Author ORCID Identifier
https://orcid.org/0000-0003-1851-3632
Date of Graduation
5-2023
Document Type
Dissertation (PhD)
Program Affiliation
Genetics and Epigenetics
Degree Name
Doctor of Philosophy (PhD)
Advisor/Committee Chair
Richard R Behringer, PhD
Committee Member
Michelle C. Barton, PhD
Committee Member
George T. Eisenhoffer Jr., PhD
Committee Member
Rachel K. Miller, PhD
Committee Member
Pierre D. McCrea, PhD
Abstract
Mammals, including humans, develop progenitor tissues for both male and female reproductive tract organs before they fully differentiate into a male or female tract. The progenitor tissue for the male reproductive tract is known as the Wolffian duct (WD), and the progenitor tissue for the female reproductive tract is the Müllerian duct (MD). The WD further differentiates into the vas deferens, epididymis, and seminal vesicle, while the MD differentiates into the oviduct, uterus and upper vagina. An essential step in sex differentiation for males is the regression of the MD. This regression initiates with anti-Müllerian hormone (Amh) transcription in the fetal testes during embryonic development and approaches completion near birth. MD regression requires AMH to bind, after it is released from the fetal testes, to its primary receptor AMHR2 in the MD mesenchyme. This is evidenced by experiments in which male knockout mice that did not express either Amh or Amhr2 did not undergo MD regression. Thus, Amh/Amhr2 are essential for male sex differentiation. A lack of MD regression results in a male simultaneously possessing a male reproductive tract and a female reproductive tract. This condition is known as Persistent Müllerian Duct Syndrome (PMDS), a difference in sex development (DSD). In addition to Amh and Amhr2, several genes have been identified to be essential for proper MD regression.
Based upon previous studies in the field, we have described a gene regulatory network (GRN) for MD regression. Further analysis of our GRN for MD regression illuminated an apparent a lack of known enhancers for genes active in the MD mesenchyme. To identify the MD mesenchyme specific transcriptional enhancers, we utilized bacterial artificial chromosome (BAC) transgenic mice. Osx and Amhr2 are two genes expressed in the MD mesenchyme which are essential for proper regression. Both genes are located on the same chromosome within regions covered by two adjacent BACs. Transgenic mice generated using the two adjacent BACs, were utilized for reporter assays to identify specific regions of enhancer activity. I produced reporter lines with genomic regions 5’ and within the Amhr2 gene, but MD mesenchyme activity was not observed in these regions, implying that the Amhr2 enhancer is not located in these anticipated regions. Transgenic mice produced with the Osx containing BAC also drove lacZ reporter activity in the male MD mesenchyme. I then observed reporter activity in a different BAC transgenic line covering a greatly reduced portion of the Osx containing BAC, which expressed throughout the MD mesenchyme. This study identifies distinct targets for enhancer activity validated by in vivo experimentation, which can inform study to further elucidate the GRN for MD regression.
Keywords
Müllerian duct, Amhr2, Osx, Gene Regulatory Network, Development, Reproductive tract, Embryonic Development, Mouse, Transgenic, Bacterial Artificial Chromosome (BAC), lacZ