| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
ARTICLES |
a Center for Animal Biotechnology and Genomics, and Department of Animal Science, Texas A&M University, College Station, Texas 77843-2471
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
), progesterone receptor (PR), and prolactin receptor (PRL-R) expression were characterized by in situ hybridization (ISH), immunohistochemistry, or both. The most striking feature of neonatal uterine development was the genesis and development of glands in the intercaruncular areas of endometrium. After birth, endometrial glandular epithelium (GE) budded and differentiated into the underlying stroma from the luminal epithelium (LE) between PNDs 1 and 7. Between PNDs 14 and 56, extensive coiling and branching morphogenesis of nascent endometrial glands occurred. By PND 56, the uterine wall appeared to be histoarchitecturally mature. At birth, nuclear PCNA protein was strongly detected in LE. Between PNDs 7 and 56, high levels of PCNA, ER-, and PR gene expression were detected in both nascent and developing GE. Higher levels of PCNA and ER- expression were detected in GE at the tips of developing glands as well as in the surrounding stroma. Progesterone was below detectable limits in serum. Serum estradiol-17ß levels were high on PND 1, increased from PNDs 14 to 28, and declined from PND 42 to PND 56. Serum PRL levels increased from PNDs 1 to 14 and declined thereafter. Using ISH and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis, expression of mRNAs for short and long forms of the ovine PRL-R were first detected in nascent GE on PND 7 and increased between PNDs 7 and 56 in proliferating and differentiating GE. These results indicate that 1) uterine gland genesis is initiated between PNDs 1 and 7 after birth and is essentially completed by PND 56; 2) neonatal uterine morphogenesis involves temporal and spatial alterations in cell proliferation and ER-, PR, and PRL-R gene expression; 3) PRL-R expression is a unique marker of GE differentiation and proliferation; and 4) serum estradiol-17ß and PRL levels increase during the onset of GE tubular branching morphogenesis. Results support the hypothesis that neonatal ovine uterine development involves epithelial PRL-R and ER- activation to stimulate and maintain endometrial gland genesis and branching morphogenesis.
developmental biology, estradiol, estradiol receptor, female reproductive tract, hormone action, pituitary hormones, progesterone, progesterone receptor, steroid hormone receptors, steroid hormones, uterus
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
The dichotomous nature of adult ovine endometrium, which consists of both aglandular caruncular areas and glandular intercaruncular areas, provides an excellent model for the study of mechanisms that underlie establishment of divergent structural and functional areas within a single, mesodermally derived organ [9]. Uterine histogenesis has been partially described and incompletely characterized in fetal and neonatal ewes [9–14]. As in other mammals, the ovine uterus develops as a specialization of the paramesonephric ducts, which give rise to the infundibula, oviducts, uterus, cervix, and anterior vagina [2]. Paramesonephric duct fusion occurs between Gestational Days 34 and 55 in sheep, is partial, and produces a bicornuate uterus [9–11]. Postnatal uterine development is dramatic and includes the appearance and proliferation of uterine glands, development of endometrial folds and, to a lesser extent, differentiation of stromal layers and growth of the caruncular areas and myometrium [9, 13]. In an elegant study by Bartol et al. [14], the withdrawal from progesterone at birth was found to be an endocrine cue for the onset of endometrial gland genesis in neonates. Progestin inhibition of endometrial adenogenesis involves suppression of epithelial estrogen receptor alpha (ER-) expression and specific growth factors and their receptors involved in epitheliomesenchymal interactions [15]. Indeed, prolonged exposure of neonatal ewe lambs to a progestin from birth prevents epigenetic endometrial gland development in the ovine uterus, thereby producing a unique adult endometrial phenotype that is characterized by the absence of uterine glands—the ovine uterine gland knockout (UGKO) ewe [8, 16]. UGKO ewes are infertile and the uterus cannot support the establishment or maintenance of pregnancy [16]. In adults, endometrial glands are the sole source of several expressed genes [8] and secretory proteins such as uterine milk proteins or serpins [17] and osteopontin [18]. During pregnancy, endometrial glands grow substantially in length and width during placentation prior to maximal increases in fetal growth [1, 17]. Histotroph produced by endometrial glands is important because ablation or reduction in uterine glands in sheep [16] and pigs [19], respectively, is associated with peri-implantation embryonic mortality. Thus, endometrial adenogenesis represents a critical period of uterine morphogenesis, which establishes, in a large part, the embryotrophic potential and functional capacity of the adult uterus [16, 19, 20].
Mechanisms that regulate postnatal endometrial adenogenesis in mammals are not well-defined. Based on regulatory mechanisms that govern gland development in other epitheliomesenchymal organs, uterine gland morphogenesis is proposed to involve 1) site-specific alterations in cell proliferation; 2) actions of specific hormones, growth factors, and their receptors; and 3) paracrine cell-cell and cell-extracellular matrix interactions [19, 20]. Depending upon photoperiod, serum prolactin (PRL) concentrations are either high or low at birth in ewe lambs and then increase substantially between Postnatal Days (PNDs) 14 and 35 [21]. In adult ovine uterus, PRL receptors (PRL-R) are expressed exclusively in endometrial GE [17], suggesting that this receptor may play a role in endometrial GE morphogenesis in neonates as well as in adults.
At birth, the neonatal ovine reproductive tract has developed to a state similar to that of Gestational Day 100 in humans [22]. Therefore, postnatal development of uterine glands makes the neonatal uterus an attractive model for studying normal developmental processes such as patterning, branching morphogenesis, and functional differentiation as well as pathological conditions such as dysplasia and neoplasia. In order to begin investigations into the hormonal, cellular, and molecular mechanisms governing uterine wall development and, specifically, the ontogeny of endometrial glands, uteri were obtained from neonatal ewes between birth and PND 56 to determine effects of age on histoarchitecture, cell proliferation, and expression of receptors for ER-, progesterone (PR), and PRL.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
All experiments and surgical procedures were performed in accordance with the Guide for the Care and Use of Agriculture Animals and approved by the University Laboratory Animal Care Committee of Texas A&M University (Animal Use Protocol 9-206).
Experimental Design, Tissue Collection, and Histology
Cross-bred Rambouillet ewe lambs were assigned randomly at birth (PND 0) to be necropsied on PND 1 (n = 6), 7 (n = 5), 14 (n = 5), 21 (n = 4), 28 (n = 5), 42 (n = 5), or 56 (n = 5). Prior to necropsy, blood samples were collected from each ewe by jugular venipuncture. The uterus was obtained and trimmed free of the broad ligament, oviduct, and cervix. Sections (
1 cm) from the mid-portion of each uterine horn were fixed in fresh 4% paraformaldehyde in PBS (pH 7.2), embedded in Paraplast Plus (Oxford Labware, St. Louis, MO), sectioned (4–6 µm), and stained with hematoxylin and eosin as described previously [16]. Additional uterine tissues for histology were obtained at necropsy from ewes at PND 0 (n = 5).
Radioimmunoassay
Blood samples were allowed to clot for 1 h at room temperature. Serum was then collected and stored at -20°C for hormone analysis. Concentrations of estradiol-17ß in 200 µl of serum, after solvent extraction, were determined by radioimmunoassay (RIA) [23]. Average extraction efficiency was 75%. Assay sensitivity was 1.5 pg per tube, and the intra-assay and interassay coefficients of variation were 5% and 9%, respectively. Concentrations of PR in serum were determined using an Active Progesterone RIA Kit (Diagnostic Systems Laboratories, Inc., Webster, TX) as described previously [16]. Assay sensitivity was 0.1 ng/ml, and the intraassay and interassay coefficients of variation were 5% and 10%, respectively. Concentrations of PRL in serum were determined using reagents for the ovine PRL RIA provided by Dr. A.F. Parlow and the National Hormone and Pituitary Program of the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK). Purified ovine PRL (NIDDK-oPRL-I-3) was iodinated using the chloramine T reaction, and the assay was conducted using methods provided by the NIDDK Pituitary Hormones and Antisera Center. The assay was fully validated for parallelism and added mass recovery. Assay sensitivity was 0.1 ng/ml; the intraassay and interassay coefficients of variation were 5% and 12%, respectively. Assay results were calculated using the AssayZap program (Biosoft, Ferguson, CA).
In Situ Hybridization
Location of ER-, PR, and PRL-R mRNAs in uterine tissue sections were determined by in situ hybridization as described previously [8, 16, 17]. Deparaffinized, rehydrated, and deproteinated tissue sections (5–7 µm) were hybridized with radiolabeled sense or antisense ER-, PR, or PRL-R cRNA probes that were generated from linearized plasmid templates using in vitro transcription with [-35S]uridine 5'-triphosphate (UTP). Plasmid templates were partial cDNAs for ovine ER- and ovine PR [23] and a full-length cDNA for the bovine long PRL-R (clone 26353) [24]. After hybridization and digestion with ribonuclease A, slides were dipped in Kodak NTB-2 liquid photographic emulsion, exposed at 4°C for 2 wk (ER- and PR) or 5 wk (PRL-R), developed in Kodak D-19 developer, counterstained with hematoxylin, dehydrated, and protected with coverslips.
Immunohistochemistry
Expression of immunoreactive proliferating cell nuclear antigen (PCNA), ER-, and PR protein were detected in uterine tissue cross-sections (5–7 µm) using specific antibodies and a Super ABC Mouse/Rat Immunoglobulin G (IgG) Kit (Biomeda, Foster City, CA). Mouse monoclonal antibody to PCNA was purchased from DAKO (Carpinteria, CA). Rat monoclonal antibody to human ER- (H222) was kindly provided by Dr. Geoffrey Greene (University of Chicago, Chicago, IL). Mouse monoclonal antibody to the human PR (MA1-411) was purchased from Affinity Bioreagents (Golden, CO). The final working antibody concentration was 1.9 µg/ml for PCNA and 5 µg/ml for both ER- and PR. Negative controls were performed in which the primary antibody was substituted with the same concentration of purified normal mouse IgG (PCNA and PR) or rat IgG (ER-) from Sigma Chemical Company (St. Louis, MO). For PCNA and PR immunolocalization, antigen retrieval with the use of a boiling citrate buffer was performed according to the manufacturer's recommendations. For ER-, antigen retrieval using limited pronase digestion was performed as described previously [23].
Relative staining intensity for immunoreactive nuclear PCNA, ER-, and PR protein expression were visually assessed in multiple tissue sections from each uterine horn from each ewe by two independent observers and scored as follows: absent (-; that is, no nuclear staining), weak (+), moderate (++), or strong (+++). Intercaruncular endometrial tissues were scored if they were histologically discernable (including LE, stroma, GE, caruncular endometrial tissues [including LE and stroma], and myometrial tissues).
Semi-Quantitative RT-PCR
Temporal alterations in the expression of mRNAs for short and long forms of the ovine PRL-R were determined by RT-PCR using methods described previously [17]. Briefly, cDNA was synthesized from total cellular RNA (5 µg) that was isolated from neonatal uteri using random and oligo-dT primers and SuperScript II Reverse Transcriptase (Life Technologies, Gaithersburg, MD). Newly synthesized cDNA was acid-ethanol precipitated, resuspended in 20 µl water, and stored at -20°C. The cDNAs were diluted (1:10 or 1:100) with sterile water prior to use in PCR reactions. The PCR reactions were performed using AmpliTaq DNA polymerase (Perkin Elmer, Foster City, CA) and Optimized Buffer D (Invitrogen, Carlsbad, CA) for ß-actin or Optimized Buffer F (Invitrogen) for short and long PRL-R according to manufacturers' recommendations.
The PRL-R primers were ovine PRL-R sense (base pairs [bp] 709–729) 5'-TTC CCA GTG AAG GAT ACA AGC-3', ovine long PRL-R antisense (bp 1018–1000) 5'-GTT CTT TGG AGG GGT GTG G-3' (GenBank accession number AF041257), and ovine short PRL-R antisense (bp 894–873) 5'-CTA TTA AAA CAC AGA CAC AAG G-3' (GenBank accession number AF041977) [25]. Using the ovine ß-actin mRNA sequence (GenBank accession number U39357), the ß-actin primers were sense (bp 274–295) 5'-CAT CCT GAC CCT CAA GTA CCC-3' and antisense (bp 694–674) 5'-CCG ATG TCG AAG TGG TGG TG-3'. ß-Actin PCR reactions contained 5 µl cDNA (1:100), whereas short and long PRL-R reactions had 5 µl cDNA (1:10).
The ß-actin PCR conditions were 30 cycles at 95°C for 30 sec, 55°C for 1 min, and 72°C for 1 min. Short and long PRL-R PCR conditions were the same, but with an annealing temperature of 54°C. The PCR conditions and amount of template cDNA used in each reaction were optimized for each primer set to ensure linear amplification of the target as described previously [15, 17]. PCR products were separated on a 2% agarose gel and visualized by ethidium bromide staining. The amount of DNA was quantitated by measuring the intensity of light emitted from correctly sized bands under ultraviolet light using an AlphaImager (Alpha Innotech Corporation, San Leandro, CA). Values are presented as relative light units.
Photomicroscopy and Imaging
Photomicrographs were taken using a Zeiss Axioplan 2 photomicroscope (Carl Zeiss, Inc., New York, NY) fitted with a Hamamatsu chilled three-color CCD camera (Hamamatsu Corporation, Bridgewater, NJ). Digital images were assembled using Adobe PhotoShop (Adobe Systems, Seattle, WA). Constant image acquisition parameters were used to ensure image equality. Black-and-white prints were made with a Kodak DS8650 color printer.
Statistical Analyses
RIA and RT-PCR data were subjected to least squares regression analysis using the Statistical Analysis System [26]. The ß-actin values were used as a covariate in statistical analyses of RT-PCR data to correct for differences in amounts of RT cDNA analyzed for each uterus. Serum estradiol-17ß values were log-transformed prior to homogeneity of regression analysis. All data are presented as untransformed least squares means (LSM) with standard errors (SEM).
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Histomorphogenesis of the ovine uterine wall between birth and PND 56 was dramatic (Fig. 1). Endometrium from PNDs 0 and 1 was devoid of uterine glands and consisted of a simple columnar epithelium supported by stroma. In presumptive intercaruncular endometrium between caruncular nodules, the LE was slightly ruffled and pseudostratified columnar. On PND 7, endometrial LE in all areas appeared to be pseudostratified columnar and the endometrium contained distinct, shallow tubular glands that invaginated slightly into the stroma. By PND 14, many simple, slightly coiled tubular glands were apparent and penetrated approximately a third to half of the distance from the uterine lumen to the inner circular layer of myometrium. Between PNDs 14 and 28, the intercaruncular endometrial areas expanded and there was extensive glandular development and growth. On PND 21, some endometrial glands extended to the myometrium. Although the glands in the upper stroma were tubular, the glands in the lower half of the stroma appeared branched and coiled. The intercaruncular LE was again simple columnar, whereas the caruncular LE was cuboidal. On PND 28, complex, coiled tubular glands were present throughout the intercaruncular endometrial stroma, and most of the glands extended to the adluminal border of the myometrium. On PNDs 42 and 56, complex, coiled, and branched tubular glands were present throughout the stroma. Between PNDs 28 and 56, growth of the endometrial GE appeared to be primarily in the lower coiled and branched glands. By PND 56, the uterine wall appeared to be essentially mature, with the intercaruncular endometrial area exhibiting two distinct stromal areas that were invested with numerous coiled and branched glands that extended radially from the LE through the stroma to the inner circular layer of myometrium.
|
Intercaruncular endometrial folds were apparent on PND 14, increased by PND 28, and were well-developed features of the uterine wall by PND 56. The LE in caruncular areas was simple columnar. The caruncular areas of the uterus and, to a lesser extent, the myometrium also appeared to have grown between birth and PND 56. Intensely vascularized caruncular areas were observed in PND 56 uteri. Regardless of neonatal age, many immune cells were distributed throughout the uterine wall and were frequently interspersed among the LE and GE.
PCNA Protein Expression
Immunoreactive PCNA expression was used as a marker for cell proliferation. PCNA is a highly conserved accessory protein of DNA polymerase 
, which is synthesized during the late G1 and S phases of the cell cycle, is essential for DNA synthesis, and is correlated with cellular proliferation [27]. As expected, PCNA protein was detected in the nuclei of most cells in the developing neonatal ovine uterus (Fig. 2). The complex temporal and spatial alterations in immunoreactive PCNA staining intensity of individual uterine tissues and cell types is summarized in Table 1.
|
|
At birth (PND 0) and on PND 1, PCNA expression was strong in LE and moderate in stroma and myometrium. On PND 7, LE and nascent GE expressed high levels of PCNA protein, whereas PCNA expression in stroma and myometrium was weak or barely detectable. Between PNDs 14 and 56, the neck regions of the glands in the upper stroma expressed low to moderate levels of PCNA protein, whereas the tip regions of emerging and developing glands contained abundant nuclear PCNA protein that was expressed in a more uniform pattern. In addition, stromal cells in the immediate vicinity of the tips of developing glands expressed higher levels of nuclear PCNA protein compared with stromal cells beneath the LE surrounding the necks of developing glands or in the caruncules. In contrast to epithelia and stroma, PCNA expression in the myometrium after PND 1 was weak or not detectable. It is interesting that PCNA expression was abundant in immune cells in uteri from all days studied (results not shown).
Radioimmunoassay of Estradiol-17ß and Progesterone
As illustrated in Figure 3, serum estradiol-17ß concentrations were affected by postnatal day of age (P < 0.03, cubic). Serum estradiol-17ß concentrations were high on PND 1 (71 ± 17 pg/ml), increased between PND 14 (72 pg/ml) and PND 28 (200 pg/ml), and then declined between PND 42 (174 pg/ml) and PND 56 (91 pg/ml). In contrast, progesterone was not detectable by RIA in any serum samples from PND 1 to 56 (results not shown).
|
ER- mRNA and Protein Expression
Temporal and spatial alterations in ER- expression of individual uterine tissues and cell types are summarized in Table 2. In situ hybridization and immunohistochemical analysis revealed parallel patterns of ER- mRNA and protein expression in developing uterine tissues (Fig. 4) and, therefore, both will be discussed together.
|
|
Intercaruncular endometrium
At birth, ER- gene expression was low or not detectable in LE and low in stroma. On PND 1, immunoreactive ER- protein was uniformly detected at low to moderate levels in nuclei of all uterine wall tissues. On PND 7, ER- was strikingly abundant in newly differentiated GE. Although ER- expression in LE was variegated, uniform and intense staining was observed in nuclei at the invaginating tips of emerging glands. On PND 14, ER- expression was uniformly strong in the developing slightly coiled, tubular glands. Between PNDs 21 and 56, abundant ER- expression was detected in the middle and tips of the coiled, branched, tubular glands, whereas expression was strikingly lower in the necks of developed glands. In contrast to developing GE, ER- expression in LE was moderate to weak on PNDs 0 to 42, and weak or undetectable on PND 56.
Stromal cell nuclear staining for ER- was weak or moderate on PNDs 0 to 14 and moderate to strong on PNDs 21 to 56. On PNDs 28 to 56, ER- expression in stromal cells underneath developing glands and near myometrium was higher than in those near LE.
Caruncular endometrium
At birth, ER- gene expression was weak or absent in LE, but moderate in stroma. On PND 1 and thereafter, ER- in LE was expressed at low to moderate levels. Stromal cells expressed low levels of ER- protein from birth to PND 21. Thereafter, ER- was expressed at moderate levels.
Myometrium and vasculature
Myometrial ER- expression was low at birth and weak or undetectable thereafter. Nuclear ER- expression was constitutively detected in endothelial cells that lined blood vessels. No nuclear staining was observed when irrelevant rat IgG was substituted for the H222 primary antibody or when primary antibody was omitted.
Progesterone mRNA and Protein Expression
In situ hybridization and immunohistochemical analysis revealed differential patterns of PR mRNA and protein expression in developing uterine tissues (Fig. 5). Although abundant PR mRNA was detected in stroma, immunoreactive PR protein was relatively weak. In contrast, low levels of PR mRNA were detected in endometrial epithelia, but there were moderate to abundant levels of immunoreactive PR protein in the same epithelia. These differences in expression of PR mRNA and protein were observed consistently in all uteri from ewes regardless of age at hysterectomy. Temporal and spatial alterations in immunoreactive PR protein expression of individual uterine tissues and cell types are summarized in Table 3.
|
|
Intercaruncular endometrium From PNDs 0 to 7, immunoreactive PR expression was detected in LE and appeared variegated from strong to weak levels in nuclei. In striking contrast, there was strong nuclear PR staining in all LE cells on PND 14. On PND 7, PR expression in emerging GE was variegated and strong at the tips of invaginating glands. On PND 14 and thereafter, PR expression was strong in LE and in all emerging and developed glands. Stromal cell nuclear staining for PR was moderate to weak at birth and weak thereafter.
Caruncular endometrium At birth, PR protein was detected in caruncular LE and was variegated. In contrast, nuclear PR staining was detected at high levels in LE on PND 7 and all days thereafter. Stromal cell nuclear staining for PR was moderate to abundant at birth and weak to moderate thereafter.
Myometrium and vasculature In myometrium, PR staining was abundant at birth, declined to moderate levels on PND 1, and was weak or undetectable thereafter. PR gene expression was not detected in blood vessels. No nuclear staining was observed when irrelevant mouse IgG was substituted for the MA1-411 primary antibody or when primary antibody was omitted.
Radioimmunoassay of Prolactin
As illustrated in Figure 6, serum PRL concentrations were affected by postnatal day of age (P < 0.01, cubic). Serum PRL concentrations increased between PND 1 (98 ± 33 ng/ml) and PND 14 (500 ng/ml) and then declined to PND 56 (147 ng/ml).
|
In Situ Hybridization Analysis of Prolactin Receptor mRNA in Ovine Uterus
As shown in Figure 7, expression of PRL-R mRNA was not detected in uteri from ewes on PNDs 0 and 1. No specific hybridization signal was detected in endometrial LE, stroma, immune cells, blood vessels, or myometrium as judged from comparison to control serial-sectioned slides that were hybridized with sense PRL-R cRNA probe. In contrast to PNDs 0 and 1, specific PRL-R mRNA expression was detected at low levels in newly differentiated endometrial GE buds on PND 7. Between PNDs 7 and 56, PRL-R mRNA expression was detected only in nascent and developing endometrial glands. It is interesting that PRL-R mRNA expression was much higher in coiled and branched glands from uteri of ewes on PNDs 28, 42, and 56. In the PND 14, PND 42, and sense darkfield photmicrographs, the intensely white cells are melanocytes and do not express PRL-R.
|
RT-PCR Analysis of Prolactin Receptor mRNA Expression
As illustrated in Figure 8A, primers were designed to amplify regions of mRNAs that encode the short and long forms of ovine PRL-R based on the alternative splicing pattern of PRL-R mRNAs produced from a single gene [25]. A single forward primer was designed within exon 9, which is contained in both splice variants. One reverse primer was designed within the last 39 bp of the short PRL-R mRNA and another within exon 10 of the long PRL-R mRNA. Use of these specific primers with the exon 9 common primer was predicted to yield different size cDNA products for the long (310 bp) and short (207 bp) PRL-R mRNA alternative splice variants. Primers were also designed to amplify a region of ovine ß-actin mRNA (420 bp) as a control for cDNA concentration. Preliminary experiments were conducted to ensure that the amount of PCR product derived from a given amount of cDNA amplified by PCR was directly proportional to the concentration of target RT cDNA in the sample (results not shown).
|
Consistent with the histological absence of endometrial glands on PNDs 0 and 1 and in situ hybridization results, PRL-R mRNA was not detected in total uterine RNA in ewes on PND 1 by RT-PCR, but PRL-R mRNA expression was first detected in total uterine RNA from PND 7 (Fig. 8B). As expected, both long (310 bp) and short (207 bp) forms of ovine PRL-R mRNA were present in neonatal uteri from PND 7 to PND 56. Two specific PCR products (310 bp and 350 bp) were consistently detected using the long PRL-R mRNA primers. The expected 310-bp product was identical to the long ovine PRL-R cDNA, whereas sequence analysis of the 350-bp cDNA product derived from the long PRL-R primers revealed that the cDNA included the 39-bp exon of the short PRL-R mRNA [17]. Sequence analyses of the PRL-R cDNAs confirmed their identity (results not shown). The ratio of the 350 bp to 310 bp long PRL-R cDNA products did not change with day of age (P > 0.1). Furthermore, the relative ratio of short to long PRL-R mRNAs revealed no effect of day (P > 0.1). As illustrated in Figure 8C, regression analysis indicated that relative levels of short and long PRL-R mRNA expression in total uterine RNA were affected by neonatal age and increased (P < 0.01, quadratic) between PNDs 7 and 14, and then remained relatively high on PND 56.
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Overall patterns of PCNA staining were similar to patterns of [3H]thymidine labeling reported for neonatal ovine endometrium at birth and on PNDs 13 and 26 [14]. Most uterine cell types contained immunoreactive PCNA protein. In contrast, PCNA expression in myometrium was either low or absent during this period, which is consistent with evidence that myometrial morphogenesis and development occurs primarily during fetal life and is essentially complete at birth [9]. High levels of PCNA expression in LE at birth foreshadowed differentiation and emergence of endometrial gland buds between PNDs 1 and 7. On PNDs 0 to 7, high levels of nuclear PCNA protein were detected in LE and emerging GE. Whether or not GE bud invagination into the subepithelial stroma requires cell proliferation remains to be determined. Gray et al. [15] reported that progestin-induced ablation of endometrial gland genesis in neonatal ovine uterus did not involve direct suppression of epithelial cell proliferation. In developing lung epithelium, bud outgrowth is not accompanied by induction of localized cell proliferation [30], but appears to involve remodeling of the basement membrane extracellular matrix [31]. This concept is supported by histochemical studies of the epithelial basement membrane in neonatal ovine uterus [13] and porcine uterus [32].
Previous results from studies of neonatal ovine [15, 19], porcine [32], and rodent [33] uteri as well as regenerating postmenstrual primate uterus [34, 35] suggest that uterine gland morphogenesis is supported and regulated through interactions between epithelia and stroma. These interactions establish developmentally critical tissue microenvironments that are conducive to support and maintain spatially focused alterations in DNA synthesis and progressive development of the glandular intercaruncular endometrium [19, 20]. The focused area of PCNA expression by tips of the developing GE and surrounding stroma suggest that the local microenvironment favors proliferation of both cell types. The concept that GE-stromal interactions are required for overall endometrial morphogenesis and establishment of proper histoarchitecture is supported by elegant tissue recombination studies of mouse uterus [36, 37] and studies of uterine gland knockout (UGKO) ewe uterus [8, 15, 16]. The endometrium of UGKO ewes is much thinner and the stroma lacks delineation into upper stratum compactum and lower stratum spongiosum. Given that UGKO uteri lack endometrial glands, it is inferred that GE-stromal interactions are required for proper intercaruncular endometrial morphogenesis in the developing ovine uterus. These interactions may also affect overall uterine wall morphogenesis because the strategy of progestin-induced uterine gland ablation in the neonatal ewe results in an adult ewe, which possesses an aglandular uterus that weighs less and has shorter horn length than uteri of comparable normal adult ewes [38].
The initial site of endometrial GE budding and differentiation from the LE occurred in endometrial clefts between caruncular nodules between PNDs 1 and 7. The mechanisms that specify differentiation of LE into GE in the clefts between caruncular nodules remain unknown but are of particular interest. In several other mammalian species, including pig [32, 39], mouse [33, 40], and rat [41], uterine endometrial glands are also absent at birth but develop rapidly after birth. In ewes, removal from a progesterone-dominated environment, as a consequence of birth, seems to be the endocrine cue for uterine gland genesis, because endometrial adenogenesis is inhibited both acutely and permanently in sheep by exposure to Norgestomet, a potent synthetic 19-norprogestin, from birth [8, 14–16, 38]. In neonatal pigs and sheep, endometrial adenogenesis proceeds normally for a period of time following ovariectomy at birth [14, 39]. Although ovarian and uterine weights increase between birth and PND 28 [12] in ewes, progesterone was not detectable in peripheral circulation after birth in the present study and is below detectable limits until puberty [12, 42, 43]. Initiation of endometrial gland development in neonatal ovine uterus is ovary- and ovarian steroid-independent because ovariectomy at birth did not alter uterine histoarchitecture on PND 13 [14]. However, ovariectomy at birth did affect gross uterine growth after PND 28 in ewes [12] and after PND 60 in pigs [39]. In the present study, estradiol-17ß was detected at relatively high levels in ewe lambs on PND 1, increased between PNDs 14 and 28, and then declined between PNDs 42 and 56. The origin of estradiol-17ß may be from the ovary as well as the adrenal gland [12]. Large numbers of growing and vesicular follicles are present in ovary at birth and on PND 28, both of which decline substantially between PNDs 28 and 84 [12]. In that study, estrogens could not be detected after birth in urine of ewe lambs, but the assay using thin-layer chromatography was rather insensitive compared with the RIA used in the present study.
In the present study, neonatal ovine uterine gland morphogenesis was accompanied by abundant expression of ER- in emerging, proliferating, and developing GE. The initial increase in ER- expression between PNDs 0 and 1 is likely due to a decrease in circulating concentrations of PR in neonatal ewes because progestins suppress epithelial ER- gene expression [15]. Genesis of endometrial glands in porcine [39] and rodent [44–47] uteri also involves coordinated changes in epithelial phenotype, which is marked by ER- expression in nascent endometrial GE. In those species, the stroma is ER--positive at birth followed by ER--positive LE and then GE. Although estrogen is not detectable in serum during the early neonatal period in pigs and rodents, ER- are functional, inasmuch as exogenous administration of estrogens to neonatal gilts [48, 49] and rodents [50] induces precocious endometrial gland development and proliferation. In addition to direct ligand-dependent activation of epithelial ER-, the proliferative effects of inappropriate estrogen on epithelial proliferation appear to be mediated primarily by stromal ER- via production of paracrine stromal-derived growth factors, such as epidermal growth factor (EGF) and insulin-like growth factor I (IGF-I) [51]. In developing neonatal pig uterus, activated ER- is required for gland genesis because administration of the ER- antagonist, ICI182,780, inhibits or severely retards endometrial adenogenesis [49]. It is not known whether ER- antagonists will ablate endometrial adenogenesis in the neonatal ewe. Homozygous ER- null mice (ERKO) have hypoplastic uteri that contain all characteristic cell types in reduced proportions [52] and ERKO mouse uteri contain reduced numbers of uterine glands [53]. Collectively, studies with ERKO mice suggest that ER- is not essential for fetal organogenesis, but is essential for normal postnatal uterine growth and development. In neonatal rat uterus, gland genesis is initiated between PNDs 10 and 15 [50]. Endogenous serum estrogens increase beginning on PND 9 to PND 11 [54], and antiestrogens inhibit uterine gland genesis [55]. While critical experiments remain to be conducted, gland morphogenesis in neonatal ovine endometrium is most likely an ER--dependent phenomenon that requires ligand-dependent and, perhaps, ligand-independent activation, as in the neonatal pig [39, 49] and rat [55].
The high levels of PR expression in LE and nascent and proliferating GE may be the consequence of ER- activation. In addition to effects of serum estradiol-17ß, ligand-independent activation may occur within nascent and proliferating GE because the EGF-like growth factor, heregulin, appears to stimulate proliferation and PR gene expression in an ER--dependent manner in MCF-7 cells [56]. In the present study, PR mRNA was abundantly expressed by stromal cells, whereas immunoreactive PR protein was low in these cells. This finding agrees with previous observations on PR gene expression in ovine uterus on PND 28 [15]. The mechanism underlying the regulation of PR gene expression in the stroma is not known, but may involve post-transcriptional effects. A requirement for ER- in ovine uterine adenogenesis is supported by the finding that progestin-induced ablation of endometrial gland genesis in neonatal ewes is mediated by suppression of epithelial ER- expression [15]. Ablation of endometrial gland genesis in neonatal gilts treated with ICI 182,780 [49] and in neonatal ewes treated with Norgestomet from birth [15] may reflect the loss or attenuation of ER- signaling. Studies to determine the role of serum estrogens, specific growth factors, and ER- activation in neonatal ovine uterine morphogenesis are currently underway.
In addition to a role for ER-, PRL also appears to play a role in neonatal ovine endometrial adenogenesis. In other epitheliomesenchymal organs, PRL stimulates differentiation and development of the epithelium [57]. Using mammary glands from PRL-R null mice, Brisken et al. [29] demonstrated that PRL directly controls lobuloalveolar development, but not alveolar bud formation or ductal side branching. In agreement with a previous report [21], serum PRL concentrations increased between PNDs 1 and 14 and then declined to PND 56, which correlates with the onset and maintenance of GE proliferation in the developing uterine wall. The PRL-R mRNAs for the short and long PRL-R proteins were expressed in nascent and proliferating endometrial GE, and the relative levels of expression increased sevenfold between PNDs 7 and 56. It is interesting that the majority of PRL-R gene expression was in the branching and terminally differentiating GE between PNDs 21 and 56. In the adult endometrium of sheep, humans, and primates [17, 58, 59], the PRL-R gene is also expressed only in the endometrial glands and, in ewes, the increase during pregnancy is highly correlated with endometrial gland hyperplasia and hypertrophy [17]. Collectively, evidence from the present study and others supports the idea that PRL stimulates PRL-R in nascent and proliferating GE. In GE, PRL may increase ER- expression [60]. In addition, PRL actions via the short and long PRL-Rs stimulates the mitogen-activated protein kinase (MAPK) signaling cascade [61] and may result in serine phosphorylation and activation of ER- in a ligand-independent manner [62] to stimulate and maintain endometrial gland morphogenesis. The sustained activation of MAPK and ER- signaling pathways may stimulate and maintain endometrial gland morphogenesis in developing neonatal ovine uterus. Higher levels of PRL-R mRNA are localized in endometrial glands undergoing the most intense proliferation and differentiation as detected by PCNA staining. Although the PRL-R gene is preferentially expressed in the lower coiled and branched glands of neonatal and adult endometrium, it remains to be determined if PRL in neonatal ewes plays a role in terminal gland development or in bud formation and ductal branching. This hypothesis is supported by observations that hyperprolactinemia causes uterine glandular hypertrophy in adult mouse, rabbit, and pig [63–65], and that intrauterine administration of placental lactogen, a PRL-like hormone stimulates proliferation of endometrial glands and, in particular, the terminal ends of coiled and branched glands in the lower stroma of adult ewes [66]. Given the central role ascribed to PRL-R in mammary gland morphogenesis and function [29], PRL-R in nascent GE of developing uterus may play a similar, albeit undiscovered role. Other growth factors, such as EGF and IGF-I, may also act on endometrial glands via specific Type I tyrosine kinase receptors to stimulate MAPK and activate ER- in a ligand-independent manner. Studies are currently underway to determine the precise roles of prolactin, estrogen, PRL, ER-, and PR in endometrial adenogenesis and glandular morphogenesis in sheep.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
1 Supported in part by U.S. Department of Agriculture NRI competitive grant 98-35203-6322 and NIH grant P30 ES09106. 
2 Correspondence: Thomas E. Spencer, Center for Animal Biotechnology and Genomics, 442 Kleberg Center, 2471 TAMU, Texas A&M University, College Station, TX 77843-2471. FAX: 979 862 2662; tspencer{at}ansc.tamu.edu
Accepted: June 1, 2000.
Received: April 7, 2000.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
knockout mouse. Biol Reprod 1998; 59:470–475 and ß in the rat corpus luteum of pregnancy: regulation by prolactin and placental lactogens. Endocrinology 1998; 139:2432–2442This article has been cited by other articles:
|
|
 |
|
  K. Hayashi, A. R O'Connell, J. L Juengel, K. P McNatty, G. H Davis, F. W Bazer, and T. E Spencer Postnatal uterine development in Inverdale ewe lambs Reproduction, March 1, 2008; 135(3): 357 - 365. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. K Lewis, J. L Farmer, R. C Burghardt, G. R Newton, G. A Johnson, D. L Adelson, F. W Bazer, and T. E Spencer Galectin 15 (LGALS15): A Gene Uniquely Expressed in the Uteri of Sheep and Goats that Functions in Trophoblast Attachment Biol Reprod, December 1, 2007; 77(6): 1027 - 1036. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M G Gnanalingham, P Williams, V Wilson, J Bispham, M A Hyatt, A Pellicano, H Budge, T Stephenson, and M E Symonds Nutritional manipulation between early to mid-gestation: effects on uncoupling protein-2, glucocorticoid sensitivity, IGF-I receptor and cell proliferation but not apoptosis in the ovine placenta Reproduction, October 1, 2007; 134(4): 615 - 623. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. C. Satterfield, K. A. Dunlap, K. Hayashi, R. C. Burghardt, T. E. Spencer, and F. W. Bazer Tight and Adherens Junctions in the Ovine Uterus: Differential Regulation by Pregnancy and Progesterone Endocrinology, August 1, 2007; 148(8): 3922 - 3931. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Hayashi, R. C. Burghardt, F. W. Bazer, and T. E. Spencer WNTs in the Ovine Uterus: Potential Regulation of Periimplantation Ovine Conceptus Development Endocrinology, July 1, 2007; 148(7): 3496 - 3506. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. C. Satterfield, F. W. Bazer, and T. E. Spencer Progesterone Regulation of Preimplantation Conceptus Growth and Galectin 15 (LGALS15) in the Ovine Uterus Biol Reprod, August 1, 2006; 75(2): 289 - 296. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Hayashi and T. E. Spencer WNT Pathways in the Neonatal Ovine Uterus: Potential Specification of Endometrial Gland Morphogenesis by SFRP2 Biol Reprod, April 1, 2006; 74(4): 721 - 733. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. G. W. Fleming, T. E. Spencer, S. H. Safe, and F. W. Bazer Estrogen Regulates Transcription of the Ovine Oxytocin Receptor Gene through GC-Rich SP1 Promoter Elements Endocrinology, February 1, 2006; 147(2): 899 - 911. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Song, T. E. Spencer, and F. W. Bazer Cathepsins in the Ovine Uterus: Regulation by Pregnancy, Progesterone, and Interferon Tau Endocrinology, November 1, 2005; 146(11): 4825 - 4833. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Hayashi and T. E. Spencer Estrogen Disruption of Neonatal Ovine Uterine Development: Effects on Gene Expression Assessed by Suppression Subtraction Hybridization Biol Reprod, October 1, 2005; 73(4): 752 - 760. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Hu and T. E. Spencer Carbonic Anhydrase Regulate Endometrial Gland Development in the Neonatal Uterus Biol Reprod, July 1, 2005; 73(1): 131 - 138. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Yin and L. Ma Development of the Mammalian Female Reproductive Tract J. Biochem., June 1, 2005; 137(6): 677 - 683. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Hayashi, K. D Carpenter, T. H Welsh Jr, R. C Burghardt, L. J Spicer, and T. E Spencer The IGF system in the neonatal ovine uterus Reproduction, March 1, 2005; 129(3): 337 - 347. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S Pearce, H Budge, A Mostyn, E Genever, R Webb, P Ingleton, A M Walker, M E Symonds, and T Stephenson Prolactin, the prolactin receptor and uncoupling protein abundance and function in adipose tissue during development in young sheep J. Endocrinol., February 1, 2005; 184(2): 351 - 359. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Hayashi, K. D. Carpenter, and T. E. Spencer Neonatal Estrogen Exposure Disrupts Uterine Development in the Postnatal Sheep Endocrinology, July 1, 2004; 145(7): 3247 - 3257. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Hu, C. A. Gray, and T. E. Spencer Gene Expression Profiling of Neonatal Mouse Uterine Development Biol Reprod, June 1, 2004; 70(6): 1870 - 1876. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. E. Spencer and F. W. Bazer Uterine and placental factors regulating conceptus growth in domestic animals J Anim Sci, January 1, 2004; 82(13_suppl): E4 - 13. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Kwon, G. Wu, F. W. Bazer, and T. E. Spencer Developmental Changes in Polyamine Levels and Synthesis in the Ovine Conceptus Biol Reprod, November 1, 2003; 69(5): 1626 - 1634. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Hayashi, K. D. Carpenter, C. A. Gray, and T. E. Spencer The Activin-Follistatin System in the Neonatal Ovine Uterus Biol Reprod, September 1, 2003; 69(3): 843 - 850. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. D. Carpenter, K. Hayashi, and T. E. Spencer Ovarian Regulation of Endometrial Gland Morphogenesis and Activin-Follistatin System in the Neonatal Ovine Uterus Biol Reprod, September 1, 2003; 69(3): 851 - 860. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. D. Carpenter, C. A. Gray, T. M. Bryan, T. H. Welsh Jr., and T. E. Spencer Estrogen and Antiestrogen Effects on Neonatal Ovine Uterine Development Biol Reprod, August 1, 2003; 69(2): 708 - 717. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. D. Carpenter, C. A. Gray, S. Noel, A. Gertler, F. W. Bazer, and T. E. Spencer Prolactin Regulation of Neonatal Ovine Uterine Gland Morphogenesis Endocrinology, January 1, 2003; 144(1): 110 - 120. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Palmarini, C. A. Gray, K. Carpenter, H. Fan, F. W. Bazer, and T. E. Spencer Expression of Endogenous Betaretroviruses in the Ovine Uterus: Effects of Neonatal Age, Estrous Cycle, Pregnancy, and Progesterone J. Virol., December 1, 2001; 75(23): 11319 - 11327. [Abstract] [Full Text]
|
|
|
|
|
|
C. A. Gray, F. F. Bartol, B. J. Tarleton, A. A. Wiley, G. A. Johnson, F. W. Bazer, and T. E. Spencer Developmental Biology of Uterine Glands Biol Reprod, November 1, 2001; 65(5): 1311 - 1323. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Ka, L. A. Jaeger, G. A. Johnson, T. E. Spencer, and F. W. Bazer Keratinocyte Growth Factor Is Up-Regulated by Estrogen in the Porcine Uterine Endometrium and Functions in Trophectoderm Cell Proliferation and Differentiation Endocrinology, June 1, 2001; 142(6): 2303 - 2310. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. A. Gray, K. M. Taylor, W. S. Ramsey, J. R. Hill, F. W. Bazer, F. F. Bartol, and T. E. Spencer Endometrial Glands Are Required for Preimplantation Conceptus Elongation and Survival Biol Reprod, June 1, 2001; 64(6): 1608 - 1613. [Abstract] [Full Text]
|
|
|
|
|
|
K. M. Taylor, C. Chen, C. A. Gray, F. W. Bazer, and T. E. Spencer Expression of Messenger Ribonucleic Acids for Fibroblast Growth Factors 7 and 10, Hepatocyte Growth Factor, and Insulin-Like Growth Factors and Their Receptors in the Neonatal Ovine Uterus Biol Reprod, April 1, 2001; 64(4): 1236 - 1246. [Abstract] [Full Text]
|
|
|
|
|
|
C. A. Gray, F. W. Bazer, and T. E. Spencer Effects of Neonatal Progestin Exposure on Female Reproductive Tract Structure and Function in the Adult Ewe Biol Reprod, March 1, 2001; 64(3): 797 - 804. [Abstract] [Full Text]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
