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Articles |
a Center for Animal Biotechnology and Genomics, Albert B. Alkek Institute of Biosciences and Technology, Texas A&M University System Health Science Center, and Department of Animal Science, Texas A&M University, College Station, Texas 77843-2471
b Department of Animal and Dairy Sciences, Auburn University, Auburn, Alabama 36849-5415
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(PGF) with luteolysis and behavioral estrus. During the estrous cycle, plasma concentrations of progesterone in intact control and UGKO ewes were not different during metestrus and diestrus, but levels did not decline in many UGKO ewes during late diestrus. Peak peripheral plasma concentrations of PGF metabolite, in response to an oxytocin challenge on Day 15, were threefold lower in UGKO compared to control ewes. Intact UGKO ewes bred repeatedly to intact rams did not display evidence of pregnancy based on results of ultrasound. Collectively, results indicate that 1) transient, progestin-induced disruption of ovine uterine development from birth alters both structural and functional integrity of the adult endometrium; 2) normal adult endometrial integrity, including uterine glands, is required to insure a luteolytic pattern of PGF production; and 3) the UGKO phenotype, characterized by the absence of endometrial glands and a compact, disorganized endometrial stroma, limits or inhibits the capacity of uterine tissues to support the establishment and/or maintenance of pregnancy.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Uterine endometrial glands are characteristic features of all mammalian uteri. Endometrial gland development (adenogenesis) in sheep, cattle, and pigs occurs rapidly after birth [10–13]. Withdrawal of fetal tissues from a progesterone-dominated prenatal environment at birth was proposed to be an endocrine cue for adenogenesis in the neonatal ovine uterus [10]. Subsequently, Bartol et al. [14] demonstrated that exposure of ewe lambs to the synthetic progestin norgestomet from birth to postnatal day (PND) 13 inhibited endometrial adenogenesis. Removal of the progestin block to adenogenesis on PND 13 permitted glands to develop by PND 26. However, these glands were not well developed and were histologically abnormal.
Recently, chronic exposure of neonatal lambs to progestins for 32 weeks from birth was shown to prevent uterine adenogenesis and to induce a unique, stable adult endometrial phenotype characterized by the absence of uterine glands, which is the ovine uterine gland knockout (UGKO) phenotype [9,12,15]. The ovine UGKO model system is a unique tool with which to study mechanisms regulating endometrial organization and adenogenesis in the neonate, as well as the functional role of endometrial glands in adult ewes [9]. Objectives of the present studies were to determine 1) the duration of postnatal progestin exposure from birth necessary to produce the UGKO phenotype; 2) if neonatally Nor-treated, UGKO ewes would attain puberty and exhibit normal estrous cycles; 3) if the UGKO phenotype affects uterine prostaglandin generating potential or relative patterns of endometrial expression in situ for progesterone, estrogen, or oxytocin receptors (PR, ER
, and OTR, respectively); and 4) if pregnancy could be established and maintained in adult UGKO ewes.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Experimental and surgical procedures complied with the Guide for Care and Use of Agriculture Animals and were approved by the Institutional Agricultural Animal Care and Use Committee of Texas A&M University (Animal Use Protocol 7–176).
Experimental Design
Thirty-five crossbred Rambouillet/Suffolk ewe lambs were assigned randomly at birth to one of six treatment groups (Fig. 1). Ewes received a single Synchromate B norgestomet implant (Sanofi, Overland Park, KS) every 2 weeks for either 8 (Group I), 16 (Group II), or 32 (Groups III and IV) weeks. The implants were inserted subcutaneously in the periscapular area of ewe lambs within 12 h of birth; they release approximately 6 mg of norgestomet (Nor; 17-acetoxy-11ß-methyl-19-norpreg-4-ene-3,20 dione), a potent synthetic 19-norprogestin, over a 14-day period [14]. Ewes not exposed to Nor served as controls (Groups V and VI). At 16 weeks of age, ewes in Groups I (n = 5), II (n = 6), III (n = 4), and V (n = 8) were subjected to midventral laparotomy, hemihysterectomy (Hhx), and removal of the ipsilateral oviduct and ovary. Once the ewes attained puberty and exhibited several estrous cycles, control (Group V) and Nor-treated ewes (Groups I, II, III) were synchronized to estrus using two injections (0700 h and 1900 h) of 10 mg prostaglandin F2 (PGF; Lutalyse, Upjohn, Kalamazoo, MI). Ewes were then assigned randomly to have the remaining uterine horn, ovary, and oviduct removed on either Day 9 or Day 15 postestrus. At hysterectomy, several sections (~0.5 cm) from the mid-portion of the uterine horn were fixed in fresh 4% paraformaldehyde in PBS (pH 7.2). After 24 h, fixed tissues were changed to 70% ethanol for 24 h and then were dehydrated and embedded in Paraplast Plus (Oxford Labware, St. Louis, MO) for histology and immunohistochemistry.
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All ewes were monitored daily for estrous behavior during the breeding season using vasectomized rams. After control ewes exhibited puberty and at least two estrous cycles of normal duration (~16–18 days), all ewes were given two intramuscular injections (0700 h and 1700 h) of 10 mg PGF. PGF injections were repeated 9 days later to synchronize estrus. Blood samples were then collected from intact Nor-treated (Group IV) and control (Group VI) ewes every 2 days after estrus (Day 0) for a full estrous cycle. Blood samples were collected via jugular venipuncture into Vacutainer Evacuated Blood Collection Tubes with sodium heparin (Becton-Dickinson, Franklin Lakes, NJ), and plasma was stored at -20°C until use.
After puberty and at 2 years of age, intact control (Group VI) and Nor-treated, UGKO ewes (Group IV) were synchronized to estrus as described above. At estrus (Day 0) and 12 h and 24 h later, all ewes were bred to intact rams of proven fertility. All ewes were monitored daily for estrous behavior during the breeding season using vasectomized rams. Ewes not returning to estrus were subjected to transabdominal ultrasound on Days 25 and 35 postbreeding to confirm pregnancy by detection of placentome formation. This experiment was repeated three times using intact, Nor-treated UGKO ewes.
Progesterone Radioimmunoassay
Concentrations of progesterone in plasma samples were determined using an Active Progesterone Radioimmunoassay kit (Diagnostic Systems Laboratories, Inc., Webster, TX). Assay sensitivity was 0.1 ng/ml, and the intra- and interassay coefficients of variation were 5.4% and 10.1%, respectively. Assay results were calculated using the AssayZap program (Biosoft, Ferguson, CA).
Oxytocin Challenge and 13,14-Dihydro-15-Keto-PGF Metabolite (PGFM) Radioimmunoassay
Intact Nor-treated (Group IV) and control (Group VI) ewes were synchronized to estrus with PGF as described above. On Day 15, ewes were challenged intravenously with oxytocin (30 IU; Sigma, St. Louis, MO) in 1 ml sterile saline. Blood samples were collected via jugular venipuncture into Vacutainer Evacuated Blood Collection Tubes with sodium heparin (Becton-Dickinson) at -10, 0 (oxytocin administration), 10, 20, 30, 40, 50, 60, 70, 80, and 90 min. Plasma was stored at -20°C. Concentrations of PGFM were determined by radioimmunoassay as described previously [16–17]. Rabbit anti-ovine PGFM antibody (J53) was generously provided by Dr. William W. Thatcher (University of Florida, Gainesville, FL). Assay sensitivity was 10 pg/ml, and the intra- and interassay coefficients of variation were 5.3% and 12.6%, respectively. Assay results were calculated using the Biosoft AssayZap program.
Histology
Embedded tissues were sectioned (4 µm), deparaffinized, and hydrated prior to staining with Hematoxylin Stain, Gill's Formulation #3 (Fisher Scientific, New Jersey, NJ). Destaining was performed using a 1.0% acid ethanol solution. Slides were developed in a 3% ammonium hydroxide solution, counterstained with 1% Eosin Y (Statlab Medical Products, Lewisville, TX), and destained in 95% ethanol followed by dehydration through a series of alcohol and xylene.
Immunohistochemistry
Immunoreactive PR and ER proteins were detected in uterine tissue cross sections using specific antibodies and a Super ABC Rabbit or Mouse/Rat IgG Kit (Biomeda, Foster City, CA) as described previously [17]. Mouse monoclonal antibody to the human PR (MA1–411) was purchased from Affinity Bioreagents (Golden, CO). Rat monclonal antibody to human ER (H222) was generously provided by Dr. Geoffrey Greene (Abbott Laboratories, Chicago, IL). Each antibody was used at a final concentration of 5 µg/ml. Negative controls included substitution of the primary antibody with purified mouse IgG (PR) or rat IgG (ER) at a final concentration of 5 µg/ml. Tissue sections from both uterine horns of each ewe were processed as sets within an experiment. Staining intensities and patterns were assessed independently by two observers.
In Situ Hybridization
OTR mRNA was localized in uterine tissue sections (6 µm) by in situ hybridization analysis as described previously [9]. Radiolabeled sense or antisense ovine OTR cRNA probes were generated from a linearized ovine OTR cDNA [18] using in vitro transcription with [-35S]UTP. After hybridization and ribonuclease A digestion, slides were then dipped in NTB-2 liquid photographic emulsion (Kodak, Rochester, NY), stored at 4°C for 1 week, and developed in Kodak D-19 developer. Slides were then counterstained with Harris modified hematoxylin (Fisher Scientific) and dehydrated through a graded series of alcohol to xylene.
Photomicroscopy
Photomicrographs were taken using a Zeiss Axioplan2 photomicroscope (Carl Zeiss, Inc., Thornwood, NY) fitted with a Hamamatsu chilled 3CCD color camera (Hamamatsu Corporation, Bridgewater, NJ). 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 were subjected to least-squares ANOVA (LS-ANOVA) using the general linear models (GLM) procedures of the Statistical Analysis System [19]. For progesterone and PGFM measurements, statistical models initially included the main effects of treatment (control, UGKO), ewe within treatment, day (progesterone) or time (PGFM), and appropriate interactions. Tests of significance were based on expectations of the error mean squares. For PGFM measurements, the time of collection of the sample containing the highest concentration of PGFM for each ewe was defined as the time to peak value and peak value, respectively. These values were then analyzed by one-way LS-ANOVA. Homogeneity of regression analysis was used to evaluate differences among treatments over day or time. Covariate analysis was used to correct for differences in basal values among ewes. Data are presented as least-square means (LSM) with pooled standard errors (SE).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Extensive glandular development was observed in the intercaruncular endometrial areas of uterine horns obtained from Group V control ewes at 16 weeks of age (Fig. 2A), as well as in adults from this group (Fig. 2B). In contrast, exposure of newborn ewe lambs to Nor for 8, 16, or 32 weeks from birth prevented endometrial adenogenesis and induced the UGKO phenotype (Fig. 2, C–H). Histological analyses demonstrated three consistent phenotypes in the endometrium of Nor-treated ewes: 1) total absence of glands (panels C and D), 2) slight glandular invaginations into the stroma (panels E and F), and 3) infrequent cyst- and gland-like structures in the stroma (panels G and H). The most common phenotype observed was total absence of uterine glands. The frequency of these phenotypes was not affected by length of progestin exposure or age at hemihysterectomy. In a number of UGKO uteri, a ruffled LE was observed in presumptive intercaruncular endometrial areas (Fig. 2F). This ruffled LE appeared to contain nascent GE buds that barely penetrated the stroma.
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In addition to lacking endometrial glands, the adult endometrium from Nor-treated or UGKO ewes lacked clear delineation of stromal zones characteristic of intercaruncular endometrial areas which, in uteri from normal control ewes, is defined by a dense stratum compactum and loose stratum spongiosum (Fig. 2, D, F, and H). In fact, the presumptive intercaruncular endometrial areas of UGKO ewes appeared to contain only compact stroma and was strikingly similar to the caruncular areas of control uteri. Interestingly, melanocyte distribution patterns were also different in control as compared to UGKO endometrium. If present, endometrial melanocytes were particularly concentrated in the subepithelial stratum compactum in control ewes; however, they were often distributed randomly in the compact stroma of UGKO endometrium. No distinct differences in myometrial histology were observed. The UGKO uteri contained both inner circular and outer longitudinal myometrial layers.
Interestrous Interval and Peripheral Plasma Concentrations of Progesterone
All control and UGKO ewes attained puberty at approximately 8 months of age and exhibited behavioral estrus. The interestrous interval (IEI) for control ewes was normal (16–18 days), whereas the IEI in UGKO ewes was highly variable, ranging from 17 to 43 days. Variable estrous cycle lengths in UGKO ewes were observed repeatably in both Hhx and intact Nor-treated ewes. The IEI in UGKO ewes was not affected by length of postnatal Nor exposure (data not shown). However, all Hhx and intact UGKO ewes responded similarly to exogenous PGF. No differences in length from PGF injection to onset of estrus was noted between control and UGKO ewes. In these ewes, luteolysis was confirmed by a precipitous decline in peripheral plasma concentrations of progesterone after PGF injection (data not shown) and exhibition of behavioral estrus.
Concentrations of progesterone in plasma obtained at 2-day intervals postestrus were affected by treatment (treatment x day, quartic, P < 0.10). Figure 3 illustrates patterns of change in peripheral plasma progesterone concentrations for individual ewes representative of intact control (Group VI) and UGKO (Group IV) groups. In all intact ewes, plasma concentrations of progesterone increased linearly from Day 0 (estrus) to maximal levels between Days 8 and 10 of the estrous cycle. In all control ewes, circulating progesterone concentrations declined precipitously during late diestrus after Day 14 (Fig. 3A). However, this decline, an indicator of functional and structural luteolysis, did not occur consistently in UGKO ewes (Fig. 3, B and C).
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Endometrial Hormone Receptor Expression
Progesterone receptor (PR) protein In uteri from Day 9 Hhx control and UGKO ewes, nuclear staining indicative of PR protein was evident at moderate to high levels in the endometrial epithelium and at lower levels in the stroma and myometrium (Fig. 4A). In Day 9 UGKO uteri, moderate to high levels of nuclear staining indicative of PR protein were also detected in the LE, while lower levels were observed in the stroma (Fig. 4C). In caruncular areas of UGKO uteri, nuclear staining for PR was uniformly positive in LE. In contrast, the ruffled LE of presumptive intercaruncular endometrial areas in uteri from Day 9 UGKO ewes were not as consistently PR positive and exhibited a variegated staining pattern (Fig. 4E). In Day 15 control uteri, PR were undetectable in the LE and superficial GE (Fig. 4B). Low to moderate levels of nuclear PR immunostaining was detected in the deeper GE, stroma, and myometrium (data not shown). Likewise, PR protein in Day 15 UGKO uteri was absent in LE but present at low levels in the stroma and myometrium (Fig. 4D).
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Estrogen receptor (ER) protein
In uteri from Day 9 Hhx control and UGKO ewes, nuclear staining for ER protein was not detected in endometrial epithelium, but it was evident at very low levels in the stroma and myometrium (Fig. 4F). In uteri from Day 15 control ewes, nuclear ER protein was detected in the LE and GE, stroma and myometrium (Fig. 4G). Strong staining for ER protein was detected in the caruncular stroma of uteri from both control and UGKO ewes. In Day 15 UGKO uteri (Fig. 4H), ER immunostaining patterns were similar to those observed in Day 15 control uteri, except for the variegated nature of ER protein expression in the ruffled LE of presumptive intercaruncular endometrial areas (Fig. 4J).
Oxytocin receptor (OTR) mRNA OTR mRNA was not detected in endometrial LE or in GE within the shallow stratum compactum (cGE) of uteri from control or UGKO ewes on Day 9 postestrus (Fig. 5). In contrast, distinct signal indicative of OTR mRNA was detected specifically in LE, cGE, and GE penetrating adluminal regions of the stratum spongiosum (sGE) of uteri from Day 15 cyclic control ewes. In uteri from Day 15 cyclic UGKO ewes, OTR mRNA was also detected in the endometrial LE. No distinct differences in the intensity of the hybridization signal was detected in the endometrial LE between control and UGKO ewes. No variations in the pattern of OTR mRNA expression in the presumptive intercaruncular endometrial LE were observed (data not shown). In the Day 15 control uterus probed with sense ovine OTR cRNA, the black melanocytes displayed an intense white signal in darkfield images, but they were not OTR mRNA positive.
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Oxytocin Challenge and Peripheral Concentration of Plasma PGFM
All intact control and UGKO ewes responded to oxytocin with increased plasma concentrations of PGFM on Day 15 postestrus (Fig. 6), but the response to oxytocin differed between control and UGKO ewes (treatment x time, quartic, P < 0.05). The time required to reach peak peripheral plasma concentrations of PGFM in response to oxytocin was not different between control and UGKO ewes (P > 0.10, 20 vs. 22 ± 1.5 min). However, peak peripheral plasma concentrations of PGFM were more than threefold greater in control compared to UGKO ewes (P < 0.04; 20 876 vs. 6261 ± 1511 pg/ml).
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Effects of UGKO Phenotype on Establishment of Pregnancy
Intact UGKO ewes bred repeatedly to intact rams of proven fertility either failed to become pregnant or failed to maintain pregnancy. Although UGKO ewes did not return to estrus following mating as monitored daily using vasectomized rams, no placentome formation was detected using ultrasonagraphy on Days 25 and 35 postmating. This result was observed in three independent experiments utilizing all intact UGKO ewes. All intact control ewes were mated to the same intact rams, and 80% of these matings produced a successful pregnancy on first service.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Uteri of Nor-exposed ewes at both 16 weeks and in the adult were essentially glandless. Most of these uteri displayed only a ruffled or corrugated LE and compact stroma in intercaruncular areas in which numerous coiled, branched glands are normally found. LE surface undulations were observed in the fetal Day 145 ovine uterus and suggested to be GE buds, which foreshadow uterine gland formation [10]. Thus, Nor may only affect certain processes underlying GE proliferation and branching morphogenesis that are independent of those involved in budding. In the differentiating mouse lung epithelium, Nogawa et al. [24] demonstrated that bud outgrowth was not triggered by induction of localized cell proliferation, suggesting that different mechanisms are involved in epithelial budding and branching morphogenesis. On the other hand, uterine epithelial corrugation in mice was suggested to be a specific morphogenetic response to chronic administration of natural and synthetic progestins [25]. Other than the absence of GE, the only other distinct difference in endometrial morphology observed between UGKO and control ewes was a general loss of loosely arranged stroma characteristic of the stratum spongiosum that was accompanied by increased stromal compaction and abnormal distribution of melanocytes. Thus, the UGKO endometrium appears histologically to consist of caruncular and intercaruncular areas both of which contain densely organized stroma supporting a simple LE. These observations suggest that antiadenogenic conditions imposed by Nor exposure from birth also disrupt organizational processes necessary for stromal zonation and establishment of histologically distinct areas typically defined by the shallow stratum compactum and deeper stratum spongiosum. Although relevant measurements were not made in the present study due to potential complications arising from hemihysterectomy, results from a previous study indicated that total length of uterine horns and uterine wet weight were less in intact adult UGKO ewes than in normal control ewes [12,15]. Thus, exposure of the developing neonatal uterus to a progestin and/or a lack of endometrial glands during prepubertal uterine morphogenesis potentially disrupts epitheliomesenchymal interactions required for normal uterine wall growth and development.
The mechanism by which chronic progestin exposure inhibits endometrial adenogenesis in the neonatal ovine uterus is not well defined. In the mouse uterus, progestins are well established inhibitors of uterine epithelial cell proliferation [26]. In the developing neonatal ovine uterus, Nor exposure for 13 days from birth suppressed proliferation of both intercaruncular epithelium and stroma [14]. The actions of progestins on both epithelium and stroma in the neonatal ovine uterus may be direct, since PR expression was detected immunohistochemically in both cell types as early as PND 1 in ewe lambs [27]. In breast cancer cells, synthetic progestins inhibit proliferation by arrest of cells in G1 phase of the cell cycle. This arrest appears to be mediated by decreased cyclin-dependent kinase (CDK) activity through decreased cyclin expression and increased CDK inhibitor association [28]. In the mouse uterus, Tong and Pollard [29] showed that progesterone inhibited estrogen-induced CDK activation, cyclin expression, and phosphorylation of retinoblastoma (Rb) in the GE. In addition to effects on the cell cycle, a classic effect of progesterone is to suppress expression of ER in the endometrial epithelium and stroma of the adult ovine uterus [30–31]. In both the pig and sheep, appearance of endometrial glands between birth and PND 14 involves development of ER-positive phenotype by, and increased DNA synthesis in, nascent GE buds [14,27,32;ch33]. Endometrial adenogenesis appears to require activated ER, because gland genesis in newborn pigs is inhibited by administration of the ER antagonist ICI 182,780 [34]. Interestingly, ICI administration to neonatal gilts inhibited GE cell proliferation and increased stromal cell compaction. Alterations in the stroma could potentially disrupt cell-cell interactions required for GE budding, proliferation and branching morphogenesis. In the developing mouse uterus, the mesenchyme is necessary to organize and induce patterns of epithelial development, and the epithelium is required to support the organization and differentiation of the endometrial stroma and myometrium [35,36]. In addition, epitheliomesenchymal interactions have been implicated in development of endometrial glands in the neonatal ovine and porcine uterus [12,32]. Collectively, available results suggest that progestins could block endometrial adenogenesis by 1) inhibiting cell cycle transition in the LE and stroma, 2) suppressing ER gene expression, and/or 3) altering critical epitheliomesenchymal interactions.
In this study, all intact and Hhx control and UGKO ewes attained puberty and exhibited normal estrous behavior by 8 months of age. However, Hhx and intact UGKO ewes displayed large variations in the interval to next estrus. It is possible that, in addition to disruption of endometrial organization and inhibition of endometrial adenogenesis, chronic progestin exposure from birth affected development of one more components of the hypothalamic-pituitary-ovarian axis negatively. Treatment of intact UGKO ewes with PGF did lyse the functional CL and cause ewes to return to estrus. Although subsequent interestrous intervals varied in length, UGKO ewes responded to exogenous PGF consistently and repeatedly with behavioral estrus that was preceded by structural and functional luteolysis as evidenced by a decline in peripheral plasma concentrations of progesterone after Day 14. Moreover, no differences in ovarian function were detected between control and UGKO ewes during metestrus or early diestrus of PGF-induced estrous cycles as reflected by peripheral plasma progesterone concentrations. Thus, luteinization and CL function after ovulation appeared to be normal in UGKO ewes. Collectively, these observations strongly suggest that development and function of the brain, hypothalamus, pituitary, or ovary in adult UGKO ewes was not affected adversely by chronic neonatal progestin exposure necessary to create the UGKO phenotype. The variability in estrous cycle length was then hypothesized to be due to inadequate development of the endometrial luteolytic prostaglandin generating mechanism.
The estrous cycle of sheep is uterine-dependent because the endometrial epithelium produces the PGF, the natural luteolysin. The luteolytic mechanism is controlled by carefully orchestrated changes in PR, ER, and OTR gene expression in the endometrial LE and sGE [30,37;ch39]. In cyclic ewes, the endometrial epithelium contains abundant ER and PR at estrus (Day 0) due to exposure of the endometrium to high levels of estrogen produced by the ovulatory follicle. With ovulation and luteinization, progesterone levels increase and act through epithelial PR to suppress expression of ER and OTR gene expression in the endometrial LE and cGE. This block to ER and OTR expression by progesterone is limited, because chronic exposure of the endometrium to progesterone for 10 to 12 days negatively autoregulates PR gene expression [17,30]. Down-regulation of epithelial PR allows for transcriptional up-regulation of ER and then OTR gene expression in the endometrial LE and cGE [17]. After Day 11 of the estrous cycle, PR mRNA and protein expression is undetectable in the LE and cGE. In the absence of functional PR, epithelial ER mRNA and protein expression increases between Days 11 and 13 followed by increases in OTR mRNA and protein expression on Days 14 and 15 in the LE and cGE [38,39]. After Day 15, OTR expression spreads to the GE of middle and deep endometrium. Available results indicate that endometrial OTR gene expression is increased by estrogen acting through ER [17,40]. Expression of cyclooxygenase-1 (COX-1) and COX-2 are already at peak levels by Day 14–15 [41]. Collectively, these events allow oxytocin, which is released from the CL and posterior pituitary beginning on Day 10, to stimulate OTR on the endometrial LE and cGE. Thus, the endometrium releases luteolytic pulses of PGF on Days 15–16 [42,43]. Consequently, functional and structural regression of the CL occurs between Days 15 and 16, and the ewe returns to estrus on Day 17.
Consistent with previous findings [30], PR protein expression was high in LE of both intact control and UGKO ewes on Day 9 of the estrous cycle, whereas ER protein could not be detected. On Day 15 of the estrous cycle, progesterone appeared to have down-regulated PR expression in LE and cGE, because no PR protein was detected in these epithelia. However, ER protein was detected in the LE of both control and UGKO ewes. These results suggest that steroid hormone regulation of epithelial PR and ER expression in the uterus of the adult UGKO ewe was not affected significantly by neonatal progestin exposure. The variegated nature of epithelial PR and ER staining in the uteri of UGKO ewes suggests that some of the LE or GE cells may have an altered or transformed phenotype due to chronic exposure of the neonatal uterine epithelium to Nor. As expected, OTR mRNA was not detected in endometrial epithelia of uteri from Day 9 cyclic control ewes, or in UGKO ewes. In uteri from Day 15 control ewes, abundant OTR mRNA was located in both the LE and cGE. Likewise, abundant OTR mRNA was evident in endometrial LE of uteri obtained from UGKO ewes on Day 15 postestrus. The total OTR content in the endometrium is probably lower in UGKO uteri, because they lack glands and appear to have a lower total amount of LE [CA Gray and TE Spencer, unpublished results]. Results of the oxytocin challenge supported this contention. The pattern of PGFM release after oxytocin administration was not different between intact control and UGKO ewes. However, peak peripheral plasma concentrations of PGFM in response to oxytocin was three-fold lower in UGKO compared to control ewes on Day 15 of the estrous cycle. Similar results were obtained in adult heifers exposed chronically to progesterone plus estradiol benzoate (PE) for approximately 200 days from birth [12]. Compared to unexposed control heifers, uteri of PE-exposed adult heifers were smaller, contained few or no endometrial glands, had less endometrial OTR numbers, and produced lower peak levels of PGFM in response to oxytocin. The amount of PGF produced by the uterus is directly related to the number of OTR present in the endometrium [44]. In this light, results of the present study can be interpreted to indicate that the endometrium of UGKO ewes that fail to cycle lacks sufficient functional mass and, therefore, contains insufficient OTR to produce a luteolytic PGF signal in response to oxytocin. Failure of UGKO ewes to cycle regularly may also stem from the specific absence of superficial glands that may be necessary to generate the luteolytic PGF signal. In addition, the number of functional OTR in endometrial LE of UGKO ewes may be altered as suggested by inconsistent patterns of ER and PR, as well as OTR expression in this cell compartment. In any event, results of the present study are consistent with the idea that OTR expressed in GE are required for endometrial production luteolytic pulses of PGF in reponse to oxytocin. Repeated breeding of intact UGKO ewes failed to produce a successful pregnancy. These results suggest that UGKO ewes are unable to establish and/or maintain pregnancy. Three UGKO ewes were bred at estrus and the uterine horns flushed on Day 13 postmating [CA Gray and TE Spencer, unpublished results]. The uterine flush of all ewes contained no morphologically recognizable conceptus tissues, although two of the three flushes contained significant amounts of antiviral activity, an indicator of the presence of interferon tau, the pregnancy recognition signal. If confirmed, these results would suggest that UGKO ewes exhibit an inability to support periattachment conceptus growth and development. This observation would lend strong support to the idea that secretions from the endometrial glandular epithelium influence preattachment conceptus development and onset of pregnancy recognition signals. In rodents, successful embryo implantation requires at least two exclusively GE synthesized and secreted proteins, leukemia inhibitory factor [45] and calcitonin [46]. A reduction or absence of one of these factors renders the mouse infertile because the embryo is unable to implant into the uterine wall. Spencer et al. [9] recently demonstrated that many differences in gene expression exist between normal and UGKO uteri. The majority of these differences are related to the absence of endometrial glands in UGKO ewes. Discovery and identification of the genes expressed by uterine glands may help to define which components of uterine histotroph are required to support periattachment conceptus development.
Collectively, results presented here support the idea that a properly organized endometrium and uterine endometrial glands are indispensable components of the uterine wall and are required for proper uterine function in both cyclic and pregnant ewes. The ovine UGKO phenotype provides a unique model with which to study developmental mechanisms regulating endometrial adenogenesis in the neonate, as well as function of endometrial glands in the adult. This study and others [12,14] provide evidence that the ewe is a useful model for evaluation of the effects of endocrine-active compounds on female urogenital tract development, and the consequences of such developmental disruption on subsequent reproductive performance. Physiological, biochemical, and molecular comparisons of neonatal and adult uterine tissues from normal and UGKO ewes should enable the identification of factors affecting uterine function, developmental determinants of uterine integrity, biological markers of exposure to endocrine disrupters, and uterine genes essential for gland development and function [see 9,12].
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported in part by NRI Competitive Grants Program/USDA Grants 98-35203-6322 to T.E.S. and 95-37203-1995 and 98-35203-6198 and to F.F.B. 
2 Correspondence: Thomas E. Spencer, Center for Animal Biotechnology and Genomics, 444 Kleberg Center, Texas A&M University, College Station, TX 77843-2471. FAX: 409 862 2662; tspencer{at}cvm.tamu.edu
3 Current address: Department of Animal and Veterinary Science, University of Idaho, Moscow, ID 83844.
Accepted: September 21, 1999.
Received: July 28, 1999.
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and other cytokines in early pregnancy. Reprod Fertil Dev 1997; 9:355–380.[CrossRef][Medline] release by the oestradiol and oxytocin. J Reprod Fertil 1986; 76:425–433. inhibits estrogen receptor up-regulation and estrogen-induced luteolysis in cyclic ewes. Endocrinology 1995; 136:4932–4944.[Abstract] and progesterone on oestrogen-stimulated expression of receptors for oestrogen, progesterone and oxytocin in the endometrium of ovariectomized ewes. Reprod Fertil Dev 1996; 8:843–853.[CrossRef][Medline]This article has been cited by other articles:
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  P. A.R. Hawken, T. J. d. St. Jorre, J. Rodger, T. Esmaili, D. Blache, and G. B. Martin Rapid Induction of Cell Proliferation in the Adult Female Ungulate Brain (Ovis aries) Associated with Activation of the Reproductive Axis by Exposure to Unfamiliar Males Biol Reprod, June 1, 2009; 80(6): 1146 - 1151. [Abstract] [Full Text] [PDF]
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 J. Kim, G. Song, H. Gao, J. L. Farmer, M. C. Satterfield, R. C. Burghardt, G. Wu, G. A. Johnson, T. E. Spencer, and F. W. Bazer Insulin-Like Growth Factor II Activates Phosphatidylinositol 3-Kinase-Protooncogenic Protein Kinase 1 and Mitogen-Activated Protein Kinase Cell Signaling Pathways, and Stimulates Migration of Ovine Trophectoderm Cells Endocrinology, June 1, 2008; 149(6): 3085 - 3094. [Abstract] [Full Text] [PDF]
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 T. E Spencer, O. Sandra, and E. Wolf Genes involved in conceptus-endometrial interactions in ruminants: insights from reductionism and thoughts on holistic approaches Reproduction, February 1, 2008; 135(2): 165 - 179. [Abstract] [Full Text] [PDF]
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 J. L. Farmer, R. C. Burghardt, F. D. Jousan, P. J. Hansen, F. W. Bazer, and T. E. Spencer Galectin 15 (LGALS15) functions in trophectoderm migration and attachment FASEB J, February 1, 2008; 22(2): 548 - 560. [Abstract] [Full Text] [PDF]
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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]
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R. G Lea and O. Sandra Immunoendocrine aspects of endometrial function and implantation Reproduction, September 1, 2007; 134(3): 389 - 404. [Abstract] [Full Text] [PDF]
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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]
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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]
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C. A. Gray, C. A. Abbey, P. D. Beremand, Y. Choi, J. L. Farmer, D. L. Adelson, T. L. Thomas, F. W. Bazer, and T. E. Spencer Identification of Endometrial Genes Regulated by Early Pregnancy, Progesterone, and Interferon Tau in the Ovine Uterus Biol Reprod, February 1, 2006; 74(2): 383 - 394. [Abstract] [Full Text] [PDF]
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 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]
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T. E Spencer, G. A Johnson, F. W Bazer, and R. C Burghardt Implantation mechanisms: insights from the sheep Reproduction, December 1, 2004; 128(6): 657 - 668. [Abstract] [Full Text] [PDF]
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T. E. Spencer, G. A. Johnson, R. C. Burghardt, and F. W. Bazer Progesterone and Placental Hormone Actions on the Uterus: Insights from Domestic Animals Biol Reprod, July 1, 2004; 71(1): 2 - 10. [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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G. A. Johnson, R. C. Burghardt, F. W. Bazer, and T. E. Spencer Osteopontin: Roles in Implantation and Placentation Biol Reprod, November 1, 2003; 69(5): 1458 - 1471. [Abstract] [Full Text] [PDF]
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B. J. Tarleton, T. D. Braden, A. A. Wiley, and F. F. Bartol Estrogen-Induced Disruption of Neonatal Porcine Uterine Development Alters Adult Uterine Function Biol Reprod, April 1, 2003; 68(4): 1387 - 1393. [Abstract] [Full Text] [PDF]
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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]
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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]
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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]
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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]
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C. Chen, T. E. Spencer, and F. W. Bazer Fibroblast Growth Factor-10: A Stromal Mediator of Epithelial Functionin the Ovine Uterus Biol Reprod, September 1, 2000; 63(3): 959 - 966. [Abstract] [Full Text]
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K. M. Taylor, C. A. Gray, M. M. Joyce, M. D. Stewart, F. W. Bazer, and T. E. Spencer Neonatal Ovine Uterine Development Involves Alterations in Expression of Receptors for Estrogen, Progesterone, and Prolactin Biol Reprod, April 1, 2000; 63(4): 1192 - 1204. [Abstract] [Full Text]
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