Fetal resorption coincides with dysregulated LH secretion in AMH-overexpressing mice.

Female anti-Müllerian hormone (AMH) overexpressing (Thy1.2-AMHTg/0) mice experience fetal resorption (miscarriage) by mid-gestation. This study examined whether the ovary, uterine implantation sites and hypothalamus are potential sites of AMH action, as AMH type-2 receptor (AMHR2) expression is reported in each tissue. Pregnancy in Thy1.2-AMHTg/0 mice was compared to wild-type (WT) mice via histological examination of implantation sites, hormone assays, embryo culture and embryo transfer. Uterine AMH and AMHR2 expression was examined by RT-qPCR and immunohistochemistry. The first signs of fetal resorption in the Thy1.2-AMHTg/0 dams occurred at embryonic day 9.5 (E9.5) with 100% of fetuses resorbing by E13.5. Cultured embryos from Thy1.2-AMHTg/0 dams had largely normal developmental rates but a small proportion experienced a minor developmental delay relative to embryos from WT dams. However, embryos transferred from WT donor females always failed to survive to term when transferred into Thy1.2-AMHTg/0 dams. Amh and Amhr2 mRNA was detected in the gravid uterus but at very low levels relative to expression in the ovaries. Progesterone and estradiol levels were not significantly different between WT and Thy1.2-AMHTg/0 dams during pregnancy but luteinizing hormone (LH) levels were significantly elevated in Thy1.2-AMHTg/0 dams at E9.5 and E13.5 relative to WT dams. Collectively, these experiments suggest that AMH overexpression does not cause fetal resorption through an effect on oocytes or preimplantation embryo development. The Thy1.2-AMHTg/0 fetal resorption phenotype is nearly identical to that of transgenic LH overexpression models, suggesting that neuroendocrine mechanisms may be involved in the cause of the miscarriage.

Anti-Müllerian hormone overexpression restricts preantral ovarian follicle survival.

Anti-Müllerian hormone (AMH) is an ovarian regulator that affects folliculogenesis. AMH inhibits the developmental activation of the dormant primordial follicles and the oocyte within. In more mature follicles, AMH reduces granulosa cell sensitivity to follicle-stimulating hormone (FSH). We examined the effects of AMH overexpression on the stages of ovarian folliculogenesis, and the development of embryos, with a transgenic mouse that overexpresses human AMH in central nervous system neurons under the control of the mouse Thy1.2 promoter (Thy1.2-AMHTg mice). These mice are severely sub-fertile, despite relatively normal ovulation rates. The embryos of Thy1.2-AMHTg females exhibited delayed preimplantation development and extensive mid-gestation fetal resorption. Young Thy1.2-AMHTg mouse ovaries exhibited only a slight reduction in the rate of primordial follicle activation but large declines in the number of developing follicles surviving past the primary stage. It was expected that Thy1.2-AMHTg mice would retain more primordial follicles as they aged, but at 5 months, their number was significantly reduced relative to wild-type females. These data indicate that moderate elevations in AMH levels can severely restrict reproductive output and the number of developing follicles in the ovary. This evidence suggests that early antral follicles are a target for AMH signaling, which may regulate early follicle survival.

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.

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.