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Immunohistochemical localization of relaxin in the reproductive system of the female opossum (Didelphis virginiana)
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Immunohistochemical localization of relaxin in the reproductive system of the female opossum
(Didelphis virginiana) William
J. Krause and Debra M. Sherman*
Department of Anatomy and Neurobiology, School of Medicine, University of Missouri, Columbia, Missouri 65212, USA, and *Department of Botany and Plant Pathology, Purdue University, West Lafayette, Indiana, USA
Summary. Relaxin-immunoreactivity was demonstrated in the cytoplasm in the luteal cells from pregnant and lactating opossums. Immunoreactivity for relaxin was not demonstrated elsewhere in the ovary, in the reproductive tract or in the placenta. The corpus luteum is thought to be the primary source of relaxin in Didelphis and in this regard is similar to several eutherian mammals including man. Key words: Relaxin-immunoreactivity - Corpus luteum Opossum
Following intracellular synthesis, relaxin is stored at least temporarily in small, electron-dense, membrane-bound granules prior to being released. Relaxin containing granules have been identified in luteal cells of the pig (Kendall et al. 1978; Fields and Fields 1985), rat (Anderson and Sherwood 1984; Fields 1984) and mouse (Anderson et al. 1984), in cells of guinea pig endometrial glands (Larkin et al. 1983) and in placental syncytiotrophoblast cells of the rabbit (Eldridge and Fields 1986). This study reports the first immunohistochemicallocalization of relaxin in the female reproductive system of a metatherian species, the North American opossum.
Introduction Relaxin, a peptide hormone that plays a role during pregnancy, parturition and lactation in mammals, acts primarily on connective tissue elements at parturition to relax pelvic ligaments and dilate the cervix. In some species this peptide functions to maintain pregnancy by inhibiting the contractions of uterine smooth muscle. Relaxin also is thought to influence other reproductive tissues but these effects are less well characterized (Bryant-Greenwood 1982; Sherwood 1988).
The highest levels of relaxin are produced by the female reproductive tract late in pregnancy. In some species (sheep, cow, pig, rat, mouse, human) the corpus luteum is the primary source of relaxin (Mathieu et al. 1981; Anderson 1982; Anderson and Sherwood 1984; Anderson et al. 1984; Vaupel et al. 1985; Ali et al. 1986) whereas in other forms (cat, rabbit, horse) the placenta is the primary source (Eldridge and Fields 1985; Sherwood 1988). In yet other mammals such as the guinea pig, the uterus is the source of relaxin (Pardo et al. 1980; Pardo and Larkin 1982).
~I
Ann. Anat. (1992) 174: 341-344 Gustav Fischer Verlag Jena
Materials and methods Nine adult female opossums (Didelphis virginiana) were examined in this study and consisted of pregnants and animals 1 week into lactation. Each group consisted of at least three animals. Approximate ages of pregnancies were determined from timed pregnancies. Female opossums were stripped of their litters and placed in breeding pens with continuous access to males. A sperm positive date was determined by examining smears taken from the urogenital sinus each morning. Females 10112 and 11 Y2 days into pregnancy (opossums have a 12112 day gestation period) as well as the lactating animals used were killed by an intra-cardial injection of Ketamine followed by exsanguination. Ovaries, oviducts, uteri, lateral vaginal canals as well as placentae from pregnant animals were removed as quickly as possible and fixed by immersion in Bouin's solution for a period of 24 hours. Tissues were processed routinely for paraffin embedding, sectioned at 5!tm and mounted on acidcleaned slides. The mounted sections were deparaffinized in xylene and rehydrated to water prior to incubation in 1.0 % H20 2 to remove endogenous peroxide. After blocking with normal goat serum, the sections were incubated with the primary antiserum (anti-porcine
relaxin, dilution 1: 10000) for 24 hours at 4°C. Immunohistochemical visualization was accomplished by the avidin-biotin-perioxidase complex (ABC) procedure (Hsu et al. 1981) using a Vecta-Iab "elite" kit. Peroxidase activity was demonstrated by incubation with 3,3' diaminobenzidine tetrahydrochloride containing 0.01 % H 20 2 in 0.1 M Tris-Hel buffer. Controls consisted of pre-absorption of the primary antiserum with the corresponding antigen as well as omission of the primary antiserum. Sections of Bouin's fixed porcine corpora lutea served as positive controls. Treated sections were then dehydrated, cleared in xylene and coverslipped for examination. Occasional treated sections also were lightly counterstained with Mayer's haematoxylin prior to being coverslipped.
2). The immunoreactive staining is confined to the cytoplasm of most, but not all, luteal cells and is not observed in adjacent endothelial cells or fibroblasts (Figs. 3, 4). Likewise, relaxin-immunoreactivity is not observed in tissues comprising either the oviduct, uterus, lateral vaginal canals or the placenta of Didelphis. Specific staining is not observed in control sections incubated with normal goat serum .or porcine relaxin antiserum pre-absorbed with porcine relaxin. Luteal cells from porcine corpora lutea stain intensely for relaxin, further supporting the immunospecificity of the relaxin antiserum used.
Discussion
Results
In Didelphis as well as other but not all marsupial species,
Corpora lutea from pregnant and lactating opossums show intense immunoreactive staining with porcine antiserum to relaxin. Immunoreactivity for porcine relaxin is not observed in adjacent follicles or other regions of the ovary (Figs. 1,
the luteal phase comprises about 60 % of the oestrous cycle. Regression of the corpus luteum coincides with birth but it disappears slowly if lactation occurs (Tyndale-Biscoe and Renfree 1987). Luteal cells comprising the corpus luteum of
Fig. 1. A region of ovary containing three corpora lutea (L) and a secondary follicle (F). Note the intense immunoreactive staining for porcine relaxin in the corpora lutea and the absence of immunoreactive staining in the secondary follicle. Opossum 11 Y2 day of pregnancy. X 15. Fig. 2. A portion of a corpus luteum demonstrates that the relaxin-immunoreactivity is confmed to luteal cells . A primary follicle (F) and the adjacent stroma are unstained . Opossum 1ll-'2 day of pregnancy. X 100. Fig. 3 . Increased magnification demonstrates that the majority but not all luteal cells of a given section show intense immunoreactive staining for porcine relaxin. Opossum 11 Y2 day of pregnancy. X 300. Fig. 4. A region of corpus luteum from an opossum 11l-'2 days into pregnancy demonstrating relaxin-immunoreactivity (arrows) that was counterstained with Mayer's haematoxyIin illustrates in greater detail its general morphology. X 300.
342
the opossum are derived exclusively from granulosa cells of the previous follicle (Martinez-Esteve 1942). Corpora attain maximum size by postestrous day 7, begin to decline after day 8 and often appear shrunken by day 12 immediately prior to birth (Fleming and Harder 1983). Peripheral plasma levels of progesterone, the major steroid hormone of Didelphis (Cook and Nalbandov 1968), correspond closely with morphological changes that occur in the corpus luteum during the oestrous cycle and pregnancy (Harder and Fleming 1981). No significant differences with regard to progesterone levels in Didelphis could be detected between non-pregnant and pregnant animals. Corpora lutea are necessary for both parturition (Hartman 1925; Renfree 1974) and mammogenesis in Didelphis as well as other marsupials (TyndaleBiscoe and Renfree 1987; Hinds 1988). Corpora lutea of the brushtailed opossum, Trichosurus vulpecula and tammar wallaby, Macropus eugenii have been reported to contain high concentrations of relaxin as measured by mouse pubic-symphysis bioassay (Tyndale-Biscoe 1969, 1981). What role relaxin plays in corpora lutea of marsupials or how it influences the pseudo-vaginal (median) canal is less clear. Relaxin is thought to act in concert with progesterone and estrogen in the preparation of the birth canal of most mammals prior to parturition. Whether or not this interaction occurs in the formation and physiological control of the unique birth canal of marsupials (the median-vaginal canal) is unknown. The presence of immunoreactivity for relaxin in most, but not all, luteal cells in Didelphis may be related to the production and/or concentration of relaxin at levels below the resolution of the light microscope or may be due to secretory exhaustion of some cells. Relaxin-immunoreactivity is not observed in the uterine epithelium, uterine glands or placentae of Didelphis and in this way resembles serveral eutherian species such as the sheep, cow, pig, rat, mouse and human in which the corpus luteum is the primary source of relaxin (Mathieu et al. 1981; Anderson 1982; Anderson and Sherwood 1984; Anderson et al. 1987; Vaupel et al. 1985; Ali et al. 1986). The mechanism of action of relaxin on the reproductive tract in Didelphis or other marsupial forms is unknown. Acknowledgements. We would like to thank Dr. M. J. Fields and Dr. P. A. Fields for their generous gifts of porcine relaxin and antiporcine relaxin. We also would like to acknowledge Midge M. Lind for the preparation of the manuscript.
References Ali SM, McMurtry JP, Bagnell CA, Bryant-Greenwood GO (1986) Immunocytochemical localization of relaxin in corpora lutea of sows throughout the estrous cycle. Bioi Reprod 34: 139-143 Anderson LL (1982) Relaxin localization in porcine and bovine ovaries by assay and morphological techniques . Adv Exp Med Bioi 143: 1-67 Anderson MB, Sherwood 00 (1984) Ultrastructural localization of relaxin immunoreactivity in corpora lutea of pregnant rats . Endocrinology 114: 1124-1127
Anderson MB, Vaupel MR, Sherwood 00 (1984) Pregnant mouse corpora lutea: Immunocytochemical localization of relaxin and ultrastructure. Bioi Reprod 31 : 391- 397 Bryant-Greenwood GO (1982) Relaxin as a new hormone. Endocrin Rev 3: 62-94 Cook B, Nalbandov AV (1968) The effect of some pituitary hormones on progesterone synthesis in vitro by the luteinized ovary of the common opossum (Didelphis marsupialis virginiana) . J Reprod Fert 15: 267-275 Eldridge RK, Fields PA (1984) Rabbit placental relaxin: Purification and immuno-histochemical localization. Endocrinology 117 :
2512-2519 Eldridge RK, Fields PA (1986) Rabbit placental relaxin: Ultrastructurallocalization in secretory granules of the syncytiotrophoblast using rabbit placental relaxin antiserum. Endocrinology 115:
606-615 Fields P A (1984) Intracellular localization of relaxin in membranebound granules in the pregnant rat luteal cell. BioI Reprod 30:
753-762 Fields PA, Fields MJ (1985) Ultrastructural localization of relaxin in the corpus luteum of the nonpregnant, pseudopregnant, and pregnant pig. Bioi Reprod 32: 1169-1179 Fleming MW, Harder JD (1983) Luteal and follicular populations in the ovary of the opossum (Didelphis virginiana) after ovulation. J Reprod Fert 67: 29-34 Harder JD, Fleming MW (1981) Estradiol and progesterone profiles indicate a lack of endocrine recognition of pregnancy in the opossum. Science 212: 1400-1402 Hartmann CG (1925) The interruption of pregnancy by ovariectomy in the aplacental opossum: A study in the physiology of implantation. Am J Physiol 71: 436-454 Hinds LA (1988) Hormonal control oflactation. In: The Developing Marsupial. Models for Biomedical Research: CH TyndaleBioscoe and PA Janssens, eds. Springer Berlin, New York, London, pp 55-67 Hsu SM, Raine L, Fanger A (1981) Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques. J Histochem Cytochem 24: 577-580 Kendall JZ, Plopper LG, Bryant-Greenwood GA (1978) Ultrastructural immunoperoxidase demonstration of relaxin in the corpora lutea from pregnant sows. Bioi Reprod 18: 94-98 Larkin LH, Pardo RJ, Randall HR (1983) Imrnunoelectron microscopic localization of relaxin in endometrial gland cells of the guinea pig. Anat Rec 205 : I06A Mathieu P, Rabier J, Thomas K (1981) Localization of relaxin in human gestational corpus luteum. Cell Tissue Res 219: 213-216 Martinez-Esteve P (1942) Observations on the histology of the opossum ovary. Contrib Embryol 30: 17-26 Pardo RJ, Larkin LA, Fields P A (1980) Immunocytochemical localization of relaxin in endometrial glands of the pregnant guinea pig . Endocrinology 107: 2110-2112 Pardo RJ, Larkin LH (1982) Localization of relaxin in endometrial glands of the pregnant, lactating and ovariectomized hormonetreated guinea pigs. Am J Anat 164: 79-90 Renfree MB (1974) Ovariectomy during gestation in the American opossum, Didelphis marsupialis virginiana. J Reprod Fert 39 :
127-130 Sherwood DO (1988) Relaxin. In: The Physiology of Reproduction, E. Knobil and J. Neill, eds . Raven Press, New York, pp 585-673 Tyndale-Biscoe CH (1969) Relaxin activity during the oestrus cycle of the marsupial, Trichosurus vulpecula (Kerr). J Reprod Fert 19:
191-193 Tyndale-Biscoe CH (1981) Evidence for relaxin in marsupials. In:
343
Relaxin, G Bryant-Greenwood, H Niall and F Greenwood, eds. North Holland/Elsevier Biomedical Press, Amsterdam, pp 225-232 Tyndale-Biscoe CH, Renfree MB (1987) Reproductive Physiology of Marsupials. Cambridge University Press, New York
Vaupel MR, Sherwood OD, Anderson MB (1985) Immunocytochemical studies of relaxin in ovaries of pregnant and cycling mice. J Histochem Cytochem 33: 303-308 Accepted April 18, 1990
344
Immunohistochemical localization of relaxin in the reproductive system of the female opossum
(Didelphis virginiana) William
J. Krause and Debra M. Sherman*
Department of Anatomy and Neurobiology, School of Medicine, University of Missouri, Columbia, Missouri 65212, USA, and *Department of Botany and Plant Pathology, Purdue University, West Lafayette, Indiana, USA
Summary. Relaxin-immunoreactivity was demonstrated in the cytoplasm in the luteal cells from pregnant and lactating opossums. Immunoreactivity for relaxin was not demonstrated elsewhere in the ovary, in the reproductive tract or in the placenta. The corpus luteum is thought to be the primary source of relaxin in Didelphis and in this regard is similar to several eutherian mammals including man. Key words: Relaxin-immunoreactivity - Corpus luteum Opossum
Following intracellular synthesis, relaxin is stored at least temporarily in small, electron-dense, membrane-bound granules prior to being released. Relaxin containing granules have been identified in luteal cells of the pig (Kendall et al. 1978; Fields and Fields 1985), rat (Anderson and Sherwood 1984; Fields 1984) and mouse (Anderson et al. 1984), in cells of guinea pig endometrial glands (Larkin et al. 1983) and in placental syncytiotrophoblast cells of the rabbit (Eldridge and Fields 1986). This study reports the first immunohistochemicallocalization of relaxin in the female reproductive system of a metatherian species, the North American opossum.
Introduction Relaxin, a peptide hormone that plays a role during pregnancy, parturition and lactation in mammals, acts primarily on connective tissue elements at parturition to relax pelvic ligaments and dilate the cervix. In some species this peptide functions to maintain pregnancy by inhibiting the contractions of uterine smooth muscle. Relaxin also is thought to influence other reproductive tissues but these effects are less well characterized (Bryant-Greenwood 1982; Sherwood 1988).
The highest levels of relaxin are produced by the female reproductive tract late in pregnancy. In some species (sheep, cow, pig, rat, mouse, human) the corpus luteum is the primary source of relaxin (Mathieu et al. 1981; Anderson 1982; Anderson and Sherwood 1984; Anderson et al. 1984; Vaupel et al. 1985; Ali et al. 1986) whereas in other forms (cat, rabbit, horse) the placenta is the primary source (Eldridge and Fields 1985; Sherwood 1988). In yet other mammals such as the guinea pig, the uterus is the source of relaxin (Pardo et al. 1980; Pardo and Larkin 1982).
~I
Ann. Anat. (1992) 174: 341-344 Gustav Fischer Verlag Jena
Materials and methods Nine adult female opossums (Didelphis virginiana) were examined in this study and consisted of pregnants and animals 1 week into lactation. Each group consisted of at least three animals. Approximate ages of pregnancies were determined from timed pregnancies. Female opossums were stripped of their litters and placed in breeding pens with continuous access to males. A sperm positive date was determined by examining smears taken from the urogenital sinus each morning. Females 10112 and 11 Y2 days into pregnancy (opossums have a 12112 day gestation period) as well as the lactating animals used were killed by an intra-cardial injection of Ketamine followed by exsanguination. Ovaries, oviducts, uteri, lateral vaginal canals as well as placentae from pregnant animals were removed as quickly as possible and fixed by immersion in Bouin's solution for a period of 24 hours. Tissues were processed routinely for paraffin embedding, sectioned at 5!tm and mounted on acidcleaned slides. The mounted sections were deparaffinized in xylene and rehydrated to water prior to incubation in 1.0 % H20 2 to remove endogenous peroxide. After blocking with normal goat serum, the sections were incubated with the primary antiserum (anti-porcine
relaxin, dilution 1: 10000) for 24 hours at 4°C. Immunohistochemical visualization was accomplished by the avidin-biotin-perioxidase complex (ABC) procedure (Hsu et al. 1981) using a Vecta-Iab "elite" kit. Peroxidase activity was demonstrated by incubation with 3,3' diaminobenzidine tetrahydrochloride containing 0.01 % H 20 2 in 0.1 M Tris-Hel buffer. Controls consisted of pre-absorption of the primary antiserum with the corresponding antigen as well as omission of the primary antiserum. Sections of Bouin's fixed porcine corpora lutea served as positive controls. Treated sections were then dehydrated, cleared in xylene and coverslipped for examination. Occasional treated sections also were lightly counterstained with Mayer's haematoxylin prior to being coverslipped.
2). The immunoreactive staining is confined to the cytoplasm of most, but not all, luteal cells and is not observed in adjacent endothelial cells or fibroblasts (Figs. 3, 4). Likewise, relaxin-immunoreactivity is not observed in tissues comprising either the oviduct, uterus, lateral vaginal canals or the placenta of Didelphis. Specific staining is not observed in control sections incubated with normal goat serum .or porcine relaxin antiserum pre-absorbed with porcine relaxin. Luteal cells from porcine corpora lutea stain intensely for relaxin, further supporting the immunospecificity of the relaxin antiserum used.
Discussion
Results
In Didelphis as well as other but not all marsupial species,
Corpora lutea from pregnant and lactating opossums show intense immunoreactive staining with porcine antiserum to relaxin. Immunoreactivity for porcine relaxin is not observed in adjacent follicles or other regions of the ovary (Figs. 1,
the luteal phase comprises about 60 % of the oestrous cycle. Regression of the corpus luteum coincides with birth but it disappears slowly if lactation occurs (Tyndale-Biscoe and Renfree 1987). Luteal cells comprising the corpus luteum of
Fig. 1. A region of ovary containing three corpora lutea (L) and a secondary follicle (F). Note the intense immunoreactive staining for porcine relaxin in the corpora lutea and the absence of immunoreactive staining in the secondary follicle. Opossum 11 Y2 day of pregnancy. X 15. Fig. 2. A portion of a corpus luteum demonstrates that the relaxin-immunoreactivity is confmed to luteal cells . A primary follicle (F) and the adjacent stroma are unstained . Opossum 1ll-'2 day of pregnancy. X 100. Fig. 3 . Increased magnification demonstrates that the majority but not all luteal cells of a given section show intense immunoreactive staining for porcine relaxin. Opossum 11 Y2 day of pregnancy. X 300. Fig. 4. A region of corpus luteum from an opossum 11l-'2 days into pregnancy demonstrating relaxin-immunoreactivity (arrows) that was counterstained with Mayer's haematoxyIin illustrates in greater detail its general morphology. X 300.
342
the opossum are derived exclusively from granulosa cells of the previous follicle (Martinez-Esteve 1942). Corpora attain maximum size by postestrous day 7, begin to decline after day 8 and often appear shrunken by day 12 immediately prior to birth (Fleming and Harder 1983). Peripheral plasma levels of progesterone, the major steroid hormone of Didelphis (Cook and Nalbandov 1968), correspond closely with morphological changes that occur in the corpus luteum during the oestrous cycle and pregnancy (Harder and Fleming 1981). No significant differences with regard to progesterone levels in Didelphis could be detected between non-pregnant and pregnant animals. Corpora lutea are necessary for both parturition (Hartman 1925; Renfree 1974) and mammogenesis in Didelphis as well as other marsupials (TyndaleBiscoe and Renfree 1987; Hinds 1988). Corpora lutea of the brushtailed opossum, Trichosurus vulpecula and tammar wallaby, Macropus eugenii have been reported to contain high concentrations of relaxin as measured by mouse pubic-symphysis bioassay (Tyndale-Biscoe 1969, 1981). What role relaxin plays in corpora lutea of marsupials or how it influences the pseudo-vaginal (median) canal is less clear. Relaxin is thought to act in concert with progesterone and estrogen in the preparation of the birth canal of most mammals prior to parturition. Whether or not this interaction occurs in the formation and physiological control of the unique birth canal of marsupials (the median-vaginal canal) is unknown. The presence of immunoreactivity for relaxin in most, but not all, luteal cells in Didelphis may be related to the production and/or concentration of relaxin at levels below the resolution of the light microscope or may be due to secretory exhaustion of some cells. Relaxin-immunoreactivity is not observed in the uterine epithelium, uterine glands or placentae of Didelphis and in this way resembles serveral eutherian species such as the sheep, cow, pig, rat, mouse and human in which the corpus luteum is the primary source of relaxin (Mathieu et al. 1981; Anderson 1982; Anderson and Sherwood 1984; Anderson et al. 1987; Vaupel et al. 1985; Ali et al. 1986). The mechanism of action of relaxin on the reproductive tract in Didelphis or other marsupial forms is unknown. Acknowledgements. We would like to thank Dr. M. J. Fields and Dr. P. A. Fields for their generous gifts of porcine relaxin and antiporcine relaxin. We also would like to acknowledge Midge M. Lind for the preparation of the manuscript.
References Ali SM, McMurtry JP, Bagnell CA, Bryant-Greenwood GO (1986) Immunocytochemical localization of relaxin in corpora lutea of sows throughout the estrous cycle. Bioi Reprod 34: 139-143 Anderson LL (1982) Relaxin localization in porcine and bovine ovaries by assay and morphological techniques . Adv Exp Med Bioi 143: 1-67 Anderson MB, Sherwood 00 (1984) Ultrastructural localization of relaxin immunoreactivity in corpora lutea of pregnant rats . Endocrinology 114: 1124-1127
Anderson MB, Vaupel MR, Sherwood 00 (1984) Pregnant mouse corpora lutea: Immunocytochemical localization of relaxin and ultrastructure. Bioi Reprod 31 : 391- 397 Bryant-Greenwood GO (1982) Relaxin as a new hormone. Endocrin Rev 3: 62-94 Cook B, Nalbandov AV (1968) The effect of some pituitary hormones on progesterone synthesis in vitro by the luteinized ovary of the common opossum (Didelphis marsupialis virginiana) . J Reprod Fert 15: 267-275 Eldridge RK, Fields PA (1984) Rabbit placental relaxin: Purification and immuno-histochemical localization. Endocrinology 117 :
2512-2519 Eldridge RK, Fields PA (1986) Rabbit placental relaxin: Ultrastructurallocalization in secretory granules of the syncytiotrophoblast using rabbit placental relaxin antiserum. Endocrinology 115:
606-615 Fields P A (1984) Intracellular localization of relaxin in membranebound granules in the pregnant rat luteal cell. BioI Reprod 30:
753-762 Fields PA, Fields MJ (1985) Ultrastructural localization of relaxin in the corpus luteum of the nonpregnant, pseudopregnant, and pregnant pig. Bioi Reprod 32: 1169-1179 Fleming MW, Harder JD (1983) Luteal and follicular populations in the ovary of the opossum (Didelphis virginiana) after ovulation. J Reprod Fert 67: 29-34 Harder JD, Fleming MW (1981) Estradiol and progesterone profiles indicate a lack of endocrine recognition of pregnancy in the opossum. Science 212: 1400-1402 Hartmann CG (1925) The interruption of pregnancy by ovariectomy in the aplacental opossum: A study in the physiology of implantation. Am J Physiol 71: 436-454 Hinds LA (1988) Hormonal control oflactation. In: The Developing Marsupial. Models for Biomedical Research: CH TyndaleBioscoe and PA Janssens, eds. Springer Berlin, New York, London, pp 55-67 Hsu SM, Raine L, Fanger A (1981) Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques. J Histochem Cytochem 24: 577-580 Kendall JZ, Plopper LG, Bryant-Greenwood GA (1978) Ultrastructural immunoperoxidase demonstration of relaxin in the corpora lutea from pregnant sows. Bioi Reprod 18: 94-98 Larkin LH, Pardo RJ, Randall HR (1983) Imrnunoelectron microscopic localization of relaxin in endometrial gland cells of the guinea pig. Anat Rec 205 : I06A Mathieu P, Rabier J, Thomas K (1981) Localization of relaxin in human gestational corpus luteum. Cell Tissue Res 219: 213-216 Martinez-Esteve P (1942) Observations on the histology of the opossum ovary. Contrib Embryol 30: 17-26 Pardo RJ, Larkin LA, Fields P A (1980) Immunocytochemical localization of relaxin in endometrial glands of the pregnant guinea pig . Endocrinology 107: 2110-2112 Pardo RJ, Larkin LH (1982) Localization of relaxin in endometrial glands of the pregnant, lactating and ovariectomized hormonetreated guinea pigs. Am J Anat 164: 79-90 Renfree MB (1974) Ovariectomy during gestation in the American opossum, Didelphis marsupialis virginiana. J Reprod Fert 39 :
127-130 Sherwood DO (1988) Relaxin. In: The Physiology of Reproduction, E. Knobil and J. Neill, eds . Raven Press, New York, pp 585-673 Tyndale-Biscoe CH (1969) Relaxin activity during the oestrus cycle of the marsupial, Trichosurus vulpecula (Kerr). J Reprod Fert 19:
191-193 Tyndale-Biscoe CH (1981) Evidence for relaxin in marsupials. In:
343
Relaxin, G Bryant-Greenwood, H Niall and F Greenwood, eds. North Holland/Elsevier Biomedical Press, Amsterdam, pp 225-232 Tyndale-Biscoe CH, Renfree MB (1987) Reproductive Physiology of Marsupials. Cambridge University Press, New York
Vaupel MR, Sherwood OD, Anderson MB (1985) Immunocytochemical studies of relaxin in ovaries of pregnant and cycling mice. J Histochem Cytochem 33: 303-308 Accepted April 18, 1990
344