 1975), we serendipitously observed that gonadotrophic hormone injection into proestrous rats in which the endogenous LU and FU surges were blocked by pentobarbital (PB) caused high FH serum values on the morning of estrus (chwartz, unpublished observations). We had previously seen that subjecting PBblocked rats to cardiac puncture caused ovulation in some; these rats also showed high terminal serum FH at estrus (Proudfit and Accepted December 8, Received August 12, A preliminary report of some of these data appeared in the program of the Ninth Annual Meeting of the ociety for the tudy of Reproduction, 1976 (Abstr. No. 28). chwartz, 1974). These findings suggested that it might be profitable to investigate further whether gonadotrophin injection into PBblocked rats at proestrus induced endogenous FH and/or LH secretion. The present study describes a series of experiments performed to answer this question. MATERIAL AND METHOD Rats were obtained from prague-dawley (AR), Madison, WI and housed in L14:D1O (midnight = midpoint of dark period). Each rat was kept under these lighting conditions for 1 week prior to the onset of daily vaginal smearing and exhibited at least 2 4-day cycles before the cycle of treatment. All treatments were initiated during the afternoon of proestrus. Autopsy Procedures Animals were quickly anesthetized with ether and a terminal blood sample taken by cardiac puncture. Blood was centrifuged and the serum was snapfrozen after separation into appropriate aliquots for 82

2 LH AND FI-1 INDUCE FH ECRETION 821 This set of experiments was done in different animal quarters and started about 6 months after Experihormone assays and then stored at -2 C until assay. Oviducts were observed under the dissecting microscope for evidence of recent ovulation; in all cases where the gross examination was ambiguous and in many cases where it was not, serial sections of one or both ovaries was carried out (azan stain). Uterine intraluminal water and wet and dry weight were measured as described in Armstrong (1968) and chwartz and Ely (197). Pituitary glands were weighed and quick frozen; LH content was measured on most of them. Hormone Assays erum LH was measured using the : method with NIU-LH-14 as the standard (Niswender et al., 1968); pituitary LU was measured by homogenizing glands (individually) in egg white-pb, diluting to 1/5 and running several different volumes in the radioimmunoassay. erum FH was assessed using the R:R system, with FH-RP-1 as the standard (Proudfit and chwartz, 1974). erum estradiol and progesterone were measured using Niswender antibody #244 and #869, respectively, without column separation, as described and validated previously (Nequin et al., 1975;Campbell et al., 1977). Hormone measurements were corrected for procedural losses, but not for values of the serum blanks (serum from rats which had been ovariectomized and adrenalectomized for 4 days). Average serum blank levels for estradiol were 13 pg/mi; for progesterone.56 ng/ml. Experiment I. Hormone Injections Into Rats Treated with Pentobarbital: Autopsy Estrus 1 h Experiment IA. This experiment is from the previous ovulation study (chwartz et al., 1975). All injections were intraperitoneal (i.p.). At 135 h rats received either saline (1 ml/1 g) or PB (3 mg/1 g); at 143 h each received saline again; at 15 h the saline controls again received saline; the PB injected rats received saline, NIH-LH-14 (approximately 25 tg/ rat) or NIH-FH-54 (4 Mg/rat). Experiment lb. A dose response test of hormone effects were carried out. PB was injected at 3 mg/1 g i.p. at 1345 h. At 14 h, saline (.5 ml) was injected via tail vein (in 3 seconds) as an injection control; equivolume doses of hormones were injected via the same route. LH (NIH-LH-14) was given in doses of 1, 2, 4 and 8 sg; FH (NIH-FH-4) was injected in doses of 2.5, 5, 1 and 2 i.tg. Experiment IC. This experiment tested the effects of saline, LH or FH injection at 1415 h in PBblocked rats (3 mg/1 g at 133 h) after the rats were laparotomized, adrenalectomized, ovariectomized or both at 14 h. All injections were via the tail vein in.5 ml; the dose of NIH-LU-14 was 8 j.lg; the dose of NIH-FH-4 was 2 big. In the above experiments (IA, B, C), pituitary LH content was measured in 2-7 rats/group; ovulation, uterine intraluminal water and terminal serum LH and FH were measured in all rats (N = 3-7). Experiment II. LH Injections into Rats Treated with Pentobarbital Autopsy at Varying Intervals ment IC. Pilot studies revealed that 3 mg/1 g PB did not prevent ovulation in all controls. Dr. J. W. Everett (personal communication) informed us that an effective blockade could be obtained in his colony by 2 injections of PB, 3.15 mg/1 g at 133 h and.75 mg/1 g at 15 h. This treatment blocked ovulation in 1% of the animals with 4-day cycles and was used in subsequent experiments. The purpose of Experiment II was to follow the time course of response to LH injection. Three treatments were used: saline i.p. at 133 h and 15 h (saline controls); PB at 133 h and 15 h (PB controls); PB at 133 h and 15 h, plus LH (8 ig NIH-LF1-14 or -16 via tail vein) at 153 h. Autopsies were performed (N = 6/group except for a larger N at 1 h estrus) at 163, 183, 23 h proestrus and 4 and 1 h estrus. Terminal serum LU, FH, estradiol and progesterone were measured on each rat at each time and complete autopsies were performed as described above. Pituitary LU content was measured at 1 h. Experiment III. Progesterone Injections into Rats Treated with Pentobarbitall Autopsy at Various Intervals This experiment was performed to test the effects of progesterone injection on PB-blocked rats. PB injections were given as just described; at 163 h rats received peanut oil (.5 ml s.c.) or progesterone in oil (2.5 mg/.5 ml s.c.) (N = 5 for each autopsy group). Terminal samples of blood were taken at 183 and 23 h proestrus, and 4 and 1 h estrus, for hormone measurements and autopsies were performed as described above. Experiment I. REULT LH, FH and Ovulation Data erum FH and ovulation data from the previous experiment (IA) are shown in Fig. 1. PB lowered estrous serum FH values from control levels and either LH or FU significantly increased the hormone, while inducing ovulation. The effect of PB in reducing values of serum FH during the night between proestrus and estrus had previously been seen (Daane and Parlow, 1971; Butcher et al., 1974). erum LU (on the morning of estrus) was not altered by treatment. ince it is known that pituitary LU content on the morning of estrus drops in control rats (chwartz, 1969) or in rats induced to ovulate by cardiac puncture in the presence of PB blockade (Proudfit and chwartz, 1974), this variable was also measured. Neither LU nor FH injection caused pituitary gland LU depletion. These data suggested that FH may have been released in the absence of LU and led us to examine possible dose response aspects of the phenomenon (Experiment IB). Each dose

3 822 CHWARTZ AND TALLEY z I (I) U- lij (I) AUTOPY AT ETRU 1 HR. PB LH (25,u.g) AUTOPY AT E.TRU IHR PB FH (4Og) HORMONE I.P PROETRU 15 HR. FIG. 1. erum FH and ovulation data at 1 h estrus (Experiment IA). PB = pentobarbital (3 mg/ 1 g) or saline injected at 135 h of proestrus. response curve of serum FH was analyzed by analysis of variance. Injection of ovine LU in subovulatory doses did not induce a rise in terminal FH (Fig. 2), but higher, ovulatory doses did. FU injection, even at a dose small enough to induce ovulation in only 1/5 rats, also in- creased terminal FH (Fig. 2). Again, terminal serum LU values did not differ among treatments nor did pituitary LII content drop. LU or FH treatment caused loss of intraluminal uterine water in the 2 experiments just described when doses were high enough, suggesting that changes in steroid secretory rates (drop in estradiol, plus rise in progesterone) were probably being induced by the gonadotrophin (Armstrong, 1968; Kennedy and Armstrong, 1975) since pentobarbital alone generally resulted in retention of the water (chwartz, 1969). It seemed of interest, therefore, to repeat the highest hormone dose used in Experiment lb in the absence of adrenals and/or the ovaries. The effects of these treatments on serum FH on the morning of estrus are summarized in Fig. 3. Laparotomy itself, or adrenalectomy, did not interfere with the effects of LU or FU on terminal FH or ovulation (compare Fig. 3 with 2). However, ovariectomy by itself induced a rise in serum FH which was not further increased at 1 h by hormone injection, nor altered by simultaneous adrenalectomy (Fig. 3). erum or pituitary LU values again were not altered by hormone injection or surgical ablation. Adrenalectomy alone caused very high uterine intraluminal water contents (>1 mg), much higher than laparotomized controls. ince in all 3 of the above experiments, some rats were injected with ovine FH (from h), how could we know that the serum FH being measured at 1 h of estrus was endogenously secreted rfh, not the injected ofh? Four observations suggest that what was measured was indeed endogenously AUTOPY AT ETRU HR z I U- UJ U, HORMONE IV PROETRU 14 HR FIG. 2. erum FITI and ovulation data at 1 h estrus (Experiment IB). PB injected at 1345 h of proestrus. Effects of various doses of LH and FH. URGERY I4tu HR, I4)RP.4E IV 1415 HR PROTRU FIG. 3. erum FH and ovulation data at 1 h estrus (Experiment IC). PB injected at 133 h. Time of surgery and injection seen on figure.

4 LII AND FH INDUCE FII ECRETION 823 secreted FH: a) the metabolic clearance rate for FI-I in the rat is fast enough so that most should have cleared by 19 h (Larochelle and Freeman, 1974); b) ofh cross reacts poorly with iodinated rfh (McNeilly et al., 1976); c) the dose response curve in Fig. 2 for injected FH does not suggest a good correlation between the dose injected and the final FH concentration; d) even 2 pg ofu superimposed on ovariectomy (Fig. 3) did not increase serum FH further than surgery alone. 4O.5 z 3O U ct1qr U o/me Contra/s.-.mpfi Controls Experiment II and III. Go nadotrophin, teroid and Ovulation Data After LH or Progesterone Injection In the saline (non-pb-blocked) controls, serum LU was showing surge values (>1 ng/ ml) in 3/6 rats at 163 h and in all (6/6) by 183 h (Fig. 4). At the 3 last sampling times, only 1 rat still showed a surge value of LH. erum FU data (Fig. 5) also showed all animals with surge values (>45 ng/ml) at 183 h, but levels of the hormone fell slowly until they reached 29 ng/ml at 1 h estrus (within the same range as controls in Fig. 1). teroid values for the saline controls are seen in Figs. 6 and 7. erum progesterone was significantly elevated at 183 h and 23 h and dropped to baseline values by 4 h of estrus. Conversely, estradiol (Fig. 7) dropped from around 5 pg/ml at 163 h to near 2 pg/mi by 183 h and remained low throughout the rest of the night. FIG. 5. erum FH as a function of LII treatment and time of day of proestrus-estrus (Experiment II). ee legend to Fig. 4 for further details. The PB-treated rats showed no LU surge values at any time (Fig. 4) (Nequin et al., 1974). erum FU rose over initial values in these rats (Fig. 5), as also demonstrated by Daane and Parlow (1971). The PB controls showed no coordinated progesterone rise (Fig. 6), but estradiol in the PB rats dropped significantly from 23 h to 1 h of estrus. However, the hormone was still higher than values in the saline controls. These changes in steroid levels resemble those reported after PB blockade by Butcher et al., (1974). uperimposition of 8 pg of LB i.v. at 153 h produced a high serum LH value at 163 h, which had dropped to baseline by 183 h. One rat at 4 h showed a value of LH which was above surge level (25 ng/ml). Thus, the serum LU data between proestrus and estrus support the pituitary content data in failing to demon- #{176}-#{176}ol,ne Contra/s #{149}---sPB fr.,/$ #{176}-#{176}olrne Covva ls ---*P#{216} Coo/rots P8rIH I ;5 z oo 22 moo mo PBFBLHA A A A FIG. 4. erum LU as a function of LH treatment and time of day of proestrus-estrus (Experiment II). First PB injection = 3.15 mg/1 g; second PB injection =.75 mg/1 g. aline controls received saline at time of PB. A = time of autopsy. LH given in 8 g dose i.v. at 153 h. Mean ± EM shown; N = 6-11/ point. 2O 15-1.oQs ojoo PBPBLHA A A A A FIG. 6. erum progesterone as a function of LU treatment and time of day of proestrus-estrus (Experiment II). ee legend to Fig. 4 for further details.

5 824 CHWARTZ AND TALLEY I OoI,ne t Coo/rats Cord-a/s TT loc( I, 2* 2 2*.4. 4 PBPBLHA A A A A FIG. 7. erum estradiol as a function of LH treatment and time of day of proestrus-estrus (Experiment II). ee legend to Fig. 4 for further details. strate release of LH after injection. erum FH (Fig. 5) rose slowly in the LU-injected rats; the values were lower than saline controls at 183 h, equaled that level at 23 h, synchronously peaked at 4 h and dropped toward normal at 1 h estrus. The FH values for the 3 treatment groups at 1 h resemble those seen in corresponding groups in Experiment I (Figs. 1-3). Progesterone rose more abruptly in the LH injected rats than in the controls, probably reflecting the earlier LH rise (Fig. 4), peaked at 183 h and then fell slowly throughout the rest of the time periods. erum estradiol levels (Fig. 7) in the LH injected rats closely resembled the controls at every time period. In the saline controls, ovulation was beginning at 4 h. ome ova in cumulus were found in the oviducts of most rats autopsied at this time, but there were still follicles containing oocytes; the granulosa cells surrounding the oocytes had loosened and the cumulus mass was moving toward the stigma. The LU-injected rats were in the same stage of partial ovulation at this time. By 1 h, ovulation was complete in controls and complete or almost so in LUinjected rats. While this time of ovulation is later than previously reported for 4-day rats (Everett, 1961), it resembles a recent study of 4-day cycling rats in our colony (Nequin, Alvarez and chwartz, unpublished observations). In all rats in Experiment II, regardless of treatment, uterine intraluminal fluid was >1 mg ( ballooned ) before 1 h, by which time the water had been released in most rats. The early rise in serum progesterone after LU injection (Fig. 6), coupled with the failure of LU to enhance FH increase after ovariectomy (Fig. 3), induced us to test the effects of progesterone injection (Experiment Ill) itself on serum FU, since we have shown that the adrenal releases a progesterone surge after the stress of ovariectomy (Campbell et al., 1977). The results of this final experiment are seen in Figs Rats treated with PB alone in this experiment showed essentially the same time course of hormones as seen in the PB treated rats in Experiment II: LH - no change (Fig. 4 vs 8); FH - some rise and then a fall (Fig. 5 vs 9); progesterone - a decline from h (Fig. 6 vs 1);estradiol -gradual decline from 183 h to 1 h (Fig. 7 vs 11). Progesterone treatment did not induce LH secretion (Fig. 8). FU was significantly (twoway analysis of variance) higher from 23-1 h, after progesterone injection (Fig. 9), but did not reach the FU elevation seen after LH injection, at 4 h and 1 h (Fig. 5), in spite of our success in elevating serum progesterone levels (Fig. 1) close to the pattern seen after LU injection (Fig. 6). Progesterone significantly (two-way analysis of variance) suppressed serum estradiol values (Fig. 11) to levels close to those which were seen after the natural surge or the LU injection (Fig. 7), however, the drop occurred after 183 h, rather than before that time, as it had in the former experiment (Fig. 7). The progesterone injection at 163 h did not induce ovulation, except for 1 progesteroneinjected rat which had ovulated by 1 h. No other rat in this group showed stimulated follicles or ova. Uterine intraluminal water was not reduced below 1 mg before 4 h follow- 4 ( fr---p8. PROG P8.-Oil boo T.o4 2 22oo, 242 * o4*o 4o I2 IcJ,po PB PBPROG A A A A FIG. 8. erum LU as a function of progesterone treatment and time of day of proestrus-estrus (Experiment III). PB injections as described in legend to Fig. 4. Progesterone (2.5 mg) or peanut oil injected s.c. at 163 h. Mean ± EM shown; N = 5/point.

6 LU AND FH INDUCE FH ECRETION A-A P8,,1 #{163}---.&P9,. PROG 8 7 A- P8.-Oil P8#PRO& 4O.5 z * 2 Ui I;; i4 4 3 * 2 1 jmoj #{176}r#{176} IOd 22 22j24 oooo o?o PB PBPROG A A A A FIG. 9. erum FI-I as a function of progesterone treatment and time of day of proestrus-estrus (Experiment II). ee legend to Fig. 8 for further details. ing progesterone, but at 4 h 2/5 animals were below this criterion and 5/5 were below at 1 h estrus, suggesting a latency for progesterone of about 12 h (Kennedy and Armstrong, 1975). DICUION The lack of change in terminal pituitary LH content after LU or FH injection or in serum LH after LU injection (Fig. 4), combined with the rise in serum FH (Figs. 1-3, 5) suggest very strongly that the gonadotrophic hormones, when injected in a single ovulatory dose, trigger endogenous FH release in the absence of endogenous LH release. While the half life of rat LU PB PBPROG A A A.. FIG. 1. erum progesterone as a function of progesterone treatment and time of day of proestrusestrus (Experiment III). ee legend to Fig. 8 for further details. A PBPBPROG A A A A FIG. 11. erum estradiol as a function of progesterone treatment and time of day of proestrus-estrus (Experiment III). ee legend to Fig. 8 for further details. is only 2-3 mm in comparison to rat FH, which is mm (Larochelle and Freeman, 1974), we are confident that had there been a significant release of LH it would have been detected either by pituitary or serum sampling. A priori there are 2 levels of action by which an artificial gonadotrophin surge (or the normal surge) could trigger FH release: a) via a central effect on the hypothalamic-pituitary axis or b) via an indirect effect on the adrenal and! or ovary, altering the level of some feedback steroidal or nonsteroidal signal to the pituitary or hypothalamus. With respect to the hypothesis that the LU might elicit FU release by a central effect, it should be noted that the search for such internal feedback of gonadotrophins has either not elicited evidence of its existence (Gay, 1974) or has demonstrated a negative feedback of LU or FH (Motta et al., 1969;Turgeon and Barradough, 1976). The recent evidence (Chappel and Barraclough, 1976) that FH can be released by hypothalamic stimulation without LU, supports present observations of independence of release, but does not suggest a site of action of the injected LU. The observation that neither LU nor FU can enhance FH release in the absence of the ovary (Fig. 3), suggests that the hormones may act via the ovaries to reduce a negative feedback signal or that the pituitary was already secreting FH at a maximal acute rate as the result of ovariectomy (Nequin et al., 1975; Campbell et al., 1977) and could not respond further at that time. The data in Experiment IIC (Fig. 3) rule out mjoo

7 826 CHWARTZ AND TALLEY the adrenal as necessary for the LH induction of FH secretion. ince the adrenal releases progesterone on the day of proestrus (Nequin et al., 1975; Campbell et al., 1977) the enhancement of water retention seen in these rats at 1 h (>1 mg), suggests that secretion of progesterone from the adrenal, as well as the ovary, may normally be responsible for the loss of this water between proestrus and estrus (Kennedy and Armstrong, 1975). Thus, we are led to the hypothesis that LH or FH induce FH secretion indirectly via the ovary. LU can cause completion of oocyte maturation (Tsafriri et al., 1976b), ovulation (chwartz et al., 1975), luteinization (chwartz and Ely, 197; Channing, 197), increase or decrease of estradiol secretion (chwartz et al., 1975; Mills, 1975; Leiberman et al., 1975; Uillensjo et al., 1976; Katz and Armstrong, 1976; Fig. 7), increased progesterone secretion (Fig. 6; Hillensjo et al., 1976; Uchida et al., 1968), increased androstenedione and testosterone production (Mills, 1975; Lieberman et al., 1975; Hillensjo et al., 1976;Armstrong and Papkoff, 1976) or decreased testosterone conversion by aromatase to estrogen (Katz and Armstrong, 1976; Armstrong and Papkoff, 1976). The apparent discrepancy in the effects of LH on estradiol secretion is dependent on how mature follicles are when the LH is tested. The mature follicle, stimulated by high surge LH levels, shows consistent enhancement of progesterone secretion and decreased androgen and estradiol secretion (Fig. 7), whereas LU induction of increased estrogen production is probably a function of a smaller, growing follicle and also of lower LU levels (chwartz and Ely, 197; Lieberman et a!., 1975; Hillensjo et al., 1976; Armstrong and Papkoff, 1976). With respect to the effects of FH (Figs. 1-3) on the proestrous ovary, while the hormone may be less potent than LU in inducing ovulation and estrogen and progesterone secretion, it does appear to have enough intrinsic activity to replicate the effects of LH on these events (chwartz et al., 1975; Mills, 1975; Tsafriri et al., 1976a; trickland and Beers, 1976). If LU or FH increase FU secretion indirectly via the ovary (Fig. 3), which of the ovarian signals just discussed seems most probable as the intermediary? The present data suggest that the increase in progesterone is not the signal for this function (Fig. 9). Is the drop in estradiol which occurs following the natural surges or the injection of LH (Fig. 7) responsible for the rise in serum FU? As in other studies (Daane and Parlow, 1971; Butcher et a!., 1974), the present one showed that pentobarbital neither blocked the estradiol drop completely (Figs. 7, 11) nor prevented the secondary FH rise completely (Figs. 5, 9). However, several observations suggest that the lowering of estradiol is not the signal. Progesterone, superimposed on pentobarbital, significantly suppressed estradiol to control levels (Fig. 11), without elevating FH to levels seen after the control surges or the artificial LU surge. Furthermore, ovariectomy on the day of proestrus strongly stimulates FU secretion, in the presence of normal estradiol levels, because of the adrenal secretion of estradiol (Nequin et al., 1975; Campbell et a!., 1977). A single injection of estradiol benzoate (1 g) in intact rats elevated FH secretion, rather than inhibiting it (Kaira et al., 1973). Finally, Chappel and Barraclough (1977) could not inhibit LHinduced FH secretion, in a test model similar to that of the present study, by estradiol injection. With respect to serum testosterone as a possible intermediate signal, Gay and Tomacari (1974) observed that antiserum to testosterone on the afternoon of the day before proestrus blocked the secondary, but not the primary, elevation of FH. These authors suggested that the elevation of serum testosterone seen early on proestrus, in contrast to other days of the cycle (Dupon and Kim, 1973) may be necessary for the secondary rise in FU. A single injection of testosterone propionate (small doses) does tend to elevate serum FU in the ovariectomized rat (Kalra et al., 1973). On the other hand, acute ovariectomy plus adrenalectomy induce rapid serum FH rises while serum testosterone becomes undetectable (Campbell, chwartz, Firlit, unpublished observations, Fig. 3). It may be that the mechanism of the antiserum to testosterone (Gay and Tomacari, 1974) is not by removing circulating testosterone, but by altering intraovarian events in which testosterone may be involved, with the peripheral signal to the hypothalamus-pituitary axis being another substance. It is possible that another ovarian steroidal or nonsteroidal signal is involved, both with the LH induction of FH release (Fig. 5), as well as the FU increase which follows ovariectomy (Fig. 3; Campbell et a!., 1977). Follicular fluid inhibits the spontaneous oocyte meiosis

8 LH AND FH INDUCE FH ECRETION 827 which occurs when the oocyte is removed from the follicle; LU can overcome this inhibitory effect (Tsafriri et al., 1976a). We have shown (chwartz and Channing, 1977) that steroid free porcine follicular fluid injected into a rat immediately after LH injection (as in Experiment 11), completely blocked the rise in serum FU seen at 4 h estrus and also that such fluid can prevent the acute, postovariectomy rise in serum FU (Marder et a!., 1977). Thus, both LU (Fig. 5) and ovariectomy (Fig. 3) may enhance FU secretion by altering or suppressing a nonsteroidal follicular signal which serves as a negative feedback on FH secretion rate. It is possible that antiserum to testosterone could have locally enhanced the secretion of this signal (Gay and Tomacari, 1974). Regardless of the mechanism(s) by which exogenous gonadotrophic hormone or the endogenously released abrupt, short-lived surge of LU and FH, cause the delayed, prolonged FH release, the adaptive significance of the secondary FH release appears to be that of preparing cohorts of follicles to develop for the following cycle(s) (chwartz, 1969; chwartz et a!., 1973; Welschen and Dullaart, 1976). This mechanism can have particular importance in short cycle animals like the polyestrous rodents. An additional implication of the prolonged FH secretion may be to add a failsafe mechanism in completion of ovulation (trickland and Beers, 1976). AC KNOWLEDGMENT We wish to thank Julian Alvarez and Brigitte G. Mann for excellent technical assistance. We would like to acknowledge Dr. Gordon Niswender for supplies of antibody #15 for : LH RIA, antibody #244 for estradiol RIA and antibody #869 for progesterone RIA; Dr. Leo Reichert, Jr., for ovine LU for iodination; NIAMDD for NIH-LH-14 and 16 used for injection and assay standards and the R:R FH assay kits. Most of the support for these studies came from PH UD 754 and NIH Research Contract We would like to thank Dr. Marianne Firlit, who designed and executed Experiment IC. REFERENCE Armstrong, D. T. (1968). Hormonal control of uterine lumen fluid retention in the rat. Am. J. Physiology 214, Armstrong, D. T. and Papkoff, U. (1976). timulation of aromatization of exogenous and endogenous androgens in ovaries of hypophysectomized rats in vivo by follicle-stimulating hormone. Endocrinology99, Butcher, R. L., Collins, W. E. and Fugo, N. W. (1974). Altered secretion of gonadotropins and steroids resuiting from delayed ovulation in the rat. Endocrinology 96, Campbell, C.., chwartz, N. B. and Firlit, M. G. (1977). The role of adrenal and ovarian steroids in the control of serum LH and FH. Endocrinology 11, Channing, C. P. (197). Influences of the in vivo and in vitro hormonal environment upon luteinization of granulosa cells in tissue culture. Rec. Prog. Horm. Res. 26, Chappel,. C. and Barraclough, C. A. (1976). Hypothalamic regulation of pituitary FU secretion. Endocrinology 98, Chappel,. C. and Barraclough, C. A. (1977). Further studies on the regulation of FH secretion. Endocrinology 11, Daane, T. A. and Parlow, A. F. (1971). Periovulatory patterns of rat serum follicle stimulating hormone and luteinizing hormone during the normal estrous cycle. effects of pentobarbital. Endocrinology 88, Dupon, C. and Kim, M. H. (1973). Peripheral plasma levels of testosterone, androstenedione, and oestradiol during the rat oestrous cycle. J. Endocrinol. 59, Everett, J. W. (1961). The mammalian female reproductive cycle and its controlling mechanisms. 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