An Immunoassay to Detect Human Mullerian Inhibiting Substance in Males and Females during Normal Development*

Abstract

An enzyme-linked immunosorbent assay has been developed to measure human Müllerian inhibiting substance (MIS) in biological fluids. The enzyme-linked immunosorbent assay is specific for MIS, with a sensitivity in human serum to 0.5 ng/ml and does not recognize transforming growth factor-beta 1 or -beta 2, LH, or FSH. It similarly fails to recognize other proteins secreted from the cell type into which the MIS gene was cloned. MIS was detected in the serum of normal newborns, infants, children, and adults. In males the serum level of MIS is 10-70 ng/mL at birth. The level increases slightly after birth, and then decreases to a basal level of 2-5 ng/mL after the first 10 yr of life. Newborn male urine contains minimal amounts of MIS (0.5 ng/mL). In females MIS is barely detectable in serum at birth, but rises to the basal level equal to that seen in males after 10 yr of age. Similar basal levels of MIS were found in adult ovarian follicular fluid. MIS levels were high in the serum of a female patient with a sex cord tumor (3200 ng/mL), but fell to 100 ng/mL after multiple excisional operations. In addition, a serum MIS level of 20 ng/mL was detected in a patient with an ovarian granulosa cell tumor. A sensitive assay for MIS could be useful in the diagnosis of patients with congenital abnormalities of sexual development and patients with Sertoli cell and/or other MIS-producing neoplasms. Other applications may also be recognized as the biology of MIS in both males and females is further elucidated.

Endometrial cancer is a receptor-mediated target for Mullerian Inhibiting Substance

“…The CHO cells were alternately maintained in 5% female FBS (FFBS)-containing media and a serum-free defined media. MIS levels in MIS gene-transfected cells were measured in conditioned media by using a human MIS-specific ELISA (20).…”

“…Cells were maintained in media containing 25-100 g͞ml hygromycin for 3-4 weeks. MIS secreted into the conditioned media of these clones was measured by the MIS ELISA (20). The cells were then stained with crystal violet, and the number of colonies Ͼ50 cells in size was counted for each transfection and empty-vector control.…”

“…In the female, ovarian granulosa cells synthesize MIS; hormone levels slowly increase after birth until adolescence, after which a steady-state low serum level persists throughout reproductive life. MIS levels then decrease and become negligible at menopause (20,21). Although the role of MIS in the postgestational human is unclear, mouse MIS-knockout models provide evidence of its role in controlling growth.…”

Müllerian Inhibiting Substance lowers testosterone in luteinizing hormone-stimulated rodents

“…After puberty, the expression of MIS is greatly reduced in males to a level more like that of females, which express low levels of MIS at puberty (5)(6)(7)(8). The physiological roles of MIS after Müllerian duct regression are currently being investigated and, based on experiments with mouse transgenics and knockouts, indicate that MIS is important for maintaining gonadal competence.…”

Identification of large-scale characteristic genes of Müllerian inhibiting substance in human ovarian cancer cells

“…MIS is produced in fetal and postnatal Sertoli cells in the testes at high levels which persist until puberty, after which, the concentration of MIS drops to basal levels, where it persists throughout life. In females, MIS is undetectable in the fetal and early postnatal ovary or in serum but becomes detectable in the granulosa cells of the developing ovarian follicles and in the serum of the adolescent and adult, and is never detected after menopause in healthy women (4)(5)(6)(7). Mutations in MIS or the MIS type II (MISRII) receptor in humans cause persistent Müllerian duct syndrome, in which males retain Müllerian ducts in the presence of testes and the Y chromosome (8,9).…”