DOI: 10.5061/dryad.jdfn2z3dr
title: Data from: Discovery of prolactin-like in lamprey: Role in osmoregulation and new insight into the evolution of the growth hormone/prolactin family

description: These data were generated to characterize prolactin-like (PRL-L) and
 growth hormone (GH) in sea lamprey (Petromyzon marinus) in
 hormone-receptor binding, tissue distribution, and gene expression
 patterns at various lifecycle stages, in metamorphosis, and in
 acclimations to hyperosmotic and hypoosmotic conditions, as well as to
 detect the hormonal effects on their signaling systems and branchial
 osmoregulation-related ion transporters. For the hormone-receptor binding
 assay, recombinant PRL-L and GH were radioactively labelled with 125iodine
 and applied for determination of binding affinities with recombinant sea
 lamprey GH receptor and PRL receptor expressed by HEK293 cell line.
 Relative abundance of each gene in the cDNA samples synthesized upon the
 RNAs of various tissues of sea lamprey was assessed using quantitative
 real-time PCR (qPCR). Semiquantification of immunoreactive staining in
 percent area of proximal pars distalis was conducted to compare the
 hormone levels in the pituitary gland of sea lamprey under hypoosmotic
 condition.
description: <strong>Ligand-receptor binding assay:
 </strong>A binding assay was set up in a 1.5-mL centrifuge
 tube, containing 35-40 μg
 membrane proteins (PRLR-HEK, GHR-HEK or non-HEK) without or with unlabeled
 ligand (<em>E. coli</em>-produced, refolded rPRL-L or rGH)
 in a series of concentrations, ranging from 8 to 800
 nM. Binding
 in the presence of 1000 nM unlabeled ligand was taken as nonspecific
 binding. Each binding ran in duplicate. Typically, 20,000 cpm of
 radioactive iodine (<sup>125</sup>I) labeled ligand was added
 to each tube in the final step. The binding was performed at 25 °C for 3
 hours in water bath with gently shaking. The membrane proteins were
 collected at the bottom of the tube by centrifugation at 15, 000 g for 15
 min, and tip of the tube containing the protein pellet was cut and
 transferred to gamma tube for determination of bound radioactivity in
 Wizard Detector gamma counter
 (PerkinElmer). <strong>Real-time quantitative PCR (qPCR) analysis: 
 </strong>The qPCR reaction (10 µl total volume)
 included SSoAdvanced Universal SYBR Green Supermix (Bio-Rad Laboratories),
 500 nM primers, and the diluted cDNA or standard DNA of 2 µl for a
 reference gene [elongation factor 1 a (EF1a)] or 4
 µl for other genes. The
 standard DNA of EF1a, PRL-L, GH, GHR and PRLR were dilutions
 of linearized plasmids (the pGEM-T vector with inserts of relevant genes).
 The standards for other genes were serial dilutions of pooled cDNA
 samples. The qPCR reaction was carried out in a 96-well plate using the
 CFX 96 Real Time System
 (Bio-Rad Laboratories). A melting curve analysis was
 performed for each qPCR assay and showed a single peak, which confirmed
 PCR specificity. Quantification cycle
 (Cq) values and log(copy numbers of the standards) were plotted for linear
 regression. Amplification efficiency was calculated on the slope of the
 regression. Copy numbers in the samples were calculated from the standard
 curves and normalized to the reference gene EF1a, which had stable mRNA
 abundance among the treatments and
 tissues. <strong>Immunohistochemistry
 (IHC):</strong> The sea lamprey heads were fixed,
 dehydrated, and embedded in paraffin. The tissue blocks were sectioned
 (thickness of 6 µm) and placed on microscope
 slides. The tissue sections were processed for IHC using
 VECTASTAIN Elite ABC-HRP kit (Vector Laboratories), and the DAB substrate
 kit (Vector Laboratories) was used for staining. The immunoreactive staining was
 semi-quantified with Fiji-ImageJ
 software. <strong>Statistical analysis: 
 </strong>Kolmogorov-Smirnov tests were used to determine normality
 distribution of the treatment groups, and Levene’s median test or F test
 were used to determine equality of variances. One-way ANOVA followed by a Tukey post-hoc test was applied
 for comparisons at various stages of lifecycle and metamorphosis. Two-way
 ANOVA followed by a Bonferroni’s multiple comparisons test were applied
 for analysis. Unpaired t-test with two-tailed <em>P</em>
 values was used for comparison of two groups at the same time, and for
 comparison of the saline-injected FW-control and SW-control in the
 <em>in vivo</em> treatment. Statistical analyses were made
 with GraphPad Prism 9. Differences were considered significant at
 <em>P</em>&lt;0.05 (*), <em>P</em>&lt;0.01
 (**) and <em>P</em>&lt;0.001
 (***).