Longitudinal association of prepubertal urinary phthalate metabolite concentrations with pubertal progression among a cohort of boys.

BACKGROUND: Epidemiological studies have reported associations of anti-androgenic phthalate metabolite concentrations with later onset of male puberty, but few have assessed associations with progression. OBJECTIVES: We examined the association of prepubertal urinary phthalate metabolite concentrations with trajectories of pubertal progression among Russian boys. METHODS: At enrollment (ages 8-9 years), medical history, dietary, and demographic information were collected. At entry and annually to age 19 years, physical examinations including testicular volume (TV) were performed and spot urines collected. Each boy's prepubertal urine samples were pooled, and 15 phthalate metabolites were quantified by isotope dilution LC-MS/MS at Moscow State University. Metabolites of anti-androgenic parent phthalates were included: butylbenzyl (BBzP), di-n-butyl (DnBP), diisobutyl (DiBP), di(2-ethylhexyl) (DEHP) and diisononyl (DiNP) phthalates. We calculated the molar sums of DEHP, DiNP, and all AAP metabolites. We used group-based trajectory models (GBTMs) to identify subgroups of boys who followed similar pubertal trajectories from ages 8-19 years based on annual TV. We used multinomial and ordinal regression models to evaluate whether prepubertal log-transformed phthalate metabolite concentrations were associated with slower or faster pubertal progression trajectories, adjusting for covariates. RESULTS: 304 boys contributed a total of 752 prepubertal urine samples (median 2, range: 1-6) for creation of individual pools. The median length of follow-up was 10.0 years; 79% of boys were followed beyond age 15. We identified three pubertal progression groups: slower (34%), moderate (43%), and faster (23%) progression. A standard deviation increase in urinary log-monobenzyl phthalate (MBzP) concentrations was associated with higher adjusted odds of being in the slow versus faster pubertal progression trajectory (aOR 1.47, 95% CI 1.06-2.04). None of the other phthalate metabolites were associated with pubertal progression. CONCLUSIONS: On average, boys with higher concentrations of prepubertal urinary MBzP had a slower tempo of pubertal progression, perhaps attributable to the disruption of androgen-dependent biological pathways.

A metagenomic study of the preventive effect of Lactobacillus rhamnosus GG on intestinal polyp formation in ApcMin/+ mice.

AIMS: To investigate the in vivo effects of Lactobacillus rhamnosus GG (LGG) on intestinal polyp development and the interaction between this single-organism probiotic and the gut microbiota therein. METHODS AND RESULTS: The ApcMin/+ mouse model was used to study the potential preventive effect of LGG on intestinal polyposis, while shotgun metagenomic sequencing was employed to characterize both taxonomic and functional changes within the gut microbial community. We found that the progression of intestinal polyps in the control group altered the community functional profile remarkably despite small variation in the taxonomic diversity. In comparison, the consumption of LGG helped maintain the overall functional potential and taxonomic profile in the resident microbes, thereby leading to a 25% decrease of total polyp counts. Furthermore, we found that LGG enriched those microbes or microbial activities related to short-chain fatty acid production (e.g. Roseburia and Coprococcus), as well as suppressed the ones that can lead to inflammation (e.g. Bilophila wadsworthia). CONCLUSIONS: Our study using shotgun metagenomics highlights how single probiotic LGG may exert its beneficial effects and decrease polyp formation in mice by maintaining gut microbial functionality. SIGNIFICANCE AND IMPACT OF THE STUDY: This probiotic intervention targeting microbiota may be used in conjugation with other dietary supplements or drugs as part of prevention strategies for early-stage colon cancer, after further clinical validations in human.

Effect of Immigration Status on Outcomes in Pediatric Kidney Transplant Recipients.

Kidney transplantation is the optimal treatment for children with end-stage renal disease. For children with undocumented immigration status, access to kidney transplantation is limited, and data on transplant outcomes in this population are scarce. The goal of the present retrospective single-center study was to compare outcomes after kidney transplantation in undocumented children with those of US citizen children. Undocumented residency status was identified in 48 (17%) of 289 children who received a kidney transplant between 1998 and 2010. In undocumented recipients, graft survival at 1 and 5 years posttransplantation was similar, and mean estimated glomerular filtration rate at 1 year was higher than that in recipients who were citizens. The risk of allograft failure was lower in undocumented recipients relative to that in citizens at 5 years posttransplantation, after adjustment for patient age, donor age, donor type, and HLA mismatch (p < 0.04). In contrast, nearly one in five undocumented recipients who reached 21 years of age lost their graft, primarily because they were unable to pay for immunosuppressive medications once their state-funded insurance had ended. These findings support the ongoing need for immigration policies for the undocumented that facilitate access to work-permits and employment-related insurance for this disadvantaged group.

Prediction of en-route complications during interfacility transport by outcome predictive scores in ED.

OBJECTIVE: The objective was to determine the accuracy of the outcome predictive scores (Modified Early Warning Score [MEWS]; Hypotension, Low Oxygen Saturation, Low Temperature, Abnormal ECG, Loss of Independence [HOTEL] score; and Simple Clinical Score [SCS]) in predicting en-route complications during interfacility transport (IFT) in emergency department. DESIGN: This was a retrospective cohort study. METHODS: All IFT cases by ambulances with either nurse-led or physician-led escort, occurring between 1 January 2011 and 31 December 2012, were included. Obstetric and pediatric cases (age < 18 years) were excluded. The condition of patients was quantified by using the predictive scores (MEWS, HOTEL, and SCS) at triage station and on ambulance departure. The accuracy of predictive scores was compared by the receiver operating characteristic (ROC) curves. RESULTS: A total of 659 cases were included. Seventeen cases had en-route complications (2.6%). The complication rate in physician-escorted transport (2.2%) was similar to that in nurse-escorted transport (2.6%). None of the 57 intubated cases had en-route complications. The area under the ROC curve for MEWS was 0.662 (triage) and 0.479 (departure). The accuracy of MEWS at triage was better than that at departure (P = .049). The area under the ROC curve for HOTEL was 0.613 (triage) and 0.597 (departure), and that for SCS was 0.6 (triage) and 0.568 (departure). In general, the predictive scores at triage were better than those on departure. CONCLUSION: None of the scores had good accuracy in prediction of en-route complications during IFT. MEWS at triage was among the best one already but was not ideal.

Steinernema innovationi n. sp. (Panagrolaimomorpha: Steinernematidae), a new entomopathogenic nematode species from South Africa.

Morphological and molecular sequence data were combined with cross-hybridization studies and used to identify a new Steinernema sp. from Free State, South Africa. Molecular and morphological data indicate that the new species belongs to the 'glaseri-group' of Steinernema spp. Key morphological diagnostic characters for S. innovationi n. sp. include the morphometric features of the third-stage infective juveniles: total body length = 1054 (1000-1103) µm, tail length = 108 (97-117) µm, location of the excretory pore = 88 (82-91) µm, and D% = 58 (54-63), E% = 115 (104-137) and H% = 43 (37-46). Additionally, the morphology of the spicules and gubernaculum of the first-generation males are considered key diagnostic traits. Steinernema innovationi n. sp. was also characterized by analysis of both rDNA and mitochondrial gene sequence data, which further indicate the uniqueness of this Steinernema species.

Breathers in lattices with alternating strain-hardening and strain-softening interactions.

This work focuses on the study of time-periodic solutions, including breathers, in a nonlinear lattice consisting of elements whose contacts alternate between strain hardening and strain softening. The existence, stability, and bifurcation structure of such solutions, as well as the system dynamics in the presence of damping and driving, are studied systematically. It is found that the linear resonant peaks in the system bend toward the frequency gap in the presence of nonlinearity. The time-periodic solutions that lie within the frequency gap compare well to Hamiltonian breathers if the damping and driving are small. In the Hamiltonian limit of the problem, we use a multiple scale analysis to derive a nonlinear Schrödinger equation to construct both acoustic and optical breathers. The latter compare very well with the numerically obtained breathers in the Hamiltonian limit.

An investigation of intracellular glycosylation activities in CHO cells: effects of nucleotide sugar precursor feeding.

Controlling glycosylation of recombinant proteins produced by CHO cells is highly desired as it can be directed towards maintaining or increasing product quality. To further our understanding of the different factors influencing glycosylation, a glycosylation sub-array of 79 genes and a capillary electrophoresis method which simultaneously analyzes 12 nucleotides and 7 nucleotide sugars; were used to generate intracellular N-glycosylation profiles. Specifically, the effects of nucleotide sugar precursor feeding on intracellular glycosylation activities were analyzed in CHO cells producing recombinant human interferon-gamma (IFN-gamma). Galactose (+/-uridine), glucosamine (+/-uridine), and N-acetylmannosamine (ManNAc) (+/-cytidine) feeding resulted in 12%, 28%, and 32% increase in IFN-gamma sialylation as compared to the untreated control cultures. This could be directly attributed to increases in nucleotide sugar substrates, UDP-Hex ( approximately 20-fold), UDP-HexNAc (6- to 15-fold) and CMP-sialic acid (30- to 120-fold), respectively. Up-regulation of B4gal and St3gal could also have enhanced glycan addition onto the proteins, leading to more complete glycosylation (sialylation). Combined feeding of glucosamine + uridine and ManNAc + cytidine increased UDP-HexNAc and CMP-sialic acid by another two- to fourfold as compared to feeding sugar precursors alone. However, it did not lead to a synergistic increase in IFN-gamma sialylation. Other factors such as glycosyltransferase or glycan substrate levels could have become limiting. In addition, uridine feeding increased the levels of uridine- and cytidine-activated nucleotide sugars simultaneously, which could imply that uridine is one of the limiting substrates for nucleotide sugar synthesis in the study. Hence, the characterization of intracellular glycosylation activities has increased our understanding of how nucleotide sugar precursor feeding influence glycosylation of recombinant proteins produced in CHO cells. It has also led to the optimization of more effective strategies for manipulating glycan quality.

Mullerian inhibiting substance: a gonadal hormone with multiple functions.

Mullerian inhibiting substance (MIS) is the gonadal hormone that causes regression of the Mullerian ducts, the anlagen of the female internal reproductive structures, during male embryogenesis. MIS is a member of the large transforming growth factor-beta (TGF beta) multigene family of glycoproteins that are involved in the regulation of growth and differentiation. The proteins in this gene family are all produced as dimeric precursors and undergo posttranslational processing for activation, requiring cleavage and dissociation to release bioactive C-terminal fragments. Similarly, the 140 kilodalton (kDa) disulfide-linked homodimer of MIS is proteolytically cleaved to generate its active C-terminal fragments. The sexually dimorphic expression of MIS in Sertoli cells of the testis and granulosa cells of the ovary is critical for normal differentiation of the internal reproductive tract structures. A number of extra-Mullerian functions such as control of germ cell maturation and gonadal morphogenesis, induction of the abdominal phase of testicular descent, suppression of lung maturation, and growth inhibition of transformed cells have also been proposed for this growth-inhibitory hormone and will be discussed. This article will summarize the current understanding of the biology and multiple functions of MIS including its activation, regulation, and mechanism of action and discuss areas of interest in ongoing research.

Incidence of neonatal chlamydial conjunctivitis and its association with nasopharyngeal colonisation in a Hong Kong hospital, assessed by polymerase chain reaction.

OBJECTIVES: Using polymerase chain reactions, this study aimed to evaluate the incidence of neonatal chlamydial conjunctivitis in our region of Hong Kong and explore any association between such conjunctivitis and nasopharyngeal colonisation with Chlamydia trachomatis. DESIGN: Prospective epidemiological study. SETTING: Regional hospital, Hong Kong. PATIENTS: Consecutive patients with neonatal conjunctivitis presenting to our hospital were recruited from May 2004 to April 2005 inclusive. Both eyes were investigated separately for Chlamydia trachomatis by polymerase chain reaction, direct immunofluorescent assay, and cell culture by two assigned ophthalmologists. Neonates diagnosed to have ocular Chlamydia trachomatis infection were subjected to additional nasopharyngeal investigations. Complete sets of ocular and nasopharyngeal investigations were also undertaken 1 week after oral erythromycin treatment to confirm complete eradication of Chlamydia trachomatis. RESULTS: Of 192 patients with neonatal conjunctivitis, 24 were diagnosed to have chlamydial conjunctivitis. Fifteen of them had nasopharyngeal colonisation with Chlamydia trachomatis. Among the 20 neonatal chlamydial conjunctivitis patients that completed the follow-up study, one suffered treatment failure. None had clinically diagnosed systemic Chlamydia trachomatis infection 3 months after oral erythromycin. CONCLUSIONS: The incidence of neonatal chlamydial conjunctivitis in our region of Hong Kong was 4 in 1000 live births, of whom 63% had nasopharyngeal presence of Chlamydia trachomatis. Owing to the high rate of nasopharyngeal isolation and possibility of treatment failure, post-treatment ocular and nasopharyngeal polymerase chain reaction testing for Chlamydia trachomatis may be considered to confirm complete eradication.

Alternative therapies used by women with breast cancer in four ethnic populations.

BACKGROUND: Interest in alternative therapies is growing rapidly in the United States. We studied the types and prevalence of conventional and alternative therapies used by women in four ethnic groups (Latino, white, black, and Chinese) diagnosed with breast cancer from 1990 through 1992 in San Francisco, CA, and explored factors influencing the choices of their therapies. METHODS: Subjects (n = 379) completed a 30-minute telephone interview in their preferred language. Logistic regression models assessed factors associated with the use of alternative therapies after a diagnosis of breast cancer. RESULTS: About one half of the women used at least one type of alternative therapy, and about one third used two types; most therapies were used for a duration of less than 6 months. Both the alternative therapies used and factors influencing the choice of therapy varied by ethnicity. Blacks most often used spiritual healing (36%), Chinese most often used herbal remedies (22%), and Latino women most often used dietary therapies (30%) and spiritual healing (26%). Among whites, 35% used dietary methods and 21% used physical methods, such as massage and acupuncture. In general, women who had a higher educational level or income, were of younger age, had private insurance, and exercised or attended support groups were more likely to use alternative therapies. About half of the women using alternative therapies reported discussing this use with their physicians. More than 90% of the subjects found the therapies helpful and would recommend them to their friends. CONCLUSIONS: Given the high prevalence of alternative therapies used in San Francisco by the four ethnic groups and the relatively poor communication between patients and doctors, physicians who treat patients with breast cancer should initiate dialogues on this topic to better understand patients' choices with regard to treatment options.

Breast cancer risk in women with abnormal cytology in nipple aspirates of breast fluid.

BACKGROUND: We previously showed that women with abnormal cytology in breast fluid obtained by nipple aspiration had an increased relative risk (RR) of breast cancer compared with women from whom fluid was not obtained and with women whose fluid had normal cytology. This study extends the follow-up in the original study group (n = 4046) and presents the first follow-up for a second group of women (n = 3627). METHODS: We collected nipple aspirate fluid from women in the San Francisco Bay Area during the period from 1972 through 1991, classified the women according to the most severe epithelial cytology observed in fluid specimens, and determined breast cancer incidence through March 1999. We estimated RRs for breast cancer using Cox regressions, adjusting for age and year of study entry. All statistical tests were two-sided. RESULTS: For women in the first and second study groups, the median years of follow-up were 21 years and 9 years, respectively, and breast cancer incidences were 7.8% (285 cases in the 3633 women for whom breast cancer status could be determined) and 3.5% (115 of 3271), respectively. Compared with women from whom no fluid was obtained, whose incidences of breast cancer were 4.7% (39 of 825) and 3.3% (65 of 1950) for those in group 1 and group 2, respectively, incidences and adjusted RRs were 8.1% (34 of 422), with RR = 1.4 (95% confidence interval [CI] = 0.9 to 2.3), and 0% (0 of 31), respectively, for those with unsatisfactory aspirate specimens and 8.2% (148 of 1816), with RR = 1.6 (95% CI = 1.1 to 2.3), and 3.1% (25 of 811), with RR = 1.2 (95% CI = 0.8 to 2.0), respectively, for those with normal cytology in aspirates. Compared with women from whom no fluid was obtained, incidences and adjusted RRs for women in group 1 with epithelial hyperplasia and atypical hyperplasia in aspirates were 10.8% (52 of 483), with RR = 2.4 (95% CI = 1.6 to 3.7), and 13.8% (12 of 87), with RR = 2.8 (95% CI = 1.5 to 5.5), respectively, while those for women in group 2 were 5.5% (25 of 457) and 0% (0 of 22), respectively, with a combined RR = 2.0 (95% CI = 1.3 to 3.3). CONCLUSION: The results obtained with the newly followed women independently confirmed previous findings that women with abnormal cytology in nipple aspirates of breast fluid have an increased risk of breast cancer.

Characterization of a highly conserved gene (OS4) amplified with CDK4 in human sarcomas.

Amplification and overexpression of genes involved in cellular growth control occur frequently in human cancers. Here, we report characterization of the full length OS4 cDNA derived from 12q13-q15 (Su et al., Proc. Natl. Acad. Sci. USA, 91: 9121-9125, 1994), a region frequently amplified in sarcomas and brain tumors. This cDNA consists of 4833 base pairs (bp) encoding an open reading frame (ORF) of 283 amino acids. The ORF predicts a water-soluble acidic (pI 5.50) polypeptide with a molecular weight of 31759. Database searches revealed highly significant similarity between OS4 and eight proteins predicted from genomic sequences of Caenorhabditis elegans, Schizosaccaharomyces pombe, and Saccharomyces cerevisiae. Thus, OS4 defines a novel evolutionarily conserved gene superfamily. Northern and database analyses revealed OS4 transcripts in numerous human tissues demonstrating its ubiquitous expression. We also observed overexpression of OS4 in three cancer cell lines with amplification of this gene. Furthermore, we detected OS4 amplification in 5/5 primary sarcomas with known amplification of the closely linked marker CDK4. These results demonstrate that the highly conserved OS4 gene is frequently included in the 12q13-q15 amplicon and may contribute to the development of a subset of sarcomas.

Cell-substrate adhesion and metastatic potential of cultured mesothelioma cells induced by asbestos.

Cell-substrate adhesion was quantified for two cultured mesothelioma cell lines (epitheliomatous and sarcomatous) on glass, fibronectin and laminin substrates. Interference reflection microscopy (IRM) was used to image the adhesion patterns of cells and a grey level analysis was employed to quantify adhesion. Sarcomatous cells demonstrated marked adhesion to glass and fibronectin-coated substrates but not to laminin-coated substrate, with the greatest adhesion occurring on the fibronectin-coated surface. This adhesion was accompanied by cytoplasmic spreading. By contrast, epitheliomatous cells showed little tendency to adhere to any of the substrates and only showed significant spreading when in contact with the laminin substrate (P < 0.01). A bioassay was used to determine the metastatic potential of each of the cell lines. Via the intravenous route, the sarcomatous cells killed the host rats in 24.7 +/- 1.5 (S.D.) days compared to 27.3 +/- 0.9 (S.D.) days for the epitheliomatous cells (P < 0.01). After subcutaneous inoculation of tumour cells, the sarcomatous cells killed the host rats in 54.7 +/- 0.7 (S.D.) days compared to 48.5 +/- 0.5 (S.D.) days for the epitheliomatous cells (P < 0.01). We conclude that the results of the metastasis bioassays were consistent with the predicted behavior of these cell lines based on their ability to adhere to substrates in the in vitro adhesion assays.

A study of surface property changes in rat mesothelial cells induced by asbestos using aqueous two-phase polymer solutions.

Intraperitoneal (i.p.) injection of crocidolite asbestos was used to induce mesotheliomas in rats. The morphological changes of the mesothelial cells were studied by light and electron microscopy and by cytologic examination of peritoneal washings. After injection, the asbestos fibres stimulated an acute inflammatory response and were rapidly phagocytosed by the mesothelial cells and incorporated into the submesothelial tissues. At 7 days, the normal microvillous surface of the mesothelium was replaced with a syncytium of proliferating mesothelial cells showing extensive loss of microvilli. Nine months or so later, multifocal mesothelial tumours arose within the peritoneal cavity. The surface thermodynamic properties of normal, asbestos-stimulated mesothelial cells and of mesothelial tumour cells in culture were studied using an aqueous two-phase system containing 4% Dextran T-2000 and 4% poly (ethylene glycol) (PEG) w/w. After asbestos stimulation, there was a significant (P < 0.01) increase in contact angle between the dextran-rich phase and the mesothelial cell surface. These changes were even greater for the mesothelial tumours. The results indicate that the work of adhesion for asbestos-stimulated mesothelial cells and mesothelial tumours is lower than in normal tissue. These findings may be relevant to the process of tumour spread in the serosal cavities and to the development of distant metastases.

Assessment of mitral annulus velocity by Doppler tissue imaging in the evaluation of left ventricular diastolic function.

OBJECTIVES: This study assessed the clinical utility of mitral annulus velocity in the evaluation of left ventricular diastolic function. BACKGROUND: Mitral inflow velocity recorded by Doppler echocardiography has been widely used to evaluate left ventricular diastolic function but is affected by other factors. The mitral annulus velocity profile during diastole may provide additional information about left ventricular diastolic function. METHODS: Mitral annulus velocity during diastole was measured by Doppler tissue imaging (DTI) 1) in 59 normal volunteers (group 1); 2) in 20 patients with a relaxation abnormality as assessed by Doppler mitral inflow variables (group 2) at baseline and after saline loading; 3) in 11 patients (group 3) with normal diastolic function before and after intravenous nitroglycerin infusion; and 4) in 38 consecutive patients (group 4) undergoing cardiac catheterization in whom mitral inflow velocity and tau as well as mitral annulus velocity were measured simultaneously. RESULTS: In group 1, mean +/- SD peak early and late diastolic mitral annulus velocity was 10.0 +/- 1.3 and 9.5 +/- 1.5 cm/s, respectively. In group 2, mitral inflow velocity profile changed toward the pseudonormalization pattern with saline loading (deceleration time 311 +/- 84 ms before to 216 +/- 40 ms after intervention, p < 0.001), whereas peak early diastolic mitral annulus velocity did not change significantly (5.3 +/- 1.2 cm/s to 5.7 +/- 1.4 cm/s, p = NS). In group 3, despite a significant change in mitral inflow velocity profile after nitroglycerin, peak early diastolic mitral annulus velocity did not change significantly (9.5 +/- 2.2 cm/s to 9.2 +/- 1.7 cm/s, p = NS). In group 4, peak early diastolic mitral annulus velocity (r = -0.56, p < 0.01) and the early/late ratio (r = -0.46, p < 0.01) correlated with tau. When the combination of normal mitral inflow variables with prolonged tau (> or = 50 ms) was classified as pseudonormalization, peak early diastolic mitral annulus velocity < 8.5 cm/s and the early/late ratio < 1 could identify the pseudonormalization with a sensitivity of 88% and specificity of 67%. CONCLUSIONS: Mitral annulus velocity determined by DTI is a relatively preload-independent variable in evaluating diastolic function.

A Novel Catastrophic Presentation of X-Linked Adrenoleukodystrophy.

OBJECTIVE: We report a novel presentation of childhood cerebral X-linked adrenoleukodystrophy: status epilepticus followed by abrupt and catastrophic neurologic deterioration. METHODS: A description of the clinical presentation, laboratory evaluation, and imaging findings leading to a diagnosis of X-linked adrenoleukodystrophy. RESULTS: A 3-year-old male with prior history of autism presented with fever, diarrhea, and status epilepticus requiring a pentobarbital coma. Admission labs were notable only for a glucose level of 22 mg/dL, which stabilized after correction. The child never returned to his prior neurologic baseline, with complete loss of gross motor, fine motor, and speech skills. Serial brain magnetic resonance imaging (MRI)/magnetic resonance spectroscopy (MRS) was notable for progressive diffuse cortical signal changes with swelling, diffusion restriction, and ultimately laminar necrosis. Nine months after presentation, CSF (cerebrospinal fluid) protein and MRS lactate were persistently elevated, concerning for a neurodegenerative disorder. This led to testing for mitochondrial disease, followed by lysosomal and peroxisomal disorders. Very long-chain fatty acids were elevated. Identification of a pathogenic ABCD1 mutation confirmed the diagnosis of X-linked adrenoleukodystrophy. CONCLUSIONS: Boys with childhood cerebral X-linked adrenoleukodystrophy typically present with gradual behavioral changes. Rare reports of boys presenting with transient altered mental status or status epilepticus describe a recovery to their pre-presentation baseline. To our knowledge this is the first X-ALD patient to present with status epilepticus with abrupt and catastrophic loss of neurologic function. X-linked adrenoleukodystrophy should be suspected in young males presenting with seizures, acute decline in neurologic function, with persistently elevated CSF protein and MRS lactate.

Mullerian inhibiting substance messenger ribonucleic acid expression in granulosa and Sertoli cells coincides with their mitotic activity.

In males, Mullerian inhibiting substance (MIS) mRNA was first detected on the medial aspect of the urogenital ridge early on the morning of day 13 of gestation before testicular differentiation was evident, and localized to the more obvious Sertoli cells later on embryonic day 13. MIS transcripts remained at maximal levels between 14.5 and 17.5 days gestation, while the Mullerian duct involutes, and remained high until birth. MIS gene expression decreased progressively after birth and, as germ cell meiosis increased, became barely detectable in the Sertoli cells of the seminiferous tubules. In female rats, MIS mRNA was first detected in the single layer of cuboidal granulosa cells surrounding larger primary follicles 3 days after birth, coincident with the initiation of follicular growth. As follicular growth progressed, MIS mRNA expression was high in preantral and small antral follicles, especially in those granulosa cells closest to the oocyte. MIS mRNA expression decreased gradually in larger antral follicles, remaining prominent only in the cumulus cells and the dividing population of granulosa cells closest to the lumen. MIS gene expression was absent in follicles with features of atresia and in the larger antral follicles. The expression of MIS mRNA in actively dividing Sertoli and granulosa cells correlates with the stages of germ cell division. These findings are suggestive of a role for MIS in the control of germ cell maturation.

Müllerian inhibiting substance production and cleavage is modulated by gonadotropins and steroids.

Analysis of the ontogeny and localization of the amino (N)-terminal and carboxy (C)-terminal cleavage products of Müllerian Inhibiting Substance (MIS) and their modulation by hormones of the hypothalamic-pituitary gonadal axis by immunohistochemistry and Northern analysis led to the discovery of a novel mode of posttranslational regulation of this differentiating agent. Antibody to both holo- and C-terminal MIS identically stained the cytosol of testicular Sertoli cells from 21-day fetal rats, whereas staining of antibody to N-terminal MIS localized to the basement membrane of seminiferous tubules. In addition, when studied longitudinally, basement membrane staining for N-terminal MIS persisted; cytosolic staining for C-terminal MIS was no longer detectable in post-natal testes, but marked basement membrane staining for the N-terminal fragment could still be observed in the testes of untreated 7-day postnatal animals. When 19-day fetuses were injected with FSH, testes collected 2 days later showed less immunohistochemical staining for holo-, N-, and C-terminal MIS, and less MIS messenger RNA. This suggested that FSH downregulates MIS transcription, as had been shown previously in neonatal testes treated with FSH. Testes collected at 21 days from fetuses treated at day 19 in utero with human CG or testosterone, also showed less staining for holo-MIS, but, surprisingly, increased staining for the N- and C-terminal fragments. These changes in MIS protein were accompanied by no or minimal changes in MIS messenger RNA levels, indicating that human CG and testosterone do not affect transcription, but may regulate the cleavage and/or dissociation of MIS. This study describes a form of post-translational regulation of MIS and shows that both transcription and processing of MIS may be differentially modulated by gonadotropins and sex steroids.

Developmentally regulated polyadenylation of two discrete messenger ribonucleic acids for müllerian inhibiting substance.

Mullerian inhibiting substance (MIS) is a 140-kilodalton homodimeric glycoprotein that causes regression of the Mullerian ducts in male embryos, and may also have a role in both males and females in the regulation of germ cell maturation. We examined the ontogeny of MIS messenger RNA (mRNA) in rat testes from midgestation through adulthood and found two discrete MIS mRNA species that are developmentally regulated. The larger 2.0-kilobase species is abundant at embryonic day 14, then decreases in late gestation, and is barely detectable after birth. The smaller 1.8-kilobase species is first noted at embryonic day 18 and is the major species detected postnatally. Both species are abundant just prior to birth, at embryonic day 21, then decrease markedly after birth. This variation in MIS mRNA levels correlates with the developmental expression of MIS protein. A series of oligonucleotide-directed ribonuclease H mapping experiments determined that the two mRNA species differ at their 3' ends in the extent of polyadenylation. Thus, differential polyadenylation of MIS mRNA may be an additional mechanism for regulating MIS expression during fetal and postnatal development.

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.