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Department of Biology, Sinsheimer Laboratories (G.T., G.O.G., B.N., F.T.), University of California, Santa Cruz, California 95064; Lawrence Berkeley Laboratory (S.G.), University of California, Berkeley, California 94720; and Morehouse School of Medicine, Department of Physiology (R.S.), Atlanta, Georgia 30310
Address all correspondence and requests for reprints to: Frank Talamantes, Department of Biology, Sinsheimer Laboratories, University of California, Santa Cruz, California 95064. E-mail: prolactin{at}aol.com
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Abstract |
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Introduction |
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At the time when the placenta is taking over the role of the pituitary in regulating the function of the CL, a large peak of androstenedione (AD) and a smaller peak of testosterone (T) appear in the circulation (7). The androgen surges coincide precisely with the large peak of PL-I in the circulation (7, 8). It is not known whether the androgens are important for maintaining the CLs in the midpregnant mouse. Based on studies in the rat, it was suggested that the androgens were important only as substrates for estrogen synthesis, and that the estrogens, in turn, were the essential component of the luteotropic complex (9). However, other studies have indicated that androgens may have direct luteotropic activity. For example, studies on cultured granulosa cells from mice (10) and rats (11) and rat luteal cells (12) have shown that the nonaromatizable androgen dihydrotestosterone (DHT) is as effective in stimulating P4 synthesis as AD or T, whereas estrogens did not enhance the release of P4.
In this study, we examined 1) whether the three mouse (m) lactogens (mPRL, mPL-I, and mPL-II) show significantly different potency in stimulating P4 release from mouse CL cells, and whether the potency of the lactogens was dependent on the stage of pregnancy the CL cells were obtained; 2) whether the lactogens and the androgens interact to regulate P4 release from the CLs of the pregnant mouse; and c) some pathways by which the androgens might exert their effect on stimulating P4 release from the CLs.
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Materials and Methods |
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Reagents
Recombinant mPL-I was generated in Chinese hamster ovary cells
and purified according to previously published procedures (13). mPL-II
and mPRL were purified as described previously (14, 15). Fadrozole and
hydroxyflutamide were generously provided as gifts from Ciba-Geigy
(Basle, Switzerland) and Schering-Plough Research Institute
(Kenilworth, NJ), respectively. The following reagents were from Sigma
Chemical Co. (St. Louis, MO): 4-androstene-3–17-dione (AD),
testosterone (T), 17ß-estradiol (E2), 5-cholestene-3ß,
22[R]-diol (22R-OHC), 5
-dihydrotestosterone (DHT), cycloheximide,
actinomycin D, fibronectin (bovine plasma), gentamicin, DMEM-Ham’s
nutrient mixture F12 (1:1) with 15 mM HEPES (DME/F12), HBSS
without calcium and magnesium, BSA, fraction V (BSA), deoxyribonuclease
from bovine pancreas type I (DNase), rabbit IgG (IgG), and trypan blue
(0.4% wt/vol solution). Nutridoma NS was purchased from Boehringer
Mannheim (Indianapolis, IN). Collagenase, type II from
Clostridium histolyticum (CLS 2) was obtained from
Worthington Biochemical Co. (Malvern, PA). Flat-bottomed tissue culture
plates (96-well) were from Becton Dickinson Co. (Lincoln Park, NJ)
Luteal cell dissociation
The entire cell dissociation procedure was carried out in
calcium- and magnesium-free HBSS containing 15 mM HEPES and
50 µg gentamicin/ml. The luteal tissue was first washed and then
transferred to a dissociation flask containing 0.1% CLS 2 collagenase
and 0.004% DNase in 10 ml medium. The enzyme digestion was carried out
at 37 C in a rotating water bath (
130 rpm) for 1 h. At the end
of the digestion period, the tissue pieces were further dispersed by
withdrawing and expelling the cell suspension with a 10-ml pipette
until mostly individual cells were obtained. Undissociated clumps of
cells were allowed to precipitate, and the supernatant, containing the
individual cells, was removed. The undigested tissue was incubated in
PBS solution containing 0.02% (wt/vol) EDTA and 2% BSA for 10 min at
37 C in a rotating water bath (130 rpm). The cell suspension was
then agitated by pipetting action as described above and filtered
through a 150-µm Nitex (Tetko, Co., Elmsford, NY) mesh. The resulting
filtrate containing individual cells was combined with the cell
suspension from the first digestion, and the pooled cells were
centrifuged at 100 x g for 10 min. The supernatant was
aspirated, and the cells resuspended in 8 ml HBSS and then layered onto
a 2-ml cushion of 44% Percoll in a 15-ml polypropylene tube and
centrifuged at 400 x g for 20 min. The luteal cells
that banded at the interface between the Percoll and the HBSS were
harvested, washed, and resuspended in 6–8 ml DME/F12 containing 50
µg/ml gentamicin, 0.5 µg/ml 22R-OHC, and Nutridoma NS diluted 1:100
(basic culture medium). The cells were then counted and the viability
assessed using trypan blue exclusion. Viability varied from
85–95%.
Luteal cell culture
We used our previously developed ovarian cell culture system
(16) with slight modifications. Briefly, the cells were cultured in
96-well plates that had been coated with bovine fibronectin. For the
coating, the fibronectin was diluted to 50 µg/ml in basic culture
medium, and 50 µl of that solution were dispensed into each well. The
plates were then incubated for 2 h at 37 C, followed by aspiration
of the coating medium. After counting, the cells were diluted to
5.0 x 105 to 7.5 x 105 cells/ml and
plated at a density of 1.0 x 105 to 1.5 x
105 cells/well in 200 µl basic culture medium. The cells
were incubated at 37 C in 99% humidity and an atmosphere of 5%
CO2, 95% air for the initial 24 h, at which time the
plating medium was discarded and fresh medium, containing the various
treatments, was added. The cells were cultured in the different
treatment media for time periods ranging from 15 min to 48 h.
Treatments of cultured luteal cells
AD, DHT, T, and E2 were all administered at the
concentrations of 5 x 10-9 M, 5 x
10-8 M, 5 x 10-7
M, and 5 x 10-6 M, with and
without 100 ng/ml of mPL-I. In addition, E2 was used at the
concentrations of 5 x 10-12 M, 5 x
10-11 M, and 5 x 10-10
M. mPRL, mPL-I, and mPL-II were administered to the
cultured cells at the concentrations of 1 ng/ml, 10 ng/ml, 100 ng/ml,
and 1000 ng/ml in the presence or absence of 5 x
10-6 M of AD. Hydroxyflutamide and fadrozole
were both used at the concentration of 5 x 10-5
M. All of these treatments were continued for 48 h,
and the medium changed every 24 h. The concentration of P4 was
measured in the medium collected after the last 24 h of culture.
For the time-course of P4 release, the luteal cells were incubated in
the presence of 5 x 10-6 M of AD, and
the cultures were terminated 15, 30, 60, and 120 min after the
treatment commenced. Actinomycin D and cycloheximide were used at the
concentrations of 1 µg/ml and 10 µg/ml, respectively. The
treatments were continued for 2 h for both actinomycin D and
cycloheximide.
RIA
P4 concentrations in the culture media were measured by an RIA
kit obtained from Diagnostic Products Co. (Los Angeles, CA). This P4
RIA has been modified and validated for the use with mouse serum and
culture medium from mouse ovarian cells (16). All the reagents that
were used in the cultures were tested for cross-reactivity in the RIA.
None of these reagents showed significant cross-reactivity at the
concentrations used in the cultures.
Statistical analyses
All the experiments were repeated at least two times using
different preparations of cells. In each experiment, the treatments and
the controls were replicated eight times. The results from one culture
for each treatment or experimental condition are presented here. This
was done because of the inherent difficulty in combining data from
cultures carried out over a long period of time. For example, although
we consider this luteal cell culture system very consistent, we saw a
3-fold difference in the basal release of P4 when the results from
cultures carried out over a period of 8 months were compared. However,
the effects of each treatment or experimental condition were in all
cases reproducible. The effects of the various treatments on the P4
release from the cultured CL cells were analyzed by one-way ANOVA and
Fisher’s protected least significant difference test. The interaction
between the lactogens and the androgens to stimulate P4 release was
assessed by two-factor ANOVA with the two hormonal treatments as main
effects. Differences between groups were considered significant when a
P value of < 0.05 was obtained. All the analyses were
carried out using the SuperANOVA program from Abacus Concepts
(Berkeley, CA).
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Results |
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). The related lactogens
mPL-II and mPRL had effects on the P4 release very similar to that of
mPL-I, and this similarity in potency between the three lactogens was
not dependent on the stage of pregnancy at which the CL cells were
obtained (Table 1). The stimulatory effects of mPL-I,
mPL-II, and mPRL on the P4 release were, however, influenced by the
stage of pregnancy at which the CL cells were obtained, as was the
effect of AD when administered alone or with the lactogens. Cells
obtained on day 10 of pregnancy showed the highest susceptibility to
the hormonal stimulation, but the effects of the hormones were reduced
in cells from day 14 of gestation and declined further in cells
obtained on day 18 of gestation (Table 1).
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). T had very similar effects as AD on P4 release (data
not shown). In addition, the nonaromatizable androgen DHT stimulated
the P4 release to a similar extent as the aromatizable androgens (Fig. 3). When an androgen (aromatizable or nonaromatizable)
was administered concomitantly with mPL-I, the two hormones acted
synergistically to enhance the P4 release (Figs. 2 and 3). The P4
release from the cultured CL cells was not increased by E2,
regardless of whether the estrogen was administered alone or in
combination with mPL-I. In fact, the highest concentration of
E2 that was used in these experiments (5 µM)
caused a significant reduction in the P4 release as compared with
controls (Fig. 4).
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). Similarly, administration of the intracellular
androgen receptor antagonist hydroxyflutamide at a concentration of
1 x 10-5 M had no inhibitory effect on
the AD-stimulated release of P4 (Fig. 6).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We did see a gradual decrease in the responsiveness of the luteal cells to all hormonal stimuli the later in pregnancy the cells were collected. Similar results have been found for AD-stimulated P4 release from cultured rat luteal cells (12). We do not know what caused the gradual reduction in the hormone-stimulated P4 release. It may be caused by physiological alterations of the CL evoked by pregnancy-associated factors. However, it is also possible that isolated luteal cells obtained at later stages of pregnancy tolerate the culture conditions less well than cells from earlier stages of pregnancy and that, in turn, diminishes the responsiveness of the cells to hormonal stimuli.
We found in this study that androgens, when administered alone, are effective stimulators of P4 release. In addition, the androgens synergize with lactogens (mPL-I, mPL-II, and mPRL) to stimulate P4 release. Androgens have been known for some time to stimulate P4 release from cultured ovarian cells (10, 11). It has been suggested that the androgens may be acting indirectly through conversion to estrogens (9). However, growing evidence now indicates that conversion to estrogen is not required for the action of androgens in the ovaries. For example, it was shown that DHT is approximately equipotent to AD and T in stimulating P4 release from rat and mouse ovarian cells (10, 11, 12). Because DHT is not a substrate for estradiol synthesis, these results indicate a direct effect of the androgens. Similarly, E2, when administered to cultured ovarian cells, did not mimic the effect of androgens on P4 release (10, 11, 12). We have, in the present study, confirmed that DHT has activity comparable with that of the other androgens to stimulate P4 release, when administered alone, and in addition, we have shown that it acts synergistically with lactogens to stimulate P4 release. We also found that E2 was ineffective in stimulating P4 release regardless whether it was administered alone or concomitantly with a lactogen. Further supporting a direct effect of the androgens was our finding that the nonsteroidal aromatase inhibitor fadrozole did not have any significant inhibitory effect on the androgen-stimulated P4 release. Therefore, the results presented here, together with previous findings, strongly attest to direct stimulatory effects of the androgens on P4 release. How the androgens are acting is, on the other hand, not known, but several lines of evidence indicate that it is not acting through the nuclear androgen receptor. It was previously shown that the action of the androgens to stimulate P4 release from rat luteal cells is rapid, occurring within minutes after androgen administration (12). We found, in the present study, that 15 min of exposure of luteal cells to AD increased the P4 release more than 2-fold over control cultures, and this level of increase in P4 release relative to control cultures remained similar after prolonged incubation periods. These findings indicated that protein synthesis was not needed for the androgen stimulatory effect on the P4 release, a finding supported by the fact that cycloheximide and actinomycin D did not prevent the stimulatory activity of AD on P4 release.
These results, therefore, imply that the stimulatory effects of the androgens on the P4 release were not mediated through activating the intracellular androgen receptor and, consequently, regulation of gene expression. Additional support for the presumption that the androgens were not acting through binding to the intracellular androgen receptor was provided by our finding that the androgen receptor antagonist hydroxyflutamide had no inhibitory effect on the androgen-stimulated P4 release. It is not new to find steroid activity that does not concur with the classical genomic steroid action. In fact, the number of reports describing nongenomic actions of various steroids, including androgens, has been growing (18, 19, 20, 21). The findings reported here add to the list of steroid activities that deviate from the well-established mode of steroid action. We do not know at the present time how the androgens exert their effects on the P4 release, but it has been suggested that nongenomic steroid activities may be mediated through routes such as changes in the cell membrane fluidity or, more interestingly, through interaction of the steroids with plasma membrane receptors resulting in activation of second-messenger systems (19). An indication that the action of the androgens may be of physiological importance is our finding that the basal release of P4 did not appear to be dependent on the day of pregnancy from which the luteal cells were obtained, whereas the hormonal effects, both those of the lactogens and those of the androgens, were significantly dependent on when during pregnancy the cells were collected.
The synergistic effect of the androgens and the lactogens on the P4 release found in this study is an intriguing phenomenon. This interaction could be of fundamental importance to maintain sufficient P4 release at midgestation, when the placenta is replacing the pituitary as the main source of hormones for regulating the ovarian functions. We do not at this time understand the nature of the synergism between the androgens and the lactogens. However, interaction between a steroid and a peptide hormone to regulate a physiological function is a common phenomenon. For example, it has been shown that androgens synergize with FSH to stimulate P4 release from cultured rat granulosa cells (22, 23). In our continuous effort to understand further how the placenta regulates the ovarian function in the latter half of pregnancy in the mouse, we will emphasize studies that will elucidate the mechanism by which the androgens exert their effects in the ovaries, and the nature of the interaction between the androgens and the lactogens.
In summary, lactogens and androgens stimulated P4 release from cultured luteal cells obtained from pregnant mice when administered alone, and they acted synergistically when administered together. The potency of individual hormones to stimulate P4 release was not dependent on when during pregnancy the ovarian tissue was collected, but the responsiveness of the luteal cells to hormone-stimulated P4 release was reduced in tissue from late-pregnant mice. The action of the androgens was direct, but not mediated through conversion to E2, it was not exerted by interaction with the intracellular androgen receptor, and it was independent of de novo protein synthesis.
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Acknowledgments |
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Received December 26, 1996.
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