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Introduction: Research on the Human Endometrium ERLIO GURPIDE Department of Obstetrics, Gynecology & Reproductive Science Mount Sinai School of Medicine New York, New York 10029
CARL0 BULLETTI Reproductive Medicine Unit Department of Obstetrics & Gynecology University of Bologna Via Massarenti I3 40138 Bologna, Italy
This volume of the Annals of the New York Academy of Sciences is the proceedings of an International Conference on the Primate Endometrium, held at the Mount Sinai Medical Center, New York City, May 28-30, 1990 in response to the accelerated increase in the number of reports describing novel, often surprising, properties of this tissue. Early fundamental experiments revealed uterotropic actions of estrogens and effects of progestins on the endometrium of rodents, rabbits and primates. Although the human tissue is strongly affected by these hormones, it differs from the endometrium of rabbits and rodents in several aspects regarding hormonal regulation of enzymatic activities, secretory products and decidua formation, as could be expected from species differences in reproductive strategies and temporal patterns of ovarian hormone production. In contrast, the Rhesus monkey has menstrual cycles similar to those of humans and has been successfully used as an experimental model providing conditions which cannot be reproduced in women. Hence the focus of the Conference on the primate endometrium. The attractiveness of endometrial research derives from both its clinical relevance and the opportunity it offers to investigate topics of fundamental importance in cell biology. TABLE1 outlines some of the clinical and biological topics relevant to endometrial research, most of which are expertly covered in this volume. There are characteristics of the human endometrium which make this tissue unique as a research model. Endometrial tissue is commonly available as surgical specimens obtained by curettage or after hysterectomies. The endogenous hormonal environment at which the portion of tissue available for in vitro research was exposed in vivo can be determined from histologic features or from analysis of blood samples at the time of surgery. In fact, regulation of biochemical parameters by either estradiol or progesterone, or lack of hormonal regulation, is suggested by changes observed in tissue samples obtained at different days of the menstrual cycle. Another attractive feature of endometrial research is the variety of endometrial samples that can be collected. Although most of the curettage specimens are histologically normal,s samples of 1
2
ANNALS NEW YORK ACADEMY OF SCIENCES
TABLE 1. Clinical and Biological Topics Relevant to Endometrial Research CLINICAL RELEVANCE OF STUDIES ON HUMAN ENDOMETRIUM Infertility In virro fertilization Contraception Menstruation, dysfunctional uterine bleeding Endometriosis Hormonal replacement therapy Preterm laborldelivery Endometrial cancer OF CELLBIOLOGY/BIOCHEMISTRY RELEVANT TO ENDOMETRIAL STUDIES TOPICS Differentiation Stromal-epithelial interactions Embryo-endometrium interactions Extracellular matrix functions Mechanisms of steroid hormone actions Cell proliferation; growth factors Cytokines, eicosanoids, polyamines Autocrine and paracrine regulatory mechanisms Tumorigenesis, invasiveness, progression Immunology
hyperplastic and neoplastic tissue can also be obtained, allowing interesting biochemical comparisons with normal tissue exposed to similar endogenous hormonal environments. Furthermore, endometrium of pregnancy can be collected during surgical abortions or by scraping fetal membranes obtained after vaginal or cesarean deliveries. Development of procedures for the separation and culture of stromal and epithelial cells allowed the identification of drastic differences in the characteristics and hormonal responsiveness of these endometrial cell types. Highly homogeneous preparation of decidual cells from gestational endometrium were also obtained, making possible studies in which their properties and secretory capabilities could be compared with those of the stromal cells of proliferative or early secretory endometrium. In fact, stromal cells isolated from proliferative endometrium can now be decidualized in vitro to yield morphologically distinct cells showing, as far as currently tested, the same products of differentiation detected in decidual cells of pregnancy. Of great interest are recent findings on cells of bone marrow origin which reside, and can even proliferate, in the endometrium. Various endometrial adenocarcinoma cell lines, some of them responsive to steroid hormones and growth factors, are widely used for studies on regulation of cancer cell proliferation, providing information which might be relevant to normal epithelial endometrial cells and useful in the evaluation of possible aberrations associated with neoplastic transformation. Most of the experimental results presented by the contributors to these proceedings have been generated either by direct analysis of endometrial tissue, collected at various physiologic or drug-induced in vivo conditions, or by in vitro manipulation of endometrial tissue. Different preparations are available for in vitro studies to suit specific experimental purposes. For instance, realization of the importance of paracrine com-
GURPIDE & BULLETTI: INTRODUCTION
3
munication among the various types of endometrial cells indicates that preservation of the cellular architecture of the tissue during in virro studies is desirable when the emphasis is placed on physiologic relevance. The preparation that may be most appropriate for this purpose is based on extracorporeal perfusion of whole uteri through arterial vessels catheterized after hysterectomy, and collection of effluents and endometrial samples. Such perfusions can be prolonged from 48-72 h with retention of endometrial viability, as evaluated by morphologic and biochemical parameters,a7and applied to test for actions of agents added to the perfusion medium, to evaluate synthetic and metabolic activities and to study interactions with human trophoblastic cells in attempts to simulate endometrial interactions with the trophectoderm during embryo implantation. Endometrial fragments offer another acceptable level of preservation of tissular structure and have been successfully used to test for hormonal effects, as in the early experiments on actions of progestins on polarized glycogen accumulation in glandular epithelial cells8 or quantitative studies on estrogen metabolism.9J0 Tissue fragments placed on grids at the surface of the medium in culture dishes, retain morphologic viability and responsiveness to hormones for 2 to 4 days, or longer if appropriate hormonal support is provided. Products secreted into the culture medium can be identified, rates of production can be measured and cells of origin can be determined by using immunochemical techniques, Northern blot analysis of mRNA extracted from the tissue, labeled thymidine incorporation into nucleic acids, and in siru hybridization procedures. Structurally preserved endometrial glands, isolated from tissue fragments by enzymatic dispersion and removal of stromal cells, offer a well-defined system for biochemical studies, albeit at the expense of losing information on possible influences of stromal cells. Dispersion and resolution of endometrial cells, leading to near homogeneous cell preparations, allow the identification of biochemical characteristics and responsiveness to bioactive agents specific to each cell type. It has been recognized, however, that the behavior of cells depends on the culture conditions and most particularly on the nature of the substrate to which they are attached. Stromal cells grown on plastic surfaces show a distorted cytoskeletal distribution; epithelial cells, which also become flat when attached to the plastic surface, lose the ability to establish their characteristic intercellular junctional structures. In contrast, cells cultured on collagen or other extracellular matrix components maintain their morphologic and functional polarity. Co-cultures of cells of different types, including decidual cells, provide reasonable models to study paracrine intercellular communications and the postulated role of the stroma as a mediator in actions of estradiol on epithelial cells. Of particular current interest, reflected by several contributions in these proceedings, is the study of decidual cells of the luteal phase endometrium and of the decidua parietalis. These cells have unique properties, made evident more than a decade ago by the recognition that they were a source of prolactinIlJ2 and, more recently, by reports on their capability to produce cytokines.I3.l4 Furthermore, the effects of prolactin, cytokines and neuropeptides of decidual origin on neighboring cells and fetal tissues may be important in the regulation of processes related to the onset of labor. The design of experimental conditions under which stromal cells isolated from proliferative endometrium can be “decidualized in virro under the influence of ovarian horm o n e ~has ~ ~provided an important experimental tool for the study of the decidual
4
ANNALS NEW YORK ACADEMY O F SCIENCES
differentiation process and for the distinction of true decidual cells from endometrial granulated lymphocytes, macrophages and other resident lymphoid cells, also capable of producing cytokines. Specimens of primary endometrial adenocarcinoma, collected after hysterectomy for determination of grade, hormone receptor status, ploidy, oncogene expression and the presence of marker antigens, are also available for research purposes. Direct tests for responsiveness of cancer tissue to progestins and estrogens provide information of potential prognostic value and are possibly useful in the prediction of responses to hormonal treatment or cytotoxic therapy. l6 Tumor tissue fragments may be used for in vitro studies, may be implanted into nude mice to form tumors or may serve to start cell cultures, some of which can lead to the establishment of endometrial cancer cell lines. Such preparations have been useful for research purposes, particularly in studies on cell proliferation and its regulation by hormones, growth factors, cytokines and other mitogenic or growth supporting agents. Although similar studies have been conducted for many years using human breast cancer cell lines and breast tumors transplanted into nude mice, biochemical differences between human breast and endometrial adenocarcinoma cells, such as their responses to tamoxifen and hydroxytamoxifen,” justify and enhance the interest in studies with cells from both sites. The studies on the endometrium presented in this volume exemplify the mutual dependence of the “basic science” and Winical” approaches to research, each providing to the other the stimulus, inspiration, rationale and methodology that make progress possible. REFERENCES
I . CORNER, G. W. & W. M. ALLEN.1929. Physiology of the corpus luteum. 11. Production of a special uterine reaction (progestational proliferation) by extracts of the corpus lutem. Am. J. Physiol. 88: 326. JR. 1961. Action of estrogens and progesterone on the reproductive 2. HISAW,F. L. & F. L. HISAW tract of lower primates. In Sex and Internal Secretions, W. C. Young & G. W. Corner, Eds. Vol. I: 556-589. The Williams & Williams Co. 3. PSYCHOYOS, A. 1973. Endocrine control of egg implantation. In Handbook of Physiology, R. 0. Greep & E. B. Astwood, Eds. Section 7, Part 2: 187-216. Am. Physiol. SOC. 4. KATZENELLENBOGEN, B. S. & J. GORSKI.1975. Estrogen actions and synthesis of macromolecules in target cells. In Biochemical Actions of Hormones. G. Litwack, Ed. Vol. 111: 188-238, Academic Press. 5. VELLIOS,F. 1984. Endometrial hyperplasia and carcinoma in-situ. Gynecol. Oncol. 2: 152. 6. BULLETTI, C., V. M. JASSONI, L. LUBICZ,C. FLAMIGNI & E. GURPIDE.1986. Extracorporeal perfusion of the human uterus. Am. J. Obstet. Gynecol. 154: 683. 7. BULLETTI, C., V. M. JASSONI,P.M. CIOTTI,S. TABANELLI, S. NALDI& C. FLAMIGNI. 1988. Extraction of estrogens by human perfused uterus. Effect of membrane permeability and binding by serum proteins on differential influx into endometrium and myometrium. Am. J. Obstet. Gynecol. 159: 509. 8. CSERMELY, T., L. M. DEMERS & E. C. HUGHES.1969. Organ culture of human endometrium. Effects of progesterone. Obstet. Gynecol. 34: 252. 9. GURPIDE, E. & M. WELCH.1969. Dynamics of uptake of estrogens and androgens by human endometrium. J. Biol. Chem. 244: 5159. 10. TSENG,L., A. STOLEE& E. GURPIDE.1972. Quantitative studies on the uptake and metabolism of estrogens and progesterone by human endometrium. Endocrinology 90: 390. 11. RIDDICK, D. H. & M. A. KUSMIK.1977. Decidua: A possible source of amniotic fluid prolactin. Am. J. Obstet. Gynecol. 127: 187.
GURPIDE & BULLETTI: INTRODUCTION
5
12. GOLLANDER, A., HURLEY, T., BARRET, J. & S. HANDWERGER. 1979. Synthesis of prolactin by human decidua in vitro. J. Endocrinol. 82: 263. 13, CASEY,M. L., S. M. Cox, B. BEUTLER, L. MILEWICH & P. C. MACDONALD. 1989. Cachectidtumor 14.
15.
16.
17.
necrosis factor-alpha formation in human decidua: Potential role of cytokines in infectioninduced preterm labor. J. Clin. Invest. 83: 430. ROMERO, R., Y.K.Wu, D. T,BRODY,E. OTARZUN, G. W. DUFF& S. K. DURUM.1989. Human decidua: A source of interleukin-I. Obstet. Gynecol. 73: 31. HUANG, J. R., L. TSENG,P. BISCHOF& 0. A. JANNE.1987. Regulation of prolactin production by progestin, estrogen and relaxin in human endometrial stromal cells. Endocrinology U1:2011. KAUPPILA, A,, H. E. ISOTALO, T. KIVINBEN & R. J. VIHKO.1986. Prediction of clinical outcome with estrogen and progestin receptor concentrations and their relationships to clinical and histopathological variables in endometrial cancer. Cancer Res. 46: 5380. GOTTARDIS. M. M., S. P. ROBINSON, P. G. SATYASWAROOP & V. C. JORDAN.1988. Contrasting actions of tamoxifen on endometrial and breast tumor growth in the athymic mouse. Cancer Res. 48: 812.
Introduction: Research on the Human Endometrium ERLIO GURPIDE Department of Obstetrics, Gynecology & Reproductive Science Mount Sinai School of Medicine New York, New York 10029
CARL0 BULLETTI Reproductive Medicine Unit Department of Obstetrics & Gynecology University of Bologna Via Massarenti I3 40138 Bologna, Italy
This volume of the Annals of the New York Academy of Sciences is the proceedings of an International Conference on the Primate Endometrium, held at the Mount Sinai Medical Center, New York City, May 28-30, 1990 in response to the accelerated increase in the number of reports describing novel, often surprising, properties of this tissue. Early fundamental experiments revealed uterotropic actions of estrogens and effects of progestins on the endometrium of rodents, rabbits and primates. Although the human tissue is strongly affected by these hormones, it differs from the endometrium of rabbits and rodents in several aspects regarding hormonal regulation of enzymatic activities, secretory products and decidua formation, as could be expected from species differences in reproductive strategies and temporal patterns of ovarian hormone production. In contrast, the Rhesus monkey has menstrual cycles similar to those of humans and has been successfully used as an experimental model providing conditions which cannot be reproduced in women. Hence the focus of the Conference on the primate endometrium. The attractiveness of endometrial research derives from both its clinical relevance and the opportunity it offers to investigate topics of fundamental importance in cell biology. TABLE1 outlines some of the clinical and biological topics relevant to endometrial research, most of which are expertly covered in this volume. There are characteristics of the human endometrium which make this tissue unique as a research model. Endometrial tissue is commonly available as surgical specimens obtained by curettage or after hysterectomies. The endogenous hormonal environment at which the portion of tissue available for in vitro research was exposed in vivo can be determined from histologic features or from analysis of blood samples at the time of surgery. In fact, regulation of biochemical parameters by either estradiol or progesterone, or lack of hormonal regulation, is suggested by changes observed in tissue samples obtained at different days of the menstrual cycle. Another attractive feature of endometrial research is the variety of endometrial samples that can be collected. Although most of the curettage specimens are histologically normal,s samples of 1
2
ANNALS NEW YORK ACADEMY OF SCIENCES
TABLE 1. Clinical and Biological Topics Relevant to Endometrial Research CLINICAL RELEVANCE OF STUDIES ON HUMAN ENDOMETRIUM Infertility In virro fertilization Contraception Menstruation, dysfunctional uterine bleeding Endometriosis Hormonal replacement therapy Preterm laborldelivery Endometrial cancer OF CELLBIOLOGY/BIOCHEMISTRY RELEVANT TO ENDOMETRIAL STUDIES TOPICS Differentiation Stromal-epithelial interactions Embryo-endometrium interactions Extracellular matrix functions Mechanisms of steroid hormone actions Cell proliferation; growth factors Cytokines, eicosanoids, polyamines Autocrine and paracrine regulatory mechanisms Tumorigenesis, invasiveness, progression Immunology
hyperplastic and neoplastic tissue can also be obtained, allowing interesting biochemical comparisons with normal tissue exposed to similar endogenous hormonal environments. Furthermore, endometrium of pregnancy can be collected during surgical abortions or by scraping fetal membranes obtained after vaginal or cesarean deliveries. Development of procedures for the separation and culture of stromal and epithelial cells allowed the identification of drastic differences in the characteristics and hormonal responsiveness of these endometrial cell types. Highly homogeneous preparation of decidual cells from gestational endometrium were also obtained, making possible studies in which their properties and secretory capabilities could be compared with those of the stromal cells of proliferative or early secretory endometrium. In fact, stromal cells isolated from proliferative endometrium can now be decidualized in vitro to yield morphologically distinct cells showing, as far as currently tested, the same products of differentiation detected in decidual cells of pregnancy. Of great interest are recent findings on cells of bone marrow origin which reside, and can even proliferate, in the endometrium. Various endometrial adenocarcinoma cell lines, some of them responsive to steroid hormones and growth factors, are widely used for studies on regulation of cancer cell proliferation, providing information which might be relevant to normal epithelial endometrial cells and useful in the evaluation of possible aberrations associated with neoplastic transformation. Most of the experimental results presented by the contributors to these proceedings have been generated either by direct analysis of endometrial tissue, collected at various physiologic or drug-induced in vivo conditions, or by in vitro manipulation of endometrial tissue. Different preparations are available for in vitro studies to suit specific experimental purposes. For instance, realization of the importance of paracrine com-
GURPIDE & BULLETTI: INTRODUCTION
3
munication among the various types of endometrial cells indicates that preservation of the cellular architecture of the tissue during in virro studies is desirable when the emphasis is placed on physiologic relevance. The preparation that may be most appropriate for this purpose is based on extracorporeal perfusion of whole uteri through arterial vessels catheterized after hysterectomy, and collection of effluents and endometrial samples. Such perfusions can be prolonged from 48-72 h with retention of endometrial viability, as evaluated by morphologic and biochemical parameters,a7and applied to test for actions of agents added to the perfusion medium, to evaluate synthetic and metabolic activities and to study interactions with human trophoblastic cells in attempts to simulate endometrial interactions with the trophectoderm during embryo implantation. Endometrial fragments offer another acceptable level of preservation of tissular structure and have been successfully used to test for hormonal effects, as in the early experiments on actions of progestins on polarized glycogen accumulation in glandular epithelial cells8 or quantitative studies on estrogen metabolism.9J0 Tissue fragments placed on grids at the surface of the medium in culture dishes, retain morphologic viability and responsiveness to hormones for 2 to 4 days, or longer if appropriate hormonal support is provided. Products secreted into the culture medium can be identified, rates of production can be measured and cells of origin can be determined by using immunochemical techniques, Northern blot analysis of mRNA extracted from the tissue, labeled thymidine incorporation into nucleic acids, and in siru hybridization procedures. Structurally preserved endometrial glands, isolated from tissue fragments by enzymatic dispersion and removal of stromal cells, offer a well-defined system for biochemical studies, albeit at the expense of losing information on possible influences of stromal cells. Dispersion and resolution of endometrial cells, leading to near homogeneous cell preparations, allow the identification of biochemical characteristics and responsiveness to bioactive agents specific to each cell type. It has been recognized, however, that the behavior of cells depends on the culture conditions and most particularly on the nature of the substrate to which they are attached. Stromal cells grown on plastic surfaces show a distorted cytoskeletal distribution; epithelial cells, which also become flat when attached to the plastic surface, lose the ability to establish their characteristic intercellular junctional structures. In contrast, cells cultured on collagen or other extracellular matrix components maintain their morphologic and functional polarity. Co-cultures of cells of different types, including decidual cells, provide reasonable models to study paracrine intercellular communications and the postulated role of the stroma as a mediator in actions of estradiol on epithelial cells. Of particular current interest, reflected by several contributions in these proceedings, is the study of decidual cells of the luteal phase endometrium and of the decidua parietalis. These cells have unique properties, made evident more than a decade ago by the recognition that they were a source of prolactinIlJ2 and, more recently, by reports on their capability to produce cytokines.I3.l4 Furthermore, the effects of prolactin, cytokines and neuropeptides of decidual origin on neighboring cells and fetal tissues may be important in the regulation of processes related to the onset of labor. The design of experimental conditions under which stromal cells isolated from proliferative endometrium can be “decidualized in virro under the influence of ovarian horm o n e ~has ~ ~provided an important experimental tool for the study of the decidual
4
ANNALS NEW YORK ACADEMY O F SCIENCES
differentiation process and for the distinction of true decidual cells from endometrial granulated lymphocytes, macrophages and other resident lymphoid cells, also capable of producing cytokines. Specimens of primary endometrial adenocarcinoma, collected after hysterectomy for determination of grade, hormone receptor status, ploidy, oncogene expression and the presence of marker antigens, are also available for research purposes. Direct tests for responsiveness of cancer tissue to progestins and estrogens provide information of potential prognostic value and are possibly useful in the prediction of responses to hormonal treatment or cytotoxic therapy. l6 Tumor tissue fragments may be used for in vitro studies, may be implanted into nude mice to form tumors or may serve to start cell cultures, some of which can lead to the establishment of endometrial cancer cell lines. Such preparations have been useful for research purposes, particularly in studies on cell proliferation and its regulation by hormones, growth factors, cytokines and other mitogenic or growth supporting agents. Although similar studies have been conducted for many years using human breast cancer cell lines and breast tumors transplanted into nude mice, biochemical differences between human breast and endometrial adenocarcinoma cells, such as their responses to tamoxifen and hydroxytamoxifen,” justify and enhance the interest in studies with cells from both sites. The studies on the endometrium presented in this volume exemplify the mutual dependence of the “basic science” and Winical” approaches to research, each providing to the other the stimulus, inspiration, rationale and methodology that make progress possible. REFERENCES
I . CORNER, G. W. & W. M. ALLEN.1929. Physiology of the corpus luteum. 11. Production of a special uterine reaction (progestational proliferation) by extracts of the corpus lutem. Am. J. Physiol. 88: 326. JR. 1961. Action of estrogens and progesterone on the reproductive 2. HISAW,F. L. & F. L. HISAW tract of lower primates. In Sex and Internal Secretions, W. C. Young & G. W. Corner, Eds. Vol. I: 556-589. The Williams & Williams Co. 3. PSYCHOYOS, A. 1973. Endocrine control of egg implantation. In Handbook of Physiology, R. 0. Greep & E. B. Astwood, Eds. Section 7, Part 2: 187-216. Am. Physiol. SOC. 4. KATZENELLENBOGEN, B. S. & J. GORSKI.1975. Estrogen actions and synthesis of macromolecules in target cells. In Biochemical Actions of Hormones. G. Litwack, Ed. Vol. 111: 188-238, Academic Press. 5. VELLIOS,F. 1984. Endometrial hyperplasia and carcinoma in-situ. Gynecol. Oncol. 2: 152. 6. BULLETTI, C., V. M. JASSONI, L. LUBICZ,C. FLAMIGNI & E. GURPIDE.1986. Extracorporeal perfusion of the human uterus. Am. J. Obstet. Gynecol. 154: 683. 7. BULLETTI, C., V. M. JASSONI,P.M. CIOTTI,S. TABANELLI, S. NALDI& C. FLAMIGNI. 1988. Extraction of estrogens by human perfused uterus. Effect of membrane permeability and binding by serum proteins on differential influx into endometrium and myometrium. Am. J. Obstet. Gynecol. 159: 509. 8. CSERMELY, T., L. M. DEMERS & E. C. HUGHES.1969. Organ culture of human endometrium. Effects of progesterone. Obstet. Gynecol. 34: 252. 9. GURPIDE, E. & M. WELCH.1969. Dynamics of uptake of estrogens and androgens by human endometrium. J. Biol. Chem. 244: 5159. 10. TSENG,L., A. STOLEE& E. GURPIDE.1972. Quantitative studies on the uptake and metabolism of estrogens and progesterone by human endometrium. Endocrinology 90: 390. 11. RIDDICK, D. H. & M. A. KUSMIK.1977. Decidua: A possible source of amniotic fluid prolactin. Am. J. Obstet. Gynecol. 127: 187.
GURPIDE & BULLETTI: INTRODUCTION
5
12. GOLLANDER, A., HURLEY, T., BARRET, J. & S. HANDWERGER. 1979. Synthesis of prolactin by human decidua in vitro. J. Endocrinol. 82: 263. 13, CASEY,M. L., S. M. Cox, B. BEUTLER, L. MILEWICH & P. C. MACDONALD. 1989. Cachectidtumor 14.
15.
16.
17.
necrosis factor-alpha formation in human decidua: Potential role of cytokines in infectioninduced preterm labor. J. Clin. Invest. 83: 430. ROMERO, R., Y.K.Wu, D. T,BRODY,E. OTARZUN, G. W. DUFF& S. K. DURUM.1989. Human decidua: A source of interleukin-I. Obstet. Gynecol. 73: 31. HUANG, J. R., L. TSENG,P. BISCHOF& 0. A. JANNE.1987. Regulation of prolactin production by progestin, estrogen and relaxin in human endometrial stromal cells. Endocrinology U1:2011. KAUPPILA, A,, H. E. ISOTALO, T. KIVINBEN & R. J. VIHKO.1986. Prediction of clinical outcome with estrogen and progestin receptor concentrations and their relationships to clinical and histopathological variables in endometrial cancer. Cancer Res. 46: 5380. GOTTARDIS. M. M., S. P. ROBINSON, P. G. SATYASWAROOP & V. C. JORDAN.1988. Contrasting actions of tamoxifen on endometrial and breast tumor growth in the athymic mouse. Cancer Res. 48: 812.
