Biol Reprod Keystone Symposia Conference on Frontiers in Reproductive Biology & Regulation of Fertility.
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Biology of Reproduction 62, 1779-1789 (2000)
© 2000 Society for the Study of Reproduction, Inc.

Articles

Prolactin Receptor and Uterine Milk Protein Expression in the Ovine Endometrium During the Estrous Cycle and Pregnancy1

M. David Stewarta, Greg A. Johnsona, C. Allison Graya, Robert C. Burghardtc, Linda A. Schulerd, Margaret M. Joycea, Fuller W. Bazera,b, and Thomas E. Spencer2,a,b

a Center for Animal Biotechnology and Genomics, Albert B. Alkek Institute of Biosciences and Technology, b Texas A&M University System Health Science Center and Department of Animal Science, Texas A&M University, College Station, Texas 77843-2471 c Department of Veterinary Anatomy and Public Health, College of Veterinary Medicine, Texas A&M University, College Station, Texas 77843 d Department of Comparative Biosciences, University of Wisconsin, Madison, Wisconsin 53706


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Lactogenic hormones regulate epithelial proliferation, differentiation, and function in a variety of epitheliomesenchymal organs. During pregnancy, the ovine uterus is a potential site for endocrine and paracrine actions of lactogenic hormones in the form of pituitary prolactin (PRL) and placental lactogen (PL). These studies determined temporal and spatial alterations in PRL receptor (PRL-R) and expression of uterine milk proteins (UTMP), a marker of endometrial secretory activity, in the ovine endometrium during the estrous cycle and pregnancy. Slot-blot hybridization analysis indicated that steady-state levels of endometrial PRL-R mRNA increased during pregnancy. In situ hybridization and immunohistochemical analyses indicated that PRL-R mRNA and protein were exclusively expressed in the endometrial glandular epithelium (GE). No PRL-R mRNA expression was detected in luminal epithelium, stroma, myometrium, or conceptus trophectoderm. Reverse transcription-polymerase chain reaction analyses determined that the endometrial GE expressed both long and short alternative splice forms of the ovine PRL-R gene. Slot-blot hybridization analysis indicated that steady-state levels of intercaruncular endometrial UTMP mRNA increased about 3-fold between Days 20 and 60, increased another 3-fold between Days 60 and 80, and then declined slightly to Day 120. In pregnant ewes, UTMP mRNA expression was restricted to the endometrial GE in the stratum spongiosum (sGE), increased substantially between Days 15 and 17, and, between Days 17 to 50 of gestation, was markedly higher in upper than lower sGE. After Day 50, hyperplasia of the sGE was accompanied by increased UTMP mRNA expression by all sGE. Collectively, results indicate that 1) endometrial sGE is a primary target for actions of lactogenic hormones and 2) UTMP mRNA expression is correlated with PL production by the trophectoderm and state of sGE differentiation during pregnancy. It is proposed that activation of PRL-R signal transduction pathways by PRL and PL plays a major role in endometrial GE remodeling and differentiated function during pregnancy in support of conceptus growth and development.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In ungulates, establishment and maintenance of pregnancy require integration of endocrine and paracrine signals from the ovary, conceptus, and uterus [1]. Establishment of pregnancy requires that the preimplantation ovine conceptus (embryo/fetus and associated membranes) enter a receptive uterus and develop sufficiently to synthesize and release interferon tau (IFN{tau}), the pregnancy recognition signal [2]. After pregnancy recognition, maintenance of pregnancy requires reciprocal communication between the conceptus and endometrium during implantation and synepitheliochorial placentation [1]. In sheep, superficial implantation and placentation constitute a lengthy process that begins on Days 15–16 and is not completed until Days 50–60 of pregnancy [3]. During this period, the ovine uterus grows substantially in order to accommodate rapid conceptus development and growth in the latter half of pregnancy. In addition to placentomal development in the caruncular areas of the endometrium and changes in vascularity, the intercaruncular endometrial glands grow substantially in length (4-fold) and width (10-fold) during pregnancy in ewes [3]. These uterine glands synthesize, secrete, or transport a variety of enzymes, growth factors, cytokines, lymphokines, hormones, transport proteins, and other substances that are collectively termed histotroph [4–6]. Available evidence strongly supports the theory that secretions from the endometrial epithelia influence conceptus development, onset of pregnancy recognition signals, and growth of the conceptus in species with an epitheliochorial type of placentation [7].

The hormonal, cellular, and molecular mechanisms regulating ruminant endometrial gland morphogenesis and function during pregnancy are not understood. In other epitheliomesenchymal organs, such as the mammary gland and pigeon crop sac, prolactin (PRL) induces mitogenesis and alters postdifferentiation gene expression programs [8–10]. In the rabbit and pig, PRL may have a role in pregnancy through direct actions on the endometrium, because hyperprolactinemia causes endometrial hypertrophy, glandular differentiation, and alterations in secretions found in the uterine lumen [11–15]. The placentae of a number of species, including rodents, humans, nonhuman primates, and ruminants, secrete hormones structurally related to pituitary PRL and growth hormone (GH) that are termed placental lactogens (PLs) [16–18]. Ovine PL is produced by binucleate cells of the conceptus trophectoderm beginning on Day 16 of pregnancy [19], is detected in maternal serum by Day 50, and reaches peak levels between Days 120 and 130 of gestation [20–22]. The PRL receptor (PRL-R) transduces signals by PRL and PL, because bovine PL activated signal transduction through the long form of the PRL-R [23]. Recently, Cassy et al. [24] reported that PRL-R expression in the sheep uterus is specifically expressed in the endometrial glandular epithelium (GE) and increases during mid to late pregnancy. Transcription of the ovine PRL-R gene generates two alternative splice forms encoding short and long forms of PRL-R proteins [25]. However, detailed analyses of PRL-R mRNA and protein expression in the ovine endometrium throughout the estrous cycle and pregnancy have not been reported.

Our working hypothesis is that endocrine and paracrine actions of lactogenic hormones on the endometrium influence GE morphogenesis and secretory function. Spencer et al. [26] recently found that intrauterine infusions of recombinant ovine PL (oPL) increased endometrial GE proliferation and enhanced mRNA expression of two GE secretory proteins, uterine milk proteins (UTMP) [27, 28] and osteopontin (OPN) [29, 30]. UTMP are members of the serpin family of serine protease inhibitors [31] and serve as an excellent marker for endometrial secretory capacity during pregnancy in the sheep [32]. Therefore, objectives of the present study were to 1) determine temporal and spatial alterations in PRL-R and UTMP mRNA expression in the ovine uterus during the estrous cycle and pregnancy and 2) characterize the mRNA splice variants of the PRL-R gene expressed in the ovine uterus.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals and Experimental Design

Mature ewes of primarily Rambouillet breeding were observed daily for estrus using vasectomized rams. All ewes exhibited at least two estrous cycles of normal duration (~16–18 days). Experimental and surgical procedures involving animals were approved by the Agricultural Animal Care and Use Committee of Texas A&M University (Animal Use Protocol 7-286). At estrus, ewes were assigned randomly to cyclic or pregnant status. Ewes assigned to pregnant status were bred to intact rams at estrus and at 12 h and 24 h post-estrus.

Study 1 Fifty-two ewes were assigned randomly to be ovariohysterectomized (n = 4 ewes/day) on Days 1, 3, 5, 7, 9, 11, 13, or 15 of the estrous cycle (Day 0 = estrus) and Days 11, 13, 15, 17, or 19 of pregnancy (Day 0 = mating) as described previously [33]. The uterine lumen of pregnant ewes assigned to be hysterectomized on Days 11–17 was flushed with 20 ml sterile saline. Pregnancy was confirmed by presence of an apparently normal conceptus in the uterine lumen (observed in uterine flushings on Days 11–17), as well as extension of the interestrous interval (Day 19).

At hysterectomy, several sections (~0.5 cm) from the midportion of each uterine horn were fixed in fresh 4% paraformaldehyde in PBS (pH 7.2). In monovulatory pregnant ewes, care was taken to mark uterine tissue samples as contralateral or ipsilateral to the ovary bearing the corpus luteum (CL) that contained the conceptus. After 24 h, fixed tissues were changed to 70% ethanol for 24 h and then dehydrated and embedded in Paraplast-Plus (Oxford Labware, St. Louis, MO). Several sections (1–1.5 cm) from the middle of each uterine horn were embedded in Tissue-Tek OCT compound (Miles, Oneonta, NY), frozen in liquid nitrogen vapor, and stored at -80°C. The remaining endometrium was physically dissected from myometrium, frozen in liquid nitrogen, and stored at -80°C for RNA extraction. In monovulatory pregnant ewes, uterine tissue samples were marked as contralateral or ipsilateral to the ovary bearing the CL. No contralateral uterine samples were used for this study.

Study 2 Forty-four ewes were assigned randomly to be ovariohysterectomized (n = 4 ewes/day) on Days 25, 30, 35, 40, 45, 50, 55, 60, 80, 100, or 120 of pregnancy (Day 0 = mating). At hysterectomy, the uterus was trimmed free of cervix and oviduct and opened along the mesometrial border. Several sections (~0.5 cm) of both interplacentomal and placentomal uterine wall regions from the midportion of each uterine horn were fixed in fresh 4% paraformaldehyde in PBS (pH 7.2). Placentomes were then removed by physical dissection, and remaining intercaruncular or interplacentomal endometrium was dissected from the myometrium. Endometrial samples were frozen in liquid nitrogen and stored at -80°C for RNA extraction. In monovulatory pregnant ewes, care was taken to mark uterine tissue samples as contralateral or ipsilateral to the ovary bearing the CL. No contralateral uterine samples were used for analyses of gene expression.

Analysis of RNA

RNA isolation Total cellular RNA was isolated from ipsilateral endometrium (study 1) and intercaruncular or interplacentomal endometrium (study 2) using Trizol (Gibco-BRL, Grand Island, NY) according to manufacturer's recommendations.

Complementary RNA probes Radiolabeled riboprobes were synthesized for slot-blot hybridization analyses by in vitro transcription using a MAXIscript Kit (Ambion, Austin, TX) according to manufacturer's recommendations. Antisense bovine PRL-R cRNA probes were generated by linearizing the pBS-SK-plasmid containing the bovine long PRL-R cDNA (clone 26353) [34] with EcoRI and in vitro transcription with T3 RNA polymerase. Sense cRNA probes were generated using NotI and T7 RNA polymerase.

A partial ovine UTMP (oUTMP) cDNA was generated by reverse transcription-polymerase chain reaction (RT-PCR) using methods described previously [35]. The sense primer (base pairs [bp] 331–349) 5'-ACAATGAGCACCAACCACC-3' and antisense primer (bp 830–810) 5'-ACCATCGTAGCAAATAGCTCC-3' were derived from the oUTMP mRNA coding sequence (GenBank accession no. J04484) [31]. The RT-PCR product (500 bp) was cloned into pCR II (Invitrogen, San Diego, CA) and fully sequenced in both directions to confirm identity and directionality. Antisense cRNA probes were generated by linearizing the pCR II-oUTMP plasmid with NotI and in vitro transcription with SP6 RNA polymerase. Sense cRNA probes were generated using BamHI and T7 RNA polymerase.

Slot-blot hybridization analysis Because of the large number of endometrial total RNA samples, steady-state levels of PRL-R mRNA were assessed using slot-blot hybridization as described previously [35]. Denatured total endometrial RNA (20 µg) from each ewe was analyzed using radiolabeled antisense bovine PRL-R and oUTMP cRNA probes. To correct for total RNA loading differences, a duplicate RNA slot membrane was hybridized with radiolabeled antisense 18S rRNA cRNA (pT718S; Ambion). After washing, nonspecific hybridization was removed by RNase A digestion [36]. The radioactivity associated with each slot was quantitated by electronic autoradiography using an Instant Imager (Packard Instruments, Meridian, CT).

Semiquantitative RT-PCR

Temporal alterations in the expression of short and long PRL-R isoforms were determined by semiquantitative RT-PCR. Complementary DNA was synthesized from total cellular RNA isolated from intercaruncular endometrial total RNA samples obtained in study 2. The RT reaction consisted of 5 µg total RNA, 0.2 µg oligo-dT primers, 1 µg random primers (Life Technologies, Gaithersburg, MD), 12 U RNasin (Promega, Madison, WI), 10 mM dithiothreitol (Life Technologies), 5 mM dNTPs, and 100 U SuperScript II Reverse Transcriptase (Life Technologies) in a total volume of 20 µl first strand buffer (Life Technologies).

Total RNA and oligo-dT primers were annealed by heat denaturation at 70°C for 10 min followed by 2 min at room temperature. All other components were added; the mixture was incubated at 42°C for 60 min, then heated to 95°C for 2 min, and placed on ice. EDTA (1 µl; 0.5 M) was added to terminate enzymatic activity prior to extraction with 20 µl phenol chloroform isoamyl alcohol. Newly synthesized cDNA was acid ethanol precipitated, resuspended in 20 µl water, and stored at -20°C. The cDNAs were diluted (1:10) with sterile water for use in PCR reactions.

PCR reactions were performed using AmpliTaq DNA polymerase (Perkin Elmer, Irvine, CA) and optimized buffer D (Invitrogen) for ß-actin or optimized buffer F (Invitrogen) for short and long PRL-R according to manufacturer's recommendations. Each PCR reaction consisted of 100 ng primer and 1 mM dNTPs in a final volume of 50 µl. The PRL-R primers were PRL-R sense (bp 709–729) 5'-TTCCCAGTGAAGGATACAAGC-3'; long PRL-R antisense (bp 1018–1000) 5'-GTTCTTTGGAGGGGTGTGG-3' (GenBank accession no. AF041257); and short PRL-R antisense (bp 894–873) 5'-CTATTAAAACACAGACACAAGG-3' (GenBank accession no. AF041977) [25]. Using the ovine ß-actin mRNA sequence (GenBank accession no. U39357), the ß-actin primers were sense (bp 274–295) 5'-CATCCTGACCCTCAAGTACCC-3' and antisense (bp 694–674) 5'-CCGATGTCGAAGTGGTGGTG-3'. ß-Actin PCR reactions contained 1 µl cDNA; short PRL-R reactions had 5 µl cDNA; and long PRL-R reactions had 2 µl cDNA. ß-actin PCR conditions were 30 cycles of 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. PCR conditions and amount of template cDNA used in each reaction were optimized for each primer set to ensure linear amplification of target.

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 UV light using a AlphaImager (Alpha Innotech, San Leandro, CA). Values are presented as relative light units. The ß-actin values were used as a covariate in statistical analyses to correct for differences in amounts of cDNA used for each endometrial sample. All RT-PCR products were cloned into pCR II (Invitrogen) and fully sequenced in both directions to confirm identity.

In Situ Hybridization Analyses

PRL-R and UTMP mRNAs were localized in uterine tissue sections by in situ hybridization as described previously [37]. Deparaffinized, rehydrated, and deproteinated uterine tissue sections (5–7 µm) were hybridized with radiolabeled antisense or sense bovine PRL-R cRNA or oUTMP riboprobes generated from linearized plasmid templates using in vitro transcription with [{alpha}-35S]UTP. Autoradiographs of slides were prepared using NTB-2 (Eastman Kodak, Rochester, NY) liquid photographic emulsion. Slides were stored at 4°C for 1–2 wk, developed in Kodak D-19 developer, counterstained with Harris' modified hematoxylin (Fisher Scientific, Fairlawn, NJ), dehydrated through a graded series of alcohol to xylene, and then protected with a coverslip.

Immunocytochemical Analysis

Frozen sections (4–8 µm) of uterine tissues embedded in OCT compound in study 1 were cut with a cryotome (Lipshaw Manufacturing, Detroit, MI) and mounted on Superfrost/Plus microscope slides (Fisher Scientific). Conceptuses were present in the uterine lumen of sections from Day 19 pregnant ewes, as these uteri were not flushed prior to uterine tissue collection. Sections were fixed in -20°C methanol for 10 min, permeabilized with 0.3% Tween 20 in 0.02 M PBS, and then blocked in antibody dilution buffer (two parts 0.02 M PBS, 1.0% BSA, 0.3% Tween 20 [pH 8.0] and one part glycerol) containing 5% normal goat serum for 1 h at room temperature. Sections were rinsed in PBS and incubated overnight at 4°C with 20 µg/ml rabbit anti-bovine PRL-R IgG [23] or 20 µg/ml normal rabbit IgG (Sigma, St. Louis, MO) as a control. After three rinses in PBS for 10 min each, sections were incubated with fluorescein-conjugated goat anti-rabbit IgG (Zymed, San Francisco, CA) for 1 h at room temperature and again washed in PBS three times for 10 min each. Sections were then overlaid with a coverslip and Prolong Antifade mounting reagent (Molecular Probes, Eugene, OR) and viewed with a Zeiss Photomicroscope III (Carl Zeiss, Thornwood, NJ) equipped with a fluorescein isothiocyanate filter set.

Photomicroscopy and Digital Imaging

Photomicrographs of representative fields of immunofluorescence slides were taken with a Zeiss Photomicroscope III and Kodak T-MAX 3200 film. Photomicrographs of in situ hybridization slides were taken under brightfield and darkfield illumination using a Zeiss Axioplan2 photomicroscope fitted with a Hamamatsu (Bridgewater, NJ) chilled 3CCD color camera. Digital images were captured and/or assembled using Adobe Photoshop 4.0 (Adobe Systems, Seattle, WA) and a MacIntosh PowerMac G3 computer (Apple Computer, Cupertino, CA). Black-and-white prints were electronically printed using a Kodak DS8650 color printer.

Statistical Analyses

Data from slot-blot hybridization and RT-PCR analyses were subjected to least squares ANOVA using the General Linear Models procedures of the Statistical Analysis System [38]. For both studies, effects of day on steady-state levels of endometrial PRL-R mRNA were examined by regression analysis. For study 1, data from Days 11, 13, and 15 were examined by two-way ANOVA considering variation due to day post-estrus/mating, status (cyclic vs. pregnant), and their interaction. Hybridization data (total counts) were normalized for differences in sample loading using the 18S rRNA data as a covariate in ANOVA. RT-PCR data (relative light units) were normalized using ß-actin data as a covariate in ANOVA. All tests of significance were performed using the appropriate error terms according to the expectations of the mean squares [39]. Data are presented as least square means (LSM) total counts with SE.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Steady-State Levels of Endometrial PRL-R mRNA

Steady-state levels of PRL-R mRNA in the endometrium were affected by day of the estrous cycle and pregnancy (Fig. 1). In cyclic ewes, endometrial PRL-R mRNA levels were affected (P < 0.09, cubic) by day, but changes were not dramatic (Fig. 1A). Between Days 11 and 19 of pregnancy (study 1), endometrial PRL-R mRNA increased (P < 0.01, quadratic) almost 2-fold, with the largest increase between Days 17 and 19 (Fig. 1A). On Days 11–15, endometrial PRL-R mRNA levels were not different between cyclic and pregnant ewes (P > 0.10, day x status). In study 2, slot-blot hybridization analyses indicated that steady-state levels of PRL-R mRNA in the intercaruncular endometrium increased linearly (P < 0.01) from Days 20 to 120 of pregnancy (Fig. 1B).



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  		FIG. 1. Slot-blot analysis of steady-state levels of PRL-R mRNA in the endometrium of cyclic and pregnant ewes. Blots were hybridized with antisense radiolabeled cRNA probes generated from a bovine long PRL-R cDNA and then digested with RNase A. A duplicate slot blot was probed with a radiolabeled human 18S rRNA cRNA probe and used as a covariate in statistical analyses. Slot blots were quantitated by electronic autoradiography, and data are expressed as LSM total counts with overall SE. A) In cyclic ewes, PRL-R mRNA levels increased after Day 11 (P < 0.09). On Days 11 to 15, PRL-R mRNA levels were not different between cyclic and pregnant ewes (day x status, P > 0.10). However, PRL-R mRNA levels increased (P < 0.01) between Days 11 and 19 in pregnant ewes. B) PRL-R mRNA levels increased linearly (P < 0.01) from Days 20 to 120 of pregnancy

RT-PCR Analysis of PRL-R mRNA Expression

As illustrated in Figure 2A, primers were designed to amplify regions of the short and long ovine PRL-R mRNAs 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. It was predicted that use of these specific primers with the exon 9 common primer would yield different-sized 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 the ovine ß-actin mRNA (420 bp) as a control for cDNA concentration. The dilution and volume of cDNAs used in PCR reactions were determined to be in the linear range to ensure that the amount of PCR product derived from a given amount of total RNA in a sample was directly proportional to the concentration of target mRNA in the sample (data not shown).



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  		FIG. 2. RT-PCR analysis of PRL-R mRNA in endometrial total RNA from pregnant ewes. A) Schematic illustrating the approach used to amplify the long (310 bp) and short (207 bp) alternative splice forms of the ovine PRL-R gene used in RT-PCR analyses. S, Stop codon. B) Representative RT-PCR analysis of PRL-R mRNAs and ß-actin in intercaruncular endometrial total RNA samples from pregnant ewes. For the negative control, RT cDNA was omitted from the PCR reaction (-RT). The cDNAs were separated in a 2% agarose gel. Measurements of cDNA synthesized in PCR reactions were made from ethidium bromide-stained agarose gels using an Alpha Innotech imaging system

As shown in Figure 2B, both long (310 bp) and short (207 bp) forms of the ovine PRL-R mRNA were present in the intercaruncular endometrium of Day 20 to 120 pregnant ewes from study 2. Two specific PCR products (310 bp and 350 bp) were consistently detected using the long PRL-R mRNA primers. Sequence analysis of the two long PRL-R PCR products indicated that the 310-bp product was identical to the long ovine PRL-R cDNA, whereas sequence analysis of the unexpected 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. The 207-bp product generated by the short PRL-R primers was found to be the short PRL-R mRNA and included the 39-bp alternatively spliced exon. The ratio of the 350-bp to 310-bp long PRL-R cDNA products did not change with day of pregnancy (P > 0.10). Further, the relative ratio of short to long PRL-R mRNAs revealed no significant effect of day (P > 0.10).

In Situ Hybridization Analysis of PRL-R mRNA in the Ovine Uterus

PRL-R mRNA expression was restricted to the endometrial glands in uteri from all cyclic and pregnant ewes (Fig. 3). Specifically, PRL-R expression was detected in GE of glands in the lower stratum spongiosum (sGE), but not the upper dense stratum compactum. No specific hybridization signal was detected in the endometrial luminal epithelium (LE), superficial GE, stroma, immune cells, blood vessels, or myometrium as judged from comparison to control slides hybridized with sense PRL-R cRNA probe. Consistent with slot-blot hybridization results, PRL-R mRNA expression increased in endometrial GE during early pregnancy. PRL-R mRNA was not detected in placental membranes between Days 17 and 19 of pregnancy. During later pregnancy (Fig. 4), abundant PRL-R mRNA expression remained restricted to endometrial sGE in both the upper and lower areas of the stratum spongiosum. No PRL-R mRNA was detected in the maternal caruncular or placental cotyledonary tissues composing the placentomes.



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  		FIG. 3. In situ localization of PRL-R mRNA in the endometrium of cyclic and early-pregnant ewes. Cross sections of the uterine wall from cyclic (C) and pregnant (Px) ewes were hybridized with {alpha}-35S-labeled antisense or sense bovine PRL-R cRNA probes. Hybridized sections were digested with RNase A, and protected transcripts were visualized by liquid emulsion autoradiography. Developed slides were counterstained lightly with hematoxylin, and photomicrographs were taken under brightfield or darkfield illumination. Car, Caruncle; L, luminal epithelium; cG, stratum compactum (superficial or shallow) glandular epithelium; sG, stratum spongiosum (deep) glandular epithelium; S, stroma; M, myometrium; P, placenta. x260



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  		FIG. 4. In situ localization of PRL-R mRNA in the endometrium from Day 20 to 120 pregnant ewes. Cross sections of the uterine wall from pregnant ewes were hybridized with {alpha}-35S-labeled antisense or sense bovine PRL-R cRNA probes. Hybridized sections were digested with RNase A, and protected transcripts were visualized by liquid emulsion autoradiography. Developed slides were counterstained lightly with hematoxylin, and photomicrographs were taken under brightfield or darkfield illumination. Car, Caruncle; Cot, cotyledon; L, luminal epithelium; cG, stratum compactum (superficial or shallow) glandular epithelium; sG, stratum spongiosum (deep) glandular epithelium; M, myometrium; P, placenta; S, stroma; V, blood vessel. x260

Immunohistochemical Analysis of PRL-R Protein in the Ovine Uterus

The location of PRL-R protein in the cyclic and pregnant ovine uteri from study 1 was assessed using immunofluorescence antibody analysis of frozen uterine sections (Fig. 5). Immunoreactive PRL-R protein was present exclusively in the apical aspect of the endometrial sGE of both cyclic and pregnant uteri. As expected from in situ hybridization results, no specific signal was detected in endometrial LE, superficial GE, stroma, or myometrium. In pregnant ewes, no specific signal was detected in the chorioallantoic placenta (data not shown). Negative controls, in which normal rabbit IgG was substituted for rabbit anti-bovine PRL-R IgG, showed no specific staining in endometrial glands.



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  		FIG. 5. Immunofluorescence localization of PRL-R protein in frozen uterine sections from Day 15 and 19 pregnant (Px) ewes. Rabbit anti-bovine PRL-R IgG or rabbit IgG (Control) was used in immunofluorescence procedures. Intense immunoreactivity was located specifically on the apical aspect of the endometrial glands, but was not present in other uterine cell types or the conceptus (data not shown). L, luminal epithelium; G, glandular epithelium. x300

Steady-State Levels of Endometrial UTMP mRNA

Steady-state levels of UTMP mRNA in the intercaruncular endometrium were affected (P < 0.01, cubic) by day of pregnancy (Fig. 6). Endometrial levels of UTMP mRNA increased approximately 3-fold between Days 20 and 60, increased another 3-fold between Days 60 and 80, and then declined to Day 120.



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  		FIG. 6. Steady-state levels of UTMP mRNA in the endometrium of pregnant ewes. A slot blot containing 20 µg endometrial total RNA for each ewe in study 2 was hybridized with antisense radiolabeled cRNA probes generated from an oUTMP partial cDNA and then digested with RNase A. A duplicate slot blot was probed with a radiolabeled human 18S rRNA cRNA probe and used as a covariate in statistical analyses. Both slot blots were quantitated by electronic autoradiography, and data are expressed as LSM total counts with overall SE. UTMP mRNA expression was affected by day of pregnancy (P < 0.01, cubic)

In Situ Hybridization Analysis of UTMP mRNA in the Ovine Uterus

In cyclic ewes, UTMP mRNA was not detected between Days 1 and 11 of the estrous cycle, but very low levels of UTMP mRNA were detected in the endometrial GE on Days 13 and 15 (Fig. 7). Similarly, very low levels of UTMP mRNA were detected in endometrial GE on Days 13 and 15 of pregnancy; however, abundant UTMP mRNA was present in sGE of the upper stratum spongiosum on Days 17 and 19. A marked dichotomy in sGE UTMP mRNA expression was detected as UTMP mRNA expression in the deep sGE was lower than in the upper sGE. No UTMP mRNA was detected in the LE, stroma, myometrium, blood vessels, or immune cells in uteri of any cyclic or pregnant ewes.



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  		FIG. 7. In situ localization of UTMP mRNA in the endometrium of cyclic and pregnant ewes. Cross sections of the uterine wall from cyclic (C) and pregnant (Px) ewes were hybridized with {alpha}-35S-labeled antisense or sense oUTMP cRNA probes. Hybridized sections were digested with RNase A, and protected transcripts were visualized by liquid emulsion autoradiography. Developed slides were counterstained lightly with hematoxylin, and photomicrographs were taken under brightfield or darkfield illumination. Car, Caruncle; L, luminal epithelium; cG, stratum compactum (superficial or shallow) glandular epithelium; sG, stratum spongiosum (deep) glandular epithelium; S, stroma; M, myometrium; TE, trophectoderm; V, blood vessel. x260

As illustrated in Figure 8, marked differences in UTMP mRNA expression in sGE persisted between Days 20 and 50 of pregnancy. In the upper sGE, expression of UTMP mRNA was high, whereas UTMP mRNA expression was at low to moderate levels in the lower sGE near the myometrium. Between Days 50 and 60 of pregnancy, significant hypertrophy in endometrial glands was detected, and this event was temporally coincident with loss of differences in UTMP mRNA expression between upper and lower sGE. Between Days 60 and 120 of pregnancy, UTMP mRNA was expressed at high levels in all sGE.



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  		FIG. 8. In situ localization of UTMP mRNA in the endometrium of pregnant (Px) ewes. Cross sections of the uterine wall from pregnant (Px) ewes were hybridized with {alpha}-35S-labeled antisense oUTMP cRNA probes. Hybridized sections were digested with RNase A, and protected transcripts were visualized by liquid emulsion autoradiography. Developed slides were counterstained lightly with hematoxylin, and photomicrographs were taken under brightfield or darkfield illumination. L, Luminal epithelium; cG, stratum compactum (superficial or shallow) glandular epithelium; sG, stratum spongiosum (deep) glandular epithelium; S, stroma; M, myometrium; P, placenta; V, blood vessel. x260


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Expression of PRL-R by uterine endometrium has been reported for the cow [23, 34], human [40–42], pig [13], rat [43, 44], rabbit [12], and ewe [24, 45]. As in the cyclic human uterus, PRL-R expression was expressed specifically in the endometrial GE. Results from this study and others [26] extend those of Cassy et al. [24] in sheep and Jones et al. [42] in the human by suggesting that lactogenic hormones play key roles in regulation of GE function. The present study showed that the initial increase in expression of the PRL-R gene in sGE occurs between Days 17 and 19. This period coincides with the time of onset of PL secretion by trophectoderm [19] and down-regulation of progesterone receptor (PR) expression in sGE [46]. Effects of PL on PRL-R gene expression in the endometrium are not known, but Galsgaard et al. [47] found that both PRL and GH increased PRL-R gene expression in insulin-producing cells.

Results of the present study indicated that both short and long forms of ovine PRL-R mRNAs were expressed by GE of endometrium from pregnant ewes and that the ratio of long to short PRL-R mRNAs did not change between Days 20 and 120 of pregnancy. Signal transduction by PRL and perhaps PL is initiated by hormone-induced receptor homodimerization that activates signal transducer and activator of transcription (STAT) and/or mitogen-activated protein kinase (MAPK) pathways [48, 49]. PRL binds to the long or intermediate forms of the PRL-R and stimulates specific gene transcription via STATs (mainly STAT5a and STAT5b and possibly STATs 1 and 3) through interaction with tyrosine kinase Janus kinase 2 (JAK2) as well as the MAPK pathway. The short form of the PRL-R lacks the cytoplasmic region required for JAK-STAT activation, but stimulates the MAPK signal transduction pathway. The ability of oPL to activate the MAPK signaling pathway has not been reported. Bovine PRL and PL stimulate expression of a reporter gene containing a PRL-responsive element through the long form, but not the short form, of the bovine PRL-R [23]. Human PL appears to use a unique signal transduction pathway different from that for human PRL and human GH [50]. Recently, oPL was shown to activate a heterodimer of the PRL-R and GH-R in addition to a homodimer of PRL-R (Arieh Gertler, personal communication). Although the precise signal transduction pathway is not determined for oPL, the expression of both the long and short PRL-R mRNAs in ovine endometrial GE suggests that lactogenic hormones may activate pathways leading to both proliferation and enhanced production of secretory proteins such as UTMP and OPN [26].

In the present study, overall changes in steady-state levels of intercaruncular endometrial UTMP mRNA during pregnancy closely paralleled changes in PL production by the placenta [19–22] and release of newly synthesized proteins, including UTMP, by intercaruncular endometrial explants from pregnant ewes [32]. Spencer et al. [26] reported that intrauterine administration of recombinant oPL increased proliferation of sGE and enhanced UTMP and OPN expression in the sGE; they suggested that PL acts in a paracrine manner on PRL-R-positive sGE to increase UTMP gene expression and perhaps the transport and/or synthesis of other GE-specific secretory products.

Previous studies indicated that progesterone induces UTMP expression by ovine endometrium [27, 28, 32, 51]. For example, treatment of ovariectomized ewes with progesterone for 6 days induced low levels of UTMP mRNA and protein whereas treatment with progesterone for 14 or 30 days greatly enhanced UTMP expression [28]. The protracted nature of this progesterone effect is not typical of genes regulated by progesterone through PR in a classic transcriptional manner. In fact, loss of PR gene expression in GE appears to be required for progesterone induction of secretory gene expression by GE. For example, administration of estrogen with progesterone induced PR expression in endometrial GE and concomitantly ablated effects of progesterone alone to induce UTMP and OPN mRNA expression in the sGE [26]. The contention that loss of epithelial PR is required for endometrial GE function during pregnancy is supported by studies of PR gene expression in endometrium from cyclic and pregnant ewes [46, 52]. During early pregnancy, PR expression is detectable in LE and GE on Day 11, but PR are undetectable in LE and superficial GE from Days 13 to 19, and are present only in stromal cells and myometrium after Day 25 of gestation in ewes [46] (unpublished results). Loss of PR expression by GE appears to be required for GE remodeling and differentiation and also to prevent inhibition of these events by progesterone [53]. Available evidence supports the concept that progesterone decreases PR expression in GE to allow pituitary PRL and/or PL to increase UTMP mRNA expression. This may explain why UTMP synthesis and secretion require long-term progesterone therapy in ovariectomized ewes [27, 28, 32, 51]. In study 1, progressive down-regulation of PR in GE after Day 11 may account for low levels of UTMP mRNA in those epithelia on Days 13 and 15 of the estrous cycle and pregnancy.

In the present study, marked differences in UTMP mRNA expression were apparent in sGE between Days 17 and 50 of pregnancy, with higher levels of UTMP mRNA in the upper sGE compared to the lower sGE near the myometrium. This dichotomy in expression may be related to the fact that deeper glands of the intercaruncular endometrium undergo proliferation during early pregnancy [3], which precedes maximal levels of protein synthesis by the intercaruncular endometrium [32]. Indeed, intrauterine administration of recombinant oPL specifically stimulates proliferation of deep sGE [26]. Likewise, GE proliferation occurs between Days 17 and 50 of pregnancy. Once fully developed, uterine glands enlarge between Days 55 and 80, a process presumably stimulated by PRL, PL, and placental GH [26, 54]. In support of this concept, all endometrial sGE expressed abundant levels of UTMP mRNA after Day 50 of pregnancy, which correlates with large amounts of PL secreted by the placenta and appearance of PL in maternal serum. In addition, temporal changes in PL production by the conceptus closely parallelled changes in total protein synthesized and secreted by the intercaruncular endometrium during pregnancy [32, 55].

It is hypothesized that secretions from the endometrial GE influence conceptus development, onset of pregnancy recognition signals, and growth of the fetus and placenta in both pigs and sheep [4–6]. Results from the present studies and others [26] support the hypothesis that members of the lactogenic hormone family stimulate endometrial gland development and function during pregnancy to facilitate implantation and placentation of the conceptus. The pregnant ovine endometrium is sequentially exposed to estrogen, progesterone, PRL, IFN{tau}, PL, and placental GH. These hormones may constitute a sequentially activated "servomechanism" that induces and maintains endometrial remodeling, secretory function, and uterine growth during gestation [26]. Since short and long PRL-R gene expression in the endometrial GE increase during pregnancy, pituitary PRL and/or PL may play central roles in endometrial gland remodeling and stimulation of differentiated function of GE necessary for optimal maternal support of conceptus growth and development throughout pregnancy.


   ACKNOWLEDGMENTS
 
The authors thank Dr. Shawn W. Ramsey and Mr. Todd Taylor of the Texas A&M University Sheep and Goat Center for care and management of ewes. Photomicrographs and digital images were prepared using facilities in the Texas A&M University College of Veterinary Medicine Image Analysis Laboratory, which is supported, in part, by NIH Grant P30 ES09106.


   FOOTNOTES
 
First decision: 5 January 2000.

1 This work was supported in part by BARD Grant US-2643–95. Back

2 Correspondence: Thomas E. Spencer, Center for Animal Biotechnology and Genomics, 444 Kleberg Center, College Station, TX 77843-2471. FAX: 409 862 2662; tspencer{at}ansc.tamu.edu Back

Accepted: January 18, 2000.

Received: December 9, 1999.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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