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a AgResearch, Wallaceville Animal Research Centre, Upper Hutt, New Zealand
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-inhibin subunit, and the ßA- and ßB-activin/inhibin subunits were localized in ovaries of ewes with 0 (++), 1 (I+), or 2 (II) copies of the FecXI gene (n = 4–9 animals per genotype per gene) using in situ hybridization. Ontogeny of expression of all mRNAs examined was similar between ++ and I+ ewes. Expression of c-kit mRNA was observed in the oocyte of all follicular types present in ++, I+, and II ewes. Moreover, granulosa cells of type 2 (II) and type 2 and larger follicles (++, I+) expressed SCF mRNA. The mRNAs encoding FSH-R, follistatin, -inhibin subunit, and ßB-activin/inhibin subunit were identified in type 3 and larger follicles of ++ and I+ ewes but not in follicles of II ewes that were only at the type 1, 1a, or 2 stages of development. However, the cells within the oocyte-free nodules of II ewes expressed all of these genes. The mRNAs encoding c-kit and ßA-activin/inhibin subunit were not observed in granulosa cells until antrum formation (type 5 follicles) or in the nodules of II ewes. Tumors from 4 ewes were obtained and classified as cystic, semisolid, or solid structures containing granulosa-like cells or as solid structures containing predominately fibroblast- and luteal-like cells. Often, two tumors were present on the same ovary. Tumors containing granulosa-like cells (n = 3–4 per gene) expressed the mRNAs encoding -inhibin subunit, ßA-, and ßB-activin/inhibin subunits, follistatin, and the FSH-R but did not contain detectable amounts of mRNA for c-kit or SCF. Tumors composed predominately of fibroblast- and luteal-like cells expressed very low levels of SCF mRNA; of the other mRNAs examined, none were detected. Also, none of the genes examined were found to be expressed by the surface epithelium, theca externa, fibroblast, or vascular cells within the ovary of animals of any genotype. These findings are consistent with the hypothesis that the somatic cells in oocyte-free nodules and tumor-like tissue in II ewes originate from the granulosa cells of the small follicles.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Various growth factors, including members of the transforming growth factor ß superfamily such as inhibin, activins, and their receptors and binding proteins, are thought to play a role in the early growth of follicles [6, 7]. In addition, stem cell factor (SCF) and its receptor, c-kit, are known to be essential for normal follicular development [8]. Interestingly, the morphological features of the II Inverdale ovaries are strikingly similar to those of the ovaries found in mice with certain mutations at the locus for SCF (Steel; Sl) or its receptor c-kit (white-spotting). For example, the mutant steel alleles, Sl+ and Slpan/Slpan, both result in phenotypes in which the growth of ovarian follicles is arrested when they are surrounded by a single layer of cuboidal granulosa cells [9–11]. Finally, while not crucial for preantral follicular growth, FSH is known to facilitate early growth [12]. We and others have reported that follistatin, -inhibin subunit, ßA- and ßB-inhibin/activin subunits, and SCF and its receptor c-kit, as well as the FSH receptor (FSH-R), are expressed by ovine follicles in a cell- and stage-specific manner [13–21]. While these genes are not candidates for the Inverdale mutation [22–28], it is possible that FecXI could be affecting their expression.
In addition to the primordial (type 1), transitory (type 1a), and primary (type 2) follicles observed in the ovaries of II ewes, small oocyte-free "nodules" of granulosa-like cells and other abnormal structures resembling tumors are often present. These tumor-like structures can be fluid-filled semisolid or solid entities and, in some instances at least, secrete immunoreactive inhibin but not steroid [5]. In II ewes it seems that follicular growth begins and the oocyte enlarges without a concomitant increase in the number of granulosa cells [4, 5]. Thereafter, it seems that the enlarged oocyte degenerates, resulting in the formation of oocyte-free structures that have been termed nodules. These nodules are present in streak ovaries during late fetal and early neonatal life and in adult animals [4, 5]. It has been speculated that the nodules coalesce and the cells within these nodules proliferate to form the large tumor-like structures often visible on the surface of the ovary [5]. However, apart from the morphology of these structures there is no evidence to support the notion that nodules or the large tumor-like masses are of granulosa cell origin. If the abnormal ovarian structures observed in II ewes are of granulosa cell origin, gene expression in these structures would be expected to be similar to that observed in granulosa cells during follicular development.
Therefore, this study on ovaries of Inverdale ewes had two aims, namely 1) to determine whether ontogeny of growth factor and hormone receptor gene expression was similar between ++ and I+ ewes and 2) to determine whether gene expression in the abnormal ovarian structures in II ewes was similar to that observed in granulosa cells of ++ ewes.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Collection of Tissue Samples
Homozygous Inverdale Romney ewes were obtained by mating progeny-tested carrier rams with the heterozygous carrier FecXI FecX+ ewes. All females of such matings were either homozygous or heterozygous carriers of the FecXI gene. The segregation of II from I+ female genotypes was made on the basis of laparoscopic observation; the former were distinguished by the presence of bilateral "streak" ovaries that were much smaller in volume than normal ovaries [2]. The control Romney ewes (++) were from a non-Inverdale Romney flock. Ovaries were recovered from ++ (n = 10), I+ (n = 7), and II (n = 9) ewes, fixed in 4% (w:v) phosphate-buffered paraformaldehyde, embedded in paraffin, and stored at 4°C until sectioned for in situ hybridization. Of the 9 II animals, 4 had abnormal structures on the surface of their ovaries, and 3 of these 4 animals had two morphologically distinct abnormal structures. Morphologically, cells in one type of tumor resembled granulosa cells, being rounded in shape with darkly staining, centrally located nuclei with scant cytoplasm (tumors containing granulosa-like cells). However, the other type of tumor contained cells that resembled fibroblast cells, being elongated with darkly staining, irregular shaped nuclei and containing scant cytoplasm. Other cells in the same tumor were morphologically similar to luteal cells, being large with abundant "foamy" cytoplasm with lighter-staining, irregularly placed nuclei. Thus, these tumors were classified as containing fibroblast- and luteal-like cells based on their morphology. At least 3 tumors containing granulosa-like cells and 3 tumors containing fibroblast- and luteal-like cells were examined for expression of each gene.
In Situ Hybridization
The in situ hybridization methodologies in ovine ovarian tissue have been reported for FSH-R [19], SCF [20], c-kit [15], and -inhibin subunit, ßA-activin/inhibin subunit, and follistatin mRNAs [18]. The ßB-activin/inhibin subunit mRNA was detected using ovine cDNA [29] after subcloning of the SphI-BamHI fragment into pGEM7Zf (Promega, Dade Diagnostics Pty Ltd, Auckland, New Zealand) using the in situ hybridization protocol described previously with minor modifications [18]. Briefly, for all in situ hybridizations, 4- to 6-µM sections were incubated overnight at 55°C in hybridization solution containing 30 000–60 000 cpm/µl of antisense RNA that had been generated with T7 or SP6 RNA polymerase using the Riboprobe Gemini system (Promega). Nonspecific hybridization of RNA was removed by RNase A digestion followed by stringent washes (double-strength SSC, 50% formamide, 65°C, and 0.2-strength SSC at 37°C; single-strength SSC is 0.15 M sodium chloride and 0.015 M sodium citrate). After washing, sections were dehydrated, air dried, and coated with autoradiographic emulsion (LM-1 emulsion; Amersham Pharmacia Biotech New Zealand, Auckland, New Zealand). The emulsion-coated slides were exposed at 4°C for 2–3 wk. Slides were then developed and fixed. The sections were stained with hematoxylin and viewed and photographed using both light- and darkfield illumination on an Olympus BH-2 (Tokyo, Japan) microscope. Nonspecific hybridization was monitored by hybridizing at least one tissue section from each genotype as well as tumor tissue with approximately equal concentrations of the sense mRNA for each gene. No specific hybridization was observed for any section hybridized with the sense mRNA for any gene.
Classification of Follicles
Ovarian follicles in the ++, I+, and II ovaries were classified from a section through the oocyte nucleolus by the configuration of granulosa cells around the oocyte. Because type 3 and 4 follicles were not often observed in the sections, these follicle types were classified using a section through the oocyte nucleus. The classification system of Lundy et al. [30] was used: type 1 (primordial follicle), one layer of flattened granulosa cells; type 1a (transitory follicles), one layer of a mixture of flattened and cuboidal granulosa cells; type 2 (primary follicles), from one to less than two complete layers of cuboidal granulosa cells; type 3 (small preantral follicles), from two to less than four complete layers of cuboidal granulosa cells; type 4 (large preantral follicles), from four to less than six complete layers of cuboidal granulosa cells. In addition, any follicle with an antrum was classified as a type 5+ follicle regardless of whether an oocyte was present in the section. Not all types of follicles were present in each animal for each gene; however, at least two animals had follicles present that spanned the type classifications at the transition point between no clear expression and clear expression.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Expression of c-kit mRNA was observed in oocytes of all follicle types examined in ++, I+, and II animals (Fig. 2, a–f, and data not shown). Granulosa cells of type 5+ follicles in ++ (Fig. 2, c and d) and I+ (data not shown) ewes also expressed c-kit mRNA. Very faint expression of c-kit mRNA in the theca interna of some type 5+ follicles (e.g., Fig. 2, c and d) was observed. However, expression was generally not observed in theca of type 5+ follicles. Thus, whether this apparent signal represents true expression of c-kit in the theca interna of type 5+ follicles or an artifact could not be determined. The rete did not contain detectable amounts of c-kit mRNA (Fig. 2, e and f, and data not shown). Expression of SCF mRNA was first clearly detected in the granulosa cells of type 2 follicles in ++ (data not shown), I+ (data not shown), and II (Fig. 3, c and d) animals and continued to be expressed in all follicle types to the 5+ stage in the ++ (data not shown) and I+ (Fig. 3, a and b) animals. Areas of tissue containing groups of type 1 and 1a follicles among rete contained mRNA encoding SCF (Fig. 3, c and d); however, whether this signal was associated with the follicles and/or the rete could not be determined. Nodules expressed SCF (Fig. 3, e and f) but not c-kit (Fig. 2, e and f) mRNA. SCF mRNA was expressed at very low levels in tumors with fibroblast- and luteal-like cells (Fig. 3, g and h) but was undetectable in tumors with mainly granulosa-like cells (data not shown). Expression of c-kit mRNA was not observed in either tumor type (Fig. 2, g and h, and data not shown). Neither SCF nor c-kit mRNA was expressed by the surface epithelium (e.g., Fig. 2, a, b, e, and f; Fig. 3, c and d), theca externa (e.g., Fig. 2, c and d; Fig. 3, a and b), fibroblast, or vascular cells in ovaries of any genotype.
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Expression of mRNAs encoding FSH-R (Fig. 4), follistatin (Fig. 5), -inhibin subunit (Fig. 6), and ßB-activin/inhibin subunit (Fig. 7) was evident in granulosa cells of type 3 and greater follicles of ++ and I+ ewes (Fig. 4, a and b; Fig. 5, a and b; Fig. 6, a and b; Fig. 7, a and b, and data not shown). None of these mRNAs was observable above background in type 1, 1a, or 2 follicles of ++, I+, or II ewes (Fig. 4, a–d; Fig. 5, a–d; Fig. 6, a and b; Fig. 7, a–d, and data not shown). However, nodules of II ewes expressed the aforementioned mRNAs (Fig. 4, e and f; Fig. 5, e and f; Fig. 6, c and d; Fig. 7, e and f). The mRNA encoding ßA-activin/inhibin subunit (Fig. 8) was not evident in granulosa cells until antrum formation (i.e., type 5 follicles) in either ++ (Fig. 8, a and b) or I+ (data not shown) ewes. Nodules of II ewes did not express mRNA encoding the ßA-activin/inhibin subunit (Fig. 8, e and f). Tumors composed mainly of granulosa-like cells expressed mRNA encoding the FSH-R (Fig. 4, g and h), follistatin (Fig. 5, g and h), -inhibin subunit (Fig. 6, e and f), and the ßA- and ßB-activin/inhibin subunits (Fig. 7, g and h; Fig. 8, g and h), whereas tumors with fibroblast- and luteal-like cells did not (Fig. 6, g and h; Fig. 7, g and h, and data not shown). The surface epithelium, theca interna/externa, fibroblast, or vascular cells of the ovaries of ++, I+, or II genotypes expressed none of the aforementioned mRNAs (e.g., Figs. 4–8). However, rete cells expressed the mRNAs encoding the ßB subunit of activin/inhibin and follistatin (data not shown).
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The localization of mRNA for c-kit, SCF, FSH-R, follistatin, -inhibin subunit, ßB-activin/inhibin subunit, and ßA-activin/inhibin subunit in the various follicular types (1–5+), nodules, and tumor types (i.e., with granulosa-like cells or fibroblast- and luteal-like cells) in ovaries from ++, I+, and II animals is summarized in Table 1. With respect to follicular types, no type 3–5+ follicles were noted in II ewes. No differences between ++ and I+ genotypes were noted with respect to the ontogeny of expression of any of the mRNAs examined; therefore, these data have been combined.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-inhibin subunit, ßA- and ßB-activin/inhibin subunits, follistatin, and FSH-R during follicular development in ++ and I+ ewes was similar to that reported for other sheep breeds [13–21]. Expression of some of the genes (i.e., SCF, FSH-R, and the ßB-activin/inhibin subunit) was first detected at a slightly later stage of development than we had previously reported [19–21]. This was most likely due to a combination of a more stringent classification of follicles and slightly lower sensitivity of the in situ hybridization because of higher background counts. With respect to the classification of follicles, those reported for the present study on types 1, 1a, and 2 follicles were based on visualization of the section of the follicle through the oocyte nucleolus, whereas in earlier reports this was not always the case. No differences were observed between ++ and I+ ewes in ontogeny of expression of any of the genes examined. Thus, it seems unlikely that a single copy of the Inverdale gene mutation is affecting early follicular growth by altering the onset of expression of these genes. The sequential and cell-specific expression of c-kit and SCF mRNA in types 1, 1a, and 2 follicles in II ewes, and the lack of expression of the other mRNAs examined, extend previous reports that follicular growth up to the primary stage of development is apparently normal in this genotype [4, 5]. The absence of type 3 or larger follicles in II ewes was not surprising, since these have not been observed in previous studies [4, 5, 31].
The question arises whether the nodules developed from type 2 follicles. If this was the case, one might expect that the cells of the nodules would express a range of genes observed in type 3 follicles. Indeed, this appears to be the case, since expression of the mRNAs examined was similar between the cells in the nodules and the granulosa cells of type 3 follicles. Interestingly, the nodules did not express mRNA for the ßA subunit of inhibin/activin or c-kit, two genes that were not expressed in the granulosa of follicles of ++ and I+ ewes until antrum formation. Thus, it seems likely that despite losing their oocytes, the cells within the nodules retain many of the exclusive characteristics of granulosa cells and represent, at least in part, a continued differentiation of granulosa cells, since none of the genes expressed in the nodules were observed in other regions of the ovary such as the theca interna/externa, interstitial cells, or surface epithelium.
In the present study there was considerable heterogeneity in the gross morphology of the tumors visible on the surface of II ovaries. Some of the tumors were solid and extensively vascularized; some were cystic or semisolid with clear or hemorrhagic fluid whereas others were solid and avascular. From histological examination, these aforementioned tumors could be classified as two general types based on the morphology of the predominant cell type(s). One type of tumor contained mainly granulosa-like cells, and these could be found in either a solid, semisolid, or cystic structure. The other type of tumor contained a mixture of fibroblast- and luteal-like cells, and these were found in the solid avascular structures. Tumors with similar morphological characteristics have previously been described in II ewes [5]. Gene expression was very different between the two classes of tumors. Those containing granulosa-like cells expressed most of the mRNAs examined, whereas those with fibroblast- and luteal-like cells did not express any of the mRNAs examined with the exception of very low levels of SCF mRNA.
The mRNAs encoding c-kit and SCF were expressed in granulosa cells of antral follicles but not tumors containing granulosa-like cells. Thus, SCF and its receptor c-kit do not appear necessary for development of tumors with granulosa-like cells in II ewes. The mRNA for c-kit as well as for SCF and/or their proteins are present in some, but not all, human ovarian tumors [32, 33]. However, in these studies granulosa cell tumors were not examined.
The expression of -inhibin subunit and the ßA- and ßB-activin/inhibin subunit genes in tumors with granulosa-like cells suggests that they secrete inhibins and/or activins. There have been several reports of inhibin secretion from benign and malignant ovarian tumors in women [34–36]. Granulosa cell tumors also express mRNA encoding the inhibin/activin subunits [37]. A report on the immunohistochemical localization of inhibin/activin subunits in epithelial and granulosa cell tumors of the ovary in women suggested that the latter express inhibin while the former probably express activin [38]. Immunoreactive inhibin was not detectable in the plasma of homozygous Inverdale ewes with streak ovaries, and the concentrations of FSH were similar to those in ovariectomized ewes [5]. However, Braw-Tal et al. [5] and McLeod et al. [39] have recorded high concentrations of immunoreactive inhibin and lower concentrations of FSH in II animals when ovarian tumors were present. Inhibin exists as a range of molecular forms, with some but not all having biological activity [40]. In previous studies in II ewes with ovarian tumors, the concentrations of plasma inhibin remained high for several weeks but fell abruptly when the structure was surgically removed, and this was associated with a corresponding increase in concentration of plasma FSH [5]. Collectively the results of the present study, showing the presence of -inhibin subunit mRNA and ßA- and ßB-activin/inhibin subunit mRNAs in the tumors with granulosa-like cells, and those of previous studies, showing high circulating levels of inhibin in II ewes with tumors together with suppressed FSH concentrations, support the notion that this tumor type secretes bioactive inhibin. At present no data are available with respect to the plasma concentrations of activin or follistatin in the II ewe. There is some debate over the role of the inhibin -subunit in tumorigenesis. In the mouse, the -inhibin gene has been shown to be a suppressor of granulosa tumorigenesis [41]. In contrast, Watson et al. [42] suggest that based on data obtained from human ovarian epithelial and granulosa cell tumors, inhibin does not function as a granulosa cell tumor suppressor gene in the human. Of note, while at any point in time approximately one third of adult II ewes have tumors visible on their ovary, evidence of metastases of these structures has never been observed. Thus, while these tumors have been observed to grow rapidly, they are invariably contained within the ovary and eventually regress [39].
Strong expression of follistatin mRNA was also observed in tumors with granulosa-like cells. Protein and mRNA encoding follistatin have been observed in both granulosa cell and ovarian epithelial tumors/cancer cell lines of humans [37, 43, 44]. The primary role of follistatin is the neutralization of activin. It is interesting to note that follistatin, through binding of activin, has been shown to stimulate [45] or inhibit [43] proliferation of ovarian cancer cells. However, none of the cell lines tested were of granulosa cell origin; thus, the potential role of follistatin in the Inverdale tumor model remains to be defined.
It is also interesting to note that tumors with granulosa-like cells strongly expressed mRNA encoding the FSH-R. Expression of FSH-R mRNA/or protein has been demonstrated in human granulosa cell tumors [46, 47]. Furthermore, survival of tumor tissue following transplantation to nude mice was supported by FSH [46]. Therefore, it could be hypothesized that granulosa-like cell tumors in II ewes are dependent on FSH and that the high levels of inhibin produced by these tumors suppress FSH secretion from the pituitary, thus causing its demise. This might explain the transitory nature of these tumors as they grow and regress in the II genotype [5, 39].
One of the tumor types morphologically resembled luteal tissue with one notable exception: luteal tissue is highly vascularized whereas the tumor tissue was not. It is important to note that while we have classified these tumors as containing fibroblast- and luteal-like cells, this classification is based solely on the morphological characteristics of the cells. Since corpora lutea do not form in II ewes, these tumors cannot be of true luteal origin. None of the genes examined were clearly expressed in tumors with predominately fibroblast- and luteal-like cells. However, ovine corpora lutea do not express mRNA for -inhibin subunit or the ßA- and ßB-activin/inhibin subunits [16] or the FSH-R [48]. Thus, if these tumors represent a tissue similar to luteal tissue, expression of these genes might not be expected. However, ovine luteal tissue expresses follistatin [49], SCF [50], and c-kit [51] mRNA; but significant expression of these genes in the fibroblast/luteal-like tumors was not observed. In addition, if these tumors were representative of luteal tissue, one would expect the tissue to be steroidogenic. A previous study of II tumors was unable to detect measurable plasma concentrations of progesterone although some steroid was detected in homogenates or cyst fluid from these structures [5]. The inability to detect steroids in the circulation may be due to the aforementioned lack of vascularization of the tumor and not an inability to synthesize steroids. Definition of the likely cellular origin and physiological phenotype of these tumors, including their steroidogenic capacity, awaits further studies.
The following model for development of the abnormal structures in the ovary of the II ewe is proposed. First, follicular development in II ewes is arrested at the type 2 (primary) stage as granulosa cell proliferation necessary to develop to the type 3 stage fails to occur. However, the oocyte continues to grow until it reaches a stage at which it is no longer supported by its surrounding somatic cell populations. The oocyte then degenerates and leaves a nodule of somatic cells with the characteristics of granulosa cells in a type 3 follicle. The growth of tumors with granulosa-like cells then develops from the coalescing of nodules and/or from proliferation of cells in the nodule(s) due to prolonged exposure to high concentrations of FSH. Subsequently, as the tumor enlarges, it secretes increasing amounts of bioactive inhibin, which in turn causes a decrease in the plasma concentrations of FSH. When exposed to decreased concentrations of FSH, the tumor then regresses or differentiates into a structure with luteal-like morphology. Since this tumor type does not secrete inhibin, the plasma concentrations of FSH increase once more. Alternatively, tumors with luteal-like morphology may be formed in times of differing endocrine status such as suppressed FSH concentration in the presence of a tumor that secretes inhibin. It is important to note that only a limited number of tumors were examined in this study, and the possibility remains that other morphologically distinct types of tumors are present in some animals.
In summary, a single copy of the Inverdale gene did not change ontogeny of expression of mRNA encoding SCF, c-kit, -inhibin subunit, ßA- and ßB-subunits of activin/inhibin, follistatin, and FSH-R from that observed in ewes not carrying the gene. In addition, gene expression in the abnormal ovarian structures in the II ewe was similar to that observed in granulosa cells of normal follicles, thus supporting the hypothesis that these structures are of granulosa cell origin. Further, many of the tumors observed in the II ewe share multiple characteristics with granulosa-cell tumors observed in humans. One notable exception is the lack of malignancy observed in the tumors of the II ewe. Gaining an understanding of the mechanisms controlling the growth and regression of the tumors in the II ewes may give insight into the mechanisms underlying the metastatic potential in human ovarian cancers. Thus, the ewe homozygous for the Inverdale gene provides a unique animal model for study of the development of ovarian tumors.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by New Zealand Foundation for Research, Science and Technology. 
2 Correspondence: Jennifer L. Juengel, Wallaceville Animal Research Centre, PO Box 40063, Upper Hutt, New Zealand. FAX: 64 4 922 1380; juengelj{at}agresearch.cri.nz
3 Current address: National Centre for Disease Investigation, Ministry of Agriculture and Forestry, PO Box 40742, Upper Hutt, New Zealand.
Accepted: January 11, 2000.
Received: November 1, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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or ßA inhibin subunit mRNA is expressed in ovine fetal ovaries in late gestation. J Mol Endocrinol 1994; 13:1–9.[Abstract]- and ßA-subunit mRNA and protein in the fetal sheep ovary throughout gestation. Mol Cell Endocrinol 1995; 107:141–147.[CrossRef][Medline] and ßA inhibin mRNA in the sheep ovary during the oestrous cycle. J Mol Endocrinol 1994; 12:181–193.[Abstract] (INHA) to chromosome 2 in sheep and cattle. Mamm Genome 1995; 6:309.-Inhibin is a tumour-suppressor gene with gonadal specificity in mice. Nature 1992; 360:313–319.[CrossRef][Medline]-inhibin locus on chromosome 2q is not a feature of human granulosa cell tumours. Gynecol Oncol 1997; 65:387–390.[CrossRef][Medline]This article has been cited by other articles:
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  K. Hayashi, A. R O'Connell, J. L Juengel, K. P McNatty, G. H Davis, F. W Bazer, and T. E Spencer Postnatal uterine development in Inverdale ewe lambs Reproduction, March 1, 2008; 135(3): 357 - 365. [Abstract] [Full Text] [PDF]
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 C. J. Guigon and S. Magre Contribution of Germ Cells to the Differentiation and Maturation of the Ovary: Insights from Models of Germ Cell Depletion Biol Reprod, March 1, 2006; 74(3): 450 - 458. [Abstract] [Full Text] [PDF]
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 J.L. Juengel and K.P. McNatty The role of proteins of the transforming growth factor-{beta} superfamily in the intraovarian regulation of follicular development Hum. Reprod. Update, March 1, 2005; 11(2): 144 - 161. [Abstract] [Full Text] [PDF]
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B. Mandon-Pepin, A. Oustry-Vaiman, B. Vigier, F. Piumi, E. Cribiu, and C. Cotinot Expression Profiles and Chromosomal Localization of Genes Controlling Meiosis and Follicular Development in the Sheep Ovary Biol Reprod, March 1, 2003; 68(3): 985 - 995. [Abstract] [Full Text] [PDF]
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K. A. Logan, J. L. Juengel, and K. P. McNatty Onset of Steroidogenic Enzyme Gene Expression During Ovarian Follicular Development in Sheep Biol Reprod, April 1, 2002; 66(4): 906 - 916. [Abstract] [Full Text] [PDF]
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