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BOR - Papers in Press, published online ahead of print January 7, 2004.
Biol Reprod 2004, 10.1095/biolreprod.103.024786
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BIOLOGY OF REPRODUCTION 70, 1349–1357 (2004)
DOI: 10.1095/biolreprod.103.024786
© 2004 by the Society for the Study of Reproduction, Inc.

Pregnancy

Uterine Androgen Receptors: Roles in Estrogen-Mediated Gene Expressionand DNA Synthesis1

Andrés A. Kowalski3, Dustin S. Vale-Cruz3, Frank A. Simmen4, and Rosalia C.M. Simmen2,4

Interdisciplinary Concentration in Animal Molecular and Cell Biology,3 University of Florida,Gainesville, Florida 32611-0910 Arkansas Children's Nutrition Center and the Department of Physiology and Biophysics,4 University of Arkansasfor Medical Sciences, Little Rock, Arkansas 72202


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The localization of androgen receptors (AR) and their ligands in the uterine microenvironment at early pregnancy suggest a role for AR in uterine physiology. We have evaluated AR expression in the pig uterine endometrium and examined whether AR ligands modulate peri-implantation uterine gene expression. Northern blot analysis demonstrated the ~10.5 kilobase AR transcript in endometrium. Endometrial levels of AR mRNA and protein were greater at early than at mid- or late pregnancy. Estrogen receptor-{alpha} mRNA levels showed similar maximal expression at early pregnancy. Immunocytochemical analysis of endometrium at early pregnancy localized AR to nuclei of glandular epithelial (GE) and stromal (ST) cells. To evaluate a role for AR in uterine gene regulation, the levels of mRNAs for insulin-like growth factor-I (IGF-I), proliferative cell nuclear antigen (PCNA), and AR itself were assessed in uterine endometrial explant cultures treated with estradiol-17ß (E), testosterone (T), and 19-nortestosterone (N). Induction by E of AR mRNA abundance occurred in endometrium from Day 10 but not from Day 12 pregnant animals and this was partially blocked by coaddition of N or T, although neither androgen alone had any effect. Abundance of IGF-I and PCNA mRNAs was increased by E and inhibited by coaddition of either T or N in Day 10 pregnant pig endometrium. In endometrium from Day 12 pregnant animals, addition of either N or T with E increased IGF-I mRNA levels over that of controls, although E alone was without effect. In contrast, PCNA mRNA abundance was suppressed by all steroid treatments in these explants. DNA synthesis in primary cultures of GE cells from endometrium at Days 10 and 12 of pregnancy was increased by E and was suppressed by T, the latter only at Day 12. E did not affect DNA synthesis in ST cells from endometrium at either pregnancy day, although T inhibited this process in an E-dependent manner in ST cells from pregnancy Day 12. Results identify AR in the pig endometrium during the window of maternal receptivity for implantation and demonstrate the functional, albeit complex, interactions of androgens and estrogens in the regulation of uterine endometrial gene expression and cell growth in vitro. Further elucidation of the role of androgens and their receptor in early pregnancy events may be relevant to an understanding of peri-implantation embryo loss.

androgen receptor, embryo, estradiol receptor, pregnancy, uterus


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The uterine endometrium is a classical target of multiple signaling molecules of endocrine, paracrine, and autocrine origins, foremost of which include the steroid hormones estrogen (E) and progesterone (P). Under the respective influences of E and P, this tissue undergoes distinct morphological and functional modifications, as manifested by alterations in the transcriptional activities of endometrial-associated genes and the subsequent production and secretion of histotroph from endometrial glands. Uterine histotroph is comprised of enzymes, growth factors, transport proteins, and many yet-to-be identified secreted proteins that are requisite for embryo development and successful implantation, especially in species with the epitheliochorial type of placentation such as in the pig [1–3]. In addition to E and P, high levels of androgens are also present in the uterine environment during early pregnancy, a seemingly common phenomenon among mammals, including humans, rats, and mice [4–6]. These androgens are presumed to derive from the circulation or, as in the case of the pig and possibly the horse, as products of the embryo-specific complement of steroidogenic enzymes [7–9]. Receptors (AR) that mediate the actions of androgens have been detected in bovine, human, primate, and porcine uteri during the estrous cycle and/or early pregnancy [10–16]. Upon interaction with its ligand(s), AR, a member of the nuclear receptor superfamily of transcription factors, regulates gene expression by binding to hormone response elements and forming functional complexes with coregulatory molecules and the transcriptional machinery [17]. Ligand-bound AR has been demonstrated to modulate uterine growth [18], stimulate prolactin secretion [19], antagonize the expression of estrogen-regulated genes [20], and support decidualization of the mouse uterine stroma [21]. High levels of serum androgens concomitant with the presence of AR in the endometrium have been associated with poor fertility in women [22].

The production by peri-implantation porcine embryos of high levels of estrogens [23], and most likely, androgens during early pregnancy [5–7], when significant embryo mortality occurs coincident with changes in uterine receptivity for implantation [24], suggests functional roles for these steroid hormones in the control of uterine function and gene expression in this species. Indeed, a possible target of androgen action is the insulin-like growth factor-I (IGF-I) gene, the temporal expression of which, in the pig uterine endometrium, parallels that of the embryonic cytochrome P450 aromatase gene [25, 26]. We have demonstrated increased IGF-I expression at the level of both mRNA and protein, in the endometrium of mature, ovariectomized pigs and in prepubertal pigs, administered estrogen in vivo [26]. Androgenic ligands may act as cofactors with estrogens in modulating uterine IGF-I gene expression because both steroids increased IGF-I mRNA abundance, albeit with distinct kinetics, in the uterus of ovariectomized rats [18, 27]. To gain further insight into the role of androgens in uterine-associated early pregnancy events, we 1) evaluated the relative expression of AR in the porcine uterine endometrium during pregnancy; 2) determined the effects of the androgens testosterone (T) and 19-nortestosterone (N), the latter presumed to be a catalytic product of the embryonic P450 aromatase enzyme [9], alone and in combination with E, in mediating gene expression in peri-implantation endometrium ex vivo; and 3) ascertained whether in vitro proliferation of endometrial glandular epithelial and stromal cells, as measured by changes in basal DNA synthesis, is modulated by T and/or N in an E-dependent manner.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials

TRIzol, minimal essential medium (MEM), and MEM vitamin solution were purchased from Gibco BRL (Grand Island, NY). Antibiotic-antimycotic (ABAM), fetal bovine serum (FBS), RPMI 1640, estradiol-17ß, testosterone, 19-nortestosterone, and diaminobenzidine were purchased from Sigma Chemical Co. (St. Louis, MO). [{alpha}-32P]Deoxycytidine triphosphate (3000 Ci/mmol), [3H]thymidine (6.7 Ci/mmol), and Bio-Trans nylon membranes were purchased from ICN Biotech. (Irvine, CA). All other molecular biology-grade reagents were purchased from Fisher Scientific (Pittsburgh, PA).

Animals

All procedures involving animals were approved by the University of Florida Institutional Animal Care and Use Committee. Prepubertal gilts were monitored daily for estrus. At the onset of estrus (Day 0 of the estrous cycle), gilts were mated with mature boars. Gilts were killed at the indicated day of pregnancy at the University of Florida Meats Processing Facility. Uterine horns were flushed with PBS-2% ABAM to remove conceptuses. Endometrium was trimmed of underlying tissue and immediately used for explant culture or cell isolation or fixed for immunolocalization studies as described below.

Endometrial Explant Cultures

Endometrial explants were isolated and cultured as previously described [25]. In brief, endometrial tissue collected from Day 10 and 12 pregnant pigs was washed in PBS-2% ABAM and minced into 1–3 mm3 sections. These pregnancy days were selected based on the documented presence of high levels (pregnancy Day 12) and low levels or lack thereof (pregnancy Day 10) of estrogens and androgens in uterine fluids, and the rapid changes in uterine endometrial gene expression between Days 10 and 18 of pregnancy [2–5, 23–26]. Tissues (250–300 mg) were cultured in 3 ml of MEM (supplemented with nonessential amino acids, glutamine [292 mg/L], MEM vitamin solution [10 ml/L], glucose [3.0 g/L], and 1% ABAM) in six-well tissue culture plates on a rocking platform at 37°C under an atmosphere of 50% N2:47.5% O2:2.5% CO2. After 2 h of incubation, explants received fresh medium supplemented with the following: estradiol-17ß (E, 50 nM), testosterone (T, 50 nM), 19-nortestosterone (N, 50 nM), E + T (50 nM each), or E + N (50 nM each); these hormone concentrations were within the range reported in uterine luminal fluids of pigs at these pregnancy days [5, 23, 24]. The explants were further incubated for 24 h, collected and stored at –80°C until analyzed. Experiments were performed with three individual pigs per pregnancy day, and three endometrial explant cultures were used per animal per treatment.

RNA Isolation, Northern Blot, Reverse Transcription- Polymerase Chain Reaction (RT-PCR), and Western Blot Analyses

Total cellular RNA was isolated from tissues and cells using the TRIzol reagent following the manufacturer's specifications. For Northern blots, total RNA (30 µg per lane) was fractionated on a 1% agarose-formaldehyde-MOPS [3-N-morpholino-propanesulfonic acid] gel, and transferred by capillary action to a Biotrans nylon membrane (ICN Biotech.). Membranes were hybridized overnight at 42°C in UltraHyb buffer (Ambion Inc., Austin, TX) with [32P]dCTP-labeled fragments prepared by nick translation. The labeled probes represent partial cDNA fragments for AR (283 base pairs [bp]), glyceraldehyde phosphate dehydrogenase (GAPDH) (593 bp), PCNA (520 bp), and IGF-I (252 bp); all were generated by RT- PCR using porcine endometrial mRNA as template and subsequently confirmed for authenticity by cloning and nucleotide sequence analysis. Hybridization signals were detected by autoradiography and band intensities were quantified by scanning densitometry using the Alpha Imager 2000 Documentation and Analysis System (Alpha Innotech Co., San Leandro, CA). Membranes were stripped of probe between hybridizations by incubation for 1 h in 1% SDS heated at 90°C.

For the RT-PCR used to quantify estrogen receptor (ER)-{alpha} transcript levels, complementary DNA was synthesized from total RNA prepared from uterine endometrium as previously described [25]. PCR amplification used intron-spanning primer sets for porcine ER{alpha} and porcine GAPDH to generate 451-bp and 593-bp DNA fragments, respectively [28]. The amount of PCR product was quantified from ethidium bromide-stained agarose gels by scanning densitometry as described above.

For Western blot analysis, nuclear extract proteins (100 µg) were resolved by 10% SDS-PAGE under reducing conditions and transferred to nitrocellulose membranes. Nonspecific sites were blocked in Tris-buffered saline (TBS, pH 7.4) containing 1% Carnation nonfat dry milk and 0.2% Tween-20, following previously described protocols [29]. Incubations with rabbit anti-human AR (0.75 µg/ml) (PG-21–40; kindly provided by Dr. Gail Prins, University of Illinois at Chicago) or mouse monoclonal anti- rat PCNA antibody (1 µg/ml) (PC10; Boehringer-Mannheim, Mannheim, Germany) were carried out in the same buffer overnight at 4°C. Following incubation with the appropriate secondary anti-rabbit (Biomeda Inc., Foster City, CA) or anti-mouse (Vector Laboratories, Burlingame, CA) immunoglobulin (Ig) G, respectively, as per manufacturers' instructions, and enhanced chemiluminescence reagent (Amersham Biosciences, Little Chalfont, UK), the filters were rinsed in five changes of TBS containing 0.2% Tween-20 at room temperature.

Immunolocalization of AR

Immunohistochemistry on porcine endometrial and ovarian tissue sections was performed using rabbit anti-human AR after antigen retrieval [30]. Tissue sections were fixed in 4% paraformaldehyde for 24 h and embedded in paraffin. Sections of 5 µm were cut, deparaffinized, and rehydrated through a series of alcohol dilutions. Sections were placed in 0.01 M citric acid (pH 6) and microwaved for 15 sec to expose cellular antigens. Nonspecific binding and endogenous peroxidase activity were blocked by incubation with protein blocker solution (Rabbit Super ABC kit; Biomeda Inc.). Rabbit anti-human polyclonal AR (PG-21–40) diluted in PBS containing 1% (w/v) BSA was added to each slide at 3 µg/ml final concentration, and incubation was carried out at 4°C overnight. Negative control was generated from corresponding tissue sections incubated with normal rabbit IgG (10 µg/ml; Biomeda) in place of the test antibody. Biotinylated anti-rabbit IgG, the secondary antibody, was used per the supplier's instructions (Biomeda), and sections were placed into a substrate solution of diaminobenzidine for staining. Photomicrographs were taken using a Zeiss Axioplan 2 microscope attached to an RGB Spot Digital camera system with corresponding software (Carl Zeiss, Inc, New York, NY). Uterine sections (n = 3 per animal) for three animals for each pregnancy day were analyzed.

Primary Cell Culture and DNA Synthesis Assay

Glandular epithelial (GE) and stromal (ST) cells were isolated from Days 10 and 12 pregnancy endometrium and cultured as previously described [31]. Primary cells were grown in RPMI 1640 supplemented with 10% FBS, 0.25 U/ml insulin, and 1% ABAM at 37°C under an atmosphere of 95% air, 5% CO2. Medium was changed every other day. At 90–95% confluence, cells were washed with Hanks balanced salt solution and cultured in serum-free, insulin-free RPMI 1640 for an additional 24 h. Cells then received fresh medium containing E and/or T, respectively, at the following concentration ratios (E:T) (nM; 0:0, 0:50; 0:500, 50:0, 50:50, 50:500, 500:0, 500:50, and 500:500). After 20 h, cells were pulse labeled with [3H]thymidine (1 µCi/well) for 4 h at 37°C. Cells were washed with PBS, fixed in methanol, and cellular DNA was precipitated with trichloroacetic acid (5%) for 10 min and recovered using NaOH (0.3 M). Incorporated labeled tritium was determined using a scintillation counter. Experiments were repeated three times, with each experiment using an individual animal and performed in triplicate wells.

Statistical Analysis

Data from endometrial explant studies were subjected to least squares ANOVA using the general linear models procedures of SAS [32]. The statistical model included treatment (hormone and dose) and experiment plus the interaction of treatment by experiment. For labeled thymidine incorporation assays, the model included E or T treatments, amounts of each steroid hormone used per treatment, and the double and triple interactions among these factors. Values were considered significant at P < 0.05 and are presented as least squares means ± SEM.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Characterization of AR Expression in the Pregnant Pig Uterus

Endometrial RNA corresponding to Days 10, 12, 14, 18, 30, 60, and 90 of pregnancy (n = 3 individual pigs for each day) was analyzed by Northern blot using porcine AR cDNA fragment as probe. As shown in the representative autoradiogram (Fig. 1A), a single transcript of approximately 10.5 kb in size and similar to that reported for AR [33] was detected. AR mRNA abundance was greatest at Day 12 > Days 10 and 18 > Days 14, 30, 60, and 90 of pregnancy (Fig. 1B). The pattern of ER{alpha} mRNA abundance was different with maximal levels at pregnancy Days 10– 14 and reduced expression at pregnancy Days 18 and 30 (Fig. 1C).



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  		FIG. 1. Androgen receptor transcripts in pig endometrium. Endometrial RNA from pigs at Days 10, 12, 14, 18, 30, 60, and 90 of pregnancy was subjected to Northern blot hybridization (30 µg/lane), using a cloned porcine AR cDNA probe. Blots were rehybridized with a porcine GAPDH cDNA probe to correct for variations in RNA loading. A) Representative Northern blot of RNA from endometrium of Days 10 (n = 2) and 12 (n = 2) pregnant pigs; top and bottom demonstrate transcripts for AR and GAPDH, respectively. B) Plot of endometrial AR mRNA abundance as a function of day of pregnancy. The ratios of AR to GAPDH hybridization signal obtained from Northern blots of endometrial RNAs (containing three individual animals per pregnancy day) were statistically analyzed and are presented as least squares means ± SEM. Superscripts designate differences (P < 0.05). C) Diagrammatic representation of levels of ER{alpha} mRNA obtained by densitometric analysis of RT-PCR product from total RNA of pig endometrium (three individual animals per pregnancy day). Values (normalized to those for GAPDH) are presented as least squares means ± SEM. Values with different superscripts differ (P < 0.05)

The presence of AR in the endometrium during early pregnancy was evaluated using a rabbit anti-human AR polyclonal antibody. An ~110 kDa protein was detected in nuclear extracts prepared from endometrium at Days 10, 12, and 14 of pregnancy (Fig. 2A). No immunoreactive protein was evident in nuclear extracts prepared from endometrium at Day 30, consistent with the relatively low level of AR mRNA transcript noted at this time (Fig. 1B). Interestingly, peak levels of AR protein were slightly delayed relative to that of its mRNA (Day 14 vs. Day 12; Figs. 1B and 2A). To correlate expression of endometrial AR with growth status, the expression of a growth-associated nuclear protein, PCNA was evaluated in the same nuclear extracts. In contrast with AR, nuclear levels of PCNA protein were low or nondetectable on pregnancy Day 10, highest on Day 12, and somewhat diminished relative to Day 12 at Days 14 and 30 of pregnancy (Fig. 2B).



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  		FIG. 2. Nuclear AR and PCNA proteins in endometrium during early pregnancy. Nuclear extracts (100 µg protein/lane) from pig endometrium at Days 10, 12, 14, and 30 of pregnancy were probed for AR (A) and PCNA (B) proteins by Western blot analysis. Each lane contains nuclear extract from an individual animal at the indicated pregnancy day. Because of the limited sample size for each day, statistical analysis was not performed

Immunolocalization of AR in Uterine Endometrium

Immunohistochemistry was used to localize expression of the AR protein to specific uterine endometrial cell types. AR was detected in the nuclear compartments of GE and ST but not luminal epithelial (LE) cells (Fig. 3, B and D). In ovarian tissue sections from a Day 12 pregnant pig (positive control), AR expression was restricted to the granulosa cells of developing follicles (Fig. 3F), consistent with previous reports [34]. Parallel serial sections of the same tissues did not show any staining when incubated with normal rabbit IgG in place of anti-human AR antibody (Fig. 3, A, C, and E).



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  		FIG. 3. Immunolocalization of AR in the uterine endometrium. Uterine sections from Day 10 (A, B) or Day 12 (C, D) pregnant pigs were incubated with normal rabbit serum (A, C) or rabbit anti-human AR antiserum (B, D) at a final concentration of 3 µg/ml. E, F) Represent staining of ovarian sections from a Day 12 pregnant pig, with normal rabbit serum or anti-human AR antiserum, respectively, the latter as a positive control for AR. Arrows indicate positive nuclear staining. GE, Glandular epithelium; LE, luminal epithelium; ST, stroma

Effects of Estrogen and Androgens on EndometrialGene Expression

Because uterine luminal fluids contain high levels of estrogens and androgens during the peri-implantation stages [5–7, 23, 24], we examined the effects of these steroid hormones, alone and in combination on uterine gene expression in vitro. In these studies, uterine endometrial explants from pigs at Days 10 and 12 of pregnancy were incubated for 24 h with E, T, or N alone or the combination of either androgen with E. The mRNA levels of three endometrial genes (AR, PCNA, and IGF-I) were evaluated by Northern blotting (Fig. 4). On Day 10 of pregnancy, AR mRNA levels were significantly stimulated by E, were unaffected by either androgen, and were diminished by N or T in the presence of E, relative to E-treated explants (Fig. 4A). Similarly, E stimulated IGF-I and, to a lesser extent, PCNA gene expression relative to untreated explants. These effects of E were abolished by the coaddition of either androgen, neither of which, when tested alone, showed demonstrable activity (Fig. 4, B and C).



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  		FIG. 4. Effects of estrogen and androgens on Day 10 pregnancy, endometrial explant gene expression. Uterine endometrial explants from Day 10 pregnant pigs were cultured in the absence (control, C) or presence of estradiol-17ß (E), testosterone (T), nortestosterone (N), or the combinations of E + T or E + N for 24 h. RNA was isolated from explants and analyzed for AR (A), IGF-I (B), and PCNA (C) transcripts by Northern blot hybridization. Bars represent hybridization intensities of transcripts normalized to those for GAPDH mRNA. Values are expressed as densitometric units (least squares means ± SEM) and were obtained from three individual animals per treatment group, with three explants analyzed per animal. Mean values without common superscripts or those designated with asterisks differ (P < 0.05)

The levels of AR and IGF-I mRNAs in endometrial explants at Day 12 of pregnancy were refractory to E, T, or N treatments (Fig. 5). In contrast, treatments with these steroid hormones, alone or in combination, inhibited PCNA gene expression relative to control explants (Fig. 5C). E + T and E + N enhanced IGF-I mRNA transcript levels but had no effect on those of AR.



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  		FIG. 5. Effects of estrogen and androgens on Day 12 pregnancy, endometrial explant gene expression. Endometrial explants from Day 12 pregnant pigs were cultured in the absence (control, C) or presence of estradiol-17ß (E), testosterone (T), nortestosterone (N), or the combinations of E + T or E + N for 24 h, as described in Figure 4. Bars represent hybridization intensities of transcripts (AR, A; IGF-I, B; PCNA, C) relative to those for GAPDH. Values are expressed as arbitrary densitometric units (least squares mean ± SEM) and were obtained from three individual animals per treatment group, with three explants analyzed per animal. Mean values designated with asterisks differ (P < 0.05) from all others

Effects of Estrogen and/or Testosterone on 3H-thymidine Incorporation in GE and ST Cells

To examine the effects of E and/or T on endometrial cell proliferation, primary cultures of GE and ST cells isolated from endometrium of Day 10 and 12 pregnant pigs were treated with E or T, alone or together in serum-free medium, at concentrations that mimicked their in vivo levels in uterine luminal fluids [5–7, 23, 24]. GE cells from Day 10 pregnant animals were responsive to E at low (50 nM) but not high (500 nM) dose and were unaffected by T (Fig. 6). In contrast, GE cells from Day 12 pregnant animals showed enhanced DNA synthesis at a high E (500 nM) concentration. Testosterone diminished DNA synthesis in these cells. Estrogen and T did not influence each other's individual effects on DNA synthesis (data not shown).



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  		FIG. 6. Effect of estrogen and testosterone on glandular epithelial cell DNA synthesis. DNA synthesis was assessed by [3H]-thymidine incorporation in primary cultures of uterine endometrial epithelial cells isolated from Day 10 (A) and Day 12 (B) pregnant pigs after treatment with estradiol-17ß (E; 50 and 500 nM) or testosterone (T; 50 and 500 nM). Values are least squares mean ± SEM of three different experiments, where each experiment represents epithelial cells isolated from a different pig, and for each pig, three replicates per treatment. *, Significantly different (P < 0.05) from control (C, untreated) cells

DNA synthesis in endometrial ST cells isolated from Day 10 pregnant pigs was unaffected by E treatment but was reduced upon treatment with T at the lower (50 nM) but not higher (500 nM) concentration (Fig. 7A). Treatments with E + T did not show any statistically significant interaction (data not shown). In contrast, while E had no effect on DNA synthesis of ST cells from Day 12 pregnant pig endometrium, a significant interaction between E and T in these cells was observed. In particular, ST cells showed decreased DNA synthesis when treated with 50 and 500 nM of T in the presence of high levels of E (500 nM) (Fig. 7B). In the absence of or at low levels of E (0 and 50 nM), T did not affect DNA synthesis.



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  		FIG. 7. Effect of estrogen and testosterone on stromal cell DNA synthesis. DNA synthesis was assessed by [3H]-thymidine incorporation in primary cultures of uterine endometrial stromal cells isolated from Day 10 (A) and Day 12 (B) pregnant pigs after treatment with estradiol-17ß (E; 50 and 500 nM) and testosterone (T; 50 and 500 nM). Values are least squares mean ± SEM of three different experiments, where each experiment represents stromal cells isolated from a different pig, and for each pig, three replicates per treatment. *, Significantly different from control (C, untreated) cells


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Previous studies reported the presence of physiological levels of androgens in the uterine luminal fluid of pigs during early pregnancy [5–7]. Although this occurs in other mammals [4, 35], including those species where embryonic steroid production is considered negligible, the physiological role of androgens in the uterine luminal environment remains unclear. Studies of androgen receptors in the peri- implantation uterus and/or corresponding embryo are relatively few in number but have added support to the potential physiological relevance of uterine and uterine luminal androgens during early pregnancy [15, 16]. Results of the present study demonstrate a) highest expression of AR in the pig endometrium during peri-implantation, which overlapped with that of ER{alpha}; b) modulation by T and N of E- regulated endometrial gene expression; and c) E-dependent androgen control of ST cell proliferation in vitro as a function of day of early pregnancy. Collectively, these observations provide further evidence to the physiological relevance of androgens and their receptors in the uterine luminal environment at peri-implantation [5–7, 24] and suggest coregulatory roles of AR and ER in endometrial gene expression and cell proliferation.

The synchronized growth and differentiation of the uterine endometrium and the developing conceptus during the peri-implantation period in the pig (Days 10–14) is requisite for successful embryo attachment [2, 3]. This period is characterized by marked differences in transcript levels for numerous genes in both tissues [26, 28, 36–42], which include those involved in growth induction (e.g., IGF-I) and growth inhibition (e.g., spermidine/spermine N1-acetyltransferase). Previous studies have shown that, while the endometrium can affect the development of the preimplantation conceptus [2, 3], genes expressed by elongating conceptuses can also influence endometrial gene expression via the intermediary role of secreted products, including steroid hormones [3, 25, 26, 39, and 41]. The present study documented the coexpression of AR and ER{alpha} in the endometrium at peri-implantation, consistent with the notion of the endometrium as a receptive target of embryonic estrogens and androgens. How these hormones modulate endometrial growth and differentiation to support successful embryo attachment/implantation remains largely unclear. In this regard, our studies demonstrate that these hormones can functionally interact at the levels of endometrial gene expression and cellular proliferation to regulate each other's activity. Cross-regulation of ER and AR, bound to their respective ligands, has been documented in other systems. For example, AR content in prostatic epithelial cells is inhibited by ER signaling [43]. Moreover, downregulation of ER{alpha} gene expression by AR was recently demonstrated in ovariectomized, postpubertal gilts injected with E + T [44]. Androgens may also functionally interact with the PR-mediated program of endometrial gene expression [45].

The androgen inhibition of E-stimulated endometrial IGF-I gene expression at Day 10 of pregnancy is consistent with other reports of the stimulatory effects of E on gene expression that can be blocked or otherwise modulated by the androgen/AR complex [20, 46]. A recent study found that the increase in luminal epithelial cell proliferation by E in immature rat uterus involves a sequential pathway in which E induction of stromal AR leads to the stimulation of IGF-I gene expression by T when complexed to AR [27]. Because our studies used endometrial explants to monitor IGF-I gene expression in response to E and T (or N) and isolated stromal and glandular epithelial cells to demonstrate the effects of these steroids on DNA synthesis, it was not possible to causally establish the connection between endometrial IGF-I synthesis and the proliferative potential of specific endometrial cell types. Nevertheless, the interaction of E and T on the proliferation of IGF-I expressing ST cells from pregnancy Day 12 endometrium, which was observed only at high E concentrations (500 nM), may be physiologically relevant because embryonic production of E and probably T is highest during this period [5, 23–25].

The present study highlighted the dynamic nature of the peri-implantation uterus, which was manifested by the distinct responses elicited by steroid hormones in this tissue within a narrow window of gestation. Thus, while at Day 10, E increased endometrial expression of AR, IGF-I, and PCNA mRNAs, it had no effect on AR and IGF-I mRNA expression and decreased that of PCNA in endometrium at Day 12 of pregnancy. The latter may be due to the prior in vivo exposure of Day 12 pregnancy endometrium to the milieu of steroid hormones (e.g., rising luminal E from embryonic synthesis), growth factors, and other molecules that can modify this tissue's responsiveness to E and T. In this regard, E induction of AR gene expression and the lack of an effect by T or N alone in endometrium from Day 10 pregnant pigs mimicked that observed in ovariectomized pigs injected with E or T [16]. Because the stimulatory effects of embryonic-derived E on uterine secretory activity are not initiated until Day 11 of pregnancy, the uterine microenvironment at pregnancy Day 10 may resemble that of the ovariectomized animal, suggesting that embryonic induction of uterine activity as mediated by E and its downstream target genes is an important parameter for successful implantation [47]. The dynamics of endometrial AR gene expression at early pregnancy, where transcript levels correlate with peak levels of estrogen production by developing embryos (Day 12) and endometrium (Day 18) [48], are consistent with E induction of AR gene expression in vivo [44]. Of note was the differing effect of E + T or E + N on endometrial IGF-I mRNA abundance at Day 10 vs. Day 12. Because endometrial nuclear AR protein levels were not different between the 2 days, the differences in response of IGF-I mRNA may reflect the lack of previous exposure of endometrium to significant E + T at Day 10, in contrast with in vivo exposure to both hormones by Day 12 [5]. Enhanced PCNA expression at Day 12 in vivo is supportive of a growth-stimulatory role for E + T via enhanced endometrial IGF-I at Day 12 [3, 25, 26]. In this regard, embryos also exhibit remarkable rates of growth at Day 12 [2, 3, 28]. However, PCNA mRNA levels were observed to decline with all steroids in the tissue explants from pregnancy Day 12, suggesting the lack of a direct relationship between IGF-I and PCNA in vitro. The basis for the differences between an earlier report that porcine AR is expressed in all endometrial cell types [16] and our present finding of detectable AR in GE and ST cells only is unclear but may be related to the different anti-AR antibodies that were used in the two studies.

To the best of our knowledge, these studies are the first to examine the effect of N on endometrial mitogenesis and gene expression and to evaluate its possible actions during pregnancy. Although the concentrations of N in uterine luminal fluids have not been accurately quantified, the recent report that a major steroid product of the stably expressed porcine P450arom type III (blastocyst) isoform is N [9] suggests physiological relevance for this androgen. N can bind to the ERß isoform, albeit with low affinity [49]. However, our findings that N behaves more like an androgen than an estrogen and the limited ERß expression in the pig endometrium during early pregnancy (unpublished data), suggest that the functional effects of N in vivo are likely to be mediated via AR.

In conclusion, androgens present in porcine uterine luminal fluid at early pregnancy may not solely serve as substrates for the production of E by peri-implantation conceptuses, but may also modulate the biological effects of E in the endometrium at peri-implantation. Thus, ligand- bound AR in concert with ER{alpha} complexed with embryonic- derived E are likely interactive partners requisite for regulating endometrial gene expression and uterine growth, both of which are obligatory for successful implantation.


   ACKNOWLEDGMENTS
 
We thank Dr. Gail Prins (University of Illinois at Chicago) for the gift of the rabbit anti-human AR antiserum, members of our laboratories for assistance with animal breeding and tissue collection, and Rijin Xiao for formatting of figures.


   FOOTNOTES
 
1 Supported, in part, by grants from the National Institutes of Health (HD21961) and USDA (98-35205-6739) and by USDA-CRIS6251-5100- 002-06S to the Arkansas Children's Nutrition Center. Back

2 Correspondence: Rosalia C.M. Simmen, Department of Physiology and Biophysics, University of Arkansas for Medical Sciences, and Arkansas Children's Nutrition Center, 1120 Marshall Street, Little Rock, Arkansas 72202. FAX: 501 364 3161; simmenrosalia{at}uams.edu Back

Received: 27 October 2003.

First decision: 14 November 2003.

Accepted: 29 December 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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