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a Center for Animal Biotechnology, Albert B. Alkek Institute of Biosciences and Technology, Texas A&M University, College Station, Texas 77843-2471
b Department of Animal Science, Auburn University, Auburn, Alabama 36849
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The process of superficial implantation undoubtedly involves a precisely orchestrated adhesion cascade that is similar to models developed from studies of lymphocyte cell adhesion [7]. Superficial implantation in sheep starts on Days 15–16 and is preceded by a prolonged preimplantation period after entry of the embryo into the uterus around Day 4 [8, 9]. Current models of implantation in domestic ungulates implicate a step-wise process involving preattachment conceptus elongation, apposition of the trophectoderm and uterine luminal epithelium (LE), adhesion of the conceptus trophectoderm to the apical portion of the endometrial LE, and syncytium formation [9]. This process may involve a precisely orchestrated adhesion cascade that culminates in the firm attachment of the trophectoderm to the endometrial LE by integrins [10]. GlyCAM-1 expressed by the ovine uterus could function as a mediator of heterologous cell-cell interactions involving the conceptus trophectoderm and endometrial LE during the periimplantation period of pregnancy. Therefore, the present studies were designed to determine 1) whether GlyCAM-1-like protein is expressed in the ovine uterus and 2) the effects of the estrous cycle and pregnancy on temporal and spatial patterns of GlyCAM-1-like protein expression in the ovine uterus.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Mature ewes of primarily Rambouillet breeding were observed daily for estrous behavior using vasectomized rams. All ewes exhibited at least two estrous cycles of normal duration (~16–18 days) before use in these studies. All 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 (Day 0), ewes were assigned randomly to cyclic or pregnant status. Ewes assigned to pregnant status were bred at estrus and at 12 h and 24 h postestrus with intact rams. Fifty-two ewes were ovariohysterectomized (n = 4 ewes per day) on Days 1, 3, 5, 7, 9, 11, 13, and 15 of the estrous cycle or Days 11, 13, 15, 17, and 19 of pregnancy. In cyclic ewes and in pregnant ewes on Days 11–17, the uterine lumen was flushed with 20 ml sterile saline at hysterectomy. Pregnancy was confirmed by presence of an apparently normal conceptus in uterine flushings (Days 11–17) or extension of the interestrous interval (Day 19). Uterine flushes were clarified by centrifugation (3000 x g for 10 min at 4°C), aliquoted, and frozen at -80°C. In Day 11–17 pregnant ewes, the conceptus was recovered from the uterine flush and fixed in fresh 4% paraformaldehyde in PBS (pH 7.2). 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. 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).
Immunohistochemistry
Immunoreactive GlyCAM-1 protein was localized in cross sections of the uterine horn (minimum of two sections per ewe per horn) using affinity-purified rabbit anti-rat GlyCAM-1 IgG (CAM02) [11] or rabbit IgG (Sigma Chemical Co., St. Louis, MO) as a negative control and a Super ABC Rabbit Kit (Biomeda, Foster City, CA) as described previously [12]. The CAM02 antibody was generated in rabbits against a peptide (CKEPSIFREELISKD) and purified by peptide column chromatography [11]. This affinity-purified IgG was kindly provided by Dr. Steven D. Rosen (University of California, San Francisco, CA). Endogenous peroxidase activity was quenched by incubating rehydrated tissue sections in methanol containing 1% hydrogen peroxide for 15 min at room temperature. Other negative controls included omission of primary and/or secondary antibodies.
Sections (5–7 µm) of the entire uterine horn from cyclic ewes and the ipsilateral uterine horn of pregnant ewes were affixed to Superfrost Plus slides (Fisher Scientific, Pittsburgh, PA). For immunolocalization, sections were deparaffinized in xylene and rehydrated to water through a graded alcohol series. Sections were then incubated with rabbit anti-rat GlyCAM-1 IgG (CAM02; 2 µg/ml in PBS containing 1% BSA) or rabbit IgG (2 µg/ml in PBS-BSA; Sigma) as a negative control for at least 16 h at 4°C. Slides were then allowed to warm to room temperature for 1 h, and immunoreactive protein was visualized in sections using an avidin-biotin-peroxidase complex according to kit instructions (Biomeda) and diaminobenzidine tetrahydrochloride (Sigma) as the color substrate. Sections were lightly counterstained with Gill's hematoxylin, dehydrated, and coverslipped with Permount (Fisher).
Uterine tissue sections from all ewes, representing each day and pregnancy status, were processed together. Staining intensities were scored visually (absent, weak, moderate, or strong) by two independent observers for three endometrial tissues (luminal epithelium, glandular epithelium, and stroma), two glandular epithelial cell types (shallow or stratum compactum and deep or stratum spongiosum), two myometrial cell types (inner circular and outer longitudinal), and the conceptus in uteri of pregnant ewes. Photomicrographs of representative fields of immunohistochemistry slides were taken in brightfield using a Zeiss (New York, NY) photomicroscope fitted with a CC10B color compensating filter (Eastman Kodak, Rochester, NY) for use of daylight film with a tungsten light source and Kodacolor Gold 100 (Eastman Kodak) color film.
Preparation of Endometrial Protein Extracts
Endometrial tissues stored at -80°C were homogenized in extraction buffer (10 mM Tris, pH 7, 1 mM EDTA, 1 mM dithiothreitol, 100 µg/ml PMSF) at a ratio of 5 ml buffer per 1 g tissue. Homogenates were sonicated for 30 sec with a Mini Ultrasonic Cell Disrupter (Sonics&Materials, Inc., Danbury, CT) and cleared by centrifugation (10 000 x g for 15 min at 4°C). Supernatants were then aliquoted and frozen at -80°C.
Western Blot Analyses
Concentrations of protein in uterine flushes and endometrial extracts were determined using the Bradford protein assay (Bio-Rad, Richmond, CA) with BSA as the standard. Proteins in uterine flushings (20 µg) or endometrial extracts (120 µg) were denatured in Laemmli buffer, separated on a 15% (total monomer) SDS-PAGE gel, and transferred to nitrocellulose as described previously [13]. Kaleidoscope prestained SDS-PAGE standards (Bio-Rad) were loaded on each gel. Blots were blocked overnight at 4°C in TBST (20 mM Tris [pH 7.5], 137 mM NaCl, 0.05% Tween 20) containing 5% dried milk. Blots were then incubated with rabbit anti-rat GlyCAM-1 IgG (CAM02; 1 µg/ml) or normal rabbit IgG (1 µg/ml) in TBST containing 2% dried milk with rocking overnight at 4°C. Blots were washed four times in TBST for 5 min each and placed in goat anti-rabbit IgG-horseradish peroxidase conjugate (1:15 000 dilution of 1 mg/ml stock; KPL, Bethesda, MD) for 1 h at room temperature with shaking. Blots were washed four times in TBST for 10 min each. Immunoreactive proteins were detected using enhanced chemiluminescence (Amersham Life Sciences, Arlington Heights, IL) and X-OMAT AR film (Eastman Kodak).
Immunoprecipitation Assay
Proteins in endometrial extracts (250 µg) or uterine flushes (100 µg) from Day 17 pregnant ewes were immunoprecipitated as described previously [13] with a few modifications. Protein extracts or uterine flushes were brought to a volume of 1 ml with IPH lysis buffer (50 mM Tris-HCl [pH 8], 150 mM NaCl, 5 mM EDTA, 0.5% Nonidet P-40, 0.1 mM PMSF). Rabbit anti-rat GlyCAM-1 IgG (CAM02; 3 µg) or rabbit IgG (3 µg) was added to relevant tubes, and incubation with rocking was performed at 4°C for 4 h. Protein A/G-Sepharose beads (20 µl; Santa Cruz Biotechnology, Santa Cruz, CA) were added, and the mixture was incubated with rocking at 4°C overnight. Beads were then pelleted by gentle centrifugation (1000 x g for 3 min) and washed with 1 ml of IPH lysis buffer for a total of four times. All centrifugation steps and washes were performed at 4°C. After the final wash, all remaining wash buffer was removed, and beads were solubilized in 40 µl SDS-PAGE loading buffer. Tubes containing beads were denatured at 70°C for 5 min, cooled on ice for 2 min, and centrifuged at 12 000 x g for 10 min. Supernatants were loaded onto a 15% SDS-PAGE gel along with endometrial extract (25 µg) or uterine flush (10 µg) and prestained molecular weight standards. After separation by electrophoresis, proteins were transferred and processed for Western blot analysis with rabbit anti-rat GlyCAM-1 IgG as described above.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Immunoreactive GlyCAM-1-like protein was detectable in uterine tissues from both cyclic and pregnant ewes (Figs. 1 and 2). In the endometrium, immunostaining patterns for GlyCAM-1 were affected by both day of the estrous cycle and pregnancy as summarized in Table 1. Staining of the LE and stratum compactum glandular epithelium (cGE) was readily detected in the shallow stroma but not in the stroma or immune cells. The smooth muscle of the endometrial vasculature, or media intima, and myometrium constitutively expressed moderate to abundant levels of immunoreactive GlyCAM-1, but there was no effect of day of the cycle or pregnancy status on staining intensity in these uterine smooth muscle cell types.
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During the estrous cycle (see Fig. 1 and Table 1), staining for GlyCAM-1-like protein in the endometrial LE and cGE increased from weak to moderate between Days 1 and 5, remained moderate in intensity on Days 5–11, and then declined to weak or absent on Day 15. Cyclic alterations in GlyCAM-1 staining intensity for the cGE paralleled those of the LE, except that low-intensity staining was detected on Day 1. In glandular epithelium in the deep stroma or stratum spongiosum glandular epithelium, GlyCAM-1 staining was not detected on Days 1 and 3, increased only slightly on Days 5–9, and was either weak or absent thereafter. Staining in the smooth muscle or media intima of the endometrial vasculature and myometrium was moderate to strong and did not change over the days of the estrous cycle. In the endometrial vasculature, staining for GlyCAM-1 was not detected in the endothelium. No significant staining was detected in control sections using normal rabbit IgG (Fig. 1) or when the secondary antibody was omitted (data not shown).
During pregnancy (see Fig. 2 and Table 1), immunoreactive GlyCAM-1-like protein was detected in the endometrial LE and cGE, trophectoderm of the conceptus, and smooth muscle of the endometrial vasculature and myometrium. In the endometrial LE and cGE, staining for GlyCAM-1 was moderate to weak on Days 11 and 13 and not different from that observed in cyclic ewes. In marked contrast, moderate to strong GlyCAM-1 staining was detected in the endometrial LE on Day 15 (Fig. 2C). Staining was particularly intense on Day 15 of pregnancy as compared to Day 15 of the cycle. This increase in GlyCAM-1 staining in the endometrial LE persisted on Days 17 and 19 of pregnancy. Weak to moderate staining for GlyCAM-1 was also detected in the conceptus trophectoderm on Days 13–19 but not in the endoderm or embryo (data not shown). As illustrated in Figure 2, GlyCAM-1 was present in the endometrial LE and glandular epithelium, as well as in the trophectoderm of the conceptus on Day 19. As observed in uteri of cyclic ewes, moderate to strong staining for GlyCAM-1 was detected in the smooth muscle of the endometrial vasculature and myometrium (data not shown), and this staining was not affected by day of pregnancy. Further, GlyCAM-1 was not detected in the stroma, endothelium, or intraepithelial immune cells (Fig. 2). No significant staining was detected in control sections with normal rabbit IgG (Fig. 2I) or when the secondary antibody was omitted (data not shown).
Western Blot Analysis of Immunoreactive GlyCAM-1 in Endometrial Extracts and Uterine Flushings
In endometrial extracts of both cyclic and pregnant ewes, Western blot analysis with affinity-purified rabbit anti-rat GlyCAM-1 IgG detected two major immunoreactive proteins of ~45 and 25 kDa and one minor protein of ~40 kDa (Fig. 3). No specific proteins were detected in a duplicate Western blot with the same concentration of normal rabbit IgG exposed at the same time (Fig. 3).
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In uterine flushes of both cyclic and pregnant ewes, Western blot analysis with GlyCAM-1 antibody detected a single immunoreactive protein of ~45 kDa (Fig. 4, A and B). Analysis of uterine flushes from all cyclic ewes detected GlyCAM-1 at very low levels. The 45-kDa species of GlyCAM-1 was present in uterine flushes of all cyclic ewes except on Days 1 and 15 (data not shown). As shown in Figure 4B, the amount of immunoreactive GlyCAM-1-like protein increased in uterine flushings of pregnant ewes after Day 13 and was abundant thereafter. No proteins were detected in a duplicate Western blot with normal rabbit IgG (Fig. 4C).
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Figure 5 illustrates results of an immunoprecipitation assay using proteins in Day 17 pregnant ovine endometrial extracts and Day 17 uterine flushes. Immunoreactive proteins were precipitated using rabbit anti-rat GlyCAM-1 IgG or rabbit IgG and protein A/G-Sepharose beads. A 5-min exposure of one tenth the protein input into each immunoprecipitation is shown; a 10-sec exposure of the immunoprecipitated proteins is shown to the right of the input lanes. Rabbit anti-rat GlyCAM-1 IgG, but not rabbit IgG, specifically immunoprecipitated a protein (~45 kDa) that was identical in size to immunoreactive GlyCAM-1 in the endometria and uterine flushes.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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During early pregnancy, staining patterns of the GlyCAM-1-like protein were affected by the conceptus. The staining intensity in the endometrial LE and cGE of Day 11 and 13 pregnant ewes was not substantially different from that observed in Day 11 and 13 cyclic ewes. In contrast, GlyCAM-1 staining was moderate to strong in the endometrial epithelium on Day 15 of pregnancy and was strong in the LE and cGE in Day 17 and Day 19 pregnant ewes. The pattern of immunoreactive GlyCAM-1 staining in endometrial epithelium paralleled changes in abundance of GlyCAM-1 in uterine flushings detected by Western blot analysis. In early-pregnant ewes, PR mRNA and protein are undetectable in endometrial LE and cGE after Day 11 [12]. Between Days 11 and 13, the conceptus undergoes elongation and begins superficial implantation on Days 15–16 [9]. Thus, GlyCAM-1 expression in the endometrial epithelium during early pregnancy may be regulated by factors from the periimplantation conceptus and is probably not influenced directly by progesterone, given the lack of PR in endometrial epithelium.
Immunoreactive GlyCAM-1-like protein was detected in the trophectoderm of Day 13–19 conceptuses. In general, intense staining was always more abundant in endometrial LE and cGE than in conceptus trophectoderm. The mechanism regulating conceptus GlyCAM-1 expression is unknown, but it may involve progesterone. The conceptus trophectoderm contains abundant levels of immunoreactive PR (unpublished results). Thus, it is possible that ovarian progesterone and conceptus-derived 5ß-dihydroprogesterone [15] may promote expression of GlyCAM-1 in the conceptus trophectoderm. The immunoreactive ~45-kDa protein detected in endometrial extracts was not different in size from the immunoreactive GlyCAM-1 protein found in Day 17 pregnant uterine flush. Collectively, results presented here indicate that an immunoreactive GlyCAM-1-like protein is present in the ovine uterus, uterine luminal fluid, and conceptus. Therefore, the increase in abundance of GlyCAM-1 in the uterine flush during pregnancy on Days 15 and 17 may represent synthesis and secretion by the endometrium and/or the conceptus. Another possible origin for GlyCAM-1-like protein in the uterus is endothelial transport or as a serum transudate as a consequence of increased uterine capillary permeability during early pregnancy.
Western blot analysis of endometrial extracts and endometrial explant culture medium detected at least three immunoreactive GlyCAM-1 protein species: two strongly reactive proteins of ~25 kDa and ~45 kDa and one minor reactive protein of ~40 kDa. The presence of the 40-kDa species was variable and may be due to partial proteolysis of mature GlyCAM-1. The ~45-kDa species detected in uterine flushes and conceptus culture medium was efficiently immunoprecipitated from endometrial extracts and uterine flushes using rabbit anti-rat GlyCAM-1 IgG. The three GlyCAM-1 species detected in the endometrium likely represent different levels of posttranslational modification, whereas only the mature 45-kDa species is secreted by the endometrial epithelium and conceptus. This idea is supported by studies of GlyCAM-1 in lymph node organ cultures [16], wherein several species of GlyCAM-1 can be detected, including forms that are unglycosylated (< 28 kDa), modified with N-acetyl-galactosamine only (28–33 kDa), and modified with sialic acid, fucose, and sulfate (40–50 kDa) but lacking L-selectin reactivity. Mature endothelial GlyCAM-1 is secreted as a 50- to 60-kDa protein and is the ligand for L-selectin [17]. It remains to be determined how the GlyCAM-1 that is present in the uterine lumen is posttranslationally modified, but sugars present in mature lymphoid GlyCAM-1 are indeed present in the sheep endometrial epithelium and conceptus during early pregnancy [18]. Whyte and Robson [18] reported that Day 14 and Day 17 pregnant sheep endometrial LE and conceptus trophectoderm reacted strongly with a lectin specific for 
-L-fucose. Further, the lectin staining intensity was reduced by trypsin digestion, suggesting that the fucose was associated with glycoprotein instead of glycolipid. The acquisition of specific glycoproteins as detected by lectins has been implicated as attachment factors and markers of uterine receptivity in the endometrium of many species [7, 9, 10].
In addition to observations in the endometrial epithelium and conceptus, moderate to abundant levels of GlyCAM-1-like protein were detected in smooth muscle cells of the uterine vasculature and myometrium. Staining for GlyCAM-1 was not affected by either day or pregnancy status, suggesting constitutive expression in these cell types. GlyCAM-1 expression has not been reported for smooth muscle in other organs, and the significance of the present observations is unclear. Moreover, this intriguing pattern of expression in the uterine wall has not been reported for any other secretory protein in the ovine uterus. Because ovine endometrial GlyCAM-1-like protein is not present in endothelium of the endometrial vasculature, it probably does not serve a role in lymphocyte trafficking as shown for lymphoid GlyCAM-1.
GlyCAM-1 expression has also been reported in rat, ovine, caprine, and bovine mammary gland, where it is localized to epithelial cells and secreted into milk [5, 6, 19]. In rat and bovine mammary gland tissue, GlyCAM-1 mRNA expression is hormonally regulated and has been found to increase during pregnancy and lactation [5, 19]. In the bovine mammary gland, the protein is called proteose peptone component 3 (PP3); it displays homology and structural conservation with mouse and rat GlyCAM-1 [20]. The secreted species of PP3 in ruminant milk is ~22 kDa [6], whereas GlyCAM-1 in rat milk is ~35 kDa [5]. In the present study, the ~45-kDa species of ovine GlyCAM-1 was closer in size to the ~50-kDa endothelial-secreted lymphoid GlyCAM-1 [17]. The posttranslational sulfate modification of endothelial GlyCAM-1, which is required for it to serve as a ligand for L-selectin, is not present in mammary gland GlyCAM-1 [5]. However, the nucleotide sequence of rat mammary gland GlyCAM-1 mRNA is identical to the sequence of that expressed in rat lymph nodes [14]. Thus, tissue-specific posttranslational modifications in the GlyCAM-1 protein can significantly alter function. Similarly, PP3 is structurally homologous to GlyCAM-1, but it is unlikely that PP3 is the functional analogue of GlyCAM-1 [20].
In lymphoid organs, lymphocyte recirculation involves a cascade of adhesion molecules that serves as a model for conceptus-maternal interactions during the periimplantation period [7, 10, 21]. GlyCAM-1 is involved in the initial interaction between lymphocytes and the endothelium by serving as a secreted endothelial ligand for L-selectin [22]. Treatment of lymphocytes with GlyCAM-1 stimulates adhesion through the ß2 integrin pathway [3]. Moreover, Giblin et al. [4] recently demonstrated that ligation of L-selectin on T lymphocytes activates ß1 integrins and promotes adhesion to fibronectin. Activation of ß1 and ß2 integrins by GlyCAM-1 facilitates firm adhesion of lymphocytes to high endothelial venules in lymph nodes [3, 4]. In the periimplantation pig uterus, Bowen et al. [23] found that the integrin subunits 4, 5, and ß1 were regulated by progesterone and abundantly expressed by the uterine epithelium and trophectoderm, whereas expression of v and ß3 by these tissues was constitutive. Selectin and integrin subunit expression in the ovine conceptus trophectoderm and endometrial epithelium during the periimplantation period is unknown. The presence and pattern of GlyCAM-1-like protein expression in cyclic and pregnant sheep endometria and conceptuses suggest a role in conceptus-maternal interactions during the periimplantation period of pregnancy. It is reasonable to speculate that GlyCAM-1 in the uterine lumen may bind to specific receptors on the conceptus and/or endometrial LE that activate certain integrins to facilitate firm adhesion of the trophectoderm to the LE at the beginning of implantation on Days 15–16 postmating. Moreover, GlyCAM-1 may mediate interactions between the uterine mucosa and intraepithelial lymphocytes in the uterine lumen. Further studies are needed to determine the biochemical nature and ligand activity of purified ovine endometrial GlyCAM-1. Such studies, combined with knowledge of selectin and integrin expression in the uterus and conceptus, should allow for determination of the true physiological function of GlyCAM-1 in the ovine uterus.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Accepted: October 10, 1998.
Received: May 8, 1998.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Z. Hou, J. P. Bailey, A. J. Vomachka, M. Matsuda, J. A. Lockefeer, and N. D. Horseman Glycosylation-Dependent Cell Adhesion Molecule 1 (GlyCAM 1) Is Induced by Prolactin and Suppressed by Progesterone in Mammary Epithelium Endocrinology, November 1, 2000; 141(11): 4278 - 4283. [Abstract] [Full Text] [PDF]
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M. D. Stewart, G. A. Johnson, C. A. Gray, R. C. Burghardt, L. A. Schuler, M. M. Joyce, F. W. Bazer, and T. E. Spencer Prolactin Receptor and Uterine Milk Protein Expression in the Ovine Endometrium During the Estrous Cycle and Pregnancy Biol Reprod, June 1, 2000; 62(6): 1779 - 1789. [Abstract] [Full Text]
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C. Chen, T. E. Spencer, and F. W. Bazer Expression of Hepatocyte Growth Factor and Its Receptor c-metin the Ovine Uterus Biol Reprod, June 1, 2000; 62(6): 1844 - 1850. [Abstract] [Full Text]
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G. A. Johnson, T. E. Spencer, R. C. Burghardt, K. M. Taylor, C. A. Gray, and F. W. Bazer Progesterone Modulation of Osteopontin Gene Expression in the Ovine Uterus Biol Reprod, May 1, 2000; 62(5): 1315 - 1321. [Abstract] [Full Text]
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G. A. Johnson, T. E. Spencer, R. C. Burghardt, M. M. Joyce, and F. W. Bazer Interferon-Tau and Progesterone Regulate Ubiquitin Cross-Reactive Protein Expression in the Ovine Uterus Biol Reprod, March 1, 2000; 62(3): 622 - 627. [Abstract] [Full Text]
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G. A. Johnson, R. C. Burghardt, G. R. Newton, F. W. Bazer, and T. E. Spencer Development and Characterization of Immortalized Ovine Endometrial Cell Lines Biol Reprod, November 1, 1999; 61(5): 1324 - 1330. [Abstract] [Full Text]
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T. E. Spencer, A. G. Stagg, M. M. Joyce, G. Jenster, C. G. Wood, F. W. Bazer, A. A. Wiley, and F. F. Bartol Discovery and Characterization of Endometrial Epithelial Messenger Ribonucleic Acids Using the Ovine Uterine Gland Knockout Model Endocrinology, September 1, 1999; 140(9): 4070 - 4080. [Abstract] [Full Text]
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