Inhibition of the LH surge in cyclic rats by stress is not mediated by opioids.

The effects of intravenous (iv) administration of the opioid antagonists naloxone and naltrexone on the restraint-induced suppression of the pro-estrous LH surge were studied in cyclic female rats. To minimize stress during repeated blood sampling, the rats were provided with a jugular vein cannula. Restraint stress for 6 hrs starting at t = -1 h (the onset of the LH surge being at t = 0 h) caused a suppression of LH levels (including peak height) during the period of the LH surge. Repeated naloxone injections, given 3 h (1 mg), 4 h (0.5 mg) and 5 h (0.5 mg) after the onset of the LH surge, did not affect the restraint-induced inhibition neither did pretreatment with 1 mg naloxone at t = -75 min (i.e. 15 min before application of restraint). Naltrexone (2 mg) administered at t = -15 min induced higher plasma LH levels at t = -6 min. When rats were subsequently subjected to restraint for 5 hrs starting at t = -5 min, the restraint-induced inhibition of surge levels of LH was not affected. The results indicate that withdrawal of opioid activity in cyclic female rats before the presumed onset of the LH surge results in a premature rise of LH levels. This is in accordance with the notion that LH levels prior to the surge are under tonic inhibition of endogenous opioid peptides (EOP). In addition, the data show that opioid receptor antagonism during or before application of restraint does not alter the restraint-induced suppression of the LH surge. It is therefore concluded that EOP do not mediate the inhibitory effect of restraint stress on the LH surge in cyclic rats.

Follicular and luteal progesterone synergize to maintain 5-day cyclicity in rats.

The length of the ovarian cycle in rat is determined by the duration of progesterone secretion from the corpora lutea (CL) during diestrus. The action of progesterone secretion from the preovulatory follicles on proestrus is also responsible for the cycle length in 4-day cyclic rats. To study whether follicular and luteal progesterone participate in the maintenance of 5-day cyclicity, the effects of the antiprogestagen RU486 (5 mg on proestrus or estrus) on estrous cycle length and on the serum concentrations of LH in 5-day cyclic rats and in 4-day cycle experimentally induced by the dopamine agonist CB154 (1 mg on estrus) were investigated. Furthermore, serum concentrations of progesterone on the day of ensuing ovulation were measured to see whether activation of the CL function after treatment with RU486 had occurred. Both 5-day and CB154-injected rats had a 3-day estrous cycle after RU486 on proestrus, while RU486 on estrus shortened by 1-day the estrous cycle length in 5-day but not in CB154-injected rats. Basal serum concentrations of LH increased and the LH surge decreased after RU486 treatment in both cycle types. Serum concentrations of progesterone rose only in 5-day rats injected with RU486. These results indicate that the actions of both follicular and luteal progesterone synergize in maintaining the length of the estrous cycle in 5-day cyclic rats and that functionally active CL increase progesterone production only under the action of a complete surge of prolactin.

Effect of CRH on the preovulatory LH and FSH surge in the cyclic rat: a role for arginine vasopressin?

The effect of intracerebroventricular (i.c.v.) injection or infusion of various doses of corticotropin-releasing hormone (CRH) on the LH and FSH surge was studied in pro-oestrous rats supplied with a jugular vein and an i.c.v. cannula. Additionally, we investigated if arginine vasopressin (AVP) was involved in the CRH-induced alterations to the surge of gonadotropins. I.c.v. injection of 10 micrograms CRH given 5 min before the presumed onset of the LH surge caused a strong inhibition of the LH surge and a slight inhibition of the FSH surge. Three to four h after CRH injection, its inhibitory effect diminished. A 6'h i.c.v. infusion of CRH started 1 h before the presumed onset of the LH surge, caused a dose-related inhibition of the LH and FSH surge. Infusion of 1 micrograms/h CRH did not suppress the surge of both hormones while infusion of 5 or 10 micrograms/h CRH inhibited the LH surge. Infusion of 10 micrograms/h CRH caused a strong suppression of plasma LH during the first 3 h of the LH surge. Despite continuation of CRH infusion, the inhibitory effect disappeared and plasma LH increased to similar levels as in controls at corresponding points of time of the LH surge. The FSH surge was also suppressed by infusion of 10 micrograms/h CRH. The surge of LH and FSH was not affected by a 9-h infusion of 10 micrograms/h CRH started 4 h before the presumed onset of the LH surge. This observation also indicates that the inhibitory effect of CRH may last for only 3-4 h. The surge of LH and FSH was not affected by i.c.v. injections of AVP-antiserum. However, pretreatment with AVP-antiserum prolonged the inhibitory effect of CRH on the LH surge. In conclusion, CRH can inhibit the pro-oestrous LH and to a lesser extent the FSH rise for only 3-4 h after the beginning of CRH administration. AVP may play a role in limiting the inhibitory effect of CRH on LH to 3-4 h.

Effect of restraint stress on the preovulatory luteinizing hormone profile and ovulation in the rat.

Plasma profiles of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) were measured during restraint stress on the day of pro-oestrus; these profiles were considered in relation to ovulation rate on the next day. Rats bearing a permanent jugular vein cannula were subjected to restraint, which was started 0, 1 or 2 h before the presumed onset of the LH surge and ended just before the beginning of the dark period. Exposure to restraint resulted in a suppression of the secretion of both gonadotrophins on the day of pro-oestrus. Suppression of the LH surge was virtually complete (plasma LH < or = 0.2 ng/ml) in 15 out of 32 stressed rats, and the ovaries of these rats contained graafian follicles with oocytes in germinal vesicle stage. In these rats, the LH surge did not occur 24 h later. In the remaining 17 rats, restraint resulted in a considerable suppression of the LH surge. Of these rats, five had an ovulation rate of 100% and four ovulated partially. In unruptured follicles of the latter, the oocyte had not resumed meiosis and the follicle wall was not luteinized. In the remaining eight rats with a reduced LH surge, ovulations had not occurred and graafian follicles were unaffected. The results of this study indicate that during pro-oestrus restraint stress suppresses and does not delay the release of preovulatory gonadotrophins. Partial suppression of LH by restraint does not result in induction of meiotic resumption without subsequent ovulation or in luteinized unruptured follicles.

Effect of hypothyroidism on the pituitary-gonadal axis in the adult female rat.

The pituitary-ovarian axis was studied after withdrawal of thyroid hormone in 131I-radiothyroidectomized adult female rats. Oestrous cycles became prolonged and irregular within 2 weeks after the supply of thyroid hormone was stopped. If an LH surge occurred in hypothyroid rats on the day of vaginal pro-oestrus it was significantly greater in rats which had been made hypothyroid for 4-5 weeks than in controls; in hypothyroid rats with an LH surge on pro-oestrus, plasma progesterone showed a rise similar to that in controls at pro-oestrus; the ovulation rate was decreased in hypothyroid rats. About half of the rats from which blood was sampled daily in the afternoon between 7 and 18 days after tri-iodothyronine (T3) withdrawal had 1 day of pro-oestrus; on this day the LH surge was higher than in controls. On days 2 and 1 before and days 1 and 2 after this pro-oestrus, plasma progesterone was similar to that of controls on days 2 and 1 before and days 1 and 2 after pro-oestrus respectively. However, progesterone was higher in the period before and after these days. The other hypothyroid rats showed no pro-oestrus and no LH surge during this period, while their plasma progesterone levels were high on all days. On the morning of day 10 after T3 withdrawal and 5 days after the preceding pro-oestrus, most hypothyroid rats had high progesterone and low oestradiol plasma levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Luteolytic and antiluteolytic effect of the antiprogestagen RU486 in pseudopregnant rats.

To study the effects of the antiprogestagen RU486 on luteal activity in pseudopregnant rats, adult female rats made pseudopregnant by sterile copulation were given daily injections with oil vehicle or with RU486 (2 mg/day) either during the entire period of pseudopregnancy (day 1 till day 14) or during the second half of pseudopregnancy (day 8 till day 14). Blood was taken every other day to measure serum concentrations of progesterone. At autopsy, on day 15, the weights of ovaries, isolated corpora lutea and pituitary glands were recorded. In a second study using the same experimental protocol, blood was taken via a jugular vein cannula on days 8, 9, 10 and 11 after induction of pseudopregnancy; on each of these days blood samples were taken at 0700, 0800 and 0900 h, and at 1700, 1800 and 1900 h to measure plasma concentrations of prolactin, LH and progesterone. Administration of RU486 from day 1 of pseudopregnancy onwards had no effect on the increasing concentrations of serum progesterone during the first half of pseudopregnancy. Thereafter progesterone concentrations of serum progesterone during the first half of pseudopregnancy. Thereafter progesterone concentrations increased further in RU486-treated rats whereas they decreased in oil-treated pseudopregnant rats. Administration of RU486 from day 8 of pseudopregnancy onwards resulted in a decline in progesterone concentrations in serum on day 10 followed by ovulation on day 11. Plasma LH concentrations in rats treated with RU486 from day 1 of pseudopregnancy were higher than those in oil-treated rats on days 8, 9, 10 and 11.(ABSTRACT TRUNCATED AT 250 WORDS)

Induction of luteinized unruptured follicles in the rat after injection of luteinizing hormone early in pro-oestrus.

The cause of formation of luteinized unruptured follicles (LUF) is unknown. Formation of LUF was studied after injection of a varying small dose of luteinizing hormone (LH) with or without subsequent injection of gonadotrophin-releasing hormone (GnRH); in addition, the effect of suppression of prolactin on LUF formation was studied. Luteinization without ovulation occurred in virtually all graafian follicles, if 0.5-1.0 microgram of LH was injected some hours before the presumed endogenous LH surge (suppressed by Nembutal); with increasing doses of LH progressively increasing numbers of ovulations were observed. If an early pro-oestrus 1 microgram of GnRH was given 4 h after 1 microgram of LH, formation of LUF was partly prevented; if the interval between LH and GnRH was 8 h or more, the great majority of graafian follicles developed into LUF. If early in pro-oestrus 1 microgram of LH was given and 8 h later 0.1 microgram of a potent GnRH analogue, about 50% of the follicles became LUF; in similarly treated rats, suppression of prolactin by ergocryptine reduced but did not prevent LUF formation. The data support the idea that deficient LH secretion in the period before ovulation may be involved in the formation of LUF.

Advancement of meiotic resumption in graafian follicles by LH in relation to preovulatory ageing of rat oocytes.

After ovulation, the fertile life of oocytes is short. In the present study, the fertile life of oocytes was studied in relation to the resumption of meiosis. Early on the day of pro-oestrus, meiotic resumption was advanced in rats by a brief infusion of LH; ovulation was induced 8 h later by Ovalyse, a GnRH analogue; rats were mated 13 h after receiving Ovalyse, that is at about the time of ovulation. Rats in which meiosis was not advanced were injected with Ovalyse and mated at various intervals after ovulation. Rats were killed 13 h after mating or on day 20 of pregnancy. In rats in which meiosis was not advanced that were mated around ovulation, fetal survival was about 90%. In rats with meiosis advanced by 8 h and mated around ovulation, only 44% of the ovulated oocytes with advanced meiosis developed into healthy fetuses; mortality before and after implantation was 37 and 19%, respectively. Rats in which meiosis was not advanced that were mated 8-9 h after ovulation had similar fetal survival and similar mortality before and after implantation. Thus ageing of the oocyte may occur either before, or after, ovulation. Preovulatory ageing is related to the resumption of meiosis.

Advancement of meiotic resumption in graafian follicles reduces fertility in the rat.

Fertile life of oocytes is usually considered to be related to ovulation time. In the present study, fertile life of rat oocytes was studied in relation to resumption of meiosis. In pro-oestrus, meiotic resumption without concomitant ovulation was induced in most graafian follicles by injection of a small amount of LH or FSH followed by Nembutal. These follicles either ovulated or developed into luteinized unruptured follicles if Ovalyse (a GnRH analogue) was given 8 h after LH or FSH. In subsequent experiments, rats were injected with FSH or saline, and Nembutal; 4 or 8 h later, Ovalyse was given to induce ovulation; the rats were mated 14 h after Ovalyse. At day 20 of pregnancy, fetal survival was 30% in rats with meiosis advanced by 8 h, against 91% and 70% in rats advanced by 0 or 4 h, respectively. Mortality occurred mainly during pre-implantation and early post-implantation. Advanced resumption of meiosis may cause pre-ovulatory ageing of oocytes; consequently, viability of these oocytes after ovulation is reduced.

The effect of decrease in body temperature with Nembutal on meiosis and ovulation after induction by gonadotrophin-releasing hormone and luteinizing hormone in adult rats.

Sodium pentobarbital (Nembutal) is often used to block the pro-oestrous luteinizing hormone (LH) surge in rats. Nembutal is also known to lower body temperature. This study was designed to investigate whether Nembutal affected the time course of meiosis and timing of ovulation induced by exogenous hormones, and whether the possible effects of Nembutal on these processes were related to temperature. Gonadotrophin-releasing hormone (GnRH), the GnRH-analogue Ovalyse, or rat luteinizing hormone (LH) were administered to trigger resumption of meiosis and ovulation; Nembutal (35 mg kg-1 body weight) or saline was given 10 or 60 min later. Plasma profiles of LH were measured and Graafian follicles were studied histologically for meiotic progress and ovulation. Nembutal suppressed the spontaneous surge of LH at pro-oestrus and caused a long-lasting decrease in body temperature. If 1000 ng GnRH was given 2 h before the pro-oestrous LH surge, most of the oocytes had extruded a polar body 10 h later and most follicles had ovulated 14 h later. Nembutal given 1 h after GnRH delayed extrusion of the polar body and ovulation by about 2 h. Nembutal caused a similar delay in ovulation when it was administered after 100 ng of Ovalyse, and it also delayed meiosis when given after 1000 ng of LH. This effect of Nembutal was prevented if body temperature was maintained at 37 degrees C. The delaying effect of Nembutal on meiosis and ovulation induced by exogenous GnRH or LH is related to a long-lasting decrease in body temperature.

Embryo loss, blastomere development and chromosome constitution after human chorionic gonadotropin-induced ovulation in mice and rats with regular cycles.

A dose of 7 IU human chorionic gonadotropin (hCG) given 14 h before the expected LH peak on proestrus significantly increased embryonic mortality in Swiss random-bred female mice to 55% of the number of corpora lutea. The use of luteinizing hormone-releasing hormone in a similar injection protocol did not induce embryonic death. The effect found in Swiss random-bred mice resembles that of a dose of 20 IU hCG in the rat. Afternoon-day-4 mouse embryos contained 39.1 +/- 12.6 nuclei after hCG-induced ovulation compared to 46.2 +/- 16.6 nuclei after spontaneous ovulation. For early-day-5 embryos of the rat, these figures were 34.2 +/- 10.1 and 31.7 +/- 8.4, respectively (mating was early on day 1). Numerical chromosome errors were estimated in secondary oocytes of the mouse and early-day-5 embryos of the rat. Compared with data from the literature, hCG seems to induce some extra meiotic nondisjunction in the rat only. Combining all genetic and physiological data, the loss of fecundity after hCG-induced ovulation is a maternal effect.

Quantification and classification of pregnancy wastage in 5-day cyclic young through middle-aged rats.

Fecundity was quantified in 4 to 11-month old rats with regular 5-day cycles. On the day following mating, less than 5% luteinized unruptured follicles occurred in either 4-month old or 11-month old rats; ovulation rate and also fertilization rate were similar in these young and middle-aged rats. At day 19 of pregnancy embryonic and fetal mortalities were quantified. Pregnancy wastage increased gradually with age; it was already significant in 7-month old rats. In rats of 9 months old it was about 30% and in 11-month old rats it was greater than 65%. Pregnancy wastage was mainly due to preimplantation and early-postimplantation mortality. Pregnancy wastage did not decrease when the pre-ovulatory surge of luteinizing hormone (LH) was reinforced and timed, by injection of the LH-releasing hormone analogue Ovalyse at noon on the day of prooestrus. The results corroborate the idea that pregnancy wastage in middle-aged rats with regular cycles is caused by an age-related reduction of the viability of ovulating eggs.

Studies on prolactin-secreting cells in aging rats of different strains. I. Alterations in pituitary histology and serum prolactin levels as related to aging.

Serum PRL levels and histologically tumor-free pituitary glands of 91 aging rats of the BN/BiRij strain, the WAG/Rij strain and their F1 hybrid were studied. In rats with pituitary glands without signs of hyperplasia, serum PRL levels were, in comparison to rats of 15-24 months, increased 25-29-month-old female BN/BiRij rats and showed a decline with further aging. This rise and decline during aging correlated with changes in the PRL cell volume density and in ultrastructural signs of their synthetic activity. Rats with hyperplastic pituitaries showed similar age-related changes in serum PRL levels, but these levels were higher. Concerning the hyperplasia, some strain differences were found. In BN/BiRij rats anti-r-PRL positive hyperplasia, and in WAG/Rij and F1 rats, anti-r-PRL negative and anti-r-PRL positive hyperplasia were present. All foci of hyperplasia were negative for anti-ACTH and anti-h-GH. In male rats no age-related changes of serum PRL levels could be established, although a decline of PRL cell volume density in the oldest rats is indicated. We conclude that the absence of a continuous age-related rise of serum PRL levels in our animals is caused by exclusion of animals with pituitary tumors.

Evidence that exteroceptive stimuli other than suckling have no major effect on plasma prolactin in lactating rats.

The ability of exteroceptive stimuli from pups to increase plasma prolactin in lactating dams was investigated. Prolactin profiles were measured during 30 min of suckling followed by complete or partial separation from pups. Prolactin profiles were also measured subsequent to complete or partial isolation from pups in dams which had been with pups permanently before the experiment. In addition, plasma prolactin was measured in dams which after a night of isolation were partially united with pups. Finally, the effect of ether stress on prolactin profiles after interruption of suckling by pups was determined. Vigorous suckling after a period of isolation induced a sharp increase of plasma prolactin. Subsequent to pup removal, either partial or complete, prolactin profiles showed a widespread variation. Also dams which before experimentation were kept permanently with pups, showed a great variation in prolactin profiles subsequent to either complete or partial separation from pups. Plasma prolactin either decreased rapidly or gradually. In several dams a rapid or a gradual decline of plasma prolactin was interrupted by one or more episodes of prolactin release. Partial reunion with pups after a night of isolation, either in mid-lactation or in late lactation, did not induce a rise of plasma prolactin. It is concluded that exteroceptive stimuli from pups are not effective as prolactin releasing signal. Because ether stress did not induce a steep rise of plasma prolactin, we conclude that the episodic rises of plasma prolactin in dams, subsequent to partial or complete removal of pups, are due to spontaneous activity of the hypothalamo-pituitary system.

The effect of a single dose of human chorionic gonadotropin at proestrus on embryonic mortality, fetal growth and gestation length in the rat.

The noxious effect of a single dose of 20 IU human chorionic gonadotropin (hCG) given at proestrus on embryonic and fetal survival as well as on fetal weight was studied. Fetal survival varied between 43.6 and 67.6%; the mortality rate was highest before implantation. The surviving fetuses of rats treated with hCG between days 17 and 20 of pregnancy weighed significantly less than controls. Embryonic mortality and fetal growth retardation could be prevented by giving anti-hCG monoclonals 28 or 45 h after hCG administration, but not when anti-hCG was given 69 or 93 h after hCG. 5 IU hCG did not induce embryonic or fetal mortality. On the other hand, 80 IU hCG increased the mortality to 100%; this was entirely due to preimplantation loss. It is speculated that due to a long metabolic half-life of hCG, the steroid metabolism is disturbed, causing implantation failure and/or delay of implantation. By day 21 of pregnancy, however, the fetuses of the hCG-treated rats had made up greatly for the growth retardation; they were born after a similar gestation length and with a similar birth weight as the pups of control rats.

Administration of human chorionic gonadotropin but not of luteinizing hormone-releasing hormone at pro-oestrus or late di-oestrus has a deleterious effect on pregnancy in the rat.

Embryonic and fetal mortality is studied, which is induced by human chorionic gonadotropin (hCG) and luteinizing hormone-releasing hormone (LHRH) given prior to ovulation. 5-day cyclic rats were injected with 20 IU hCG or with 1 microgram LHRH on day 3 of di-oestrus or on the day of pro-oestrus, and mated 4 h later. Autopsy was performed on day 3 or between days 13 and 18 of pregnancy. Advancement of ovulation by LHRH did not induce embryonic or fetal mortality. Administration of hCG at pro-oestrus or day 3 of di-oestrus induced a considerable mortality, which for the greater part occurred after day 3 of pregnancy but before implantation. This embryonic mortality could be prevented by anti-hCG serum given 21 h after hCG. It is speculated that embryonic mortality induced by hCG is caused by a relatively long-lasting disturbance of steroid metabolism, due to a long metabolic half-life of hCG. The disturbance of steroid metabolism as a possible cause of implantation failure is discussed.

Plasma prolactin in the rat during suckling without prior separation from pups.

The relation between suckling and plasma prolactin (Prl) was studied in the rat, without prior separation of the dam from its pups. When the pups were replaced by a hungry foster litter, upon renewed suckling plasma Prl showed episodic increases and decreases in individual rats. When, subsequent to litter removal, similar rats were injected with perphenazine, a significant increase of plasma Prl was observed. This indicates that a decline of plasma Prl during suckling was not caused by exhaustion of Prl stores in the pituitary. In 22 individual rats blood was sampled every other minute while observations were made on nursing behaviour of the dams. During apparent suckling, increases as well as decreases of plasma Prl occurred. However, in most cases suckling did not affect plasma Prl, i.e. it remained stable at a high or a relatively low level. On the other hand, a considerable rise of plasma Prl was frequently observed when a dam was away from the nest. The data indicate that in the physiological situation Prl secretion from the pituitary is not directly related suckling activity, though episodes of suckling are essential to maintain a high Prl secretory capacity of the pituitary gland.

The response of plasma prolactin to suckling during normal and prolonged lactation in the rat.

In dams which had been kept isolated from pups for 8-10 h, the magnitude of the suckling-induced prolactin rise in the plasma was studied in relation to intensity of suckling stimulus and lactational age of the mother. At midlactation the response of prolactin evoked by suckling was enhanced as litter size increased. Suckling of 2 pups induced a greater prolactin rise in dams adjusted to 2 pups than in dams adjusted to 8 pups. Suckling of 8 pups caused a greater prolactin rise in dams which had been adjusted to an 8-pup litter, than in rats with a 2-pup litter. At late and prolonged lactation the rise of prolactin in the plasma induced by the suckling stimulus of 8 pups was significantly lower than at midlactation. Injection of perphenazine after a period of suckling induced a moderate increase of plasma prolactin in dams at midlactation, and a similar increase in dams at late lactation and at day 42 of lactation. It is concluded that in the first half of lactation the number of pups, i.e. the intensity of the suckling stimulus, is an important factor in determining the magnitude of the prolactin response to suckling. The lower response of plasma prolactin to suckling in late lactation is neither caused by a decrease in suckling stimulus from the pups nor by an increase in prolactin clearance; it is probably due to a gradual reduction in prolactin synthesizing and releasing capacity of the pituitary, brought on by a desensitization of the neural or neuroendocrine system to suckling stimuli as lactation proceeds.

No evidence for a peripheral effect of L-DOPA on plasma prolactin in the rat.

Male and female rats with two permanently indwelling intravenous catheters were infused for 2 hours with ovine prolactin. During equilibrium conditions the effects of intravenously injected L-DOPA and benzerazide (a blocker of dopa-decarboxylase) on steady state levels of ovine prolactin were measured. A dose of 4.5 mg L-DOPA per 100 gr body weight (b.w.) caused a transient increase of plasma ovine prolactin. A dose of 0.3 mg L-DOPA/100 gr b.w. had no effect, neither in males nor in females, while benzerazide (20 mg/100 gr b.w.) had only a slight effect. The experiments suggest that L-DOPA does not affect the peripheral uptake of prolactin from the plasma.

Intraperitoneal infusion of EDTA in the rat blocks completely the prolactin rise in the plasma during suckling.

Lactating rats supplied with chronically indwelling intravenous (i.v.) and intraperitoneal (i.p.) catheters were allowed to suckle after a night of isolation from their offspring. The effect of infusions of saline and EDTA, given i.v. or i.p. on the suckling-induced rise of plasma prolactin was evaluated by radioimmunoassay. i.v. saline and EDTA did not affect the normal prolactin rise induced by suckling. However, i.p. EDTA completely blocked this rise, while the weight gain of the pups during the suckling period was only slightly depressed. i.p. EDTA had no suppressive effect on the perphenazine-induced rise of plasma prolactin and caused only a moderate inhibition of the prolactin rise in the plasma evoked by thyrotropin-releasing hormone and by brief exposure to ether. The experiments indicate, that because i.p. EDTA probably stimulates dopamine only slightly, it must inhibit prolactin secretion via another mechanism. This study, therefore, strongly supports the idea that more than one hypothalamic factors affect prolactin release during suckling.