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Estrogen affects apoptotic cell death in estrogen-responsive tissues. The purpose of the present study was to examine dynamic changes in apoptotic cell death in the anterior pituitary gland during the estrous cycle and to investigate neuroendocrine regulation of these changes in cycling female rats. Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) for in situ detection of DNA strand breaks revealed that the number of TUNEL-positive anterior pituitary cells was changing during the estrous cycle, with a maximum in the morning of proestrus and a minimum in the morning of estrus. A similar pattern was observed with Bax immunostaining; however, no difference was observed in Bcl-2 immunostaining. Most of Bax-immunoreactive cells were identified as a subpopulation of gonadotropes. Pentobarbital administered in the afternoon of proestrus attenuated the decrease in TUNEL-positive or Bax-immunoreactive cells in the morning of estrus, although estradiol treatment failed to affect it. This action of pentobarbital was reduced by simultaneous treatment with an ovulation-inducing dose of gonadotropin-releasing hormone (GnRH), but not with progesterone, an ovarian steroid also released after GnRH treatment. These results suggest that anterior pituitary cells, mostly gonadotropes, undergo a cyclic change in apoptotic cell death during the estrous cycle and that the inhibition of apoptosis on estrus is due, at least in part, to the proestrous surge of GnRH secretion., (Copyright 2002 S. Karger AG, Basel)

Estradiol induces surges of prolactin (PRL) and luteinizing hormone (LH) secretion as well as lactotrope proliferation in female rats. We examined whether these hypothalamus-dependent events require the direct action of estradiol on the anterior pituitary gland by selective blockade of its peripheral actions, using ICI182,780 (ICI), an antiestrogen that cannot cross the blood-brain barrier. Injection of ICI into ovariectomized rats, at a dose of 250 microg/day for 4 days, almost completely inhibited estradiol-induced growth of the uterus, proliferation of lactotropes as determined by bromodeoxyuridine incorporation, and afternoon surges of LH secretion. However, ICI only partially inhibited estradiol-induced surges of PRL secretion and had no effect on estradiol-induced tonic inhibition of LH secretion even at the highest dose of 1,000 microg/day. The inhibitory effects of ICI found at 250 microg/day were attributable to its selective peripheral, but not central actions since ICI did not alter hypothalamic expression of progesterone receptors, an estradiol-dependent brain process. Estradiol-induced increases in the number of progesterone receptor-immunoreactive cells in the hypothalamic ventromedial nucleus and the medial preoptic area were not inhibited by this dose of ICI but were inhibited by 500 microg/day tamoxifen, an antiestrogen that can cross the blood-brain barrier. Treatment of cycling female rats with 250 microg/day ICI beginning from diestrus day 2 was also effective in blocking estrous lactotrope proliferation and preovulatory surges of LH secretion but not PRL secretion. Finally, in ovariectomized estradiol-treated pup-deprived lactating rats, ICI did not affect suckling-induced PRL secretion but completely blocked lactotrope proliferation. These results suggest that a direct estradiol action on the anterior pituitary gland is required for lactotrope proliferation and the positive feedback action on LH secretion but not for the secretory surges of PRL or for negative feedback., (Copyright 2002 S. Karger AG, Basel)

The proestrous surge of prolactin (PRL) secretion and subsequent proliferation of lactotropes at estrus have been suggested to be induced by a common hypothalamic hormone. We investigated changes in lactotrope proliferation at other reproductive stages of female rats when PRL secretion was stimulated. To assess proliferative activity of lactotropes, incorporation of 5-bromo-2'-deoxyuridine (BrdU) into DNA was measured by double immunostaining for PRL and BrdU. BrdU-labeling indices, determined by BrdU injections at 10.00 h, revealed low levels of proliferative activity of lactotropes at the reproductive stages including diestrus, days 6 and 13 of pregnancy, and day 6 of lactation while high levels were detected on estrus and the day of parturition. When BrdU-labeling indices were determined at 3-hour intervals through day 6 of pregnancy to find an increase in lactotrope proliferation which might occur at times other than 10.00 h, proliferative activity of lactotropes remained at low levels with a slight increase in the afternoon. Such a diurnal change as observed in early pregnancy was not detected on day 13 of pregnancy. In contrast, short-interval determinations of BrdU-labeling indices during a period from day 20 of pregnancy to day 2 of lactation revealed a marked increase in proliferative activity on the day of parturition with a peak at 18.00 h, which was comparable to that observed at estrus. To investigate involvement of ovarian steroids in suppression of lactotrope proliferation as observed during early pregnancy and lactation, ovariectomized and pup-removed lactating rats were given one of treatment combinations of estradiol and suckling. In pup-removed lactating rats, estradiol treatment alone induced neither a PRL surge nor an increase in BrdU-labeling indices. Suckling stimuli, which were effective in increasing serum PRL concentrations irrespective of estradiol treatment, elicited a marked increase in BrdU-labeling indices in the presence of estradiol but not in its absence. These results suggest that proliferative activity of rat lactotropes does not necessarily correlate with PRL secretion during pregnancy and lactation. In contrast to PRL release, lactotrope proliferation requires both a hypophysiotropic stimulatory input from the hypothalamus and a sensitizing action of estradiol, an observation which may account for the fact that proliferation does not occur during pregnancy and lactation in spite of elevated PRL release., (Copyright 2000 S. Karger AG, Basel.)

Dopaminergic agonists are effective in vivo in inhibiting lactotrope proliferation and prolactin (PRL)-secreting pituitary tumors. The purpose of the present study was to demonstrate in vitro actions of dopaminergic agents on proliferation and cell shape of rat lactotropes. Anterior pituitary cells cultured with serum-free, chemically defined medium were treated with dopaminergic agents and were labeled with 5-bromo-2'-deoxyuridine (BrdU) for 3 h before the end of culture. BrdU-labeling indices indicative of the proliferation rate of lactotropes were determined by double immunofluorescence staining for BrdU and PRL. Treatment with dopamine for 21 h decreased BrdU-labeling indices of lactotropes in a dose-dependent manner with a nadir at 3 x 10(-7) M. The inhibitory action of 10(-5) M dopamine appeared 15 h after the initiation of treatment and became pronounced with time up to 33 h. The dopamine action was mimicked by treatment with the D2 receptor agonist bromocriptine at concentrations over 10(-9) M. Phase-contrast microscopy revealed that the flat polygonal cell shape of cultured lactotropes had changed to a round refractive cell shape after treatment with dopamine or bromocriptine, and that these changes in cell shape exactly paralleled those in the BrdU-labeling index. The changes in cell shape of lactotropes were accompanied by changes in subcellular distribution of actin filaments. Pretreatment with 10(-7) M eticlopride, a D2 receptor antagonist, blocked the dopamine- or bromocriptine-induced changes in both BrdU-labeling index and cell shape. These results suggest that (1) the in vitro experimental system established in the present study is a good model for studying the mechanism of the antiproliferative action of dopamine and (2) D2-receptor-mediated inhibition of proliferation of lactotropes in serum-free culture is closely related to changes in actin organization and cell shape.

Substance P (SP) which is synthesized and secreted by anterior pituitary cells has been suggested to alter pituitary functions as a local modulator. We determined which cell type(s) of rat anterior pituitary gland secretes SP. A new technique, named the sandwich cell immunoblot assay (CIBA), was developed to identify two protein substances that are secreted simultaneously from the same cells. Monodispersed anterior pituitary cells were sandwiched with pairs of transfer membranes and incubated to absorb secretory substances on the membranes. Small reference points for identifying the location of cell blots were simultaneously put on the pairs of transfer membranes after the incubation. The validity of the sandwich CIBA was revealed by the following: (1) cell blots were detected at the same locations on the pairs of transfer membranes that were immunostained with the same antisera against the anterior pituitary hormones, and (2) the areas of these cell blots detected at the same locations on different membranes correlated well. When one of the pair of transfer membranes was immunostained for SP and the remaining immunostained for one of the anterior pituitary hormones, it was found that 78 and 27% of SP-immunoreactive cell blots showed also GH and TSH immunoreactivity, respectively. None of the SP-immunoreactive cell blots showed immunoreactivity for PRL, ACTH, LH or FSH. These results suggest that the sandwich CIBA is useful to identify two substances cosecreted from a cell and that a subpopulation of rat somatotropes or thyrotropes cosecretes SP.

The functions of tuberoinfundibular dopaminergic (TIDA) neurons after long-term estradiol treatment were investigated in Fischer 344 (F344) rats which have high susceptibility to estradiol-induced prolactin (PRL)-secreting pituitary tumors. Dopamine synthesis in and release from TIDA neurons were determined in vitro by 3,4-dihydroxyphenylalanine (DOPA) accumulation in the median eminence following incubation with a DOPA decarboxylase inhibitor and endogenous dopamine release from the median eminence, respectively. The concentration of serum PRL and the weight of the anterior pituitary in ovariectomized F344 rats were markedly increased 3 weeks after a single injection of 2 mg estradiol valerate (EV) and decreased thereafter, but still showed higher levels at 15 and 24 weeks than control ovariectomized rats. Dopamine contents in the median eminence were decreased 3 weeks and unchanged 24 weeks after EV treatment. DOPA accumulation and basal dopamine release in the median eminence of F344 rats were decreased 3 weeks and increased 15 and 24 weeks after EV treatment, similarly to those of Wistar rats as reported previously. However, EV treatment, which caused similar increases in the concentrations of serum PRL and estradiol in F344 and Wistar rats, decreased KCl-induced dopamine release in Wistar rats at 3 weeks, but failed to do so in F344 rats. KCl-induced dopamine release 24 weeks after a single EV injection in F344 rats was greater than that in control rats, whereas the dopamine release 24 weeks after the last treatment of 4 injections at 3-week intervals was not different from that in control rats.(ABSTRACT TRUNCATED AT 250 WORDS)

To clarify the regulatory mechanisms for the secretion of somatostatin (SRIF) from the hypothalamus, the effects of intracerebroventricular (i.c.v.) administration of growth hormone-releasing factor (GRF) and corticotropin-releasing factor (CRF) on SRIF secretion into hypophysial portal blood were examined in pentobarbital-anesthetized male rats. Neither the concentration of SRIF in portal plasma nor the secretion rate of SRIF was changed after i.c.v. administration of 0.9% saline. Administration of 10 ng or 5 micrograms human GRF i.c.v. produced a significant increase in the portal plasma concentration and secretion rate of SRIF. Likewise, 5 micrograms CRF significantly increased the portal plasma concentration and secretion rate of SRIF. These results suggest that the neuropeptides GRF and CRF centrally influence SRIF secretion into hypophysial portal blood.

To determine the involvement of the midbrain in the control of the proestrous surge of gonadotropins and prolactin, various transections were acutely placed in proestrous rats. Half-dome shaped transections which interrupted the neural fibers in the ventromedial part of the midbrain (VMM transection), as well as lesions in the ventral tegmental area, blocked ovulation and the proestrous surges of gonadotropins and prolactin whereas sham transections or the other transections, either dorsal or lateral to VMM transections, had no effect on ovulation. VMM transections, however, affected neither the postcastration rise in serum gonadotropins in males nor the secondary rise in serum FSH in estrous females. These results suggest that in the ventromedial part of the midbrain there exists a common neural mechanism which controls the proestrous surges of gonadotropins and prolactin.

Bilateral lesions in the medial basal part of the suprachiasmatic area (MBSC) lying immediately rostral to the suprachiasmatic nuclei caused persistent vaginal cornification. Estradiol benzoate (EB) injections induced a surge in serum prolactin, luteinizing hormone (LH), and follicle-stimulating hormone (FSH) levels between 15.00 and 18.00 h in ovariectomized rats bearing sham lesions in the MBSC. The EB-induced surge of LH and FSH release was eliminated in ovariectomized animals with lesions in the MBSC. Serum prolactin levels showed consistently low levels during 24 h 3 out of the 7 lesioned animals and considerable fluctuations but no surge in the remaining 4 animals. Progesterone injections into the EB-treated rats with MBSC lesions did not elicit the marked surge in serum LH and FSH which was observed in sham-lesioned animals. In contrast, the EB-treated rats with MBSC lesions showed a significant rise of serum prolactin levels after progesterone injections, similar to that of sham lesioned animals. These results suggest that MBSC plays an essential role in the neural control of the steroid-induced surges of prolactin and gonadotropin release.

The present study was undertaken to determine the role of the hypothalamic periventricular (PV) region in the regulation of the secretory pattern of growth hormone (GH) in adult male rats. The PV regions were destroyed stereotaxically by means of a modified Halasz's knife which was lowered through the midline and rotated several times at the level of the region. In sham-operated controls, the knife was lowered to the same region but no rotation was performed. Serial blood samplings were performed at 10-min intervals for 10 h (10.00-20.00 h), via an intracardiac cannula, at 2, 4 and 6 weeks postoperatively. In 7 sham-operated controls, GH was secreted with a surge period of about 3.5 h, with peak levels of 120-170 ng/ml whole blood and basal levels of about 10 ng/ml whole blood. In 11 PV-lesioned animals, the basal levels of GH were unaltered but the surge period decreased to about 3.0 h and peak levels increased to about twice as high as in sham-operated controls. Somatostatin concentrations in the median eminence of PV-lesioned rats were significantly decreased to about 32% of that of sham-operated rats. The results suggest that, via somatostatin neurons, the PV region plays an inhibitory role in the regulation of periodicity and peak levels of GH secretory bursts.

To address the issue of whether, after morphine treatment, the reduced release of dopamine (DA) into portal blood is entirely responsible for the increased prolactin (PRL) release, the following study was conducted. The concentration of DA in plasma from a single portal vessel of untreated, ovariectomized rats was 1.85 +/- 0.33 ng/ml (mean +/- SE). Treatment of ovariectomized rats with alpha-methyl-p-tyrosine (alpha MT) caused a 91% reduction in the concentration of DA in portal plasma. Infusion of DA (0.4 microgram/min/kg BW) into a jugular vein of rats pretreated with alpha MT restored the DA concentration in portal plasma to that seen in untreated rats. Injection of morphine sulfate elicited a marked increase in the concentration of PRL in plasma. Infusion of DA at rates of 0.4 and 0.8 microgram/min/kg BW suppressed by 52 and 75%, respectively, the secretion of PRL after morphine treatment. Infusion of DA had no effect on the release of PRL induced by intracerebroventricularly administered beta-endorphin. Although treatment of rats with alpha MT caused release of PRL that was similar to that seen in rats treated with morphine, infusions of DA at 0.4 and 0.8 microgram/min/kg BW into alpha MT-treated, ovariectomized rats suppressed the secretion of PRL by 89 and 96%, respectively. Thus, after morphine treatment, the decreased release of DA into portal blood is not in itself sufficient to account for the increase seen in the secretion of PRL. It is suggested that morphine and opiate-like peptides induce the release of a hypothalamic substance(s) that stimulates PRL release.

A new method for estimation of in vitro neurosecretory activity of tuberoinfundibular dopaminergic (TIDA) neurons was developed by measuring the rate of synthesis of dihydroxyphenylalanine (DOPA) in the median eminence of hypothalamic slices. Sagittal hypothalamic slices of ovariectomized rats were incubated in a medium containing 3-hydroxybenzylhydrazine (NSD 1015), an inhibitor of DOPA decarboxylase. DOPA accumulated in the median eminence following incubation with NSD 1015 was determined by high-performance liquid chromatography with electro-chemical detection. The amount of DOPA accumulated in vitro in the median eminence was maximal in a medium containing 10 mM NSD 1015 and linear up to 120 min at 37 degrees C. Increasing the concentration of tyrosine in medium stimulated the synthesis of DOPA in the median eminence. The synthesis of DOPA was blocked by 1 mM alpha-methyltyrosine, an inhibitor of tyrosine hydroxylase. The rate of in vitro synthesis of DOPA in the median eminence was 33% of that of in vivo synthesis. Incubation in a medium containing 50 mM K+ to depolarize neurons caused a 2.4-fold increase in DOPA synthesis in the median eminence. The high K+-induced increase in DOPA synthesis was blocked by omission of Ca2+ and addition of 1 mM EGTA into the medium, suggesting Ca2+ dependency of depolarization-activated DOPA synthesis. These results indicate that this in vitro assay is a useful means to study the regulatory mechanisms of TIDA neurons.