Abstract
The roles of androgens in male reproductive development and function in zebrafish are poorly understood. To investigate this topic, we employed CRISPR/Cas9 to generate cyp11c1 (11β-hydroxylase) mutant zebrafish lines. Our study confirms recently published findings from a different cyp11c1−/− mutant zebrafish line, and also reports novel aspects of the phenotype caused by loss of Cyp11c1 function. We report that Cyp11c1-deficient zebrafish display predominantly female secondary sex characteristics, but may possess either ovaries or testes. Moreover, we observed that cyp11c1−/− mutant male zebrafish are profoundly androgen- and cortisol-deficient. These results provide further evidence that androgens are dispensable for testis formation in zebrafish, as has been demonstrated previously in androgen-deficient and androgen-resistant zebrafish. Herein, we show that the testes of cyp11c1−/− mutant zebrafish exhibit a disorganised tubular structure; and for the first time demonstrate that the spermatic ducts, which connect the testes to the urogenital orifice, are severely hypoplastic in androgen-deficient zebrafish. Furthermore, we show that spermatogenesis and characteristic breeding behaviours are impaired in cyp11c1−/− mutant zebrafish. Expression of nanos2, a type A spermatogonia marker, was significantly increased in the testes of Cyp11c1-deficient zebrafish, whereas expression of markers for later stages of spermatogenesis was significantly decreased. These observations indicate that in zebrafish, production of type A spermatogonia is androgen-independent, but differentiation of type A spermatogonia is an androgen-dependent process. Overall, our results demonstrate that whilst androgens are not required for testis formation, they play important roles in determining secondary sexual characteristics, proper organisation of seminiferous tubules, and differentiation of male germ cells.
Introduction
The roles of androgens in zebrafish sex differentiation, development of male sexual characteristics, and maintenance and function of the adult testes are poorly understood. Laboratory strains of zebrafish lack sex chromosomes and exhibit polygenic sex determination (Liew et al. 2012). Prior to gonadal differentiation zebrafish develop a juvenile ovary; this is maintained in presumptive females and continues to develop, whereas presumptive males undergo juvenile ovary-to-testis transformation (Uchida et al. 2002, Wang et al. 2007). This process is highly sensitive to sex steroids. Treatment of developing fish with oestrogens results in feminisation (Andersen et al. 2003, Brion et al. 2004, Orn et al. 2016); whilst mutation of cyp19a1a, crucial for oestrogen production, causes robust masculinisation (Lau et al. 2016, Yin et al. 2017). Conversely, treatment of developing zebrafish with androgens results in robust masculinisation (Larsen & Baatrup 2010, Morthorst et al. 2010, Lee et al. 2017). These findings suggest that androgen-deficiency or androgen-resistance might cause robust feminisation; however, this is not the case.
Recent studies have characterised androgen-deficient and -resistant zebrafish (Crowder et al. 2017, Yong et al. 2017, Tang et al. 2018, Li et al. 2020, Oakes et al. 2019). These fish share similar phenotypes, exhibiting primarily female secondary sex characteristics. Despite their appearance, these fish may possess either ovaries or testes, indicating that androgens are dispensable for testis differentiation. Androgen-deficient or -resistant male zebrafish are infertile in standard breeding scenarios, however, their sperm may fertilise eggs collected from WT females in IVF experiments. Several factors appear to contribute to this phenotype, including disorganised testicular structure and impaired breeding behaviour and spermatogenesis.
Steroid 11β-hydroxylase (CYP11B1) is crucial for the conversion of 11-deoxycortisol to cortisol in the final stage of glucocorticoid biosynthesis in humans (Miller & Auchus 2011). CYP11B subfamily enzymes are located at the inner-mitochondrial membrane, where they are supplied with electrons by NADPH via ferredoxin and ferredoxin reductase, to allow substrate hydroxylation (Schiffer et al. 2015). In zebrafish, the final stage of glucocorticoid biosynthesis is catalysed by the zebrafish homolog of 11β-hydroxylase, Cyp11c1 (Fig. 1) (Tokarz et al. 2015). Genomic analysis of CYP11 genes suggests that the CYP11C genes in fish and the CYP11B genes in terrestrial mammals are orthologous, which is consistent with studies on the evolution of adrenal and sex steroidogenic enzymes (reviewed in Baker et al. (2015)). Unlike the situation in mammals, Cyp11c1 is thought to play an important role in gonadal androgen synthesis in zebrafish (Fig. 1) (Oakes et al. 2019). In the zebrafish testes Cyp11c1 is found in the steroidogenic Leydig cells, as well as in certain germ cell stages (Caulier et al. 2015). The principal androgens in humans are testosterone and 5α-dihydrotestosterone, whereas in zebrafish the principal androgen is 11-ketotestosterone (Tokarz et al. 2015). This is due to the fact that zebrafish favour production of 11-oxygenated androgens from androstenedione, rather than conversion of androstenedione to testosterone (de Waal et al. 2008).
Cyp11c1 activity depends on electron transfer from the steroidogenic cofactor Fdx1b (Griffin et al. 2016). Our recent work has established Fdx1b-deficient zebrafish as a model of combined androgen- and cortisol-deficiency. Fdx1b-deficient zebrafish are infertile and exhibit disorganised testis structure and impaired spermatogenesis, as well as reduced stereotypical breeding behaviours (Oakes et al. 2019).
Almost simultaneously with the submission of this manuscript, another paper was published describing a different zebrafish cyp11c1-mutant line (Zhang et al. 2020). This study focussed primarily on the phenotypic characteristics of cyp11c1-mutant zebrafish during development. Herein, we present cyp11c1-mutant zebrafish lines as novel models for research into the roles of steroid deficiency on sex differentiation and adult reproductive processes. Our study confirms key findings of the previously published cyp11c1-mutant line and also presents several novel, previously undescribed phenotypes. Our results confirm that Cyp11c1-deficient zebrafish are cortisol and androgen deficient, exhibit infertility and impaired breeding behaviour, as well as reduced spermatogenesis. Novel findings in our study include characterisation of steroid precursor concentrations, disorganised testis morphology in adult mutants and impaired locomotor behaviour. In addition to this, we have performed in-depth molecular investigation into the nature of impaired spermatogenesis in cyp11c1−/− mutant zebrafish. We also demonstrate, for the first time, that androgens are crucial for development or maintenance of key anatomical reproductive structures such as the spermatic duct, the structure linking the testes and urogenital orifice.
Materials and methods
Zebrafish husbandry and ethics
Adult zebrafish were maintained in a recirculating system (ZebTECTM, Tecniplast®, Kettering, UK) at 28.5°C on a 10 h light:14 h darkness photoperiod. Zebrafish were bred from an AB WT background. Fish were aged between 96 and 154 days post fertilisation (dpf) at the time of experimentation. Fish were euthanized by administration of the anaesthetic tricaine mesylate (Pharmaq, Fordingbridge, UK). All experiments with animals were performed under licence from the UK Home Office and approved by the University of Sheffield Animal Welfare and Ethical Review Body (AWERB).
Targeted genetic disruption of cyp11c1 by CRISPR/Cas9
Mutation of cyp11c1 was achieved using the SygRNA system (Sigma). A crRNA was designed to target exon 2 of cyp11c1 (ENSDART00000185978.1). Approximately 1 nL of a 4 µL mixture containing 0.1 µM crRNA, 0.1 µM tracrRNA (Sigma), 1 µL phenol red and 1 µL Cas9 (NEB, Ipswich, Massachusetts, USA) was injected into 1-cell stage embryos. The Cas9 cut site overlapped a BslI restriction site, allowing screening for mutant alleles lacking sensitivity to BslI (Supplementary Fig. 1, see section on supplementary materials given at the end of this article). CRISPR/Cas9-injected embryos were raised and outcrossed to unrelated WT fish. The resulting progeny were screened for disruption of cyp11c1, and out-of-frame mutations were identified by DNA sequencing.
Steroid quantification by LC-MS/MS
Adult zebrafish were euthanized, transferred to a silanised test tube, and snap-frozen on dry ice. Sample preparation was as previously described except for omission of the final Phree column elution (Oakes et al. 2019). Steroids were separated and quantified using an Acquity UPLC System (Waters, Milford, CT, USA) coupled to a Xevo TQ-S tandem mass spectrometer (Waters) as previously described (O’Reilly et al. 2017).
Fertility and behavioural analysis, IVF and sperm release
cyp11c1−/− mutant male zebrafish were outcrossed with unrelated WT females on three separate occasions using the pair mating technique, breeding was deemed successful if fertilised eggs were produced (Westerfield 2000). Breeding behaviour was analysed as previously described (Oakes et al. 2019). For open field tests fish were transferred individually to an opaque test tank and movement was tracked for 5 min using Zebralab software (Viewpoint, Lyon, France), this was repeated on three occasions with at least 3 days between trials. Fish were deemed to exhibit fast swimming behaviour at speeds of greater than 10 cm/s.
For in vitro fertilisation (IVF) and sperm counting, testes were dissected and lightly homogenised in a 50× mass:volume dilution of Hank’s balanced salt solution (HBSS). Eggs were obtained by gentle abdominal palpation of anaesthetised WT female fish. In total, 50 µL of sperm solution was added to a clutch of eggs, followed by 400 µL of aquarium water; after 2 min a further 2 mL of aquarium water was added (Westerfield 2000). Fertilisation was confirmed under a dissecting microscope. For sperm counting, 10 µL of sperm solution was transferred to each chamber of an improved Neubauer haemocytometer (Hawksley, Sussex, UK). A minimum of 200 sperm was counted in each chamber and the number of sperm/nL was multiplied by the dilution factor (50×) to obtain sperm counts. Gonadosomatic index was calculated using the formula GSI = (gonad weight/total tissue weight) × 100.
To assess sperm release, cyp11c1−/− mutant and WT sibling zebrafish were anaesthetised, and semen was collected by stroking the abdomen with blunt-ended forceps (Millipore), followed by aspiration of expelled fluid into a microcapillary tube and transfer to 25 µL of ice-cold HBSS (Westerfield 2000). Presence of mature sperm was confirmed by visualisation under a 20× objective.
Histology
Preparation of samples and H&E staining was performed as previously described (Oakes et al. 2019).
Gene expression analysis by quantitative PCR (qPCR)
For larval gene expression analysis, the progeny of a cyp11c1+/− incross were sorted by visual background adaption (VBA) at 4–5 dpf (Griffin et al. 2016). Larvae were housed in dark conditions for 1 h, followed by a 20 min light exposure. Larvae were subsequently sorted into lightly (VBA+) and darkly (VBA−) pigmented groups. Sorted larvae were pooled into groups of 20 and snap-frozen on dry ice. For adult gene expression analysis, fish were euthanized, dissected, and organs collected by snap freezing on dry ice.
Total RNA was isolated using Trizol (Ambion). cDNA was prepared using SuperScript II (Thermo Fisher Scientific) with 20mer oligo(dT) primers (IDT, Coralville, IA, USA) and 1 µg of RNA. GoTaq qPCR master mix (Promega) was utilised in reactions containing 1 µL cDNA synthesis product and specific primers (Supplementary Table 1) at 1000 nM. Reactions were run on a 7900HT Fast Real-Time PCR System (Applied Biosystems). Data were analysed by the Livak method (Livak & Schmittgen 2001) with ef1a as reference gene. Fold changes in gene expression are displayed relative to expression in WT male tissue.
Infertility in cyp11c1−/− mutant male zebrafish.
Allele | Genotype | Total number of crosses | Number of crosses resulting in fertilised eggs |
---|---|---|---|
11bp deletion | +/+ (n = 4) | 12 | 11 (92%) |
−/− (n = 4) | 12 | 0 (0%) | |
47bp deletion | +/+ (n = 4) | 12 | 8 (66%) |
−/− (n = 4) | 12 | 0 (0%) |
cyp11c1−/− mutant (n = 4) and WT sibling males (n = 4) were outcrossed with WT females on three separate occasions. No crosses involving cyp11c1−/− mutant males from either the 11 bp or 47 bp deletion alleles produced any fertilised embryos; their WT siblings produced fertilised embryos in 92% and 66% of crosses, respectively.
Statistical methods
Statistical analysis was performed in Graphpad Prism (GraphPad Software). Data normality was assessed using inbuilt tests. Normally distributed biometric and qPCR data were analysed using unpaired t-tests, non-normal data were analysed by Mann–Whitney tests. Behavioural data were analysed using multiple t-tests (Holm–Sidak method). Scatter plot error bars represent the s.e.m. Statistical significances are reported using asterisks: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns, not significant.
Results
Generation of cyp11c1 mutant alleles by CRISPR/Cas9
Exon 2 of cyp11c1 was targeted using the SygRNA two-part system (see ‘Methods’ section). CRISPR-injected fish were outcrossed at ~10 wpf, and heritable mutations were identified in their progeny. Several out-of-frame mutations were identified, including 11 bp (c.312_322del, p.Glu105Profs*33, allele number SH548) and 47bp (c.285_331del, p.Met96Hisfs*30, allele number SH547) deletions. Both are predicted to produce a truncated protein ~25% the size of the WT isoform (Supplementary Fig. 2). Inheritance of cyp11c1 mutant alleles did not significantly deviate from expected Mendelian ratios.
Cortisol-deficient zebrafish larvae exhibit impaired VBA – the ability to adapt pigmentation to light conditions. We found that VBA was impaired in cyp11c1−/− mutant larvae at 5 dpf, and expression of glucocorticoid responsive genes fkbp5 and pck1 was significantly decreased, suggesting reduced cortisol production due to loss of Cyp11c1 function (Supplementary Fig. 3 and Supplementary Table 2).
Proportion of cyp11c1−/− mutant and WT sibling sperm samples producing fertilised embryos in IVF experiments.
Genotype | Proportion producing fertilised embryos |
---|---|
cyp11c111bp+/+ (n = 4) | 4/4 (100%) |
cyp11c111bp−/− (n = 4) | 3/4 (75%) |
cyp11c147bp+/+ (n = 4) | 4/4 (100%) |
cyp11c147bp−/− (n = 4) | 4/4 (100%) |
Testes were dissected from male zebrafish and homogenised in a 50× mass:volume dilution of HBSS. Sperm solutions were used to fertilise eggs collected from WT females.
cyp11c1−/− mutant zebrafish exhibit female secondary sex characteristics but may possess either ovaries or testes
Morphological secondary sex characteristics in zebrafish include body shape, fin and body pigmentation, and genital papilla prominence. Male zebrafish are streamlined in shape, have palely pigmented dorsal fins, and orange striped anal fins. Female fish have a more rounded abdomen, green-yellow pigmented dorsal fins and little orange pigmentation in the anal fin (Fig. 2). Female zebrafish have a large and prominent genital papilla; in males this structure is much smaller and mostly hidden from view.
Upon raising the progeny of cyp11c1+/− incrosses, it was apparent that all homozygous mutant fish displayed predominantly female secondary sex characteristics (Fig. 2). Close inspection revealed that some cyp11c1−/− mutant fish had prominent genital papillae like WT females, whereas others had small hidden genital papillae like those of WT males. Dissection of cyp11c1−/− mutant fish revealed that they could possess either testes or ovaries, and this was accurately predicted by the presence or absence of a prominent genital papilla. The ratio of males:females (testes:ovary) in populations of WT and cyp11c1−/− zebrafish did not significantly differ.
Additionally, biometric data was also collected. Males from both cyp11c1−/− mutant lines were significantly longer and heavier than WT siblings (Fig. 2).
Adult cyp11c1−/− mutant male zebrafish exhibit profound cortisol and 11-ketotestosterone deficiency
In order to assess the impact of cyp11c1 mutation on interrenal and testicular steroidogenesis, we employed LC-MS/MS to quantify steroid concentrations in samples prepared from whole adult zebrafish males. Cortisol concentrations were profoundly decreased by mutation of cyp11c1 (Fig. 3), whereas concentrations of its precursor, 11-deoxycortisol, were significantly increased. Thus, we have demonstrated the in vivo importance of Cyp11c1 function for the conversion of 11-deoxycortisol to cortisol in glucocorticoid biosynthesis. Concentrations of the sex steroid precursor androstenedione were significantly increased in cyp11c1−/− mutant male zebrafish, probably due to blocking of the androgen synthesis pathway; shunting of glucocorticoid precursors into the sex steroid pathway may also contribute to increased androstenedione concentrations. Blockade of the androgen synthesis pathway was evidenced by undetectable concentrations of 11-ketotestosterone and its precursor 11-ketoandrostenedione (Fig. 3). Concentrations of testosterone were not affected by mutation of cyp11c1 (Fig. 3).
Expression of the glucocorticoid-responsive genes fkbp5 and pck1 (Griffin et al. 2016, Eachus et al. 2017) was significantly reduced in cyp11c1−/− mutant male zebrafish livers compared to WT siblings, thus demonstrating the systemic consequences of glucocorticoid deficiency. An apparent decrease in the expression of the proposed androgen-responsive gene cyp2k22, which is postulated to play a role in androgen metabolism, (Fetter et al. 2015, Siegenthaler et al. 2017) did not achieve statistical significance (Fig. 4).
Disruption of cyp11c1 results in infertility and impaired breeding behaviour
Androgen-resistant and androgen-deficient male zebrafish are infertile (Crowder et al. 2018, Oakes et al. 2019) and the incidence of stereotypical breeding behaviours is decreased (Yong et al. 2017). To investigate the impact of Cyp11c1-deficiency on breeding behaviour in male zebrafish, we analysed two well-characterised breeding behaviours. In all trials, the number of intimate contacts, where fish touch or cross one another, and the duration of chasing, where one fish closely follows the other, were significantly reduced in cyp11c1−/− mutant lines compared to WT siblings (Fig. 5). The proportion of trials resulting in the production of fertilised embryos was also recorded. Outcrosses of WT females and WT sibling males from the 11 and 47 bp alleles produced fertilised embryos in 92% and 66% of crosses, respectively. No fertilised embryos were observed in any crosses with cyp11c1−/− mutant zebrafish (Table 1). Despite exhibiting infertility in normal breeding scenarios, the sperm of cyp11c1−/− mutant zebrafish were able to fertilise eggs collected from WT females by IVF (Table 2).
Whilst conducting breeding experiments on cyp11c1−/− mutant zebrafish it was noted that they appeared to exhibit reduced locomotor activity. In order to quantify this, mutant and WT male zebrafish were exposed to open field tests. This revealed that the total distance swam, as well as the duration of fast swimming, was significantly and consistently reduced in cyp11c1−/− mutant males (Fig. 6). Freezing duration, the duration for which the fish was stationary in the tank, was also recorded. Freezing duration was consistently greater in trials involving cyp11c1−/− mutant zebrafish; however, these results were not statistically significant, presumably due to the extremely high variability with which fish express this phenotype (Fig. 6).
Cyp11c1 disruption results in testicular disorganisation and reduced spermatogenesis
In order to examine the impact of cyp11c1 mutations on testis morphology, we collected coronal sections through whole adult zebrafish and performed H&E staining. The testes of WT males appeared to be well organised, with defined seminiferous tubule structures clearly visible (Fig. 7). In contrast, the structure of cyp11c1−/− mutant testes was generally disorganised, with defined seminiferous tubule structures rarely in evidence. The seminiferous tubules of WT testes comprised clusters of developing spermatogonia, spermatocytes and spermatids lining the perimeter, with mature spermatozoa in the central lumen (Fig. 7). The testes of cyp11c1−/− mutant zebrafish also contained cells at all stages of spermatogenesis; however, the proportion of developing germ cells to mature sperm appeared to be much greater. This was accompanied by a qualitative reduction in the amount of mature spermatozoa; this finding was later quantified by sperm counting (Fig. 7). No difference in gonadosomatic index, the percentage contribution of the gonads to body weight, was recorded for either cyp11c1 −/− mutant allele.
Cyp11c1 is crucial for development of the spermatic duct
In zebrafish, sperm is conducted from the testes to the urogenital orifice via the spermatic duct (Menke et al. 2011). As testicular tubule structure was found to be disorganised in cyp11c1−/− mutant zebrafish, we investigated the possibility that the spermatic duct may also exhibit impaired development or maintenance.
The structure of the spermatic duct was examined ventral to the spermatogenic tissue of the testes and dorsal to the genital orifice and was found to occupy the region posterior to the intestine and anterior to the renal collecting duct (Fig. 8). The spermatic ducts of WT zebrafish comprised an extensive tubular structure, with tubules containing spermatozoa (Fig. 8A and D). In contrast, the spermatic ducts of cyp11c1−/− mutant zebrafish appeared as severely hypoplastic structures immediately posterior to the intestine. cyp11c1 −/− mutant spermatic ducts either contained no sperm (11 bp deletion: 4/5, 47 bp deletion: 2/5) (Fig. 8B and E) or existed as a slightly more extensive structure containing some mature spermatozoa (Fig. 8C and F).
In order to determine if hypoplasia of the spermatic duct resulted in impaired sperm release, we subjected cyp11c1 −/− mutant zebrafish and WT siblings to manual gamete expression (Westerfield 2000). Cyp11c1-deficient zebrafish exhibited profoundly impaired sperm release, although spermatozoa were observed in samples obtained from some cyp11c1 47bp−/− mutant zebrafish in one of two trials (Table 3).
Proportion of fish producing sperm samples during gamete expression experiments.
Genotype | Number of fish producing sperm sample | |
---|---|---|
First trial | Second trial | |
cyp11c1 11bp+/+ | 6/6 | 8/8 |
cyp11c1 11bp−/− | 0/6 | 0/7 |
cyp11c1 47bp+/+ | 6/6 | 6/6 |
cyp11c1 47bp−/− |
6/9a | 0/6 |
a2/6 samples from fish which produced sperm contained negligible sperm numbers.
Reduced expression of pro-male and spermatogenic genes in the testes of cyp11c1−/− zebrafish
Steroid hormones act via their cognate nuclear receptors to regulate gene transcription (de Waal et al. 2008). In order to understand the impact of altered steroid concentrations on gene expression in the testes, and gain insight into the molecular mechanisms underlying the observed phenotype, we used qPCR to measure the expression of genes related to gonadal function.
Igf3 and Insl3 are important factors in zebrafish spermatogenesis; specifically, they are involved in regulating the proliferation and differentiation of type A spermatogonia (Nobrega et al. 2015, Assis et al. 2016, Morais et al. 2017). Both igf3 and insl3 were significantly downregulated in cyp11c1 −/− mutant zebrafish, potentially suggesting impairment of early stages of spermatogenesis in these mutants (Fig. 9).
The expression of dmrt1 and sox9a, both of which play important roles in male sex differentiation (Sun et al. 2013, Webster et al. 2017) was unaffected by Cyp11c1-deficiency (Fig. 9). Expression of the androgen receptor, via which 11-ketotestosterone exerts its effects on gene expression, was significantly upregulated in the testes of cyp11c1 −/− mutant zebrafish (Fig. 9). This indicates a potential compensatory mechanism involving increased androgen receptor expression to scavenge for reduced androgens. Inhibins exert negative feedback on the hypothalamus-pituitary-gonadal axis, and may also play a role in Sertoli cell proliferation and spermatogenesis in vertebrates (Gregory & Kaiser 2004, Poon et al. 2009, Cai et al. 2011). We observed significant down-regulation of inha in Cyp11c1-deficient zebrafish (Fig. 9).
Expression of spermatogenesis marker genes suggests a crucial role for androgens in the differentiation of type A spermatogonia into meiotic spermatocytes
Spermatogenesis comprises a series of cell division and differentiation events whereupon spermatogonial stem cells give rise to primary spermatocytes, which then enter meiosis, and eventually give rise to mature haploid spermatozoa. Having observed a reduction in the numbers of mature spermatozoa by histology and sperm counting, we endeavoured to deepen our understanding of the impact of androgen deficiency on spermatogenesis. To this end, we measured the expression of marker genes for several stages of spermatogenesis (Fig. 10). nanos2 and piwil1 are expressed in type A spermatogonia (Beer & Draper 2013, Chen et al. 2013, Safian et al. 2016). Significantly increased expression of nanos2 was observed in cyp11c1 −/− mutant zebrafish. Increased expression of piwil1 was observed in cyp11c1 −/− mutant zebrafish carrying the 11 bp deletion allele, but not in those carrying the 47bp deletion allele (Fig. 10). No change in the expression of the type B spermatogonia marker dazl (Chen et al. 2013) was observed; however, expression of the spermatocyte marker sycp3 (Ozaki et al. 2011) and spermatid marker odf3b (Yano et al. 2008, Nobrega et al. 2015) was significantly reduced in cyp11c1 −/− mutant zebrafish, indicating a reduced proportion of cells at the later stages of spermatogenesis (Fig. 10). Taken together, these results indicate an accumulation of type A spermatogonia in cyp11c1 −/− mutant testes, due to the blockade or impairment of the transformation of spermatogonia into spermatocytes and subsequently spermatozoa.
Discussion
Herein, we described the phenotype of androgen- and cortisol-deficient cyp11c1 mutant zebrafish, paying particular attention to the roles of these steroids in the development, maintenance and function of the male reproductive system. We produced cyp11c1 −/− mutant alleles using CRISPR/Cas9 to target exon 2 of ENSDART00000185978.1 (Supplementary Fig. 2), whereas the previously published cyp11c1-mutant zebrafish line used CRISPR to target exon 3 of this transcript (Zhang et al. 2020). These mutations are predicted to produce similar truncated and functionally inactive protein products.
Cyp11c1 is important for the production of cortisol and 11-ketotestosterone (11KT), the principal zebrafish androgen (de Waal et al. 2008). Cyp11c1-deficient zebrafish exhibit profound deficiencies of both steroids, confirming the crucial role of Cyp11c1 in steroidogenesis (Fig. 3) (Zhang et al. 2020). In addition to measuring cortisol and 11KT, we measured the concentrations of several intermediate steroid hormone precursors. We observed accumulation of 11-deoxycortisol and androstenedione (Fig. 3); precursors that may enter other steroidogenic pathways such as the oestrogen biosynthetic pathway. The phenotypic impact of the shunting of steroid precursors into alternative pathways remains unknown. Unchanged concentrations of testosterone in Cyp11c1-deficient zebrafish suggest that excess androstenedione was not converted to testosterone; this provides in vivo evidence for previous in vitro findings indicating that conversion of androstenedione to testosterone, followed by 11β-hydroxylation of testosterone by Cyp11c1 to produce the 11KT precursor 11β-hydroxytestosterone, is a minor pathway to 11KT production in zebrafish (de Waal et al. 2008).
Decreased cortisol concentrations were reflected in decreased expression of the glucocorticoid responsive genes fkbp5 and pck1 (Griffin et al. 2016, Eachus et al. 2017) in cyp11c1 −/− mutant male liver tissue, demonstrating systemic glucocorticoid deficiency (Fig. 4). cyp2k22 has been proposed as an androgen-responsive gene in zebrafish (Fetter et al. 2015, Siegenthaler et al. 2017), and is robustly downregulated in the livers of androgen-deficient fdx1b −/− mutant zebrafish (Oakes et al. 2019). An apparent reduction in the expression of cyp2k22 in the livers of cyp11c1 −/− mutant zebrafish was not significant (Fig. 4). The high variability in the expression of this gene, particularly in WT fish, is a likely explanation for this finding, and suggests that it may also be regulated by other factors in addition to androgen signalling.
As with other zebrafish lines carrying mutations resulting in impaired androgen signalling, cyp11c1 −/− mutant zebrafish exhibit primarily female pigmentation patterns (Crowder et al. 2018, Zhai et al. 2018, Oakes et al. 2019). Feminisation of anal fin pigmentation appears to be to more pronounced in our study compared to Zhang et al. (2020). This variability may arise from differences in time of analysis and age of fish. In addition to analysis of the anal fin, we have also described feminisation of dorsal fin pigmentation in Cyp11c1-deficient males, this was not formally assessed in the study of Zhang et al., but appeared to be the case in the representative fish presented in their paper. Overall, these findings suggest that androgens may induce the expression of genes important for fin colour patterning during development.
Cyp11c1-deficient adult male zebrafish were infertile in normal breeding scenarios (Table 1); nevertheless, we observed that their sperm could fertilise eggs collected from WT female zebrafish by IVF (Table 2). These findings confirm the results of Zhang et al. (2020), and indicate that Cyp11c1-deficient zebrafish are able to produce mature sperm but are infertile due to another factor, such as impaired breeding behaviour or spermatogenesis, or morphological disruption of the testes or male reproductive tract resulting in impaired sperm release.
Breeding behaviours are decreased in both androgen-deficient and androgen-resistant zebrafish (Yong et al. 2017, Oakes et al. 2019). In this regard, the phenotype of our new cyp11c1 −/− mutant zebrafish lines closely resembles that of fdx1b−/− mutant zebrafish (Fig. 5) (Oakes et al. 2019). This finding also confirms similar results obtained by Zhang et al. (2020), although different behavioural assays were used. A key difference is our experimental design, in which behavioural trials were repeated several times with the same fish to control for novelty and habituation. Importantly, our results were similar irrespective of trial number, indicating that the phenotype remains the same despite habituation to the technique (Fig. 5).
In addition to analysis of breeding behaviour, we have demonstrated altered locomotor behaviour in Cyp11c1-deficient adult zebrafish. To our knowledge, this is the first time adult androgen- and glucocorticoid-deficient zebrafish have been revealed to exhibit such a behavioural phenotype. Cyp11c1-deficient male zebrafish exhibited decreased locomotor activity compared to WT siblings (Fig. 6). Decreased locomotor activity may affect readouts of multiple breeding behaviours; for example, slow swimming fish may have less opportunity for intimate contacts with their tank mate and may not be able to participate effectively in chasing behaviour. Locomotor activity and freezing behaviours have been linked to stress and glucocorticoid signalling in zebrafish; glucocorticoid receptor mutants are known to exhibit freezing behaviour and slower average swim velocities (Ziv et al. 2013). Cortisol deficiency may be responsible for the impaired locomotor behaviour seen in Cyp11c1-deficient zebrafish, but may not fully explain the reduction in breeding behaviours, as mutation of the androgen receptor also produces a similar phenotype (Yong et al. 2017). Overall, the behavioural phenotype of Cyp11c1-deficient zebrafish is likely to result from disruption of both glucocorticoid- and androgen-regulated processes.
As previously described in androgen-deficient and androgen-resistant zebrafish lines, we show that androgen signalling is dispensable for definitive testicular differentiation (Crowder et al. 2018, Oakes et al. 2019). In the other recently reported cyp11c1 −/− mutant zebrafish line, histological examination was restricted to the stage when gonadal differentiation is taking place. Zhang et al. (2020) showed that Cyp11c1-deficiency resulted in prolonged juvenile ovary-to-testis transformation, with degenerating oocytes present long after the normal period of testis differentiation. In contrast to Zhang et al., we examined the histological testicular phenotype of adult cyp11c1 −/− mutant male zebrafish. Our histological examination of adult cyp11c1 −/− mutants revealed that their testes were highly disorganised: seminiferous tubules were poorly defined, and the quantity of spermatozoa was reduced (Fig. 7). The histological appearance of cyp11c1 −/− mutant testes was similar to that described in other zebrafish models of disrupted androgen signalling (Crowder et al. 2018, Oakes et al. 2019), thus providing further confirmation that androgens are required for correct organisation and morphological development or maintenance of the testes. Tubular structure formation in the testes appears to occur during the latter stages of, or after, the period of testicular differentiation in zebrafish, as tubules are not clearly visible until well after the gonad is committed to testis development (van der Ven & Wester 2003). Overall, it appears that the crucial roles for androgens in zebrafish testicular development are temporal control of gonadal differentiation (Zhang et al. 2020), and subsequent formation and maintenance of correct seminiferous tubule organisation in the juvenile and adult testis.
We previously postulated that Sertoli cell dysfunction may be responsible for the testicular phenotype observed in androgen-deficient zebrafish; several Sertoli cell expressed genes, such as sox9a and inha, were downregulated in fdx1b −/− mutant zebrafish, which exhibit a similar phenotype to that described in the present study (Oakes et al. 2019). Sox9a may be of importance in testis tubulogenesis, as a role in this process has been proposed for this gene in a related teleost (Nakamoto et al. 2005). Sox9a expression was unaffected by mutation of cyp11c1, whereas inha was significantly downregulated (Fig. 9). The mechanism by which androgens control appropriate testis tubule morphogenesis or maintenance remains elusive, and is an exciting topic for further study.
Zhang et al. (2020) described that, despite their infertility, Cyp11c1-deficent zebrafish could produce morphologically normal spermatozoa. However, when Cyp11c1-deficient males were subjected to manual gamete expression, a reduced volume of semen was produced in comparison to WTs, indicating impaired spermatogenesis or sperm release in the cyp11c1 −/− mutants (Zhang et al. 2020). In our Cyp11c1-deficient zebrafish, whole-testes sperm counts were significantly lower than in WT siblings, also indicating impaired spermatogenesis (Fig. 7). To further investigate impaired spermatogenesis we performed novel and in-depth characterisation of spermatogenic defects in Cyp11c1-deficient zebrafish by measuring the expression of spermatogenic factors and spermatogenic stage-specific marker genes. We observed significant down-regulation of igf3 and insl3 in Cyp11c1-deficient zebrafish (Fig. 9); both genes are important for the differentiation and proliferation of type A spermatogonia (Nobrega et al. 2015, Assis et al. 2016, Morais et al. 2017). This was reflected by increased expression of nanos2, a marker for type A spermatogonia (Beer & Draper 2013, Safian et al. 2016), and decreased expression of sycp3 and odf3b, markers of later stages in spermatogenesis (Fig. 10) (Yano et al. 2008, Ozaki et al. 2011, Nobrega et al. 2015). Histological examination also suggested a qualitative increase in the proportion of developing sperm to mature sperm in the testes (Fig. 7). Taken together, these findings support the proposition that, whilst production of type A spermatogonia is normal in cyp11c1 −/− mutant testes, the subsequent differentiation of type A spermatogonia is highly androgen-dependent.
In addition to impaired spermatogonial differentiation, entry of type B spermatogonia into meiosis may also be disrupted in cyp11c1−/− mutant zebrafish. Whilst expression of the type B spermatogonia marker dazl was unchanged by mutation cyp11c1, expression of the spermatocyte marker sycp3 was downregulated, as was that of the spermatid marker odf3b (Fig. 10). Spermatogenic arrest or delay during meiosis has previously been reported in androgen receptor mutant zebrafish (Yu et al. 2018). Sycp3 is a component of the synaptonemal complex, which plays important roles during meiotic prophase, including regulation of chromosome recombination (Page & Hawley 2004, Syrjänen et al. 2014). Thus, reduced expression of sycp3 in cyp11c1−/− mutant spermatocytes could contribute to the impairment of meiosis.
Expression of a small number of genes was characterised at the adult stage in the previously published cyp11c1 −/− mutant zebrafish line (Zhang et al. 2020). Both the previous mutant and the mutant described herein exhibited decreased expression of the spermatogenic factor insl3. In contrast to observations described by Zhang et al., no change of dmrt1 expression was observed in our cyp11c1 −/− mutants (Fig. 9), which is consistent with our previously reported findings in androgen- and cortisol-deficient fdx1b −/− mutants (Oakes et al. 2019).
Attempts to manually collect semen from Cyp11c1-deficient zebrafish revealed that sperm release may be impaired. However, this technique may not accurately replicate natural ejaculation, and therefore, successful sperm release in natural breeding conditions cannot be ruled out. As sperm release appeared to be impaired, we investigated the structure of the spermatic duct and found that it to be severely hypoplastic in Cyp11c1-deficient zebrafish. Little is known about the development of this structure; however, we have shown here for the first time that its development is highly steroid dependent, and this is likely to be mediated by 11-ketotestosterone. This structure may be comparable to Wolffian duct structures in mammals, however these structures are of different embryological origins and may be analogous in function alone (Shaw & Renfree 2014, Matthews et al. 2018). Nevertheless, both structures appear to be highly dependent on androgens for their development, as abnormal Wolffian duct structures are frequently seen in complete androgen insensitivity syndrome (Hannema et al. 2006, Barbaro et al. 2007). Wolffian duct structures are also absent in AR knock-out mice (Yeh et al. 2002).
Herein, we have described novel zebrafish lines carrying mutation of cyp11c1, which is crucial for 11-ketotestosterone and cortisol biosynthesis. In addition to confirming several results described in a recently published cyp11c1 zebrafish mutant (Zhang et al. 2020), our study describes novel phenotypes, including testicular disorganisation, hypoplastic spermatic ducts, and impaired locomotor function, as well as characterising spermatogenic defects through the measurement of marker gene expression. As such, our work represents a significant and novel contribution to the literature regarding the roles of steroids in regulation of zebrafish reproduction. cyp11c1 −/− mutant zebrafish exhibit a phenotype characteristic of androgen deficiency and represent a novel tool for the investigation of the roles of androgens in male reproductive development and function. The discovery that androgens are essential for spermatic duct morphogenesis in zebrafish is a particularly exciting finding, and will pave the way for further research into this poorly characterised structure.
Supplementary materials
This is linked to the online version of the paper at https://doi.org/10.1530/JOE-20-0160.
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 work was supported by the International Fund Congenital Adrenal Hyperplasia 2017 research grant (to N P K and V T C) and the Deutsche Forschungsgemeinschaft (KR 3363/3-1).
Author contribution statement
V T C and N P K contributed equally to this work.
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