Diverse Regulation but Conserved Function: SOX9 in Vertebrate Sex Determination

Abstract

Sex determination occurs early during embryogenesis among vertebrates. It involves the differentiation of the bipotential gonad to ovaries or testes by a fascinating diversity of molecular switches. In most mammals, the switch is SRY (sex determining region Y); in other vertebrates it could be one of a variety of genes including Dmrt1 or dmy. Downstream of the switch gene, SOX9 upregulation is a central event in testes development, controlled by gonad-specific enhancers across the 2 Mb SOX9 locus. SOX9 is a 'hub' gene of gonadal development, regulated positively in males and negatively in females. Despite this diversity, SOX9 protein sequence and function among vertebrates remains highly conserved. This article explores the cellular, morphological, and genetic mechanisms initiated by SOX9 for male gonad differentiation.

Keywords: SOX9; evolution; sex determination; transcription factor; transdifferentiation.

Figure 5

The evolutionary history of SOX9 was inferred using the Neighbour-Joining method [98]. The optimal tree is shown. The tree is drawn to scale, with branch lengths in the units of the number of amino acid substitutions per site to infer evolutionary distance, computed using the Poisson correction method [99]. This analysis involved the amino acid sequences of the SOX9 protein from 28 species aligned using NCBI COBALT [90]. All ambiguous positions were removed for each sequence pair (pairwise deletion option). Phylogenetic tree generated using MEGA X software [100].

Figure 1

Morphological changes of the differentiating mouse testis: Between embryonic day (E)8.75 and E9.5, germ cells migrate dorsally towards the developing genital ridge. The testis-determining factor SRY activates Sox9 expression to drive Sertoli cell (blue) differentiation from E10.5. Proliferating Sertoli cells begin to compartmentalize to form testis cords around E12.5. Following Sertoli cell proliferation, fetal Leydig cells (purple) and peritubular myoid cells (green) differentiate. By E15.5, the majority of testis cell types have differentiated, and a distinct mouse testis has formed with typical testis cord structure and interstitial space. The Sertoli cells and peritubular myoid cells secrete various extracellular matrix (ECM) proteins to form the basal lamina, which surrounds the testis cords and maintains their structural integrity. The testis cords are composed of mitotically arrested germ cells enclosed by Sertoli cells, with an outer layer of peritubular myoid cells and extracellular matrix. The interstitial space comprises steroidogenic fetal Leydig cells, mesenchyme, and a prominent blood vasculature.

Figure 2

SOX9 upstream regulatory region towards KCNJ2 gene. Green regions represent the enhancers that control SOX9 expression within the testes. Yellow boxes behind the structure indicate regions commonly associated with PRS (Pierre Robin Syndrome) and craniofacial formation, or CMPD (Campomelic Dysplasia) and chondrogenesis. Enhancers eALDI and hTESCO are calculated from Croft et al., [48]. Sox9up7 is identified from bioinformatic analysis by Ohnesorg et al., 2016 [66].

Figure 3

Human protein structure: including the dimerization domain (DIM), DNA-binding HMG-box, transactivating domain in the middle of the protein (TAM), the proline, glutamine, and alanine rich region (PQA) and the proline, glutamine, and serine rich region (PQS), both required for transactivation. Adapted from Symon & Harley, 2017 [60].

Figure 4

Multiple sequence alignment of SOX9 amino acid sequences across 28 species, compared to human SOX9 reference. Human SOX9 protein along the top of the multiple sequence alignment, indicating the dimerization domain (DIM), DNA-binding HMG-box, transactivating domain in the middle of the protein (TAM), the proline, glutamine, and alanine rich region (PQA) and the proline, glutamine, and serine rich region (PQS). In the multiple sequence alignment, grey indicates identical sequence to human SOX9 at each residue, red indicates different residue, and blue indicates an insertion. Images created through NCBI COBALT (Constraint-based multiple alignment tool) with 28 sequences selected through the NCBI Orthologues feature, NCBI Multiple Alignment Sequence Alignment Viewer, Version 1.19.1 (https://www.ncbi.nlm.nih.gov/gene/6662 (accessed on 16 February 2021)).

Figure 6

Human SOX9 protein, indicating the dimerization domain (DIM), DNA-binding HMG-box, transactivating domain in the middle of the protein (TAM), the proline, glutamine, and alanine rich region (PQA) and the proline, glutamine, and serine rich region (PQS). Dashes underneath indicate missense single base-pair mutations causing a triplet change and subsequent amino acid change, causing various phenotypes [82,101,128,131,134,135,136,137,138,139,140,141,142,143,144,145,146,147,148,149,150,151,152,153,154,155,156].

Figure 7

Molecular mechanisms of mammalian sex determination leading to male or female supporting cell fate.