The role of Amh in sexual differentiation and gametogenesis in teleost fish

Author(s)

Marcos Antonio de Oliveira

• Document type: Doctoral Thesis
• Press: Jaboticabal. 2019-03-13.
• Institution: Universidade Estadual Paulista (Unesp). Faculdade de Ciências Agrárias e Veterinárias. Jaboticabal
• Defense date: 2019-01-31
• Advisor: Rafael Henrique Nóbrega; Manfred Schartl

Abstract

The decision whether the undifferentiated gonad will develop in a testicle or an ovary is a critical step in the sexual differentiation of vertebrates. This process consists of a complex genetic cascade that culminates in the differentiation of the somatic cells present in the undifferentiated gonad. In this context, the anti-Müllerian hormone is an important effector in this decision. Studies with the medaka hotei fish, in which the Amh type 2 receptor (amhrII) is mutated, show high gonadosomatic index, and sexual reversion from male to female. Based on this information, it is postulated that the Amh regulates the number of germ cells, and directly or indirectly this fact may influence the decision of the bipotential gonad to become a testicle or ovary. Therefore, the objective of the present study was to evaluate the signaling of Amh during sexual differentiation in medaka. For that, studies of loss of gene function were employed, in which the amh gene was deleted through the CRISPR / Cas9 system. We also evaluated the expression of genes that act in the window of sexual differentiation by RT-qPCR. We initially identified two types of mutation in F1 heterozygous (amh -/+) animals. The most interesting mutation was the deletion of 759 base pairs between exons 6 and 7 which comprises part of the Amh_N and TGF-β domains. In the F2 generation, the homozygous amh (-/-) mutants XY males presented 100% of sexual reversion. In addition, the amh (-/-) animals presented gonadal hypertrophy. We did not perform any histological analysis, but considering the literature data, it is suggested that the absence of Amh in our mutants also led to an excessive proliferation of germ cells. Thus, our work shows that Amh is involved in the proliferation of germ cells, being a crucial effector for testicular formation. This result corroborates the temporal expression data of the amh and some key genes of the differentiation process during different stages of development of wild medakas. amh transcripts were highly expressed in males than in females during all stages of development. Its differential expression begins in stage 34 that precedes the window of sexual differentiation in medaka. Interestingly, the expression of other genes involved in the cascade of male sexual differentiation, as dmrt1, amhrII and gsdf was later than amh. This data suggests that Amh acts as an upstream effector of the male differentiation cascade, being responsible, directly or indirectly, for activating genes such as dmrt1 and gsdf. These results are in line with the data found in this work for the amh (-/-) mutant medakas, wherein the absence of Amh, all XY males undergone sex reversal to females. In the absence of Amh, the downstream cascade effectors would not be activated, which would lead to female gonadal differentiation. In general, this work demonstrates that Amh plays a crucial role in the process of sexual differentiation of medaka fish. In addition, Amh acts as an effector of cascade of male sexual differentiation and also regulates the proliferation of germ cells in this species. (AU)

• FAPESP's process: 14/25313-4 - The role of AMH on sex differentiation and gametogenesis of teleost fish
• Grantee: Marcos Antônio de Oliveira
• Support Opportunities: Scholarships in Brazil - Doctorate