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  • Author: Rüdiger W Schulz x
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Diego Safian, Jan Bogerd and Rüdiger W Schulz

Follicle-stimulating hormone (Fsh) is a major regulator of spermatogenesis, targeting somatic cell functions in the testes. We reported previously that zebrafish Fsh promoted the differentiation of type A undifferentiated spermatogonia (Aund) by stimulating the production of factors that advance germ cell differentiation, such as androgens, insulin-like peptide 3 (Insl3) and insulin-like growth factor 3 (Igf3). In addition, Fsh also modulated the transcript levels of several other genes, including some belonging to the Wnt signaling pathway. Here, we evaluated if and how Fsh utilizes part of the canonical Wnt pathway to regulate the development of spermatogonia. We quantified the proliferation activity and relative section areas occupied by Aund and type A differentiating (Adiff) spermatogonia and we analyzed the expression of selected genes in response to recombinant proteins and pharmacological inhibitors. We found that from the three downstream mediators of Fsh activity we examined, Igf3, but not 11-ketotestosterone or Insl3, modulated the transcript levels of two β-catenin sensitive genes (cyclinD1 and axin2). Using a zebrafish β-catenin signaling reporter line, we showed that Igf3 activated β-catenin signaling in type A spermatogonia and that this activation did not depend on the release of Wnt ligands. Pharmacological inhibition of the β-catenin or of the phosphoinositide 3-kinase (PI3K) pathways revealed that Igf3 activated β-catenin signaling in a manner involving PI3K to promote the differentiation of Aund to Adiff spermatogonia. This mechanism represents an intriguing example for a pituitary hormone like Fsh using Igf signaling to recruit the evolutionary conserved, local β-catenin signaling pathway to regulate spermatogenesis.

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Nilli Zmora, Yukinori Kazeto, R Sampath Kumar, Rüdiger W Schulz and John M Trant

Due to the lack of purified, native gonadotropins (GtH) for almost all species of fish, we designed a system for the production of recombinant bioactive luteinizing hormone (LH) and follicle stimulating hormone (FSH) using the channel catfish (Ictalurus punctatus) as a model animal. The strategy was to produce the three subunits composing FSH and LH, i.e. the common α-subunit (α-glycoprotein hormone (α-GP)), β-FSH, and β-LH subunit, individually in stable recombinant insect cells (S2) with C-terminal His-tag. This expression system was also used to co-express the α-subunit without the His-tag with each of the His-tagged β-subunits. The recombinant S2 cells were capable of secreting FSH and LH heterodimers and α-GP in abundance; however, expression of the individual β-subunits was much less successful. The recombinant GtHs were partially purified from the cell medium by immobilized metal affinity chromatography to ~15% purity with a yield of 7 and 4 mg per liter of medium for FSH and LH respectively. These recombinant GtHs activated their receptors in vitro, enhanced estrogen secretion, up-regulated several steroidogenic enzyme genes in channel catfish ovarian follicles, and increased androgen secretion from African catfish testis. Interestingly, the FSH and LH dose–response curves for each of these biological activities clearly demonstrate differences in their cellular action and physiological roles. This expression system may be an important development for the production of species-specific GtHs so that FSH- and LH-specific mechanisms of actions within the reproductive endocrine processes can finally be examined with homologous, albeit recombinant, hormones.

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Michelle C Melo, Eva Andersson, Per Gunnar Fjelldal, Jan Bogerd, Luiz R França, Geir Lasse Taranger and Rüdiger W Schulz

The Atlantic salmon shows substantial life cycle plasticity, which also applies to the timing of puberty. While it is characterized by the activation of the brain–pituitary–gonad axis, many morphophysiological aspects of puberty and the influence of environmental conditions, such as water salinity, are not well understood in fish. Here, 12-month-old Atlantic salmon coming from an out-of-season smoltification regime in December were exposed to freshwater (FW) or seawater (SW) at 16 °C to stimulate puberty under a 24-h constant light (LL) or 12 h light:12 h darkness (LD) photoperiod. These four treatment groups (FWLL, SWLL, FWLD, and SWLD) were studied from January to March. Next to 11-ketotestosterone (11-KT) plasma levels, the expression of pituitary genes (gnrhr4, fshb, and lhb) and spermatogenesis was quantified. When spermatogonial proliferation started, fshb mRNA levels increased steeply and began to decrease when spermatogonial mitosis approached completion and most germ cells had reached meiotic or post-meiotic stages. Conversely, lhb mRNA levels increased progressively during spermatogenesis. Most males in all treatment groups matured, but exposure to SW resulted in the strongest stimulation of the onset of spermatogenesis and elevation of pituitary gnrhr4 and fshb mRNA levels. Later on, the LD photoperiod accelerated, irrespective of the salinity, the completion of spermatogenesis, associated with higher lhb mRNA and 11-KT plasma levels than in the LL groups. We find that both salinity and photoperiod modulated different aspects of spermatogenesis, and resulted in a differential activation of pituitary and testis functions; SW stimulating the onset and the shorter photoperiod the completion of spermatogenesis.

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Paul P de Waal, Marcelo C Leal, Ángel García-López, Sergio Liarte, Hugo de Jonge, Nathalie Hinfray, François Brion, Rüdiger W Schulz and Jan Bogerd

Androgens can induce complete spermatogenesis in immature or prepubertal teleost fish. However, many aspects of the role of androgens in adult teleost spermatogenesis have remained elusive. Since oestrogens inhibit androgen synthesis, we used an oestrogen-induced androgen depletion model to identify androgen-dependent stages during adult zebrafish spermatogenesis. Exposure to 10 nM 17β-oestradiol (E2) in vivo at least halved the mass of differentiating germ cells (from type B spermatogonia to spermatids), while type A spermatogonia accumulated. Studies on the cellular dynamics revealed that a reduction of spermatogonial proliferation together with an inhibition of their differentiation to type B spermatogonia were the basis for the oestrogen-mediated disturbance of spermatogenesis. The capacity of the zebrafish testis to produce 11-ketotestosterone as well as the expression of steroidogenesis-related genes was markedly decreased after in vivo oestrogen exposure. Moreover, the androgen-release response to recombinant zebrafish Lh was lost after oestrogen exposure. We conclude that oestrogen exposure caused a state of androgen insufficiency in adult male zebrafish. Since the downregulation of the steroidogenic system as well as the disturbance of spermatogenesis in testicular explants exposed to E2 ex vivo was much less severe than after in vivo exposure, the main inhibitory effect appears to be exerted via feedback inhibition of gonadotropin release. This experimental set-up helped to identify spermatogonial proliferation and their differentiation as androgen targets in adult zebrafish spermatogenesis.