The mechanisms underlying the effects of AMH on Müllerian duct regression in male mice

Abstract

Anti-Müllerian hormone (AMH) produced in the developing testis induces the regression of the Müllerian duct, which develops into the oviducts, uterus and upper vagina. In our true hermaphrodite mouse with an ovary on one side and a testis on the other (O/T), the oviduct and uterus are present only on the ovary side, and nothing derived from the Müllerian duct is present on the testis side. Here, we investigate the mechanism underlying the unilateral Müllerian duct regression and the mode of AMH signaling, by performing immunohistology, Western blotting, and organ culture analyses. The histological analysis revealed that during the start of the Müllerian duct regression, the duct in the O/T mice was clearly regressed on the AMH-positive testis side compared to the AMH-negative ovary side. The immunohistochemistry showed a diffuse immunoreaction of AMH in the interstitium surrounding the testis cord and boundary region between the testis and mesonephros, especially in the cranial portion. Western blotting revealed that the amount of AMH in the cranial half of the mesonephros was larger than that in the caudal half. AMH injected into the gonads in organ culture induced the regression of the Müllerian duct via the interstitium of the organ. These results suggest that AMH acts on the Müllerian duct in male mice by exuding into the interstitium surrounding the testis cord and infiltrating through the cranial region from the testis to the mesonephros.

Keywords: AMH (anti-Müllerian hormone); Müllerian duct regression; secretion manner; sexual differentiation; true hermaphrodite.

Fig. 1.

Expression of AMH in sagittal sections of the gonad at 14.5 dpc in a normal male (A), normal female (B) and B6N-XY^POS mouse (C–H). Sertoli cells in the testis cord expressed AMH in the normal testis (A), and the normal ovary was negative for AMH expression (B). In the B6N-XY^POS mice, AMH was detected in a portion of both gonads (C and D), detected only in a portion of the right gonad (E and F) or rarely detected (G and H). Scale bars=100 µm.

Fig. 2.

The phenotypes of the gonads of a normal female (A), a normal male (B) and an O/T mouse (C and D) under a stereomicroscope at 14.5 dpc. The ovary (A) and testis (B) can be distinguished visually, because the testis is thickened and forms testis cords (B), and the ovary is not thickened and does not form testis cords (A). The ovary in the O/T mouse lacked testis cords (C) and was almost indistinguishable from the normal ovary (A). The testis in the same O/T mouse contained cord-like structures (D) and was similar in appearance to a normal testis (B).

Fig. 3.

The transverse sections of the cranial, middle and caudal portions of the ovary (A–C) and testis (D–F) of an O/T mouse at 14.5 dpc. Immunoreactivity of Foxl2, a marker of granulosa cells (A and D), Sox9, a marker of Sertoli cells (B and E) and AMH (C and F) in the gonads of the O/T mouse are shown, with the appearance shown in Fig. 2C and 2D. In the ovary from the O/T mouse, Foxl2 was expressed along the whole length (A) and AMH was negative (C). In the testis from the O/T mouse, Sox9 was detected through the entire length, and the testis cords were clearly visualized especially in the middle portion (E), whereas AMH was detected in some of the Sox9-positive regions (F). Scale bars=100 µm.

Fig. 4.

The expression of AMH at 14.5 dpc in the normal ovary (A), normal testis (B), ovary in O/T mice (C, I and K) and testis in O/T mice (D, J and L), and the respective Müllerian ducts in transverse sections (E–H and M–P). In the normal males, the diameter of the Müllerian ducts was small and the mesenchymal cells surrounding the Müllerian ducts were condensed and formed a swirl pattern (F), whereas those in the normal females did not exhibit these features (E). The mesenchymal cells surrounding Müllerian ducts of the ovary (G, M and O) and testis (H, N and P) in the O/T mice were similar to those of the normal phenotype (E and F), respectively. In addition, the ratio of the Müllerian duct diameter to the width of the mesonephros on the testis side (H, N and P) was smaller than that on the ovary side (G, M and O). The dashed line indicates the basal line of the Müllerian duct epithelium. Scale bars=20 µm.

Fig. 5.

Immunoreaction of AMH secretion in a sagittal section of a normal testis at 13.5 (A and B) or 14.5 dpc (C) and transverse sections of a normal testis at 13.5 dpc (D–F). AMH was strongly detected in Sertoli cells and weakly detected in the interstitium of the testis (A–F). In addition, a diffused immunoreaction was observed in the boundary region between the testis and mesonephros in one part of the cranial region (dashed line in A’–C’ and D). In the sagittal sections, the left side of each panel is the cranial region and the right side is the caudal region. Scale bars=100 µm.

Fig. 6.

A: AMH expression in the cranial/caudal mesonephroi at 13.5 and 14.5 dpc in normal males by Western blotting. Normal female gonad-mesonephros complexes were used as a negative control for AMH, and normal male gonads were used as a positive control for AMH. AMH bands (12.5 kDa) were detected in male gonads and cranial/caudal mesonephros, and GAPDH bands (37 kDa) were visualized in all samples. B: The amounts of AMH in the cranial and caudal mesonephros. The band intensity of AMH was quantified by using the value of the GAPDH bands. When the amount of AMH in the cranial mesonephros is set at 100, the amount in the caudal mesonephros is less than half as large at both 13.5 and 14.5 dpc. F: female gonad-mesonephros complexes; M: male gonads; Cr: cranial half of the male mesonephroi; Ca: caudal half of the male mesonephroi.

Fig. 7.

HE staining for the urogenital ridges of normal male and normal female in an organ culture (A–H) and magnified images focusing on the Müllerian ducts (A’–H’). The Müllerian ducts with AMH (B, D, F and H) are clearly more regressed than those with normal saline (A, C, E and G) at the same height of the mesonephros, respectively. Scale bars=100 µm.