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

Abstract

The current view of the control of spermatogenesis by Fsh and Lh in non-mammalian vertebrates is largely based on studies carried out in teleosts with cystic and cyclic spermatogenesis. Much less is known concerning the specific actions of gonadotropins during semicystic germ cell development, a type of spermatogenesis in which germ cells are released into the tubular lumen where they transform into spermatozoa. In this study, using homologous gonadotropins and a candidate gene approach, for which the genes' testicular cell-type-specific expression was established, we investigated the regulatory effects of Fsh and Lh on gene expression during spermatogenesis in Senegalese sole (Solea senegalensis), a flatfish with asynchronous and semicystic germ cell development. During early spermatogenesis, Fsh and Lh upregulated steroidogenesis-related genes and nuclear steroid receptors, expressed in both somatic and germ cells, through steroid-dependent pathways, although Lh preferentially stimulated the expression of downstream genes involved in androgen and progestin syntheses. In addition, Lh specifically promoted the expression of spermatid-specific genes encoding spermatozoan flagellar proteins through direct interaction with the Lh receptor in these cells. Interestingly, at this spermatogenic stage, Fsh primarily regulated genes encoding Sertoli cell growth factors with potentially antagonistic effects on germ cell proliferation and differentiation through steroid mediation. During late spermatogenesis, fewer genes were regulated by Fsh or Lh, which was associated with a translational and posttranslational downregulation of the Fsh receptor in different testicular compartments. These results reveal that conserved and specialized gonadotropic pathways regulate semicystic spermatogenesis in flatfish, which may spatially adjust cell germ development to maintain a continuous reservoir of spermatids in the testis.

Toward developing recombinant gonadotropin-based hormone therapies for increasing fertility in the flatfish Senegalese sole

“…The histological evaluation of the testis from fish treated with rFsh and a low rLh dose revealed visible changes occurring in the cortical part of the testis, a region that encloses the seminiferous tubules, which often do not have the central lumen formed and usually contain germ cells at all developmental stages (spermatogonia, spermatocytes, and spermatids), but with very few spermatozoa [19]. In Group 1, many spermatogonia were packed within the tubules and some spermatocytes and spermatids could be observed after 4 weeks of treatment (Fig 4A).…”

“…Expression of 17β-hydroxysteroid dehydrogenase ( hsd17b ) in the testis, as a marker of Leydig cells, was carried out by FISH using specific DIG-labeled hsd17b riboprobes [19] following previously described methods [18, 43]. In this case, the green fluorescence was obtained by using Alexa Fluor 488-conjugated goat anti-mouse IgG secondary antibodies (1:500) in TBST for 2 h at room temperature.…”

“…Amounts of the target transcripts were normalized to those of the elongation factor ef1a1 . The sequence of the specific primers for all the genes has been previously described [19], and their efficiency was estimated by the generation of a standard curve for each primer pair from 10-fold serial dilutions (from 10 to 0.0001) of a pool of mixed samples from testes at time 0. For the assays, each sample was determined in duplicate on 384-well plates using the ABI PRISM 7900HT sequence detection system.…”

“…As in mammals, spermatogenesis in the Senegalese sole might be regulated by the pituitary gonadotropins follicle-stimulating (Fsh) and luteinizing (Lh) hormones through the binding to their cognate receptors in the testis, the Fsh receptor (Fshr) and the Lh/chorionic gonadotropin receptor (Lhcgr) [16, 17]. For the investigation of the gonadotropic regulation of testis development in sole, biologically active homologous single-chain recombinant gonadotropins (rFsh and rLh) have recently been produced in Chinese hamster ovary (CHO) cells, which can stimulate the synthesis and release of the major androgen 11-ketotestosterone (11-KT) both in vitro and in vivo [18, 19]. The studies in vitro suggest that Fsh is primarily involved in early spermatogenesis through the activation of the Fshra in both the Sertoli and the steroidogenic Leydig cells, whereas Lh preferentially regulates the later stages of germ cell development and the differentiation of spermatozoa through the Lhcgrba present in Leydig cells as well as in spermatids [18, 20].…”

Reproduction, olfaction and dominance behaviour in Senegalese sole (Solea senegalensis)

“…The current study suggests that a similar role may be played by urine from mature females in the sole; a brief exposure of the olfactory system of mature males to female urine provoked a sustained (at least 30 minutes) increase in plasma [LH]. This is therefore likely to have similar effects on spermatogenesis and milt production in the sole (Chauvigné et al, 2014a;Chauvigné et al, 2014b). Given the difference in urinary odorants between mature and immature males, it is possible that females use this chemical information in mate-choice, similar to MHC-related odorants in sticklebacks (Aeschlimann et al, 2003;Milinski et al, 2005;Milinski et al, 2010) or the anal glands in blennies (Barata et al, 2008).…”