Abstract
Thyroid hormones are crucial mediators of many aspects of vertebrate life, including reproduction. The key player is the biologically active 3,5,3’-triiodothyronine (T3), whose local bio-availability is strictly regulated by deiodinase enzymes. Deiodinase type 2 (Dio2) is present in many tissues and is the main enzyme for local T3 production. To unravel its role in different physiological processes, we generated a mutant zebrafish line, completely lacking Dio2 activity. Here we focus on the reproductive phenotype studied at the level of offspring production, gametogenesis, functioning of the hypothalamic–pituitary–gonadal axis and sex steroid production. Homozygous Dio2-deficient zebrafish were hypothyroid, displayed a delay in sexual maturity and the duration of their reproductive period was substantially shortened. Fecundity and fertilization were also severely reduced. Gamete counts pointed to a delay in oogenesis at onset of sexual maturity and later on to an accumulation of oocytes in mutant ovaries due to inhibition of ovulation. Analysis of spermatogenesis showed a strongly decreased number of spermatogonia A at onset of sexual maturity. Investigation of the hypothalamic–pituitary–gonadal axis revealed that dysregulation was largely confined to the gonads with significant upregulation of igf3, and a strong decrease in sex steroid production concomitant with alterations in gene expression in steroidogenesis/steroid signaling pathways. Rescue of the phenotype by T3 supplementation starting at 4 weeks resulted in normalization of reproductive activity in both sexes. The combined results show that reproductive function in mutants is severely hampered in both sexes, thereby linking the loss of Dio2 activity and the resulting hypothyroidism to reproductive dysfunction.
Introduction
Vertebrate reproduction is controlled by several endocrine factors, among which thyroid hormones (THs) are indispensable. They play an essential role in gametogenesis, steroidogenesis, embryogenesis, fetal development and growth, always with 3,5,3’-triiodothyronine (T3) as a key factor (Jana & Bhattacharya 1994, Kirby et al. 1996, Tovo-Neto et al. 2018). Balanced T3 levels are therefore of crucial importance, since both too high or too low levels can have detrimental consequences for reproduction/fertility, as shown from fish to humans (Beamer et al. 1981, Carr & Patino 2011, Mintziori et al. 2016).
The link between THs and reproduction is being investigated in different animal models, including zebrafish. Gametogenesis in teleosts, being oviparous and anamniote vertebrates, differs somehow from mammals. Oogonia continue to proliferate throughout adulthood, oocytes store a large amount of yolk during vitellogenesis and postvitellogenic follicles contain no antrum (Jalabert 2005). Spermatogenesis occurs in cysts, enveloping a single germ cell clone, and cyst-forming Sertoli cells retain their capacity to proliferate throughout adulthood (Schulz et al. 2010). Zebrafish are sexually mature from approximately 3 months onward, they can spawn all year round and evidence for TH action in the gonads is evident: mRNA for both TH receptors (thraa and thrb) has been detected in zebrafish ovaries and testes (Morais et al. 2013, Marelli et al. 2016) and specifically in males it was shown that T3 stimulates the formation of new cysts via proliferation of Sertoli cells and undifferentiated spermatogonia A, mediated by insulin-like growth factor 3 (Igf3) (Morais et al. 2013).
An often used approach to study the role of THs in fish reproduction is goitrogen treatment. These chemicals lower systemic TH levels by blocking hormone production in the thyroid gland. This adversely affects reproductive success, as shown by decreased fecundity and gonadosomatic index, altered sex steroid levels and decreased sperm count (Mukhi & Patino 2007, Naderi et al. 2014, Forsatkar et al. 2018). Next to systemic TH levels, deiodinases are important players in regulating tissue TH content. By intracellular activation and inactivation of THs, these enzymes function to adapt the amount of active TH to the local needs. Deiodinases are structurally and functionally conserved among vertebrates and exist in three different types (Darras & Van Herck 2012). Deiodinase type 2 (Dio2) is the most important one for catalyzing the conversion of the prohormone 3,5,3’,5’-tetraiodothyronine (T4) into T3, whereas deiodinase type 3 (Dio3) is the main inactivator of T3. Deiodinase type 1 (Dio1) can both activate and inactivate THs but has a lower substrate affinity (Darras & Van Herck 2012).
We recently generated Dio2-knockout (KO) zebrafish (dio2 − / −) and initial characterization of the phenotype revealed a severe disruption of reproduction (Houbrechts et al. 2016). This is in strong contrast to DIO2KO mice that do not show any obvious reproductive defects (Schneider et al. 2001). On the other hand, reproduction is severely hampered in both male and female DIO3KO mice (Hernandez et al. 2006), showing that in mammals too, deiodinase deficiency can have detrimental effects on reproduction. One factor that could play a role in the divergence between the three models are the differences observed in TH status. dio2 − / − mutant zebrafish are severely hypothyroid (Houbrechts et al. 2016) while DIO2KO mice are euthyroid (Schneider et al. 2001). DIO3KO mice experience thyrotoxicosis in early life, but this shifts to hypothyroidism from postnatal day 15 onward throughout adulthood (Hernandez et al. 2006). These data raise the question about the relative importance of deiodinases versus inappropriate systemic TH levels in the regulation of reproduction.
In the present study we investigated the contribution of Dio2 in reproduction, using our dio2 − / − mutant zebrafish. These animals are permanently Dio2 deficient, suffer from severe hypothyroidism and reproduce poorly (Houbrechts et al. 2016). We first studied their reproductive phenotype at different levels and subsequently investigated whether reversing their overall hypothyroidism by T3 supplementation was able to rescue this phenotype.
Materials and methods
Zebrafish husbandry
Our research group recently generated two dio2 − / − zebrafish lines (Houbrechts et al. 2016). For the present study we used the deletion mutant (lv1) which is missing 3 conserved amino acids upstream of selenocystein, leading to a complete abolishment of Dio2 activity. Wild type (WT), heterozygous and homozygous mutant zebrafish were maintained in circulating freshwater aquaria at 27.5 ± 1°C under a 14L/10D regimen. Fish were fed ad libitum with formulated feed in the morning and Artemia salina larvae in the evening. Embryos and larvae (until 7 days post fertilization) were kept in 0.3 × Danieau’s medium after spawning and incubated at 28 ± 0.5°C. Animal experiments were approved by the Institutional Ethical Committee of the KU Leuven (P239/2013 and P091/2017) and executed in strict accordance with the European Council Directive (2010/63/EC).
Fecundity and fertilization rate assessment
Sexually mature fish were allowed to spawn either via natural group matings or via individual mating set-ups. After 3 h the eggs were collected and counted. Fertilized/unfertilized eggs were discerned under a stereomicroscope and fertilization percentages were determined. Group spawning experiments were conducted as previously described (Houbrechts et al. 2016). For individual mating set-ups five male and five female dio2 +/+ and dio2 − / − siblings were paired alternately with the same and the opposite genotype.
Thyroid hormone measurements
For TH extractions, 3 pools of 3–4 ovaries per genotype and 4–7 pools of 10–14 testes per genotype were collected from 1-year-old fish and snap-frozen on dry ice. THs were extracted and measured by radioimmunoassay as previously described (Reyns et al. 2002).
RNA isolation and quantitative PCR
Ovaries, testes, liver and brain were dissected from 7-month-old and 1-year-old WT and dio2 − / − fish and snap-frozen on dry ice. For pooled samples, 3 replicates (pools of 3 fish) were used per genotype. When analyzing individual samples, quantitative PCR (qPCR) was performed on 5–7 replicates per genotype. vtg1 (liver), igf3, ar, esr2a, esr2b, hsd11b2, hsd17b3, cyp19a1b (gonads), gnrh2, gnrh3, kiss1 and kiss2 (brain) mRNA was quantified using SYBR Green-based qPCR as explained in detail in Supplementary data and Supplementary Table 1 (see section on supplementary data given at the end of this article).
Histology
Adult WT and dio2 − / − fish were killed using 0.1% tricaine. Gonads were sampled, fixed in 4% phosphate-buffered paraformaldehyde at 4°C, dehydrated, embedded in paraffin and sectioned at 7 µm. Three different stages were investigated for females: 3/4 months (i.e. onset of sexual maturity of WT and dio2 − / − respectively), 1 year (i.e. age that dio2 − / − no longer reproduce but WT still do), and for WT also 2 years (after reproductive arrest). Testis histology was performed at 3/4 months and 1 year. All sections were stained using hematoxylin (1 min) and 1% acidified eosin (5 s) and surface measurements and gamete counts were performed. Details on histological analysis can be found in Supplementary data. For each genotype and sex, gonads from 3 to 4 fish were analyzed.
Steroid hormone measurements
We measured sex steroid levels (estradiol (E2), 11-ketotestosterone (11-KT) and testosterone) in gonads from 1-year-old dio2 +/+ and dio2 − / − fish. Ovaries were collected individually (n = 12 for dio2 +/+; n = 9 for dio2 − / −). For testes we pooled tissues from 3 fish per replica (n = 7 for dio2 +/+; n = 10 for dio2 − / −). Gonad tissue was extracted twice with 4 volumes of diethyl ether. The combined supernatant was dried and dissolved in 200 µL buffer after which enzyme-linked immunosorbent assay (ELISA; kits from Cayman Chemical Company) was performed. These kits have previously been used for sex steroid measurement in zebrafish (Liu et al. 2009, Felix et al. 2013). Additionally, sex steroid secretion in the holding water was determined. Seven-month-old sexually active dio2 +/+ and dio2 − / − fish (7 males, 7 females) were placed individually in a glass container with 110 mL system water for 4 h without visual contact. Hormones were extracted from the holding water and ELISA was performed according to the methods described by Felix et al. (2013) and Morthorst et al. (2013) for 11-KT and E2 respectively.
T3 rescue treatment
Two separate batches of dio2 +/+ (n = 41–48) and dio2 − / − (n = 13–44) 4-week-old juveniles (F2 generation from siblings) were divided in half and placed in 8 L tanks. One group received T3 supplementation (10 nM) while the other group served as control (10−5 N NaOH). Solutions were refreshed 3 times a week and treatment continued until 6 months of age. Fecundity and fertilization rates were assessed starting from 3 months of age. Fish were set up to reproduce once weekly.
Statistical analysis
Statistical analysis for qPCR, gamete counts and surface measurements was performed using GraphPad Prism for Windows version 5.00 (GraphPad Software). If data passed the Kolmogorov–Smirnov normality test, they were analyzed by unpaired Student’s t test (2 groups) or one-way ANOVA with Tukey post hoc test (>2 groups). When data did not follow Gaussian distribution, the non-parametric Mann–Whitney U test (1 variable) or Kruskal–Wallis test (2 variables) was performed. Data from mating set-ups were analyzed by repeated-measures two-way ANOVA in GraphPad. A P value ≤0.05 was considered statistically significant for all analyses.
Results
General reproductive phenotype
WT zebrafish typically start reproducing around the age of 3 months. The onset of sexual maturity in dio2 − / − fish was delayed by approximately 2 months. Furthermore, WT zebrafish can reproduce for 18 months or more, while the reproductive period of dio2 − / − fish was severely shortened to 2–3 months. This implies that by 8 months of age, mutants normally no longer show sexual activity. Moreover, more than half of the mutants systematically failed to spawn during this 2- to 3-month period, which renders them infertile. Survival rates in embryos/larvae of fertilized mutant eggs were similar to those for WT. We did however observe a clear shift toward a male population. Sex determination of 179 fish (combination of 2 batches) after heterozygous intercross showed the following male-to-female ratios: dio2 +/+: 51:49; dio2 +/−: 68:32; dio2 − / −: 76:24.
Thyroid status in the gonads
T3 is the most active TH and is mainly produced peripherally by the Dio2 enzyme. Therefore, tissue T3 levels are the most representative for local thyroid status. T3 levels measured in 1-year-old ovaries and testes were significantly reduced by 94 and 79% in dio2 − / − zebrafish respectively (Fig. 1A and B). For ovaries we also measured T4 levels, which were reduced by 48% (0.96 ± 0.23 pmol/g for dio2 − / − vs 1.83 ± 0.18 pmol/g for dio2 +/+; P = 0.04). T4 data for testes are not available due to lack of material. These data show that the gonads of dio2 − / − zebrafish are in a hypothyroid status, but that the effect is most pronounced in females where T3 levels are extremely reduced.
TH status in gonads of 1-year-old dio2 +/+ and dio2 − / − zebrafish. (A and B) T3 levels (pmol per gram wet weight) in ovary (3 replicas of 3–4 ovaries) and testis (4–7 replicas of 10–14 testes) extracts of WT and dio2 − / − fish. (C and D) qPCR analysis in WT and dio2 − / − fish, showing relative mRNA expression of dio1, dio3a, dio3b, thraa and thrb in ovary and testis. Data were normalized based on the mean expression of three housekeeping genes (eef1a1l1, rpl13a and eif1b for ovary and eef1a1l1, rpl13a and hprt1 for testis). Average WT ( dio2 +/+ ) values were set to 1 and dio2 − / − values were rescaled accordingly. All data are expressed as mean + s.e.m. (three replicates with three animals pooled per replicate). Per tissue and per gene, differences between both genotypes were analyzed by an unpaired Student’s t test. *P < 0.05; **P < 0.01.
Citation: Journal of Endocrinology 241, 2; 10.1530/JOE-18-0549
Effects on the expression of other genes regulating TH levels and action were investigated by qPCR in 1-year-old fish. In the ovaries we found a significant upregulation of dio1 and thraa expression in dio2 − / − mutants compared to WT, while dio3a, dio3b and thrb expression were unaffected (Fig. 1C). For testes a significant downregulation was observed in dio1 and thrb expression (Fig. 1D).
Effects on spawning
Our previous study briefly mentioned that reproduction is hampered in dio2 − / − mutants (Houbrechts et al. 2016). We now followed spawning in three batches, containing different male-to-female ratios (i.e. 22:9, 5:12 and 15:15). Regardless of the sex ratio, matings of dio2 − / − fish yielded significantly less eggs compared to WT (17 ± 5 for dio2 − / − vs 152 ± 17 for dio2 +/+) and fertilization percentages were significantly reduced (21 ± 5% for dio2 − / − vs 76 ± 4% for dio2 +/+).
Since these results suggested that fertility might be hampered in both sexes, we subsequently crossed individual dio2 +/+ and dio2 − / − siblings (Fig. 2). As expected, the best reproductive performance was obtained when pairing WT males and females. For the mating between mutant males and females both egg production and fertilization were significantly reduced. Combination of WT fish with the opposite mutant sex also adversely affected fecundity and fertilization with values between those of pure WT or dio2 − / − combinations.
Spawning experiment of dio2 +/+ and dio2 − / − siblings. Five males and five females of each genotype were individually crossed in two combinations: male and female of the same genotype or male and female of different genotypes. (A) Fecundity: total number of eggs per spawning; (B) fertilization percentages. Values represent mean + s.e.m. Data were analyzed by repeated-measures ANOVA with Tukey post hoc test. Groups with no common letter are significantly different (P < 0.05).
Citation: Journal of Endocrinology 241, 2; 10.1530/JOE-18-0549
Effects on gonad size and gametogenesis
Since weight but not length of female dio2 − / − mutants increases drastically from sexual maturity onward (Houbrechts et al. 2016), we hypothesized this was caused by the accumulation of eggs in the abdomen. We determined the gonad section surface area of mutant and WT fish at 3/4 months and 1 year as a proxy for ovary/testis size. Ovary size was significantly increased in dio2 − / − fish at both ages (Fig. 3A) while testis size was not affected (Fig. 3E). Representative pictures of ovaries and testes are shown in Fig. 4. To corroborate our surface measurements, we also weighed gonads of a different 1-year-old batch. These data substantiate the surface measurements, showing significantly increased ovarian weight in dio2 − / − fish (30.1 ± 3.2 mg for WT vs 85.1 ± 13.1 mg for mutant; n = 5 per genotype; unpaired Student’s t test; P = 0.008), while testis weight did not differ (2.7 ± 0.3 mg/animal for WT (n = 5, pools of 6) vs 2.9 ± 0.3 mg/animal for mutant (n = 4, pools of ≥5); Mann–Whitney U test; P = 0.206).
Gametogenesis stage counting in dio2 +/+ and dio2 − / − fish. (A) Ovary size, presented as average ovarian section surface (mm²); Percentage of primary (B), vitellogenic (C) and mature (D) oocytes determined on five sections per female at 3/4 months (3/4 M) and 1 year (1 Y); (E) Testis size, presented as average testicular section surface (mm2); Percentage of spermatogonia A (F), spermatozoa (G) and remaining area (H) determined on four sections per male at 3/4 months and 1 year. Data are expressed as mean + s.e.m. (n = 3–4) and were analyzed by Mann–Whitney U test. * P < 0.05.
Citation: Journal of Endocrinology 241, 2; 10.1530/JOE-18-0549
Representative pictures of gonads of dio2 +/+ and dio2 − / − fish at 1 year of age. (A and B) Ovaries showing primary oocytes, vitellogenic oocytes (cortical alveolus stage + vitellogenesis) and mature oocytes; (C and D) Testes showing spermatogonia A and spermatozoa. For counts, all four types of spermatogonia A (1 undifferentiated and 3 differentiated) were included.
Citation: Journal of Endocrinology 241, 2; 10.1530/JOE-18-0549
Counting different oocyte stages, we only observed significant differences at 3/4 months showing a higher percentage of primary oocytes and a lower percentage of vitellogenic oocytes in dio2 − / − compared to WT (Fig. 3B and C). To make an accurate comparison at the stage of reproductive arrest, we also investigated 2-year-old WT females. This showed a strong decrease in oocyte number in WT fish when they stopped reproducing: 49, 59 and 43% reduction from 1 towards 2 years for primary, vitellogenic and mature oocytes, respectively. This is in strong contrast to the also non-active 1-year-old mutants, where oocyte numbers remained high, confirming they have problems with ovulation/egg deposition.
For testes, comparison between genotypes revealed a significant reduction in the percentage of area covered by spermatogonia A at 3/4 months in dio2 − / − fish, while the opposite was observed at 1 year (Fig. 3F). Spermatozoa area on the other hand was significantly increased in dio2 − / − fish at reproductive onset, but significantly decreased at 1 year compared to WT (Fig. 3G). The remaining area (containing spermatogonia B, spermatocytes, spermatids, Leydig and Sertoli cells) was significantly lower at 3/4 months in dio2 − / − fish (Fig. 3H).
Effects on the hypothalamic–pituitary–gonadal axis
To identify potential disturbances in the functioning of the hypothalamic–pituitary–gonadal (HPG) axis, we measured the expression levels of relevant genes in tissues of 7-month-old sexually active siblings (Fig. 5). Two primary components were measured: kisspeptin (kiss1 and kiss2) and gonadotropin-releasing hormone (gnrh2, gnrh3). No differences were observed for both sexes, except for a significant increase in kiss2 in male dio2 − / − brain (Fig. 5B). Due to lack of biological material, we were not able to obtain data on gonadotropins in the pituitary gland. Therefore, gonadal expression of igf3, a gonadotropin-regulated gene, was measured, revealing a significant upregulation in dio2 − / − mutants of both sexes (Fig. 5A and B).
Components of HPG-axis in 7-month-old dio2 +/+ and dio2 − / − siblings. Expression of gnrh2, gnrh3, kiss1, kiss2 (brain), igf3 (gonad) and vtg1 (liver) in female (A) and male (B) fish. Data were normalized based on the mean expression of different housekeeping genes (eef1a1l1, rpl13a, and eif1b for brain, liver and ovary; eef1a1l1, rpl13a and hprt1 for testis). Average WT ( dio2 +/+) values were set to 1 and dio2 − / − values were rescaled accordingly. Data are expressed as mean + s.e.m. (5–7 individual animals per condition). Per tissue and per gene, differences between both genotypes were analyzed by unpaired Student’s t test (Gaussian distribution) or Mann–Whitney U test (non-parametric). *P < 0.05; **P < 0.01.
Citation: Journal of Endocrinology 241, 2; 10.1530/JOE-18-0549
Next, we measured intra-gonadal sex steroid levels in 1-year-old WT and dio2 − / − fish (F1 generation from siblings). E2 levels in ovarian extracts were significantly reduced in dio2 − / − fish (Fig. 6A). Similarly, 11-KT and testosterone levels were significantly decreased in mutant testis extracts (Fig. 6A). Since these sex steroids also act as pheromones that could also be involved in spawning failure, we determined the amount of E2 (females) and 11-KT (males) secreted into the water by WT fish and infertile mutants at 7 months of age (F1 generation from siblings). E2 as well as 11-KT secretion was significantly reduced in dio2 − / − fish (Fig. 6B).
Sex steroid profiles from dio2 +/+ and dio2 − / − fish (F1 generation from siblings). (A) Intra-gonadal levels (pg/mg tissue) of E2 in ovarian tissue (n = 9–12) and 11-KT and testosterone in testicular tissue (n = 7–12); (B) Concentration (pg/mL) of E2 and 11-KT in the holding water of female and male fish respectively (n = 7 individual fish per genotype). (C and D) esr2a, esr2b, ar, hsd11b2, hsd17b3 and cyp19a1b expression in ovary and testis. Data were normalized based on the mean expression of different housekeeping genes (eef1a1l1, rpl13a, and eif1b for ovary; eef1a1l1, rpl13a and hprt1 for testis). Average WT ( dio2 +/+) values were set to 1 and dio2 − / − values were rescaled accordingly (n = 5–7 individual animals per condition). Data are expressed as mean + s.e.m. Differences between both genotypes were analyzed either by unpaired Student’s t test (Gaussian distribution) or Mann–Whitney U test (no Gaussian distribution). *P < 0.05; **P < 0.01; ***P < 0.001.
Citation: Journal of Endocrinology 241, 2; 10.1530/JOE-18-0549
To find out the reason and consequences of the decreased sex steroid levels, we tested a set of genes belonging to the steroid biosynthesis and signaling pathway: 2 estrogen receptors (esr2a, esr2b), the androgen receptor (ar), the aromatase enzyme-converting testosterone into E2 (cyp19a1b), and 2 enzymes involved in 11-KT production (hsd11b2 and hsd17b3), the most abundant and most potent androgen in fish. Steroidogenesis and steroid signaling seemed to be similarly affected in dio2 − / − ovaries and testes compared to WT siblings (Fig. 6C and D). A general trend toward upregulation was observed, with a significant increase for ar (both sexes), esr2b (ovary) and hsd17b3 (testis). One remarkable exception was the significant reduction in hsd11b2 levels in both sexes. Hepatic expression of vtg1, a major component of egg yolk, showed a significant upregulation in dio2 − / − fish (Fig. 5A). We also analyzed heterozygous individuals and found that these fish showed expression profiles very similar to their WT siblings (data not shown).
The effect of T3 supplementation on reproductive status
dio2 − / − zebrafish suffer from severe peripheral hypothyroidism. To find out if the defects caused by insufficient Dio2-dependent T3 production in the gonads and/or hypothalamus/pituitary could be overcome by systemic hormone treatment, T3 was added to the holding water of developing WT and mutant zebrafish (F2 generation from siblings) from 4 weeks post fertilization. This treatment did not only normalize the onset of sexual maturity in dio2 − / − fish to 3 months instead of 5 months, but both egg production and fertilization rates also increased up to levels of WT fish (Fig. 7). As expected, mutants receiving a vehicle treatment only started to reproduce after 5 months of age for a duration of 2 months or less.
Rescue by T3 supplementation. (A) fecundity and (B) fertilization percentages of dio2 +/+ and dio2 − / − fish (F2 generation from siblings) treated or not with 10 nM T3. Statistical analysis was performed by one-way ANOVA with Tukey post hoc test. Groups with no common letter are significantly different (P < 0.05).
Citation: Journal of Endocrinology 241, 2; 10.1530/JOE-18-0549
Discussion
Deiodinases are important to ensure the correct TH balance throughout the body and activity-reducing deiodinase polymorphisms have a negative impact on different aspects of human health (Verloop et al. 2014). This has however not yet been thoroughly studied in relation to reproduction. Data from mouse studies show that reproduction is severely hampered in DIO3KO mice while fertility is unimpeded in DIO2KO mice (Schneider et al. 2001, Hernandez et al. 2007). We studied reproduction in a Dio2-deficient zebrafish line where adults, in contrast to euthyroid DIO2-deficient mice, display severe hypothyroidism throughout the body. Our results clearly demonstrated adverse effects of Dio2 deficiency and the resulting hypothyroid status on reproduction but also showed that reversing systemic hypothyroidism prior to gonad maturation rescues reproductive activity.
dio2 − / − zebrafish show a delay in onset of reproduction
The onset of sexual maturity is delayed in dio2 − / − zebrafish. Female mutants only start laying eggs around 5 months of age. At 3/4 months, corresponding to the onset of sexual maturity, we found a shift in oocyte development, with more primary oocytes at the expense of vitellogenic oocytes, indeed pointing to a delay. This shift may be due to a delay in vitellogenin incorporation in hypothyroid dio2 − / − females. It was indeed found in Japanese eel that T3 accelerates vitellogenin incorporation and increases the percentage of vitellogenic oocytes (Kayaba et al. 2008). Therefore we would expect hepatic vtg1 expression to be lower in mutants but unfortunately, we do not have expression data at this early age. Analysis at 7 months showed a significant increase in vtg1 expression, which points in a different direction and should be further investigated.
Pronounced hypothyroidism is known to disturb spermatogenesis, as observed for instance in young goitrogen-treated tilapia showing complete absence of spermatids and spermatozoa (Matta et al. 2002). The effects of Dio2 deficiency in young zebrafish were less severe, showing a reduced percentage of spermatogonia A but an increased percentage of spermatozoa in mutants right before the onset of apparent sexual activity. One possibility is that the available pool of spermatogonia in hypothyroid dio2 − / − fish is reduced. Since stimulation of spermatogonial proliferation by T3 occurs via Igf3 (Morais et al. 2017) and igf3 expression is increased in the mutants (although only measured at a later stage), this is doubtful. Alternatively, high Igf3 levels might stimulate differentiation of spermatogonia toward mature sperm cells (Nobrega et al. 2015), which would fit with the observed shift in relative proportion of spermatogonia/spermatozoa.
Following the definition of teleostean puberty, which starts with the onset of gametogenesis and ends with first reproduction (Schulz & Goos 1999), we can hypothesize that dio2 − / − fish are delayed in puberty since they did not reproduce at 3 months of age. This corresponds to the observed delay in sexual activity in hypothyroid mammals (Bakke et al. 1976, Weber et al. 2003). Another zebrafish study similarly showed that goitrogens delay male pubertal development (Sharma & Patino 2013). In contrast to hypothyroid DIO3KO mice with delayed puberty, we did not find indications for a lack of testis growth spurt (Martinez et al. 2016). Rather, diminished production of sex steroids (specifically 11-KT in males) could be the underlying cause (Schulz & Goos 1999).
Reproduction remains impaired at later age
The apparent delay in oogenesis was caught up at later age, since relative oocyte counts at 1 year were very similar for WT and mutant fish. The maturation process of the oocytes is thus fully implemented in dio2 − / − females, which corresponds to the significant upregulation at 7 months of ovarian igf3, a growth factor with a potent stimulatory action on oocyte maturation (Li et al. 2015), as well as ar, which positively regulates oocyte maturation and fecundity in females (Crowder et al. 2018). In contrast, loss of Ar function results in fewer mature oocytes in zebrafish as well as mice (Shiina et al. 2006, Walters et al. 2010).
While mutant females possess high numbers of (mature) oocytes, they spawn poorly and completely fail to spawn from the age of 8 months. When comparing fish in reproductive arrest (i.e. 2 year for WT; 1 year for mutants), we found that oocyte numbers were drastically decreased in WT fish while numbers remained high in dio2 − / − fish, indicating that females cannot deposit their eggs and have ovulatory problems. It has been shown in many non-mammalian vertebrates that egg laying is a parameter susceptible to hypothyroidism, proportional to the degree of severity (Mukhi & Patino 2007, Chen et al. 2008, Dumont 2008). This may be linked to the decrease in E2 in dio2 − / − mutants, since sex steroids play an essential role in several aspects of the reproductive process and are modulated by THs (Cyr & Eales 1988). E2 is known to peak before the ovulatory phase, as demonstrated in zebrafish via intra-ovarian measurements (Lister & Van Der Kraak 2009). The significant decrease in intra-ovarian as well as secreted E2 in our dio2 − / − females observed at different stages may thus in part explain the ovulatory problems and account for the significant upregulation of esr2b. Furthermore, egg deposition requires the contractile action of muscles and we already showed in our previous work that muscle development/contraction and locomotor behavior is adversely affected by Dio2 deficiency (Bagci et al. 2015, Houbrechts et al. 2016). Lastly, the combination of male sexual behavior and pheromone secretion normally triggers female ovulation (Hisoaka & Firlit 1962). Sex steroids are known to control courtship behavior in zebrafish (Pradhan & Olsson 2015) and their production/secretion is in general stimulated by THs, either directly or indirectly via the HPG axis (Swapna & Senthilkumaran 2007). Sex steroid production and release is disturbed in dio2 − / − mutants and this indeed seemed to diminish male-typical courtship behavior (personal observations), potentially leading to an inadequate stimulation of female ovulation.
Unlike oogenesis, mutant spermatogenesis still shows dysregulation at 1 year of age when they are in reproductive arrest. In contrast to the situation at 3/4 months, the percentage of spermatogonia A was now increased in dio2 − / − males compared to WT. This could be related to their androgen insufficiency (low 11-KT levels) which was previously shown in zebrafish to induce accumulation of spermatogonia A (de Waal et al. 2009). The premature reproductive arrest in males was also accompanied by a significantly decreased percentage of spermatozoa. Similar findings occurred in zebrafish and another teleost, the fathead minnow, exposed to goitrogens. They displayed reduced T3 and T4 levels (Noyes et al. 2013, Naderi et al. 2014), accompanied by a decreased number of mature spermatozoa, leading to male infertility (Muirhead et al. 2006, Naderi et al. 2014). Another known thyroid-blocking chemical, 2,3,7,8-tetrachlorodibenzo-p-dioxin, also impaired zebrafish reproductive capacity by decreasing spermatozoa number with a concurrent increase in spermatogonial cells (Baker et al. 2016).
Despite some changes in spermatogenesis, dio2 − / − mutants still possess a substantial amount of spermatozoa. This is in strong contrast to the recurring fertilization problems. Fertilization percentages are low in sexually active fish and from 8 months onward reproduction is halted. This could reflect problems and (at later stages) even defects in sperm ejaculation. Moreover, reduced sperm quality and/or motility can cause additional complications. The latter hypothesis is substantiated by the fact that, even when paired with WT females, mutant male fertilization percentages on average did not increase drastically (from 40 to 53%). It has been shown in different mammalian species that hypothyroidism lowered the quality and motility of spermatozoa (while spermatogenesis was only sporadically affected), often in combination with reduced testosterone levels (Chandrasekhar et al. 1985, Mallem et al. 2006). The stronger impact of Dio2 deficiency on spermatogenesis compared to oogenesis may relate to differences in local TH signaling. 1-year-old ovaries show a possible compensatory response to the reduction in T3 availability by a significant increase in thraa expression. The opposite effect is found in testis where the decrease in thrb expression may further reduce TH action.
It thus seems that the spermatogenic process in the dio2 − / − mutants continues at later stages while reproductive behavior is absent. This is similar to the phenotypic characteristics observed in mutant zebrafish and medaka with deficiencies in steroidogenic enzymes (Sato et al. 2008, Zhai et al. 2018). Fertile males (normal spermatogenesis) were compromised in their male-typical reproductive behavior, caused by insufficient sex steroid production. Our sex steroid measurements were performed at 7 months and 1 year of age, corresponding to sexually active and non-active fish respectively. Although the values represent either secretion (water) or production (intra-gonadal) levels, the difference seems much larger in older fish. This could mean that sex steroid production is progressively decreasing with age and once it falls below a minimum (maybe around 8 months) it can no longer sustain reproductive behavior and sexual activity ceases. In this context it would be interesting to find out if sex steroid supplementation could rescue sexual behavior in the dio2 − / − mutants at later age.
The upregulation of ar and hsd17b3 found in dio2 − / − males might be a compensatory reaction to the low intra-testicular 11-KT levels in order to stimulate the reproductive axis. This is comparable to findings in ar− / −-mutant zebrafish and ARKO mice, displaying upregulated expression levels of steroidogenic genes such as hsd17b3 (De Gendt et al. 2005, Tang et al. 2018). Moreover, mRNA levels of hypothalamic genes were not markedly changed in the ar − / − zebrafish model, while these fish, similar to our 3/4-month-old mutants, displayed significantly reduced 11-KT levels together with reduced numbers of spermatogonia (Tang et al. 2018).
Elucidating the reproductive phenotype at different levels of the HPG axis
The clear decrease in gonadal steroids is likely to result in a reduced negative feedback on different regulators in the HPG axis. Except for an increase in kiss2 in male mutants, we found no apparent dysregulation at the hypothalamic level. This is not necessarily surprising, since the kiss/gnrh system is considered less crucial in zebrafish. Null mutants for gnrh3, kiss1+kiss2 and gnrh3+kiss1+kiss2 display normal gonad maturation and reproductive capacity (Tang et al. 2015, Liu et al. 2017). We think alterations rather occur at the pituitary level for the gonadotropins. As Igf3, a growth factor controlled by Fsh in males (Nobrega et al. 2015), is clearly upregulated, we expect this gonadotropin to be elevated. This could reflect a situation of primary hypogonadism, like in male DIO3KO mice characterized by an increase in Fsh/FSH and Lh/LH, but a decrease in testosterone (Martinez et al. 2016).
Sex steroid production in the gonads and subsequent secretion in the water is diminished by Dio2 deficiency, especially in males. The low secretion levels of 11-KT in sexually active dio2 − / − males implies impaired pheromone production from the Leydig cells. It was shown in zebrafish that this specific cell type is TH responsive through the presence of Thrb (Morais et al. 2013). In zebrafish ovaries, both thraa and thrb were detected, but no cell types were specified (Marelli et al. 2016). Zebrafish steroidogenesis is thus positively correlated to T3 levels as demonstrated in different fishes and mammals, where hypothyroidism leads to a reduction in 11-KT and testosterone tissue levels and/or circulating testosterone and E2 levels (Swapna et al. 2006, Sahoo et al. 2008, Tomza-Marciniak et al. 2014).
Recovery of reproductive capacity after T3 supplementation
Reproductive activity in Dio2-deficient fish was remarkably well recovered by systemic T3 supplementation starting at 4 weeks. This suggests that although Dio2 is expressed in zebrafish ovary and testis (unpublished data), and therefore, normally contributes to T3 availability in these organs, the lack of local T3 production can be overcome by providing enough external T3, implying the local presence of one or more transporters with good affinity for T3. Mct8 is a likely candidate, but its presence in adult zebrafish gonads remains to be confirmed. In mouse, several TH transporters have been identified in testis and ovary (Kinne et al. 2011) and also in another teleost, the fathead minnow, mct8, mct10 and especially oatp1c1 were found to be expressed in the gonads (Muzzio et al. 2014).
Although the present rescue experiment suggests that the negative impact of Dio2 deficiency on reproduction can be overcome by correcting systemic hypothyroidism, a few questions remain. We started T3 supplementation prior to sexual maturity when maturation of the gonads was still ongoing. It would be interesting to test whether treating fully mature zebrafish is also capable of normalizing reproductive activity. If not, this would point to persistent defects originating during gonad development and maturation. We also found a skewed sex ratio with male predominance in the offspring of dio2 − / − fish. Interestingly, this is exactly the opposite of what has been observed due to hypothyroidism in other non-mammalian studies. In fish and Xenopus, lower T4 and/or T3 levels resulted in a female-skewed sex ratio, while male predominance was found when T4 levels were increased (Goleman et al. 2002, Zhong et al. 2005, Sharma & Patino 2013, Naderi et al. 2014). Another dissimilarity compared to hypothyroidism is found in the ovarian size. Ovaries of dio2 − / − fish more or less doubled in size, in contrast to available data from hypothyroid animals (fish and mammals) showing reduced ovarian weight (Dijkstra et al. 1996, Noyes et al. 2013). These differences suggest that local Dio2 activity may still have a role of its own. Last but not least, we need to identify changes at the level of the hypothalamus and pituitary in more detail to find out whether they are the cause or rather the consequence of impaired gonadal function. This is essential in unraveling the molecular mechanisms underlying the negative impact of impaired T3 signaling on reproduction.
Conclusion
Permanent disruption of Dio2 activity hampers both male and female fertility at different levels of the reproductive axis. Counteracting the hypothyroid status by T3 supplementation from early stages remarkably well recovered reproductive function. This implies that gonadal TH activation by Dio2 is not indispensable when T3 can be supplied via an alternative way. It also explains the striking difference between the dio2 − / − zebrafish reproductive phenotype and normal reproduction in the euthyroid DIO2KO mice. Some subtle differences with the effects of goitrogen-induced hypothyroidism remain, suggesting that local Dio2 action might be needed at some stages of gonadal development and function. This needs to be further investigated, for instance by starting T3 rescue treatment at different stages, such as immediately after fertilization or only during the reproductive period. Generating a conditional KO line would also be extremely helpful in this regard, something that should be possible in the near future thanks to the rapid evolution in techniques for genome editing.
Supplementary data
This is linked to the online version of the paper at https://doi.org/10.1530/JOE-18-0549.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Funding
This research was supported by grants from the IBSA (Institut Biochimique SA) foundation for scientific research (Switzerland), the Research Council of KU Leuven (PDM/17/087) and the Research Foundation-Flanders (FWO) (G.0528.10).
Author contribution statement
A M H performed all experiments, acquired and analyzed the data and was responsible for writing the manuscript. J V H assisted in the histological studies. V M D (supervisor) designed the experiments together with A M H and participated in writing the manuscript.
Acknowledgements
The authors thank Lut Noterdaeme, Arnold Van Den Eynde and Véronique Brouwers for technical assistance, Prof A Canario and Prof G Van Der Kraak for their advice on steroid measurements, Pieter Vancamp for his useful critique on the manuscript draft and the IBSA for supporting A M H.
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