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Departments of Molecular & Integrative Physiology Pathology and Laboratory Medicine University of Kansas Medical Center Kansas City, Kansas 66160
Address all correspondence and requests for reprints to: T. Rajendra Kumar, Ph.D., Department of Molecular & Integrative Physiology, University of Kansas Medical Center, Kansas City, Kansas 66160. E-mail: tkumar{at}kumc.edu.
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Introduction |
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 Top Introduction Transgenic Mouse Models to...Major Findings of Burdette...Final Remarks and Future...References |
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Several hypotheses have been put forward to explain the mechanistic basis for development of the ovarian epithelial cancer, including incessant ovulations as one of the important factors contributing to the onset of this cancer (6, 7, 8, 9, 10, 11). A model that emerged based on this hypothesis is that chronic ovulations cause invaginations of ovarian surface epithelial cells, leading to formation of inclusion cysts that serve as precursors for more malignant epithelial cancers. However, this hypothesis has not been rigorously tested in a genetic model system. Furthermore, the characteristics of cysts and the key players involved in cyst formation have not been well studied.
In this issue of Endocrinology, Burdette et al. (12) exploit a combination of pharmacological and in vivo approaches, and provide evidence that gonadotropin-induced multiple ovulations cause ovarian epithelial inclusion cysts in a unique strain of transgenic mice.
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Transgenic Mouse Models to Study Ovarian Epithelial Inclusion Cysts |
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TopIntroductionTransgenic Mouse Models to...Major Findings of Burdette...Final Remarks and Future...References |
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The common activins are either homo- or heterodimers of ßA- or ßB-subunits and inhibins are heterodimers consisting of an 
-subunit and either ßA- or ßB-subunit (14, 15, 16, 17). Members of the TGF-ß superfamily typically act through the serine/threonine kinase signaling pathway that involves a cascade of phosphorylations, followed by transcriptional responses generated by specific combinations of homologs of both the Caenorhabditis elegans protein SMA and the drosophila protein, mothers against decapentaplegic (SMAD) proteins (18, 19, 20).
In the first mouse model, rat inhibin -subunit is overexpressed, and this has likely caused a sequestration of activin subunits resulting in a loss of activin signaling (13). In a second mouse model, a dominant-negative form of Smad2 (Smad2DN) also caused loss of activin signaling by blocking both SMAD2 and SMAD3 (9). These are typical SMADs through which activins normally signal in many tissue/cell types including ovary (15, 20, 21). Both of the above transgenic mouse models have been generated using promoter sequences of Müllerian-inhibiting substance [the coding sequences encode MIS (also known as anti-Müllerian hormone, AMH) is also a TGF-ß family member] that direct transgenes specifically to the gonads (9, 22).
Strikingly, female mice of the above two strains develop, as early as 3 months of age, cysts that are continuous with the ovarian surface epithelium (9, 13). This remarkable recapitulation of the human endosalpingiosis phenotype in these transgenic mice suggests that the cyst cells may be derived from the highly plastic ovarian surface epithelial cell layer. In the present study, Burdette et al. (12) performed elegant experiments to define the characteristics of ovarian inclusion cysts that develop in Smad2DN transgenic mice. However, it is not clear why they chose the second mouse model over the first one.
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Major Findings of Burdette et al. (12): An Insight into Morphometric and Functional Aspects of Ovarian Epithelial Inclusion Cysts |
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TopIntroductionTransgenic Mouse Models to...Major Findings of Burdette...Final Remarks and Future...References |
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First, the authors have compared the effect of chronic superovulation of Smad2DN transgenic mice (on a CD1 genetic background) and normal CD1 mice. Chronic superovulation of mice at 42 d of age was achieved by periodic injections of equine chorionic gonadotropin followed by human chorionic gonadotropin. Superovulation with hormones continued for approximately 4.5 months and subsequently, the cyst number was quantified. Interestingly, chronic superovulation did not significantly increase cyst number when noninjected and hormone-treated CD1, or Smad2DN mouse groups were compared. However, when cyst number in noninjected CD1 group was compared with that in superovulated Smad2DN group, a significant increase was found. Most importantly, the cysts were all cytokeratin 8-immunopositive indicating that the cysts had originated from epithelium.
Second, the authors have identified that chronic superovulation of Smad2DN mice caused a location-dependent cyst accumulation in the ovary. These labor-intensive and quantifiable morphometric data revealed that the highest percentage of cysts was observed in the center of the ovary. These observations are consistent with earlier published data that demonstrated chronic superovulation resulted in greater number of cysts located in the center of the ovary in normal mice (23, 24). Whether this phenomenon is purely a stochastic event or an extracellular matrix-driven regulated process remains to be investigated.
Third, the effect of superovulation on cyst area, a criterion that is predicted to vary between genotypes and hormone treatments, was assessed. These studies indicated that total cyst area is dramatically increased in Smad2DN group compared with that observed in noninjected or superovulated CD1mice. However, the cyst area was decreased when each of the superovulated groups was compared with the corresponding noninjected control groups, respectively (i.e. superovulated vs. noninjected CD1 mice and superovulated vs. noninjected Smad2DN mice). Thus, superovulation caused a reduction in average cyst area, presumably due to accelerated formation of new cysts that are relatively small to begin with.
Fourth, the hypothesis that activins/TGF-ß are antiproliferative in many cells, generated based on several in vitro studies, was directly tested. In vivo 5-bromo-2-deoxyuridine labeling experiments using the Smad2DN mouse model were designed. The rationale was that blocking activin/TGF-ß signaling, by directing a dominant-negative form of SMAD2 selectively to the ovarian surface epithelium, would be expected to increase cell proliferation and thus might explain rapid cyst formation in superovulated Smad2DN mice. Data from this elaborate set of experiments confirmed that neither the proliferation rate of cells lining the ovarian cysts nor that of the ovarian surface epithelia cells was significantly enhanced. Thus, although cells within the cystic structures are highly proliferative (high 5-bromo-2-deoxyuridine-labeling index), the overall rate of proliferation did not change with superovulation.
One reason for the rapid proliferation of cells within the cysts could be prolonged stimulation by excess steroids, notably estrogen, produced as a result of normal or superovulation. Supporting this theory is the observation that these cells express both estrogen and progesterone receptors (7, 10). An additional source of estrogen would be the growing follicles stimulated in response to superovulation (7, 10).
Finally, how does SMAD2 phosphorylation in surface epithelium and cells within the inclusion cyst correlate in ovaries of Smad2DN mice? Experiments to address this issue provided intriguing and exciting data. Undetectable SMAD2 phosphorylation was observed in ovarian surface epithelium of 60% of transgenic mice, whereas in only 30% of CD1 mice. Interestingly, comparison within the same ovary indicated that the cysts, presumably originated from SMAD2-negative surface epithelium, contained more SMAD2 phosphorylation.
The regain of SMAD2 phosphorylation in the cysts, but its absence in surface epithelium of the majority of the transgenic mice may be explained as follows. Because the endogenous Amh promoter is active in ovarian surface epithelium, expression of SMAD2DN form would block phosphorylation of SMAD2 in these cells in transgenic mice. Although expression of MIS (produced from the endogenous mouse promoter) itself was retained in the cysts of normal mice, it was lost in those in ovaries of Smad2DN transgenic mice. This would suggest that the cysts in transgenic mice lose Amh promoter activity leading to loss of expression of SMAD2DN form, and thus SMAD2 phosphorylation is regained. It is also possible that Amh promoter may be temporally regulated in the cysts of transgenic mice; the promoter may be active during initial stages of cyst formation to produce SMAD2 DN and subsequently, its expression is lost as the cysts continue to grow.
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Final Remarks and Future Directions |
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TopIntroductionTransgenic Mouse Models to...Major Findings of Burdette...Final Remarks and Future...References |
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), similar to the "multiple hit" scenario in case of human ovarian epithelial cancers (32, 33, 34).
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What other signaling pathways interact to induce cyst formation? For example, Wnt signaling has been implicated in human ovarian epithelial cancers (35, 36, 37) and perhaps regulation of this pathway can be evaluated in cyst formation in Smad2DN mice. Would superovulations of Smad2DN mice on a tumor suppressor null background (such as p53, Rb, Pten, Brca1) cause more advanced neoplastic changes or even cancer (Fig. 1
)? Genetic intercrosses with Smad2DN and the available tumor suppressor null mice can be performed.
Why is SMAD2 phosphorylation in Smad2DN mice variable (undetectable in only 60% of mice)? Recently, Hox genes that specify regional identity in the reproductive tract have been implicated to cause variations in human ovarian epithelial cancers and in their appearance of Müllerian-like features (38). Because the transgene in the present study is expressed from the Amh promoter, this may explain the variability of SMAD2 phosphorylation in Smad2DN transgenic mice.
Finally, because the cyst formation occurs progressively, it will be feasible to "laser capture" cells within the cyst at different stages of development (39, 40, 41). This powerful genetic resource should be useful for large-scale expression profiling to identify both mRNAs and microRNAs that are developmentally regulated in cysts (39, 40, 41). Data from these experiments could identify several known and unknown gene interaction networks. It is hoped that such studies with the existing genetic model, coupled with comparative genomics with human tissue samples would ultimately help to accurately model human ovarian epithelial cancer in mice (32, 33). This knowledge would in turn be useful to design novel strategies for diagnosis and treatment of human ovarian epithelial cancer.
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Acknowledgments |
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Footnotes |
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Abbreviations: SMAD, Homologs of both the Caenorhabditis elegans protein SMA and the drosophila protein, mothers against decapentaplegic; Smad2DN, dominant-negative form of Smad2.
Received May 1, 2007.
Accepted for publication May 2, 2007.
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References |
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TopIntroductionTransgenic Mouse Models to...Major Findings of Burdette...Final Remarks and Future...References |
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-subunit. Endocrinology 142:5005–5014
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