Prenatal vitamin E treatment improves lung growth in fetal rats with congenital diaphragmatic hernia.

PURPOSE: Congenital diaphragmatic hernia (CDH) is associated with pulmonary hypoplasia. To discover factors that would accelerate fetal lung growth, the authors developed models of hypoplasia, found that antioxidants improved lung growth in vitro, and then proceeded to in vivo studies. METHODS: Timed-pregnant rats were fed nitrofen (100 mg) on gestational day 9.5 (term, 22), and fetal lungs were harvested at day 13.5 and placed in organ culture in serum-free media with (n = 10) or without (n = 9) additional vitamin E (0.134 IU/mL). Camera lucida tracings were made daily on live, unstained lungs for 4 days, scanned, digitized, and analyzed for multiple growth parameters. Similar nitrofen-exposed rats were fed an optimized total dose of 150 IU vitamin E (n = 19) or olive oil (n = 13) from days 16.5 to 20.5, and fetal lungs were harvested at day 21.5, weighed and fixed for histology, or homogenized and biochemically analyzed. RESULTS: Vitamin E accelerated hypoplastic fetal lung growth in vitro as measured by area, perimeter, lung bud count, perimeter over square root area, and fractal dimension. In vivo vitamin E significantly increased lung weights, total DNA, and protein contents. CONCLUSIONS: Vitamin E accelerates hypoplastic fetal rat lung growth and complexity in vitro, and prenatal vitamin E treatment in vivo improves pulmonary hypoplasia in fetal rats with CDH.

Requirement for phosphatidylinositol-3'-kinase in cytokine-mediated germ cell survival during fetal oogenesis in the mouse.

Apoptosis is responsible for primordial germ cell (PGC) attrition in the developing fetal ovary. In monolayer cultures of murine PGC, stem cell factor (SCF) and leukemia inhibitory factor (LIF) independently promote survival in vitro; however, the relevance of these data to fetal ovarian oogonium and oocyte survival, as well as the intracellular events involved in transducing the antiapoptotic actions of these cytokines in germ cells, remain to be elucidated. In this report, we investigated the effects of SCF and LIF, alone and in combination, on the survival of oogonia and oocytes, and elaborated on components of the signal transduction pathway used by these molecules, after validating a method of culturing fetal mouse ovaries. We further employed this system to also test the hypothesis that insulin-like growth factor-I (IGF-I), a classic antiapoptotic molecule, and transforming growth factor-beta (TGF-beta), a classic pro-apoptotic molecule, interact with the SCF/LIF pathway and function in a reciprocal fashion to precisely regulate germ cell numbers during fetal oogenesis. Freshly isolated embryonic day 13.5 ovaries contained nonapoptotic germ cells, as determined by histologic analysis of cellular morphology and in situ 3'-end-labeling of DNA integrity. In vitro culture of fetal ovaries without tropic support for 24, 48, and 72 h resulted in a time-dependent induction of germ cell apoptosis, such that most oogonia and oocytes present after 72 h were apoptotic. Morphometric analysis of serially sectioned ovaries indicated that the numbers of nonapoptotic germ cells remaining after 24, 48, and 72 h of culture were 78%, 38%, and 10%, respectively, of the number present before culture (P < 0.05 for all time points vs. 0 h). Inclusion of SCF (100 ng/ml) together with LIF (100 ng/ml) in the culture medium significantly attenuated germ cell apoptosis, with the SCF/LIF-treated ovaries retaining 5.5-fold more oogonia and oocytes after 72 h of culture as compared with control ovaries deprived of tropic support (P < 0.05). However, SCF or LIF, when added separately, had no (SCF) or little (LIF) inhibitory effect on germ cell apoptosis. Provision of 50 ng/ml IGF-I maintained survival of approximately two-thirds of the germ cells in cultured ovaries (P < 0.05), whereas a combination of all three growth factors (SCF, LIF, IGF-I) completely preserved the fetal ovary in culture to that resembling a freshly-isolated gonad. Cotreatment with 25 ng/ml TGF-beta partially reversed the survival actions of IGF-I or SCF/LIF, such that only one-third of the starting number of oogonia/oocytes remained after 72 h of culture (P < 0.05). Lastly, the antiapoptotic effects of SCF/LIF or IGF-I were almost entirely eliminated by cotreatment of fetal ovaries with either one of two inhibitors of phosphatidylinositol-3'-kinase (PI3K), LY294002 (5 microM) or wortmannin (50 nM), whereas cotreatment with an inhibitor of p70 S6 kinase (rapamycin, 25 ng/ml) was without effect. These data indicate that the combined actions of SCF, LIF, and IGF-I are required for maximal inhibition of apoptosis in germ cells of fetal mouse ovaries, and that the PI3K signaling pathway is an essential component of cytokine-mediated female germ cell survival. Moreover, TGF-beta can partially override the antiapoptotic actions of SCF/LIF or IGF-I in oogonia and oocytes, suggesting the existence of a complex signaling network that ultimately determines fetal ovarian germ cell fate.

Prenatal glucocorticoids improve pulmonary morphometrics in fetal sheep with congenital diaphragmatic hernia.

PURPOSE: Prenatal glucocorticoids reverse pulmonary immaturity in rodents with pharmacologically induced congenital diaphragmatic hernia (CDH). The authors applied quantitative stereologic morphometric techniques to test whether these effects could be reproduced in large animals (sheep) with surgically created CDH. METHODS: Diaphragmatic hernias were created surgically in fetal lambs at gestational day 80. The fetuses were treated with intravenous cortisol (n = 6) or normal saline control (n = 5) from days 133 to 135. Lungs distended at 15 cm pressure from each group were harvested at day 136, processed histologically, and studied by brightfield microscopy at 400 x using a 42-point equidistant counting grid. Ten morphometric parameters (Mean +/- SEM) were measured by point-counting 60 fields/lung, and analysis of variance was performed. RESULTS: The CDH-cortisol-treated lungs showed striking significant maturational improvements when compared with lungs of CDH-normal saline controls by seven of ten morphometric parameters. CONCLUSIONS: (1) Prenatal glucocorticoids accelerate lung maturity in fetal lambs with CDH by seven quantitative morphometric parameters. (2) The observation that prenatal glucocorticoid therapy improves measures of maturity for both CDH rodent and sheep models encourages proceeding with a Phase I human clinical trial in ultrasound-confirmed CDH.

Müllerian inhibiting substance inhibits branching morphogenesis and induces apoptosis in fetal rat lung.

Müllerian inhibiting substance (MIS) is a glycoprotein hormone required for normal male reproductive tract development; it is presumed to signal through a heteromeric complex of type I and type II receptors. MIS exposure produces a paracrine-mediated regression of the embryonic Müllerian duct with histological changes consistent with apoptosis. MIS has also been shown to inhibit fetal lung development in vitro and in vivo, although the mechanism of this inhibition is unknown. The primordial lung and gonad are anatomically proximate on embryonic day 13.5, raising the possibility of a paracrine-mediated influence of MIS in male embryos on lung as well as MIS effecting dissolution of the Müllerian duct. We hypothesized that a negative regulatory event(s) might occur in the lung, as occurs in the duct, at the onset of MIS protein expression; thus, apoptosis and branching morphogenesis were studied in explanted fetal rat lungs incubated with proteolytically activated MIS. MIS exposure resulted in reduced total lung bud number as well as lung perimeter length. Explanted lungs exposed to MIS also exhibited numerous apoptotic bodies. To assess whether this MIS-induced phenomenon in lung might be mediated by the MIS type II receptor (MIS RII), reverse transcriptase-PCR performed on multiple fetal rat lung RNA samples using oligonucleotide primers designed from the 3'-untranslated region of rat MIS RII complementary DNA showed a product of the expected size that when sequenced was nearly identical to rat MIS RII. Northern blot analysis using polyadenylated fetal rat lung RNA and a 3'-MIS RII probe revealed a 2-kilobase transcript that was also seen in testicular messenger RNA. These studies show that the putative ligand binding receptor for MIS is expressed in embryonic lung, where MIS negatively modulates branching and activates apoptosis. We speculate that the mechanism of MIS-induced inhibition of lung development in the male fetus begins with MIS binding to the MIS RII, followed by a signaling cascade resulting in delayed airway branching temporally associated with enhanced apoptosis.

Prenatal hormonal therapy improves pulmonary morphology in rats with congenital diaphragmatic hernia.

The high mortality of congenital diaphragmatic hernia (CDH) is due to associated pulmonary hypoplasia, which resembles that seen in premature newborns with respiratory distress syndrome (RDS). By use of successful therapies extrapolated from RDS, quantitative stereologic morphometry techniques were applied to evaluate pulmonary development following prenatal hormonal therapy in rats with nitrofen-induced CDH. Antenatal hormonal therapy was administered on Days 18.5 and 19.5 prior to delivery on Day 21.5 (term = Day 22), using dexamethasone (Dex), thyrotropin-releasing hormone (TRH), Dex-TRH, or normal saline (NS) as vehicle control. Lungs from CDH rats (n = 5) and non-nitrofen-fed controls (n = 5) were studied, and 10 morphometric airspace parameters were determined by point counting 18-30 fields/lung/animal. Indices of maturation, including total internal surface area (SA), airspace volume fractions (V(Valv)), duct fractions (V(Vducts)), and radial alveolar count (RAC), were improved by Dex and Dex-TRH compared with NS-CDH controls (P = 0.0001), as were five other morphometric airspace parameters (P < 0.05). Strikingly, Dex and Dex-TRH treatment corrected average airspace volume (AAV) and the volume fraction of air-conducting elements (V(Vducts)) toward normal values seen in non-nitrofen-fed control animals. TRH therapy alone had minimal beneficial effects. Prenatal steroid +/- TRH thus improved multiple morphometric parameters of lung maturity in CDH rats, supporting the potential use of in utero hormonal therapy to treat humans with antenatally diagnosed CDH.

Prenatal hormonal therapy improves pulmonary compliance in the nitrofen-induced CDH rat model.

Neonates with congenital diaphragmatic hernia (CDH) experience a high mortality despite intensive medical and surgical management. The associated pulmonary hypoplasia is accompanied by an underlying biochemical deficiency that bears similarity to respiratory distress syndrome (RDS) in the premature newborn. Using therapies extrapolated from those used to treat RDS, the authors have previously shown correction of the immature pulmonary biochemical indices in the nitrofen rat CDH model. This study investigates the functional and histological outcome of prenatal hormone therapy on CDH rats. Compared with saline-treated CDH controls, dexamethasone-treated CDH animals achieved significant increases in lung distensibility (P = .0006) and functional residual capacity (P = .004); CDH rats treated with combined dexamethasone and thyrotropin-releasing hormone (TRH) showed improved functional residual capacity (P = .043) and alveolar stability (P = .025); CDH animals treated with TRH alone (TRH-CDH) showed no improvement in any parameter tested. Histologically, the lungs from dexamethasone- and dexamethasone-TRH-treated CDH animals showed changes that included narrow septal walls, increased air saccule size, and thinning of the pulmonary interstitium compared with the lungs of saline or TRH-CDH rats, which were developmentally arrested at the canalicular stage. Lung weights and lung weight-body weights ratios were similar in all CDH rats, confirming that treatment did not impair pulmonary growth. These results support the potential clinical use of prenatal pharmacological therapies to treat human fetuses with prenatally diagnosed CDH.

Cleavage of Müllerian inhibiting substance activates antiproliferative effects in vivo.

Müllerian inhibiting substance (MIS), an inhibitor of growth and development of the female reproductive ducts in male fetuses, requires precise proteolytic cleavage to yield its biologically active species. Human plasmin is now used to cleave and, thereby, activate immunoaffinity-purified recombinant human MIS at its monobasic arginine-serine site at residues 427-428. To avoid the need for exogenous enzymatic cleavage and to simplify purification, we created an arginine-arginine dibasic cleavage site (MIS RR) using site-directed mutagenesis to change the serine at position 428 (AGC) to an arginine (cGC). The mutant cDNA was then stably transfected into a MIS-responsive ocular melanoma cell line, OM431, followed by cloning for amplified expression to test its biological activity in vitro and in vivo. Media from each clone were assayed for production of MIS RR by a sensitive ELISA for holo-MIS, and high- and low-producing clones were selected for further study. Media from the highest MIS RR producer caused Müllerian duct regression in an organ culture bioassay. Other transfections were done with an empty vector (pcDNAI Neo) or a construct lacking the leader sequence and thus failing to secrete MIS, to serve as controls. The OM431 clones containing the MIS RR mutant were growth inhibited in monolayer culture. The high- and low-producing MIS RR OM431 clones, along with transfected OM431 controls, were injected into the tail veins of immunosuppressed severe combined immunodeficiency mice for in vivo analyses. Four to 6 weeks later, pulmonary metastases were counted in uniformly inflated lungs. OM431 clones containing the more easily cleaved MIS RR displayed a significant dose-dependent reduction in pulmonary metastases when compared to the lungs of animals given injections of OM431 clones containing empty vector, leaderless MIS, or wild-type MIS that requires activation by plasmin cleavage. Since the purification protocol of MIS RR is less complicated than that for wild-type MIS, which requires subsequent enzymatic activation, MIS RR can be used for scale-up production with increased yields for further therapeutic trials against MIS-sensitive tumors.

Embryonic cell death patterns associated with nitrofen-induced congenital diaphragmatic hernia.

The offspring of pregnant Sprague-Dawley rats exposed to nitrofen on gestational day 9.5 develop left-sided congenital diaphragmatic hernia (CDH). Twenty-four hours after treatment, on day 10.5, supravital staining with Nile blue sulfate and histological examination showed bilateral excessive cell death in cervical somites 2 through 4. After 48 hours, on day 11.5, cell death was absent in the cervical somites but was apparent in the mesoderm adjacent to the somites in the septum transversum and in the developing sympathetic ganglia adjacent to the dorsal aortae. Cell death was not apparent in the foregut or lung primordia on either day 10.5 or 11.5. The incidence of nitrofen-exposed embryos with such patterns of cell death closely paralleled that of left-sided CDH in similarly treated day 21.5 fetuses. Control animals treated with olive oil had normal programmed cell death patterns in the regions of interest and had no evidence of CDH on day 21.5. It is possible that these patterns of excessive cell death early in gestation may play a role in the genesis of diaphragmatic hernia. Mesoderm derived from cervical somites 3 through 5 contributes to the diaphragmatic anlage and forms the major portion of the muscle of the diaphragm. Because nitrofen damages mesodermal cell populations in cervical somites 2 through 4 and in the mesenchyme adjacent to the septum transversum 24 to 48 hours after administration, the authors propose that damage to these populations may reduce progenitor cells needed to populate the diaphragmatic anlage, thereby hindering pleuro-peritoneal canal closure.(ABSTRACT TRUNCATED AT 250 WORDS)

Müllerian inhibiting substance is present in embryonic testes of dogs with persistent müllerian duct syndrome.

Müllerian Inhibiting Substance (MIS) causes regression of the Müllerian ducts during a critical period in embryonic development in male mammals. In Persistent Müllerian Duct Syndrome (PMDS), an autosomal recessive trait in humans and dogs, the Müllerian ducts fail to regress in otherwise normal males. Previously we reported that PMDS-affected dogs produce bioactive testicular MIS postnatally. The purpose of the present study was to determine whether PMDS-affected canine embryos appropriately express MIS mRNA and protein during the critical period for Müllerian duct regression. Homozygous (PMDS-affected) and normal canine embryos were removed from timed pregnancies. Gonadal sex and the degree of Müllerian duct regression were determined from histologic sections. Positive immunohistochemical staining for MIS was found in testis sections of PMDS-affected and normal male embryos. A 1.8-kb MIS mRNA transcript was detected in testes of PMDS-affected males and normal male embryos and neonates. Furthermore, equal amounts of MIS mRNA transcript were detected in testes of PMDS-affected embryos and normal male littermates during the critical period for Müllerian duct regression. These data support a hypothesis of target organ resistance, such as an abnormality in the putative MIS receptor, as the etiology of the defect in this dog model.

Mullerian duct regression and antiproliferative bioactivities of mullerian inhibiting substance reside in its carboxy-terminal domain.

A 25-kilodalton dimeric carboxy-terminal fragment of the recombinant human Mullerian inhibiting substance protein (rhMIS) was produced by proteolytic cleavage with plasmin and purified by size-exclusion chromatography. The identity of the isolated dimer as the carboxy-terminal fragment was confirmed by gel electrophoresis and Western analysis. As was true of every sample of the holo molecule, all preparations of the carboxy-terminal domain of rhMIS (n = 10), when added in the 0.5-5.0 micrograms/ml range, exhibited a dose-dependent partial to complete regression of the 14.5-day fetal rat Mullerian duct in an organ culture assay. The carboxy-terminal dimer also inhibited, in a dose-dependent manner, the growth of A431 cells in monolayer cultures. Daily addition of 5, 10, or 20 micrograms carboxy-terminus for 3 days resulted in 0%, 25%, and 100% inhibition of cell proliferation, respectively. Similar and higher doses of holo rhMIS had no or inconsistent antiproliferative activity (0-34% inhibition), even though the preparations caused Mullerian duct regression. All amino-terminal fragments prepared using this separation protocol were found to be inactive in these assays. These findings suggest that the bioactivity of rhMIS as a regressor of fetal Mullerian ducts and an inhibitor of A431 cell growth resides in its carboxy-terminal domain. These results indicate that the urogenital ridge tissue, but not A431 cells in culture, may be capable of cleaving intact MIS to a biologically active conformation.

Mullerian inhibiting substance binding and uptake.

Mullerian inhibiting substance (MIS) is a 140,000 M(r) Sertoli cell derived glycoprotein with a critical regulatory role in the male fetus initiated presumably by ligand binding with receptor. To localize this binding species we performed time course incubations of cultured fetal rat lungs or control tissues with MIS, applied rabbit anti-MIS IgG, and fluorescein conjugated anti-rabbit IgG, and examined specimens with laser confocal microscopy. Punctate surface fluorescence followed by cytosolic and nuclear localization in lung consistent with specific adsorptive endocytosis was seen. Confocal imaging also detected MIS binding to the Mullerian duct in the urogenital ridge. Crosslinking of 125I-MIS with plasma membranes revealed a high molecular mass binder with signal displaceable by excess unlabeled ligand. These data support the hypothesis that a specific plasma membrane binding protein for MIS exists.

The critical period for mullerian duct regression in the dog embryo.

The embryonic period during which Mullerian duct regression and Mullerian Inhibiting Substance (MIS) secretion occur was determined in canine embryos removed from timed pregnancies (32, 36, 37, 39, 42, and 46 days gestation). Sex chromosomes of each embryo were identified in metaphase spreads prepared from fibroblast cultures. Testicular differentiation, defined by seminiferous tubule formation and the presence of Sertoli cells and Leydig cells, and the degree of Mullerian duct regression were determined by careful morphologic analysis of histologic sections of canine embryonic gonads (n = 20) and Mullerian ducts (n = 20). MIS was detected immunohistochemically in embryonic testes using avidin-biotin complex enhancement of a specific rabbit polyclonal anti-MIS antibody. Testicular differentiation was observed at 36 days gestation. The earliest evidence of Mullerian duct regression in male embryos was observed at 36 days gestation, and regression was completed by 46 days gestation. Positive staining for MIS was present in testes from 36 to 46 days (n = 9). Staining was absent in the undifferentiated testis (n = 1) at 32 days gestation and in ovaries at all ages tested (n = 10). Thus, MIS is normally present throughout the critical period for Mullerian duct regression in the embryonic male dog.

Mullerian inhibiting substance ontogeny and its modulation by follicle-stimulating hormone in the rat testes.

Mullerian Inhibiting Substance (MIS) production in rat testes from the late fetal to the adult period and its modulation by gonadotropins in neonatal testes were studied using immunohistochemistry, northern analysis, and a graded organ culture bioassay for MIS. The intense immunohistochemical staining for MIS seen in fetal and newborn testes began to decrease gradually after the third postnatal day, then decreased dramatically on the fifth postnatal day. MIS immunohistochemical activity was then present at a low level until about the 20th postnatal day, after which it was barely detectable. The testes from rats treated with FSH at birth showed a considerable drop in MIS immunohistochemical activity on the third postnatal day to 29% of control testes, and a less profound decrease on the second and fourth postnatal days to 46% and 61% of control, respectively; thereafter MIS levels were the same in treated and untreated animals. With shorter courses of FSH treatment, immunohistochemical staining showed less depression of MIS on the third day, and no difference by the fourth postnatal day, indicating that the inhibitory effect on testicular MIS production may require continued FSH exposure. Three-day testes that had been treated with FSH for 2-1/2 days had less MIS messenger RNA compared to control testes of the same age, suggesting that the inhibitory effect of FSH on MIS production could be transcriptionally mediated. In contrast LH treatment produced no difference in either messenger RNA expression or immunohistochemical staining for MIS. These findings suggested that FSH may be a modulator of MIS production in neonatal testes.

Sex-specific fetal lung development and müllerian inhibiting substance.

Male neonates develop respiratory distress syndrome (RDS) with a greater incidence and mortality than do female neonates; the cause of this male disadvantage remains obscure. Male fetuses are exposed to higher levels of androgens and Müllerian inhibiting substance (MIS). Androgens have been shown to inhibit fetal lung maturation, and recent evidence in vitro indicates that MIS, a Sertoli cell-derived glycoprotein made early in ontogeny of the testis, may also inhibit lung development. To study whether this fetal regressor might inhibit maturation of the fetal lung in vivo, we injected human recombinant MIS (rMIS) into fetal rats, measured serum levels of rMIS using an enzyme-linked immunosorbent assay, and analyzed fetal lung tissue histologically and for protein, glycogen, DNA, and disaturated phosphatidylcholine content. Peak serum levels of recombinant MIS were measured at 6 h, with an apparent elimination half-life of 3 h, and without leakage into adjacent littermates injected with vehicle alone. Female fetal rat lung tissue exposed to recombinant MIS (10(-9) M, 10(-8) M) revealed depressed disaturated phosphatidylcholine content both 48 and 72 h after injection compared with female vehicle-injected littermates. Male lungs of the same gestational age appeared inhibited at a higher (10(-8) M) rMIS dose. These inhibitory effects observed in vivo confirm those previously seen in vitro and suggest that MIS, as well as androgens, may play a causative or important ancillary role in the sexual dimorphism that characterizes the neonatal respiratory distress syndrome.

Müllerian inhibiting substance is present in testes of dogs with persistent müllerian duct syndrome.

Breeding studies in a strain of miniature schnauzer dogs with Persistent Müllerian Duct Syndrome (PMDS) indicate this syndrome is inherited as an autosomal recessive trait, as it is in man. Testes of neonatal dogs affected with PMDS and normal male littermates were examined for Müllerian Inhibiting Substance (MIS) production by immunohistochemistry and bioassay. MIS immunoactivity was detected in Sertoli cells of normal and affected pups using an avidin-biotin complex-enhanced method. Rat embryonic Müllerian ducts regressed when cocultured with testis fragments of both normal and affected pups in a graded organ culture bioassay, demonstrating that the MIS produced was bioactive. These findings indicate that Müllerian duct persistence in affected dogs is not due to a mutation in the structural gene for MIS, but rather, by inference, to a failure of response to MIS at the receptor level.

Mullerian inhibiting substance in the adult rat ovary during various stages of the estrous cycle.

Mullerian inhibiting substance (MIS) in the adult rat ovary can be detected by immunohistochemistry in the granulosa cells of growing preantral follicles and in the cumulus oophorus and periluminal granulosa cells of antral follicles in estrus, metestrus, diestrus, and proestrus. Neither the corpus luteum nor atreitic follicles stained for MIS. During proestrus, dramatic changes occurred in the large preovulatory antral follicles, which early in the day manifest intense MIS-specific staining in the granulosa cells located close to the oocyte, however, at 2300 h, just before ovulation when the germinal vesicle was extruded to indicate resumed meiotic division, MIS staining waned. When the morphology of the late preovulatory stage was created experimentally in 26-day-old immature ovaries by stimulating 48 h earlier with hCG, the intense staining of the granulosa cells surrounding the oocytes from untreated ovaries was lost in the cumulus cells of such hCG-treated animals. This temporal pattern of MIS staining and the prior demonstration that MIS could inhibit in vitro meiosis of oocytes from untreated immature rats suggest that this regressor, well characterized in the fetal testis, might function in the ovary as a regulator of oocyte maturation and follicular development during the adult reproductive cycle.

Cellular localization of müllerian inhibiting substance in the developing rat ovary.

The ontogeny of Müllerian Inhibiting Substance (MIS) production was studied in the immature developing rat ovary using immunohistochemistry and bioassay. In a graded organ culture assay, in which regression of the Müllerian duct of the 14 1/2-day rat fetus was used as a measure of bioactivity, MIS could not be detected in ovarian fragments from fetal rats. After birth, however, MIS bioactivity first became detectable at 4 days of age. Fragments from ovaries of rats 7 days of age and older contained moderate levels of MIS activity which remained detectable throughout the prepubertal period, although extreme individual variability was characteristic. A rabbit polyclonal antibody against human recombinant MIS was used to localize MIS in rat ovarian tissue. Avidin-biotin enhanced immunoperoxidase staining could not detect MIS in the 15-day fetal or 1 day postnatal ovary, where no follicular growth was noticed. In ovaries from rats 1 week or older, where follicular growth was apparent, MIS could be localized specifically and exclusively in the cytoplasm of granulosa cells. MIS was found more in the innermost layers of granulosa cells than in the peripheral layers in preantral follicles. In antral follicles, MIS was found predominantly in the cumulus oophorus cells and periantral cells. In these developing ovaries, MIS could not be found in follicles with features of atresia.

Proteolytic processing of mullerian inhibiting substance produces a transforming growth factor-beta-like fragment.

Mullerian inhibiting substance (MIS) is a differentiation factor that causes the Mullerian duct to regress during the development of the male reproductive tract. The active form is a disulfide-linked dimer consisting of two identical 70-kDa subunits. Recently, the amino acid sequence for MIS was deduced from its gene sequence and revealed that the carboxyl-terminal region shares homology with transforming growth factor (TGF)-beta. Since TGF-beta is produced as a large latent precursor that requires proteolytic activation for activity, we sought to determine if MIS might undergo a similar processing event. Here we demonstrate that typically 5 to 20% of the protein in MIS preparations is cleaved at a site 109 amino acids from the carboxyl terminus. Concurrent cleavages from both chains of the MIS dimer produces a 25-kDa TGF-beta-like fragment and a high molecular mass complex derived from the amino terminus of the protein. Although the two fragments are noncovalently linked, they remain tightly associated after cleavage, and thus are structurally organized like TGF-beta within its precursor. The same cleavage products also can be generated by limited proteolysis with plasmin, which provides a simple method for converting the entire preparation into the cleaved form. The plasmin-digested MIS is fully active in the organ culture assay.

Müllerian inhibiting substance depresses accumulation in vitro of disaturated phosphatidylcholine in fetal rat lung.

Respiratory distress syndrome and associated pulmonary surfactant deficiency are more common in male neonates. Androgens have been shown to depress surfactant production. We tested the hypothesis that müllerian inhibiting substance, another fetal testicular product, might inhibit lung maturation measured as disaturated phosphatidylcholine accumulation. Initially, female fetal rat lungs were incubated with fetal testis or ovary or nanomolar concentrations of bovine müllerian inhibiting substance. Cultured lungs produced less disaturated phosphatidylcholine after incubation for 5 days with either testis (p = 0.012) or müllerian inhibiting substance than after coculture with ovary. In more comprehensive experiments, female lung fragments of 17.5 days' gestation, when incubated with nanomolar concentrations of bovine müllerian inhibiting substance or picomolar concentrations of human recombinant müllerian inhibiting substance, showed significant suppression of disaturated phosphatidylcholine accumulation (p less than 0.004) when compared with incubation with vehicle buffer. The suppression of surfactant accumulation produced in vitro by müllerian inhibiting substance, a potent male-specific fetal regressor, may be a factor in the increased susceptibility of male infants to respiratory distress syndrome.

Human recombinant mullerian inhibiting substance inhibition of rat oocyte meiosis is reversed by epidermal growth factor in vitro.

Mullerian inhibiting substance (MIS), a glycoprotein responsible for the regression of Mullerian duct in the male mammalian embryo, was recently localized not only in the fetal and newborn testis, but also in the older ovary throughout reproductive life. Bovine MIS purified from newborn testicular tissue inhibited spontaneous oocyte meiosis in vitro in the rat. These studies show that partially purified recombinant MIS produced from a human MIS genomic construct caused inhibition of oocyte meiosis, but when purified to homogeneity, the effect was lost. Addition of low concentrations of the detergent Nonidet P-40, used to maintain stability in the purified bovine preparations, however, restored the MIS inhibitory effect to the human preparation, which could, in turn, be blocked by a polyclonal antibody raised to human recombinant MIS; Nonidet P-40 itself caused no inhibition. Since we have shown that epidermal growth factor (EGF) and MIS are antagonistic in a number of other systems, we tested the effect of EGF on the ability of MIS to inhibit oocyte meiosis. EGF added to the medium at a dose that caused no effect on oocytes (25 ng/ml) blocked the MIS inhibitory action on spontaneous rat oocyte meiosis, while EGF had no effect on a known oocyte meiosis inhibitor, 3-isobutyl-1-methylxanthine. These data indicate that human recombinant MIS can inhibit oocyte meiosis in the ovary and that its regulatory effect can be modulated by EGF, which appears to be an antagonist of MIS.