Termination of pregnancy after accelerated lactation in the rat. IV. Relationship to 20alpha-hydroxysteroid dehydrogenase activity and plasma progesterone concentration.

The activity of 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD) was assayed in the ovaries of rats after accelerated lactation to determine its relationship to the decrease in progesteron secretion that occurs. When rats were sjbjected to accelerated lactation on Day 9 of pregnancy, activity of the enzyme was only slightly increased by Day 10, but had risen to twice the control level by Day 11, and three times the control level by Day 12. Administration of LH or progesterone prevented the increase in enzyme activity. Progesterone concentration had decreased considerably before the time at which any significant increase in 20alpha-HSD activity was detected. These findings are discussed in relation to the role of 20alpha-HSF in regulating progesterone levels in the rat.     

Failure of two progesterone antagonists, mifepristone and onapristone, to affect luteal activity in lactating rats

The progesterone antagonists, mifepristone (RU-38,486) and onapristone (ZK-98,299), given as 2 mg daily, did not markedly affect lactation in rats. Both litter growth and time spent by 10-pup litters attached to their mothers were similar in antagonist-treated mothers and in solvent-treated controls. The progesterone antagonists did not affect the steroid content in corpora lutea remaining from the preceding pregnancy. Corpora lutea formed after post-partum ovulation also showed nearly normal function throughout the first 17 days of lactation. It is concluded that progesterone itself plays no role in the initiation or maintenance of luteal function when prolactin secretion is governed through an action independent of the ovaries, as through suckling. Antagonist-treated rats ovulated around Day 13 of lactation despite suckling. This ovulation was not associated with a decrease of progesterone production by the corpora lutea formed after post-partum ovulation. Apparently, elimination of progesterone action may protect corpora lutea from luteolysis. The latter finding indicates a possible role of progesterone in luteolysis and deserves further analysis.     

Termination of pregnancy after accelerated lactation in the rat. II. Relationship to number of young, day of pregnancy and length of nursing.

Pregnancy is terminated in rats that have mated at the postpartum oestrus if they are allowed to suckle a large litter after implantation. For consistent termination, the number of sucking young must be at least nine, the accelerated lactation must be begun before Day 10 of pregnancy, and must be continued for at least 2 days.     

Prolactin binding in rat mammary gland during pregnancy and lactation.

Lactogenic hormones from the placenta and pituitary are primarily responsible for the growth and function of the mammary gland during pregnancy and lactation. In the present study we described the optimal conditions for the measurement of 125I-labeled ovine prolactin binding to mammary gland slices of pregnant and lactating rats. Prolactin binding is saturable (Kd approx. 2.36 - 10(-9) M), hormone specific and destroyed by proteases. The hormonal environments of pregnancy and lactation dramatically influence the availability and measurement of prolactin binding sites. Whereas binding consistently appears to be low in mammary glands removed from rats during pregnancy, binding levels rise 7--8-fold shortly after birth and remain high during the 22 days of lactation. However, the removal of the ovaries and gravid uteri at specific times during pregnancy results in a prompt 3--6-fold increase in prolactin binding. Elevated levels in potential prolactin binding capacity appear in mammary tissue coincident with the reported rise in serum rat placental lactogen between the eighth and eleventh days. We suggest that high levels of this lactogenic hormone promote the appearance of prolactin binding sites during pregnancy and mask the sites such that they are not available for measurement in vitro.     

Serum levels of prolactin during the ovarian cycle,pregnancy, and lactation in the common marmoset (Callithrix jacchus)

A homologous double-antibody radioimmunoassay developed for humans was used to measure serum prolactin, progesterone, and estradiol in common marmosets. In the ovarian cycle of common marmosets, serum progesterone began to increase after an estradiol surge, attained a peak level, and then declined before the ensuing pre-ovulatory rise in estradiol. During the luteal phase, the change in serum concentrations of estradiol was synchronized with that of progesterone. During the ovarian cycle there was no consistent change in serum prolactin concentrations. During the last 75 days of pregnancy the prolactin level was higher than during the ovarian cycle and the first 70 days of pregnancy. Moreover, during lactation, mothers with suckling twin infants had a higher prolactin level than during the final stage of pregnancy.     

Suckling-induced prolactin release potentiates mifepristone-induced lactogenesis in pregnant rats

Suckling, starting at 19:00 h on Day 18 of pregnancy, induced a significant increase in serum prolactin concentration at 20:00 h on Day 19 of pregnancy, but no increase in mammary gland casein or lactose content. Mifepristone (2 mg/kg) injection at 08:00 h on Day 19 of pregnancy induced significant increases in casein, but not in lactose, 24 h after administration. Mifepristone alone did not induce prolactin secretion, indicating that lactogenesis was induced by placental lactogen in the absence of progesterone action. When mifepristone was injected into suckling rats, serum prolactin concentrations were higher than in the untreated suckling rats. Casein in these rats increased significantly 12 h after mifepristone administration and lactose at 24 h after. If the suckling mifepristone-treated rats were given two injections of bromocriptine (1.5 mg/kg) at 12:00 h on Days 18 and 19 of pregnancy, serum prolactin concentrations were not increased by suckling, but casein and lactose concentrations in the mammary gland showed values similar to those obtained in the mifepristone-treated non-suckling rats. Mifepristone can therefore potentiate suckling-induced prolactin release in pregnant rats, demonstrating a direct central inhibitory action of progesterone on prolactin secretion. This suckling-induced prolactin secretion, unable to induce casein or lactose synthesis in the presence of progesterone, enhanced significantly synthesis of these milk components in the absence of progesterone action (rats treated with mifepristone). Fatty acid synthase, which is stimulated by the suckling stimulus in lactating rats, was not modified by mifepristone or suckling in pregnant rats.     

Effect of mammogenic hormones on the expression of FGF7, FGF10 and their receptor in mouse mammary gland

To investigate the regulation of estrogen, progesterone and prolactin stimulating the development of mammary gland, the Kunming mice were used as experimental animals in this study. Through the experiment in vitro, the effect of mammogenic hormones were systematically investigated on expression of FGF7 and FGF10 and their receptor in different periods. The results are as follows: in mammary glands of mice, 17 beta-estradiol increased the expression of FGF7; progesterone did not affect the expression of FGF7; prolactin up-regulated the expression of FGF7 significantly in pregnancy and lactation. 17 beta-estradiol increased the expression of FGF10; progesterone and prolactin reduced the expression of FGF10 significantly in virgin; prolactin significantly increased the expression of FGF10 in pregnancy. When 17 beta-estradiol in the body was in relatively high proportion, it would lower the expression of KGFR; while 17 beta-estradiol in the body was in relatively low proportion, it would increase the expression of KGFR. Low concentration of progesterone increased the expression of KGFR and high progesterone did not affect the expression of KGFR. Prolactin increased the expression of KGFR significantly in pregnancy and lactation.     

Regulation of ghrelin secretion during pregnancy and lactation in the rat: possible involvement of hypothalamus

We investigated the plasma concentration of ghrelin peptide during pregnancy and lactation in rats. Plasma ghrelin levels on days 10 and 15 of pregnancy were significantly lower than those of the non-pregnant rats. Thereafter, the plasma ghrelin levels on day 20 of pregnancy sharply increased to levels comparable with those in non-pregnant rats. Ghrelin peptide concentrations in the stomach did not change significantly during pregnancy. In the hypothalamus, ghrelin mRNA levels were significantly lower on day 15 of pregnancy than in the non-pregnant rats. Also, plasma ghrelin levels were significantly lower in lactating dams than non-lactating controls on days 3 and 8 of lactation. We examined the possible involvement of prolactin and oxytocin in the regulation of plasma ghrelin concentrations during lactation. Although plasma prolactin levels were decreased by the administration of bromocriptine, plasma ghrelin levels did not differ significantly between vehicle- and drug-treated lactating rats. Administration of haloperidol produced a marked increase in plasma prolactin levels as compared with the non-lactating controls. However, plasma ghrelin levels were not significantly different between vehicle- and drug-treated rats. Administration of an oxytocin antagonist into the lateral ventricle significantly inhibited the increase in the plasma oxytocin level induced by acute suckling. However, plasma ghrelin levels did not significantly between the groups. These observations indicated that the decrease in serum ghrelin is caused by a loss of the contribution of hypothalamic ghrelin. Furthermore, the present results suggested that the suckling stimulus itself, but the release of prolactin or oxytocin, is the factor most likely to be responsible for the suppression of ghrelin secretion during lactation.     

Lactational [correction of Lacational] anovulation in rats and its dependency on progesterone

The relationship between prolactin (PRL) secretion and anovulation in lactating rats was studied. Normal lactating rats and lactating rats treated with antiserum against luteinizing hormone-releasing hormone at the time of postpartal ovulation were used. Normal lactating rats were treated with either a dopamine agonist (CB-154, 150 micrograms/rat) on Day 10 or 13, or pups removal on Day 7 or 10, and thereafter luteolysis and inhibition on PRL secretion were assessed. With the CB-154 treatment, the incidence of luteolysis increased as the lactational period advanced (42% vs 72%), whereas it decreased (73% vs 14%) with the pups removal. Thus, dopamine effectively inhibited PRL secretion during the later lactational stage, but could not do so during the earlier stage when there were mechanisms other than dopamine stimulating PRL secretion. Following luteal regression induced by CB-154, ovulation did not occur if the rats were treated with CB-154 on Day 10, whereas 50% of the rats ovulated within 4 days if treated on Day 13. Furthermore, in the lactating rats treated with anti-luteinizing hormone-releasing hormone serum during late pregnancy, ovulation was not observed until Day 10 of lactation. Since the serum progesterone levels were low in these rats due to the absence of ovulation and lactational corpora lutea, the blockade of ovulation was not due to elevated circulating progesterone during the early lactational period. The mechanism of ovulation blockade during lactation thus seems to shift from being progesterone independent to progesterone dependent at a similar period when the neuroendocrine control of PRL secretion shifts from dopamine independent to dependent.     

Proceedings: Neurohormonal control of prolactin release

Neurohomonal control of prolactin release was studied in pseudopregnant and pregnant rats. Nembutal administered at 1300 hours on Day 3 of pseudopregnancy prevented prolactin release which normally occurred at 1700 hours of the same day. Antiestrogen administered the day before did not prevent prolactin release but ovariectomy did. Estrogen administered immediately after ovariectomy did not restore prolactin secretion; however, progesterone on Day 3 in the ovariectomized-estrogen treated induced an increase in prolactin at 1700 hours. Progesterone was capable of increasing prolactin release the first 5 days of pseudopregnancy but not Days 6-12 when prolactin values were low. A similar effect was seen the first 7 days of pregnancy. Progesterone, but not estrogen, modified prolactin values on Day 9 at 1700 hours. Ovariectomy on Day 19 of pregnancy induced prolactin release within 4 hours and persisted for 58 hours. Progesterone administration immediately after ovariectomy prevented prolactin release for a few hours. These results suggest that the regulation of prolactin release by the central nervous system depends on the circulating estrogen/progesterone ratio, since estrogen facilitated prolactin release when plasma progesterone was low and progesterone induced prolactin release when adequated levels of estrogen existed, but exerted an inhibitory action when estrogen was not present.     

In vivo hormonal environment leads to differential susceptibility of the corpus luteum to apoptosis in vitro

We evaluated the involvement of the in vivo hormonal environment on the ability of the rat corpus luteum (CL) to undergo apoptosis. Gel electrophoretic DNA fragmentation analysis revealed no apoptosis in CL isolated either the 2 last days of pregnancy (Days 21 and 22) or throughout the 4 days following parturition, suggesting that the number of cells undergoing apoptosis at the same time is not sufficient to allow for visualization of DNA breakdown. In contrast, CL incubated in serum-free medium underwent significant apoptosis, as evaluated by chromatin condensation and DNA fragmentation, regardless of their developmental stage in pregnancy. However, CL obtained on Day 7 of pregnancy and on Day 4 postpartum demonstrated higher sensitivity to apoptosis in vitro, but lactation reduced significantly the capacity of the CL to undergo apoptosis when maintained in culture. These data suggest that the exposure of the CL to different hormonal environments throughout pregnancy and after parturition is responsible for the differential susceptibility to apoptosis observed in vitro. We have previously shown that progesterone is a direct factor for survival of the CL. Prolactin stimulates luteal progesterone production; therefore, we examined whether prolactin prevents apoptosis in luteal cells independently of its stimulatory action on progesterone production. We used a luteal cell line (GG-CL) that expresses the prolactin receptor but does not produce progesterone. These cells undergo apoptosis under conditions of serum starvation, and addition of prolactin to the culture medium significantly reduced DNA fragmentation. These results indicate that the extent of luteal cell death induced by incubation of CL under serum-free conditions depends on the hormonal environment to which this endocrine gland is exposed in vivo. These results also indicate an important role for lactation in preventing apoptosis, which is further supported by the antiapoptotic activity of prolactin observed in luteal cells.     

Clinical, biological and hormonal study of mid-pregnancy termination in cats with aglepristone

In order to evaluate the efficacy, the safety and the variation in plasma concentrations of estrogens, progesterone, PGFM, oxytocin, cortisol and prolactin after mid-pregnancy termination induced by aglepristone, 61 pregnant queens (33.3 + 4.2 days), were injected subcutaneously with 15 [corrected] mg/kg aglepristone, (Alizine) [corrected] repeated once 24 h later. Five queens served as control and received a placebo. The efficacy of aglepristone was 88.5% and termination of pregnancy was achieved in 50% of the queens within 3 days. Brief periods of depression and anorexia were noted in 9.3% of the queens before fetal expulsion (these symptoms were attributed to the phenomenon of fetal expulsions). Not one of the queens that aborted developed uterine disease. There were no changes in plasma concentrations of estrogen, prostaglandin, prolactin or oxytocin following aglepristone administration. However, there were significant increases in plasma concentrations of progesterone and cortisol 60 and 30 h, respectively, after aglepristone administration. Termination of pregnancy occurred with high plasma progesterone concentrations. Fetal expulsion was characterised by an increase in estrogen, PGFM and oxytocin concentrations, whereas prolactin and cortisol levels remained at a basal level.     

The failure of a prostaglandin F2 alpha analogue, cloprostenol, to initiate lactation in cattle

An experiment was designed to determine if the analogue of prostaglandin F2 alpha, cloprostenol, at a dose sufficient to cause luteolysis, was lactogenic in cattle. The mammary glands of eight Friesian heifers were developed by treatment with progesterone plus oestrogen. Lactation was then initiated by administration of cloprostenol and subsequent milk production was compared to that of heifers lactating after a normal pregnancy. Injection of cloprostenol failed to initiate lactation. The level of prolactin but not cortisol in blood was substantially elevated following treatment. The results cast further doubt on the importance of prolactin in the lactogenic process but indicate the likely involvement of glucocorticoids.     

Cytological and immunocytochemical studies of the anterior pituitary gland of the beagle bitch during various reproduction phases]

The changes in anterior pituitary (AP) of pregnant and lactating dogs as compared with pituitary of animals in metestrus-anestrus phase are described with special reference to the relative proportion, topography and morphology of prolactin cells, somatotrophs, corticotrophs, thyrotrophs and gonadotrophs. For demonstration of these cells, suitable histological, histochemical and immunoenzyme cytochemical methods are used. The prolactin cells show progressive hyperplasia during pregnancy, so that at the end of this phase and during lactation, they comprise the most predominant glandular cells in AP. At the same time, they reveal massive hypertrophy with marked morphological features of high secretory activity, After transient or continous interruption of the suckling stimulus they show signs of functional inhibition on involution. The corticotrophs appear at 20. and 30. days of pregnancy to be relatively increased in number. While in the last third of pregnancy and during lactation, they only seem to be more active than those in pituitary of metestrus-anestrus dogs. The somatotrophs appear to be progressively reduced in relative number during pregnancy and lactation. However, they show some morphological signs of active secretion. The thyrotrophs did not show any morphological alterations during the different reproductive phases. The gonadotrophs reveal during pregnancy, especially at 30. day morphological signs of stimulation. On the contrary they appear atrophied during lactation. This may be a result of suckling stimulus and morphological expression of the inverse relationship in the secretion pattern of gonadotrophins and prolactin in dogs during suckling. The estrogen and progesterone levels in plasma as well as the changes in their relative concentrations may largely account for the structural changes on AP of pregnant dogs. However, neuroendocrine reflexes (e.g. suckling stimulus) seem to be of a great importance for the maintainance of stimulation of prolactin cells during lactation.     

Effect of mammogenic hormones on the expression of dFGF7, FGF10 and their receptor in mouse mammary gland

To investigate the regulation of estrogen, progesterone and prolactin stimulating the development of mammary gland, the Kunming mice were used as experimental animals in this study.Through the ex-periment in vitro, the effect of mammogenic hormones were systematically investigated on expression of FGF7 and FGF10 and their receptor in different periods.The results are as follows:in mammary glands of mice, 17 beta-estradiol increased the expression of FGF7;progesterone did not affect the expression of FGF7;prolactin up-regulated the expression of FGF7 significantly in pregnancy and lac-tation.17 beta-estradiol increased the expression of FGF10;progesterone and prolactin reduced the expression of FGF10 significantly in virgin;prolactin significantly increased the expression of FGF10 in pregnancy.When 17 beta-estradiol in the body was in relatively high proportion, it would lower the ex-pression of KGFR;while 17 beta-estradiol in the body was in relatively low proportion, it would increase the expression of KGFR.Low concentration of progesterone increased the expression of KGFR and high progesterone did not affect the expression of KGFR.Prolactin increased the expression of KGFR significantly in pregnancy and lactation.     

Role of insulin receptors in the changing metabolism of adipose tissue during pregnancy and lactation in the rat.

Changes in the volume, the rates of fatty acid synthesis and synthesis of the glycerol moiety of acylglycerols, the activity of lipoprotein lipase, and the number and affinity of insulin receptors of adipocytes, and concentrations of serum insulin, prolactin and progesterone were determined in virgin rats and in rats at various stages of pregnancy and lactation. Changes in the metabolic activities of adipose tissue appeared to be synchronized and primarily comprised a marked decrease in anabolic activity around parturition. In contrast, the number of insulin receptors (Kd 1.5 nM) per adipocyte doubled during pregnancy before returning to normal values around parturition. It is postulated that the increase in the number of insulin receptors is an adaptation to counteract the effects of insulin-antagonistic hormones during pregnancy and that the decrease in the number of receptors is primarily responsible for the loss of anabolic activity around parturition.     

Termination of mid-gestation pregnancy in bitches with aglepristone, a progesterone receptor antagonist

Six pregnancies were terminated in mid-gestation with aglépristone, a progesterone receptor antagonist, in 5 beagle bitches in order to determine the effects of aglépristone on plasma concentrations of prolactin and progesterone, the duration of the luteal phase, and the interestrous interval. In addition, the effects of aglépristone on the condition of the uterus and fetuses were examined by ultrasonography. After confirmation of pregnancy by ultrasonography, the dogs received 10 mg, s.c. aglépristone per kg body weight on 2 consecutive days at about 30 d post ovulation. Before, during and after treatment with aglépristone, plasma samples were collected for determination of the concentrations of prolactin and progesterone. The condition of the uterus and fetuses was assessed by ultrasonography the day before and at least 3 times a week for at least 2 wk after aglépristone administration. Termination of pregnancy occurred within 4 to 7 d after the start of aglépristone treatment, which was well tolerated, with no side-effects except slight vaginal discharge. The results of ultrasonographic examination indicated that aglépristone leads to abortion but not to fetal resorption. Elevated plasma concentrations of prolactin were observed during aglépristone treatment, while plasma progesterone levels remained unchanged. Pregnancy termination with aglépristone resulted in premature cessation of luteal function. In addition, the interestrous interval was shortened. The latter effects may be the consequence of actions of the progesterone receptor antagonist at the hypothalamus-pituitary level. In conclusion, aglépristone proved to be a safe and effective abortifacient in mid-gestation in the bitch. The results of the present study also indicated that aglépristone directly or indirectly influences pituitary function.     

Effects of ergocryptine on prolactin secretion druing concurrent pregnancy and lactation in the rat

Ergocryptine (2 mg/kg) caused short- and long-term reduction of prolactin secretion in rats experiencing concurrent lactation and pregnancy. The long-term effects of the drug lasted at least 60 days and resulted in reduced milk secretion and termination of pregnancy. Prolactin replacement therapy at a low dose (5 i.u./day) was unsuccessful in overcoming these effects but a higher dose (up to 60 i.u./day) increased milk production and maintained pregnancy. One possible explanation of these results is that prolactin, rather than the suckling stimulus, was responsible for the suppression of oestrous cycles, because ergocryptine brought about a resumption of oestrous vaginal smears in all treated rats in spite of continued suckling.     

Regulation of 6-phosphofructo-1-kinase from the epithelial cells of rat small intestine during pregnancy and lactation

The regulation of 6-phosphofructo-1-kinase (PFK) in the epithelial cells of rat small intestine was studied during pregnancy and lactation. The total activities and activity ratios (activity at 0.5 mM fructose 6-phosphate at pH 7.0/activity at pH 8.0 (nu 0.5/V] of the partially purified mucosal PFK were found to increase initially in early pregnant rats (11-12 days of gestation) and to fall back to normal in late pregnant rats (19-20 days of gestation). These changes in enzyme activity during pregnancy were associated with similar changes in the circulating levels of progesterone. The maximal activity and activity ratio (nu 0.5/V) were increased in male and female rats injected with progesterone. An increase in the total activity and activity ratio of mucosal PFK was also obtained in lactating rats. However, the enzyme was not strongly activated by inorganic phosphate, fructose 2,6-bisphosphate or glucose 1,6-bisphosphate either in early pregnant or lactating rats. These results indicate that mucosal PFK is already present as an active form during early pregnancy and lactation. Therefore, it is suggested that female sex hormones increase the circulating levels of insulin during early pregnancy which, in turn, positively affect the activity of mucosal PFK which could be also stimulated by the increased levels of fructose 2,6-bisphosphate. The increased activity of PFK in the peak lactating rats could be possible because of an increased demand for lactate production from glucose together with the stimulation of PFK by the increased concentrations of fructose 2,6-bisphosphate which therefore increases the rate of glycolysis.     

Induction of mammary prolactin receptors and lactose synthesis after ovariectomy in the pregnant mouse.

The development of prolactin receptors in the mammary gland after ovariectomy was investigated in pregnant KA mice. Mice were ovariectomized on day 13 of pregnancy and used for the determination of the amount of specific binding of 125I-labelled prolactin to the mammary tissue, and the contents of lactose and nucleic acids in the mammary gland 0, 8, 24, and 72 hr after the operation. The specific binding of 125I-labelled prolactin, lactose and RNA contents in the mammary gland remained low until 8 hr, sharply increased 24 hr and decreased 72 hr after ovariectomy. When ovariectomized mice were treated with 0.2 mg progesterone, pregnancy was maintained and an increase (1.5-fold) in the amount of specific binding was observed with an increase of lactose content. Five mg progesterone completely inhibited lactose synthesis. Cortisol administered with progesterone did not show any specific change at the dose used (0.5 to 10 mg). Although the amount of specific binding was also increased after hysterectomy, this increase (2-fold) did not fully cover the increase after ovariectomy (3-fold). These results suggest that the recepter site for prolactin is induced before the initiation of lactose synthesis caused by ovariectomy during pregnancy.