Activities of Amphioxus GH-Like Protein in Osmoregulation: Insight into Origin of Vertebrate GH Family

Abstract

GH is known to play an important role in both growth promotion and osmoregulation in vertebrates. We have shown that amphioxus possesses a single GH-like hormone (GHl) gene encoding a functional protein capable of promoting growth. However, if GHl can mediate osmoregulation remains open. Here, we demonstrated clearly that GHl increased not only the survival rate of amphioxus but also the muscle moisture under high salinity. Moreover, GHl induced the expression of both the ion transporter Na⁺-K⁺-ATPase (NKA) and Na⁺-K⁺-2Cl^- cotransporter (NKCC) in the gill as well as the mediator of GH action IGFl in the hepatic caecum, indicating that GHl fulfills this osmoregulatory activity through the same mechanisms of vertebrate GH. These results together suggest that the osmoregulatory activities of GH had emerged in the basal chordate amphioxus. We also proposed a new model depicting the origin of pituitary hormone family in vertebrates.

Figure 1

The survival rates of amphioxus. The animals were injected with saline, rGHl, rzGH, or rzPRL by intracoelomic injection, followed by culture in seawater with 38‰ salinity (a) or 15‰ salinity (b). Survival was recorded every 24 h. Data are from two independent experiments.

Figure 2

Identification of nka and nkcc. (a) The expression profiles of nka in the different tissues including the gill, hindgut, hepatic caecum, skin, notochord, and muscle. (b) The expression profiles of nkcc in the different tissues. (c) Expression of nka in the gill of amphioxus cultured under 25‰ or 30‰ salinity. (d) Expression of nkcc in the gill of amphioxus cultured under 25‰ or 30‰ salinity. The ef1α was chosen as internal control for normalization. Data were from 3 independent experiments and expressed as mean ± SEM. The symbol ∗ shows p < 0.05.

Figure 3

Induction of nka and nkcc expressions by rGHl. (a) Expression of nka in the gill cultures in response to rGHl or rzGH (concentrations ranging from 10 ng/ml to 1000 ng/ml). (b) Expression of nkcc in the gill cultures in response to rGHl or rzGH (concentrations ranging from 10 ng/ml to 1000 ng/ml). (c) Expression of nka in the gill of amphioxus injected with saline, rGHl, or rzGH. (d) Expression of nkcc in the gill of amphioxus injected with saline, rGHl, or rzGH. (e) NKA activity in the gill of amphioxus injected with saline, rGHl, or rzGH. One unit of enzymatic activity was defined as 1 μmoles ADP released per hour. Data were from 3 independent experiments and expressed as mean ± SEM. The symbol ∗ shows p < 0.05.

Figure 4

Muscle moisture of amphioxus. (a) Muscle moisture of amphioxus cultured under 25‰ or 30‰ salinity. (b) Muscle moisture of amphioxus injected with saline, rGHl, or rzGH. The animals were cultured in natural seawater. Data were from 3 independent experiments and expressed as mean ± SEM. The symbol ∗ shows p < 0.05.

Figure 5

Induction of gh/prllbp expression by rGHl. (a) Expression of gh/prllbp in the cultures of the hepatic caecum in response to rGHl or rzGH (concentrations ranging from 10 ng/ml to 1000 ng/ml). (b) Expression of gh/prllbp in the cultures of the gill in response to rGHl or rzGH (concentrations ranging from 10 ng/ml to 1000 ng/ml). (c) Expression of gh/prllbp in the hepatic caecum of amphioxus injected with saline, rGHl, or rzGH. (d) Expression of gh/prllbp in the gill of amphioxus injected with saline, rGHl, or rzGH. Data were from 3 independent experiments and expressed as mean ± SEM. The symbol ∗ shows p < 0.05.

Figure 6

Induction of igfl expression by rGHl. (a) Expression of igfl in the cultures of the hepatic caecum in response to rGHl or rzGH (concentrations ranging from 10 ng/ml to 1000 ng/ml). (b) Expression of igfl in the cultures of the gill in response to rGHl or rzGH (concentrations ranging from 10 ng/ml to 1000 ng/ml). (c) Expression of gh/prllbp in the hepatic caecum of amphioxus injected with saline, rGHl, or rzGH. (d) Expression of igfl in the gill of amphioxus injected with saline, rGHl, or rzGH. Data were from 3 independent experiments and expressed as mean ± SEM. The symbol ∗ shows p < 0.05.

Figure 7

Expression of ghl/igfl axis genes of amphioxus cultured under 25‰ or 30‰ salinity. (a) Expression of ghl in the Hatschek's pit. (b) Expression of ghl in the gill. (c) Expression of gh/prllbp in the hepatic caecum. (d) Expression of gh/prllbp in the gill. (e) Expression of igfl in the hepatic caecum. (f) Expression of igfl in the gill. Data were from 3 independent experiments and expressed as mean ± SEM. The symbol ∗ shows p < 0.05.

Figure 8

A proposed model for the evolution of GH/PRL family. (a) Ancestral GH/GHR system already emerged in the basal chordate, and PRL/PRLR originated with the advent of Gnathostomata. (b) The ancestral gene, like amphioxus GHl gene, generated two genes by gene duplication early in vertebrate evolution. One of the resulting genes evolved directly into GH gene in modern Gnathostomata, while the other gene evolved into PRL gene, possibly due to adaptation of FW habitats, by genetic innovations and/or mutation after split of agnathan/Gnathostomata.