SUMMARY. at metoestrus and dioestrus, and could not be demonstrated at oestrus. to the

Transcription

1 A PRIMING EFFECT OF LUTEINIZING HORMONE RELEASING FACTOR ON THE ANTERIOR PITUITARY GLAND IN THE FEMALE RAT M. S. AIYER, SHARON A. CHIAPPA and G. FINK Department of Human Anatomy, South Parks Road, Oxford, 0X1 3QX (Received 14 December 1973) SUMMARY The possibility that luteinizing hormone releasing factor (LH-RF) not only stimulates the secretion of luteinizing hormone (LH) but also has the ability to prime the anterior pituitary gland so that further exposure to LH-RF enhances the responsiveness of the gonadotrophs has been investigated. The effect of two successive i.v. injections of the same dose of LH-RF (50 ng/100 g body weight) on the concentration of LH in plasma was determined in rats anaesthetized with sodium pentobarbitone. Blood samples were collected from the external jugular vein immediately before and at frequent intervals after the injections of LH-RF, and plasma concentrations of LH and, in some samples, follicle-stimulating hormone (FSH) were measured by radioimmunoassay. In rats anaesthetized with sodium pentobarbitone at h of pro-oestrus and injected i.v. with two successive doses of LH-RF, separated by an interval of 30, 60, 120 or 240 min, the LH response to the second injection was significantly greater than that to the first. The LH response was greatest when the two doses were separated by an interval of 60 min. The FSH response to the second injection of LH-RF given 60 min after the first was not significantly different from that to the first injection. Compared with pro-oestrus, the priming effect of LH-RF was much less at metoestrus and dioestrus, and could not be demonstrated at oestrus. to the Ovariectomy on the morning of dioestrus reduced the LH responses first and second injections of LH-RF given 60 min apart on the afternoon of pro-oestrus, an effect which was partially reversed by administration of oestradiol benzoate shortly after ovariectomy. These findings together with the fact that administration of oestradiol benzoate at metoestrus increased that the the magnitude of the priming effect of LH-RF at dioestrus suggest response at pro-oestrus is dependent upon the rise in plasma oestradiol-17\g=b\ which reaches a peak on the morning of pro-oestrus. Although oestrogen plays an important role in determining the magnitude of the priming effect of LH-RF, it appears that none of the steroids secreted by either the ovaries or the adrenal glands mediates this effect since the profile and magnitude ofthe LH responses to the two doses of LH-RF injected

2 at pro-oestrus into rats adrenalectomized and ovariectomized before the first injection of LH-RF were comparable to those in control animals. The possible role which the priming effect of LH-RF may play in the development of the preovulatory surge of LH in the rat and man is discussed. INTRODUCTION In a previous study (Aiyer, Fink & Greig, 1973, 1974) on the increase in sensitivity of the anterior pituitary gland to synthetic luteinizing hormone releasing factor (LH-RF) which occurs at pro-oestrus, it was noted that during the period of the preovulatory surge of luteinizing hormone (LH) there was a significant positive correlation between the levels of LH in plasma before and the maximal concentrations of LH in plasma after injection of the factor. It was found also that in animals anaesthetized with sodium pentobarbitone at h of pro-oestrus the LH response to an injection of LH-RF at h of the same day was significantly lower than in animals anaesthetized 30 min before injection of the factor at h. These obser vations suggested that endogenous LH-RF acting on an anterior pituitary gland sensitized by sex steroid hormones may exert a priming effect on the pituitary gonadotrophs so that further exposure to the factor is followed by a marked increase in response. This possibility was supported by preliminary findings that the LH response to the second of two injections of synthetic LH-RF administered during the afternoon of pro-oestrus was significantly greater than the LH response first injection (Aiyer, Chiappa, Fink & Greig, 1973). These studies have been extended, and the results reported in this paper indicate that the priming effect of LH-RF on the anterior pituitary gland together with the potentiating effect of sex steroid to the hormones (Aiyer & Fink, 1974) may play an important role in the development of the preovulatory surge of LH in the rat. MATERIALS AND METHODS Adult female Wistar rats, weighing g, supplied by Charles River (U.K.) Ltd, were maintained as described in a preceding paper (Aiyer & Fink, 1974). Synthetic LH-RF was stored and prepared for injection as described in the pre ceding paper (Aiyer & Fink, 1974). After the induction of anaesthesia by sodium pentobarbitone (Veterinary Nembutal, Abbott Laboratories Ltd, 36 mg/kg body wt, i.p.) two doses of LH-RF (each containing 50 ng/100 g body wt in ml 0-9% saline) were injected into the external jugular vein at the intervals mentioned under Results. Small volumes of blood (approx ml) were withdrawn from the external jugular vein immediately before and 20 min after the first injection of LH-RF as well as before and at 15, 20, 30 and 60 min after the second injection of the factor. Each blood sample was collected in a heparinized 1-ml disposable plastic syringe and centrifuged. The plasma was stored at 15 C until shortly before assay. The determination of plasma LH and follicle-stimulating hormone (FSH) by radio immunoassay and statistical analysis of results are described in the preceding paper (Aiyer & Fink, 1974) with the exception that plasma FSH was estimated using single 200 A aliquots.

3 Priming effect of LH-RF 575 RESULTS Effect of two successive injections of LH-RF on the afternoon of pro-oestrus On the LH response Groups of rats were anaesthetized at h of pro-oestrus and min later were injected i.v. with 50 ng LH-RF/100 g body wt. The same dose of LH-RF was injected again 30, 60, 120 or 240 min after the first injection. The effect of successive injections of LH-RF on plasma LH concentrations in the various groups of animals (Fig. 1) was that 20 min after the first injection there was an increase in plasma LH concentration which subsequently declined. After (i 30 i V 60 i i 120 i ; 240 min i Time (min) Fig. 1. Mean plasma luteinizing hormone (LH) concentrations (ng NIH-LH-S13/ml) after two successive i.v. injections of 50 ng luteinizing hormone releasing factor (LH-RF)/100 g body weight. The first dose of LH-RF was injected min after the administration of sodium pentobarbitone at h of pro-oestrus and the second dose of LH-RF was injected either 30 ( - ), 60 (O-O), 120( - ) or 240 min ( - } after the first. Vertical bars indicate + s.e.m. the second injection of LH-RF there were further increases in plasma LH con centration in each group. The profiles of the mean plasma LH concentrations after the second injection were similar in the four groups of rats, reaching a peak value min after injection and thereafter declining. The mean maximal increments in plasma LH concentration after the two injections of LH-RF are shown in Table 1. There were no significant differences between the groups in response to the first injection of LH-RF, and in all groups the LH responses to the second injection of LH-RF were significantly greater than those which followed the first injection (see Table 1 for levels of significance). The response to the second injection in the group injected with LH-RF 60 min after the first (individual LH profiles shown in Fig. 2)

4 576 M. S. Aiyer, Sharon A. Chiappa and G. Fink was significantly greater compared with those after the second injection given 30 or 240 min after the first (P < 0-05 and < 0-01, respectively). There were no significant increases in plasma LH concentrations after the i.v. injection of 0-5 ml 0-9% saline either 60 min before or after the injection of LH-RF (Table 2). Table 1. Maximal increments in plasma luteinizing hormone (LH) concentration (ng NIH-LH-S13/ml) and ovulation after two successive i.v. injections of 50 ng lutein izing hormone releasing factor (LH-RF)/100 g body wt separated by an interval of 30, 60, 120 or 240 min. The first dose of LH-RF was injected min after the administration of sodium pentobarbitone at h of pro-oestrus. The animals were killed on the next morning and number of ova in the oviducts counted (group means ± S.E.M.) Interval between two injections of LH-RF (min) No. of rats Maximal increments in plasma LH concn (ng/ml) after: First injection 21-3 ± Second injection * *** ** *** No. of rats ovulating No. of ova/ ovulating rat * P < 0-05; ** P < 0-01; *** P < 0-001: Significance of the difference between the responses to the first and second injections of LH-RF in each group of animals, determined by the paired i-test. Significance of the difference between the means of the maximal increments after the second injection of LH-RF (Duncan's multiple range test): P < 005: 60 min v. 30 min. P < 0-01: 60 min v. 240 min. Table 2. Maximal increments in plasma luteinizing hormone (LH) concentration (ng NIH-LH-S13/ml) and ovulation after either two i.v. injections of 50 ng luteinizing hormone releasing factor (LH-RF)/100 g body wt separated by an interval of 60 min or the i.v. injection of 0-5 ml saline 60 min before or after a single injection of 50 ng LH-RF/100 g body wt. The first injection of LH-RF or saline was given min after the administration of sodium pentobarbitone at h of pro-oestrus. The animals were killed on the next morning and the number of ova in the oviducts counted (group means + s.e.m.) First injection LH-RF Saline LH-RF Treatment Second injection LH-RFf LH-RF Saline No. of rats Maximal increments in plasma LH concn (ng/ml) after: First injection ND Second injection *** 19-3 ± 40** ** No. of rats ovulating 9 6 No. of ova/ ovulating rat ** P < 0-01; *** P < 0-001: Significance of the difference between the responses to the first and second injections in each group of animals, determined by the paired i-test. Significance of the difference between the means of the maximal increments (Duncan's multiple range test): After the first injection P < 0 001: Saline-LH-RF v. other two groups. After the second injection P < 0-001: LH-RF-LH-RF v. other two groups. t Data for this group taken from Table 1. ND = < 1 ng/ml in all animals of the group.

5 Priming effect of LH-RF 577 Ova were found in the oviducts of each animal on the morning after the injection of LH-RF. The means of the number of ova per ovulating rat in the various groups were not significantly different from each other (see Tables 1 and 2) Time (min) Fig. 2. Plasma luteinizing hormone (LH) concentrations (ng NIH-LH-S13/ml) in individual animals injected i.v. with two successive doses of 50 ng luteinizing hormone releasing factor (LH-RF)/100 g body wt, 60 min apart. The first dose of LH-RF was injected min after the administration of sodium pentobarbitone at h of pro-oestrus. On the FSH response The volume of plasma that remained after the assay for LH was insufficient to permit a meaningful determination of plasma FSH concentrations in the majority of these samples. Therefore, another group of six rats was used to compare the effect of two successive injections of LH-RF on plasma FSH and LH concentrations. Animals were anaesthetized with sodium pentobarbitone at h of pro-oestrus

6 578 M. S. Aiyer, Sharon A. Chiappa and G. Fink 60 min Time (min) Fig. 4 Fig. 3. Plasma (i) follicle-stimulating hormone (FSH) (ng NIAMD-rat-FSH-RPl/ml) and (ii) luteinizing hormone (ng NIH-LH-S13/ml) concentrations in individual animals (solid lines) injected i.v. with two doses of 50 ng luteinizing hormone releasing factor (LH-RF)/100 g body wt. The mean plasma hormone concentrations are shown by the interrupted line. The first dose of LH-RF was injected min after the administration of sodium pentobarbitone at h of pro-oestrus. The letters on the right of the profiles indicate the individual animal, permitting a comparison of the FSH and LH responses in the same rat. Fig. 4. Mean plasma luteinizing hormone (LH) concentrations (ng NIH-LH-S13/ml) after two successive i.v. injections of 50 ng luteinizing hormone releasing factor (LH-RF)/100 g body wt, 60 min apart. The first dose of LH-RF was injected min after the administration of sodium pentobarbitone at h on each day of the cycle. Vertical bars indicate + s.e.m., pro-oestrus; O, oestrus;, metoestrus; x, dioestrus. Nine animals studied at pro-oestrus; six animals in other groups.

7 Priming effect of LH-RF 579 and min later were injected with LH-RF; the same dose of the factor was injected 60 min after the first. Blood samples were collected immediately before and 30 min after each injection of LH-RF as well as 60 min after the second in jection. Since fewer samples were collected, a larger volume of blood ( ml) was obtained at each interval so that there was sufficient plasma to measure both LH and FSH in the same samples. Table 3. Maximal increments in plasma luteinizing hormone (LH) concentration (ng NIH-LH-S13/ml) and ovulation after two i.v. injections of 50 ng luteinizing hormone releasing factor (LH-RF)/100 g body wt 60 min apart. The first dose of LH-RF was injected min after the administration of sodium pentobarbitone at h of each day of the oestrous cycle. The animals were killed on the morning after the injections of LH-RF and the number of ova in the oviduct were counted (group means ± S.E.M.) Maximal increments in plasma LH concn (ng/ml) after: No. of No. of No.,-*-, rats ova/ovulating Stage of cycle of rats First injection Second injection ovulating rat Dioestrus ** 3f 11-3 ± 1-2 Pro-oestrusî ± *** Oestrus ± Metoestrus ± 5-1* 0 * P < 002; ** P < 0-01; *** P < 0-001: Significance of the difference between the first and second injections of LH-RF in each group of animals, determined by the paired i-test. Significance of the difference between the means of the maximal increments (Duncan's multiple range test) : After the first injection P < 0-01: Oestrus v. dioestrus. P < 0-005: Pro-oestrus v. metoestrus. P < : Pro-oestrus v. dioestrus. After the second injection P < 0-005: Pro-oestrus v. oestrus; metoestrus; dioestrus. t One animal in this group died overnight. J Data taken from Table 1. Figure 3 shows the profiles of plasma FSH and LH in individual animals indicating that the second injection of LH-RF had a more marked effect on LH than on FSH concentrations. Plasma LH levels increased above preinjection values 30 min after the first injection of LH-RF and had decreased after 60 min. In contrast, plasma FSH levels were greater 60 min after the first injection compared with the level after 30 min. Thirty minutes after the second injection of LH-RF the mean concentration of both gonadotrophins in plasma was greater than the levels immediately before this injection of the factor, although the increase was significant (P < 0-001) only for LH. Plasma levels of LH and FSH were lower 60 min after the second injection compared with those 30 min earlier. The mean maximal increment in LH ( (s.e.m.) ng/ml) after the second injection of LH-RF was significantly greater (P < 0-001) compared with that ( ng/ml) after the first, whereas the mean maximal increments in FSH after each injection of LH-RF (221-9 ± 36-1 and ng/ml after the first and second injections respectively) were not significantly different from each other.

8 580 M. S. Aiyer, Sharon A. Chiappa and G. Fink Effect of two successive doses of LH-RF on other days of the oestrous cycle The effect of two successive injections of LH-RF was also examined at other stages of the oestrous cycle, when the hormonal environment before induction of anaesthesia was different from that on the afternoon of pro-oestrus. Rats were anaesthetized with Nembutal at h of oestrus, metoestrus and dioestrus. Thirty to sixty minutes after the induction of anaesthesia the standard dose of LH-RF was injected i.v. followed 60 min later by a second injection of the same dose. The profiles of plasma LH concentration after the two injections of LH-RF on the 4 days of the cycle (Fig. 4) show that in all groups there was an increase in plasma LH concentration after each injection of LH-RF, and that the magnitude of these responses was greatest on the day of pro-oestrus. Table 3 shows the mean maximal increment in plasma LH concentration after two injections of LH-RF in each group of rats. There was a significant increase in the LH response to the second in jection of LH-RF compared with that to the first in the pro-oestrous, metoestrous and dioestrous groups of animals (P < 0-001, < 0-02 and < 0-01, respectively) but not in the oestrous animals. Whereas the LH response to the first injection of LH-RF was significantly greater (P < 0-01) in the oestrous compared with that in the dioestrous group of animals, the mean LH response to the second injection was lower, although not significantly, in the rats at oestrus compared with that in the dioestrous animals. On the morning after the injection of the two doses of LH-RF the rats were killed and their reproductive tracts examined. In the animals given LH-RF at dioestrus, ova were found in the oviducts of three out of five rats which survived until the rat = (s.e.m.)). In following morning (mean number of ova/ovulating all five rats the vaginal smear consisted of nucleated epithelial cells, and the uterus was not distended with fluid. No ova were found in rats injected with LH-RF at oestrus or metoestrus, and in these animals the uterus was not distended with fluid, and the vaginal smear consisted predominantly of leucocytes. These results suggested that changes in the concentration of sex steroid hormones in blood may play an important role in determining the magnitude of the LH re sponses to the two injections of LH-RF and, therefore, the effects of administration of oestrogen and progesterone were examined. Effect of manipulation of sex steroid hormones on the LH responses to two successive doses of LH-RF After the administration of oestrogen or progesterone at metoestrus Groups of rats were injected s.c. with either 0-2 ml vehicle (ethyl oléate : arachis oil, 1:4, v/v), 10 fig oestradiol benzoate or 2-5 mg progesterone at h of met oestrus. The following day the animals were anaesthetized with Nembutal at h and then injected i.v. with two successive doses of LH-RF separated by an interval of 60 min. Figure 5 shows that in each group an increase in plasma LH followed both the first and second injections of LH-RF. The mean maximal increments in plasma LH concentration after two injections of LH-RF are presented in Table 4

9 Priming effect of LH-RF 581 and show that in each group the response to the second injection was significantly greater compared with that to the first. However, the LH responses to both the first and second injections were significantly greater (P < 0-001) in animals pretreated with oestradiol benzoate compared with those in animals receiving either progesterone or vehicle. There were no significant differences in the responses to the first or the 60 min Time (min) Fig. 5. Mean plasma luteinizing hormone (LH) concentrations (ng NIH-LH-S13/ml) after two successive i.v. injections of 50 ng luteinizing hormone releasing factor (LH-RF)/100 g body wt, 60 min apart. The animals were injected s.c. with 0-2 ml oil ( x ), 10 fig oestradiol benzoate (O) or 2-5 mg progesterone (0) at h of metoestrus. The first dose of LH-RF was injected min after the administration of sodium pentobarbitone at h of dioestrus. Vertical bars indicate + s.e.m. Table 4. Maximal increments in plasma luteinizing hormone (LH) concentration (ng NIH-LH-S13/ml) and ovulation after two successive i.v. injections of 50 ng lutein izing hormone releasing factor (LH-RF)/100 g body wt 60 min apart. The animals were injected s.c. with 0-2 ml vehicle, 10-0 ig oestradiol benzoate (OB) or 2-5 mg progesterone (Prog.) at h of metoestrus. The first dose of LH-RF was injected min after the administration of sodium pentobarbitone at h of dioestrus. The animals were killed on the next morning and the number of ova in the oviducts counted (group means ± S.E.M.) Vehicle OB Prog. Treatment No. of rats 6 e Maximal increments in plasma LH concn (ng/ml) after: First injection Second injection ** 48-0 ± 4-5*** ** ^ No. of rats ovulating No. of ova/ovulating rat ** P < 0-01; *** P < 0-001: Significance of the difference between the responses to the first and second injections of LH-RF in each group of animals, determined by the paired i-test. Significance of the difference between the means of the maximal increments (Duncan's multiple range test): After both first and second injection P < 0001: OB v. oil; prog. 15, 5

10 582 M. S. Aiyer, Sharon A. Chiappa and G. Fink second injections of LH-RF between animals receiving either progesterone or vehicle although the response to the second injection was lower in the progesterone-treated animals. On the following morning no ova were found in the oviducts of any of the rats treated with oestradiol benzoate; in these animals the uterus was ballooned and the vaginal smear consisted entirely of cornified cells. In rats treated with vehicle alone, ova were found in two (15 and 5 ova) out of six animals, and in all of them the vaginal smear consisted of nucleated cells; the uterus was not ballooned in any of these rats. In rats pretreated with progesterone no ova were found in the oviducts, the uterus was not distended with fluid and the vaginal smear consisted of a mixture of nucleated cells and leucocytes. After ovariectomy, with and without oestrogen replacement, at dioestrus Rats were subjected to bilateral ovariectomy, under ether anaesthesia between and h of dioestrus. Immediately after the operation they were injected s.c. with either 0-2 ml vehicle (ethyl oléate : arachis oil; 1:4, v/v) or 2-5 / g oestradiol benzoate. On the following day (expected day of pro-oestrus) the rats were anaes thetized with Nembutal and min later were injected with two doses of LH-RF given 60 min apart. In rats treated with oestradiol benzoate the LH response to the first and second injections of LH-RF ( and ng/ml respectively; n = 6) were significantly greater (P < 0-01) compared with the corresponding responses ( and ng/ml, respectively; n = 5) in animals treated with vehicle alone. In both groups the LH response to the second injection was significantly greater (P < 0-01) compared with that to the first. In all rats treated with oestradiol benzoate and killed on the morning after the injection of LH-RF, the vaginal smear consisted entirely of cornified cells and the uterus was distended with fluid. In rats which received the vehicle alone leucocytes predominated in the vaginal smear and the uterus was not distended with fluid. Do steroid hormones mediate the augmented LH response to LH-RF? The possibility that the augmented response to the second injection of LH-RF may have been mediated by either ovarian steroids secreted as a consequence of the increase in plasma LH concentration after the first injection and/or adrenal steroids secreted in response to the stress of operation and blood loss was examined. Effects of ovariectomy and adrenalectomy on the LH responses to two doses of LH-RF at pro-oestrus Rats were subjected to either bilateral ovariectomy or sham ovariectomy im mediately before the first injection of LH-RF and were injected with the second dose 60 min later. Table 5 shows that in both ovariectomized and sham-operated rats the LH response to the second injection was significantly greater compared with that to the first injection. The LH response to the second injection was greater, though not significantly, in the ovariectomized group compared with that in the shamoperated animals.

11 Priming effect of LH-RF 583 The possible role of adrenal steroid hormones was investigated by subjecting rats, which had previously shown at least two regular 4-day cycles, to either bilateral adrenalectomy or sham adrenalectomy under ether anaesthesia on the day of metoestrus (at which time there is the least interference with the duration of the oestrous cycle). The rats were allowed to recover from the operation and were used only after they had shown at least one 4-day cycle after the operation. During the postoperative period, the sham-adrenalectomized rats were permitted to drink a solution containing 1 g chlortetracycline/litre tapwater and the adrenalectomized animals were maintained on a solution containing 1 g chlortetracycline/litre 0-9 % NaCl solution. On the day of pro-oestrus the animals were anaesthetized with Nembutal at h and min later were injected with LH-RF followed by a second injection 60 min later. In each rat the concentration of corticosterone in the plasma of the blood sample collected 60 min after the second injection of LH-RF was determined by a competitive protein-binding assay. In animals subjected to Table 5. Maximal increments in plasma luteinizing hormone (LH) concentration (ng NIH-LH-S13/ml) and ovulation after two successive i.v. injections of 50 ng lutein izing hormone releasing factor (LH-RF)/100 g body wt 60 min apart. The animals were ovariectomized, sham ovariectomized, adrenalectomized or sham adrenalectomized, either singly or in various combinations as indicated. Adrenalectomized and shamadrenalectomized rats were used only after they had shown at least one 4-day cycle after the operation. Ovariectomy and sham ovariectomy were carried out immediately before the first injection of LH-RF which was given min after the administration of sodium pentobarbitone at h of pro-oestrus. The number of ova in the oviducts of those animals which survived until the following morning were counted (group means ± S.E.M.) Maximal increments in plasma No. LH concn (ng/ml) after: No. of No. of of,-*-, rats ova/ovulating Treatment rats First injection Second injection ovulating rat Ovariectomy **** Sham ovariectomy ± ** Adrenalectomy Sham adrenalectomy *** Adrenalectomy plus ovariectomy ± * Adrenalectomy plus sham ovariectomy ** All adrenalectomized animals died during the night following the period of experimentation. * P ** < 005; P < 002; *** P **** < 001; P < 0001: Significance of the difference between the LH responses to the first and second injections of LH-RF in each group of rats, determined by the paired i-test. sham adrenalectomy the concentration of corticosterone in plasma was / g/100 ml whereas plasma corticosterone was not detectable ( < 5 / g/100 ml) in any of the adrenalectomized rats. The LH responses to the two injections of LH-RF in the sham-adrenalectomized and adrenalectomized animals are shown in Table 5. In both groups the LH response to the second injection of LH-RF was greater than that to the first. Table 5 also includes the results obtained in two further groups of

12 584 M. S. Aiyer, Sharon A. Chiappa and G. Fink adrenalectomized rats which were either bilaterally ovariectomized or sham ovari ectomized immediately before the first injection of LH-RF. The LH response to the second injection was significantly greater than that to the first in both groups of rats. Table 5 shows that there were no significant differences between the means of the maximal increments either after the first or after the second injection of LH-RF among the various groups of animals. The variability in the magnitude of the responses of the adrenalectomized rats is shown by the relatively large s.e.m. in these groups. Effect of progesterone on the LH responses to two injections of LH-RF at pro-oestrus An increased secretion of progesterone from the adrenal glands and the ovaries is known to occur before and during the LH surge (e.g. Feder, Brown-Grant & Corker, 1971). To determine directly the effects of this steroid, 2-5 mg progesterone, dissolved in 0-2 ml propylene glycol, was injected i.v. within 2 min of the first injection of LH-RF; control animals received 0-2 ml vehicle alone. The first dose of LH-RF was injected min after the administration of Nembutal at h of pro-oestrus and the second dose of the factor was injected 60 min after the first. The LH responses to either the first or the second injections of LH-RF ( and ng/ml, respectively; n 5) in animals treated with progesterone = were not significantly different from the corresponding responses ( and ng/ml, respectively; n 5) in the control = group. The LH response to the second injection was significantly greater compared with that to the first in both progesterone-treated and control rats (P < 0-02, < 0-002, respectively). The number of ova found in the oviducts on the next morning was 13-3 ± 0-6 in animals which received progesterone and in rats which were treated with propylene glycol alone. DISCUSSION These results indicate clearly that, in the rat, LH-RF not only stimulates the secretion of LH but also has the ability to prime the anterior pituitary gland so that further exposure to this factor enhances the responsiveness of the gonadotrophs. The magnitude of the priming effect of LH-RF depends on the stage of the oestrous cycle and on the interval between two injections of the factor: the greatest response occurred on the afternoon of pro-oestrus and when the two doses of LH-RF were separated by 60 min. The significantly greater response at pro-oestrus compared with those on the other days of the cycle appears to be dependent mainly upon the rise in plasma con centrations of oestradiol-11ß which reaches a peak on the morning of pro-oestrus (Brown-Grant, Exley & Naftolin, 1970; Shaikh, 1971), for while ovariectomy on the morning of dioestrus decreased the LH responses to the two injections of LH-RF given at pro-oestrus, the responses to both injections were increased significantly by treatment with 2-5 / g oestradiol benzoate immediately after ovariectomy. Additional support for this view is provided by the fact that oestradiol benzoate administered on the morning of metoestrus potentiated the priming effect of LH-RF at dioestrus. While oestrogen may play an important role in determining the magnitude of the

13 Priming effect of LH-RF 585 priming effect of LH-RF it is unlikely that it mediates this effect, since ovariectomy immediately before the first injection did not alter significantly the LH responses to either of the two injections of LH-RF. Furthermore, it seems unlikely that any other steroid (including progesterone) secreted by the ovaries or adrenal glands mediates the priming effect of LH-RF, for the profile and magnitude of plasma LH concentration which followed two injections of the factor in rats adrenalecto mized and ovariectomized before the first injection were comparable to those in control animals. Progesterone given immediately after the first injection of LH-RF did not appreciably affect the LH responses to either the first or second injections of the factor. The variability in the responses of the adrenalectomized groups of rats may have been due to the metabolic changes which follow a decrease in circulating glucocorticoids; it is relevant to mention here that Feder et al. (1971) have shown that treatment with dexamethasone after adrenalectomy increases the number of animals which exhibit regular oestrous cycles. We have not excluded the possibility that LH released in response to the first injection of LH-RF may have sensitized the gonadotrophs to the second injection. However, Gay, Niswender & Midgley (1970) have shown that exogenous LH does not alter significantly the LH responses to extracts of hypothalamic tissue injected into ovariectomized rats treated with oestrogen and progesterone. Indeed, the only available evidence in favour of an effect of LH on its own secretion suggests that it is of an inhibitory nature (Motta, Fraschini & Martini, 1969; Ojeda & Ramirez, 1969). Thus it seems likely that LH-RF itself sensitizes the gonadotrophs. The failure of Gay et al. (1970) to demonstrate convincingly a priming effect of LH-RF may be due to the fact that in their experiments extracts of hypothalamic tissue were injected into long-term ovariectomized rats treated with oestrogen and progesterone, and that the effects on plasma LH were examined only when the injections were separated by2h. The FSH response to the second injection of LH-RF was not significantly greater than that to the first when the two doses of the factor were given 1 h apart on the afternoon of pro-oestrus. While this does not exclude the possibility that under other conditions LH-RF may exert a priming effect on the FSH gonadotrophs, the temporal characteristics of such an effect are apparently different from those which apply to the LH gonadotrophs. What is the physiological significance ofthe priming effect of LH-RF on the pituitary gland? The finding that the magnitude of this effect was most marked on the after noon of pro-oestrus suggests that it plays an important role in the development of the second phase of increased pituitary sensitivity to LH-RF which occurs during the period of the preovulatory surge of LH (Aiyer et al. 1974). We suggested that this phase of increased sensitivity is important in determining the timing and magnitude of the LH surge. It is likely, therefore, that the priming effect of LH-RF is a major link in the chain of events which results in the LH surge. The concept that the LH surge is initiated by an increased secretion of LH-RF at nerve terminals in the median eminence has been accepted for many years (see reviews by Everett, 1964; Flerkó, 1966; Schally, Arimura & Kastin, 1973). The finding that LH-RF primes the LH gonadotrophs permits the speculation that only a relatively slight increase in LH- RF secretion may be necessary to initiate the LH surge. It is conceivable that during

14 586 M. S. Aiyer, Sharon A. Chiappa and G. Fink the critical period on the afternoon of pro-oestrus the priming effect of LH-RF is being established and that once the pituitary gland has been sufficiently primed the consequent increase in pituitary sensitivity results in the LH surge. The blockade of the surge by Nembutal, administered before the onset of the critical period, may, therefore, be the result of interference with the priming effect of LH-RF consequent on a decrease in the secretion of endogenous LH-RF. This possibility is supported by the finding that the administration of Nembutal immediately before the onset of the critical period inhibits the development of the second phase of increased sensitivity (Aiyer et al. 1974). It is of interest to note that it takes about 1 h for the priming effect to be fully established and that it may be elicited up to 4 h later, for these time periods are remarkably similar to the duration of the critical period and the LH surge respectively. Recent studies in man suggest that the priming effect of LH-RF may also be of clinical importance. The responsiveness of the pituitary gland to LH-RF is consider ably enhanced in patients with hypergonadotrophic hypogonadism (Roth, Kelch, Kaplan & Grumbach, 1972; Gual, 1973; Isurugi, Wakabayashi, Fukutani, Takayasu, Tamaoki & Okada, 1973; Siler & Yen, 1973) and is lowered in prepubertal males and in some, but not all, patients with hypogonadotrophic hypogonadism (Roth et al. 1972; Bell, Spitz, Slonim, Perlman, Segal, Palti & Rabinowitz, 1973; Isurugi et al. 1973; Yen, Rebar, VandenBerg & Judd, 1973; Zarate, Kastin, Soria, Canales & Schally, 1973). Roth et al. (1972) have suggested that the levels of endogenous LH-RF are raised in patients with hypergonadotrophic hypogonadism and are depressed in prepubertal males and cases of hypogonadotrophic hypogonadism. From this they infer that "The degree of prior exposure of the gonadotropes to endogenous LRF appears to affect both the magnitude and the nature of the FSH and LH responses to an acute dose of synthetic LRF". If a similar phenomenon occurs during the menstrual cycle, it is likely that in man, as in the rat, the priming effect of LH-RF may play a major role in the development of the preovulatory surge of LH. Our studies indicate that in determining the LH and FSH responses to two successive injections of LH-RF in man, the time-interval between the injections should be much shorter than the 24 and 48 h periods employed by Thompson, Arfania & Taymor (1973) and Roth, Kelch, Kaplan & Grumbach (1973) in their investigations of women with secondary amenorrhoea and children before and at puberty, respectively. It is of interest that in adult male subjects given three successive injections of 25 / g LH-RF at intervals of 60 min the concentrations of LH after the second and third injections are greater than after the first injection, although the three increments in LH do not seem to be appreciably different from each other (Wagner, Böckel, Hrubesch & Grote, 1973). The finding that ovulation was advanced by 24 h in five out of 11 rats not pretreated with sex steroid hormones and injected with two doses of LH-RF at dioestrus was unexpected, since it has been reported (van Rees, van Dieten, Bijleveld & Müller, 1968) that the amount of human chorionic gonadotrophin needed to induce ovulation at dioestrus-1 of a 5-day cycle is approximately 80 times greater than that required at pro-oestrus. In the rats in which ovulation was advanced, the maximal concen trations of LH after injection (16-0, 40-0, 35-5, 40-0 and 25-6 ng/ml) were not much greater than that suggested as the lowest ovulatory concentration of plasma LH

15 Priming effect of LH-RF 587 during the LH surge (Naftolin, Brown-Grant & Corker, 1972). This reinforces pre vious suggestions (Greig & Weisz, 1973 ; Aiyer et al. 1974) that the ovulatory threshold level of LH may be related to the total amount of the hormone reaching the ovaries rather than to the peak concentration of LH in plasma alone. However, ovulation was not advanced in any animal pretreated with oestradiol benzoate at metoestrus, although the LH responses (and hence, presumably, the amount of LH reaching the ovaries) were significantly greater than those in control rats, and this may have been due to the impairment of follicular growth which is caused by this steroid (Krey & Everett, 1973). We are grateful to Drs H. Gregory and A. L. Walpole (I.C.I. Ltd, Pharmaceuticals Division, Macclesfield) for the gift of synthetic LH-RF, and to Drs G. D. Niswender, L. E. Reichert, Jr and A. F. Parlow and the National Institute of Arthritis and Metabolic Diseases, Bethesda for generously providing materials used in the radioimmunoassays. This work was supported by a grant (G971/598/B) from the Medical Research Council to G.F. One of us (M.S. A.) thanks the Wellcome Trust for personal support. REFERENCES Aiyer, M. S., Chiappa, S. A., Fink, G. & Greig, F. (1973). A priming effect of luteinizing hormone releasing factor on the anterior pituitary gland in the female rat. J. Physiol., Lond. 234, 81P-82P. Aiyer, M. S. <fe Fink, G. (1974). The role of sex steroid hormones in modulating the responsiveness of the anterior pituitary gland to luteinizing hormone releasing factor in the female rat. J. Endocr. 62, Aiyer, M. S., Fink, G. & Greig, F. (1973). Sensitivity of the anterior pituitary gland to luteinizing hor mone releasing factor (LRF) during pro-oestrus in the rat. J. Physiol., Lond. 231, 32P-34P. Aiyer, M. S., Fink, G. & Greig, F. (1974). Changes in the sensitivity of the pituitary gland to luteinizing hormone releasing factor during the oestrous cycle of the rat. J. Endocr. 60, Bell, J., Spitz, I., Slonim, A., Perlman, A., Segal, S., Palti, Z. & Rabinowitz, D. (1973). Heterogeneity of gonadotropin response to LHRH in hypogonadotropie hypogonadism. J. clin. Endocr. Metab. 36, Brown-Grant, K, Exley, D. & Naftolin, F. (1970). Peripheral plasma oestradiol and luteinizing hormone concentrations during the oestrous cycle of the rat. J. Endocr. 48, Everett, J. W. (1964). Central neural control of reproductive functions of the adenohypophysis. Physiol. Rev. 44, Feder, H. H., Brown-Grant, K. & Corker, C. S. (1971). Pre-ovulatory progesterone, the adrenal cortex and the 'critical period' for luteinizing hormone release in rats. J. Endocr. 50, Flerkó, B. (1966). Control of gonadotropin secretion in the female. In Neuroendocrinology, Vol. 1, pp Eds L. Martini & W. F. Ganong. New York and London: Academic Press. Gay, V. L., Niswender, G. D. & Midgley, A. R., Jr (1970). Response of individual rats and sheep to one or more injections of hypothalamic extract as determined by radioimmunoassay of plasma LH. Endocrinology 86, Greig, F. <fc Weisz, J. (1973). Preovulatory levels of luteinizing hormone, the critical period and ovu lation in rats. J. Endocr. 57, Gual, C. (1973). Clinical effects and uses of hypothalamic releasing and inhibiting factors. In Frontiers in neuroendocrinology, pp Eds W. F. Ganong & L. Martini. New York, London and Toronto : Oxford University Press. Isurugi, K, Wakabayashi, K., Fukutani, K, Takayasu, H, Tamaoki, B.-I. & Okada, M. (1973). Responses of serum luteinizing hormone and follicle-stimulating hormone levels to synthetic luteinizing hormone-releasing hormone (LH-RH) in various forms of testicular disorders. J. din. Endocr. Metab. 37, Krey, L. C. & Everett, J. W. (1973). Multiple ovarian responses to single estrogen injections early in rat estrous cycles: impaired growth, luteotropic stimulation and advanced ovulation. Endocrinology 93, Motta, M., Fraschini, F. & Martini, L. (1969). "Short" feedback mechanisms in the control of anterior pituitary function. In Frontiers in neuroendocrinology, pp Eds W. F. Ganong & L. Martini. New York, London and Toronto: Oxford University Press. 38 EN D 62

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