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In the course of pregnancy maternal tissues become increasingly more insensitive to insulin. As 6 days of euglycaemic hyperinsulinaemic clamping, from day 8 until 14 of gestation, ameliorates total glucose consumption, we analysed the contribution of individual tissues in this phenomenon. We measured not only glucose consumption, but also concentrations of the glucose transporter protein GLUT4 in selected tissues. On day 15 of pregnancy in saline-infused (pregnant control) rats. F-18-fluoro-2-deoxy-D-glucose (FDG) consumption, as measured by positron emission tomography, as well as GLUT4 content were diminished in heart (P

Pregnancy is associated with increased glucose-stimulated insulin secretion and increased pancreatic is in-cell proliferation. In the present study it was investigated whether increased food intake, as occurs during pregnancy, Is Involved in the regulation of these phenomena. From Day 0 of pregnancy, rats received each day the mean amount of food they consumed daily during the estrous cycle prior to conception. This food restriction regime resulted in lower maternal body weight, and in lower fetal weight on Day 20 of gestation, but did not affect fetal survival. Food-restricted rats showed decreased insulin responses to an i.v. glucose challenge on Day 13, and lower islet-cell replication rates on Day 14 of pregnancy than pregnant rats fed ad lib. Plasma lactogenic activity in food-restricted animals was increased on Days 11 and 13; plasma progesterone levels were unchanged, but plasma leptin concentrations declined progressively during food restriction. Glucose tolerance was normal, suggesting that food restriction improved insulin action. On Day 20 of pregnancy, insulin responses were similar in food restricted and ad lib-fed rats; glucose tolerance was Still unchanged. It thus seems that the improved insulin action as present on Day 13 had disappeared on Day 20. Also on Day 20, lactogenic activity as well as progesterone concentrations were similar in food-restricted and ad lib-fed rats. It was concluded that increased food intake plays an important role in the stimulation of islet-cell proliferation and insulin secretion, as well as in the diminished insulin action during the second week of rat pregnancy. (C) 1999 Elsevier Science Inc.

Genetic conflicts in pregnancyIn a recent paper, Haig (1993) applied the evolutionary theoryofparent–offspringconflict(Trivers,1974),tothefetal–maternalrelationship.Basictothisviewistherecognitionthattheinterestsof the genes of the mother do not necessarily agree with theinterests of the genes of the offspring, as the genes active inthe fetus, essentially a combination of ‘own’ (maternal) and‘strange’ (paternal) genes, will be selected to promote anythingthat is necessary to serve their fetus’ development, whilst thegenes active in the mother will be selected to guard the interestsof the mother. Hence, according to Haig, any fetal–maternalrelationship is characterized by ‘genetic conflict’.To prevent ‘escalation’ of this conflict, the mother and thefetus should interact adequately, that is, they should sendadequate signals to each other and respond accordingly. This,however, clearly does not always proceed impeccably, as thereare various conditions in which there is obviously no optimaltuning between the maternal and fetal interests. One of theseconditionsispre-eclampsia,aprinciplecauseofmaternalmortal-ity and of iatrogenic pre-term delivery, which may contribute toneonatal deaths from immaturity (MacGillivray, 1983). As thesigns of pre-eclampsia generally disappear quickly once preg-nancy is terminated (Redman, 1991), pre-eclampsia can beregardedasaninadequateresponseofthemothertothepresenceof her conceptus. This raises various questions, to be discussedin this paper, the most relevant ones being: (i) to what is themother responding?; (ii) what is the nature of this response?;and (iii) why is this type of response so common in the human?Pre-eclampsiaPre-eclampsia is a disease which in the Western world affects~6% of all pregnancies (though preferably first pregnancies;MacGillivray,1958).Itis clinicallydefinedbyhypertensionandproteinuria (Redman and Jefferies, 1988), but the disease mayalso be associated with abnormalities of the central nervoussystem and the blood (Redman, 1990), as well as with dissemin-ated intravascular coagulation (DIC) (Bonnar et al., 1971). DICmay result in renal lesions which may eventually cause renalfailure (Easterling and Benedetti, 1989). Also the liver may beaffected (Steegers et al., 1995); in a special group of pre

To partly or completely satisfy the increasing demand for insulin, pregnant rats were infused SC with human insulin (2.4 or 4.8 IU/day) from day 14 to day 20 of gestation. Cyclic control rats underwent the same procedure of 6 days of insulin-treatment. During the treatment all,stoups of rats were hypoglycaemic, but foetal survival was not affected. The low dose treatment prevented the characteristic rise of the insulin response to a glucose challenge during pregnancy, both in vivo and in vitro, while the high dose treatment suppressed the insulin response, as well as the pancreatic insulin content. The insulin responses and insulin contents of pregnant rats were higher than those of the corresponding cyclic control rats. These results support the hypothesis that during gestation the increased insulin demand, due to the actions of placental hormones, is the cause of the increased insulin secretion. However, it cannot be excluded that direct effects of placental hormones on the islets of Langerhans are also involved.

Can the endotoxin-induced inflammatory response, underlying experimental pre-eclampsia, in pregnant rats be inhibited by corticosterone?On day 10 of pregnancy, rats were implanted with pellets containing 25% corticosterone and 75% cholesterol (n = 10) or with 100% cholesterol-pellets (n = 10). On day 14 of pregnancy, rats were infused with either endotoxin (1.0 microg/kg bw) or saline. Three days later, they were sacrificed. Cryostat kidney sections were immunohistologically stained for the presence of neutrophils (PMN) and monocytes (MO) and the expression of inflammation-associated adhesion molecules.In cholesterol-treated rats, endotoxin significantly increased glomerular numbers of PMN and MO, glomerular expression of ICAM-1 and VCAM-1 and glomerular numbers of LFA-1 and VLA-4-positive cells as compared with saline. Corticosterone treatment significantly inhibited glomerular infiltration of PMN, MO and LFA-1 positive cells after endotoxin infusion. It did not affect glomerular ICAM-1 or VCAM-1 expression or numbers of VLA-4 positive cells.It is concluded that pre-treatment with corticosterone inhibits the low dose endotoxin-induced glomerular inflammatory reaction in pregnant rats, most likely by inhibiting LFA-1 expression, thereby decreasing the adhesiveness of inflammatory cells for activated endothelial cells.

The effect of follicle stimulating hormone (FSH) treatment on the pituitary response to gonadotrophin-releasing hormone (GnRH) was studied in rats in various reproductive conditions. A 3-day treatment of cycling rats with FSH (Metrodin(R); 10 IU/injection) lowered the spontaneous pre-ovulatory LH-surge and suppressed the pituitary luteinizing hormone (LH) response to GnRH. FSH also suppressed the LH response of pseudopregnant (PSP) rats on day 8 of pseudopregnancy, but not that of day-8 PSP rats which had been ovariectomized on day 4 (OVX-PSP rats). GnRH induced self priming in cycling, PSP and OVX-PSP rats. Oestradiol strongly augmented the pituitary LH-response to GnRH injection in PSP and OVX-PSP rats, but not in cycling rats; probably because in these latter animals the LH response to GnRH was already augmented by endogenous oestradiol. FSH suppressed the LH response to GnRH in oestradiol-treated PSP and cycling rats; in these latter rats the suppression of the LH response was as strong as that in cycling rats not treated with oestradiol. FSH did not suppress the LH response of oestradiol-treated OVX-PSP rats. The effect of FSH was not associated with changes in plasma oestradiol and progesterone concentrations. Analysis of the data revealed that FSH specifically suppressed the augmentative effect of oestradiol, but did not affect the GnRH-self priming effect. It is concluded that under the influence of FSH, the ovaries produce a factor which suppresses the augmentative effect of oestradiol on the GnRH-induced LH response of the pituitary gland. It is suggested that this effect of FSH underlies the suppression of the spontaneous LH-surges of FSH-treated cycling rats. As the present putative 'oestrogen-antagonizing factor' did not suppress the GnRH-self priming effect, it is suggested that this factor is not identical to gonadotrophin surge inhibiting factor.

Treatment of cyclic and pregnant rats with progesterone stimulates cell proliferation within the islets of Langerhans. It was investigated whether this effect of progesterone depends on sex and/or the presence of the gonads or the presence of oestradiol. For this purpose, Silastic tubes containing progesterone were inserted s.c. in intact and gonadectomized male and female rats, and in gonadectomized female rats treated with oestradiol. After 6 days of progesterone treatment, rats were infused for 24 h with 5-bromo-2'-deoxyuridine (BrdU) and dividing cells were identified in pancreatic sections by immunostaining for BrdU. Progesterone treatment increased islet-cell proliferation in intact male and female rats (P < 0.05), but not in gonadectomized male and female rats or in gonadectomized female rats supplemented with oestradiol. Furthermore, in intact male and female rats, progesterone treatment also stimulated cell proliferation in extra-islet pancreatic tissue (P < 0.05). Identification of the proliferating cells, by double-immunocytochemistry, revealed that progesterone treatment stimulated proliferation of both alpha and beta cells within the pancreatic islets. In extra-islet pancreatic tissue, progesterone treatment stimulated proliferation in both duct (cytokeratin 20-immunoreactive) and non-duct cells. Progesterone treatment did not increase the number of single glucagon or insulin-containing cells outside the pancreatic islets, nor that of cytokeratin 20/insulin double-positive cells, suggesting that progesterone treatment did not stimulate differentiation of duct cells into endocrine cells. Progesterone treatment did not affect insulin responses to an i.v. glucose load (0.5 g/kg body weight). It is concluded that progesterone stimulates pancreatic cell proliferation indirectly; gonadal factor(s), not identical to oestradiol, is (are) probably involved.

The effect of pregnancy on lactation was studied during the third week of lactational pregnancy in postpartum pregnant rats with a delay in implantation of only 1 day (1d-LP rats). In an experimental design in which the suckling litter was prevented from consuming solid food, lactational performance was estimated by weighing the ten-pup suckling litters on days 16-21 of lactation or by measuring maternal weight loss after a nursing spell on day 21. In 1d-LP rats, food consumption as well as lactational performance was lower than it was in nonpregnant lactating rats (L rats) and pregnant-lactating rats with a normal long delay of implantation of at least 6 days (LP rats). The time spent by the pups sucking at the nipples was not different among the three groups, but the number of milk ejections was diminished in 1d-LP dams. Restriction of daily food supply during days 16 to 21 of lactation diminished lactational performance more strongly in 1d-LP rats than it did in L rats; 1d-LP rats conserved protein stores and mobilized fewer minerals than did L rats. The weight and composition of the litter in vitro were not affected by the food restriction. In pregnant-lactating rats (LP and 1d-LP rats), the number of early resorptions was increased in comparison with pregnant rats, showing that lactation can affect the earlier stages of pregnancy. It was concluded that late pregnancy does not affect nursing behaviour, but suppresses lactation by restricting maternal food intake and mobilization of maternal stores. Measurements in serum indicate a causative role for oestradiol, but not for leptin.

The effect of progesterone (P) on pancreatic islet-cell proliferation and function of cyclic and pregnant rats was investigated in vivo. Silastic tubes containing P were inserted s.c. in cyclic rats for 7 or 14 days and in pregnant rats from day 7 to 14, from day 14 to 21 or from day 7 to 21 of pregnancy. 5-Bromo-2-deoxyuridine (BrdU) was infused during the last 24 h of the treatment; the proportion of dividing islet-cells was determined in pancreatic sections, which were immunostained for BrdU. Islet-cell function was determined by measuring glucose and insulin response to a standard intravenous glucose challenge. P treatment increased P and 20 alpha-dihydroprogesterone (20 alpha-OHP) levels in cyclic rats; in pregnant rats, only the plasma levels of 20 alpha-OHP were elevated. Both 7 and 14 days of P treatment stimulated islet-cell proliferation in cyclic rats. In pregnant rats, P treatment increased islet-cell proliferation on day 14, but not on day 21 after either 7 or 14 days of P treatment. P did not affect plasma lactogenic activity in pregnant rats; plasma concentrations of prolactin were decreased after 14 days of P treatment in cyclic rats, but were not affected in pregnant rats. P treatment had no effect on glucose tolerance and glucose-stimulated insulin secretion in any of the groups. It was concluded that: 1, in vivo P stimulates islet-cell proliferation, but does not affect islet-cell function, 2. the stimulatory effects of P are indirect and possibly mediated by the P metabolite 20 alpha-OHP and 3. at the end of gestation, stimulation of islet-cell proliferation is inhibited by some factor, which is not identical to P.

During pregnancy in the rat the insulin sensitivity of the maternal tissues decreases, thereby increasing the demand for insulin (Leturque et al. 1989). At the same time the insulin production and release increases (Koiter et al. 1989). These phenomena are associated with a transient increase in islet-cell proliferation (Dunger et al. 1989). It has been suggested that placental lactogens directly stimulate β-cell function and the proliferation of islet cells (Nielsen et al. 1986; Parsons et al. 1992). Alternatively, placental lactogens, by increasing the demand for insulin (Freinkel 1985), indirectly cause the increase in insulin production and cell-proliferation in the islets. This hypothesis of increased insulin demand was tested by investigating how insulin production and cell-proliferation change when exogenous insulin was infused in pregnant rats at such a rate that the demand for insulin was fully met.

To study the effect of moderately elevated gonadotrophin releasing hormone (GnRH) baseline concentrations during the luteal and the follicular phase, pseudopregnant rats were infused s.c. with GnRH at several doses for 5 days. These rats were also treated with oestradiol or sham-treated during the last 3 days of GnRH treatment. GnRH infusions started on day 7 or day 3 of the luteal phase of the ovulatory cycle; in the rat, the luteal phase or pseudopregnancy lasts about 10 days. Luteinizing hormone (LH) and follicle stimulating hormone (FSH) responses were induced by i.v. injection of GnRH on days 12 (after expected luteolysis) or on day 8 (before expected luteolysis). In normal rats the LH and FSH responses induced by GnRH on day 12 were higher than on day 8 (approximately 160 and approximately 50% respectively). In GnRH-infused rats the LH and FSH responses were not increased. In these rats the luteal phase was extended (the plasma progesterone concentrations remained high) and the onset of the follicular phase was postponed (plasma oestrogen concentrations did not increase). Oestradiol increased the day 12 LH and FSH responses; this effect of oestradiol was suppressed by GnRH infusion. On day 8, exogenous oestradiol also increased the LH and FSH responses, but again the effect of oestradiol was suppressed when the animals were concomitantly infused with GnRH. These data may suggest that in the rat, GnRH baseline concentrations participate in the neuroendocrine system controlling gonadotrophin secretion and hence the ovulatory cycle.

Pancreatic beta-cell function was studied in adult female rats, in which endogenous insulin demand was fully met by SC infusion of human insulin (4.8 IU/24 h) for 6 days, resulting in hyperinsulinaemia and severe hypoglycaemia. The amount of pancreatic endocrine tissue declined by 40%, (pro)insulin mRNA, as determined by in situ hybridisation by 95%, and the amount of stored insulin by 90%. Islet-cell proliferation as determined by 24 h of BrdU infusion declined by 60%. Basal glucose levels normalized within 2 days after the insulin treatment was ended, whereas about 1 week was needed to restore the amount of pancreatic insulin, glucose-induced insulin release, and glucose tolerance to normal values. The amount of endocrine tissue recovered within 48 h and mRNA abundance within 96 h after discontinuation of the insulin infusion, whereas at that time islet-cell proliferation still showed a sixfold increase, before returning to control levels after 1 week. These results show that after a period of suppression of beta-cell function, recovery of insulin synthetic capacity does not immediately result in normalization of insulin stores and insulin release. Under these conditions, episodes of hyperglycaemia may occur, which may act as a stimulus for islet-cell proliferation.

The effect of treatment with estradiol valerate (6 days, 2-6 mg/day) on basal levels of LH and FSH and on response of LH and FSH levels to GnRH challenge (2 × 25 μg GnRH, iv) were investigated in women with "hypothalamic amenorrhea", but without other endocrine disorders. Three groups were studied: 11 women with primary amenorrhea, 10 women exhibiting secondary amenorrhea related with weight loss, and 7 women with normal weight and with amenorrhea persisting after a period of severe weight loss. Before treatment with estradiol valerate the estradiol concentrations in all women were at the lower limit of the follicular phase of a normal ovulatory cycle. In addition, there were no differences between the groups in basal LH and FSH levels and in responses to GnRH challenges. Treatment with estradiol valerate suppressed the basal levels of FSH but not of LH in all women. Estradiol did not affect the response to GnRH challenge in women with primary amenorrhea, weakly augmented the response in women with secondary amenorrhea associated with weight loss, and strongly increased the response in secondary amenorrheic women who had regained normal weight. The results are interpreted in the light of the well-established fact that estrogen augments the gonadotropin response only if the pituitary gland is not exposed to high concentrations of GnRH. It is hypothesized that the differential response to GnRH of the present patients after estrogen treatment reflects differences in GnRH exposure of the pituitary gland, with patients with primary amenorrhea having the highest level of GnRH exposure.

The influence of short-term exposure to LRH on the responsiveness to LRH and on the LH content of the pituitary gland was studied in the 2-weeks ovariectomized rat. Rats were infused with LRH at the rate of either 208 or 1040 ng/h from 12.00 h to 15.00 h on day 0, and injected with 3 μg of oestradiol-benzoate (OeB) or oil at 09.00 h on either day 1 or day 3. LH-responses were induced by a 21-h lasting infusion of LRH at the rate of 104 ng/h, starting at 12.00 h on the day after the OeB/oil treatment. Control rats received only one LRH treatment: they were injected with OeB or oil on day—1 and LH-responses were induced on day 0. Results were evaluated on the basis of two parameters: total amounts of LH released during exposure to LRH (1) and release efficiency, i.e. total amounts of LH released in relation to pituitary LH content (2). The findings obtained indicate that exposure to relatively low LRH concentrations causes a greater loss of release efficiency than exposure to relatively high ones. Loss of release efficiency may even increase long after the blood has been cleared of the releasing hormone. From the present experiments it can thus be inferred that exposure of the pituitary gland to LRH leads to either a marked loss of release efficiency and a relatively small LH depletion (1), or a smaller loss of release efficiency and a relatively large LH depletion (2). The two variables both influence the state of responsiveness of the pituitary gland and the present results indicate that only in situation 2 the LH-responsiveness recovers effectively. However, when after exposure to LRH rats are treated with OeB, the consequences, in terms of total amounts of LH released, of differences in degrees of loss of release efficiency and in depletion of LH are eliminated. It is suggested that during the ovulatory cycle this effect of oestrogen plays an important role in the process of recovery of the pituitary gland from diminished responsiveness to LRH induced by LRH.

The bacterial microflora was examined in the vagina of cyclic female rats kept under normal laboratory conditions. Large variations occurred during the cycle with high numbers of bacteria (10(5)-10(8) per vagina) during proestrus and estrus and low numbers (10(1)-10(4) per vagina) during the diestrus period. Histological analysis of in situ vaginal tissue and transplanted vaginal tissue revealed an association of high bacterial numbers with the presence of large amounts of cellular debris in the vaginal lumen during the period of epithelial keratinization. Absence of phagocytosis in leucocytes at mestestrus suggested that leucocytes did not play an active role in reduction of bacterial numbers between estrus and metestrus. Accurate measurement of the pH in the vaginal lumen failed to reveal differences which could explain the reduction in bacterial numbers between estrus and metestrus. The cyclic changes in the bacterial population-consisting of species which are normally present in the intestinal flora-- seem to be controlled by cyclic changes in the amounts of cellular debris in the vaginal lumen.

The effect of oestradiol benzoate (OeB; 3 μg sc) on LH-RH-induced LH-secretory responses was investigated in: (1) long-term ovariectomized (OVX) rats; (2) intact (i.e. sham-OVX) and 24 h-OVX persistent oestrous (PO) rats (intact PO rats do not possess corpora lutea); (3) intact and 24 h-OVX cyclic (dioestrous) rats (intact cyclic rats possess "not-activated" corpora lutea), and (4) intact and 24 h-OVX phase-2-pseudo-pregnant (phase-2-PSP) rats (phase 2 of PSP lasts from day 2 to day 10. Experiments were performed on days 7-8. Intact phase-2-PSP rats possess "activated" corpora lutea). A "positive" effect of OeB was observed in: (1) long-term OVX rats; (2) intact and 24 h-OVX PO rats; (3) 24 h-OVX cyclic rats and (4) 24 h-OVX phase-2-PSP rats. In intact cyclic and phase-2-PSP rats OeB was completely ineffective. From these observations it is concluded that only in the absence of corpora lutea OeB can exert a "positive" effect on LH-RH-induced LH-secretory responses. Ovariectomy alone, however, may have a negative impact on LH-RH-induced LH-secretory responses. This holds for PO- and phase-3-PSP rats (phase 3 of PSP begins once the corpora lutea come to lysis, i.e. on or shortly before day 10, and lasts until the end of PSP), but not for phase-2-PSP rats. In cyclic rats, on the other hand, ovariectomy performed on dioestrus-1 "only" interferes with the normal development of pituitary responsiveness to LH-RH and this can be nullified by OeB. Our observations suggest that (1) relatively long-acting products of corpora lutea "stabilize" (phase-2-PSP rats), c.q. limit the (almost certainly oestrogen-dependent) development of pituitary responsiveness to LH-RH (cyclic and phase-3-PSP rats); (2) in ovariectomized rats the influence of these products vanishes and that (3) this results in a situation in which OeB can become effective. It is also suggested that in the course of the ovulatory cycle and phase 3 of PSP a similar disappearance of corpora luteal products occurs "spontaneously" as a result of the process of luteolysis with as a consequence that the pituitary responsiveness to LH-RH, under the influence of endogenous oestrogens, develops continuously until, on pro-oestrus, an LH-surge occurs. Evidence is presented that the corpora luteal products, postulated in this paper, are not identical to progesterone.

LH responses induced in the long-term ovariectomized rat by GnRH or GnRH agonistic analogue are augmented by E2. The augmentation by E2 does not occur during, but after termination of GnRH pretreatment. In this study it was investigated whether the augmenting effect of E2 develops also in the GnRH-pretreated rat when the animals were treated with GnRH antagonistic analogue. Two weeks after ovariectomy rats were treated for 10 days with 250 ng GnRH/h (GnRH-rats), released by sc implanted osmotic minipumps. Control rats received a Silastic 'sham pump'. Rats were simultaneously treated with solvent (oil) or estradiol benzoate (EB, 3 μg, sc). Each group of rats was divided into two subgroups, one receiving solvent, the other the GnRH antagonist, Org 30093 (ANT, 100 μg/injection) on 3 consecutive days. In Experiment 1, the pituitary LH content and the LH secretion following stimulation with the agonistic GnRH analogue buserelin, were measured, in Experiment 2, the plasma concentrations of LH before and after cessation of ANT treatment. The effects of treatment with GnRH, EB and ANT were studied on the basis of 1) the height of the maximal LH response and 2) the halfmaximally effective dose (ed 50) of buserelin. Experiment 1 revealed that GnRH depleted the pitutiary gland to about 42% of its original LH content. In EB-treated GnRH-rats the depletion was even stronger (to 14%). After ANT treatment, the pituitary glands of the GnRH-rats were (partly) repleted (oil: to 65%; EB: to 31%). ANT and EB had no effect on the pituitary LH content in control rats. EB increased the maximal LH response in control rats but not in GnRH-rats. ANT increased the maximal LH response to buserelin in oil-injected control rats as well as in oil- and EB-treated GnRH-rats. In these latter two groups, the increase of the maximal LH response was equally large. However, there was an effect of EB on the ed 50 of buserelin during ANT treatment, the pituitary gland of the EB-treated GnRH-rats had become more sensitive to GnRH. Experiment 2 revealed that GnRH pretreatment reduced the plasma LH concentrations to about 49% of the control levels. EB and ANT, too, lowered the plasma LH concentrations (to about 25%). Neither EB nor ANT, alone or in combination, changed the plasma LH concentrations in GnRH-rats. After cessation of ANT treatment, the plasma LH levels of the oil-injected control rats slowly returned to pretreatment levels, but those of the EB-treated control rats remained suppressed. In the GnRH-rats the reverse was seen: after cessation of ANT treatment, peaks of LH appeared in the plasma of the EB-treated rats, but not in the oil-injected. It is concluded that 1) treatment with ANT is not fully equivalent to termination of GnRH administration because it antagonizes the effects of GnRH only in part: 2) ANT has an intrinsic augmenting effect on the pituitary GnRH-responsiveness, and 3) owing to E2-induced sensitization of the pituitary gland, peaks of LH appear in the plasma of GnRH-rats but not of control rats after termination of ANT treatment.

Hemi-pituitary glands of ovariectomized rats were superfused for 4 h with either LHRH or the analog buserelin (HOE 766) at several concentrations, and thereafter with medium only for another 1.5 h. In a further experiment glands were exposed for 2.5 h to LHRH or buserelin at a single concentration (5 ng/ml) and subsequently for another 2.5 h to either the same agonist (LHRH or buserelin) alone (5 ng/ml), the agonist plus an LHRH-antagonist (ORG 30093, 1000 ng/ml), the LHRH- antagonist alone, or medium alone. LHRH and buserelin stimulated gonadotropin release equally well. After cessation of this stimulation, the gonadotropin release by the buserelin-treated pituitary glands and the glands, treated with the highest dose of LHRH (1000 ng/ml), continued, while the release by the glands, treated with the lower doses of LHRH, declined. The LHRH-antagonist completely blocked the release of LH, stimulated by buserelin or LHRH, as well as the prolonged activation of the release, caused by buserelin pre-treatment. In a superfusion experiment with pituitary cell aggregates of 14-day-old intact female rats, buserelin stimulated the release of LH much more effectively than LHRH itself. Moreover, the release caused by buserelin declined more slowly after cessation of the stimulation. Finally, in a pituitary cell monolayer culture the Kd's of LHRH, buserelin and the antagonist were determined as 4.7 X 10(-9) M, 2.4 X 10(-10) M and 4.6 X 10(-9) respectively. It was concluded that the estimates of the potencies of LHRH and buserelin depend on the choice of the test-system. It is suggested that the long duration of action of buserelin is at least partly due to prolonged binding to the LHRH-receptor.

LH-RH and the potent agonistic analogue (D-Ser(But)6-des-Gly10)-LH-RH(1-9)-ethylamide (HOE-766 or buserelin) were at several doses either infused or injected intravenously in 5-weeks-ovariectomized rats, which had been treated with either 3 micrograms estradiol-benzoate (EB) or with oil, 24 h previously. Blood samples for assay of LH were taken during the subsequent 24 h. Pituitary glands were removed at the end of the experiments. Buserelin, when infused, was slightly more effective than LH-RH on releasing LH. When injected, however, buserelin was at the higher dose ranges increasingly more effective as an LH-releasing agent than LH-RH. EB-treatment increased the LH response of the pituitary gland to both peptides in an identical way. It was concluded that buserelin derives its high potency not from its intrinsic LH-releasing activity, which is only slightly greater than that of LH-RH, but from a longer duration of action.

Secretion of LH in response to an LRH infusion (104 ng/h during 21 h), secretion of progesterone (P) and 20 α-dihydroprogesterone (DHP), as well as the 24 h rhythm of prolactin (Prl) secretion were investigated in rats rendered persistently oestrous by exposure to constant light and in which subsequently pseudopregnancy (PSP) had been induced by cervical stimulation after induction of ovulation by hCG. Similar persistently oestrous rats, but otherwise untreated, served as controls. LRH was infused through an intra-jugular cannula and blood samples for assay of LH were taken via an intra-carotid cannula. LH responses were judged on the basis of the mean maximal height (MH; ng LH ml plasma) and the rate of decrease or half life (t½ of the plasma LH concentrations after 2 h of LRH infusion. For assay of P and DHP blood samples were taken from the orbitalplexus. In another series of animals blood, for assay of Prl, was sampled every 2 h for 24 h from a cannula with the tip in the right atrium. It was observed that in persistently oestrous rats LH-responses were high (MH = 3326 ± 305) and steep (t½ = 65 ± 10 min). On the day after the night of ovulation (day 0 of PSP) and on days 1, 5 and 9 of PSP the responses were lower (MH between 1315 and 1041) and more blunted (t½ varied between 236 and 142 min), whilst on day 12 of PSP they were again higher (MH = 4416 ± 575) and steeper (t½ = 101 ± 12 min). P and DHP concentrations were low in persistently oestrous rats and high during days 5 and 8 of PSP. On day 12 of PSP P concentrations were low again, while the DHP concentrations had increased further. In persistently oestrous rats the plasma Prl levels were low with incidental elevations (up to 141 ng Prl/ml aAntoni van Leeuwenhoekhuis The Netherlands Cancer Institute, Amsterdam, The Netherlands plasma). During days 8 – 9 of PSP Prl was secreted at irregular intervals (1 to 3 peaks/24 h) with peak levels of up to 400 ng Prl/ml plasma. On days 11 – 12 of PSP, Prl peaks were lacking almost completely: only a few minor elevations (up to 41 ng Prl/ml plasma) were measured. It is concluded that during days 0 to 10 of pseudopregnancy under constant light the endocrine state of the animals (in terms of LRH-responsiveness and P and DHP secretion) differs significantly form that during the previous and the following days. As similar changes are observed in PSP rats maintained under standard lighting conditions, it is apparent that the lighting conditions do not influence these changes. The pattern of Prl secretion during PSP, on the other hand is affected by the lighting conditions.

The mid-pregnancy rescue of corpora lutea can be mimicked in the pseudopregnant rat by induction of decidual tissue in the uterus: in such rats, around day 10, there is neither luteolysis, nor resumption of follicle-development or increase of the pituitary responsiveness to LHRH. The results suggest that the mid-pregnancy rescue of corpora lutea is caused by a maternal factor.

LH-secretory responses, induced by (standard) infusion of LRH in the pro-oestrous rat, are much greater than in the oestrous rat. In the present investigation the factors which accomplish the transition from the pro-oestrus to the oestrus state of responsiveness to LRH of the pituitary were studied in the pro-oestrous pseudo-pregnant (PSP) rat. It was considered that the change in responsiveness might be caused by either the cyclic release of LRH by the hypothalamus (which results in a depletion of pituitary LH content and an altered state to further stimulation), the peri-ovulatory changes in the endocrine milieu, or some combination of these factors. It was observed that LH-secretory responses induced 24 h after ovariectomy (OVX) on pro-oestrus, take an intermediate position between the pro-oestrus and the oestrus responses. However, when treated with oestradiol benzoate (OeB), such animals maintain the pro-oestrus state of responsiveness. Regardless of the presence of OeB or of the ovaries, following the simulation of a 'pre-ovulatory' LH-surge by a 3 h lasting infusion of LRH, the pituitary gland passes into the oestrus state of responsiveness to LRH. When, however, ovulation was induced by hCG, the pituitary passed into the same state of responsiveness as after OVX, i.e. the intermediate position. It is concluded that the transition from the pro-oestrus to the oestrus state of responsiveness of the pituitary results from the exposure of the gland to the pro-oestrous LRH-surge.

The biological effects of LH-RH and the agonist [D-Ser(But)6-des Gly10]-LH-RH(1-9)-ethylamide (buserelin) were compared during 8 h of incubation with female rat hemi-pituitary glands. Similar dose-response relationships were found for LH-RH and buserelin as concerns the release of luteinizing hormone (LH) by pituitary glands from intact and ovariectomized rats. Also the LH secretion patterns from glands of intact rats were similar: an initial low response was followed by a fast increase (priming effect) after which the response declined again (desensitization). In a subsequent experiment pituitary glands from ovariectomized rats were first exposed to LH-RH or buserelin for 4 h and then further incubated in medium only. After discontinuation of the stimuli the rate of LH release decreased in all cases, but this decrease was significantly greater when the glands had been exposed to LH-RH. Short-term (1/2, 1 or 2 h) exposures to LH-RH or buserelin followed by an intervening period (1 1/2, 1 or 0 h, respectively) of incubation in medium only resulted in an almost similar, significant increase in the subsequent protein synthesis-independent LH response to LH-RH (priming effect). Only preincubation with LH-RH for 2 h was significantly more effective. The results demonstrate equal intrinsic activities for LH-RH and buserelin. Differences in the biopotencies for LH-RH and buserelin in vivo and in vitro may occur only after discontinuation of the external stimuli.

LH secretion and the depletion of the pituitary LH stores caused by continuous infusion of LRH, were studied in the 5-weeks ovariectomized rat. After 1 day of infusion at the rate of 416 ng/h, pituitary LH content decreased to about 40% of its initial value. Upon continuation of the infusion the LH content increased slowly until, after 7 days, it increased to about 54% of the original value. The plasma LH concentrations, after having reached peak values of 7122 ± 291 ng/ml after 2 h of infusion, declined to a plateau at about 300 ng/ml but it took 5 days to reach this value. When after either 2 or 8 days of LRH infusion at the rate of 416 ng/h the infusion was changed to a 'supramaximal' rate of 10 400 ng/h for 24 h, a transient secondary rise of LH secretion was induced. The magnitude of the secondary response, was essentially the same after 2 or 8 days of LRH infusion, but these secondary reponses were significantly smaller than the LH-response during a similar LRH-infusion in previously untreated ovariectomized rats.

In ovariectomized rats the desensitization of the LH cells in vivo, which develops during constant rate infusion of LHRH, 1) does not depend on a concomitant depletion of the pituitary LH stores, 2) proceeds normally when the hypothalamo-pituitary connection has been severed and 3) is a process in which LH itself is not involved.

Ovariectomized rats were infused with varying doses of luteinizing hormone-releasing-hormone (LRH). Some of the rats were also treated with oestradiol benzoate (EB). The effects of these pre-treatments on the in vitro release of luteinizing hormone (LH) were studied. The following parameters of in vitro LH release were measured: a) the autonomous secretion rate; b) the secretion rate following maximum stimulation with LRH, and c) the total quantity of LH released during the 6-hour experiment. The in vivo pre-treatments with LRH and EB dose-dependently decreased the pituitary LH content as well as all three of the above parameters of in vitro LH secretion. There was a linear relationship between the pituitary LH content and the three parameters of in vitro LH release. These parameters were therefore expressed as percentage of the pituitary LH content to give the relative LH secretion rates. The three parameters were thereby corrected for LRH/EB-induced changes in the pituitary LH content. In this way we obtained information on the effects of LRH and EB on the state of the LH release mechanisms of the gonadotropes. EB potentiated the LRH-induced depletion of the pituitary LH stores at all in vivo LRH infusion rates. The effect of EB on the quantity of LH released during perifusion in vitro, however, varied with the previous LRH infusion rates. After LRH infusion rates lower than about 120 ng/h (which establishes plasma concentrations of about 70 ng/l) EB enhanced the stimulated quantity of LH released. After higher rates of LRH infusion, EB lowered the amount of LH released. The effect of EB on the relative secretion of LH in vitro, i.e. on the LH release mechanisms, however, was positive irrespective of the prior in vivo LRH infusion rates, although the effect of EB was greater at the lower rates of LRH infusion. The effect of EB on the autonomous, in vitro, LH secretion rate was positive irrespective of the prior in vivo LRH infusion rates. The positive effect of EB on the mechanism underlying this component of LH secretion was LRH-independent. The effect of EB on the mechanism underlying the LRH-stimulated component of LH release appeared to be strongly LRH-dependent. The effect of EB was maximal if the LRH infusion rate had been lower than about 50 ng/h. With higher infusion rates it became increasingly smaller and was zero at the rate of about 180 ng/h or more. The LRH infusion rates of 50 and 180 ng/h establish plasma LRH concentrations of about 30 and 90 ng/l. Thus, the positive effect of EB on the LRH-stimulated component of LH secretion can be regulated by LRH at the plasma concentration interval of 30–90 ng/l. This study demonstrates that the 'overall' effect of EB on the LH secretion rate is determined by the 'balance' between the effect of EB on the pituitary LH content (the potentiation of the LRH-induced depletion of the LH stores) and the effect of EB on the LH release mechanisms (which effect, in the case of the LRH-stimulated component of LH secretion, can be suppressed by LRH). If the former effect dominates, the effect of EB on the secretion of LH is negative, if the latter dominates, the effect of EB is positive. The LRH concentration at which the positive effect turns into the negative effect is about 70 ng/l. We suggest that the ability of LRH and EB to influence each others' effect on the pituitary gland at physiological concentrations of the two hormones, plays a role in the regulation of the secretion of LH.

In a first series of experiments plasma concentrations of luteinizing hormone (LH) were measured at 10 min intervals during 2 h of constant rate infusion of luteinizing hormone-releasing hormone (LRH; 104 ng/ h) in phenobarbitone-anaesthetized long-term ovariectomized (OVX) rats, treated with oil or oestradiol-benzoate (OeB). From these data the mean LH secretion rates during the sampling intervals were calculated using a one-compartment model for the elimination of LH from the plasma. It was found in the OeB-primed OVX rats that during the initial 30–40 min of infusion the LH release is high but constant. Thereafter it shows a further increase. In the oil-treated OVX rats a similar biphasic LH release pattern was found, but in these animals it was preceded by an initial phase of very high LH release, lasting a few minutes. In another series of experiments a second LRH infusion (again 104 ng/h) was given to OeB-primed OVX rats, starting 1.5 h after the discontinuation of a first LRH infusion lasting either 1, 3.5 or 20 h. The resulting secondary LH responses were smaller the longer the first infusion had lasted, but the LH secretion pattern was similar with all three time schedules and resembled the triphasic pattern observed during the first experiment in the oil-treated OVX rats, rather than the biphasic pattern of the OeB-primed OVX rats. These results indicate that the LH response to LRH of OVX rats (either treated with OeB or oil), like that of the cyclic rats, exhibits a phase of constant LH release. It is generally assumed that during this period conditions, necessary for the subsequent further increase of the LH secretion, are generated. It is concluded that these conditions largely disappear during a 1.5 h non-stimulus period. It is also concluded that the short initial phase of very high LH secretion is due to recent exposure of the LH-secretory system to stimulatory amounts of LRH.

The LH and FSH responses to a standard infusion of LHRH were studied on days 8, 12, 15, 17, 19, 21 and 22 after conception as well as on day 23, i.e. after parturition. Groups of rats were also killed on days 8, 15, 19, 22 and 23 and on the day of pro-oestrus of the 4-day cycle for the assay of progesterone, 20α-dihydroprogesterone (DHP), oestradiol-17β, LH and FSH. Finally, the post-partum surges of LH and FSH were compared with those at pro-oestrus in 4-day cyclic rats. The LH and FSH responses to LHRH were relatively low on days 8 and 12, twice as high on days 15, 17 and 19, had increased further on day 21 and reached maximal values on day 22. The gonadotrophin responses were low on day 23. The post-partum surges of LH and FSH were much higher than the pro-oestrous surges. Pituitary contents of LH and FSH were higher than on pro-oestrus of the 4-day cycle. On day 23, however, the pituitary contents had declined by 60–80%. No apparent relationship was found between plasma concentrations of LH and FSH and LHRH responsiveness during pregnancy. Concentrations of oestradiol-17β on day 22 were higher than on all other days of pregnancy, but lower than on pro-oestrus of the 4-day cycle. Concentrations of progesterone were high until day 19 and low on days 22 and 23; the concentration of DHP was low until day 19 and very high on days 22 and 23. It was concluded that the increase of the gonadotrophin response to LHRH half-way through pregnancy, in contrast to the increase at the end of pregnancy, cannot be explained by an increase in the concentration of oestradiol-17β, while the pituitary LH and FSH contents play, if any, only a permissive role. The effect of s.c. implants of progesterone on various hormone parameters during the last week of pregnancy was also investigated. It was found that progesterone not only delayed parturition but also prevented the rise of the gonadotrophin response at the end of pregnancy. Concentrations of oestradiol-17β and DHP were also lower on day 23 than in untreated rats, while the pituitary gonadotrophin contents were higher. It is suggested that delay of luteolysis (in the present study by progesterone treatment) at the end of pregnancy prevents the occurrence of a series of interrelated events: a rise in oestradiol-17β production, a rise in the gonadotrophin responses to LHRH, parturition and the generation of the post-partum gonadotrophin surges. J. Endocr. (1987) 115, 247–254

The effect of exogenous progesterone (P) on the corpus luteum function (in terms of the secretion of P and 20-α-dihydroprogesterone (DHP), on the secretion of prolactin (Prl) and on the pituitary responsiveness to LHRH was studied in pseudopregnant (PSP) rats kept in alternating and constant lighting conditions (LD-PSP and LL-PSP rats, respectively). Rats were rendered pseudopregnant by appropriately timed stimulation of the cervix uteri (LL rats first received an ovulatory dose of hCG). LH responses were induced by constant rate infusion of LHRH (104 ng/hfor 21 h). P was delivered by subcutaneously inserted Silastic implants; control rats received sham implants. In both LD- and LL-PSP rats the plasma P and DHP levels were high on day 8 of PSP. On day 12, however, the plasma P levels had fallen but the DHP levels had risen, demonstrating that between days 8 and 12 functional luteolysis had occurred and that neither the production of P and DHP, nor the timing of luteolysis are under the control of the lighting conditions. On day 12 of PSP the pituitary responsiveness to LHRH was much higher than on day 8. Moreover, on days 8/9 of PSP peaks of Prl were seen in all rats, but on days 11/12 such peaks were largely absent. In LD-PSP rats ‘nocturnal’ Prl peaks were seen on days 8/9 in all 9 experimental animals, but ‘diurnal’ peaks were seen in only 4 of these animals. Also, the diurnal peaks were on average much lower than the nocturnal peaks. In LL-PSP rats we saw on days 8/9 over 24 h 2–3 irregularly timed peaks of Prl, which were not as high as the nocturnal peaks but higher than the diurnal peaks of LD-PSP rats. After implantation of two Silastic P implants into LD- and LL-PSP rats on day 6 of PSP, i.e. before functional luteolysis, the peaks of Prl and a low pituitary LHRH responsiveness were still present on day 12. Also, on day 12 the plasma concentrations of DHP were not increased in P-implanted rats. P implants inserted on day 11 (i.e. after functional luteolysis) did not prevent cessation of the appearance of Prl peaks and the rise of DHP secretion in both LD- and LL-PSP rats. Also, in LL-PSP rats which were on day 6 of PSP both ovariectomized and implanted with P, peaks of Prl were still observed on days 11/12, but in animals with sham implants peaks were absent. It thus appears that P is the corpus luteum factor which provides a permissive environment for the neural signal which causes the secretion of peaks of Prl. It is concluded that (1) during PSP and until functional luteolysis the relatively high P levels play a role in the maintenance of the secretion of peaks of Prl, and that (2) maintenance of the corpus luteum function by exogenous P (and thereby the maintenance of the state of low LHRH responsiveness) is due to P postponement of luteolysis.

The LH-secretory response (LH-S.R.), caused by (standard) infusion of LH-RH, was estimated throughout pseudopregnancy (PSP) as well as on dioestrus – 1 and –2 of the 4-day oestrous cycle. The LH-S.R. was judged according to 3 parameters: (1) mean maximal plasma LH concentration ("height", expressed in ng LH-RP-1/ml plasma), (2) mean area under the curve (AUC, expressed in "area units"), and (3) a composite, dimensionless, parameter: RQ ("response quotient"), defined as height/AUC. This latter parameter informs on the shape of the curve. The LH-S.R.'s on day 1 of PSP and dioestrus-1 were both relatively low (about 350 ng LH-RP-1/ml plasma) and characterized by the same RQ about 12). On days 2, 5, 7 and 9 of PSP the LH-S.R.'s were essentially the same and appeared to have characteristics of the LH-S.R.'s of both PSP-1 and dioestrus-1 (RQ) and dioestrus-2 (height; about 1000 ng LH-RP-1/ml plasma). From day 10 on the LH-S.R.'s became increasingly higher (PSP 11: about 1900 ng LH-RP-1/ml plasma; PSP 12: about 3300 ng LH-RP-1/ml plasma) and steeper (RQ equals about 22). At the end of PSP the spontaneous LH-surges were measured. These appeared to be very high if compared to those which are generated in 4-day cyclic rats (about 5800 ng LH-RP-1/ml plasma). The results indicate that a period of PSP is not simply an ovulatory cycle of which the dioestrous phase is extended by about a week and it is suggested that "active" corpora lutea secrete, next to progesterone, substances which play a key role in the regulation of the secretion of LH in the course of PSP.

With prolonged constant rate infusion of luteinizing hormone-releasing hormone (LRH), the LH secretion rate of the rat pituitary gland changes continuously until a steady state of relative desensitization has developed. Recovery from this state can occur independently from changes in the pituitary's LH content.

The effect of a combined in vivo pre-treatment with luteinizing hormone-releasing hormone (LRH) and oestradiol benzoate (EB) on the autonomous and the 'supra-maximally' LRH-stimulated in vitro release of LH and FSH by pituitary glands of 2 weeks ovariectomized (OVX) rats was studied using a perifusion system. The concentration of LRH in the perifusion medium was 1 μg/ml. Pre-treatment with LRH during 6 days was effected by means of sc implanted Alzet® osmotic minipumps (MP). Control rats received a piece of silastic with the dimesions of a minipump ('sham-pump'; Sh-P). EB, 3 μg/injection or solvent (arachis oil) was sc injected on days –3 and –1 (day of perifusion: day 0). Of the pituitary glands of EB-injected, Sh-P-implanted rats both the autonomous and the LRH-stimulated secretion of LH and the LRH-stimulated secretion of FSH were significantly higher than those of the oil-injected, Sh-P-implanted rats without EB administration. Pretreatment with LRH for 6 days had a suppressing effect on the autonomous and the LRH-induced depletion of the pituitary LH and FSH stores. In combination with EB, the suppressing effect of LRH pre-treatment on the LRH-stimulated secretion of LH and FSH was still greater: the pituitary gland appeared to be fixed in a relatively unresponsive state with very low autonomous LH and FSH secretion. It is discussed that increase of pituitary LRH-responsiveness due to EB demands withdrawal of the pituitary gland from the influence of LRH, an effect which is in vivo achieved by the negative feedback of oestrogen on the hypothalamus.

Estradiol sensitizes the pituitary gland for the gonadotropin-releasing activity of LHRH. LHRH desensitizes the pituitary gland and does so in a dose-dependent manner. Moreover, LHRH dose-dependently suppresses the sensitizing effect of EB. In rats with an LHRH concentration in the plasma of about 90 pg/ml, the sensitizing effect of estradiol is absent.

Recovery of the pituitary gland from LRH-induced refractoriness to LRH was studied in 5-weeks ovariectomized rats. Rats first received over a period of 48 h an infusion of LRH at a rate of 416 ng/h. After discontinuation of this infusion pituitary LH content had decreased to about 50% of its original value and the rate of LH secretion decreased markedly. Over a period of 72 h after discontinuation of the infusion plasma LH values rose to pre-infusion values but pituitary LH content showed such a recovery only partly. In another series of experiments a second infusion of LRH was given during 24 h and again at a rate of 416 ng/h, starting 3, 12, 24 or 72 h after discontinuation of the first infusion. It induced rises of LH secretion which were smaller than those following the first infusion, indicating refractoriness of the pituitary gland to LRH. However, the LH responses became progressively larger with increasing time intervals after the end of the first infusion. This stimulation of LH release was accompanied by a further decrease of pituitary LH content which was still depressed after the first infusion. Comparison of plasma LH levels and pituitary LH content indicated that pituitary responsiveness to LRH can recover independently from pituitary LH content. It can also be concluded that the absolute value of the blood LRH concentration can not be the sole determining factor of LH secretion.

In the long-term ovariectomized rat, both estradiol benzoate (EB) and clomiphene citrate enhance the release of LH induced by luteinizing hormone-releasing hormone (LHRH). EB also enhances the release of FSH. In rats pretreated with LHRH, EB strongly depresses the LHRH-induced LH/FSH release, but clomiphene enhances this release, regardless of the presence of EB.

The mechanism of increased sensitivity for endotoxin in pregnancy as reflected by the formation of microthrombi in renal glomeruli is unknown. It has been shown that reduced glomerular diphosphatase (ADPase) activity in the rat kidney greatly increases the intraglomerular thrombotic tendency. We now studied experimental intraglomerular thrombosis ex vivo in association with glomerular ADPase activity in pregnant and nonpregnant control rats after infusion of either endotoxin or saline solution. Each animal (Wistar rat) was equipped with a permanent vena jugularis catheter and received either endotoxin (1.0 micrograms/kg body weight) (n = 6) or saline solution (n = 5) 7 days before being killed; nonpregnant rats were also treated with endotoxin (n = 5) or saline solution (n = 4). On day 21, before the animals were to be killed, they were anesthetized and their left kidneys were perfused with adenosine diphosphate solution (10 micrograms/ml) and platelet-rich plasma (1 x 10(9) cells/ml). Perfused kidneys were processed for light microscopy, electron microscopy, enzyme cytochemistry at the ultrastructural level, and immunohistology. The results showed decreased ADPase activity exclusively in the glomerular basement membrane of kidneys of pregnant rats treated with endotoxin in contrast to the findings in control rats. In addition, exclusively in the group of endotoxin-treated pregnant rats, significantly increased intraglomerular platelet aggregation could be detected after alternate perfusion ex vivo. We suggest that, in the present model, enhanced susceptibility of glomerular ADPase for endotoxin is due to pregnancy-associated factors that have yet to be identified. This increased susceptibility may promote in situ formation of intraglomerular microthrombi.

We studied the glucose tolerance in freely moving rats throughout pregnancy and lactation and during the first week after weaning. Dioestrous virgin rats served as controls. Basal glucose and insulin levels were determined after a 2-hr fasting period. Subsequently, the changes of the insulin and the glucose levels were determined during ingestion of a mixed ad lib meal or a 2 g mixed test meal, or during infusion of glucose (7.4 mg/min for 20 min) into the vena cava. Basal glucose levels were high during early pregnancy, low during late pregnancy, and in the normal range throughout lactation and after weaning. Basal insulin levels were decreased at the end of lactation. The results of the ad lib meal and test meal experiments were essentially the same. Glucose tolerance during meals was somewhat decreased early in pregnancy. The corresponding insulin responses greatly increased during the last week of pregnancy. Glucose tolerance during IV infusion of glucose was normal during pregnancy, but increased during lactation. Insulin responses to the infusion were increased during pregnancy and decreased during lactation. We concluded that glucose tolerance is hardly affected by pregnancy and even increases in the course of lactation. This is effected by an increased responsiveness of the B-cells to glucose during late pregnancy and by an increased turnover of glucose during lactation. We discuss to what extent the actions of progesterone, placental lactogen and prolactin may explain these adaptions of maternal metabolism.

Influx of leucocytes in the vagina at metoestrus occurs in germfree mice and also in sterile isotransplants of vaginal tissue in conventional mice. In contrast to the situation in rats, bacteria thus do not seem to be required for the production of postovulatory leucotactic stimuli in the vagina.

The hypothesis that corpora lutea (CL) secrete substances which prevent oestrogens from influencing the state of responsiveness of the pituitary gland to LRH (RESP) was tested in rats rendered persistently oestrous (PO) by exposure to permanent illumination. In these rats ovulation and hence the presence of a single set of CL, was induced by exogenous gonadotrophin, hCG. The RESP of the animals was judged on 3 parameters based on the surge-like LH-secretory responses which were induced by 21-h long constant rate LRH infusions (104 ng/LRH/h). These 3 parameters were: a) the maximal height (MH) of the responses; b) the 'area under the curve' (AUC) of the LH values; and c) the constant β (or alternatively the t½) which characterizes the rate of decrease of the plasma LH concentrations after 2 h of infusion. Four experiments were performed, all with PO rats: 1) rats were injected with either oestradiol benzoate (OeB; 3 μg sc) or oil on day 0 (the day treatments were started was always denoted as day 0); 2) rats were treated similarly, but they also received an ovulatory dose of hCG (50 IU/100 g b.w. ip) on day 0; 3) rats were injected with hCG on day 0 and with OeB or oil on day 3; and 4) rats were injected with hCG on days 0 and 4, on which latter day they also received OeB or oil. In all 4 experiments the LRH infusions were started 21 h after administration of OeB or oil. Blood samples for LH determinations were taken at times apparent from 'Results and Discussion'. It is observed that: 1) after administration of OeB the MH and thereby the AUC increased significantly but the β of the LH-secretory responses was unchanged; 2) after administration of an ovulatory dose of hCG the LH-secretory responses changed also; of these responses, however, the β had decreased and the AUC had increased, whilst the MH remained unchanged; 3) when given together, OeB and hCG exhibited their effect simultaneously: the two effects are additive; 4) in the presence of 3-day old CL OeB is ineffective; and 5) in the presence of 4-day old CL neither OeB nor hCG is able to affect the RESP. It is concluded: 1) OeB and 'hCG' probably influence a different substrate of the LH-release mechanism; and 2) that these results confirm the hypothesis that CL secrete substances which prevent oestrogen from affecting the RESP for at least 4 days. If this hypothesis is extended to the cyclic rat, CL, arisen after the previous ovulation, may still be of importance on the day of the next pro-oestrus by exerting a significant influence on the RESP.

The LH releasing activities of LRH and the LRH-analogue buserelin® (HOE 766; (D-Ser (But)6-LRH(1–9)nona peptide-ethylamide) were compared in intact and short- and long-term castrated male rats, pre-treated (either 1 or 3 days) with oestradiol benzoate (EB) or oil. LRH and buserelin were infused iv at the constant rate of 104 ng/h for 21 h. Blood samples were taken from an intracarotid cannula. LH responses were judged on the basis of the mean maximal height of the LH concentration (MH; ng LH/ml plasma) and a parameter of total LH release, i.e. the area under the curve of LH concentrations plotted against time ('area under the curve', AUC; expressed in 'area units'). The release efficiency of LRH and buserelin, E (see for a definition: Materials and Methods), which informs on the total quantity of LH released in relation to pituitary LH content, was calculated by dividing the AUC × 100 by the pituitary LH content at the beginning of stimulation. Maximal plasma LH concentrations were observed between t= 1.5 and t=3 h after LRH and between t= 1.5 and t=9 after buserelin treatment. Both with LRH and buserelin the rise of LH secretion was greater the longer the animals were castrated and/or pre-treated with EB. The buserelin-induced LH response (with the exception of the responses induced in the EB-pre-treated, 4-weeks castrated rat) were about 2–2.5 times higher (MH) and larger (AUC) than the corresponding LRH-induced responses. The buserelin/LRH potency ratio, therefore, is about 2–2.5. EB-pre-treatment did not change the pituitary LH content. It therefore enhanced the efficiency of release of LH of both LRH and buserelin. Castration, on the other hand, caused an increase of the pituitary LH content: after 4 weeks it was raised by a factor 4. Since, however, the LH responses induced by LRH and buserelin were proportionally higher and larger, castration did not significantly change the efficiency of LH release. The results indicate that the efficiency of LH release can be changed by changes in the endocrine environment in the experimental animals, whilst for the magnitude of LH responses the pituitary LH content is also important. It is therefore suggested that the responsiveness of the pituitary gland to LRH (and agonistic analogues) is determined by (1) the state of the LH secretion mechanism and (2) the pituitary LH content.

Administration of oestradiol benzoate (OeB) on the second day of dioestrus of 5-day cyclic rats may advance ovulation by 24 h. The threshold dose of OeB necessary to achieve this effect varies with the time of administration. At 09.00 h, the 50% effective dose (ED-50) of OeB for advancing ovulation was 3.1 μg; at 17.00 h it was 31 μg. In the present study OeB was injected at either 09.00 or 17.00 h at doses about 3 times the corresponding ED-50's: 10 and 100 μg, respectively. Though both regimens of OeB administration resulted in advancement of ovulation, neither had more than a moderate effect on the development of pituitary responsiveness to LRH. In neither group could OeB-induced LH-surges be distinguished from "normal" 4-day rat-LH-surges and both differ from 5-day rat-LH-surges. It is concluded that (1) in the "Everett-model" OeB acts almost exclusively on the central nervous system; (2) the oestrogen-induced surge of LH is an all-or-none effect; and (3) there exists no relationship between the blood oestrogen concentrations and the characteristics of the induced LH-surges.

The effect of exogenous progesterone (P) on the corpus luteum function (in terms of the secretion of P and 20-alpha-dihydroprogesterone (DHP), on the secretion of prolactin (Prl) and on the pituitary responsiveness to LHRH was studied in pseudopregnant (PSP) rats kept in alternating and constant lighting conditions (LD-PSP and LL-PSP rats, respectively). Rats were rendered pseudopregnant by appropriately timed stimulation of the cervix uteri (LL rats first received an ovulatory dose of hCG). LH responses were induced by constant rate infusion of LHRH (104 ng/h for 21 h). P was delivered by subcutaneously inserted Silastic implants; control rats received sham implants. In both LD-and LL-PSP rats the plasma P and DHP levels were high on day 8 of PSP. On day 12, however, the plasma P levels had fallen but the DHP levels had risen, demonstrating that between days 8 and 12 functional luteolysis had occurred and that neither the production of P and DHP, nor the timing of luteolysis are under the control of the lighting conditions. On day 12 of PSP the pituitary responsiveness to LHRH was much higher than on day 8. Moreover, on days 8/9 of PSP peaks of Prl were seen in all rats, but on days 11/12 such peaks were largely absent. In LD-PSP rats 'nocturnal' Prl peaks were seen on days 8/9 in all 9 experimental animals, but 'diurnal' peaks were seen in only 4 of these animals. Also, the diurnal peaks were on average much lower than the nocturnal peaks.(ABSTRACT TRUNCATED AT 250 WORDS)

The patterns of LH secretion during constant stimulation of the pituitary glands of estradiol-treated ovariectomized rats with a maximally stimulating amount of LH-RH in vivo and in vitro correspond with each other qualitatively and quantitatively. In vitro the changes with time of the LH secretion rate are somewhat retarded, especially the occurrence of desensitization.

In the ovariectomized rat we investigated the effects of prolonged treatment with oestradiol and 'physiological' pulses of exogenous GnRH on the GnRH-responsiveness of the pituitary gland. Rats were for 20 h pre-treated with either GnRH (3-ng pulses of 3 min duration every 20 min) or saline and for 19 h with either oestradiolbenzoate (OB; 3 μg/sc injection) or oil. Then LH and FSH responses were evoked by continuous test infusions of GnRH at the rates of either 60, 100, 150 or 1000 ng/h, lasting 10 h. It appeared that, irrespective of pre-treatment with either GnRH or saline, OB caused an increase of the maximal LH and FSH responses (that is the responses to the maximal stimulus of 1000 ng GnRH/h), and an increase of the GnRH-sensitivity of the pituitary gland, as far as the FSH secretion is concerned. GnRH pulses caused, irrespective of pre-treatment with OB or oil, a decrease of the GnRH-sensitivity of the pituitary gland, as well as a decrease of the maximal FSH response and of the pituitary FSH content. It was concluded that low-dose pulses of exogenous GnRH desensitize the pituitary gland and deplete the pituitary FSH stores, but do not change the positive effect of oestradiol on the maximal gonadotropin response.

The LH and FSH release-stimulating (experiment 1) and -blocking (experiment 2) effects of LH-releasing hormone (LHRH) and of the LHRH analogue d-Ser(But)6-des-Gly10-LHRH-ethylamide (buserelin), as well as the effect of combined treatment with LH RH and oestradiol benzoate (OB; experiment 3) on the 'supra-maximally' LHRH-stimulated release of LH and FSH were studied in rats ovariectomized for 2 weeks. Pretreatment with LHRH (250 or 500 ng/h) or buserelin (250 ng/h) for 6 days was effected by means of subcutaneously implanted Alzet osmotic minipumps; control rats received a 'sham pump', i.e. a piece of silicone elastomer with the dimensions of a minipump. Oestradiol benzoate (3 μg/injection) or solvent was injected subcutaneously 75 and 27 h before the induction of LH/FSH responses. Experiment 1 revealed that after infusion of LHRH and buserelin, both at the rate of 1 μg/h, plasma LHRH concentrations were established which were about twice as low as the plasma buserelin concentrations. This might suggest that buserelin has a longer half-life than LHRH. As an LH and FSH release-stimulating substance, however, it appeared that buserelin was about as effective as LHRH. Experiment 2, however, suggested that as an LH/FSH release-blocking agent buserelin was much more effective than LHRH. In addition, after buserelin pretreatment the pituitary glands contained much less LH and FSH than after LHRH pretreatment at both dose levels used. However, this may also (at least partly) be due to the fact that buserelin has a longer half-life so that after infusion of buserelin and LHRH at the same rate the plasma concentrations of buserelin are higher than those of LHRH; after buserelin infusion the pituitary gland is therefore stimulated at a higher intensity. Experiment 3 showed that in OB-injected, sham-implanted rats the LHRH-stimulated secretion of LH and FSH was significantly higher than in the oil-injected, sham-implanted rats. In the LHRH-pretreated rats (LHRH: 250 ng/h for 6 days), however, the already depressed LHRH-stimulated secretion of LH and FSH was still further depressed by OB treatment. These latter results suggest that the increase of the pituitary LHRH responsiveness during exposure to oestrogen requires a reduction of the LHRH stimulation, which is normally caused by the negative feedback of oestrogen on the hypothalamus. J. Endocr. (1984) 103, 301–309

The effects of steroid-free bovine follicular fluid (bFF) and sodium phenobarbitone on spontaneous LH releasing hormone (LHRH)-induced secretion of FSH and LH were studied in ovariectomized rats. Luteinizing hormone releasing hormone was administered by infusion to rats anaesthetized with phenobarbitone. Bovine follicular fluid reduced FSH release and synthesis. Luteinizing hormone release remained unaffected after bFF treatment. Phenobarbitone reduced both FSH and LH release. The observed suppressive effects of bFF and phenobarbitone on FSH secretion were additive, suggesting that the basal release of FSH has an LHRH-dependent and an LHRH-independent component. Furthermore, bFF did not affect pituitary responsiveness of LH secretion to LHRH and reduced the responsiveness of FSH secretion only when administered some time before the LHRH challenge. The present observations support the view that in the ovariectomized rat the pituitary gland is the only site of action of inhibin-like activity as present in bFF.