A time-less function for mouse timeless.

The timeless (tim) gene is essential for circadian clock function in Drosophila melanogaster. A putative mouse homolog, mTimeless (mTim), has been difficult to place in the circadian clock of mammals. Here we show that mTim is essential for embryonic development, but does not have substantiated circadian function.

[Vascular clamping in cirrhotic liver surgery]. / Il clampaggio vascolare nella chirurgia del fegato cirrotico.

Different vascular clamp methods in liver surgery have led to less complications. The aim of this study was to evaluate the results after hepatic resection involving different vascular clamping methods and liver function outcome. Our study examined 46 patients who underwent surgery for liver lesions, developed on cirrhotic and noncirrhotic livers, applying the technique of selective clamping and pedicular clamping. There was one death (1/17; 5.9%) due to postoperative liver failure which occurred in a cirrhotic liver patient who underwent left hepatectomy with pedicular clamping. Complication rate was higher, but not significant (4/7; 57.1%) in the group with selective clamping compared to those with pedicular clamping (3/10; 30%). Hemorrhagic complications were observed in a higher rate among patients with selective clamping (3/7; 42.9%) compared to those with pedicular clamping (1/10; 10%). Selective clamping seems to find major indications in patients with chronic liver disease undergoing minimal hepatic resections. Intermittent pedicular clamping seems to be more effective in regards to blood loss and postoperative hepatic function.

Surgical approach to nipple discharge: a ten-year experience.

BACKGROUND AND OBJECTIVES: The actual relationship between malignancy and secreting breast has not yet been extensively verified, mainly in patients with nipple discharge but without evidence of a breast lump. This study was carried out in 1,251 consecutive patients to evaluate the reliability of cytology combined with galactography in order to assess the relationship of malignant and premalignant lesions with discharge without the presence of a breast lump. METHODS: Those patients with bilateral discharge were approached endocrinologically, whereas the patients (433) with unilateral secretion were evaluated by cytology, mammography, fine needle biopsy, and galactography. Of 194 patients without a breast lump, 94 with positive cytology were surgically treated after mammogalactography. Surgical treatment included ductgalactophorectomy in 53, segmentectomy in 23, microdochectomy in 13, and mastectomy in 5 patients. RESULTS: Pathologic findings showed a solitary papilloma in 49 cases, minimal breast cancer in 20, fibrocystic disease in 11, papillomatosis in 7, lobular cancer in 5, and, finally, a duct ectasia in 2. CONCLUSIONS: In the patients with secreting breast but without lump, cytological analysis in addition to galactography seems to be useful in identifying minimal breast cancer and in detecting premalignant lesions like papillomatosis.

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.

Blind versus open approach to laparoscopic cholecystectomy: a randomized study.

Intraabdominal structures may be damaged during blind introduction of the first trocar for laparoscopic operations. In this study, 150 patients with gallbladder lithiasis who underwent laparoscopy were randomly assigned to two groups, a blind (V group) or an open (H group), in order to compare the results and the rate of complications. No mortality was observed. Major complications occurred in 3/75 (4%) patients of the V group and in 1/75 (1.3%) patient of the H group (p < 0.05). Minor complications occurred in 5/75 (6.7%) patients of either group. The achievement of pneumoperitoneum required 4.5+/-0.4 min in the V group and 3.2+/-0.2 min in the H group (p < 0.05). The open laparoscopic technique is safer and faster than the blind approach; therefore, it is proposed that this approach be routinely used in all laparoscopic procedures.

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.

The immunophilin FKBP12 functions as a common inhibitor of the TGF beta family type I receptors.

The immunophilin FKBP12 is an evolutionarily conserved abundant protein; however, its physiological roles remain poorly defined. Here we report that FKBP12 is a common cytoplasmic interactor of TGF beta family type I receptors. FKBP12 binds to ligand-free TGF beta type I receptor, from which it is released upon a ligand-induced, type II receptor mediated phosphorylation of the type I receptor. Blocking FKBP12/type I receptor interaction with FK506 nonfunctional derivatives enhances the ligand activity, indicating that FKBP12 binding is inhibitory to the signaling pathways of the TGF beta family ligands. Overexpression of a myristylated FKBP12 in Mv1Lu cell specifically inhibits two separate pathways activated by TGF beta, and two point mutations on FKBP12 (G89P, I90K) abolish the inhibitory activity of FKBP12, suggesting that FKBP12 may dock a cytoplasmic protein to the type I receptors to inhibit TGF beta family mediated signaling.

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)

[The role of necrosectomy and continuous peritoneal lavage in the treatment of acute necrotic-hemorrhagic pancreatitis]. / Il ruolo della necrosectomia e del lavaggio peritoneale continuo nel trattamento della pancreatite acuta necrotico-emorragica.

Acute necrotizing pancreatitis involves high mortality. When diagnosed, the disease implies a choice of suitable timing and proper technique of surgical approach. The experience on 16 patients with acute necrotizing pancreatitis, 9 males and 7 females, mean age of 54.7 +/- 3.3 years, is presented in this study. Necrosectomy and continuous local lavage of abdominal collections and pancreatic necrotic surfaces was the most appropriate surgical treatment. The method seems able to remove necrosis and active biological compounds and would appear to achieve a limited mortality and morbidity. Necrosectomy and postoperative local lavage represent a therapeutic effective procedure.

Müllerian inhibiting substance production associated with loss of oocytes and testicular differentiation in the transplanted mouse XX gonadal primordium.

The mouse XX gonadal primordium develops seminiferous-like tubules after transplantation into the renal subcapsular site of the adult male or female mouse. We examined the ontogeny of Sertoli cell differentiation in XX gonadal grafts by immunocytochemical staining and organ culture bioassay for Müllerian Inhibiting Substance (MIS). During normal in situ development of the XY gonad, MIS staining was first detected in fetal Sertoli cells at 12 days of gestation (d.g.) and remained intense until 4 days postpartum (d.pp.), after which it gradually diminished with progressive testicular development. In the normal in situ XX gonad, MIS was detected in granulosa cells of growing follicles at 7 d.pp. and thereafter. When the XX gonad at 12 d.g. was grafted beneath the renal capsule, a few testicular cords composed of MIS-positive cells appeared on Day 7 post-transplantation (equivalent to 19 d.g.), much earlier than the normal appearance of MIS production in the intact XX ovary. The ovarian region containing germ cells at the meiotic prophase was unstained for MIS in the same sections. The incidence of XX gonadal grafts containing MIS-positive testicular cords and the number of such cords per gonadal graft steadily increased from Day 7 to Day 14 post-transplantation. Germ cells were absent or scarce inside the MIS-positive testicular cords. The MIS bioactivity in both control gonads and gonadal grafts coincided with the immunocytochemical staining for MIS. These results support the hypothesis that XX cells differentiate into Sertoli cells as a consequence of oocyte loss in the gonadal graft.

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.

[The use of fibrin glue in the surgery of breast carcinoma]. / L'impiego della colla di fibrina nella chirurgia del carcinoma della mammella.

Twenty-four patients operated on for breast carcinoma with associated axillary node dissection were randomly assigned to two protocols. In the first group fibrin glue was applied intraoperatively, in the second group no complementary treatment was accomplished. The aim of the study was to evaluate the effect of fibrin glue in reducing postoperative axillary sero-lymphatic secretion. In the fibrin glue group a significant reduction of postoperative axillary secretion was observed.

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.