| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Experimental Studies |
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 IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
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 |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
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
-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) 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 |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
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
[-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 |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
). In contrast, nuclear
staining for ER was absent in CL during the ML (Fig. 1A) and LL phases
of the cycle. ER was also undetectable in CL on days 18, 25, and 32
(Fig. 1B) of pregnancy and was not different from the negative control
value (Fig. 1C).
|
). Several of these cells also
demonstrated specific cytoplasmic staining for 3ßHSD (Fig. 2A). Both
PR and 3ßHSD staining was reduced in CL from the LL phase, becoming
almost undetectable in a regressing CL (Fig. 2B). Specific staining for
PR was present in baboon endometrium (Fig. 2D); however, 3ßHSD
staining was undetectable.
|
). 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. 2A
and 3),
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.
|
). 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).
|
). 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).
|
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. 6). 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. 4), was reduced in CL from the LL phase, mRNA
levels remained elevated during this stage (Fig. 6).
|
). 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. 8. 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.
|
|
3.6, 3.0, and 1.7 kb) for aromatase were
detected in total RNA from CL of the cycle and early pregnancy
(Fig. 9). 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.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
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 |
|---|
|
Footnotes |
|---|
2 Recipient of National Research Scientist Award HD-07508339. Current
address: BIOQUAL, Inc., 9600 Medical Center Drive, Rockville, Maryland
20850.
Received April 9, 1996.
Revised October 31, 1996.
Accepted November 15, 1996.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
5-3ß-hydroxysteroid dehydrogenase 5–4
isomerase systems of human placenta and corpus luteum of pregnancy. Gynecol Invest. 4:106–120.[Medline]
5–4 isomerase
from placenta: expression in nonsteroidogenic cells of a protein that
catalyzes the dehydrogenation/isomerization of C21 and C19 steroids. Endocrinology. 126:2493–2498.5–4 isomerase
messenger ribonucleic acids in the primate corpus luteum. Endocrinology. 131:2065–2070.This article has been cited by other articles:
 |
|
 |
|
  C. Wang, P. A. Mavrogianis, and A. T. Fazleabas Endometriosis Is Associated with Progesterone Resistance in the Baboon (Papio anubis) Oviduct: Evidence Based on the Localization of Oviductal Glycoprotein 1 (OVGP1) Biol Reprod, February 1, 2009; 80(2): 272 - 278. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Z. A. Radi and N. K. Khan Comparative Expression and Distribution of c-fos, Estrogen Receptor{alpha} (ER{alpha}), and p38{alpha} in the Uterus of Rats, Monkeys, and Humans Toxicol Pathol, June 1, 2006; 34(4): 327 - 335. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W C. Duncan, E. Gay, and J. A Maybin The effect of human chorionic gonadotrophin on the expression of progesterone receptors in human luteal cells in vivo and in vitro Reproduction, July 1, 2005; 130(1): 83 - 93. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Simard, M.-L. Ricketts, S. Gingras, P. Soucy, F. A. Feltus, and M. H. Melner Molecular Biology of the 3{beta}-Hydroxysteroid Dehydrogenase/{Delta}5-{Delta}4 Isomerase Gene Family Endocr. Rev., June 1, 2005; 26(4): 525 - 582. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. A Maybin and W C. Duncan The human corpus luteum: which cells have progesterone receptors? Reproduction, October 1, 2004; 128(4): 423 - 431. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. A. Goyeneche, R. P. Deis, G. Gibori, and C. M. Telleria Progesterone Promotes Survival of the Rat Corpus Luteum in the Absence of Cognate Receptors Biol Reprod, January 1, 2003; 68(1): 151 - 158. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C.-H. Chiang, K. W. Cheng, S. Igarashi, P. S. Nathwani, and P. C. K. Leung Hormonal Regulation of Estrogen Receptor {alpha} and {beta} Gene Expression in Human Granulosa-Luteal Cells in Vitro J. Clin. Endocrinol. Metab., October 1, 2000; 85(10): 3828 - 3839. [Abstract] [Full Text]
|
|
|
|
|
|
B. R. Rueda, I. R. Hendry, W. J. Hendry III, F. Stormshak, O.D. Slayden, and J. S. Davis Decreased Progesterone Levels and Progesterone Receptor Antagonists Promote Apoptotic Cell Death in Bovine Luteal Cells Biol Reprod, February 1, 2000; 62(2): 269 - 276. [Abstract] [Full Text]
|
|
|
|
 |
|
 W. C. Duncan, G. M. Cowen, and P. J. Illingworth Steroidogenic enzyme expression in human corpora lutea in the presence and absence of exogenous human chorionic gonadotrophin (HCG) Mol. Hum. Reprod., April 1, 1999; 5(4): 291 - 298. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. A. McCracken, E. E. Custer, and J. C. Lamsa Luteolysis: A Neuroendocrine-Mediated Event Physiol Rev, April 1, 1999; 79(2): 263 - 323. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. J. Zeleznik In vivo responses of the primate corpus luteum to luteinizing hormone and chorionic gonadotropin PNAS, September 1, 1998; 95(18): 11002 - 11007. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. F. Benyo and A. J. Zeleznik Cyclic Adenosine Monophosphate Signaling in the Primate Corpus Luteum: Maintenance of Protein Kinase A Activity throughout the Luteal Phase of the Menstrual Cycle Endocrinology, August 1, 1997; 138(8): 3452 - 3458. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
), 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.
