Adult male zebrafish between 4 and 12 months of age were used in this study. Six to eight animals were used per experiment. All experiments carried out in this study followed the Dutch National regulations for animal care and use in experimentation, and the experimental protocols have been submitted to, and were approved by, the Utrecht University Experimental Animal Committee (2015.I.857.013 and AVD108002015333).

To study the regulation of igfbp transcript levels, adult zebrafish testes were dissected for tissue culture experiments using a previously described system (26), in which one testis was incubated under control conditions, the other testis under experimental conditions.

Zebrafish testes were incubated for 5 days under basal conditions or in the presence of recombinant zebrafish Fsh (25, 50, 100, or 1,000 ng/mL) (6). In a second series of experiments, zebrafish testes were incubated under basal conditions or in the presence of Fsh (100 ng/mL) for 1, 3, 5, or 7 days.

To study the effect of Igf3 on igfbp expression, zebrafish testes were incubated in the absence or presence of recombinant zebrafish Igf3 (100 ng/mL) (9) for 3 or 7 days. Based on the slow effect of Igf3 on igfbp expression, testes were incubated for 5 or 7 days in the presence of Fsh (100 ng/mL) with or without NVP-AEW541 [10 µM; Selleckchem (25)], an inhibitor of Igf1 receptors; incubation media for the control and experimental groups contained the same final concentration of dimethyl sulfoxide (0.1%).

In a different set of experiments, zebrafish testes were incubated under basal conditions or in the presence of 11-KT [200 nM in ethanol (0.01%); Sigma] for 3 or 7 days (10), or in the presence of 11-KT (200 nM) with or without 10 µM NVP-AEW541 for 7 days. The reason to carry out this experiment was the previously reported, slight stimulatory effect of 11-KT on igf3 transcript levels (9). At the end of the incubation period, testis tissue was snap-frozen in liquid nitrogen and stored at −80°C until RNA extraction.

We have reported previously that 100 ng/mL Igf3 stimulated the proliferation of type A spermatogonia and increased the transcript levels of marker genes associated with spermatogonial differentiation (9). To further study Igf3 effects and the role of Igfbps on zebrafish spermatogenesis, we incubated testes under basal conditions or in the presence of 25 or 100 ng/mL Igf3 for 3 days. The lower dose was expected to have no/little effect on its own, based on a previous dose-response study (9). In addition, zebrafish testes were incubated for 3 days in the presence of 25 ng/mL Igf3 with or without NBI-31772 (10 µM; Sigma-Aldrich), an Igfbp inhibitor (27, 28); NBI-31772 alone has no effects on spermatogenesis or expression of germ cells markers (23).

The Fsh time-course experiment showed that two igfbp mRNAs were upregulated with a delay of at least 3 days, so that two experiments were designed to preferentially study these two igfbps upregulated by Fsh. To this end, zebrafish testes were incubated for 7 days with 100 ng Fsh/mL in both control and experimental groups. During the last 4 days, the experimental group was in addition exposed to 10 µM NBI-31772. In the second experiment, testes of the control group were incubated in the presence of 100 ng/mL Fsh and 10 µM NBI-31772 for 7 days, whereas in the experimental group, testes were incubated for the first 3 days under the same conditions but for the remaining 4 days, the medium contained only Fsh but no NBI-31772. At the end of the 7 days long incubation period, testis tissue was fixed for morphological analyses.

The production of biologically active steroids was blocked by including trilostane (25 µg/mL; Chemos), an inhibitor of 3β-hydroxysteroid dehydrogenase activity, in all experiment with Fsh, a potent steroidogenic hormone in fish (6).

The relative transcript levels of igfbps, germ cells markers, and other genes of interest (Table 1) were analyzed by real-time, quantitative polymerase chain reaction (qPCR) assays. The expression of igfbp3 was analyzed using a commercial available TaqMan gene expression assay (Applied Biosystems, Cat# 4351372).

Total RNA was isolated from the tissue using an RNAqueous Micro kit (Ambion), according to the manufacturer’s protocol. cDNA synthesis from total RNA and quantification of transcript levels were carried out as described previously (31). In brief, 2 µg of total RNA were reverse transcribed using 250 U of Supercript II RNase-reverse transcriptase (Life Technologies). qPCR were performed by using 2× SYBER Green assay mix (Applied Biosystems), specific qPCR primers (900 nM) and 5 µL of cDNA in a total volume of 20 µL. The quantification cycle (Cq) values were determined in a Step One Plus Real-Time PCR System (Applied Biosystems) using default settings. The relative amounts of mRNA in the cDNA samples were calculated using the arithmetic comparative method (ΔΔCt method), according to Bogerd et al. (31). Expression of the ribosomal RNA 18S (18S) transcript was stable (Figure S1 in Supplementary Material). 18S expression served as reference transcript and was analyzed using a commercially available TaqMan gene expression assay (Applied Biosystems). All results were expressed as fold change with respect to the control group.

To quantify the proliferation activity of A_und, A_diff, and B spermatogonia, 100 µg/mL of the proliferation marker 5-bromo-2′-deoxyuridine (BrdU; Sigma-Aldrich) was added to the tissue culture medium during the last 6 h of the incubation period. After fixation in methacarn (60% [v/v] absolute ethanol, 30% chloroform, and 10% acetic acid), the samples were dehydrated in graded ethanol (70, 96, and 100%), embedded in Technovit 7100 (Heraeus Kulzer) and sectioned at a thickness of 4 µm. To determine the proliferation activity, one set of sections was used to localize BrdU as described previously (26). The mitotic index was determined by analyzing 100 spermatogenic cysts (A_diff and B spermatogonia) or 100 A_und cells, discriminating between BrdU positive and negative cysts/cells, respectively.

To quantify the proportion of section area occupied by the different spermatogonial cell types, another set of sections was stained with toluidine blue and 10 randomly chosen, non-overlapping fields were photographed at ×400 magnification with a digital camera. The images were analyzed quantitatively based on the number of points counted over the germ cell types investigated (A_und, A_diff, and B spermatogonia), using the ImageJ freeware (National Institutes of Health, Bethesda, MD, USA, http://rsbweb.nih.gov/ij) with a 540-point grid.

Statistical analyses were carried out using the GraphPad Prism 5 software package (San Diego, CA, USA). Since our tissue culture system compares the two testes of a given fish incubated under control versus experimental conditions, we applied Student’s t-test for paired observation to estimate statistical significance. All data are presented as fold of basal (mean ± SEM). The individual data before normalization are provided as supplemental figures (Figure S2 in Supplementary Material). To achieve homogeneity of variance, data were log transformed when appropriate.

To study the regulation of igfbp transcript levels by Fsh, dose-response and time-course experiments were carried out. igfbp1b, 2b, 5a, and 6b transcript levels were not regulated by Fsh at any concentration or time evaluated in the present study (data not shown). From the five remaining transcripts, three (igfbp1a, 3, and 6a) were down- and two (igfbp2a and 5b) were upregulated by Fsh. The Fsh dose-response experiment was carried out using 5 days of incubation. The downregulated igfbp1a, 3, and 6a responded to the two higher concentrations of 100 and 1,000 ng/mL Fsh (Figures 1A–C). This was also the case as regards the upregulated igfbp5b, while the second upregulated igfbp2a responded to all Fsh concentrations used (Figures 1D,E).

Based on these data, we used 100 ng Fsh/mL in the time-course experiment. We found that igfbp1a expression was quickly downregulated after 1 day, while igfbp3 and 6a transcript levels had decreased significantly after 3 days of incubation (Figures 2A–C). Upregulation of igfbp2a and 5b required more time and became significant after 5 days of tissue culture (Figures 2D,E).

Since Fsh increased Igf3 release, we studied if (some of) the changes induced by Fsh are mediated by Igf3. As shown in Figure 2, Fsh modulated the transcript levels of some igfbps after a short (1 or 3 days for igfbp1a, igfbp3, and igfbp6a) and others after a longer (5 or 7 days for igfbp2a and igfbp5b) period of incubation. Therefore, testes were incubated in the presence of recombinant zebrafish Igf3 (100 ng/mL) for 3 or 7 days. Effects of Igf3 on igfbp expression were evident after 7 days of incubation only, when we found significantly decreased transcript levels of igfbp1a, 3, and 6a (Figure 3A); igfbp2a and igfbp5b expression did not change in response to Igf3 after 3 or 7 days of incubation (data not shown). To directly examine if the slow Igf3 effects on igfbp transcript levels are downstream of Fsh, we incubated testis tissue for 5 or 7 days with Fsh in the absence or presence of a pharmacological Igf receptor inhibitor. While igfbp transcript levels did not change after 5 days (data not shown), all three transcripts (igfbp1a, igfbp3, and igfbp6a) increased in response to the Igf receptor inhibitor after 7 days of incubation (Figure 3B). This data shows that the late decrease of igfbp1a, -3, and -6a transcripts specifically depends on Fsh-triggered, Igf3-dependent signaling.

In fish, other mediators of Fsh effects are androgens, considering the strong steroidogenic potency of zebrafish Fsh, for example (6). While steroid-mediated effects were neutralized by including trilostane in the incubation medium in experiments with Fsh, the next set of experiments aimed at investigating potential androgen effects. 11-KT (200 nM) upregulated the expression of igfbp2a and igfbp5b, while igfbp6a was downregulated after 7 days but not after 3 days of incubation (Figure 3C). Igfbp1a, 3, and 6a transcript levels did not response to 11-KT after 3 or 7 days of incubation (data not shown). Since igf3 expression also responds to 11-KT (9) and since igfbp transcript levels responded to 11-KT after 7 days, the experiment was repeated in the presence of the Igf receptor inhibitor for 7 days. However, the effects of 11-KT on the transcript levels of igfbps did not change in the additional presence of the Igf receptor inhibitor (data not shown), suggesting that the 11-KT effects were not mediated by Igf3.

Exposure to 25 ng/mL Igf3 did not modulate the mitotic index and proportion of spermatogonia after 3 days of incubation (Figures 4A,C,D), different from a higher concentration of Igf3 (100 ng/mL) that increased the mitotic indices of all spermatogonia (Figures 4A,E). The proportion of area occupied by A_und was reduced, while the one for A_diff and B spermatogonia increased in the presence of 100 ng/mL of Igf3 for 3 days (Figure 4B).

In order to better understand Igf3 signaling and its modulation by Igfbps in regulating spermatogenesis, we used NBI-31772, an inhibitor of Igf-Igfbp interaction. We asked if a low concentration of Igf3 not eliciting effects by itself (25 ng/mL; see Figure 4), does modulate BrdU incorporation and the proportion of section area occupied by spermatogonia when NBI-31772 was present as well. Indeed, the mitotic indices of all types of spermatogonia increased after 3 days of incubation in response to Igf3 and NBI-31772 (Figures 5A,C); also, the proportion of section surface area occupied by type A_diff and B spermatogonia increased, while the one for A_und decreased (Figure 5B). In parallel experiments, we quantified the transcript levels of selected genes to complement morphological with molecular data. Considering germ cell marker transcripts, foxa2 (a potential marker for undifferentiated spermatogonia) transcript levels decreased, whereas dazl [expressed by B spermatogonia and primary spermatocytes (30)] and piwil2 [expressed by all germ cells except A_und and spermatozoa (29)] expression was upregulated in the presence of Igf3 and NBI-31772 (Figure 5D), suggesting that the total number of germ cells has increased, associated with a shift from undifferentiated spermatogonia to B spermatogonia and spermatocytes. Transcript levels of igf1rb were also upregulated significantly (Figure 5D).

Previous work has shown that blocking Igf binding to Igfbps by NBI-31772 during 4 days of incubation with Fsh resulted in a strong pro-differentiation signal for spermatogonia and depleted undifferentiated spermatogonia (23), suggesting that Igfbps mainly restricted Igf3 bioactivity. However, the present time course and dose response experiments also showed that Fsh and 11-KT, two hormones promoting germ cell differentiation, can upregulate two igfbp transcripts with a delay of at least 3 days. It therefore seems possible that these Igfbps can support Igf3 bioactivity and contribute to the pro-differentiation signaling of Fsh and 11-KT. When the Igfbp inhibitor NBI-31772 was present only during the last 4 days of incubation (Figures 6A,B), when igfbp2a and -5b transcripts were upregulated by Fsh (Figures 2D,E) or 11-KT (Figure 3), the mitotic indices of A_und and A_diff did not change, whereas the one for type B decreased in response to Fsh (Figure 6A). The section surface area occupied by A_und increased, while the one for type B spermatogonia decreased in the presence of Fsh in combination with NBI-31772 during the last 4 days (Figure 6B). These observations suggest that blocking the “late rising” Igfbps partially inhibited spermatogonial differentiation. Inversing the experimental setting (i.e., NBI-31772 was only absent during the last 4 days of incubation) showed that the mitotic index and proportion of surface area of type B spermatogonia increased (Figures 6C,D). Under these conditions, the “early decreasing” Igfbps were blocked from the start of Fsh exposure, and the “late rising” Igfbps were allowed to bind Igf ligands. This resulted in a stronger pro-differentiation effect of Fsh, in particular for the type B spermatogonia (Figure 6D).

More than 90% of the circulating IGF is bound to IGFBPs (32); hence, locally produced IGFBPs seem primarily involved in modulating locally produced IGF bioactivity. The recently discovered Igf3 is prominently [e.g., zebrafish (33)], in certain species preferentially (22), expressed in gonadal tissue of adult fish (34–37). Previous studies showed that one possibility for Fsh to stimulate the differentiating proliferation of type A spermatogonia in an androgen-independent manner is to release Igf3 (9). This Fsh effect was strengthened, leading to a partial depletion of type A_und spermatogonia, by blocking Igfbps during a 4-day culture period, suggesting that Igfbps protected A_und from excessive differentiation via Fsh-stimulated Igf3 release (23). These recent studies highlight the importance of the Igf signaling system in modulating zebrafish spermatogenesis. Here, we report that Fsh, next to regulating igf3 and igfbp1a expression, modulated the expression of four other igfbps. While igfbp1a, igfbp3, and igfbp6a transcript levels were downregulated quickly by Fsh, or more slowly by Igf3 or 11-KT, the expression of igfbp2a and igfbp5b increased with a delay of at least 3 days in response to Fsh or 11-KT. Information on the regulation of igfbp transcript levels is scarce, and few studies have addressed igfbp expression in gonads. In rat, Igfbp2, 3, and 4 transcripts have been detected in LCs and seminiferous tubules (38) and FSH reduced Igfbp3 transcript levels in hypophysectomized rats (39). Igfbp2-6 were found in sheep testis in association with high Igf1 levels (40). In rainbow trout testis, the expression of igfbp6 was upregulated by Fsh and its levels slightly decreased in the additional presence of trilostane, suggesting that both Fsh and androgens increased igfbp6 expression in this species (41). To our knowledge, our study is the first to investigate dose and time effects of Fsh, revealing a dynamic modulation of igfbp transcript levels that is apparently relevant for modulating Igf3 bioactivity in zebrafish testis.

Igf and steroid hormones modulated igfbp expression in non-gonadal tissues in fish (42, 43). We examined if Igf3 or 11-KT, both mediators of Fsh bioactivity, were involved in the regulation of the Fsh-modulated testicular igfbps. The transcript levels of igfbp1a, -3, and -6a were modulated in the presence of Igf3 or in response to Fsh and an Igf1r inhibitor after 7 days of incubation, suggesting that Igf3 is a downstream mediator of Fsh on igfbp expression and that the faster response induced by Fsh used a different mechanism than the delayed response mediated by Igf3.

However, since Fsh increases Igf3 release (9), we can expect a fast drop of igfbp transcript levels induced by Fsh, and a continued suppression of transcript levels mediated by Igf3. The androgen 11-KT, on the other hand, selectively increased igfbp2a and -5b but reduced igfbp6a transcript levels after 7 days of incubation. Based on these results, the igfbps produced in the testis can be grouped in three categories: (1) non-responding to Fsh, Igf3, or 11-KT, (2) downregulated by Fsh, Igf3 or 11-KT, and (3) upregulated by Fsh and 11-KT but not by Igf3 (Figure 7).

The present data not only show that igfbp transcript levels respond to Fsh and downstream mediators but also open the possibility that Igfbps exert differential effects on testicular Igfs, potentially restricting or supporting Igf signaling. Still, the igfbps not modulated by Fsh, Igf3 or 11-KT should not be disregarded. In addition, Igfbps can act in an Igf-independent manner in mammals (44). Also in zebrafish, Igfbp3 blocked bone morphogenetic protein (Bmp) signaling by binding Bmp2a during embryonic development (45). Zebrafish Igfbp3, -5a, and -5b were localized also in the nucleus of U2O2 and HEK 293 cells (45, 46). Igfbp5a and -5b differ considering that Igfbp5b, but not -5a, shows transactivation activity in zebrafish (46). Since igfbp5b transcript levels are ~250-fold higher than those of igfbp5a, the latter also not being regulated by Fsh or 11-KT, it seems possible that Igfbp5b might be the more relevant form for potential nuclear functions in the testis. However, in general, the functional significances of nuclear Igfbps are still not well understood.

Previous studies have suggested that Igfbp can inhibit or enhance Igf action. While IGFBP1 and -6 generally inhibited IGF actions, IGFBP2-5 can inhibit or potentiate the IGF action, depending on the cell or tissue type, or on the physiological or experimental context (24). Due to the important role of Igf in muscle, many studies on extrahepatic IGFBP function addressed this tissue. In vascular smooth muscle cells, IGFBP2 and IGFBP4 exert an inhibitory effect on IGF1-induced DNA synthesis, while IGFBP5 potentiates the mitogenic effect of IGF1 (47, 48). Knock down of IGFBP5 impairs myogenesis and downregulated IGF2 expression in cultured myoblast cells (49). Administration of IGFBP5 in combination with a low concentration of IGF2 restored IGF2 expression and myogenic differentiation, whereas a non-functional IGFBP-5 or an IGF analog that activates the IGF1R but cannot bind IGFBPs, had no or a limited effect, respectively (49). These differential Igfbp effects seem to be conserved in fish. The transition from zero growth (achieved by food restriction) to fast growth involves upregulation of igf1, igfbp4, and -5b in Atlantic salmon skeletal muscle (50). Moreover, primary cultures of Atlantic salmon myogenic satellite cells (stem cells in muscle) respond to amino acids and/or Igf1 by expressing high levels of igf1 and igf2, and igfbp4, -5a, and -5b (42). Similarly, upregulation of igfbp2, igfbp4, igfbp5, and igf1 was recorded during muscle growth recovery after the end of a starvation period in rainbow trout (51). A dual role for the Igfbps has also been suggested in the regulation of zebrafish muscle growth and differentiation (12). Here, we have started exploring the potentially dual role of Fsh-regulated igfbps on zebrafish spermatogenesis.

Stimulatory effects of Igf3 on spermatogonial proliferation have been reported previously (9). The latter study did neither examine potential effects on type B spermatogonia nor on the volume fractions occupied by the different spermatogonia. We report here that Igf3 (100 ng/mL) promoted the proliferation of all spermatogonial cell types and also increased the areas occupied by A_diff and B spermatogonia while reducing the one occupied by A_und spermatogonia. This suggests that Fsh-stimulated Igf3 release promotes differentiation of A_und into A_diff and further into B spermatogonia. Our study also reports several findings as regards Igfbp functions in testis physiology, based on examining the effects of NBI-31772 on Igf3 activity. For example, blocking Igfbps increased the biological activity of a sub-threshold dose of Igf3, and Igf3 release stimulated by either Fsh or thyroid hormone preferentially promoted differentiation of A_und spermatogonia when Igfbps were blocked (23). These findings suggest that Igfbps protect the pool of A_und spermatogonia against excessive differentiation driven by high levels of Igf3. Our data moreover indicate that this protective effect may be mediated by the three igfbps rapidly down regulated by Fsh. Interestingly, the response to blocking Igfbps included upregulation of the expression of igf1rb. As mentioned above, zebrafish testis tissue expresses both igf1 receptor genes and the expression of igf1rb was previously upregulated under experimental conditions promoting spermatogonial proliferation (30). Altogether these results suggest that Igf3-mediated stimulation of spermatogonial proliferation and differentiation that is enhanced by blocking inhibitory Igfbps may involve upregulation of igf1 receptor expression.

In addition to the three igfbp transcripts being downregulated by Fsh, Igf3, or 11-KT, two other family members (igfbp2a and -5b) were upregulated in a delayed manner by Fsh or 11-KT. Blocking and de-blocking experiments suggested that the “late-rising” binding proteins facilitate pro-differentiation effects of Igf. Therefore, we propose that the concept of specific Igfbps either limiting or supporting Igf bioactivity, is also valid for testis tissue, where Fsh, but also downstream mediators (Igf3 and androgens), modulate igfbp gene expression. While Fsh and Igf3 or Fsh and 11-KT have similar effects as regards the direction of change, they exert their effects on igfbp transcript levels with differences in the time course, suggesting the use of different mechanisms to modulate igfbp gene expression. This may allow more sustained effects. Both, the acute as well as the delayed effects of Fsh via the Igf signaling system affected all spermatogonial cell types (A_und, A_diff, and B), suggesting that Fsh promotes spermatogonial development in a broad sense, making use of the Igf signaling system to generate different signals over time to different germ cell generations.

Several other signaling systems are also modulated by Fsh, next to the Igf/Igfbp system (11). It will be interesting to address in future studies the differentiation of the response to Fsh in space, e.g., by examining if spermatogenic cysts containing germ cells in different stages of development respond differently to a given Fsh challenge with respect to the expression of different igfbp transcripts or transcript amounts. Also in context with our previous observations (23), we propose that Igfbps negatively modulate the activity of Igf3 in the presence of a comparatively weak stimulator of Igf3 release, T₃, whereas when Fsh is present, Igfbps restricting Igf3 action (Igfbp1a, Igfbp3, and Igfbp6a) are rapidly suppressed, while the availability of Igfbps supporting Igf3 action (Igfbp2a and Igfbp5b) increases after a lag phase of 3–5 days, when suppression of the inhibitory Igfbps is also supported by downstream effectors of Fsh, such as Igf3 and androgens (Figure 7).

In conclusion, we have shown that of the nine igfbps expressed in zebrafish testis tissue, five are selectively modulated by Fsh and two Fsh downstream mediators (Igf3 and 11-KT) to promote spermatogonial differentiation. We also report that the pro-differentiation effect of Igf3 is reinforced by blocking the binding of Igf to the rapidly downregulated Igfbps, supporting the role for certain Igfbp as protecting A_und from excessive differentiation in response to Igf3.