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Endocrinology Vol. 138, No. 2 649-656
Copyright © 1997 by The Endocrine Society

Articles

The Interaction between ß-Endorphin and Gonadal Steroids in Regulation of Luteinizing Hormone (LH) Secretion and Sex Steroid Regulation of LH and Proopiomelanocortin Peptide Secretion by Individual Pituitary Cells1

Fouad R. Kandeel and Ronald S. Swerdloff

Department of Diabetes, Endocrinology, and Metabolism, City of Hope National Medical Center, Duarte, California 91010; and the Department of Medicine, Harbor-University of California-Los Angeles Medical Center, Torrance, California 90509

Address all correspondence and requests for reprints to: Fouad R. Kandeel, M.D., Ph.D., Department of Diabetes, Endocrinology, and Metabolism, City of Hope National Medical Center, 1500 East Duarte Road, Duarte, California 91010.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Reported studies using the conventional pituitary cell culture technique suggest that ß-endorphin (B-EP) produced locally in the pituitary or reaching it from the hypothalamus acts in conjunction with estradiol (E2) to initiate and in conjunction with progesterone (P4) to terminate the midcycle surge of LH. In addition, the reverse hemolytic plaque assay (RHPA) was used to investigate the effects of E2 and P4 on the secretory activity of individual pituitary cells. The results of these experiments indicate that 1) E2 enhances the secretion of LH, ACTH, and B-EP by individual pituitary cells; 2) E2 increases the number of secreting cells for each of the three hormones; 3) the rise in B-EP and ACTH secretion antecede that of LH; 4) P4 augments ACTH and B-EP secretion by individual pituitary cells; and 5) P4 has dual effects, acutely (1 h) potentiating LH secretion from already active cells and subsequently (8 h) recruiting cells that formerly had little or no secretory activities.

Collectively, the above studies support a role for steroid hormones in regulation of midcycle LH secretion at the pituitary level. The results also suggest that intrapituitary (paracrine/autocrine) and extrapituitary (endocrine) B-EP modulates gonadal steroid effects on LH secretion by pituitary gonadotrophs.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE ABUNDANT evidence for a prominent hypothalamic role for endogenous opioids in control of gonadotropin secretion has drawn attention away from the possibility of a local regulatory effect at the pituitary level. A few relatively recent studies have demonstrated the presence of such local regulatory effects. Sanchez-Franco and Cacicedo (1) used cultured male rat pituitary cells and showed that ß-endorphin (B-EP) blocks GnRH-stimulated LH release. These investigators also showed that the effect of B-EP on LH release is time dependent, and a period of 48 h is required for the manifestation of the maximal inhibitory effect (2). Independently, Blank and colleagues (3) found that morphine sulfate directly inhibits both basal and GnRH-stimulated LH release by cultured rat pituitary cells and that treatment of cells with the opioid receptor antagonist, naltrexone, or with B-EP antiserum significantly increases basal LH release. Also incubation of the pituitary cells with CRH causes a significant decrease in basal LH release, an effect that is reversible by naltrexone. Collectively, these studies suggest that intrapituitary opioid peptides could exert a paracrine regulatory action on gonadotroph cell function. This conclusion is further supported by other reports in which opioid receptors were shown to be present on gonadotroph cell surface (3), and LH was colocalized with ACTH in a small number of pituitary cells (4). ACTH and B-EP are both derived from the common precursor POMC molecule.

We used conventional pituitary cell cultures to investigate the interplay between B-EP and gonadal steroids in the control of the midcycle LH surge. Further, we used the reverse hemolytic plaque assay (RHPA) to characterize the temporal effects of gonadal estrogen and progesterone (P4) on the secretion of LH, ACTH, and B-EP by individual pituitary cells in terms of latency of cell activation and cell recruitment. The principle of RHPA depends on computerized image analysis of staphylococcal protein A-linked ovine erythrocytes hemolysis (plaque formation) in response to complement activation by antigen (hormone)-antibody binding in the immediate surrounding of an activated hormone-producing cell (4, 5). Thus, the RHPA permits characterization of the functional activity of individual pituitary cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Conventional pituitary cell cultures
Pituitary cell cultures from short term (10-day) ovariectomized Sprague-Dawley adult female rats (60 days of age at death) were prepared according to the method of Vale et al. (6) and used to examine the interaction between gonadal steroids [estradiol (E2) and P4] and B-EP on gonadotropin release in in vitro. After 2 days of preincubation of the pituitary cells in steroid-free medium, cultured wells were divided into four groups and replenished with steroid and B-EP-free medium (vehicle control), medium containing 10-9 M E2 (two groups), or medium containing 10-9 M E2 and 10-7 M B-EP (one group), respectively. Forty-eight hours later, media were removed and discarded, and each culture group was treated sequentially for two 3-h periods. In the first 3-h incubation period, each culture group was treated with medium containing the same reagent mixture used in the 48-h incubation period. In the second, 10-5 M P4 was added to the reagent mixture of one of the two E2-treated groups and to the E2-B-EP treated group. Otherwise, reagent mixtures remained the same as those during the previous incubation period. Medium collected after each of these 3-h periods was used for measurement of LH. No changes in cell viability, assessed with trypan blue staining and cell count, were observed among the different treatment groups.

RHPA
The anti-hACTH antiserum 36–121882 and the anti-hB-EP antiserum 7–41980, supplied by Dr. A. F. Parlow, and the anti-oLH antiserum AFP192279, supplied by the National Pituitary Agency, were used for the plaque assays of ACTH, B-EP, and LH, respectively. Two novel approaches were employed to examine the specificity of selected antisera. In the first approach, the antiserum, authentic hormone, or related peptides (possible cross-reactants), protein A-linked erythrocytes, and complement, all in concentrations identical to those used in the plaque assay, were infused in Cunningham’s chambers constructed on slides and incubated at 37 C for 2 h. Slides were then photographed at a magnification of x40. The average number of intact (unhemolyzed) erythrocytes in 1-cm2 area for each antigen concentration was calculated and a dose-response curve was derived. The results of these studies are shown in Fig. 1aGo, which illustrates the cross-reactivity of LH antiserum. In the second approach, the above reagents were mixed in microtubes and then incubated in a shaker water bath at 37 C, followed by electronic counting of intact erythrocytes. Figure 1bGo illustrates the dose-response relationship between antigen concentration (B-EP) and the number of intact erythrocytes in the reaction mixture. Results obtained in these experiments were in general agreement with those obtained in cross-reactivity studies by the RIA for all three antisera. The major cross-reactant with the ACTH antiserum, compared on a weight basis to ACTH-(1–39) (100%), was ACTH-(1–34) (64%). ACTH-(18–39) showed minimal cross-reactivity (<1%), and no cross-reactivity was exhibited by ACTH-(4–10), ACTH-(34–39), {alpha}MSH, or ßMSH. Major cross-reactants with the B-EP antiserum, compared on a weight basis to B-EP-(1–31) (100%), were: N-acetyl-B-EP (100%), B-EP-(1–27) (26%), and ß-lipotropin (17%). Neither {alpha}-endorphin, {gamma}-endorphin, Met-enkephalin, nor Leu-enkephalin exhibited any cross-reactivity toward B-EP antiserum. No significant cross-reactivity was noted for any LH-related peptide with the anti-oLH antiserum except for LH. Validation of the plaque assay for each hormone was performed according to the criteria of Smith and colleagues (4, 5), including loss of plaque formation when any of the assay reagents was omitted or when the antiserum was replaced with normal rabbit serum and the presence of a dose-response relationship between the concentration of secretagogue and the size of the plaque.



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  		Figure 1. a, Specificity of antiovine LH AFP192279 antiserum using the number of unhemolyzed protein A-coated sheep red blood cells in a 1-cm2 area of Cunningham’s chambers to infer the degree of cross-reactivity. b, The dose-response curve of B-EP antiserum 7–41980 calculated from the number of unhemolyzed protein A-coated sheep red blood cells in reaction mixture, See text for details.

 
Several plaque assays of pituitary cells were performed to examine the direct effect of gonadal steroids on LH, ACTH, and B-EP secretion by individual pituitary cells of short term (10-day) ovariectomized adult Sprague-Dawley female rats. These experiments were performed identically except for varying durations of estrogen and P4 treatment. In all assays, freshly dispersed pituitary cells were harvested according to the method of Vale et al. (6), plated on slides (105 cells in 0.1 ml DMEM-charcoal stripped horse serum/slide), and incubated at 37 C for 50 min to allow for cell attachment. Slides were then placed in culture dishes containing DMEM-charcoal-stripped horse serum and incubated at 37 C for 24 h. Media were mixed with vehicle, E2 alone (10-9 M), or E2 followed by the addition of P4 (10-6 M) for the designated times (see below). The doses of steroid hormones used in these plaque assay experiments and in the conventional pituitary cell cultures described above are based on physiological ranges and on earlier investigations in which these doses significantly stimulated gonadotropin secretion in conventional culture experiments (7, 8). Slides were washed with DMEM, and Cunningham chambers were constructed above the cell-containing areas. Chambers were infused with a mixture of antiserum (LH, ACTH, or B-EP), secretagogue (GnRH or CRH), protein A-coated sheep erythrocytes, and complement. After 3 h of incubation at 37 C, slides were examined microscopically to assess plaque formation. Positive plaque formation was defined as an area of hemolysis greater than the diameter of two erythrocytes surrounding an individual pituitary cell. Plaque size parameters (diameter, perimeter, and area) were then determined using a video system and Micro-Image software (Analytical Imaging Concepts, Irvine, CA). As plaque size parameters are interrelated and always change in the same direction, only the area data are presented in Results. The number of plaques formed on each slide was counted manually. At the end of the steroid treatment periods, one slide of each treatment group was used to assess cell viability. These slides were stained with trypan blue and examined microscopically for evidence of cell death. No significant differences in cell viability were noted among the different treatment groups.

Data for each experiment were assessed for homogeneity of variances among groups as well as for normality of distribution of each group. When necessary, appropriate transformations were performed before the statistical analysis with the applicable BMDP program. One or two-way ANOVA with trend analysis or multiple comparisons (Tukey two-tailed Studentized range, Bonferroni and Newman-Keuls tests) of groups were performed. When the two-way interaction was significant, a simple effects ANOVA was performed. Each experiment was repeated three to six times. As the interassay variation in plaque size parameters is large, only the results of one representative assay are given below. The results of the other assays were used to confirm the reproducibility of the experiments, i.e. the qualitative changes in hormone secretion and the number of activated cells.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of B-EP and its interaction with gonadal steroids on LH secretion by female rat pituitary cells in conventional cell cultures
Conventional pituitary cell cultures were performed as described in Materials and Methods and used to determine the effects of steroid-B-EP interactions on LH secretion. Conventional pituitary cells primed with E2-B-EP for 48 h produced significantly higher LH concentrations during the first 3-h incubation period with identical reagents than cultures treated with E2 (P < 0.01) or vehicle (P < 0.005; Fig. 2aGo). During the subsequent 3-h period, pituitary cell cultures treated with E2-P4 had LH concentrations significantly higher than all other groups (P < 0.05–0.005). Cultures treated with E2, B-EP, and P4 had LH concentrations that were not different from those in the E2 alone or vehicle-treated groups (Fig. 2bGo).



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  		Figure 2. LH secretion over 3 h (mean ± SEM) in conventional pituitary cell cultures previously exposed to E2 or E2 plus B-EP for 48 h is depicted in a. LH secretion (mean ± SEM) from pituitary cells exposed to E2 alone, E2 plus P4, or E2, P4, and B-EP for an additional 3 h is shown in b. See text for details.

 
As treatment of pituitary cell cultures with opioids was shown to result in inhibition of gonadotropin secretion, we attempted to reproduce the results of Sanchez-Franco and Cacicedo (1) using male rat pituitary cells (0.3 x 106 cells/ml·well and an incubation time of 48 h). After a preincubation period of 48 h without (vehicle) or with B-EP treatment (100 nM), cell cultures were treated with identical reagents for 3 h, and media were collected for LH measurement. Media of cells treated with B-EP had significantly lower LH concentrations than controls (mean ± SEM, 4.48 ± 0.35 vs. 7.26 ± 0.44 ng/ml; P < 0.01); these results are consistent with those of the above investigators.

Effect of E2 on ACTH secretion by individual pituitary cells
Plaque assays for ACTH were performed after pretreatment with vehicle or E2 for 24, 36, 56, or 80 h to examine the time course of the effect of estrogen treatment on ACTH secretion by individual pituitary cells. Log-transformed data on plaque area were analyzed by two-way ANOVA. The largest effect of E2 treatment on plaque size parameters was observed at 36 h (Table 1Goa). The number of plaque-forming cells for ACTH showed a progressive increase from 24 to 56 h of treatment for both the control and E2-treated groups (Table 1Gob). Statistically, there were significant main time and treatment effects (P < 0.015 and P < 0.0025, respectively) with no interaction between these two parameters, suggesting that the main effects of time and treatment are additive in nature. The linear (P < 0.03), but not the quadratic (P < 0.0502), component of the time effects was significant.


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  		Table 1. ACTH secretion by individual pituitary cells in response to estradiol treatment

 
Effects of E2 and P4 treatments on ACTH and B-EP secretion by individual pituitary cells
Separate plaque assays of ACTH and B-EP were performed on pituitary cells pretreated with vehicle, E2 alone, or E2 and P4 for a total of 56 h to examine the effect of sequential treatment with E2 and P4 on POMC-derived peptide secretion by individual pituitary cells. When applicable, the exposure to P4 was commenced 8 h before the conclusion of the treatment period. Data obtained for each hormone were analyzed by one-way ANOVA. The treatment effect was significant in each case (P < 0.001). Tukey’s multiple comparison and Newman-Keuls testing showed no differences between vehicle-treated and E2 only-treated groups, and both of these had significantly smaller plaque areas of each hormone than the E2- and P4-treated cells (P < 0.05–0.01; Table 2Go, a and b).


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  		Table 2. ACTH and ß-endorphin secretion by individual pituitary cells in response to estradiol and progesterone treatment

 
Effect of E2 treatment on LH secretion by individual pituitary cells
Plaque assays for LH were performed after pretreatment with vehicle or E2 for periods of 36, 56, or 80 h to examine the time course of estrogen treatment on LH secretion by individual pituitary cells. At 56 h, E2 induced a larger plaque area than vehicle treatment, with the opposite effect seen at 80 h (Table 3Goa). E2 also increased the number of plaque-forming cells at 56 h (Table 3Gob).


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  		Table 3. LH secretion by individual pituitary cells in response to estradiol treatment

 
The differences between vehicle and E2 treatments on plaque area were characterized by two-way ANOVA after log transformation. In this analysis, a significant time effect (P < 0.01) and a significant time by treatment interaction effect (P < 0.0001) were found. Both linear and quadratic components of the time effect were affected by treatment (P < 0.0015). Simple time effects were significant for both vehicle- and E2-treated groups (P < 0.025). The simple linear trend (increased plaque size with time) was significant for both treatment groups, whereas the quadratic trend (plaque size at 56 h was larger than at 80 h of incubation) was significant only for the E2-treated group (P < 0.05). The simple main effects of treatment were significant only for the E2-treated group at 56 and 80 h (P < 0.002), but not at 36 h. Thus, E2 stimulation of LH is time dependent up to about 56 h and is followed by a decline in hormone production by the responding cell. The actions of E2 also involve activation of cells with little or no LH secretory activity.

Effect of P4 treatment on LH secretion by E2-primed pituitary cells
The plaque assay was performed on pituitary cells treated with vehicle, E2 alone, or E2 plus P4 for a total period of 56 h (the time at which E2 induced a maximum increase in LH secretion in the above experiments). When applicable, the exposure to P4 was commenced 4, 8, 12, or 24 h before conclusion of treatment to examine the time course of the P4 effect on LH secretion by the estrogen-primed pituitary cells. One-way ANOVA was performed for each plaque area data set. The overall treatment effect was significant (P < 0.02). All steroid-treated groups were contrasted with the control group and found to be significant (P < 0.02). A trend analysis of the mean values for the steroid-treated groups showed significant quadratic and cubic P4 exposure time effects (P < 0.05). The highest mean plaque area was exhibited by the E2 only-treated group, the lowest by the control group, and the next lowest by the 8-h P4-treated group. The cubic P4 exposure time effect largely reflected the plateauing of group means between 12 and 24 h (Table 4Goa). The average plaque number for the E2 only-treated cultures was 30% higher than that in the control group, and treatment with P4 for 8 h resulted in a further increase in plaque number by 25% above that found with the E2 only treatment (Table 4Gob). This increase in plaque number after 8 h of P4 treatment may reflect a newly recruited and activated population of gonadotroph cells.


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  		Table 4. LH secretion by individual pituitary cells in response to estradiol and progesterone treatment

 
Established data in both intact animals and conventional culture experiments, as presented above, demonstrate that P4 treatment after E2 priming enhances LH secretion. Therefore, we examined the possibility that an increase in LH plaque size area may occur earlier than the shortest P4 exposure time tested (4 h). Thus, the above experimental paradigm was repeated but the P4 exposure time was limited to 1, 2, or 3 h (Table 5Go). P4 treatment of estrogen-primed cells for 1 h induced a significant increase in the surface area of LH plaques (Table 5Goa), but not in the number of LH plaque-forming cells. However, estrogen alone treatment increased both plaque size and number (Table 5Go, a and b), effects that were consistent with the results of other reported experiments.


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  		Table 5. LH secretion by individual pituitary cells in response to estradiol and progesterone treatment

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Information on hypothalamic or pituitary opioid tone during the phase of high gonadotropin release (midcycle surge) is conspicuously deficient. Studies in intact rats and monkeys showed that opioid receptor blockade with naloxone stimulates LH release during stages of high gonadal steroid milieu, except at the time of the preovulatory gonadotropin surge, when it becomes ineffective (9, 10). Although these and other animal and human studies established an inhibitory role for endogenous opioid peptides on gonadotropin release at times of the cycle other than the preovulatory phase, they did not distinguish between local hypothalamic vs. pituitary actions of these peptides, nor did they clarify their effects during the midcycle gonadotropin surge. Intuitively, the opioid peptides cannot have an unconditional inhibitory influence on gonadotropin release throughout the menstrual cycle, as this may dampen or hinder the occurrence of the midcycle surge. Some events capable of curtailing or overriding the inhibitory effect of opioids must, therefore, be taking place at this phase of the cycle. Indeed, a transient decrease in the inhibitory effect of opioids, induced by naloxone infusion for 24 h, in the preovulatory phase was shown to contribute to the initiation of the midcycle gonadotropin surge in women (11).

An alternative hypothesis that could explain the occurrence of the midcycle gonadotropin surge in the face of rising B-EP concentrations is that the opioid effects may be diverse in nature depending upon factors such as the prevailing gonadal steroid milieu and the site of action (pituitary or hypothalamic). If this was true, then at the time of midcycle surge, opioid peptides could have a permissive or even a facilitatory role for gonadotropin release. Many observations have already been made to support this contention of diverse opioid effects. Examples of these are the opioid peptide-stimulated LH release in certain experimental conditions in the rat (12, 13) and the biphasic effect (initial rise followed by a sustained fall) on LH secretion during the early follicular phase of the human female (14). Thus, there appears to be a gap in the knowledge concerning the regulation of gonadotropin secretion by endogenous opioid peptides during the preovulatory surge, for the site of action and the nature of effect. Likewise, the interactions between steroids and opioids and their modulating effects on gonadotroph cell function during the preovulatory phase are largely unknown.

The data presented in this communication confirm the previously reported inhibitory effects of opioids on LH secretion by rat pituitaries in conventional cell cultures (1, 3). These studies, however, were performed on cells obtained from male animals and in the absence of any modulating effects from gonadal steroids. More importantly, our data on female pituitary cell cultures (Exp 1) suggest an important role for the interaction between ovarian steroids and B-EP in the direct regulation of pituitary secretion of gonadotropins, which is the main objective of the present studies. The data indicate that, at least in the female, the effect of B-EP on gonadotropin secretion is dependent on the ambient steroid hormone milieu and period of exposure. Specifically, B-EP stimulates LH secretion in the presence of a high estrogen milieu. Conversely, when P4 is added, it inhibits the P4-mediated augmentation of LH secretion by estrogen-primed cells. Thus, as the pre/periovulatory phase of the female reproductive cycle exhibits sequential increases in E2, B-EP, and P4 secretion, it may be feasible to propose that B-EP produced locally in the pituitary gland or reaching the pituitary from the hypothalamus during the pre/periovulatory phase, participates in regulation of the midcycle gonadotropin surge by acting in conjunction with E2 to potentiate and in conjunction with P4 to terminate the midcycle surge of LH. Figure 3Go illustrates the proposed hypothetical interactions between B-EP and gonadal steroids in the regulation of the midcycle LH surge. It should be noted, however, that any possible regulatory effects of opioid peptides at the pituitary must occur in concert with those exerted at the hypothalamus, as in the case of gonadal steroids.



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  		Figure 3. Hypothetical interaction between B-EP and gonadal steroids in regulation of the midcycle LH surge. The preovulatory rise in B-EP potentiates E2 stimulation of LH secretion and therefore augments the magnitude of the LH surge. As luteinization commences, and circulating P4 starts to rise, B-EP reverses its action to limit the P4-induced augmentation of LH secretion and thus helps to terminate the midcycle surge of LH.

 
The concept of local paracrine regulation among the pituitary hormones is not only restricted to regulation of LH secretion by B-EP, but also applies to the regulation of other anterior pituitary hormones. For example, Ellerkmann and colleagues used the RHPA to show that N-acetylated {alpha}MSH and B-EP are capable of increasing the fraction of pituitary cells that secretes PRL, and demonstrated that both of these POMC-derived peptides act in a cooperative fashion to recruit additional cells into the PRL-producing population (15). In a separate study, the same group of investigators showed that estrogen stimulation of PRL secretion depends on the release of an activity factor from the neurointermediate lobe that is capable of augmenting the PRL secretory capacity of individual pituitary cells as well as recruiting additional PRL-producing cells into the secretory pool (16). Similarly, Weber and Calogero found that PRL treatment of anterior pituitary cell culture causes a 65% increase in the secretion of ACTH (17).

The cyclic changes in hypothalamic B-EP release (18) and peripheral blood B-EP concentrations (19) suggest a role for gonadal steroids in the regulation of B-EP secretion by B-EP-producing tissues. The pituitary is the most likely source of peripheral blood B-EP, and cyclic variation in peripheral blood concentrations of ACTH, which is known to be secreted from corticotrophs on an equimolar basis with B-EP, occurs (20, 21). Therefore, it is most likely that B-EP secretion by pituitary corticotrophs is in part regulated by gonadal steroids. Several recent studies have provided additional circumstantial support for a role for gonadal steroids in the regulation of POMC-derived peptides at the level of the pituitary. Spinedi and colleagues (22) found that pituitary cells, cultured in vitro, obtained from random cycling and ovariectomized and estrogen-replaced rats at physiological levels secrete a higher amount of ACTH and have a greater binding capacity for angiotensin II than cells from ovariectomized rats without or with estrogen replacement at supraphysiological concentrations. However, direct estrogen treatment of pituitary cell cultures had no effect on ACTH secretion in this study. Other investigators showed that P4 antagonizes glucocorticoid inhibition of B-EP (23) and CRH stimulation of ACTH and B-EP secretion (24, 25) in primary cultures. Our results in Exp 2 and 3 using the RHPA provide direct evidence for gonadal steroid regulation of POMC-derived peptides at the level of the pituitary. E2 enhanced the secretion of each of the POMC-derived peptides in a time-dependent manner by increasing both the number of hormone-producing cells and the amount of hormone produced by individual pituitary cells. Further, P4 treatment of estrogen-primed cells increased the amount of peptide produced by individual cells after the stimulating effects of estrogen alone treatment had dissipated. These results support a role for both ovarian E2 and P4 in direct stimulation of B-EP and related peptide secretion by the pituitary corticotrophs during the pre- and periovulatory periods. Our results of P4 enhancement of ACTH and B-EP secretion are in accordance with those of Abou-Samra and colleagues (23), in which P4 blocked the feedback inhibition of B-EP release by rat anterior pituitary cells. However, the apparent discrepancy in the effects of P4 observed in our experiments and those of others (24, 25) is probably related to differences in the experimental design and the prior exposure to estrogen. Further, the shorter latency for the estrogen-mediated POMC-derived peptides than for the estrogen-mediated LH secretion demonstrated in Exp 4 (36 vs. 56 h) supports the contention of a temporal role for the earlier increase in POMC-derived peptides in control of the subsequent surge in LH production. Moreover, other recent evidence suggested that a small number of pituitary cells (<0.4% of neonatal male rat pituitary cells cultured in vitro) stain for and secrete both ACTH and LH (4). Although this amount of hormone coproduction may not be significant in the neonatal male rat, its presence raises the possibility of cosecretion of structurally unrelated hormones by pituitary cells to a more significant degree in other physiological states. If this is the case, and if these cells are found in the adult female, then they most likely would be responsive to the modulating effects of gonadal steroids. In such a case the gonadotroph cell function would be influenced by B-EP secretion from three different sources representing all known regulatory mechanisms: the endocrine component of B-EP is secreted by the hypothalamus and reaches the pituitary via the portal circulation, the paracrine component is secreted by the corticotroph cells within the pituitary, and the autocrine component is secreted by the gonadotroph cell population itself (4, 20, 21, 26, 27, 28).

The results of the RHPA of LH after sequential exposure to E2 and P4 (Exp 5) are of particular interest. In contrast to the effects of E2 alone demonstrated in Exp 4, which produced a simultaneous increase in the number of hormone-producing cells and the amount of LH produced by each cell, P4 treatment of E2-primed gonadotrophs augmented the secretion of LH acutely (within 1 h) from the actively producing cells, but its action on the number of hormone-producing cells was more delayed (~8 h). The delayed increase in the number of LH-secreting cells appears to reflect the recruitment and activation of dormant gonadotrophs. This would include stimulation of gene transcription as well as translation. These processes are more time consuming than the mere increase in message translation expected to occur during the acute stimulation of hormone secretion that was observed within 1 h of P4 exposure. The significance of the new cell recruitment mediated by P4 treatment is not immediately apparent. Plausible explanations include the possibility that activation of a new cell population prevents the premature termination of the midcycle gonadotropin surge subsequent to exhaustion and down-regulation of cell secretion or to the inhibitory effects of B-EP on LH secretion in the presence of P4. Alternatively, P4-mediated gonadotroph cell recruitment may be necessary for the manifestation of the high amplitude pulse secretion of gonadotropins characteristic of the luteal phase of the normal female reproductive cycle (29–31). Figure 4Go illustrates the hypothetical contribution of the acute and delayed effects of P4 on the overall secretion of LH by E2-primed pituitary gonadotrophs.



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  		Figure 4. Hypothetical regulatory effects of P4 on LH secretion by the E2-primed pituitary gland. P4 enhances LH secretion via two mechanisms. The first is rapid and involves potentiation of the actively secreting cells. The second is slower and involves recruitment and activation of dormant gonadotrophs, possibly through switching on de novo gene transcription.

 
In summary, the results of conventional pituitary cell cultures and RHPA presented in this communication indicate that 1) E2 enhances the secretion of LH, and POMC-derived peptides by individual pituitary cells in a time-dependent manner; 2) E2 increases the number of hormone-secreting cells for these hormones also in a time-related fashion; 3) the rise in POMC-derived peptides antecede that of LH (36 vs. 56 h); 4) P4 augments ACTH, B-EP, and LH secretion by individual cells; and 5) at least in the case of LH, P4 may have dual effects, initially potentiating LH secretion from already active cells and subsequently recruiting and activating cells that formerly had little or no secretory activity.


   Acknowledgments
 
The authors thank Dr. A. F. Parlow and the National Pituitary Agency for their generous gifts of antisera, Mr. Jeff Sellers and Dr. Jimmy Neill for their significant advice in setting up the plaque assay, and Lou Ann McAdams for her assistance with statistical analysis. They also thank Pacita Tracy for her assistance in preparing the manuscript.


   Footnotes
 
1 This work was supported by New Development Award of the University of California-Los Angeles Population Research Center (2P30-HD-19445-04). The study was presented in abstract form at the Third International Pituitary Congress, Marina Del Rey, CA, 1993. Back

Received October 20, 1996.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Sanchez-Franco F, Cacicedo L 1986 Inhibitory effects of B-endorphin on gonadotropin-releasing hormone and thyrotropin-releasing hormone releasing activity in cultured rat anterior pituitary cells. Horm Res 24:55–61[Medline]
  2. Cacicedo L, Sanchez-Franco F 1985 Direct action of opioid peptides and naloxone on gonadotropin secretion by cultured rat anterior pituitary cells. Life Sci 38:617–625
  3. Blank MS, Fabbri A, Catt KJ, Dufau ML 1986 Inhibition luteinizing hormone released by morphine and endogenous opiates in cultured pituitary cells. Endocrinology 118:2097–2101[Abstract/Free Full Text]
  4. Neill JD, Smith PF, Luque EH, De Toro MM, Nagy G, Mulchahey JJ 1987 Detection and measurement of hormone secretion from individual pituitary cells. Recent Prog Horm Res 43:175–229
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