Evolutionary origin of a functional gonadotropin in the pituitary of the most primitive vertebrate, hagfish

Abstract

Hagfish, which lack both jaws and vertebrae, are considered the most primitive vertebrate known, living or extinct. Hagfish have long been the enigma of vertebrate evolution not only because of their evolutionary position, but also because of our lack of knowledge on fundamental processes. Key elements of the reproductive endocrine system in hagfish have yet to be elucidated. Here, the presence and identity of a functional glycoprotein hormone (GPH) have been elucidated from the brown hagfish Paramyxine atami. The hagfish GPH consists of two subunits, alpha and beta, which are synthesized and colocalized in the same cells of the adenohypophysis. The cellular and transcriptional activities of hagfish GPHalpha and -beta were significantly correlated with the developmental stages of the gonad. The purified native GPH induced the release of gonadal sex steroids in vitro. From our phylogenetic analysis, we propose that ancestral glycoprotein alpha-subunit 2 (GPA2) and beta-subunit 5 (GPB5) gave rise to GPHalpha and GPHbeta of the vertebrate glycoprotein hormone family, respectively. The identified hagfish GPHalpha and -beta subunits appear to be the typical gnathostome GPHalpha and -beta subunits based on the sequence and phylogenetic analyses. We hypothesize that the identity of a single functional GPH of the hagfish, hagfish GTH, provides critical evidence for the existence of a pituitary-gonadal system in the earliest divergent vertebrate that likely evolved from an ancestral, prevertebrate exclusively neuroendocrine mechanism by gradual emergence of a previously undescribed control level, the pituitary, which is not found in the Protochordates.

Fig. 1.

Molecular characteristics of hagfish GPH. Alignment of the amino acid sequences of vertebrate GPHα (A) and GPHβ (B) subunits. Gaps are inserted to minimize alignment of Cys (C) residues among GPHα subunits, and between hagfish GPHβ and lamprey GTHβ, and FSHβ, LHβ, TSHβ subunits of fish species. The two sequences of hagfish GPH are indicated with blue letters and Cys residues that are conserved for all of these sequences are shown with red letters. Putative N-glycosylation sites are indicated by asterisks. The DDBJ/EMBL/GenBank accession numbers of amino acid sequences used for the alignment are as follows: (A) Shark GPHα (AJ310342), Sturgeon GPHα (AJ310343), and Lungfish GPHα (AB050093); (B) Lamprey GTHβ (AY730276), Shark FSHβ (SCA310344), LHβ (AJ310345), Sturgeon FSHβ (AST251658), LHβ (AJ251656), TSHβ (AJ251659), and Lungfish FSHβ (AJ578040), LHβ (AJ578038), and TSHβ (AJ578039).

Fig. 2.

The molecular phylogenetic analysis was constructed using Maximum-likelihood method using 80 deduced amino acid sequences of the α- and β-subunits of FSH, TSH, and LH; GPB5 and GPA2. Sequences were aligned using the Mafft (33) sequence-alignment method in conjunction with Seaview version 4.2 (34). To complete the bootstrap analyses we used GARLI (genetic algorithm for rapid-likelihood inference) (35) and Grid computing (36) through The Lattice Project (37), which includes clusters and desktops in one encompassing system (38). Numbers on the branches indicate bootstrap probabilities following 1,000 replications in constructing the tree. The DDBJ/EMBL/GenBank accession numbers of sequences used for analysis are listed in Table S1. Note that the goldfish TSHβ forms a clade with FSHβs, not with TSHβs of other species.

Fig. 3.

Histochemical characteristics of GPH producing cells in the adenohypophysis of mature hagfish. (A and B) Cellular expression of GPHα gene detected by in situ hybridization with labeled hagfish GPHα riboprobe. (C–F) Immunohistochemical detection of GPHα- and β-producing cells on adjacent pituitary sections detected with specific antisera: (C and D) anti-hagfish GPHα; (E and F) anti-hagfish GPHβ. Note that GPHα-producing cells are well in accordance with GPHβ-producing cells (arrowheads). B, D, and F show magnified views of regions shown by rectangles in A, C, and E, respectively. AH, adenohypophysis; CT, connective tissue; NH, neurohypophysis; III, third ventricle. (Scale bars: A, C, and E, 100 μm; B, D, and F, 20 μm.)

Fig. 4.

Correlation between pituitary GPH activities and gonadal development in hagfish. (A–D) Cellular activities of GPHβ cells in the hagfish pituitary. Note that intense immunoreactions are observed in mature female (C and D), but faint reactions presented in juvenile (A and B). AH, adenohypophysis; CT, connective tissue; NH, neurohypophysis; III, third ventricle. (Scale bars: A and C, 100 μm; B and D, 20 μm.) (E and F) Relative GPHα and GPHβ gene expressions in the pituitary of female (E) and male (F) hagfish. Open bars represent GPHα gene expressions and filled bars represent GPHβ gene expressions. The two GPH mRNA levels were normalized by β-actin mRNA levels. Relative values are expressed as mean ± SE (n = 8–18). Significant differences from the juvenile are indicated by *, P < 0.05; **, P < 0.01; and ***, P < 0.001. Note that two GPH transcripts in both sexes increase in accordance with the developmental stage of the gonad.

Fig. 5.

Biological activity of native GPH from hagfish pituitary with in vitro bioassay. In vitro effects of 8-Br-cAMP (A and B, gray columns) and native GPH (C and D, black columns) on the releases of estradiol-17β (A and C) and testosterone (B and D) from organ-cultured testis. Values are expressed as mean ± SE (n = 6–8). Significant differences from the control groups (0 mM in A and B, 0 μg/mL in C and D) are indicated by *, P < 0.05; **, P < 0.01; and ***, P < 0.001. It is notable that intact GPH stimulates the release of sex steroids from cultured testis in vitro, indicating “gonadotropic action” of hagfish GPH.