Functional and Evolutionary Analysis of Flatfish Gonadotropin Receptors Reveals Cladal- and Lineage-Level Divergence of the Teleost Glycoprotein Receptor Family1

Abstract

Pituitary gonadotropins, follicle-stimulating hormone (FSH), and luteinizing hormone (LH) act via their cognate glycoprotein hormone receptors (GpHRs), FSH receptor (FSHR), and LH/choriogonadotropin receptor (LHCGR) to regulate gonad physiology. Here, we show that the flatfish Senegalese sole (Solea senegalensis) expresses functional isoforms of fshr and lhcgr, but the genomic origin, ligand activation, and tissue distribution of the receptor transcripts are more complex than expected. By integrating the molecular phylogeny of GpHRs with the syntenic loci of vertebrate orthologs, and by subsequently characterizing the physical maps with the phylogeny of flanking genes, we found that vertebrate GpHRs have undergone a divergent evolution. In Teleostei, fshr genes have a common descent and can be classified as fshra, whereas lhcgrb genes exist as alternatively coded genes even in closely related species. Structural analyses of the receptors revealed that Fshra has an elongated ligand-binding domain, containing an extra leucine-rich repeat that specifically arose in the Acanthomorpha because of exon duplication. Ectopic expression in Xenopus laevis oocytes demonstrated that sole Fshra responded to piscine Fsh and Lh, whereas Lhcgrba was preferentially activated by its cognate hormone. The expression pattern of sole fshra and lhcgrba in gonads during the reproductive cycle was consistent with earlier observations wherein Fshra regulates ovarian growth and spermatogenesis and Lhcgrb triggers gamete maturation, respectively. However, contrary to observations in other teleosts, fshra was localized exclusively in Sertoli cells of the testis, whereas lhcgrba was expressed in Leydig cells as well as in spermatids. These results demonstrate the presence of alternatively coded lhcgr isoforms (lhcgrba and lhcgrbb) in teleosts and suggest a role of the lhcgrba receptor in the differentiation of spermatids into spermatozoa in Senegalese sole.

Fsh and Lh direct conserved and specific pathways during flatfish semicystic spermatogenesis

“…The testes were removed and weighed in order to determine the gonadosomatic index (GSI; testis weight/ body weight!100). Different biopsies were deep-frozen in liquid nitrogen and stored at K80 8C, used for in vitro culture, or processed for histology, immunohistochemistry, and in situ hybridization (ISH) as described previously (Chauvigné et al 2010). The testes were classified as stages I-II (early and mid spermatogenesis respectively) or stage III (late spermatogenesis) (García-Ló pez et al 2006a) after histological analysis carried out as described previously (Agulleiro et al 2007).…”

“…Total RNA extraction from the testis, cDNA synthesis, and RT-PCR were carried out as described previously (Chauvigné et al 2010). To isolate partial cDNAs encoding Senegalese sole structural maintenance of chromosomes protein 1b (smc1b), cytochrome P450 19a1 (cyp19a1, aromatase), nuclear androgen receptor isoform alpha (ara), nuclear androgen receptor isoform beta (arb), gonadal soma-derived factor variant (gsdf), and estrogen receptor beta (erb), oligonucleotide primers were designed from the expressed sequence tags (ESTs) available in GenBank or in the Solea-DB database v2.0 (http:// www.juntadeandalucia.es/agriculturaypesca/ifapa/soleadb_ ifapa/) (Supplementary Table 1, see section on supplementary data given at the end of this article).…”

“…Immunostaining for sole gonadotropin receptors, Fshr form a (Fshra) and Lhcgr form ba (Lhcgrba) (Chauvigné et al 2010), were carried out using characterized receptor-specific affinity-purified antibodies as described previously (Chauvigné et al 2012). For immunofluorescence, sections were counterstained with 4 0 ,6-diamidino-2-phenylindole dihydrochloride (DAPI; 1:5000; Sigma-Aldrich) for 3 min before mounting.…”

“…Transcript levels in each replicate testicular explant before (time zero) and after hormone treatment in vitro were determined by real-time quantitative RT-PCR (qRT-PCR) as described previously (Chauvigné et al 2010, Zapater et al 2012. The specific primers used are listed in Supplementary Table 1.…”

“…The gonadotropic regulation of the expression of the 20 selected transcripts, plus fshra and lhcgrba, as well as radial spoke head 1 homolog (rsph1) and StAR protein-like (starl) previously characterized (Chauvigné et al 2010, 2014, was investigated in testes explants at stages I-II and III using rFsh and rLh. The results of the qRT-PCR experiments showed that the levels of all transcripts changed in response to rFsh and/or rLh except for fshra and piwil2 (Supplementary Figure 2, see section on supplementary data given at the end of this article).…”