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Regular Article |
a Departments of Pathology,
b Molecular and Human Genetics, and
c Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030
d Department of Pharmacology, Case Western Reserve University, Cleveland, Ohio 44106
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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:ß heterodimers mediate cell contacts to the extracellular matrix and initiate intracellular signaling cascades in response to a variety of factors. Integrins interact with many determinants of cellular phenotypes and play roles in controlling the development, structural integrity, and function of every type of tissue. Despite their importance, little is known about the regulation of integrin subunits in the mammalian ovary and how they function in folliculogenesis. To determine their relevance to ovarian physiology, we have studied the expression of integrin subunit mRNAs by Northern blot analysis and in situ hybridization in ovaries of wild-type, growth differentiation factor 9 (Gdf 9) knockout, FSHß (Fshb) knockout, and inhibin (Inha) knockout mice. Integrin 6 mRNA is expressed in oocytes and granulosa cells of single-layer follicles and in oocytes and theca cells of multilayer follicles. Integrin 6 is highly expressed in Gdf 9 knockout ovaries, which are enriched in oocytes and primary (single layer) follicles because of a block at this stage of follicular development. Integrin v mRNA is most highly expressed in the granulosa cells of multilayer growing follicles, and therefore only low levels of expression are detectable in the Gdf 9 knockout ovaries. Integrin ß1 mRNA exhibits a broad expression pattern in ovaries, including oocytes, granulosa cells, theca cells, and corpora lutea. Integrin ß3 mRNA is expressed in theca and interstitial cells and is upregulated in corpora lutea. It is nearly undetectable in ovaries of Fshb knockout mice, which develop preantral follicles but have no luteal cells. Integrin ß5 mRNA is predominantly expressed in granulosa cells of multilayer follicles. It is expressed at high levels in the Fshb knockout mice and in a compartmentalized manner in the granulosa cell/Sertoli cell tumors that develop in the Inha knockout mice. Specific integrins are associated with ovarian cellular phenotypes in mice, which raises intriguing possibilities as to integrin functions in oocyte competence, follicular development, luteinization, and granulosa cell proliferation.
corpus luteum, follicle, follicular development, granulosa cells, theca cells
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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subunit (120–180 kDa) and one ß subunit (90–100 kDa), which associate noncovalently. There is great complexity to the expression, dimerization, and function of integrin subunits. To date, 18 subunits and 8 ß subunits have been described. Integrin dimers attach cells to one another and to the surrounding extracellular matrix. These contacts and the intracellular signaling pathways mediated by integrin proteins are important determinants of cell migration, endocytosis, proliferation, and differentiation [1]. Integrins have pivotal roles in the development and function of all tissues and organs and have been implicated in the pathogenesis of diverse diseases. Knockout mouse models for 13 subunits and 7 ß subunits have been developed (reviewed in [2]). In reproductive biology, integrins are known to be important mediators of sperm-egg binding, uterine receptivity, and early embryonic development [3]. Moreover, in the mammary glands, specific integrins are differentially expressed in response to steroid hormones [4, 5] and during carcinogenesis [6]. Remaining largely unexplored, however, are the functions of ovarian integrins in organizing follicles and directing somatic cell proliferation, apoptosis, and differentiation.
The adult mammalian ovary is a structurally dynamic organ, filled with follicles in different stages of development and uniquely subject to regulation by a host of cell autonomous, paracrine, and endocrine factors. For ovulation of a competent oocyte to occur, primordial follicles composed of an oocyte and a single layer of squamous granulosa cells must develop into large antral follicles. Preovulatory follicle development involves granulosa cell growth and proliferation, the recruitment of the circumscribing theca cells from the interstitial cell population, the formation of a fluid-filled antrum between granulosa cell layers, and the establishment of complex communication pathways among follicular compartments and between the follicle and the central endocrine axis. Preovulatory follicles have several layers of granulosa cells surrounding the oocyte (cumulus granulosa cells) and lining the follicle wall (mural granulosa cells) in addition to an outer layer of theca cells. Rupture of the follicle wall at ovulation releases the oocyte and its most closely associated granulosa cells. Subsequently, differentiation of remnant granulosa cells and theca cells into the corpus luteum in response to LH is essential for mediating uterine receptivity and allowing for early embryonic development. Events of follicular development are reviewed in [7].
To identify alterations of cell-cell and cell-extracellular matrix connections that coincide with the morphological and functional changes of folliculogenesis, we examined the localization of integrin subunit expression in the mouse ovary. We focused on integrin subunits known to be expressed in mammalian ovaries and those that are known to bind to integrin subunits and growth factors that have been implicated in ovarian function. We studied integrin subunit mRNAs by Northern blot analysis and in situ hybridization in ovaries of wild-type, growth differentiation factor 9 (GDF9) knockout, FSHß knockout, and inhibin knockout mice. There are striking differences in the composition of the ovaries of each of these knockout models (reviewed in [8, 9]). GDF9 knockout mice are deficient in an oocyte-secreted factor that is necessary for granulosa cell proliferation, and their ovaries are filled with oocytes in follicles arrested at the primary (single cell layer) stage [10, 11]. FSHß knockout mice lack a pituitary gonadotropin essential for antral follicle formation and luteinization; their ovaries are filled with arrested multilayer preantral follicles [12]. Inhibin knockout mice lack an important intragonadal factor and develop tumors of the granulosa/Sertoli cell lineage, which completely disrupt normal ovarian architecture [13]. This use of these knockout mice allowed us to investigate aspects of integrin regulation in the mammalian ovary.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Mice were maintained as described in the NIH Guide for the Care and Use of Laboratory Animals. Methods for the generation and genotyping of mice carrying the Fshb (Fshbtm1Zuk), Gdf 9 (Gdf 9tm1Zuk), and Inha (Inhatm1Zuk) null alleles have been described [10, 12, 13]. Ovaries were collected and pooled from 12–20 adult (8–16 wk of age) C57BL/J6/129/SvEv (hybrid strain) wild-type (WT), FSHß knockout (Fshb-/-), or GDF9 knockout (Gdf 9-/-) females for total RNA isolation or tissue fixation. Wild-type mice were randomly cycling. For the in situ hybridization, ovaries were collected from six different WT, Fshb knockout, and Gdf 9 knockout mice. The ovarian tumor used in this study was collected from an inhibin knockout (Inha-/-) female developing overt signs of cachexia at 14 wk of age.
RNA Isolation, Probe Preparation,and Northern Blot Analysis
Total RNA was isolated by acid guanidinium thiocyanate-phenol-chloroform extraction using the RNA STAT-60 reagent according to the instructions of the manufacturer (Leedo Medical Laboratories, Houston, TX). Complementary DNA was prepared from wild-type ovary RNA by reverse transcription polymerase chain reaction (RT-PCR) using the Superscript system (Invitrogen, Carlsbad, CA). Sequences of selected integrins were then amplified according to the manufacturer's instructions using primers designed from the NCBI sequence database (Table 1). The PCR products were ligated into a T-vector (Promega, Madison, WI), checked by bidirectional sequencing, and used as templates for the synthesis of Northern blot and in situ hybridization probes.
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For Northern blot analyses, 15 µg of each RNA sample was electrophoresed and transferred to nylon membranes as previously described [14]. Probes radiolabeled with [32P]dATP were synthesized using the Strip-EZ kit (Ambion, Austin, TX). Autoradiography and phosphorimaging allowed for visualization and quantification of probe hybridization, respectively. After washing, membranes were exposed to X-OMAT film with the use of intensifying screens for 6 h to 1 wk (Eastman Kodak, Rochester, NY). Phosphorimaging plates were scanned and analyzed using ImageQuant software (Molecular Dynamics, Sunnyvale, CA) [15]. A background level for each membrane was subtracted, and blots were then stripped and reprobed for glyceraldehyde phosphate dehydrogenase (GAPDH) to allow for loading corrections.
In Situ Hybridization
In situ hybridization was performed essentially as previously reported [11, 16]. Ovaries or ovarian tumors were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned. Tissue was pretreated, prehybridized, probed with [35S]UTP-labeled antisense and sense riboprobes, and washed as described by Albrecht et al. [17]. Probe hybridization to endogenous mRNAs was visualized using NTB-2 radiographic emulsion (Eastman Kodak); exposure times ranged from 3 days to 3 wk. Hematoxylin counterstaining and/or periodic acid-Schiff (PAS)/hematoxylin staining of an adjacent tissue section allowed us to readily associate a hybridization pattern with a particular cell population.
Affymetrix Gene Chip mRNA Expression Analyses
Total ovarian RNA from wild-type mice was used as a template for cDNA synthesis and biotinylated antisense cRNA probe preparation as described by the manufacturers of the SuperScript System kit (Invitrogen) and the ENZO BioArray HighYield RNA labeling kit (Enzo Diagnostics, Farmingdale, NY). Unincorporated nucleotides were removed from the riboprobe preparation using the RNeasy Mini kit (Qiagen, Valencia, CA). Integrity of the riboprobe was checked by gel electrophoresis.
The murine 11K oligo array set chip (Mu11KsubA, Mu11KsubB) was hybridized, washed, and scanned using the manufacturer's equipment and protocols (Affymetrix, Santa Clara, CA). Data analysis was performed using Affymetrix software with the recommended decision matrix default parameters. Here, we report absent/presence calls based on an absolute analysis with amplification. More information on protocols and data analyses and a listing of the genes surveyed in this analysis is available at the Affymetrix website (www.affymetrix.com).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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6 mRNA Expression
To characterize ovarian expression of the integrin 6 subunit, we performed Northern blot and in situ hybridization analyses on ovarian RNA and tissue from wild-type, GDF9 knockout (Gdf 9-/-), FSHß knockout (Fshb-/-), and inhibin knockout (Inha-/-) mice. The labeled integrin 6 cDNA probe hybridized to a single mRNA species in the total ovarian RNA samples in Northern blot analysis. Integrin 6 mRNA was detectable by this method in all of the samples assessed and was very highly expressed in the ovaries of Gdf 9 knockout mice in particular (Fig. 1A). Phosphorimaging quantification indicated a 3-fold enrichment of integrin 6 mRNA in Gdf 9 knockout ovaries as compared with the wild-type control. This finding corroborates our in situ hybridization analysis, which demonstrated highest expression of integrin 6 in oocytes and the granulosa cells immediately surrounding them in single-layer follicles (Fig. 2). These single-layer follicles predominate in the Gdf 9 knockout ovaries, in which folliculogenesis beyond this stage is blocked (Fig. 2, I–L). Integrin 6 mRNA was also detected in the oocytes of multilayer growing follicles and in the theca cells circumscribing these follicles (Fig. 2, A–D). An identical pattern of expression was observed in the ovaries of Fshb knockout mice, which are filled with multilayer preantral follicles. Integrin 6 mRNA is relatively excluded from granulosa cells of these and preovulatory follicles and from cells in corpora lutea (Fig. 2, G and H). Integrin 6 mRNA was detectable by Northern blot analysis of ovarian tumor RNA recovered from an Inha knockout mouse; variations in its expression within a select tumor sample were not demonstrated by in situ hybridization (data not shown).
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Integrin v mRNA Expression
Integrin v subunit mRNA expression levels were slightly enhanced in ovaries of Fshb knockout mice (1.3-fold) and markedly lowered in Gdf 9 knockout ovaries (<1/5) by Northern blot analysis (Fig. 1B). These data agree with those of our in situ hybridization experiment, indicating that integrin v is most highly expressed in the granulosa cell populations of multilayer follicles (Fig. 3, A and B). Integrin v subunit mRNA was essentially undetectable by this method in granulosa cells of single-layer follicles (Fig. 3, C and D), and in situ hybridization of Gdf 9 knockout ovaries revealed no defined pattern of expression (data not shown). Similarly, no integrin v probe hybridization was observed in luteinized cells (Fig. 3, C and D). In contrast, the ovaries of Fshb knockout mice, which are filled with multilayer follicles with granulosa cells that do not luteinize, expressed the integrin v subunit at a high level (Fig. 3, E and F). Integrin v mRNA was expressed in Inha knockout ovarian tumors as assessed by Northern blot analysis; in situ hybridization of a select tumor sample did not show evidence of a specific expression pattern (data not shown).
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Integrin ß1 mRNA Expression
Integrin ß1 was highly expressed in all of the ovarian RNA samples studied by Northern blot analysis. No significant changes in the level of its expression were noted when these samples were compared, despite very striking differences in the composition of these ovaries (Fig. 1C). In situ hybridization analysis revealed that integrin ß1 mRNA is expressed essentially ubiquitously in the mammalian ovary. Short exposures to the photographic emulsion highlighted probe hybridization to oocytes (Fig. 4, A–F); longer exposures demonstrated the expression of integrin ß1 in other cell types, including granulosa cells, theca cells, interstitial cells, and to a lesser extent luteinized cells (Fig. 4, G–J). Negative control slides probed with sense riboprobe confirmed a low background of nonspecific riboprobe binding and emulsion exposure (Fig. 4, K and L). Hybridization was detected in every cell population in Fshb and Gdf 9 knockout ovaries and in the Inha knockout ovarian tumor sample (data not shown).
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Integrin ß3 mRNA Expression
Integrin ß3 subunit mRNA was detectable in total ovarian RNA of wild-type and Gdf 9 knockout mice by Northern blot analysis. Integrin ß3 was expressed at lower levels in the ovaries of Fshb knockout mice at the threshold of visualization given our experimental conditions (Fig. 1D). This finding corroborated our in situ hybridization results, which localized integrin ß3 subunit expression primarily to corpora lutea (Fig. 5, C and D) and to a lesser degree theca and interstitial cell populations (Fig. 5, A and B). There were no luteinized cells in ovaries of Fshb knockout mice; in situ hybridization to detect integrin ß3 mRNA in these ovaries was not sufficiently sensitive to demonstrate a specific pattern of expression (data not shown). In situ hybridization to integrin ß3 transcript in Gdf 9 knockout ovaries demonstrated expression in the interstitial cells and the nests of luteal-like cells between follicles (Fig. 5, E and F). Although integrin ß3 expression is relatively excluded from granulosa cells in the aforementioned mouse models, integrin ß3 mRNA was expressed in the granulosa cell component of the Inha knockout ovarian tumor (data not shown).
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Integrin ß5 mRNA Expression
Integrin ß5 subunit mRNA was expressed broadly in the murine ovary, as indicated by its presence in wild-type, Fshb knockout, and Gdf 9 knockout ovaries revealed by Northern blot analysis (Fig. 1E). This mRNA species was enriched nearly 2-fold in ovaries of Fshb knockout mice. After long exposure, in situ hybridization experiments revealed ubiquitous integrin ß5 expression in the ovarian samples probed, with minimal background on slides probed instead with a sense riboprobe control (Fig. 6, E and F). Shortened exposure highlighted the granulosa cell population in multilayer follicles as the dominant source of expression in wild-type (Fig. 6, A–D) and Fshb knockout mice (data not shown). A relative downregulation of integrin ß5 mRNA appeared to accompany luteinization of these cells (Fig. 6, C and D).
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Differential expression of integrin ß5 was observed in the ovarian tumor sample collected from an inhibin knockout mouse (Fig. 6, G and H). A higher level of expression of the integrin subunit mRNA was detected in a compartment of the tumor composed of relatively well-differentiated granulosa cells with microfollicular features of organization. Lower levels of expression were detected in the relatively undifferentiated tumor component, in which cells with high nucleus:cytoplasm ratios accumulated in nests and cords (Fig. 6, outside the margin).
Other Integrin Subunits and Affymetrix Chip Analysis
We amplified cDNAs corresponding to integrin IIb, integrin ß2, and integrin ß6 sequences by RT-PCR using RNA from wild-type ovaries. However, Northern blot analyses using these sequences as probes against total ovarian RNA demonstrated minimal or undetectable probe hybridization (data not shown). In addition, Affymetrix gene chip analysis using parameters specified by the manufacturer indicated that integrin 1, integrin 4, integrin 5, integrin 7, integrin M, integrin ß2, integrin ß4, and integrin ß7 subunit sequences were absent in wild-type ovarian RNA pools. These data indicate that these integrin subunits are not highly expressed in the ovaries of C57BL/J6/129SvEv mice.
Affymetrix gene chip analysis confirmed that integrin 6, integrin ß1, and integrin ß5 are expressed in the wild-type C57BL/J6/129/SvEv mouse ovary. Average difference values obtained for these integrin subunits were 1145.4, 11130.5, and 2695.8, respectively, and these values were qualitatively correlated with the signal intensities obtained with Northern blot hybridization (Fig. 1). Affymetrix analyses also demonstrated that mRNAs coding these three integrin subunits were present in ovaries of CF-1 mice and C57BL/6 x CF-1 F1 hybrids (unpublished data).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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v) [18, 19] or for postnatal survival (e.g., integrin 6) [20], whereas others exhibit functional redundancy (e.g., integrin ß5) [21]. Because of these variable effects, these knockout models do not demonstrate defined reproductive phenotypes, and little can be concluded from these studies about ovarian integrin function.
To identify integrin candidates that might mediate events of folliculogenesis, we examined the expression of integrin subunit mRNAs in wild-type mouse ovaries and in ovaries of knockout mice with major aberrations in ovarian morphology: 1) GDF9 knockout mouse ovaries, which are filled with oocytes and have no granulosa cell proliferation, 2) FSHß knockout mouse ovaries, which exhibit a later preantral stage block in folliculogenesis and have no luteinization, and 3) inhibin knockout mouse ovaries, which have uncontrolled proliferation of granulosa cells [10, 12, 13]. Our results are summarized in Table 2.
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Oocytes had high levels of expression of integrin 6 and integrin ß1 subunit mRNAs. This finding corroborates reports that integrin 6ß1 protein dimers are expressed on the surface of murine oocytes and are implicated in sperm-egg binding and fertilization [22]. Integrin 6ß1 has also been previously described in granulosa cells in human, porcine, and marmoset follicles [23, 24]. This integrin is downregulated in the marmoset in atretic tertiary follicles [24], and interference with integrin 6ß1 binding to laminin in human granulosa cells in vitro enhances the progesterone production of these cells [25, 26]. These data have led to the prediction that integrin 6ß1 expression is necessary for maintaining a healthy, nonluteal granulosa cell phenotype. However, we found a relatively restricted expression of integrin 6 mRNA in granulosa cells in the murine ovary, which was detectable by in situ hybridization in single-cell-layer follicles but not in the granulosa cells of multilayer follicles. This finding suggests that integrin 6ß1 is not likely to mediate events of later follicular development, multilayer follicle apoptosis, or luteinization in the mouse ovary. Our observation of integrin 6 mRNA in the theca layer of multilayer follicles is in agreement with results of immunocytochemistry studies [27]. Whether the 6 subunit associates with integrin ß1, integrin ß3, integrin ß5, or another ß subunit is unknown, and the roles of 6, if any, in theca layer recruitment or function remain obscure.
In this study, we demonstrated high levels of integrin v and integrin ß5 subunit expression in granulosa cells of growing multilayer follicles. Although integrin vß5 has not been described previously in the ovary, this dimer has been characterized [28]. Integrin vß5 forms a receptor for vitronectin and is implicated in cancer cell division and invasion, angiogenesis, adenoviral susceptibility, and cellular responses to insulin-like growth factor and transforming growth factor ß1 [29–33]. The expression of integrin v and integrin ß5 subunits in the murine ovary overlaps the expression of connective tissue growth factor (CTGF, Fisp12) mRNA [34]. CTGF is hypothesized to function in follicular development [35] and has been shown to bind integrins vß3 and IIbß3 [36, 37]. Whether CTGF affects granulosa cells in an autocrine manner through integrin vß5 or another integrin receptor or affects a neighboring follicular compartment by paracrine signaling remains unknown. In addition to its physiological roles in proliferating granulosa cells, integrin ß5 may be an important determinant of the cellular phenotype of tumors in inhibin knockout mice. In contrast to knockout mice lacking integrin v, which do not survive embryogenesis, knockout mice without integrin ß5 develop and reproduce normally [19, 21]. Which, if any, integrin ß subunit may compensate for the lack of the integrin ß5 subunit in preserving granulosa cell function in these mice has not been examined.
Integrin ß3 subunit mRNA was upregulated in corpora lutea and was nearly undetectable in the ovaries of FSHß knockout mice, which lack luteinized cells [16]. It is not known which subunit associates with integrin ß3 and how this dimer affects the cellular events of luteinization or the vascular and extracellular matrix changes associated with the formation of corpora lutea.
Tissue-specific transgenic models and double mutant models could be used to better appreciate the complex biological functions of integrin proteins. An understanding of roles of integrins in the ovary may bring us new insights into ovarian function and failure, methods for categorizing ovarian cancers, and a physical means of targeting specific cell populations or signaling pathways within the ovary. This characterization of integrin expression in mouse ovaries reaffirms models that associate integrin subunit mRNA regulation with particular cellular phenotypes, raises many intriguing questions pertaining to ovarian integrin function, and provides important preliminary work for future studies in mouse models.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported by NIH grants CA 60651 and HD 33438 (M.M.M.), as well as CA086387 (J.H.N.). K.H.B. is a student in the Medical Scientist Training Program and is supported in part by NIH grant T32GM07330 and by grant T32EY07102 from the National Eye Institute. G.E.O. is a student in the Medical Scientist Training Program supported by T32GM007250. 
2 Correspondence: Martin M. Matzuk, Department of Pathology, One Baylor Plaza, Baylor College of Medicine, Houston, TX 77030.FAX: 713 798 5833; mmatzuk{at}bcm.tmc.edu
Accepted: March 18, 2002.
Received: October 24, 2001.
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(5)ß(1) integrin are under developmental and ovarian steroid regulation in the normal mouse mammary gland. Endocrinology 2001 142:3214-32222ß1 integrin changes during mammary gland development by ß-oestradiol. Biochim Biophys Acta 2001 1499:232-241[Medline]-Inhibin is a tumor-suppressor gene with gonadal specificity in mice. Nature 1992 360:313-319[CrossRef][Medline]v integrins. Cell 1998 95:507-519[CrossRef][Medline]6 leads to epidermolysis bullosa and neonatal death in mice. Nat Genet 1996 13:370-373[CrossRef][Medline]6ß1 integrin is the receptor for sperm fertilin ß. Chem Biol 1999 6:1-10[CrossRef][Medline]6: possible involvement of integrin 6 in folliculogenesis. Horm Res 1996 46::24-306ß1. J Clin Endocrinol Metab 1997 82:2122-21286ß1 in ovarian functions. Horm Res 1998 50:25-296 Subunit of integrins in the development and sex differentiation of the mouse ovary. Dev Dyn 1995 202::397-404[Medline]vß5. An adhesion receptor for vitronectin. J Biol Chem 1990 265:11008-11013vß3 and vß5 integrin receptor antagonist. Cancer Res 2001 61::2232-2238vß5 integrin. J Biol Chem 2001 276:10952-10962vß5 selectively promotes adenovirus mediated cell membrane permeabilization. J Cell Biol 1994 127:257-264vß5 to promote tumor cell dissemination in vivo. J Clin Invest 1997 99::1390-1398[Medline]IIbß3. J Biol Chem 1999 274:24321-24327vß3, promotes endothelial cell survival, and induces angiogenesis in vivo. Mol Cell Biol 1999 19:2958-2966This article has been cited by other articles:
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