Toyokazu Shimotani

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

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, α and β, which are synthesized and colocalized in the same cells of the adenohypophysis. The cellular and transcriptional activities of hagfish GPHα and -β 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 α-subunit 2 (GPA2) and β-subunit 5 (GPB5) gave rise to GPHα and GPHβ of the vertebrate glycoprotein hormone family, respectively. The identified hagfish GPHα and -β subunits appear to be the typical gnathostome GPHα and -β 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.

Gonadotropin-like and adrenocorticotropin-like cells in the pituitary gland of hagfish, Paramyxine atami : Immunohistochemistry in combination with lectin histochemistry

The pituitary system of the hagfish remains an enigma. The present study has aimed to detect possible adenohypophysial hormones in the pituitary gland of the brown hagfish, Paramyxine atami, by means of immunohistochemistry in combination with lectin histochemistry. Rabbit antisera raised against ovine luteinizing hormone (LH)β, proopiomelanocortin (POMC)-related peptides, and the growth hormone/prolactin family of tetrapod and fish species were used, and 25 kinds of lectins were tested. Three different types of adenohypophysial cells were revealed in the pituitary of brown hagfish. The first was stained with both anti-ovine LHβ and several D-mannose-binding lectins, such as Lens culinaris agglutinin and Pisum sativum agglutinin. This cell type predominated in the adenohypophysis in adults with developing gonads and thus appeared to be involved in the regulation of gonadal functions. The second was negative for anti-ovine LHβ but was stained with several N-acetylglucosamine-binding lectins, such as wheat germ agglutinin and Lycopersicon esculentum lectin. This cell type exhibited a weak positive reaction with anti-lamprey adrenocorticotropin (ACTH) and thus appeared to be related to POMC-like cells. The second cell type was found in the adenohypophysis regardless of the developmental state of the gonads. The third cell type was negative for both antisera and lectins. Since this cell type was numerous in juveniles and adults without developing gonads, most cells of this type were probably undifferentiated. These findings suggest that GTH and ACTH are major adenohypophysial hormones in the hagfish.

Effects of estradiol or testosterone treatment on expression of gonadotropin subunit mRNAs and proteins in the pituitary of juvenile brown hagfish, Paramyxine atami

A single functional gonadotropin (GTH) comprising two subunits, α and β, was recently identified in the pituitary of brown hagfish (Paramyxine atami). Little is known about the feedback mechanisms that regulate these GTH subunits by sex steroids in the hagfish. The present study was designed to examine feedback effects of estradiol and testosterone on mRNA expression and protein expression of both GTHα and GTHβ subunits in the pituitary of the juvenile P. atami. Intraperitoneal administration of estradiol over the course of 27days resulted in substantial accumulation of immunoreactive (ir)-GTHα and ir-GTHβ in the adenohypophysis, but testosterone treatments over 27days had no effects on ir-GTHα or ir-GTHβ. Estradiol treatment for 1, 2, 4 or 14days had no effect on GTHα mRNA levels. In contrast, after 2days of estradiol treatment, GTHβ mRNA levels had increased significantly from baseline, while these levels were not affected after treatment over 1, 4, or 14days. After 14days of testosterone treatment, both GTHα and GTHβ mRNA levels had decreased significantly from baseline levels. These results indicate that estradiol acted primarily to suppress the secretion of GTH, and hence resulted in the accumulations of ir-GTHα and ir-GTHβ in the pituitary. On the other hand, testosterone appeared to suppress both the synthesis and the secretion of GTH. Thus, estradiol and testosterone probably differ in their effects on the regulation of pituitary GTH synthesis and secretion in juvenile hagfish.

Relationships between Plasma Concentrations of Sex Steroid Hormones and Gonadal Development in the Brown Hagfish, Paramyxine atami

The relationship between sex steroid hormone profiles in plasma and gonadal function in hagfish is poorly understood. In the present study, plasma concentrations of estradiol, testosterone, and progesterone were examined with respect to gonadal development, sexual differences, and possible function of atretic follicles in the brown hagfish, Paramyxine atami, using a time-resolved fluoroimmunoassay. Plasma concentrations of these three hormones were low in juveniles of both sexes. In females, plasma estradiol showed a significant correlation with ovarian development, with the highest concentrations in late vitellogenic adults. Plasma testosterone and progesterone also increased significantly in non-vitellogenic adult females; however, plasma testosterone showed no significant differences among adult females at different ovarian developments, while plasma progesterone was significantly lower in late vitellogenic adults than it was in non-vitellogenic adults. Vitellogenic females that possessed atretic follicles showed significantly lower concentrations of all three hormones than females that only possessed normal follicles. In males, no significant differences were found in plasma estradiol or testosterone levels among groups of different developmental stages of the testis, while plasma progesterone showed a clear inverse relationship with testicular development. Thus, differences were found in plasma sex steroid hormone profiles between male and female P. atami. Moreover, plasma estradiol showed a significant correlation with ovarian development, which suggests that estradiol is involved in the regulation of ovarian development. The present study also revealed that steroid hormone production was strongly suppressed in females that possessed atretic follicles in their ovaries.

Scoliorhapis dianthus, a new species of sea cucumber (Apodida: Chiridotidae) from the Sea of Japan

Abstract Scoliorhapis dianthus, new species, was collected from depths of 0.20–1 m in the Sea of Japan. It is distinctive in having ten tentacles with six pairs of digits of equal size. Ossicles in the body wall have a sigmoid shape and average 75 ± 5 μm in length. An identification key to all species of the genus is provided.

Immunohistochemical detection of gonadotropin-like material in the hagfish pituitary, correlated with their gonadal conditions

In the present study, immunoreactive gonadotropin (GTH)-like material was examined in the hagfish pituitary, in correlation with their gonadal conditions. For this purpose, three species of hagfish, Myxine glutinosa, Eptatretus burgeri and Paramyxine atami, were used. In all species, GTH-like cells were observed in the adenohypophysis. They were stained with several antibodies to LH-related GTHs, such as ovine LHβ, human LHβ, bullfrog LH, and salmon GTHIIβ. In the subsequent study, the relationship between the amount of GTH-like material and gonadal conditions was examined in Paramyxine atami. The adenohypophysis of adult animals exhibited well-developing features, and about half of the adenohypophysial cells were stained intensely with anti-ovine LHβ. On the other hand, the adenohypophysis of juveniles exhibited less developed features with few or no cells stained with anti-ovine LHβ. These results suggest that a GTH-gonadal system is truly present in the hagfish.

Distribution of Immunoreactivities for Adenohypophysial Hormones in the Pituitary Gland of the Polypteriform Fish, Polypterus endlicheri

Abstract Polypteriform fish constitutes the most primitive living descendent of the ancient bony fish. In polypteriform fish, only proopiomelanocortin (POMC) has been identified so far in the adenohypophysis, which is surprising in view of their evolutionary importance. In the present study, distribution of immunore-active adenohypophysial hormones was examined in juvenile individuals of Polypterus endlicheri. Antisera to tetrapod and fish adenohypophysial hormones were used as immunostaining probes. Adrenocorticotropin (ACTH)-like cells were detected by antisera to salmon POMC N-terminal peptide, porcine ACTH and mammalian α-melanotropin (MSH), and were distributed in the rostral pars distalis in close proximity to the hypophysial duct. MSH-like cells were found in the pars intermedia, and were stained by anti-salmon N-Ac-β-endorphin II as well as anti-mammalian α-MSH and anti-salmon POMC-N terminal peptide. Prolactin (PRL)-like cells were detected only after application of anti-sturgeon PRL, and were distributed in the rostral pars distalis, where PRL-positive material was found in columnar mucinous cells lining the diverticuli of the hypophysial duct. Growth hormone (GH)-like cells were stained with antisera to sturgeon GH, human GH, salmon GH and blue shark GH, and were distributed in the proximal pars distalis. Somatolactin (SL)-like cells were stained with anti-salmon SL, and were distributed in the pars intermedia. Two types of glycoprotein hormone-positive cells were detected in the proximal pars distalis. Although both types of cells were stained with several antisera to glycoprotein hormones, such as sturgeon LHβ and salmon LHβ, it was difficult to know which types of cells produce LH, FSH, or TSH. Thus, the present study revealed seven types of adenohypophysial hormone-like cells in the Polypterus pituitary gland, which may provide the morphological basis for better understanding on evolution of the pituitary gland and the adenohypophysial hormones in vertebrates.