Yuji Mishina

Requirement of Bmpr1a for Müllerian duct regression during male sexual development.

Elimination of the developing female reproductive tract in male fetuses is an essential step in mammalian sexual differentiation. In males, the fetal testis produces the transforming growth factor β (TGF-β) family member anti-Müllerian hormone (Amh, also known as Müllerian-inhibiting substance (Mis)), which causes regression of the Müllerian ducts, the primordia of the oviducts, uterus and upper vagina. Amh induces regression by binding to a specific type II receptor (Amhr2) expressed in the mesenchyme surrounding the ductal epithelium. Mutations in AMH or AMHR2 in humans and mice disrupt signaling, producing male pseudohermaphrodites that possess oviducts and uteri. The type I receptor and Smad proteins that are required in vivo for Müllerian duct regression have not yet been identified. Here we show that targeted disruption of the widely expressed type I bone morphogenetic protein (BMP) receptor Bmpr1a (also known as Alk3) in the mesenchymal cells of the Müllerian ducts leads to retention of oviducts and uteri in males. These results identify Bmpr1a as a type I receptor for Amh-induced regression of Müllerian ducts. Because Bmpr1a is evolutionarily conserved, these findings indicate that a component of the BMP signaling pathway has been co-opted during evolution for male sexual development in amniotes.

High specificity of Müllerian-inhibiting substance signaling in vivo

Female transgenic mice that ectopically express high levels of human Mullerian-inhibiting substance (hMIS) under the control of the mouse metallothionein (MT) promoter lack a uterus, oviducts, and ovaries. The loss of the uterus and oviducts is consistent with the known activities for MIS. However, it is not clear if the loss of the ovaries in these transgenic females is caused by interactions of MIS with its normal receptor signaling pathway or by abnormal interactions with other transforming growth factor-beta (TGF-beta) super family receptor signaling pathways. To address this question, female mice carrying the MT-hMIS transgene that were also homozygous for a targeted deletion of the MIS type II receptor gene were generated. Although these females had high levels of circulating hMIS, they had normal reproductive tracts and ovaries with germ cells. In addition, these females were able to become pregnant and gave birth to pups. These findings demonstrate that all of the abnormalities of the reproductive system that are found in female transgenic mice that ectopically express high levels of hMIS are caused by signaling through the MIS type II receptor. These in vivo data demonstrate a high specificity for MIS and its receptor.

Analysis of the role of Amh and Fra1 in the Sry regulatory pathway

Sry is the Y‐chromosomal gene responsible for initiating the pathway of male development in mammals, presumably by regulating downstream target genes. As a basis for examining the role played by Sry and other candidate sex‐determining genes, we have used reverse‐transcriptase PCR to produce a precise temporal profile of expression of Sry in the developing gonad. Sry expression first occurs at 10.5 days postcoitum (dpc) and is upregulated to reach a maximum level from 11.25 to 12 dpc. Expression is then steadily downregulated from 12.25 to 13.25 dpc and is completely extinguished by 13.5 dpc. Two genes implicated in sexual development are Amh, encoding anti‐müllerian hormone (AMH), and Fra1, encoding Fos‐related antigen‐1. Expression profiles of Amh and Sry in fetal gonads indicate a possible role for AMH in the downregulation of Sry expression. We show, however, that the timing of Sry downregulation is unaffected in Amh‐deficient mice, excluding a role for AMH as a negative regulator of Sry. Further, we have examined the possibility that SRY may activate Fra1 during testis determination by analysing the expression of Fra1 in mouse fetal gonads. Fra1 is not expressed at any stage at or around the critical period of sex determination when Sry transcripts are present, thus excluding a role for Fra1 in sex determination and differentiation.

The in Vivo Function of Müllerian-Inhibiting Substance During Mammalian Sexual Development

In mammals, both XX and XY individuals develop one pair of undifferentiated gonads and two pairs of genital ducts, the Mullerian ducts and the Wolffian ducts, associated with the mesonephroi during development (Fig. 1). The undifferentiated gonads are bipotent, and will give rise to either testes or ovaries depending on the sex-chromosome genotype. The Mullerian ducts have the potential to give rise to female reproductive organs, including the uterus, oviducts, and upper portion of the vagina. The Wolffian ducts are the primordia of male reproductive organs, which are the vas deferens, epididymis, and seminal vesicles. Therefore, regardless of sex-chromosome genotype, each individual has the potential to develop male and female reproductive systems. The presence of the Y chromosome determines that an individual becomes a male because of a gene termed Sry. There must be a mechanism to select only one duct system, depending on the presence or absence of Sry. Two gonadal hormones—Mullerianinhibiting substance (MIS)—and testosterone, play essential roles during this selection process (Fig. 1) (1–3) This chapter, examines the results of in vivo approaches using transgenic and targeted mutant mice to study MIS function.