ARTICLE |
Correspondence to: Firyal S. Khan-Dawood, Dept. of Obstetrics, Gynecology and Reproductive Sciences, U. of Texas Medical School at Houston, 6431 Fannin, Suite 3.204, Houston, TX 77030.
Correspondence to: M. Yusoff Dawood, Dept. of Obstetrics, Gynecology and Reproductive Sciences, U. of Texas Medical School at Houston, 6431 Fannin, Suite 3.204, Houston, TX 77030.
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Summary |
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We have recently shown the presence of E-cadherin and of - and
-catenins in human and baboon corpora lutea. These are components of adherens junctions between cells. The cytoplasmic catenins link the cell membrane-associated cadherins to the actin-based cytoskeleton. This interaction is necessary for the functional activity of the E-cadherins. Our aim therefore was to determine the presence of
-actin in the baboon corpus luteum, to further establish whether the necessary components for E-cadherin activity are present in this tissue. An antibody specific for the smooth muscle isoform of actin,
-actin, was used for these studies. The results using immunohistochemistry show that (a)
-actin is present in steroidogenic cells of the active corpus luteum, theca externa of the corpus luteum, cells of the vasculature, and the tunica albuginea surrounding the ovary. The intensity of immunoreactivity for
-actin varied, with the cells of the vasculature reacting more intensely than the luteal cells. A difference in intensity of immunoreactivity was also observed among the luteal cells, with the inner granulosa cells showing stronger immunoreactivity than the peripheral theca lutein cells. There was no detectable immunoreactivity in the steroidogenic cells of the atretic corpus luteum. However, in both the active and atretic corpora lutea,
-actin-positive vascular cells were dispersed within the tissue. (b) Total
-actin (luteal and non-luteal), as determined by Western blot analyses, does not change during the luteal phase and subsequent corpus luteum demise (atretic corpora lutea). (c) hCG stimulated the expression of
-actin and progesterone secretion by the early luteal phase (LH surge + 1-5 days) and mid-luteal phase (LH surge + 6-10 days) cells in culture, but only progesterone in the late luteal phase (LH surge + 11-15 days). The data show that
-actin is present in luteal cells and that its expression is regulated by hCG, thus suggesting that E-cadherin may form functional adherens junctions in the corpus luteum. (J Histochem Cytochem 45:71-77, 1997)
Key Words:
Primate, Baboon, Corpus luteum, Immunohistology, Western analysis, -actin, Cytoskeleton, Cell-cell communication
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Introduction |
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The steroidogenic cells of the corpus luteum of the menstrual cycle are distinguishable by size, morphology, and biochemistry (
The function of the cell adhesion- and tight junction-related molecules is determined by their association with the cytoplasmic cytoskeletal proteins, particularly actin. These interactions are important in the organization of the cytoskeleton, regulation of adhesion between cells, signal transduction, cell motility (
We therefore sought to determine (a) the presence of -actin in the baboon corpora lutea of the menstrual cycle, (b) the levels of the protein through various stages of corpus luteum development, and (c) the effect of hCG on actin expression and progesterone production in cultured luteal cells.
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Materials and Methods |
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Tissues
Baboon ovaries and corpora lutea were obtained from female cycling baboons (Papio anubis) with well-defined menstrual cycles and weighing 14-17 kg. The study was approved by the Review Board for Animal Experimentation, University of Texas Health Science Center at Houston, and was carried out in accordance with the principles and procedures described in the guide for the care and use of laboratory animals as approved by the National Institutes of Health (NIH 80-23). The baboons were kept in individual cages and in air-conditioned rooms. They were fed monkey chow and water ad libitum. Their menstrual cycles were monitored daily by the appearance and score of their perineal tumescence and serum progesterone after deturgescence (
Under general anesthesia induced with ketamine HCl (10-12 mg/kg body weight) and maintained with endotracheal intubation and nitrous oxide:oxygen 1:1, by volume with 3-4% halothane as necessary, a laparotomy was carried out through a lower abdominal midline incision. The ovaries were examined to locate the corpus luteum. A lutectomy was performed as previously described (
Chemicals
The antiserum for anti-human smooth muscle -actin was obtained from Dako (Carpinteria, CA). The immunogen used to generate the monoclonal antiserum was against the N-terminal decapeptide of smooth muscle
-actin, clone 1A4 (
-actin present. The antibody does not react with actin from fibroblasts (ß- and
-cytoplasmic) and striated muscle (
-sarcomeric) (
Immunohistochemical Localization Procedures
Tissues fixed in Bouin's solution (24 hr at 22°C) were dehydrated and embedded in Surgipath paraffin (Surgipath Medical Industries; Grayslake, IL) as previously described (
Western Analyses
Analysis of Tissue Concentrations of -actin.
Western blot analyses were performed as described (
-actin antibody at a dilution of 1 µg/ml (1:1000 dilution) for 16 hr at 4°C. The reaction products were detected using the Renaissance chemiluminescence procedure essentially as described by the manufacturer (Dupont NEN; Boston, MA).
Effect of hCG on Expression of -actin
Luteal cells from each phase (early, mid-, and late) of corpus luteum development were cultured for 4 days as previously described (
The cells were lysed in buffer (
Radioimmunoassay of Progesterone
Progesterone in the media was assayed in non-extracted samples by a previously described radioimmunoassay (-hydroxyprogesterone, 20
-dihydroprogesterone, and corticosterone is a characteristic of this antibody. The sensitivity of the assay is 5 pg/tube and media blanks have no detectable progesterone. Inter- and intra-assay coefficients of variation are 6.0 ± 1.2% and 7.0 ± 0.5%, respectively. Progesterone production in ng/100,000 cells/24 hr was expressed as a mean of triplicate samples assayed.
Analyses of Data
The Western blots of five tissue samples in the early, mid-, and late luteal phase were analyzed by determining band intensities of the electrophoresed samples using a Phosphoimager Series 400 (Molecular Dynamics; Sunnyvale, CA) and Image Quant 3.2 software. The densitometric values obtained were statistically analyzed using a one-way ANOVA to compare samples from each stage of luteal development, followed by the Student's Newman-Keuls test for determination of significance. Differences were considered to be significant at p0.001.
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Results |
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Smooth muscle -actin was localized in cardiac tissue (Figure 1), uterine tissue (not shown) used as positive controls, and ovarian tissue. In cardiac tissue in which the primary antibody was omitted (Figure 2) and in ovarian tissue that was treated with preadsorbed antiserum for the smooth muscle
-actin (Figure 3), no positive immunoreactivity was observed. Smooth muscle
-actin was detected at various intensities in the ovarian tissue. Intense immunoreactivity was observed in the theca externa (TE) surrounding the luteal cells as well as in the blood vessels (BV) that form a significant component of the corpus luteum. Comparatively lower levels of immunoreactivity were seen in the luteal cells, with the inner granulosa lutein cells (GC) showing stronger immunoreactivity compared to the peripheral theca lutein cells (TL) (Figure 4). The luteal cells of the early, mid-, and late corpus luteum (Figure 4 Figure 5 Figure 6) showed stronger immunoreactivity than the luteal cells of the atretic corpus luteum (CL) (Figure 7). The single layer of surface epithelial cells (SE) of the ovary did not show the presence of
-actin. However, a layer of cells immediately below the surface epithelium forming the tunica albuginea (TA) was strongly positive, and positive cells were present and dispersed in the ovarian stroma. The oocyte (O) and the surrounding follicular cells (F) were not positive for
-actin (Figure 7).
Western blot analyses of 25 µg of the membrane fraction indicated that the overall level of protein in the early, mid-, late and atretic corpora lutea does not change significantly (Figure 8, Lanes A-D). The level of -actin in luteal tissue was less than in cardiac tissue (Figure 8, Lane E). Cultured luteal cells in all three stages showed the presence of
-actin (Figure 9a-c, Lane A). Examination of the effect of hCG on the expression of the protein indicated that hCG stimulates the expression of
-actin in the cells of the early (Figure 9a, Lanes B and C) and mid-luteal (Figure 9b, Lanes B and C) phase cells but not those of the late luteal phase (Figure 9c, Lanes B and C; Table 1). Under the experimental conditions, a 20 ± 3% increase in the early luteal phase and a 22 ± 4% increase in the mid-luteal phase, both of which were significantly different from the control, were observed. However, progesterone levels were significantly increased by hCG in all three luteal stages (Table 1).
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Discussion |
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In the ovary, smooth muscle -actin was differentially expressed in the various compartments of this organ. The high level of
-actin in the tunica albuginea, the walls of the blood vessels, and the theca externa surrounding the corpus luteum is probably involved in the contractile function of this protein. Both actin and myosin have been identified in the ovary of the rat (
The cytoskeletal protein actin has been extensively examined in the differentiation of both the rat and human granulosa cells (-actin was localized in the steroidogenic cells of the corpus luteum at different intensities, suggesting different levels of the protein in these cells. Thus, the inner region of the corpus luteum, composed mainly of granulosa lutein cells that form the "large cell" population of the corpus luteum parenchyma, appears to have higher levels of
-actin. In contrast, the surrounding layer of theca lutein cells, which form the "small cell" population, have lower levels of this protein. In most species examined, only one cell type responds to endocrine regulators. In the human and the baboon (
-actin, which is an integral component of these junctions (
The total actin levels in the corpus luteum do not appear to change during luteal development. However, -actin in the steroidogenic luteal cells is stimulated by LH/hCG, as reflected by increased
-actin levels from early and mid-luteal phase cultured luteal cells in the presence of hCG. hCG also stimulated progesterone production in these cells. However, hCG failed to significantly increase
-actin by luteal cells from the late luteal phase, in spite of increased progesterone output.
In summary, we have demonstrated the presence of -actin in the ovary and in various stages of corpus luteum development in the baboon. The differential immunoreactive pattern observed suggests different levels of the protein in the various compartments of the ovary. The presence of the peptide in the luteal cells suggests that previously identified junctional proteins may be functional in this tissue and that hCG/LH may regulate its expression.
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Acknowledgments |
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Supported in part by a National Institutes of Health Grant HD24928.
Received for publication March 28, 1996; accepted August 22, 1996.
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