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Center for Animal Biotechnology and Genomics (K.D.C., C.A.G., S.N., F.W.B., T.E.S.), Department of Animal Science, Texas A&M University, College Station, Texas 77843-2471; Institute of Biochemistry, Food Science and Nutrition (A.G.), The Hebrew University of Jerusalem, Rehovot 76100, Israel
Address all correspondence and requests for reprints to: Thomas E. Spencer, Center for Animal Biotechnology and Genomics, 442 Kleberg Center, 2471 TAMU, Texas A&M University, College Station, Texas 77843-2471. E-mail: tspencer{at}tamu.edu.
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Introduction |
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In the neonatal ewe, pituitary prolactin (PRL), estradiol-17ß, and stromal growth factors, including fibroblast growth factors-7 and -10, hepatocyte growth factor, and IGFs I and II, with epithelial receptors have been implicated as regulatory factors controlling endometrial adenogenesis (1, 2, 3). Circulating levels of PRL are relatively high on PND 1, reach a maximum on PND 14, and then decline slightly to PND 56 (2, 8). Expression of mRNAs for the short and long PRL receptor (PRLR) proteins is restricted to the nascent uterine GE buds on PND 7 and in proliferating and differentiating GE from PNDs 14–56 (1). In the adult ovine uterus, PRLR expression is also restricted to the endometrial glands and not detected in other uterine cell types (7). PRL, a member of the helix bundle peptide hormone/cytokine superfamily (9), regulates the growth and differentiation of a number of epitheliomesenchymal organs, including the pigeon crop sac, mammary gland, prostate, and uterus (10). In the mammary gland, PRL and PRLR are required for development and differentiation of the lobuloalveolar portion of the GE (11, 12, 13). Hyperprolactinemia elicits uterine glandular hyperplasia in the adult mouse, rabbit, and pig (14, 15, 16). The precise role of PRL in neonatal ovine uterine adenogenesis has not been elucidated.
Biological responses to PRL in other model systems is mediated by the PRLR and intracellular activation of several signal transduction systems, including signal transducers and activators of transcription (STAT) proteins 1, 3, and 5, interferon regulatory factor 1 (IRF-1), and the MAPK cascade (17, 18, 19). High levels of phosporylated ERK 1 and 2 MAPKs are also detected in nascent and proliferating endometrial glands of the neonatal ovine uterus (2). Available evidence in the neonatal ewe supports the working hypothesis that PRL activates PRLR signaling pathways in the nascent and proliferating endometrial GE to stimulate and maintain their coiling and branching morphogenesis in the neonatal ovine uterus. To test this hypothesis, studies were conducted in the neonatal ewe to determine effects of: 1) hypoprolactinemia on uterine adenogenesis; 2) hyperprolactinemia on uterine adenogenesis; 3) postnatal age on expression of STATs 1, 3, and 5 and IRF-1; and 4) PRL on activation of STAT and MAPK signal transduction pathways.
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Animals
All experiments and surgical procedures were 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.
Preparation of recombinant ovine PRL (roPRL)
roPRL (GenBank accession no. M27057) was prepared in Escherichia coli cells as described by Leibovich et al. (20). The expressed protein, found in inclusion bodies, was refolded and purified to homogeneity on a Q-Sepharose column, yielding an electrophoretically pure fraction composed of over 98% monomeric protein of the expected molecular mass of approximately 23 kDa. The biological activity of the recombinant oPRL after proper renaturation was evidenced in vitro by its ability to stimulate proliferation of rat lymphoma Nb2 cells possessing PRLR, stimulate biological activity in human embryonic kidney 293 cells transiently transfected with ovine PRLR, and induce progesterone secretion in primary cultures of luteal cells obtained from midpregnant ewes (20).
Experimental design
Cross-bred Suffolk ewes were mated to Suffolk rams in the fall between the months of September and November. Pregnant ewes were maintained according to normal husbandry practices and fed hay and corn. Ewes used in the following experiments were born in the spring between the months of February and May.
Study one.
Biodegradable placebo and bromocryptine mesylate (BROMO) (100 mg) 21-d release pellets were obtained from Innovative Research of America (Sarasota, FL). Ten ewe lambs (n = 5 per treatment) were assigned randomly at birth (postnatal day or PND 0) to be implanted with a placebo pellet as a control (CX) or 100 mg BROMO pellet that releases 100 mg over a 21-d period (
4.8 mg/d). Biodegradable pellets were placed sc in the periscapular region every 20 d from birth. Blood samples were collected every 7 d beginning at birth by jugular venipuncture. On PND 56, all ewes were hemi-ovariohysterectomized. For removal of the right uterine horn and ovary, a hemostat was clamped perpendicular across the uterine horn at bifurcation of the uterine horns. A scalpel blade was used to remove the right uterine horn, oviduct and ovary. Electrocautery was used to seal the opening of the remaining portion of the uterine horn. The uterine horn piece was then trimmed free of the broad ligament, oviduct, and cervix. Sections (1 cm) from the mid-region of the uterine horn were fixed in fresh 4% paraformaldehyde in PBS (pH 7.2) for 24 h at room temperature and processed for histology.
Study two.
Cross-bred Suffolk ewes (n = 5 per treatment) were randomly assigned on PND 0 to receive twice daily injections (0700 h and 1800 h) of sterile saline vehicle as CX or roPRL (1 mg/kg body weight) from PND 1–56 to determine effects on uterine gland development. Body weight of the ewes was measured every 4 d and used to adjust treatments. Blood samples were collected every 8 d beginning on PND 1 by jugular venipuncture. On PND 14, all ewes were subjected to mid-ventral laparotomy. The right ovarian pedicle was ligated, and the ovary and oviduct removed. One-half of the ipsilateral anterior uterine horn was then removed and fixed in 4% paraformaldehyde for histology. On PND 56, all ewes were weighed and necropsied. The left ovary was trimmed free of the mesovarium and weighed. The uterus was obtained and trimmed free of the broad ligament, oviduct, and cervix. The entire left uterine horn was dissected free of the partial right uterine horn and weighed. Sections from the mid-region of the uterine horn were fixed in paraformaldehyde for histology.
Study three.
Ewes were assigned randomly on PND 0 to be necropsied (n = 5 ewes/d) on PNDs 1, 7, 14, 28, 42, or 56 to examine temporal and spatial alterations in expression of STATs 1, 3, and 5. At necropsy, the entire reproductive tract was excised, and the uterus trimmed free of the broad ligament, oviduct and cervix. Sections from the mid-region of each uterine horn were fixed in paraformaldehyde for histology.
Study four.
Ewes (n = 5) were hysterectomized on PND 28 for uterine explant cultures. The uterus was trimmed free of the broad ligament, oviduct and cervix and placed in DMEM with F-12 salts (DMEM-F12; Sigma-Aldrich) supplemented with antibiotic-antimycotic (Life Technologies, Inc., Gaithersburg, MD). The uterus from each ewe (n = 5) was weighed (3 g). In a laminar flow hood, uterine horns were separated and opened along the mesometrial side with a pair of fine surgical scissors. Uteri were then cut into small pieces (4–5 mm3), and explants placed in a 150-mm culture dish (Becton Dickinson Labware, Franklin Lakes, NJ) containing 50 ml of culture medium (serum-free DMEM/F-12 with antibiotic-antimycotic). Uterine explants were cultured in a rocking Bellco incubator (Bellco Glass Inc., Vineland, NJ) at 37 C in an atmosphere of 5% CO2/95% O2 for 3–4 h. The culture medium was then replaced with fresh medium. Uterine tissue (300 mg) was then placed in a 60 x 15-mm culture dish (Becton Dickinson Labware) containing 3 ml of culture media with 500 ng/ml of purified ovine pituitary PRL (NIDDK-oPRL-21). Explants were placed in a tissue culture incubator and cultured at 37 C in an atmosphere of 5% CO2/95% O2 for 0, 15, 30, 60, or 120 min.
At the designated time, uterine tissue was removed from the culture dishes, blotted on sterile gauze, and placed in 2 ml of freshly prepared, ice-cold lysis buffer (60 mM Tris, pH 6.8), 1 mM sodium orthovanadate, 10% glycerol, 2% sodium dodecyl sulfate, aprotinin (44 µg/ml) and PMSF (100 µg/ml). Tissue was homogenized using a PRO250 homogenizer (Pro Scientific Inc., Monroe, CT) and then ground using a 2 ml Dounce tissue grinder (Kontec Glassware Co., Vineland, NJ) with 30 strokes of the B pestle. Tissue homogenate was then clarified by centrifugation for 5 min at 20,000 x g at 4 C. The supernatant was aliquoted and frozen at –80 C for Western blot analysis.
Histology and morphometry
After 24 h of fixation in 4% paraformaldehyde, uterine tissues were changed to 70% ethanol for 24 h and then dehydrated and embedded in Paraplast Plus (Oxford Labware, St. Louis, MO). Uteri were sectioned (4–6 µm) and stained with hematoxylin and eosin as described previously (3). Sections (n = 4) of the uterus from each ewe were photomicrographed, and images were analyzed using Scion Image software (Scion Corp., Frederick, MD). Measurements were standardized using the image of a stage micrometer at the same magnification. In the intercaruncular endometrium, the thickness or width of the endometrium and myometrium (inner circular and outer longitudinal layers) was measured using the Scion Image software from multiple points (n = 3 to 4) of each uterine section. The number of superficial ductal invaginations of the GE into the stroma was counted in each section. Endometrial gland number was determined by counting the total number of uterine glands in a complete cross-section of the uterine horn using methods similar to those described previously (21). Gland number estimates were generated for at least 10 nonsequential sections from each uterine horn. The observation of a gland cross-section with a visible open lumen was counted as a gland. Intra- and inter-section repeatability estimates for determination of gland number by a single observer were 0.85 and 0.8, respectively. Data are presented as total gland number per uterine horn cross-section. Gland density was determined in the stratum compactum and stratum spongiosum of the intercaruncular endometrium. The number of glands in a 200-µm2 area was counted in four areas of four sections for each uterine horn. Data are presented as total gland number per 200-µm2 area.
Immunohistochemistry
Expression of immunoreactive STAT 1, STAT 3, STAT 5, and IRF-1 proteins was detected in uterine tissue cross-sections (5–7 µm) using the appropriate mouse STAT antibodies and rabbit IRF-1 antibody and a Super ABC Mouse/Rat IgG Kit (Biomeda, Foster City, CA) according to methods described previously (22). The mouse antibodies detect both unphosphorylated and phosphorylated forms of the STAT proteins in ovine endometrial cells as described previously (23). The final working antibody dilutions were 1:1000 for STAT antibodies and 1–20 µg/ml for IRF-1. Antigen retrieval using boiling citrate buffer was performed as described previously (1, 2, 3). The chromagen used for peroxidase localization was 3,3'-diaminobenzidine tetrahydrochloride from Sigma-Aldrich. Negative controls were performed in which the primary antibody was substituted with the same concentration of purified normal mouse IgG or normal rabbit IgG from Sigma-Aldrich. Multiple tissue sections from each ewe were processed as sets within an experiment.
Photomicroscopy
Representative photomicrographs were taken using a Nikon Eclipse 1000 photomicroscope (Nikon Instruments Inc., Lewisville, TX) fitted with a Nikon DXM1200 digital camera.
RIA
Blood samples were allowed to clot for 1 h at room temperature. Serum was then collected by centrifugation (3000 x g for 30 min at 4 C), removed and stored at -20 C for hormone analyses. Concentrations of PRL in serum were determined using reagents for the oPRL RIA provided by Dr. A. F. Parlow and the NIDDK National Hormone and Pituitary Program as described previously (1). Purified oPRL (NIDDK-oPRL-I-3) was iodinated using the chloramine-T reaction, and the assay conducted using methods and reagents provided by the NIDDK Pituitary Hormones and Antisera Center. Assay sensitivity was 0.1 ng/ml, and the intraassay and interassay coefficients of variation were 5% and 12%, respectively.
Concentrations of estradiol-17ß in the serum were determined by RIA as described previously (1). Assay sensitivity was 1 pg per tube, and the intraassay and interassay coefficients of variation were 8% and 14%, respectively. Assay results were calculated using the AssayZap Version 3.1 program (Biosoft, Ferguson, CA).
Western blot analyses
The protein concentration of the supernatant was determined by Bradford assay (Bio-Rad Laboratories, Inc., Hercules, CA) using BSA as the standard. Proteins (40 µg) from each uterine explant were separated by SDS-PAGE and transferred to nitrocellulose as described previously (23, 24). Blots were blocked for 1 h at room temperature with either 5% BSA-TBST (5% wt/vol BSA, Tris-buffered saline, 0.1% Tween-20) for phospho-specific antibodies or 5%-nonfat milk-TBST for all other antibodies. Primary antibodies were diluted according to manufacturers recommendations in either 5% BSA-TBST for phospho-specific antibodies or 2% milk-TBST for all other antibodies. Blots were incubated with primary antibody overnight at 4 C, rinsed for 30 min at room temperature with TBST, incubated with the appropriate peroxidase-conjugated secondary antibody for 1 h at room temperature, and then rinsed again for 30 min at room temperature with TBST. Immunoreactive proteins were detected using enhanced chemiluminescence (SuperSignal West Pico Luminol System, Pierce Chemical Co., Rockford, IL) according to the manufacturer’s recommendations using X-OMAT AR film (Kodak, Rochester, NY). For loading control, Western blots probed with phospho-specific antibodies were reprobed with antibodies that detected both phosphorylated and unphosphorylated protein. Western blots were quantified by scanning densitometry using a Bio-Rad Laboratories, Inc. GS-690 Imaging Densitometer and MultiAnalyst software (Bio-Rad Laboratories, Inc.) as described previously (24).
Statistical analyses
All quantitative data were subjected to least-squares ANOVA using General Linear Models procedures of the Statistical Analysis System (25). For serum PRL and estradiol-17ß measurements, statistical models included the main effects of treatment, PND, and their interaction. Statistical models for analysis of morphometry data in study one included main effects of treatment, ewe within treatment, tissue section, microscopic field within tissue section, and the appropriate interactions. Models for study two included main effects of treatment, ewe within treatment, PND, tissue section, microscopic field within tissue section, and the appropriate interactions. Initial analyses indicated that uterine wall location, tissue section, and microscopic field within section were not significant sources of variation. In some cases, data were log transformed to alleviate heterogeneity of error variance. Data are presented as least-square means (LSM) with overall SEs (SE).
Analyses of integrated optical density (Westerns) measurements included time (min post PRL treatment) and replicate as sources of variation. The initial measurement of band optical density at time 0 was used as a covariate. The LSM and SE illustrated in graphs were derived from this analysis. If a significant effect of PRL treatment was detected (P < 0.10), the data for each individual protein were then analyzed by least squares regression analysis. In these analyses, time was considered a continuous, independent source of variation with replicate as a dependent source. The initial measurement of band optical density at time 0 was used as a covariate in regression analyses. In all analyses, tests of significance were based on expectations of the error mean squares and error terms used in tests of significance were identified according to the expectation of the mean squares for error.
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Results |
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). In CX ewes, serum levels of PRL were high on PND 1, increased to a maximum on PND 14, and decreased thereafter (P < 0.10, cubic). In BROMO ewes, serum PRL levels were lower on PND 1 and increased 2-fold (P < 0.10, linear) to PND 56, but always remained markedly lower (4.5-fold) than CX ewes. Treatment with BROMO did not affect (P > 0.10) serum levels of estradiol-17ß compared with CX ewes (data not shown).
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). In contrast, the endometrium of BROMO ewes lacked the large numbers of characteristically coiled and branched glands in lower stroma of CX uteri. As summarized in Table 1, the number of primary ductal invaginations of the GE into the stratum compactum stroma was lower (P < 0.06) in the endometrium of BROMO-treated compared with CX ewes. Endometrial gland number was lower (P < 0.001) in the endometrium of BROMO-treated compared with CX ewes. Endometrial gland density was lower (P < 0.001) in the stratum spongiosum endometrium, but not in the stratum compactum, of BROMO-treated ewes. Endometrial and myometrial thickness was not affected (P > 0.10) by treatment.
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). In CX ewes, serum levels of PRL were high on PND 1, reached a maximum on PND 17, and decreased thereafter (cubic effect of day, P < 0.05). Overall, treatment of neonatal ewes with roPRL increased (P < 0.01) circulating levels of PRL. In roPRL ewes, serum levels of PRL were higher than CX ewes on PND 1 (P < 0.01, day x treatment) and increased between PNDs 1 and 56 (quadratic effect of day, P < 0.10). Treatment with roPRL did not affect (P > 0.10) serum levels of estradiol-17ß compared with CX ewes (data not shown). As summarized in Table 2, weight of the ovary and uterine horn was not affected (P > 0.10) by treatment on PND 56.
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). On PND 14, the endometrium of CX ewes contained nascent glands that were mostly tubular. On PND 56, the endometrium of CX ewes contained large numbers of coiled and branched endometrial glands, particularly in the stratum spongiosum endometrium. As summarized in Table 2
, the number of primary superficial ductal invaginations of the GE into the stratum compactum increased (P < 0.01) between PND 14 and PND 56 in CX ewes, but was not affected (P > 0.10) by treatment with roPRL regardless of PND. In contrast, endometrial gland number and density was affected (P < 0.01) by PND, treatment and their interaction. In CX ewes, endometrial gland numbers increased (P < 0.001) almost 13-fold from PND 14–56. Administration of roPRL tended to increase (P = 0.09) endometrial gland number on PND 14 and increased (P < 0.01) it by 63% on PND 56. Similarly, endometrial gland density was increased (P < 0.05) by roPRL on PND 14. On PND 56, endometrial gland density was greater (P < 0.05) in the stratum spongiosum endometrium, but not stratum compactum, in roPRL-treated compared with CX ewes. Endometrial and myometrial thickness were affected (P < 0.10) by PND, treatment, and their interaction. The thickness of the endometrium and myometrium were not different (P > 0.10) on PND 14. However, treatment with roPRL increased (P < 0.10) the thickness or width of the endometrium and myometrium on PND 56.
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). STAT 1 protein was detected in all cell types on PND 1, but was more abundant in the LE. On PND 14 and thereafter, STAT 1 protein was detected in the developing GE. STAT 3 protein was detected in the LE and stroma. Expression of STAT 3 protein was particularly abundant in the LE and nascent and developing GE. In the stroma, STAT 3 protein expression was most abundant in immune cells compared with stromal cells. STAT 5 protein was detected in all uterine cell types on PND 1 but was most abundant in the stroma and LE. STAT 5 protein was detected in the nascent and developing GE throughout development. Negligible levels of background were detected in negative controls wherein the primary antibodies were replaced with an equal amount of nonspecific mouse IgG.
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PRL stimulates phosphorylation of STATs 1 and 5, ERK 1 and 2, and JNK/SAPK (study four)
To investigate PRL signaling pathways in the neonatal ovine uterus, uteri from PND 28 ewes were explanted in serum-free medium and stimulated with 500 ng/ml of purified native oPRL. The explants were harvested at specific times, and effects of PRL on STAT, ERK 1 and 2 MAPK, and JNK/SAPK signaling pathways determined by Western blot analysis of proteins isolated from whole uterine explants (Fig. 6). Levels of tyrosine phosphorylated STAT 1 were also increased by PRL (P < 0.05, quadratic) after 30 min post treatment (Fig. 6, A and C). Treatment of uterine explants with PRL elicited a transient increase (P < 0.01, cubic) in levels of tyrosine phosphorylated STAT 5 within 15 min post treatment (Fig. 6, A and D) but had no effect (P > 0.10) on STAT 3 (Fig. 6A). The phospho-specific antibody used for this study detects both STAT 5a and STAT 5b. Within 15 min, PRL increased (P < 0.01, cubic) levels of threonine and tyrosine phosphorylated ERK 1 and 2 MAPKs (Fig. 6, B and E). Similarly, PRL also stimulated an increase (P < 0.10, cubic) in levels of threonine and tyrosine phosphorylated JNK/SAPK by 15 min post treatment (Fig. 6, B and F).
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Discussion |
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4.5-fold) circulating levels of PRL to an average of 36 ng/ml. These findings are consistent with the ability of bromocryptine to markedly inhibit, but not completely ablate, PRL release from the pituitary in sheep (11). Unfortunately, an effective antagonist of the PRLR is not available (28). Hypoprolactinemia decreased endometrial gland number and density in the PND 56 uterus. Histologically, the uteri of bromocryptine-treated ewes contained lower numbers of superficial ductal gland invaginations as well as coiled and branched endometrial glands in the endometrial stratum spongiosum near the myometrium. In the neonatal ewe, PRLR mRNA is most abundant in the proliferating, branching and differentiating GE undergoing proliferation between PNDs 21 and 56 (1, 2). These findings are interpreted to support the hypothesis that PRL is a regulatory factor controlling endometrial gland coiling and branching morphogenesis in the developing neonatal ovine uterus. However, the inability of bromocryptine treatment to completely inhibit PRL secretion may preclude determination of the precise role of PRL in the initial stages of endometrial adenogenesis. Hyperprolactinemia causes uterine glandular hyperplasia in the adult mouse, rabbit, and pig (14, 15, 16). Moreover, intrauterine administration of ovine placental lactogen, a PRL-like hormone that homodimerizes and signals through the PRLR (29), stimulates proliferation of endometrial glands in the adult ewe and, in particular, the coiled and branched glands in the stratum spongiosum of adult ewes (21). In study two, treatment of ewes with roPRL from birth to PND 14 only slightly elevated circulating PRL levels, but increased endometrial gland number and density on PND 14. Indeed, the endometrium of roPRL-treated ewes on PND 56 contained more coiled and branched endometrial glands in the stratum spongiosum, but not stratum compactum. The budding, nascent, and proliferating endometrial glands express mRNAs for the long and short PRLR forms on PNDs 7 and 14 (1). Although all budding glands express PRLR on PNDs 7 and 14, only the GE in the stratum spongiosum express PRLR on PNDs 28–56 (1). These temporal and spatial changes in PRLR mRNA expression are likely to be responsible for the differential effects of hyperprolactinemia on endometrial adenogenesis on PND 14 compared with PND 56. After the budded glands elongate to a more tubular form and begin coiling and branching morphogenesis by PND 21 (1), the ductal GE loses PRLR expression, thereby preventing it from responding to PRL. Indeed, the PRLR is expressed only in the endometrial glands of the stratum compactum in the adult ewe (7). In mice with targeted disruption of the PRL gene, the mammary glands display normal ductal tree formation, but fail to develop lobuloalveolar structure (12). Similarly, mammary gland transplants from PRLR null mice into normal mice showed normal side branching and the formation of alveolar buds, but no lobuloalveolar development (13, 30). These findings are different from the present studies and suggest that mechanisms whereby PRL regulates gland development is organ specific.
In study two, treatment of neonatal ewes with roPRL increased thickness of the endometrium and myometrium on PND 56. These effects of PRL are likely to be the result of increased numbers of endometrial glands. Development of the neonatal uterus involves reciprocal epithelial-mesenchymal interactions (see Ref. 4 for review). In the developing rodent uterus, the endometrial epithelium affects myometrial organization and growth (31). Similarly, progestin treatment of neonatal ewes epigenetically ablates endometrial adenogenesis and reduces endometrial and myometrial thickness (32). In study two, the thickness of the endometrium and myometrium increased between PND 14 and 56 in CX ewes, which correlates to coiling and branching morphogenetic development of the endometrial glands into the stratum spongiosum adjacent to the myometrium. These results support the idea that the endometrial glands regulate endometrial and myometrial growth during uterine development. Although estrogen can affect uterine growth and size in a number of species (4), the effects of bromocryptine and roPRL in studies one and two did not involve alterations in the circulating levels of estradiol-17ß. Collectively, results from studies one and two strongly support the hypothesis that PRL acts directly on the endometrial GE that express the PRLR and regulates coiling and branching morphogenesis during postnatal development of the ovine uterus.
Biological responses to PRL in other model systems are mediated by the PRLR and intracellular activation of several signal transduction systems, including STATs 1, 3, and 5 proteins, IRF-1, and the MAPK cascade (17, 18, 19). PRL binding to the PRLR activates Janus kinase 2 (JAK2) and STATs 1, 3, and 5 (33, 34). Although each of these STAT proteins have been ablated in mouse models, their roles in endometrial gland morphogenesis may be difficult to fully ascertain, because the mouse uterus lacks the extensive coiled and branched endometrial gland architecture characteristic of uteri from domestic animals and primates (4). Results from study three indicate that STATs 1, 3, and 5 are present in the developing ovine uterus and, in particular, are expressed in the nascent and developing endometrial glands. In study four, PRL was observed to increase phosphorylation of STATs 5 and 1, but not STAT 3 in uterine explants from a PND 28 ewe. Interestingly, the effect of PRL on phospho-STAT 1 levels was more protracted and not observed until 60 min. Activation of the JAK2/STAT 5 cascade by PRL probably represents the hallmark of PRL signaling (35). Functional development of the mammary gland epithelium during pregnancy depends on PRL signaling, and STAT 5a is essential for mammary gland alveolar proliferation and function (36, 37, 38). Therefore, PRL signaling via the PRLR and STAT 5 may be critical for endometrial adenogenesis in the uterus during the neonatal period. However, the precise roles of STAT 5 are not known in neonatal ovine uterine gland development or in adult uterine gland hyperplasia and hypertrophy that normally occurs in response to ovine placental lactogen in pregnant ewes (21).
In the PRL-responsive Nb2 T lymphoma cell line, PRL-mediated STAT 1 activation increases expression of the immediate-early gene IRF-1 (38, 39). In the common marmoset monkey and human, the PRLR is also expressed predominantly in the endometrial glands along with JAK2, STAT 1, and IRF-1 (34, 40). Further, treatment of proliferative phase endometrium from the monkey uterus increases expression of IRF-1 (40). In the present study, IRF-1 mRNA and protein could not be detected in the endometrial glands of the developing neonatal ovine uterus, even though IRF-1 gene expression has been detected in the adult ovine uterus in response to conceptus interferon 
, a type I interferon (26). Therefore, IRF-1 does not appear to be a mediator of PRL actions on the endometrial GE in the neonatal ovine uterus.
Results from study four indicated that three members of the MAPK family are involved in the effects of PRL in the neonatal ovine uterus. Both ERK 1 and ERK 2 (also known as p44 and p42 MAPKs) function in a protein kinase cascade that plays a critical role in regulation of cell growth and differentiation (41, 42). These two kinases share structural homology with the more recently discovered JNK/SAPK family (43, 44). A variety of extracellular stimuli activate the JNK/SAPK pathway, including inflammatory cytokines, UV light, and osmotic stress. In Nb2 cells, PRL activated JNK/SAPK, and JNK/SAPK was found to be important for mitogenic signaling and perhaps suppression of apoptosis (45). In bovine mammary gland epithelial cells, PRL stimulation of cell proliferation involves activation of JNK/SAPK and an increase in c-Jun content of the activator protein 1 transcriptional complex that leads to increased gene transactivation (46). Recently, PRL signaling in the human endometrial glands was also shown to involve activation of ERK 1 and ERK 2 (47). The results of Study 4 indicate that PRL increased phosphorylated ERK 1, ERK 2, and JNK/SAPK MAPKs. Previously, Taylor et al. (2) found high levels of phosphorylated ERK 1 and 2 in the nascent and proliferating endometrial glands that express PRLR in the developing neonatal ovine uterus. Collectively, results indicate that PRL stimulates several signaling pathways, including STATs 1 and 5, ERK 1 and 2 MAPKs, and JNK/SAPK, and is a major regulatory factor controlling endometrial gland morphogenesis in the neonatal ovine uterus.
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Acknowledgments |
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Footnotes |
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Abbreviations: BROMO, Bromocryptine mesylate; CX, control; GE, glandular epithelium; IRF-1, interferon regulatory factor 1 (IRF-1); JAK, Janus kinase; JNK, c-Jun N-terminal kinase; LE, luminal epithelium; LSM, least-square means; oPRL, ovine PRL; PND, postnatal day; PRL, prolactin; PRLR, PRL receptor; roPRL, recombinant ovine PRL; SAPK, stress-activated protein kinase; STAT, signal transducers and activators of transcription.
Received June 17, 2002.
Accepted for publication October 9, 2002.
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References |
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, placental lactogen and growth hormone on ovine endometrial function. Biol Reprod 61:1409–1418 activates multiple signal transducer and activator of transcription proteins and has complex effects on interferon-responsive gene transcription in ovine endometrial epithelial cells. Endocrinology 142:98–107 (IFN) regulation of IFN-stimulated gene expression in cell lines lacking specific IFN-signaling components. Endocrinology 142:1786–1794
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 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]
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R. M. Bigsby, A. Caperell-Grant, N. Berry, K. Nephew, and D. Lubahn Estrogen Induces a Systemic Growth Factor Through an Estrogen Receptor-Alpha-Dependent Mechanism Biol Reprod, January 1, 2004; 70(1): 178 - 183. [Abstract] [Full Text] [PDF]
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 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]
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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]
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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]
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