Highly purified müllerian inhibiting substance inhibits human ovarian cancer in vivo.

PURPOSE: Müllerian inhibiting substance (MIS) causes Müllerianduct regression in mammalian, avian, and reptilian embryos; MIS also inhibits growth in vitro of Müllerian-derived cell lines and primary cells from human ovarian carcinomas. We hypothesize that highly purified MIS delivered parenterally inhibits ovarian cancer in vivo. EXPERIMENTAL DESIGN: To test the efficacy of highly purified MIS against ovarian cancer cell lines in vivo, we treated immunosuppressed mice with MIS after implanting OVCAR 8 or IGROV 1 human ovarian cancer cells beneath the renal capsules and measured tumor volume over time. Animals were treated with daily injections of 10 micro g of purified exogenous recombinant human MIS or by endogenous MIS secreted from cells growing on biodegradable mesh. RESULTS: The average graft size ratio (change in size compared with starting size) of the OVCAR 8 tumor implants was larger in the control animals than in animals treated for 2 weeks (P < 0.019) or 3 weeks (P < 0.001) with parenteral MIS, or after treating with MIS produced from transfected cells, which impregnated the biodegradable mesh (P = 0.02). The average graft size ratio of the IGROV 1 tumors was also larger in the control animals than in those treated with injected MIS (P = 0.0174). CONCLUSIONS: Highly purified recombinant human MIS, delivered parenterally, or MIS produced endogenously causes inhibition of human ovarian cancer cell lines in vivo, providing convincing preclinical evidence to support the use of MIS as a parenteral agent for the treatment of ovarian cancer.

Expression of the CD15 differentiation antigen in the reproductive tract of the female rat during fetal and early postnatal ontogeny.

The expression of the (alpha1-->3)-fucosyl-N-acetyl-lactosamine (CD15) epitope in the genital tract of the female rat during fetal and early postnatal ontogeny was investigated by means of immunohistochemistry. CD15 was exclusively associated with epithelial cells and was mainly located along the cell membrane. The CD15 expression was characterized, firstly, by considerable differences within the various structures and even substructures of the genital tract and secondly, by the high degree of time-related changes which accompanied the morphological development. In the Mullerian duct, CD15 was present from embryonic day (E) 14 until birth on the apical membranes throughout the epithelial cell layer. In the Wolffian duct, CD15 expression was present between E16 and E19. Along the longitudinal extent of the Wolffian duct, expression intensity differed, showing moderate to high levels in the epithelial cells of the cranial and caudal parts, but without recognizable CD15 expression in the intermediate part. In the urogenital sinus, CD15 was expressed from E15 until E21. In the cranial parts, all epithelial cells were positive, whereas in the caudal parts, CD15 was present only on their apical membranes. In the ovarian tube, uterine horn, and vagina, a moderate to high CD15 expression at birth gradually diminished to very low levels during postnatal days (P) 8 and 9. After P9, re-expression of CD15 occurred in the caudal part of the ovarian tube and in the uterus, increasing to a maximum at about P32. The findings provide (indirect) evidence for a correlation between the intensity of CD15 immunoreactivity and the serum concentrations of estrogens as well as of estrogen receptors in the urogenital tract. Since steroid hormone dependency can be regarded as a gauge of the differentiation of malignancies, it would be worthwhile correlating CD15 levels with those parameters.

Persistence of müllerian ducts in male rabbits passively immunized against bovine anti-müllerian hormone during fetal life.

A female rabbit was immunized against purified bovine AMH and mated. Booster injections were given at Day 8 of pregnancy to ensure a high titer of anti-AMH antibodies at the time the rabbit fetal testis begins to produce AMH. In three consecutive litters, the immunized female produced a total of 12 males, 9 of which had persistent Müllerian duct derivatives. No other significant abnormalities were detected in these animals, which were compared to the offspring of a control saline-injected female. In particular, testicular morphology was normal in most animals, and serum FSH levels did not differ from controls. This experimental model lends no support to the hypothesis that AMH controls extra-Müllerian events of male sex differentiation, nor that of the existence of a regulatory mechanism for synthesis of AMH by Sertoli cells, but it does not definitely exclude these possibilities, inasmuch as our tentative conclusions are based upon study of only one immunized female.

Changes of Lex and dimeric Lex haptens and their sialylated antigens during development of human kidney and kidney tumors.

The carbohydrate antigen termed Lex (Gal beta 1----4[Fuc alpha 1----3]GlcNAc beta 1----R), its di- or trimeric form, and their sialylated antigens have been characterized as developmentally regulated, tumor-associated antigens in human gastrointestinal epithelia. In this paper, remarkable changes of these antigens, defined by respective monoclonal antibodies FH3, FH4, and FH6, in fetal kidney (mesonephros and metanephros) and other urogenital organs, as well as in various types of kidney tumors, have been investigated. During the development of each organ and tissue, the antigens were found to be maximally expressed at a defined period of organogenesis, and a shifting of expression from one locus to another was observed. Each antigen showed a slightly but clearly different stage of maximum expression. The following changes in metanephros development are of particular interest. Expression of the antigen defined by FH3 followed by the antigen defined by FH4 appeared only after six weeks of gestation in the convoluted tubuli at the central region of metanephros, and propagated rapidly into those at the peripheral cortex region with a simultaneous regression at the central region. The regression of FH4 antigen was more rapid than that of FH3. All three antigens were expressed in the medullar thin tubuli, which develop into the thin-limb of Henle's loop, in which only the antigens defined by FH3 and FH4 persisted and FH6 antigen disappeared. Well-differentiated, but not undifferentiated, renal adenocarcinomas strongly expressed the antigens defined by FH4 and FH6, although the antigen defined by FH6 was expressed in more differentiated tumor cells than the antigen defined by FH4. Well-differentiated cells organized into tubular structures showed a strong expression of these differentiation antigens. However, some tumor cells that were organized into tubular structures, but were characterized by undifferentiated cytomorphology (larger nucleus and smaller volume of cytoplasm), did not express FH4 and FH6 antigens. Thus, cytodifferentiation and histotypic differentiation proceed independently within kidney tumors. The fucosylated type 2 chain structures defined by these three monoclonal antibodies are useful markers that indicate the degree of tumor differentiation.

[Persistence of Mullerian ducts in the male rabbit by passive immunization against anti-mullerian hormone during fetal life]. / Maintien des canaux de Müller chez le Lapin mâle par immunisation passive contre l'hormone anti-müllérienne pendant la vie foetale.

A female Rabbit immunized against bovine anti-Müllerian hormone (AMH) was mated and gave birth to 3 litters, containing a total of 13 males. Ten of these presented with Müllerian derivatives, of variable development. Testicular position and structure, Wolffian development and virilization of the urogenital sinus were apparently normal in males with persistent Müllerian ducts.

Monoclonal antibody to Mullerian inhibiting substance.

Mullerian Inhibiting Substance (MIS) is a testicular product that causes the regression of the Mullerian duct in the developing male embryo. Antibody specific for MIS would facilitate the purification and study of this "hormone," but because of its impure status, traditional polyclonal antisera specific for MIS would be untenable. The requisite specificity, however, might be obtained by the technique of somatic fusion, regardless of the purity of the immunizing antigen. This paper describes the production of 2 monoclonal antibodies specific for MIS by the technique of somatic cell fusion. Spleen cells from mice immunized with an impure preparation of MIS were fused with myeloma cell line NS1. Culture media from the resulting hybridoma cell lines were screened for anti-MIS antibody by a sensitive RIA. Specificity for MIS was demonstrated by the adsorption of biologically active MIS on an affinity column prepared from monoclonal anti-MIS antibody. MIS activity as assessed by an organ culture assay was subsequently recovered from the affinity column in the fraction eluted with MH4SCN. Using the RIA, monoclonal anti-MIS antibody was also shown to compete favorably with a variety of potentially cross-reactive proteins.

Immuno-biochemical studies of a non-histone chromosomal protein in embryonic and mature chick oviduct.

A non-histone chromosomal 95K protein (of mol.wt. 95 000) from hen oviduct was isolated and purified for antibody induction in the rabbit. Immuno-micro-complement-fixation and biochemical techniques were used to probe the presence of 95K protein in the oviduct chromatin of the embryonic and immature chick and the hen. The antiserum against 95K protein did not react with high-mobility-group proteins 1 and 2 obtained from oviduct, brain and liver, nor with histones. After limited digestion of chromatin with nucleases, until 10% DNA was hydrolysed, a small amount of 95K protein was released. Thus the 95K protein is probably not located in the region of chromatin that is sensitive to nuclease digestion. The amount of 95K protein in immature chick oviduct chromatin is less than that in the mature hen oviduct. However, the amount of 95K protein in the immature chick oviduct was increased after oestrogen administration. The presence of 95K protein in embryonic oviduct was detected in the 10-, 12-, 15- and 18- day chick embryo. The quantity of this protein increased with the age of the embryo and reached its highest value in the chromatin of the hen oviduct.