PMID- 30576646 OWN - NLM STAT- MEDLINE DCOM- 20190219 LR - 20190320 IS - 1095-6840 (Electronic) IS - 0016-6480 (Linking) VI - 272 DP - 2019 Feb 1 TI - Medaka follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh): Developmental profiles of pituitary protein and gene expression levels. PG - 93-108 LID - S0016-6480(18)30377-0 [pii] LID - 10.1016/j.ygcen.2018.12.006 [doi] AB - The two gonadotropins follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh) are of particular importance within the hypothalamic-pituitary-gonadal (HPG) axis of vertebrates. In the current study, we demonstrate the production and validation of Japanese medaka (Oryzias latipes) recombinant (md) gonadotropins Fshbeta (mdFshbeta), Lhbeta (mdLhbeta), Fshbetaalpha (mdFshbetaalpha), and Lhbetaalpha (mdLhbetaalpha) by Pichia pastoris, the generation of specific rabbit antibodies against their respective beta subunits, and their use within the development and validation of competitive enzyme-linked immunosorbent assays (ELISAs) for quantification of medaka Fsh and Lh. mdFsh and mdLh were produced as single-chain polypeptides by linking the alpha subunit with mdFshbeta or mdLhbeta mature protein coding sequences to produce a "tethered" polypeptide with the beta-chain at the N-terminal and the alpha-chain at the C-terminal. The specificity of the antibodies raised against mdFshbeta and mdLhbeta was determined by immunofluorescence (IF) for Fshbeta and Lhbeta on medaka pituitary tissue, while comparison with fluorescence in situ hybridization (FISH) for fshb and lhb mRNA was used for validation. Competitive ELISAs were developed using antibodies against mdFshbeta or mdLhbeta, and the tethered proteins mdFshbetaalpha or mdLhbetaalpha for standard curves. The standard curve for the Fsh ELISA ranged from 97.6 pg/ml to 50 ng/ml, and for the Lh ELISA from 12.21 pg/ml to 6.25 ng/ml. The sensitivity of the assays for Fsh and Lh was 44.7 and 70.8 pg/ml, respectively. A profile of pituitary protein levels of medaka Fsh and Lh comparing juveniles with adults showed significant increase of protein amount from juvenile group (body length from 12 mm to 16.5 mm) to adult group (body length from 21 mm to 26.5 mm) for both hormones in male medaka. Comparing these data to a developmental profile of pituitary mRNA expression of medaka fshb and lhb, the mRNA expression of lhb also increased during male maturation and a linear regression analysis revealed a significant increase of lhb expression with increased body length that proposes a linear model. However, fshb mRNA expression did not change significantly during male development and therefore was not correlated with body length. In summary, we have developed and validated homologous ELISA assays for medaka Fsh and Lh based on proteins produced in P. pastoris, assays that will be used to study the functions and regulations of Fsh and Lh in more detail. CI - Copyright (c) 2018 Elsevier Inc. All rights reserved. FAU - Burow, Susann AU - Burow S AD - Department of Basic Sciences and Aquatic Medicine, Faculty of Veterinary Medicine, Norwegian University of Life Sciences, 0454 Oslo, Norway. FAU - Fontaine, Romain AU - Fontaine R AD - Department of Basic Sciences and Aquatic Medicine, Faculty of Veterinary Medicine, Norwegian University of Life Sciences, 0454 Oslo, Norway. FAU - von Krogh, Kristine AU - von Krogh K AD - Department of Basic Sciences and Aquatic Medicine, Faculty of Veterinary Medicine, Norwegian University of Life Sciences, 0454 Oslo, Norway. FAU - Mayer, Ian AU - Mayer I AD - Department of Production Animal Clinical Sciences, Faculty of Veterinary Medicine, Norwegian University of Life Sciences, 0454 Oslo, Norway. FAU - Nourizadeh-Lillabadi, Rasoul AU - Nourizadeh-Lillabadi R AD - Department of Basic Sciences and Aquatic Medicine, Faculty of Veterinary Medicine, Norwegian University of Life Sciences, 0454 Oslo, Norway. FAU - Hollander-Cohen, Lian AU - Hollander-Cohen L AD - Department of Animal Sciences, Faculty of Agriculture, Food and Environment, The Hebrew University, Rehovot 76100, Israel. FAU - Cohen, Yaron AU - Cohen Y AD - Department of Animal Sciences, Faculty of Agriculture, Food and Environment, The Hebrew University, Rehovot 76100, Israel. FAU - Shpilman, Michal AU - Shpilman M AD - Department of Animal Sciences, Faculty of Agriculture, Food and Environment, The Hebrew University, Rehovot 76100, Israel. FAU - Levavi-Sivan, Berta AU - Levavi-Sivan B AD - Department of Animal Sciences, Faculty of Agriculture, Food and Environment, The Hebrew University, Rehovot 76100, Israel. FAU - Weltzien, Finn-Arne AU - Weltzien FA AD - Department of Basic Sciences and Aquatic Medicine, Faculty of Veterinary Medicine, Norwegian University of Life Sciences, 0454 Oslo, Norway. Electronic address: finn-arne.weltzien@nmbu.no. LA - eng PT - Journal Article PT - Research Support, Non-U.S. Gov't DEP - 20181218 PL - United States TA - Gen Comp Endocrinol JT - General and comparative endocrinology JID - 0370735 RN - 0 (Recombinant Proteins) RN - 9002-67-9 (Luteinizing Hormone) RN - 9002-68-0 (Follicle Stimulating Hormone) SB - IM MH - Animals MH - Enzyme-Linked Immunosorbent Assay/*methods MH - Female MH - Follicle Stimulating Hormone/*metabolism MH - Gene Expression/*genetics MH - Luteinizing Hormone/*metabolism MH - Male MH - Rabbits MH - Recombinant Proteins/*metabolism OTO - NOTNLM OT - Enzyme-linked immunosorbent assay OT - Follicle-stimulating hormone OT - Gonadotropins OT - Luteinizing hormone OT - Oryzias latipes OT - Recombinant proteins EDAT- 2018/12/24 06:00 MHDA- 2019/03/21 06:00 CRDT- 2018/12/22 06:00 PHST- 2018/07/09 00:00 [received] PHST- 2018/11/30 00:00 [revised] PHST- 2018/12/16 00:00 [accepted] PHST- 2018/12/24 06:00 [pubmed] PHST- 2019/03/21 06:00 [medline] PHST- 2018/12/22 06:00 [entrez] AID - S0016-6480(18)30377-0 [pii] AID - 10.1016/j.ygcen.2018.12.006 [doi] PST - ppublish SO - Gen Comp Endocrinol. 2019 Feb 1;272:93-108. doi: 10.1016/j.ygcen.2018.12.006. Epub 2018 Dec 18.