Michael W. Pankhurst

Human blood contains both the uncleaved precursor of anti-Müllerian 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.

Anti-Müllerian hormone is a gonadal cytokine with two circulating forms and cryptic actions

Anti-Müllerian hormone (AMH) is a multi-faceted gonadal cytokine. It is present in all vertebrates with its original function in phylogeny being as a regulator of germ cells in both sexes, and as a prime inducer of the male phenotype. Its ancient functions appear to be broadly conserved in mammals, but with this being obscured by its overt role in triggering the regression of the Müllerian ducts in male embryos. Sertoli and ovarian follicular cells primarily release AMH as a prohormone (proAMH), which forms a stable complex (AMHN,C) after cleavage by subtilisin/kexin-type proprotein convertases or serine proteinases. Circulating AMH is a mixture of proAMH and AMHN,C, suggesting that proAMH is activated within the gonads and putatively by its endocrine target-cells. The gonadal expression of the cleavage enzymes is subject to complex regulation, and the preliminary data suggest that this influences the relative proportions of proAMH and AMHN,C in the circulation. AMH shares an intracellular pathway with the bone morphogenetic protein (BMP) and growth differentiation factor (GDF) ligands. AMH is male specific during the initial stage of development, and theoretically should produce male biases throughout the body by adding a male-specific amplification of BMP/GDF signalling. Consistent with this, some of the male biases in neuron number and the non-sexual behaviours of mice are dependent on AMH. After puberty, circulating levels of AMH are similar in men and women. Putatively, the function of AMH in adulthood maybe to add a gonadal influence to BMP/GDF-regulated homeostasis.

Enzyme-linked immunosorbent assay measurements of antimüllerian hormone (AMH) in human blood are a composite of the uncleaved and bioactive cleaved forms of AMH

OBJECTIVE To determine whether the Beckman Coulter antimüllerian hormone (AMH) Gen II enzyme-linked immunosorbent assay (ELISA) detects the uncleaved precursor (proAMH) and/or the active cleaved form (AMHN,C) of AMH. DESIGN Technical investigation. SETTING Community study. PATIENT(S) Healthy boys and male and female adult volunteers. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) Assay of AMH and Western blot analysis of captured forms of AMH. RESULT(S) In blood, AMH in blood consists of both proAMH, the inactive uncleaved precursor, and AMHN,C, the enzyme-cleaved, receptor-competent form. The Gen II AMH ELISA detected both recombinant proAMH and AMHN,C. The noncovalent association of the two cleavage fragments of AMHN,C appears to be necessary for ELISA detection because recombinant free AMHC and AMHN were undetectable. Spike-recovery experiments showed that proAMH was not completely recovered from serum unless it was prediluted 1 hour before the assay. CONCLUSION(S) The leading ELISA for AMH provides a composite value of two biologically distinct forms of AMH. It is not known whether proAMH and AMHN,C have identical relationships to ovarian reserve, antral follicle counts, or other aspects of ovarian function. Hence, future research into the physiology and clinical utility of AMH should consider the two forms separately.

Inhibin B and anti-Müllerian hormone/Müllerian-inhibiting substance may contribute to the male bias in autism

The autistic spectrum disorders have a significant male bias in incidence, which is unexplained. The Sertoli cells of the immature testes secrete supra-adult levels of Müllerian-inhibiting substance/anti-Müllerian hormone (AMH) and inhibin B (InhB), with both hormones being putative regulators of brain development. We report here, that 82 boys with an autism spectrum disorder have normal levels of InhB and AMH. However, the boys’ level of InhB correlated with their autism diagnostic interview—revised (ADI-R) scores for the social interaction (R=0.29, P=0.009, N=82) and communication domains (R=0.29, P=0.022, N=63), and with the number of autistic traits the boys exhibited (R=0.34 and 0.27, respectively). The strengths of the abovementioned correlates were stronger in the boys with milder autism (R=0.42 and 0.50, respectively), with AMH exhibiting a significant negative correlation to the ADI-R score in these boys (R=−0.44 and R=−0.39, respectively). Neither hormone correlated to the incidence of stereotyped and repetitive behaviours. This suggests that the male bias in the autistic spectrum has multiple determinants, which modulate the effects of an otherwise non-dimorphic pathology. Furthermore, AMH and InhB have opposing effects on the SMAD1/5/8 pathway, and opposing correlates to autistic traits, implicating the SMAD pathways as a putative point of molecular convergence for the autistic spectrum.

Metallothionein (MT) -I and MT-II expression are induced and cause zinc sequestration in the liver after brain injury.

Experiments with transgenic over-expressing, and null mutant mice have determined that metallothionein-I and -II (MT-I/II) are protective after brain injury. MT-I/II is primarily a zinc-binding protein and it is not known how it provides neuroprotection to the injured brain or where MT-I/II acts to have its effects. MT-I/II is often expressed in the liver under stressful conditions but to date, measurement of MT-I/II expression after brain injury has focused primarily on the injured brain itself. In the present study we measured MT-I/II expression in the liver of mice after cryolesion brain injury by quantitative reverse-transcriptase PCR (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) with the UC1MT antibody. Displacement curves constructed using MT-I/II knockout (MT-I/II−/−) mouse tissues were used to validate the ELISA. Hepatic MT-I and MT-II mRNA levels were significantly increased within 24 hours of brain injury but hepatic MT-I/II protein levels were not significantly increased until 3 days post injury (DPI) and were maximal at the end of the experimental period, 7 DPI. Hepatic zinc content was measured by atomic absorption spectroscopy and was found to decrease at 1 and 3 DPI but returned to normal by 7DPI. Zinc in the livers of MT-I/II−/− mice did not show a return to normal at 7 DPI which suggests that after brain injury, MT-I/II is responsible for sequestering elevated levels of zinc to the liver. Conclusion: MT-I/II is up-regulated in the liver after brain injury and modulates the amount of zinc that is sequestered to the liver.

The Daily Profiles of Circulating AMH and INSL3 in Men are Distinct from the Other Testicular Hormones, Inhibin B and Testosterone.

The testes secrete four hormones (anti-Müllerian hormone, insulin-like peptide 3, Inhibin B and testosterone) from two endocrine cell types. It is unknown whether anti-Müllerian hormone and insulin-like peptide 3 levels have a diurnal variation, and if so, whether they covary during the day with testosterone and InhB. Sera were obtained from 13 men at 00:00, 06:00, 09:00, 12:00, 14:00, 17:00 and 19:00 hours and the levels of their testicular hormones measured by ELISA. A second cohort of 20 men was similarly examined with blood drawn at 19:00 and the following 06:00. Anti-Müllerian hormone levels exhibited a subtle diurnal pattern with a 19:00 peak that was 4.9% higher on average than the 06:00 nadir (p = 0.004). The decrease in anti-Müllerian hormone coincided with a rise in testosterone and InhB, but there was no association between the person-to-person variation in the diurnal patterns of anti-Müllerian hormone and testosterone or Inhibin B. Insulin-like peptide 3 had no diurnal pattern, with only minor sporadic variation between time points being observed in some men. In conclusion, the diurnal and sporadic variation of each testicular hormone is distinct, indicating that the major regulation is at the level of the hormone rather than at the endocrine cell type. Consequently, the balance of the hormones being released by the testes has complex variation during the day. The physiological significance of this will vary depending on which combinations of testicular hormones that the target cells respond to.

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.

Increased circulating leukocyte numbers and altered macrophage phenotype correlate with the altered immune response to brain injury in metallothionein (MT) -I/II null mutant mice

BackgroundMetallothionein-I and -II (MT-I/II) is produced by reactive astrocytes in the injured brain and has been shown to have neuroprotective effects. The neuroprotective effects of MT-I/II can be replicated in vitro which suggests that MT-I/II may act directly on injured neurons. However, MT-I/II is also known to modulate the immune system and inflammatory processes mediated by the immune system can exacerbate brain injury. The present study tests the hypothesis that MT-I/II may have an indirect neuroprotective action via modulation of the immune system.MethodsWild type and MT-I/II-/- mice were administered cryolesion brain injury and the progression of brain injury was compared by immunohistochemistry and quantitative reverse-transcriptase PCR. The levels of circulating leukocytes in the two strains were compared by flow cytometry and plasma cytokines were assayed by immunoassay.ResultsComparison of MT-I/II-/- mice with wild type controls following cryolesion brain injury revealed that the MT-I/II-/- mice only showed increased rates of neuron death after 7 days post-injury (DPI). This coincided with increases in numbers of T cells in the injury site, increased IL-2 levels in plasma and increased circulating leukocyte numbers in MT-I/II-/- mice which were only significant at 7 DPI relative to wild type mice. Examination of mRNA for the marker of alternatively activated macrophages, Ym1, revealed a decreased expression level in circulating monocytes and brain of MT-I/II-/- mice that was independent of brain injury.ConclusionsThese results contribute to the evidence that MT-I/II-/- mice have altered immune system function and provide a new hypothesis that this alteration is partly responsible for the differences observed in MT-I/II-/- mice after brain injury relative to wild type mice.

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.

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.