Mice with either diminished or elevated levels of anti-Müllerian hormone have decreased litter sizes.

Anti-Müllerian hormone (AMH) is both a gonadal hormone and a putative paracrine regulator of neurons, the uterus, and the placenta. A mouse line with neuronal expression of AMH (Thy1.2-AMH) was generated to examine the role of paracrine AMH in the brain. The mice had normal behavior, but unexpectantly AMH was present in the circulation of the transgenic mice. Thy1.2-AMHTg/0 studs sired pups with a normal frequency, when mated with wild-type dams. In stark contrast, Thy1.2-AMHTg/0 dams rarely gave birth, with evidence of spontaneous midgestational abortion. This leads to the hypothesis that AMH influences the capacity of dams to carry concepti to term. This hypothesis was tested by mating AMH-deficient (Amh-/-), Thy1.2-AMHTg/0, and wild-type dams when 49-, 80-, and 111 days old, using proven wild-type studs. The litter sizes from the first two matings and the number of fetuses present on the 10th day of gestation of the third mating were recorded. Thy1.2-AMHTg/0 dams carried near normal numbers of midterm fetuses, but typically produced no pups, indicating that extensive late resorption of fetuses was occurring. Amh-/- dams exhibited a lesser reduction in litter size than the Thy1.2-AMHTg/0 dams, with no evidence of enhanced loss of fetuses. In conclusion, this study provides the first evidence that high AMH levels can cause a miscarriage phenotype and that the absence of AMH affects reproductive output.

Anti-Müllerian hormone signaling is influenced by Follistatin 288, but not 14 other transforming growth factor beta superfamily regulators.

The hypothesis that, in contrast to other transforming growth factor-beta (TGFß) superfamily ligands, the dose-response curve of Anti-Müllerian hormone (AMH) is unmodulated was tested by examining whether known TGFB superfamily modulators affect AMH signaling, using a P19/BRE luciferase reporter assay. AMHC and AMHN,C activated the reporter with an EC50 of approximately 0.5 nM. Follistatins (FS) produced concentration-dependent increases in AMHC - and AMHN,C -initiated reporter activity, with FS288 being more potent than FS315; however, the maximum bioactivity of AMH was not altered by either follistatin. Thirteen other TGFß regulators (Chordin, Chordin-like 1, Chordin-like 2, Differential screening-selected gene aberrative in neuroblastoma [DAN], Decorin, Endoglin, Follistatin-like 1, Follistatin-like 3, Follistatin-like 4, Noggin, α2 macroglobulin, TGFß receptor 3, Von Willebrand factor C domain-containing 2) had little or no effect. Surface plasmon resonance analysis showed no significant association between FS288 and AMHC , suggesting that FS288 indirectly regulates AMH signaling. Activin A, a direct target of FS288, did not itself induce reporter activity in P19 cells, but did prevent the FS288-induced increase in AMH signaling. Hence, local concentrations of FS288 and Activin A may influence the response of some cell types to AMH.

Human blood contains both the uncleaved precursor of anti-Mullerian hormone and a complex of the NH2- and COOH-terminal peptides.

Anti-Müllerian hormone (AMH) in blood is a marker of ovarian status in women and the presence of cryptic testes in babies. Despite this, the molecular form of AMH in blood has not been verified. AMH is synthesized as an inert proprotein precursor (proAMH), which can be cleaved to yield NH2-terminal (AMHN) and COOH-terminal (AMHC) fragments, that can complex noncovalently (AMHN,C). Developing males have 10-fold more AMH than young adults. We report here that human blood is a mixture of inactive proAMH and receptor-binding AMHN,C. The AMH in the blood of boys, men, and premenopausal women was immunoprecipitated using antibodies to the NH2- and COOH-terminal peptides. The precipitated proteins were then analyzed by Western blots, using recombinant proteins as markers. The glycosylation status of AMH was verified using deglycosylating enzymes. The NH2-terminal antibody precipitated a major protein that migrated alongside rhproAMH and was detected by anti-AMHN and anti-AMHC. This antibody also precipitated significant levels of AMHN and AMHC from all participants. Antibodies specific to AMHC precipitated rhAMHC but did not precipitate AMHC from human blood. Hence, all the AMHC in human blood appears to be bound to AMHN. Both AMHN and proAMH were glycosylated, independent of age and sex. In conclusion, boys and young adults have the same form of AMH, with a significant proportion being the inactive precursor. This raises the possibility that the endocrine functions of AMH are partly controlled by its cleavage in the target organ. The presence of proAMH in blood may confound the use of AMH for diagnosis.

The bed nucleus of the stria terminalis has developmental and adult forms in mice, with the male bias in the developmental form being dependent on testicular AMH.

Canonically, the sexual dimorphism in the brain develops perinatally, with adult sexuality emerging due to the activating effects of pubescent sexual hormones. This concept does not readily explain why children have a gender identity and exhibit sex-stereotypic behaviours. These phenomena could be explained if some aspects of the sexual brain networks have childhood forms, which are transformed at puberty to generate adult sexuality. The bed nucleus of stria terminalis (BNST) is a dimorphic nucleus that is sex-reversed in transsexuals but not homosexuals. We report here that the principal nucleus of the BNST (BNSTp) of mice has developmental and adult forms that are differentially regulated. In 20-day-old prepubescent mice, the male bias in the principal nucleus of the BNST (BNSTp) was moderate (360 ± 6 vs 288 ± 12 calbindin(+ve) neurons, p < 0.0001), and absent in mice that lacked a gonadal hormone, AMH. After 20 days, the number of BNSTp neurons increased in the male mice by 25% (p < 0.0001) and decreased in female mice by 15% (p = 0.0012), independent of AMH. Adult male AMH-deficient mice had a normal preference for sniffing female pheromones (soiled bedding), but exhibited a relative disinterest in both male and female pheromones. This suggests that male mice require AMH to undergo normal social development. The reported observations provide a rationale for examining AMH levels in children with gender identity disorders and disorders of socialization that involve a male bias.

TGF-beta2 alters the characteristics of the neuromuscular junction by regulating presynaptic quantal size.

The amount of neurotransmitter released from a presynaptic terminal is the product of the quantal content (number of vesicles) and the presynaptic quantal size (QSpre, amount of transmitter per vesicle). QSpre varies with synaptic use, but its regulation is poorly understood. The motor nerve terminals at the neuromuscular junction (NMJ) contain TGF-beta receptors. We present evidence that TGF-beta2 regulates QSpre at the NMJ. Application of TGF-beta2 to the rat diaphragm NMJ increased the postsynaptic response to both spontaneous and evoked release of acetylcholine, whereas antibodies to TGF-beta2 or its receptor had the converse effect. L-vesamicol and bafilomycin blocked the actions of TGF-beta2, indicating that TGF-beta2 acts by altering the extent of vesicular filling. Recordings of the postsynaptic currents from the diaphragm were consistent with TGF-beta2 having this presynaptic action and a lesser postsynaptic effect on input resistance. TGF-beta2 also decreased quantal content by an atropine-sensitive pathway, indicating that this change is secondary to cholinergic feedback on vesicular release. Consequently, the net actions of TGF-beta2 at the NMJ were to amplify the postsynaptic effects of spontaneous transmission and to diminish the number of vesicles used per evoked stimulus, without diminishing the amount of acetylcholine released.

Anti-Müllerian hormone level in older women: detection of granulosa cell tumor recurrence.

OBJECTIVE: To determine whether anti-Müllerian hormone (AMH) production resumes during normal late menopausal aging. Anti-Müllerian hormone has been proposed as a specific serum marker for adult granulosa cell tumors. MATERIALS AND METHODS: Serum AMH from 21 elderly postmenopausal women (mean age, 77 years) and 9 young women (mean age, 22 years) were measured by ultrasensitive immunoassay. RESULTS: Both median (0 pmol/L) and mean (0.48 pmol/L) serum AMH values for the elderly women were below the level of detection for the immunoassay kit. Three of the 21 participants had minimally detectable level of AMH (1.13-2.76 pmol/L). The cohort of young women had expected normal values of AMH as measurable by the same immunoassay kit. CONCLUSIONS: Serum AMH values were negligible for postmenopausal women older than 65 years. This extends the normative data for AMH to 108 years old, providing a reference range for the detection of granulosa cell tumors in postmenopausal women.

Müllerian inhibiting substance contributes to sex-linked biases in the brain and behavior.

Many behavioral traits and most brain disorders are common to males and females but are more evident in one sex than the other. The control of these subtle sex-linked biases is largely unstudied and has been presumed to mirror that of the highly dimorphic reproductive nuclei. Sexual dimorphism in the reproductive tract is a product of Müllerian inhibiting substance (MIS), as well as the sex steroids. Males with a genetic deficiency in MIS signaling are sexually males, leading to the presumption that MIS is not a neural regulator. We challenge this presumption by reporting that most immature neurons in mice express the MIS-specific receptor (MISRII) and that male Mis(-/-) and Misrii(-/-) mice exhibit subtle feminization of their spinal motor neurons and of their exploratory behavior. Consequently, MIS may be a broad regulator of the subtle sex-linked biases in the nervous system.

Mice with disrupted TGFbeta signaling have normal cerebella development, but exhibit facial dysmorphogenesis and strain-dependent deficits in their body wall.

The transforming growth factor betas (TGFbetas) are context-dependent regulators of neurons in vitro, but their physiological functions in the brain are unclear. Haploinsufficiency of either Tgfbeta1 or Tgfbeta2 leads to age-related deterioration of neurons, but the development of the brain is normal in the full absence of either of these genes. However, some individuals with mis-sense mutations of TGFbeta receptors are mentally retarded, suggesting that the TGFbeta isoforms can compensate for each other during brain development. This possibility was tested by generating mice (NSE x PTR) with neuron-specific expression of a dominant-negative inhibitor of TGFbeta signaling. The NSE x PTR mice with a FVBxC57Bl/6 genetic background were viable and developed normally despite strong neuronal expression of the inhibitor of TGFbeta signaling. Their cerebella were of normal size and contained normal numbers of neurons. When the genetic background of the mice was changed to C57BL/6, the phenotype of the mice became neonatal lethal, with the neonates exhibiting various malformations. The malformations correlated with sites of non-neuronal expression of the transgenes and included facial dysmorphogenesis, incomplete closure of the ventral body wall and absence of intestinal motility. The C57BL/6 Tgfbm1-3 alleles, which modulate the phenotype of Tgfbeta1(-/-) mice, were not major determinants of the NSE x PTR phenotype. The data suggest that the development of the cerebellum is insensitive to the level of TGFbeta signaling, although this may be dependent on the genetic background.

Fliih, a gelsolin-related cytoskeletal regulator essential for early mammalian embryonic development.

The Drosophila melanogaster flightless I gene is required for normal cellularization of the syncytial blastoderm. Highly conserved homologues of flightless I are present in Caenorhabditis elegans, mouse, and human. We have disrupted the mouse homologue Fliih by homologous recombination in embryonic stem cells. Heterozygous Fliih mutant mice develop normally, although the level of Fliih protein is reduced. Cultured homozygous Fliih mutant blastocysts hatch, attach, and form an outgrowing trophoblast cell layer, but egg cylinder formation fails and the embryos degenerate. Similarly, Fliih mutant embryos initiate implantation in vivo but then rapidly degenerate. We have constructed a transgenic mouse carrying the complete human FLII gene and shown that the FLII transgene is capable of rescuing the embryonic lethality of the homozygous targeted Fliih mutation. These results confirm the specific inactivation of the Fliih gene and establish that the human FLII gene and its gene product are functional in the mouse. The Fliih mouse mutant phenotype is much more severe than in the case of the related gelsolin family members gelsolin, villin, and CapG, where the homozygous mutant mice are viable and fertile but display alterations in cytoskeletal actin regulation.

The Testicular Hormones AMH, InhB, INSL3, and Testosterone Can Be Independently Deficient in Older Men.

BACKGROUND: Late-onset hypogonadism is symptomatically diverse and not fully explained by circulating testosterone level. The adult testes secrete four distinct hormones (testosterone, AMH, INSL3, and InhB) into the circulation. Testosterone and InhB have proven dynamic regulation, with limited information available for AMH and INSL3. During aging, there is cellular senescence, which may underlie the diversity of hypogonadism. This leads to the postulate that the relative levels (profile) of the four testicular hormones in older men are variable and cannot be evaluated by the measurement of one hormone. METHODS: 111 men aged 19-50 years and 98 men aged 70-90 years were examined. The circulating levels of the testicular hormones were measured using ELISAs, and the variation in the levels of hormones was analyzed by various correlative analyses. RESULTS: All four hormones were largely or totally independent. Some men were deficient in multiple hormones, but no man had multiple elevated hormones. The average hormonal levels were lower in older men, with diverse profiles of the four testicular hormones. Hence, some men had one or more hormones below the reference range, with testosterone the most conserved. Consequently, testosterone levels were not indicative of the complete state of the endocrine testes. CONCLUSIONS: The four hormones vary independently of each other, in younger and older men. This indicates that they are regulated dynamically rather than influenced by endocrine cell number. Older men exhibited diverse profiles of low levels of testicular hormones, suggesting that the testes age differently between men. Testosterone alone inadequately describes gonadal states.

Circulating anti-Müllerian hormone (AMH) associates with the maturity of boys' drawings: Does AMH slow cognitive development in males?

PURPOSE: High levels of circulating anti-Müllerian hormone are unique to developing males, but the function of anti-Müllerian hormone in boys is unknown. In mice, anti-Müllerian hormone contributes to the male biases in the brain, but its receptors are present throughout non-sexually dimorphic portions of the brain. In humans, the speed of maturation is the most overt difference between girls and boys. We postulate that this is because anti-Müllerian hormone slows the maturation of the male human brain. METHODS: One hundred and fourty three 5-year or 6-year-old boys and 38 age-matched girls drew a person and donated a blood sample. The children's drawings were blind-scored to generate a maturity index. The level of anti-Müllerian hormone and the other Sertoli cell hormone, inhibin B, were measured by ELISA. The relationship between the children's age, hormones and maturity index were examined by linear regression analysis. RESULTS: The girls drew more complex and realistic person than the boys (32%, p = 0.001), with their drawings also being larger (39%, p = 0.037) and more coloured-in (235%, p = 0.0005). The maturity index in boys correlated with age (+r = 0.43, p < 0.0005) and anti-Müllerian hormone level (-r = -0.29, p < 0.0005). The association between maturity index and anti-Müllerian hormone level persisted when corrected for age and for inhibin B (r = -0.24, p = 0.0005). The calculated effect of the median level of anti-Müllerian hormone (1 nM) was equal to 0.81 months of development. The size and colouring of the drawings did not correlate with the boys' age, anti-Müllerian hormone or inhibin B. CONCLUSIONS: This exploratory study provides the first indicative evidence that circulating anti-Müllerian hormone may influence the development of the human brain.

Acute Supplementation with High Dose Vitamin D3 Increases Serum Anti-Müllerian Hormone in Young Women.

Anti-Müllerian hormone (AMH) is a paracrine regulator of ovarian follicles. Vitamin D (Vit D) regulates AMH production in vitro, but its role as a regulator of ovarian AMH production is contentious. If Vit D influences ovarian AMH production, then an acute rise in Vit D level should lead to an acute rise in circulating AMH levels. This hypothesis was tested with a randomized double-blind design, with 18-25-year-old women recruited from the community. The study was conducted in early spring, when the marker of Vit D level (25-hydroxyvitamin D, 25(OH)D) tends to be at its nadir. The women consumed either an oral dose of 50,000 IU of Vit D3 (n = 27) or placebo (n = 22). The initial 25(OH)D ± SD value was 53.6 ± 23.3 nmol/L, with 42 of the 49 women having a value below 75 nmol/L, consistent with seasonal nadir. All women receiving Vit D3 treatment exhibited a robust increase in serum 25(OH)D within 1 day (15.8 ± 1.1 nmol/L (n = 27), p < 0.0001), with the increase sustained over the study week. Circulating levels of AMH in the women receiving Vit D3 progressively rose during the following week, with a mean increase of 12.9 ± 3.7% (n = 24, p = 0.001). The study supports the hypothesis that Vit D's positive effects on the fertility of woman may involve the regulation of ovarian AMH levels.

A specific immunoassay for proAMH, the uncleaved proprotein precursor of anti-Müllerian hormone.

The utility of serum anti-Müllerian hormone (AMH) assays in assessment of female fertility have been investigated extensively but little is known about the biological activity of the hormone being studied. ProAMH is the proprotein precursor and is incapable of binding to the AMH-specific type II receptor. Proteolytic cleavage generates receptor-competent AMHN,C which is a non-covalent complex of the N- and C-terminal cleavage fragments. Commercially available AMH assays do not differentiate the two forms of AMH. Techniques were developed to dissociate the AMHN,C complex and abolish its two-site immunoassay immunoreactivity. This allowed specific quantification of proAMH. The surfactant sodium deoxycholate (DOC) dissociated AMHN,C without disrupting binding of proAMH to the capture-antibody with an optimal concentration of 0.1-0.2%w/v. The incorporation of a DOC incubation step into the AMH Gen II ELISA detected proAMH, with AMHN,C cross-detection conservatively estimated at 6.0% ± 2.5% (mean ± S.D.). The intra-assay and inter-assay variability were estimated at 8.0%CV and 13.0%CV respectively. The levels of proAMH and total AMH were assessed in 5 boys and 5 men and the proportion of proAMH was found to be significantly higher in boys (p = 0.005). This study will facilitate further investigation of the role of proteolytic cleavage in AMH signalling.

Relative levels of the proprotein and cleavage-activated form of circulating human anti-Müllerian hormone are sexually dimorphic and variable during the life cycle.

Anti-Müllerian hormone (AMH) is a gonadal hormone, which induces aspects of the male phenotype, and influences ovarian follicular recruitment. AMH is synthesized as a proprotein (proAMH), which is incompletely cleaved to the receptor-competent AMHN ,C AMH ELISAs have not distinguished between proAMH and AMHN ,C; consequently, the physiological ranges of circulating proAMH and AMHN ,C are unknown. A novel proAMH ELISA has been used to assay serum proAMH in humans. Total AMH was also measured, enabling the AMHN ,C concentration to be calculated. Stored serum from 131 boys, 80 younger, and 106 older men were examined, with serum from 14 girls and 18 women included for comparison. The mean levels of proAMH and AMHN ,C in pM were respectively: boys (253, 526), men (7.7, 36), elderly men (5.7, 19), girls (3.3, 15), and women (5.2, 27) (boys vs. men, P < 0.001; girls vs. women, P = 0.032). The proportion of proAMH as a percentage of total AMH (API) was approximately twofold higher in boys than men (P < 0.001) with little overlap between the ranges, with girls also exhibiting lesser cleavage of their AMH than women (P < 0.001). The API varied within each population group. In young men, the API did not correlate with circulating levels of the other testicular hormones (testosterone, InhB, and INSL3). In conclusion, the cleavage of circulating AMH varies extensively within the human population, with most individuals having significant levels of proAMH The physiological and clinical relevance of circulating proAMH needs to be established.

The Anti-Müllerian Hormone Precursor (proAMH) Is Not Converted to the Receptor-Competent Form (AMHN,C) in the Circulating Blood of Mice.

Anti-Müllerian hormone (AMH) is a gonadal hormone that regulates aspects of male sexual differentiation and ovarian function. AMH is synthesized as the AMH proprotein precursor (proAMH), which is converted to a receptor-binding form (AMHN,C) by proteolytic cleavage. ProAMH appears to be the predominant species in the ovary, whereas AMHN,C is the prevalent form in circulation. The aim of this study was to determine whether cleavage of proAMH occurs before it is released from the gonad or while in circulation. The individual half-lives of the proAMH and AMHN,C were also determined, as this has important implications for understanding the mechanisms of AMH signaling. Recombinant human (rh)-proAMH or rh-AMHN,C was injected iv into mice. AMH levels were analyzed in a series of repeated blood samples using an assay that detects human, but not murine, AMH. The degree of cleavage of injected proAMH was assessed by immunoprecipitation and Western blotting. The elimination half-life curves were biphasic. The fast-phase elimination was estimated at 6 and 11 minutes for rh-proAMH and rh-AMHN,C, respectively. The slow-phase half-life estimates were 2.4 and 3.8 hours for rh-proAMH and rh-AMHN,C, respectively. Immunoprecipitation of rh-proAMH 1 hour after injection determined that no detectable conversion of proAMH to AMHN,C was occurring in circulation. The data suggest that the ratio of proAMH to AMHN,C in the circulation is not altered after it is released from the gonads and that the levels of these 2 circulating forms are likely to reflect AMH activity in the gonad.

Changes in Circulating ProAMH and Total AMH during Healthy Pregnancy and Post-Partum: A Longitudinal Study.

Circulating Anti-Müllerian hormone (AMH) is derived from the gonads, and is a mixture of the prohormone (proAMH), which does not bind to AMH receptors, and receptor-competent AMH. The functions of a hormone are partially defined by the factors that control its levels. Ovarian reserve accounts for 55~75% of the woman-to-woman variation in AMH level, leaving over 25% of the biological variation to be explained. Pregnancy has been reported to decrease circulating AMH levels, but the observations are inconsistent, with the effect of pregnancy on the bioactivity of AMH being unknown. We have therefore undertaken a longitudinal study of circulating proAMH and total AMH during pregnancy. Serum samples were drawn at 6-8 gestational time-points (first trimester to post-partum) from 25 healthy women with prior uneventful pregnancies. The total AMH and proAMH levels were measured at each time-point using ELISA. The level of circulating total AMH progressively decreased during pregnancy, in all women (p<0.001). On average, the percentage decline between the first trimester and 36-39 weeks' gestation was 61.5%, with a standard deviation of 13.0% (range 30.4-81.2%). The percentage decline in total AMH levels associated with maternal age (R = -0.53, p = 0.024), but not with the women's first trimester AMH level. The postpartum total AMH levels showed no consistent relationship to the woman's first trimester values (range 31-273%). This raises the possibility that a fundamental determinant of circulating AMH levels is reset during pregnancy. The ratio of proAMH to total AMH levels exhibited little or no variation during pregnancy, indicating that the control of the cleavage/activation of AMH is distinct from the mechanisms that control the total level of AMH.

Transport of transforming growth factor-beta 2 across the blood-brain barrier.

The blood-brain barrier acts as an interface between the brain and body through a combination of restrictive mechanisms and transport processes. Substances essential for brain function pass through the barrier either by passive diffusion or by active transport. We report here that [125I]-transforming growth factor-beta2 (TGF-beta2) passes through the blood-brain barrier and blood-nerve barriers, after intravenous, intraperitoneal or intramuscular injections. The entry of the [125I]-TGF-beta2 to the brain was rapid, saturable and inhibited by co-injection of unlabelled TGF-beta2. In contrast, co-injection of unlabelled TGF-beta2 increased the retention of [125I]-TGF-beta2 in the blood. The [125I]-TGF-beta2 transported into the brain was localised by autoradiography to the extracellular space, and was intact as judged by SDS-PAGE. The [125I]-TGF-beta2 was widely distributed throughout the brain, with the highest concentrations in the hypothalamus and nerves and the lowest in the cerebral hemispheres. The [125I]-TGF-beta2 had a half-life of 4 h in the brain. These results indicate that therapeutically relevant levels of TGF-beta2 reach the brain after peripheral administration of TGF-beta2.

The transforming growth factor-betas: multifaceted regulators of the development and maintenance of skeletal muscles, motoneurons and Schwann cells.

This review discusses the roles of the transforming growth factor-betas (TGF-betas) as part of a complex network that regulates the development and maintenance of the neuromuscular system. The actions of the TGF-betas often vary depending on which other growth factors are present, making it difficult to extrapolate results from in vitro experiments to the in vivo situation. A new approach has therefore been needed to understand the physiological functions of the TGF-betas. The behaviours (proliferation, fusion, apoptosis) of many of the cells in the neuromuscular system have a complex pattern which varies in space and time. The actions of growth factors in this system can thus be deduced based on how well their pattern of expression correlates with known cellular behaviours. Hypotheses based on this molecular anatomical evidence can then be further tested with genetically modified mice. From this type of evidence, we suggest that: (1) TGF-beta1 is an autocrine regulator of Schwann cells; (2) maternally-derived TGF-beta1 helps to suppress self and maternal immune attack; (3) TGF-beta2 regulates when and where myoblasts fuse to myotubes; (4) motoneuron survival is regulated by multiple sources of TGF-betas, with TGF-beta2 being the more important isoform. The concept of TGF-beta1 as a regulator of secondary myotube formation is not supported by either the location of the TGF-beta1 in developing muscles or by the phenotype of TGF-beta1-/- mice. The review concludes with a discussion of whether all of these of postulated functions can occur independently of each other, within the confines of the neuromuscular system.