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The Journal of Clinical Endocrinology & Metabolism Vol. 82, No. 3 955-962
Copyright © 1997 by The Endocrine Society

Experimental Studies

Expression of Steroid Receptors and Steroidogenic Enzymes in the Baboon (Papio anubis) Corpus Luteum during the Menstrual Cycle and Early Pregnancy1

Sheri Hild-Petito2 and Asgerally T. Fazleabas

Department of Obstetrics and Gynecology, University of Illinois College of Medicine, Chicago, Illinois 60612

Address all correspondence and requests for reprints to: Dr. Asgi Fazleabas, Department of Obstetrics and Gynecology, University of Illinois, 820 South Wood Street (M/C 808), Chicago, Illinois 60612-7313.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
As estrogen and progesterone are proposed regulators of luteal function, this study was undertaken to correlate the presence of receptors for these steroids with luteal function during early pregnancy. Corpora lutea (CL) were obtained from nonpregnant baboons during the midluteal [ML; days 7–8 postovulation (PO)] and late luteal (LL; days 11–12 PO) phases of the menstrual cycle or from pregnant baboons on days 18, 25, 29, or 31–33 PO. Estrogen and progestin receptors (ER and PR, respectively) and 3ß-hydroxysteroid dehydrogenase (3ßHSD) were detected by immunocytochemistry using specific monoclonal (H222 for ER; JZB39 for PR) or polyclonal (S683 for 3ßHSD) antibodies. In addition, ribonucleic acid (RNA) was extracted from CL, processed for Northern blot analysis, and probed with complementary DNAs to human PR, human 3ßHSD, and rat aromatase. Levels of messenger RNA (mRNA) for 3ßHSD were quantified by laser densitometric scanning, and the data were normalized to the expression of a housekeeping gene (glyceraldehyde-3-phosphate dehydrogenase) to correct for loading differences. CL did not demonstrate specific nuclear stain for ER at any stage of the menstrual cycle or pregnancy. In contrast, PR-positive cells were present during the ML phase, but decreased during the LL phase (P < 0.05). PR-positive cells were maintained during early pregnancy at levels comparable to the ML phase (P > 0.05). Staining for 3ßHSD was present at all stages of the cycle and pregnancy. Although the percent of 3ßHSD-positive cells appeared to decrease as pregnancy proceeded, this was not statistically different (P > 0.05). The complementary DNA to PR hybridized to multiple transcripts (~4.4, 3.1, 1.6, and 0.95 kilobases) in CL of the cycle. A single transcript (~1.8 kilobases) for 3ßHSD was present in CL at all stages of the cycle and pregnancy. The level of 3ßHSD mRNA was highest during the ML phase and declined significantly (P < 0.05) during the LL phase and early pregnancy. Three transcripts (~3.6, 3.0, and 1.7 kilobases) for aromatase were detected in CL of the cycle and pregnancy. Aromatase mRNA increased during early pregnancy. These results support the concept of PR-mediated events, but not ER-regulated processes in the primate CL. Furthermore, the data suggest that the steroidogenic enzymes 3ßHSD and aromatase are differentially regulated during early pregnancy.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IN PRIMATES, LH is clearly required to sustain progesterone secretion by the corpus luteum (CL). In addition, it has been proposed that estrogen and progesterone may play a paracrine or autocrine role in regulating luteal function. In rats and rabbits, estrogen is luteotropic (1); however, estrogen induces luteolysis in women and rhesus monkeys (2, 3) when administered exogenously. As estrogen treatment was unable to cause luteolysis in monkeys whose gonadotropin secretion was controlled by the administration of pulsatile GnRH (4), the luteolytic effect of exogenous estrogen is presumably mediated by a reduction in LH secretion. However, high levels of estrogen suppressed basal and/or gonadotropin-stimulated progesterone production by human (5) and macaque (6) luteal cells in vitro. Thus, the high levels of estrogen within the CL (7) may play a role in modulating luteal life span and function.

Rothchild (8) originally proposed that progesterone was a luteotropin that could promote its own synthesis and maintain the structural integrity of the CL. Limited data support this hypothesis. Treatment with an antiprogestin, RU 486, resulted in a dose-dependent decrease in 3ß-hydroxysteroid dehydrogenase (3ßHSD) and 17{alpha}-hydroxylase activities in human granulosa lutein cells in culture (9, 10). In addition, progesterone altered the synthesis and ratio of luteotropic (PGI2) and luteolytic (PGF2{alpha}) PGs (11) and promoted LH receptor synthesis (12) in cultured bovine luteal cells. Inhibition of endogenous progesterone production with trilostane (an inhibitor of 3ßHSD) on days 6–7 of the luteal phase in rhesus monkeys resulted in early luteolysis, as determined by morphological criteria and the inability of the CL to recover progesterone production (13). Early luteolysis occurred despite maintenance of tonic circulating LH levels and the recovery of normal pregnenolone levels after the cessation of trilostane treatment. These data suggest that progesterone is involved in promoting its own synthesis and maintaining the structural integrity of the CL.

Both estrogen and progesterone are proposed to exert their actions via receptor-mediated pathways. Specific nuclear estrogen receptors (ER) have been detected in the CL of laboratory and domestic animals (14, 15). In primates, ER has been detected in the granulosa cells of some follicles in women (16) and baboons (17); however, ER protein and messenger ribonucleic acid (mRNA) were not detected in the human or macaque CL (18, 19). Thus, the actions of estrogen may be indirect via interactions with specific enzymes, such as inhibition of 3ßHSD (20) or activation of PG synthetase (21), and not through ER.

Progestin receptors (PR) have been detected in whole ovaries of rats (22) and CL of humans (16, 23, 24) and macaques (18, 19, 25). Induction of PR mRNA and protein corresponded with LH/CG-stimulated ovulation and luteinization of rat (26, 27), rabbit (28), pig (29), and macaque (30) follicles. Functional PR was essential for luteinization of rat granulosa cells (27). Progesterone has also been shown to inhibit the proliferation of human granulosa cells, decrease the number of human granulosa cells undergoing apoptosis, and block the differentiation of human granulosa cells (31, 32, 33). Thus, progesterone appears to be involved in early luteal development and function. In addition, these data suggest that PR is regulated by LH/CG.

The current study was undertaken to determine the relative importance of estrogen and progesterone in the regulation of the CL of pregnancy in the baboon. Thus, immunocytochemistry was used to localize ER and PR in the baboon CL during the menstrual cycle and early pregnancy, and Northern blots were used to measure PR mRNA levels during the cycle. In addition, receptor expression was correlated with changes in two critical steroidogenic enzymes: 3ßHSD, which converts pregnenolone to progesterone, and aromatase, which converts androgens to estrogens. These two enzymes are indicators of luteal function and are involved in determining the amount of endogenous ligand available for interaction with ER and PR. Our results suggest that estrogen does not regulate the baboon CL of the cycle or pregnancy via a receptor-mediated mechanism. In contrast, we hypothesize that progesterone, via its receptor, is involved in the structural and functional maintenance of the CL of pregnancy. Furthermore, our results imply that the steroidogenic enzymes 3ßHSD and aromatase are differentially regulated in the baboon CL during early pregnancy.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Tissue collection

CL were obtained from adult female baboons (Papio anubis) during the cycle at the midluteal (ML) phase on days 7 and 8 postovulation (PO; n = 6) and the late luteal (LL) phase on days 11 and 12 PO (n = 6). The stage of the cycle was determined by menstrual records, sex skin tumescence, and circulating levels of estradiol and progesterone (34). Mature cycling baboons were mated with fertile males during the periovulatory period, as determined by sex skin tumescence. CL were obtained on days 18, 25, 29, and 31–33 PO (n = 16). The stage of pregnancy was confirmed by ultrasound and circulating levels of CG, estradiol, and progesterone (35, 36). Circulating levels of baboon CG increase beginning on day 12 PO, peak on day 27 PO and decline to undetectable by day 51 PO (35). The time points of CL collection correspond to before, during, and after the luteal-placental shift that occurs between days 20–25 PO in this species (37). In addition, a CL was obtained from a baboon treated with hCG to mimic early pregnancy (38). At laparotomy, CL were generally collected by lutectomy; however, in some cases both ovaries were removed. Control tissues (oviduct and endometrium) were obtained from baboons undergoing hysterectomy during the late follicular phase. Liver was obtained from an adult female baboon at necropsy.

Immunocytochemistry

Tissues were processed for indirect immunocytochemical localization of ER, PR, and 3ßHSD as previously described (18). Briefly, frozen blocks were sectioned (4–6 µm) in a Reichert-Jung 2800 Frigocut N (Cambridge Instruments, Buffalo, NY) and thaw-mounted onto slides. The sections were freeze-substituted in acetone-calcium chloride and fixed at 4 C in 0.2% picric acid plus 2% paraformaldehyde followed by 85% ethanol. After blocking nonspecific binding with normal rabbit serum, the sections were incubated overnight at 4 C with specific monoclonal antibodies against either human (h) ER (H222; 10 µg/mL) (39) or hPR (JZB39; 2.5 µg/mL) (24). Both of these antibodies recognize the occupied (activated) and unoccupied (unactivated) forms of these steroid receptors (for ER, see Refs. 40 and 41; for PR, see Ref.24). In addition, H222 and JZB39 have been previously shown to cross-react with baboon ER and PR despite elevated local or systemic estradiol and progesterone levels (17, 42). Nonspecific staining was determined in adjacent sections by substituting the receptor antibodies with purified rat IgG. The antigen-antibody complex was visualized using a Vectastain ABC Kit (Vector Laboratories, Burlingame, CA) and diaminobenzidine tetrahydrochloride (DAB; Sigma Chemical Co., St. Louis, MO) as the chromogen. The sections were counterstained with hematoxylin.

For colocalization of PR and 3ßHSD, sections immunocytochemically stained for PR (after DAB reaction) were incubated with a specific polyclonal antibody to h3ßHSD (S683; 5 µg/mL) (43). The sections were not counterstained with hematoxylin because this interfered with subsequent 3ßHSD staining. For negative controls, sections previously incubated with rat IgG and reacted with DAB (PR negative control) were incubated with rabbit IgG instead of 3ßHSD antibody. The 3ßHSD-antibody complex was visualized using a Vectastain ABC-AP kit (Vector Laboratories) with nitro blue tetrazolium as the substrate for alkaline phosphatase. Endogenous alkaline phosphatase activity was inhibited by including levamisole (1 mmol/L) in the substrate solution. To insure that immunocytochemical staining for PR did not interfere with subsequent staining for 3ßHSD, adjacent sections of CL were stained for 3ßHSD only.

The percentage of nuclei or cells staining for PR or 3ßHSD, respectively, in CL was determined by counting all positive and negative nuclei or cells within a field with the aid of an ocular grid. Three grid fields of a constant size from the peripheral and central regions of each CL were randomly selected and examined at a magnification of x400. To visualize negative nuclei, sections counterstained with hematoxylin were used to determine the percentage of PR-positive nuclei. As the double stain allowed for better visualization of cells and their nuclei and had no apparent effect on 3ßHSD staining, sections that were double stained for PR and 3ßHSD were used to determine the percentage of 3ßHSD-positive cells. Significant (P < 0.05) differences in the percentage of positive nuclei or cells relative to the stage of the cycle and pregnancy were determined by one-way ANOVA, followed by the Student-Newman-Keuls test for a significant F value. The data are presented as the mean ± SEM for each group, where n equals the number of CL per group.

RNA isolation and Northern blot analysis

Total RNA was isolated from the tissues as previously described in our laboratory (44). Briefly, whole CL (dissected from the ovary), whole ovary, or portions of oviduct or liver were homogenized in guanidinium isothiocyanate buffer, layered onto a cesium chloride gradient, and centrifuged overnight. The phenol/chloroform-extracted RNA was quantified on a spectrometer at a wavelength of 260 nm. Twenty micrograms of total RNA were electrophoresed on a 1.2% agarose formaldehyde gel under denaturing conditions, transferred to nitrocellulose, and baked for 2 h at 80 C (44).

The membranes were prehybridized at 50 C in the following solution: 6-strength standard sodium phosphate ethylenediamine tetraacetic acid buffer (SSPE), 25% formamide, 2 g dextran sulfate, 5-strength Denhardt’s solution, 100 µg/mL salmon sperm DNA, and 0.5% SDS. A complimentary DNA (cDNA) was labeled with [{alpha}-32P]deoxy-CTP (3000 Ci/mmol; Amersham International, Aylesbury, UK) using a random primer DNA labeling system (BRL, Bethesda, MD). The following cDNAs were used in this study: the full-length sequence [2.5 kilobases (kb)] of the hPR (45), the 1.2-kb insert to placental h3ßHSD (46), and the 1.2-kb insert to rat aromatase (47). The 32P-labeled cDNA was hybridized to the nitrocellulose membranes overnight at 50 C in the above solution. Stringent washes and autoradiography were performed as previously described (44).

For densitometric analysis, the 32P-labeled cDNA to 3ßHSD was removed by incubation in 0.1-strength SSPE and 10 mmol/L ethylenediamine tetraacetate at 80 C for 30 min. The membranes were then probed with a cDNA to a housekeeping gene, glycerol-3-phosphate dehydrogenase (G3PDH; Clontech Laboratories, Palo Alto, CA). After autoradiography, the 3ßHSD and G3PDH transcripts were densitometrically scanned. The autoradiographic signal for G3PDH did not differ significantly (P > 0.05, by one-way ANOVA) among CL from the cycle or pregnancy (2.61 ± 0.47, 4.75 ± 0.88, and 2.59 ± 0.49 for ML, LL, and day 25 PO, respectively; mean ± SEM; n = 3/group). The variability observed in the G3PDH autoradiographic signal of CL was similar to the that observed in the ethidium bromide staining of the Northern blot. This suggests that the differences were related to loading and running of the gel as opposed to actual differences in G3PDH expression in CL. For statistical analysis, the levels of 3ßHSD mRNA were normalized to G3PDH for individual CL, and differences among CL from the cycle and pregnancy were determined by one-way ANOVA, followed by the Student-Newman-Keuls test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Specific nuclear stain for ER was present in endometrium from a baboon during the late follicular phase, which represented a positive control tissue (Fig. 1DGo). In contrast, nuclear staining for ER was absent in CL during the ML (Fig. 1AGo) and LL phases of the cycle. ER was also undetectable in CL on days 18, 25, and 32 (Fig. 1BGo) of pregnancy and was not different from the negative control value (Fig. 1CGo).



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  		Figure 1. Immunocytochemical staining for ER in CL of the cycle and pregnancy. Nuclear localization of ER was absent in CL obtained from the ML phase (A) and day 32 of pregnancy (B). No nuclear stain was present in CL when the primary antibody (H222) was replaced with rat IgG (C). In contrast, ER was readily detectable in the glandular epithelium (ge) and stroma (s) of endometrium (D) obtained from a baboon during the follicular phase (DAB precipitate appears brown). A, B, and D are counterstained with hematoxylin. Magnification, x300.

 
Many of these cells demonstrated specific nuclear staining for PR in CL from the ML phase (Fig. 2AGo). Several of these cells also demonstrated specific cytoplasmic staining for 3ßHSD (Fig. 2AGo). Both PR and 3ßHSD staining was reduced in CL from the LL phase, becoming almost undetectable in a regressing CL (Fig. 2BGo). Specific staining for PR was present in baboon endometrium (Fig. 2DGo); however, 3ßHSD staining was undetectable.



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  		Figure 2. Colocalization of PR and 3ßHSD in CL and endometrium during the menstrual cycle. Nuclear staining (DAB precipitate appears brown) for PR and cytoplasmic staining (nitro blue tetrazolium precipitate appears blue) for 3ßHSD were present in CL obtained during the ML phase (A). Three cell types were apparent: 1) cells that were positive for PR and 3ßHSD (), 2) PR-positive cells ( ), and 3) cells positive for 3ßHSD only ). A dramatic decrease in staining for both PR and 3ßHSD was apparent in a regressing CL from the LL phase (B). No specific staining for PR or 3ßHSD was present in CL from the ML phase when primary antibodies were not used (C). Endometrium from the follicular phase demonstrated positive stain for PR, but not 3ßHSD (D). ge, Glandular epithelium; s, stroma. Magnification, x300.

 
During early pregnancy, many PR-positive cells were present in the CL (Fig. 3Go). Some of the cells of the CL continued to demonstrate staining for 3ßHSD on days 18, 25, and 32 of pregnancy. During the ML phase and throughout early pregnancy (Figs. 2AGo and 3Go), three types of cells could be identified based on the localization of PR and 3ßHSD: 1) PR- and 3ßHSD-positive cells, 2) cells that stain only for PR, and 3) 3ßHSD-positive, PR-negative cells.



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  		Figure 3. Localization of PR and 3ßHSD in CL of pregnancy. Many PR-positive cells were present in CL throughout early pregnancy (A, day 18; B, day 25; C, day 32). However, not as many cells were positive for 3ßHSD during pregnancy. , PR and 3ßHSD positive; , PR positive only; , 3ßHSD positive only. Magnificatin, x300.

 
The percentage of PR-positive nuclei was high in CL from the ML phase (Fig. 4Go). The percentage of PR-positive nuclei was significantly (P < 0.05) lower in CL from the LL phase. In CL obtained on day 25 of pregnancy, the percentage of PR-positive nuclei was greater than that during the LL phase (P < 0.05) and was comparable to that during the ML phase (P > 0.05).



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  		Figure 4. Percentage of PR-positive nuclei in CL of the cycle and early pregnancy. Bars represent the mean ± SEM for the indicated number of CL per group. Bars with different letters represent means that are significantly (P < 0.05) different. ML, n = 3; LL, n = 3. d, Day; px, pregnancy. d18px, n = 3; d25px, n = 4; d32px, n = 2.

 
The percentage of 3ßHSD-positive cells was highest in CL from the ML phase (Fig. 5Go). There was an apparent decrease in the percentage of cells staining positive for 3ßHSD during the LL phase and throughout early pregnancy. However, this decrease was not statistically significant (P > 0.05).



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  		Figure 5. Percentage of 3ßHSD-positive cells in CL of the cycle and early pregnancy. Bars represent the mean ± SEM for the indicated number of CL per group. No significant differences (P > 0.05) between groups were detected. ML, n = 3; LL, n = 3. d, Day; px, pregnancy. d18px, n = 3; d25px, n = 4; d32px, n = 2.

 
Multiple transcripts (~4.4, 3.1, 1.6, and 0.95 kb) for PR were present in CL of the cycle, ovary, and oviduct, but not liver (Fig. 6Go). These transcripts were similar in size to that reported for the hPR (48, 49). Levels of PR mRNA in CL were not as high as in the oviduct. Although PR protein content, as assessed by immunocytochemistry (Fig. 4Go), was reduced in CL from the LL phase, mRNA levels remained elevated during this stage (Fig. 6Go).



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  		Figure 6. Northern blot of total RNA from CL of the cycle probed with cDNA to hPR. Multiple transcripts (arrowheads, ~4.4, 3.1, 1.6, and 0.95 kb) for PR were present in total RNA from CL during ML (n = 3) and LL (n = 3) phases and nonluteal ovary obtained from a baboon at midcycle and an oviduct collected from a baboon during the follicular phase. No message for PR was detected in total RNA from liver. The exposure time was 48 h.

 
A single 1.8-kb transcript (approximate size) for 3ßHSD was present in CL at all stages of the cycle and pregnancy (Fig. 7Go). This transcript size corresponded to the reported size for h3ßHSD in macaque CL (50, 51). The message for 3ßHSD was also present in whole nonluteal ovary and in a CL obtained from a baboon treated with hCG to mimic early pregnancy. No 3ßHSD mRNA was detected in oviduct or liver RNA. The levels of the 1.8-kb transcript, as quantified by densitometric scan, are depicted in Fig. 8Go. The levels of 3ßHSD mRNA were highest in CL from the ML phase. Significantly (P < 0.05) lower levels of 3ßHSD mRNA were expressed in CL during the LL phase and early pregnancy.



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  		Figure 7. Northern blot of total RNA from CL of the menstrual cycle and pregnancy probed with a cDNA to h3ßHSD. A single transcript of approximately 1.8 kb was detected in total RNA from CL during the cycle (ML, n = 3; LL, n = 3) and early pregnancy (d, day; px, pregnancy; d18px, n = 2; d25px, n = 4; d29px, n = 1; d31px, n = 1). This transcript was also present in total RNA from CL obtained from a baboon treated with hCG and a nonluteal ovary (same sample as in Fig. 6Go). No mRNA for 3ßHSD was detected in total RNA from oviduct and liver. *, Total RNA from the entire CL bearing ovary on d25px (d25px CL/ovary); this lane was excluded from the analysis in Fig. 8Go. The exposure time was 8.5 h.

 


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  		Figure 8. Expression of 3ßHSD mRNA in CL of the cycle and pregnancy. Bars represent the mean ± SEM. Different letters represent means that are significantly different (P < 0.05). ML, n = 3; LL, n = 3. d, Day; px, pregnancy. d18px, n = 2; d25px, n = 3; d29–31px, n = 2.

 
Three transcripts (~3.6, 3.0, and 1.7 kb) for aromatase were detected in total RNA from CL of the cycle and early pregnancy (Fig. 9Go). The transcripts were similar in size to those reported for rat aromatase (46). Although the levels of the three aromatase transcripts varied between individual CL, the overall levels of aromatase mRNA were lower in CL of the cycle than in CL of early pregnancy.



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  		Figure 9. Northern blot of total RNA from CL of the cycle and pregnancy probed with a cDNA to rat aromatase. Multiple transcripts (arrowheads, ~3.6, 3.0, and 1.7 kb) were present in total RNA from CL of the cycle and early pregnancy. ML, n = 3; LL, n = 3. d, Day; px, pregnancy. d18px, n = 2; d25px, n = 3; d29px, n = 1; d31px, n = 1; d33px, n = 1, d25px CL/ovary, entire CL-bearing ovary, n = 1. The exposure time was 24 h.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The maintenance of PR in functional CL of the cycle and early pregnancy (present study and Refs. 18, 25, and 52) supports the hypothesis that progesterone is able to act via a receptor-mediated mechanism to regulate luteal function and life span. Localization of PR in 3ßHSD-positive and -negative cells suggests that progesterone is capable of acting as both an autocrine and a paracrine regulator. The demonstration of three potential cell types based on the expression of PR and 3ßHSD supports the concept that the primate CL is comprised of a heterogeneous population of cell types. Further research is required to elucidate the potential PR-mediated actions on primate CL.

The lack of ER present in the primate CL during the cycle or early pregnancy (present study and Refs. 16, 18, and 19) suggests that estrogen does not directly regulate luteal function via a classical receptor-mediated event. Although the existence of low levels of ER is possible, it seems unlikely because PCR techniques also failed to detect ER mRNA in the macaque CL (19). Recent studies have demonstrated the presence of a new form of nuclear ER, termed ERß, in the rodent ovary (53). It is not known whether ERß is present in the baboon CL and, if so, whether this receptor mediates estrogen action.

The lack of nuclear ER in baboon CL suggests that regulation of PR is unlike that in other target tissues. In classical target tissues (e.g. uterus), estrogen induces PR via a receptor-mediated pathway (54). Instead, recent studies suggest that LH/CG regulates ovarian PR. In rats (26, 27), rabbits (28), pigs (29), and macaques (30), the LH surge or CG treatment in stimulated cycles was associated with the induction of PR mRNA and protein in granulosa cells. These results in combination with the data from the present study suggest that CG is involved in the maintenance of PR in CL of pregnancy. PR may also be regulated at the translational level, as we and others (18, 19, 52) detected low levels of PR protein despite elevated mRNA levels. However, discrepancies between PR mRNA and protein may reflect differences in the methodology used to measure them.

The levels of mRNA and protein for 3ßHSD correlate with progesterone production by the baboon CL during the luteal phase of the menstrual cycle. However, luteal mRNA for 3ßHSD declines dramatically during early pregnancy despite increased luteal progesterone synthesis (35, 37). These changes in 3ßHSD mRNA levels are similar to those reported in macaque CL during the luteal phase (45, 46) and simulated early pregnancy (55). Although LH is essential for the maintenance of steroidogenic enzymes in macaque CL (46), CG production during early pregnancy did not maintain the mRNA for 3ßHSD. Previous work has demonstrated that CG given early in the luteal phase maintains 3ßHSD mRNA levels in the macaque CL (55). These data suggest that primate CL have an age-related responsiveness to CG, such that the CL loses its ability to maintain 3ßHSD mRNA in response to CG during the ML to LL phase (present study and Ref.55). In contrast, rat CL maintain elevated levels of 3ßHSD mRNA throughout pseudopregnancy (56). In this species progesterone production by the CL is essential for continued maintenance of pregnancy because the rat placenta does not produce progesterone. In the baboon, the decline in 3ßHSD mRNA correlates with the luteal placental shift (days 20–25 postovulation) in this species (37). However, the baboon CL contributes significant progesterone to the circulation through day 30 of pregnancy. This suggests that the CL of early pregnancy is still actively secreting progesterone despite the decline in 3ßHSD mRNA and protein levels. However, enzyme activity was not examined in this study. Other investigators (57) have demonstrated differences between enzyme levels and activity for aromatase in the rat CL. Thus, the decline in 3ßHSD mRNA and protein in the baboon CL of early pregnancy may not reflect 3ßHSD activity in the tissue.

During early pregnancy, the increase in circulating estradiol levels (35) correlates with the overall expression of the three aromatase transcripts in the baboon CL. Presumably, all three transcripts (3.6, 3.0, and 1.7 kb) encode for the open reading frame (1509 bp) and only differ in the degree of polyadenylation (47). However, the precise function of these three transcripts is unknown. Treatment of baboons with CG to mimic early pregnancy resulted in circulating estradiol levels equivalent to those of early pregnancy (38). Increased expression of aromatase mRNA was also observed in the CL of macaques treated with CG (55). These data suggest that the primate CL is the primary source of estrogen during early pregnancy and that CG induces the elevated aromatase mRNA levels in the CL. The second messenger system regulating CG-induced expression of aromatase mRNA in the primate CL has not been investigated. In the cow the aromatase gene does not contain a cAMP response element and contains different promoters for expression in the follicle, CL, and placenta (58). In the rat LH/hCG-induced luteinization results in elevated aromatase mRNA and estrogen biosynthesis in the CL that are maintained by cAMP-independent mechanisms (47). Taken together, these data suggest that CG regulation of aromatase in CL is not via cAMP. In addition, the ability of CG to differentially increase aromatase expression while 3ßHSD expression declines may be due to tissue-specific promoters.

In summary, these studies support the concept of PR-mediated events in the primate CL and suggest that PR expression is associated with functional CL of the menstrual cycle and early pregnancy. The data imply that estrogen actions on the primate CL are not mediated by a nuclear receptor. In addition, CG appears to differentially regulate enzymes in the steroidogenic pathway, increasing aromatase expression during early pregnancy while 3ßHSD expression declines.


   Acknowledgments
 
We are grateful for the generous donation of H222 and JZB39 antibodies from Dr. Geoffrey Greene, Ben May Institute of the University of Chicago (Chicago, IL), and S683 antibody from Dr. Ian Mason Southwest Medical School (Dallas, TX). The cDNAs to hER and hPR were received from Dr. G. Greene; 3ßHSD cDNA was provided by Dr. I. Mason; the cDNA to aromatase was kindly provided by Dr. JoAnne Richards, Baylor College of Medicine (Houston, TX). We also acknowledge the surgical skills of Dr. Jeffrey Fortman and the secretarial skills of Ms. Margarita Guerrero.


   Footnotes
 
1 This work was supported by NIH Grant HD-21991 (to A.T.F.). Back

2 Recipient of National Research Scientist Award HD-07508339. Current address: BIOQUAL, Inc., 9600 Medical Center Drive, Rockville, Maryland 20850. Back

Received April 9, 1996.

Revised October 31, 1996.

Accepted November 15, 1996.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Keyes PL, Gadsby JE, Yuh K-CM, Bill CH. 1983 The corpus luteum. In: Greep RO, ed. Reproductive physiology. Baltimore: University Park Press; vol 4:57–97.
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