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Data from: Discovery of prolactin-like in lamprey: Role in osmoregulation and new insight into the evolution of the growth hormone/prolactin family

Citation

Gong, Ningping et al. (2022), Data from: Discovery of prolactin-like in lamprey: Role in osmoregulation and new insight into the evolution of the growth hormone/prolactin family, Dryad, Dataset, https://doi.org/10.5061/dryad.jdfn2z3dr

Abstract

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.

Methods

Ligand-receptor binding assay: 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 (E. coli-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 (125I) 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).

Real-time quantitative PCR (qPCR) analysis:  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.

Immunohistochemistry (IHC): 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.

Statistical analysis:  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 P 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 in vivo treatment. Statistical analyses were made with GraphPad Prism 9. Differences were considered significant at P<0.05 (*), P<0.01 (**) and P<0.001 (***).

Funding

National Science Foundation, Award: 1558037