Fertility impairment with defective spermatogenesis and steroidogenesis in male zebrafish lacking androgen receptor

Fertility impairment with defective spermatogenesis and steroidogenesis in male zebrafish lacking... Abstract The pivotal role of androgen receptor (AR) in regulating male fertility has attracted much research attention in the past two decades. Previous studies have shown that total AR knockout would lead to incomplete spermatogenesis and lowered serum testosterone levels in mice, resulting in azoospermia and infertility. However, the precise physiological role of ar in controlling fertility of male fish is still poorly understood. In this study, we have established an ar knockout zebrafish line by transcription activator-like effectors nucleases. Homozygous ar mutant male fish with smaller testis size were found to be infertile when tested by natural mating. Intriguingly, a small amount of mature spermatozoa was observed in the ar mutant fish. These mature spermatozoa could fertilize healthy oocytes, albeit with a lower fertilization rate, by in vitro fertilization. Moreover, the expression levels of most steroidogenic genes in the testes were significantly elevated in the ar mutants. In contrast, the levels of estradiol and 11-ketotestosterone (11-KT) were significantly decreased in the ar mutants, indicating that steroidogenesis was defective in the mutants. Furthermore, the protein level of LHβ in the serum decreased markedly in the ar mutants when compared with wild-type fish, probably due to the positive feedback from the diminished steroid hormone levels. Introduction Androgens are among the main hormones involved in normal male reproduction in mammals [1–3]. Extensive evidence suggests the roles of androgens in spermatogenesis, in the development of secondary sex characters, and in the expression of reproductive behaviors. Testosterone alone could maintain spermatogenesis in vitro [4]. Disruption of androgen production by hypophysectomy [5], Leydig cell ablation [6], and knockout of luteinizing hormone receptor (LHR) [7] demonstrated that spermatogenesis cannot proceed to completion under these circumstances. These studies provide strong evidence that androgens are critical in maintaining the normal function of the male reproductive system [1,8–10]. Nearly all the biological actions of androgens are mediated by the specific androgen receptor (AR, NR3C4), a member of the nuclear receptor gene superfamily [11–14]. Androgen and AR have been shown to play important roles in normal male fertility [10,15]. In males, AR has been detected in Sertoli cells, Leydig cells, peritubular myoid cells and vascular smooth cells in mature testis [16,17]. It is widely accepted that spermatogenesis depends highly on autocrine and paracrine communication among these different cell types. For instance, the action of testosterone on spermatogenesis is mediated via AR on these cell types. The development of the reproductive tract and the gonad as well as sexual behavior is severely impaired in ARKO male mice. The fact that spermatogenesis in ARKO male mice is arrested at different stages [18–27] suggests a role of ar for the maintenance of a normal male reproduction in mammals. Androgens also play pivotal roles in controlling reproduction in teleosts. Two AR subtypes (arα and arβ) have been identified in fish and they are all predominantly expressed in the gonad, suggesting their functional roles on reproduction [28–30]. In addition, the plasma levels of androgens (testosterone, T; 11-ketotestosterone, 11-KT) increase gradually during the later stages of spermatogenesis in most teleost species [31–33]. Moreover, androgen treatment induces spermatogenesis in a number of teleost species. Treatment with 11-KT induced precocious spermatogonial proliferation and meiosis as well as development of secondary sexual characteristics in African catfish [34]. Using a long-term testis tissue culture system, it was shown that 11-KT could promote full spermatogenesis in the Japanese eel [35]. In prepubertal trout, androgen treatment affects the expression of a series of genes involved in spermatogenesis and steroidogenesis. Various transcription factors important for testis development were regulated by androgen treatment [33]. Although the important role of androgen and ar in male fertility of teleosts has been recognized, the precise roles of ar in male fertility are still not fully defined. To address this issue, we undertook this study to analyze the function of ar in zebrafish using transcription activator-like effectors nucleases (TALENs). Our in vivo genetic data provide valuable information for a more thorough understanding of the functional role of ar in vertebrates. Materials and methods Zebrafish husbandry All zebrafish (Danio rerio) were reared and kept in a circulated water system with a 14 h light and 10 h dark cycle at 28°C. The larval and adult zebrafish were fed with brine shrimp (hatched from 20 g eggs in 4 L salt water) twice daily. All animal experiments were conducted in accordance with the guidelines and approval of the respective Animal Research and Ethics Committees of Sun Yat-Sen University. Generation of ar gene mutant zebrafish using TALENs To obtain the zebrafish mutant lines, specific TALEN target sites were designed in the first exon of ar. Paired TALENs were constructed using the Golden Gate TALEN Kit (Addgene, Cambridge, MA, USA) as described previously [36,37]. Approximately 200–500 pg TALEN mRNAs were microinjected into one-cell stage zebrafish embryos, which were then incubated at 28.5°C. Two days after injection, 8–10 embryos were collected for DNA extraction to check whether the targeted genomic fragment was deleted. The target genomic regions were amplified by PCR and subcloned into the pTZ57R/T vector (Fermentas). Single colonies were genotyped by sequencing. To obtain germline mutations, the TALEN-injected embryos were raised to adulthood and the P0 founders were outcrossed with the wild-type fish. The F1 progeny were genotyped by sequencing. To obtain homozygous mutants, heterozygous mutant with the same mutation were obtained and self-crossed. The primers used in this study are listed in Supplemental Table 1. PCR genotyping To assess TALEN-induced mutations of the ar target region, PCR genotyping was performed. Genomic DNA extracted by phenol-chloroform method from the embryo or tail fin of F1 or F2 littermates of a heterozygote cross was employed for PCR. The PCR was conducted with specific primers as listed in Supplemental Table 1. PCR products were sequenced to resolve and identify the mutations. Morphological and histological analyses of the zebrafish mutant lines Morphological and histological analyses were performed as described before [38,39]. Briefly, the gross morphology of adult fish was analyzed at 90 days post fertilization (dpf). Fish were first anesthetized by submersion in 250 mg/L MS-222, and images were taken using a digital camera. Total body length and body weight were measured for each fish, respectively. Then, the testis was isolated from the body cavity for histological examination. The gonadsomatic index (GSI) was calculated as (gonad weight/body weight) × 100%. For gonad histology, the testis samples were fixed in Bouin's solution overnight at room temperature and then immediately dehydrated in a graded series of ethanol, immersed in xylol and finally embedded in paraffin wax. The samples were serially cut into 2–3 μm sections on a Leica microtome. After rehydration, the sections were stained with hematoxylin and eosin and mounted with Canada balsam (Sigma-Aldrich) for microscopic examination. Fertility assessment The fertility of the mutant fish was assessed by natural mating with wild-type females in a spawning tray. At 1 h before the end of the light period, adult male zebrafish (wild-type or homozygotes) were transferred to a breeding aquarium and mate with one wild-type female fish. One hour after light on in the next morning, the embryos were collected by siphoning the bottom of the tank. The number of spawned eggs produced by each female fish was recorded. Individuals that failed to spawn after at least 12 trials were considered infertile [40]. Sperm quality assessment Intact testis from adult zebrafish were carefully dissected out after anesthetization and decapitation, then placed in a tube containing 20 μL zebrafish sperm immobilizing solution (140 mM NaCl, 10 mM KCl, 2 mM CaCl2, and 20 mM HEPES titrated to pH 8.5 with 1.0 M NaOH) on ice, and homogenized by gently grinding the tube with a pipette tip. Samples were kept on ice and used within 1 h. To activate the sperms, 1 μL of semen suspension in zebrafish sperm immobilizing solution was mixed with 20 μL of aged tap water and quickly applied into a single well of a 12-well multitest slide (MP Biomedicals). The slides and coverslips were coated with 1% (wt/vol) polyvinyl alcohol to reduce sticking of sperms. Sperm quality was assessed using CASA (computer-assisted sperm analysis). Fertilization assay by in vitro fertilization Female zebrafish was selected for egg collection using a net to place it gently in a dish after anesthetization. The eggs were squeezed out from the abdomen by stroking the belly gently with fingers. Eggs were scooped out with a fine probe from the belly surface. The fish was then gently lifted and returned to the tank. Ten microliters of the semen suspension were added to the clutch of eggs, and then mixed with 50 μL of aged tap water. The mixture was then placed at 28°C for subsequent incubation. The number of fertilized/unfertilized eggs was discerned under a dissecting microscope at 4 h post fertilization. At least 50 embryos from each replicate were scored. For each fish line, the fertilization rate was accessed from five matured males and five wild-type females in three independent crosses. RNA isolation and real-time PCR Total RNA was extracted from the testis of zebrafish using TRIzol reagent (Invitrogen). The cDNA was produced from 500 ng total RNA using ReverTra Ace α-first strand cDNA Synthesis Kit (TOYOBO, Japan), and used as template in the subsequent real-time PCR analyses. The specific primers used in this study are listed in Supplemental Table 1. The transcription levels of the target genes were measured using the SYBR Green PCR Master Mix Kit (ABI, USA) carried out on an ABI Real-Time PCR Fast System (ABI, USA). The quantitative RT-PCR conditions were as follows: denaturation at 95°C for 5 min, followed by 40 cycles of 95°C for 15 s, 58°C for 15 s, 72°C for 15 s, and then 84°C for 10 s (fluorescent data collection). All mRNA quantification data were normalized to ef1a and expressed as fold differences of target gene expression relative to the control. Whole-body steroid determination Whole-body steroid determination was performed as described previously [41]. Whole-body extractions of steroids were performed to measure the amount of estradiol in each fish. First, the male fish (n = 6) were anesthetized by submersion in 250 mg/L MS-222, weighed on an analytical balance, and then homogenized in 3 mL phosphate-buffer saline (PBS) in glass tubes using a Polytron homogenizer. Steroids were extracted by adding 5 mL diethyl ether to each tube followed by vortexing for 1 min, then centrifuging at 3000 g for 2 min. Thereafter, the tubes were frozen in a methanol/dry ice bath at –30°C, and the ether layer containing the steroids was poured off into another set of tubes. The diethyl ether was then evaporated off under a flow of air. The extraction procedure was repeated twice to enhance the extraction efficiency and the extract was dissolved in PBS. The amount of each steroid (estradiol and 11-KT) was determined using Enzyme-linked immunosorbent assay (ELISA) according to the manufacturer's protocol (Cayman Chemical Company, Ann Arbor, MI, USA). All standards and samples were run in triplicates. Western blot analyses Western blot analysis was performed as previously described with slight modifications [42]. Briefly, the pituitary samples were collected from male fish (90 dpf). The samples were lysed in 60 μl cell lysis reagent (Sigma, USA) with proteinase inhibitor cocktail (1:100; Sigma, USA), mixed with 15 μL of 5× DualColor Protein Loading Buffer (Fudebio-tech) and heated at 100°C for 10 min. For the serum samples, the fish were first anesthetized by submersion in 250 mg/L MS-222, then blood was gently aspired from the heart using a 10-μL tip and centrifuged (10 000 g, 30 min, 4°C) after being held in a 4°C environment for half an hour. The supernatant was transferred to a new tube, and then diluted in PBS to stated concentrations. Thereafter, the solution was mixed with 5× DualColor Protein Loading Buffer and heated at 100°C for 10 min. The lysates or serum samples were first separated on a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel. The separated proteins were then transferred onto poly vinylidene fluoride (PVDF) membranes (Pall, USA). The membranes were blocked with 5% bovine serum albumin (BSA) in Tris-buffered saline with 0.05% Tween 20 (TBST) and then cut into two halves according to the visible marker bands (25 and 35 KD). The membranes containing the larger Mw proteins were incubated with GAPDH mouse monoclonal primary antibodies (1:4000, Proteintech, RRID: AB_2107436), and the membranes containing smaller Mw proteins were incubated with FSHβ (follicle-stimulating hormones, 1:3000, RRID: AB_2651068) or LHβ (luteinizing hormones, 1:6000, RRID: AB_2651067) rabbit polyclonal primary antibodies, as previously reported [42]. The membranes were blocked for 30 min in 5% BSA in TBST prior to the addition of the primary antibody and then incubated with secondary antibodies conjugated to horseradish peroxidase, the protein bands were visualized using enhanced chemiluminescence reagents (ThermoFisher, USA). The bands were analyzed semi-quantitively by densitometry using the ImageJ software (grayscale analysis) and normalized to their loading controls. All western blot analyses were performed twice. Statistical analyses All data were expressed as mean values ± SEM, and analyzed by Student's t-test using the GraphPad Instat software (GraphPad Software, USA). P < 0.05 was considered statistically significant. All experiments were performed at least two times to confirm the results. Results Generation of ar gene knockout zebrafish line To study the role of androgens in male fertility, the ar mutant line was generated using TALENs. The target sequences were designed based on the sequence of ar provided in NCBI. A pair of TALEN-binding sites was chosen within the first exon (Figure 1A). The mutated genotype with a 13-bp deletion was further used to establish the homozygous mutant line in this study (Figure 1A). Successful ar deletion was confirmed by sequencing (Figure 1B). This mutation could induce an open reading frame (ORF) shift, generating a truncated AR protein lacking the DNA-binding domain, hinge domain, and the ligand-binding domain (Figure 1C) [43]. Figure 1. View largeDownload slide Targeted disruption of the ar locus in zebrafish using TALENs. (A) TALEN target site and the mutated genotype. The TALEN-binding site was chosen within the first exon. A genotype with an 8-bp deletion was used to establish the ar mutant line. (B) Sanger sequencing analysis of the 8-bp deletion genotype. (C) Schematic representation of the protein functional domains of AR from the WT and the mutant. TAD: transactivation domain; DBD: DNA-binding domain; HD: hinge domain; LBD: ligand binding domain. Figure 1. View largeDownload slide Targeted disruption of the ar locus in zebrafish using TALENs. (A) TALEN target site and the mutated genotype. The TALEN-binding site was chosen within the first exon. A genotype with an 8-bp deletion was used to establish the ar mutant line. (B) Sanger sequencing analysis of the 8-bp deletion genotype. (C) Schematic representation of the protein functional domains of AR from the WT and the mutant. TAD: transactivation domain; DBD: DNA-binding domain; HD: hinge domain; LBD: ligand binding domain. Phenotypic characterization of ar −/− male zebrafish The ar mutant line was viable and developed normally. The gross morphology of adult male fish was analyzed at 90 dpf. As showed in Figure 2A, male ar mutant fish exhibited female-like appearance. The testis size was significantly decreased in the homozygotes (Figure 2A), while the body length and overall body weight were not affected (Figure 2B and C). Accordingly, a significant reduction in the GSI was found in the homozygous mutants (Figure 2D). Mating between homozygous ar mutant males and wild-type females failed to induce spawning, indicating that ar-deficient males were infertile (Figure 2E). These results suggested that both testis development and fertility were impaired in male zebrafish lacking ar. Figure 2. View largeDownload slide Phenotypic characterization of the ar−/− male zebrafish. (A) Gross and gonad morphology of the ar+/+ and ar−/–fish. The testis size was significantly decreased in the homozygotes. (B–D) Body length (B), body weight (C), and GSI (D) of the ar+/+ and ar−/–fish. (E) The reproductive capacity of males from the indicated genotypes crossed with wild-type females (n = 12). Each value represents the mean value ± SEM (n = 6) (***, P < 0.001 vs control). Figure 2. View largeDownload slide Phenotypic characterization of the ar−/− male zebrafish. (A) Gross and gonad morphology of the ar+/+ and ar−/–fish. The testis size was significantly decreased in the homozygotes. (B–D) Body length (B), body weight (C), and GSI (D) of the ar+/+ and ar−/–fish. (E) The reproductive capacity of males from the indicated genotypes crossed with wild-type females (n = 12). Each value represents the mean value ± SEM (n = 6) (***, P < 0.001 vs control). Testicular histology, sperm morphology, and sperm quality in the ar mutant fish To further analyze the causation of fertility defects observed in the male ar mutants, histological and sperm morphology assessment was performed on the testis sections from male fish at 90 dpf. The overall testicular size was reduced in the ar mutant males when compared with wild-type males (Figure 3A). Histological analysis from hematoxylin and eosin (H&E) staining revealed that germ cell development was severely disrupted. A pronounced reduction in the number of germ cells was observed in the ar mutant fish and most of the germ cells were blocked at the early development stages. Interestingly, a small amount mature spermatozoon could still be found in the ar mutants. In addition, these spermatozoa from the ar mutants were viable and their morphology was also normal (Figure 3A). The sperm quality was assessed and no significant difference in sperm motility (Figure 3B), average curvilinear velocity (VCL) (Figure 3C) and average straightness (STR) (Figure 3D) were observed in both genotypes. Finally, fertilization assays were performed by in vitro fertilization. Our data showed that sperm obtained from the ar mutants could fertilize healthy oocytes, albeit with a low fertilization rate (Figure 3E). Overall, it implies that ar plays a pivotal role in normal spermatogenesis in zebrafish. Figure 3. View largeDownload slide Testicular histology, sperm morphology, and sperm quality of the ar +/+ and ar −/− male zebrafish at 90 dpf. (A) Testicular histology and sperm morphology of adult male zebrafish of ar +/+ and ar −/–genotypes. A pronounced reduction in germ cell number was observed in the ar mutant fish, while a very small number of mature spermatozoa of normal morphology could still be found in the ar mutant fish. (B–D) Sperm motility (B), VCL (C), SRT (D) in adult male zebrafish of the indicated genotypes. (E) Fertilization rate of sperm obtained from the ar mutant fish performed by in vitro fertilization. sz: Spermatozoa; VCL: average curvilinear velocity; STR: average straightness. Each value represents the mean value ± SEM (n = 6) (***, P < 0.001 vs control). Figure 3. View largeDownload slide Testicular histology, sperm morphology, and sperm quality of the ar +/+ and ar −/− male zebrafish at 90 dpf. (A) Testicular histology and sperm morphology of adult male zebrafish of ar +/+ and ar −/–genotypes. A pronounced reduction in germ cell number was observed in the ar mutant fish, while a very small number of mature spermatozoa of normal morphology could still be found in the ar mutant fish. (B–D) Sperm motility (B), VCL (C), SRT (D) in adult male zebrafish of the indicated genotypes. (E) Fertilization rate of sperm obtained from the ar mutant fish performed by in vitro fertilization. sz: Spermatozoa; VCL: average curvilinear velocity; STR: average straightness. Each value represents the mean value ± SEM (n = 6) (***, P < 0.001 vs control). The mRNA level of selected testicular genes in the double mutants To understand the mechanism of spermatogenetic defects caused by ar deficiency, we have selected a number of spermatogenesis-related genes as candidates for real-time PCR analyses. We found that the genes expressed in the type Aund spermatogonia, spermatogonia, and spermatocyte such as nanos2, piwil1, piwil2, dazl, and sycp3l were elevated in the mutant testis relative to the wild-type (Figure 4A–E). In contrast, the expression of odf3b, a gene expressed in spermatid, was significantly decreased. The genes expressed in Sertoli cells such as amh and gsdf were significantly upregulated (Figure 4G and H), while the expression of igf3 was significantly downregulated in the ar mutants (Figure 4I). The gene expressed in Leydig cells, such as insl3, significantly decreased in the mutants (Figure 4J). We also analyzed the mRNA level of cx43 (an important gap junction protein that participates in the control of cell proliferation) and found that its expression was significantly downregulated in the mutants (Figure 4K). Taken together with the histological analyses, the number of type Aund spermatogonia, spermatogonia, spermatocyte increased, whereas the number of spermatid and proliferative cells decreased in the ar mutants when compared with wild-type zebrafish, suggesting that spermatogenesis was defective in the ar mutants. Figure 4. View largeDownload slide The mRNA level of selected testicular genes in the wild-type and mutant fish at 90 dpf. (A–K) The relative expression of spermatogenesis-related genes in the testis of wild-type and mutant lines. The mRNA levels were normalized to that of ef1a, with the level in the ar−/– fish expression as the fold change of the level in the ar+/+ fish. Each value represents the mean value ± SEM. (*, P < 0.05; **, P < 0.01; ***, P < 0.001 vs control). Figure 4. View largeDownload slide The mRNA level of selected testicular genes in the wild-type and mutant fish at 90 dpf. (A–K) The relative expression of spermatogenesis-related genes in the testis of wild-type and mutant lines. The mRNA levels were normalized to that of ef1a, with the level in the ar−/– fish expression as the fold change of the level in the ar+/+ fish. Each value represents the mean value ± SEM. (*, P < 0.05; **, P < 0.01; ***, P < 0.001 vs control). Defective steroidogenesis in ar mutant males In order to determine whether endocrine abnormalities might underlie or contribute to the ar mutant fish phenotype, the expression profile of the steroidogenic-related genes was analyzed. Real-time PCR quantification revealed that testicular star, cyp11a1, cyp11a2, cyp17a1, hsd3β1, hsd3β2, hsd17β3, and hsd11β1 mRNA levels were increased (Figure 5A–D, F, G, I and J), whereas hsd17β1 mRNA level was decreased (Figure 5H) in the ar mutants when compared with the age-matched wild-type fish. But no significant alteration in testicular mRNA levels of cyp17a1, hsd11β2, and cyp19a1a was observed in the ar mutant males when compared with wild-type males (Figure 5E, K and l). Hormone assays showed that estradiol and 11-KT levels were lower in the ar mutants (Figure 5M and N). These data indicated that the steroidogenesis pathway was affected in the ar mutant males (Figure 5O). Figure 5. View largeDownload slide Defective steroidogenesis in the ar mutant males. (A–L) The relative expression of steroidogenic pathway genes in the testis of ar+/+ and ar−/– fish. The mRNA levels were normalized to that of ef1a, with the level in the ar−/– fish expression as the fold change of the level in the ar+/+ fish. (M and N) Whole-body estradiol (M) and 11-KT (N) levels. (O) Summing of the expression profile of steroidogenic pathway genes in the ar deficient zebrafish. Each value represents the mean value ± SEM. (*, P < 0.05; **, P < 0.01; ***, P < 0.001 vs control). Figure 5. View largeDownload slide Defective steroidogenesis in the ar mutant males. (A–L) The relative expression of steroidogenic pathway genes in the testis of ar+/+ and ar−/– fish. The mRNA levels were normalized to that of ef1a, with the level in the ar−/– fish expression as the fold change of the level in the ar+/+ fish. (M and N) Whole-body estradiol (M) and 11-KT (N) levels. (O) Summing of the expression profile of steroidogenic pathway genes in the ar deficient zebrafish. Each value represents the mean value ± SEM. (*, P < 0.05; **, P < 0.01; ***, P < 0.001 vs control). The protein levels of LHβ and FSHβ in the pituitary and serum The protein levels of LHβ and FSHβ in the pituitary gland and serum of male zebrafish were analyzed using western blot. It was found that the expression levels of LHβ and FSHβ in the pituitary gland were not significantly altered in the ar mutant male fish (Figure 6A–C). The expression levels of LHβ and FSHβ in the serum were subsequently analyzed (Figure 6D and E). No change was observed in serum FSHβ, whereas serum LHβ decreased markedly in the ar mutants when compared with the age-matched wild-type fish (Figure 6F and G). Thereafter, the mRNA levels of lhr, fshr, and ers in the testis were analyzed by real-time PCR. It was found that the mRNA level of lhr was significantly downregulated in the ar mutants, while fshr did not vary markedly (Figure 6H and I). In addition, of the three er genes in zebrafish, the esr2b gene but not esr1 and esr2a exhibited significant upregulation in the testis of ar mutants (Figure 6j–L). Finally, we analyzed the expression of several neuroendocrine factors including kiss1, kiss2, gnrh2, gnrh3, gnih, th1, and th2, in the brain by real-time PCR. As showed in Supplemental Figure S1, th2 was significantly decreased in the ar mutants (Supplemental Figure S1a–g). The expression levels of the three esr genes in the brain were further analyzed and on significant changes were detected (Supplemental Figure S1h–j). Figure 6. View largeDownload slide Western blot of LHβ and FSHβ in the pituitary gland and serum from ar+/+ and ar−/− fish. (A) Western blot for LHβ and FSHβ in the pituitary gland from ar+/+ and ar−/− fish. (B and C) Densitometric analysis of blot on expression of LHβ (B) and FSHβ (C) in the pituitary gland from ar+/+ and ar−/− fish. (D and E) Western blot for LHβ (D) and FSHβ (E) in the serum from the ar+/+ and ar−/− fish. Ponceau protein stain of the transfer membrane indicating approximately equal loading across the gel. (F and G) Densitometric analysis of blot on expression of LHβ (F) and FSHβ (G) in the serum from ar+/+ and ar−/− fish. (H and I) The relative expression of lhr (H) and fshr (I) in the testis of ar+/+ and ar−/- fish. (j–l) The relative expression of esr1 (J), esr2a (K), and esr2b (L) in the testis of ar+/+ and ar−/- fish. Western blot was performed twice. Each value represents the mean value ± SEM. (*, P < 0.05; **, P < 0.01). Figure 6. View largeDownload slide Western blot of LHβ and FSHβ in the pituitary gland and serum from ar+/+ and ar−/− fish. (A) Western blot for LHβ and FSHβ in the pituitary gland from ar+/+ and ar−/− fish. (B and C) Densitometric analysis of blot on expression of LHβ (B) and FSHβ (C) in the pituitary gland from ar+/+ and ar−/− fish. (D and E) Western blot for LHβ (D) and FSHβ (E) in the serum from the ar+/+ and ar−/− fish. Ponceau protein stain of the transfer membrane indicating approximately equal loading across the gel. (F and G) Densitometric analysis of blot on expression of LHβ (F) and FSHβ (G) in the serum from ar+/+ and ar−/− fish. (H and I) The relative expression of lhr (H) and fshr (I) in the testis of ar+/+ and ar−/- fish. (j–l) The relative expression of esr1 (J), esr2a (K), and esr2b (L) in the testis of ar+/+ and ar−/- fish. Western blot was performed twice. Each value represents the mean value ± SEM. (*, P < 0.05; **, P < 0.01). Table 1. Comparison of reproductive phenotypes in T-AR−/y, G-AR−/y, S-AR−/y, L-AR−/y, PM-AR−/y mice and ar−/− zebrafish.   Overall fertility  Testis size  Mutant testis size/WT testis size(%)  Sperm count  Testosterone/11-KT level  LH (Serum)  FSH (Serum)  References    T-AR−/y  Infertile  Decreased  7  No epididymis  Decreased  Elevated  Elevated  [21, 22, 23, 26]    AR−/y  Infertile  Decreased  23.40  No sperm  Decreased  Elevated  Normal  [19, 20, 22, 23, 25, 26]  Mice  L-AR−/y  Infertile  Decreased  31.10  No sperm  Decreased  Elevated  Elevated  [23, 24]    PM-AR−/y  Normal fertility  Decreased  79.00  Decreased by 57% of WT sperm count  Normal  Normal  Normal  [27]    G-AR−/y  Normal fertility  Normal  Normal  Within normal range  Normal  Normal  Normal  [23]  Zebrafish  ar−/−  Infertile  Decreased  20.88  Very few sperm with a low fertilization rate  Decreased  Decreased  Normal  Present study    Overall fertility  Testis size  Mutant testis size/WT testis size(%)  Sperm count  Testosterone/11-KT level  LH (Serum)  FSH (Serum)  References    T-AR−/y  Infertile  Decreased  7  No epididymis  Decreased  Elevated  Elevated  [21, 22, 23, 26]    AR−/y  Infertile  Decreased  23.40  No sperm  Decreased  Elevated  Normal  [19, 20, 22, 23, 25, 26]  Mice  L-AR−/y  Infertile  Decreased  31.10  No sperm  Decreased  Elevated  Elevated  [23, 24]    PM-AR−/y  Normal fertility  Decreased  79.00  Decreased by 57% of WT sperm count  Normal  Normal  Normal  [27]    G-AR−/y  Normal fertility  Normal  Normal  Within normal range  Normal  Normal  Normal  [23]  Zebrafish  ar−/−  Infertile  Decreased  20.88  Very few sperm with a low fertilization rate  Decreased  Decreased  Normal  Present study  T-AR−/y, Total AR knockout; S-AR−/y, Sertoli cell-specific AR knockout; L-AR−/y, Leydig cell-specific AR knockout; PM-AR−/y, PMcell-specific AR knockout; G-AR−/y, Germ cell-specific AR knockout View Large Discussion Unlike the mammalian counterparts, the functional roles of ar in teleosts are not fully understood. Using TALENs, we have undertaken the present study to evaluate the function and importance of ar in zebrafish. Our data demonstrate that fertility is impaired with defective spermatogenesis and steroidogenesis in male zebrafish lacking AR. Naturally occurring androgens as well as synthetic androgens are potential ligands for ARs [44]. In fish, the principal androgens are testosterone and 11-KT, which had been shown to bind and activate fish ar in vitro [44–46]. Different from the single copy of mammalian nuclear AR gene, many teleost species possess two ar genes (arα and arβ) [28–30], arising from a teleost-specific genome duplication that occurred in the teleost lineage [47–49]. However, only a single copy of functional ar gene is found in zebrafish [45]. Actually, species such as zebrafish that possess only a single copy ar gene cannot help to elucidate the potential role complete role of ars in teleosts since many species possess two ars. As a first step, we have utilized the convenient zebrafish model because of the ease of genome-editing. As demonstrated in this study, the ar mutant males possess testes of diminished size. The testis mass was reduced by approximately 80% in the ar mutant males when compared with wild-type fish, and all male mutants were infertile. These findings are in agreement with ARKO models in mice [18–24]. These phenotypes indicate that the function of ar is well conserved from fish to mammals. To further examine whether spermatogenesis proceeded normally, histological examination of the testis was performed. It was found that loss of the ar has resulted in reduction in sperm count in the ar mutant zebrafish. Despite the presence of some sperms in the small testis, the mutant fish were infertile. This is very different from previous studies in mice that a reduced sperm count is associated with normal fertility [27,50–53]. Furthermore, ar mutant fish did produce some viable spermatozoa of normal motility, and these sperms were able to fertilize healthy oocytes although with a lower fertilization rate. It has been reported previously that androgens could mediate its action through estrogen receptors [54]. Indeed, we have detected the expression of esr genes in the testis and found that esr2b was significantly upregulated in the ar mutants, suggesting that compensation of androgen by cross-activating ER may exist in the male mutants. As a matter of fact, ar is not the only AR in the zebrafish. Membrane AR (mAR, ZIP9) [55,56] may have a compensation effect as a result of ar disruption. Moreover, the G protein-coupled receptor family C group 6 subtype A (GPRC6A) is functionally important in regulating the nongenomic actions of androgens in mice [57], indicating that androgens might exert their function by binding to and activating other receptors or factors. The finding of the present study in zebrafish is different from the phenotype in AR null mice in which spermatogenesis was arrested in the pachytene spermatocyte stage [21,58]. We have analyzed a series of genes expressed in the germ cells, Sertoli cells and Leydig cells. In concordance with the testicular histology, the genes expressed in spermatogonia and spermatocytes were upregulated, while the genes expressed in the spermatids were downregulated in the ar mutant fish. Moreover, the transcript level of amh, a gene known to inhibit the differentiation of spermatogonia, was upregulated. In contrast, the transcript level of igf3, a gene known to stimulate spermatogenesis, was downregulated. Taken together, spermatogenesis is severely impaired in the ar mutant zebrafish, in spite of the presence of a small number of viable spermatozoa. Spermatogenesis (the production of haploid germ cells) and steroidogenesis (the production of steroid hormones that support male reproductive development and function) are the major functions of the testes. Both of them are regulated in a highly coordinated manner [10]. The expression profile of the steroidogenic genes was also investigated. It was found that the expression levels of most steroidogenic genes were upregulated, concordant with what was reported in ARKO mice [26]. However, the levels of steroid hormones such as estradiol and 11-KT were significantly decreased in the ar mutants, probably explaining dramatically the small size of the testes in the mutants. In addition, 11-KT is one of the factors involved in the initiation of spermatogonial proliferation [59,60]. The low level of 11-KT also caused the pronounced reduction in the number of germ cells in the testes of the ar mutants. Furthermore, AR antagonists (flutamide and vinclozolin) also impact the reproductive process via multiple pathways related to steroidogenesis, spermatogenesis, and fertilization [61]. Therefore, the mutant fish line generated in this study is a valuable resource for investigating compounds with endocrine disrupting potential. We further observed that the protein levels of LHβ and FSHβ in the pituitary gland were not significantly affected in the ar mutant males, despite the expression of th2, code for rate-limiting enzyme in dopamine biosynthesis [62], was significantly decreased in the ar mutant males. Interestingly, both the protein level of LHβ in the serum and mRNA level of lhr in the testis were reduced in the ar mutant males, probably due to the strong positive feedback from the diminished steroid hormones levels. In fact, previous work has demonstrated that positive feedback control of LHβ (GTH-II) release is a common feature in adult teleost [63,64]. Moreover, we found that the mRNA levels of kiss1, kiss2, gnrh2, and gnrh3 were not markedly altered in the ar mutants when compared with wild-type fish. Additionally, our previous studies have demonstrated that double knockout of kiss1; kiss2 and triple knockout of kiss1; kiss2; gnrh3 exhibited no defect in the reproduction of zebrafish [38,65]. Likewise, ontogeny and reproductive activity were not altered upon disruption of gnrh2; gnrh3 in zebrafish [66]. Furthermore, a very recent study from our team revealed that E2 could directly act on the pituitary level to stimulate LHβ expression during puberty in zebrafish [67]. Taken together, it can be concluded that steroid hormones can directly regulate LHβ of the pituitary level in a kiss; gnrh system independent manner via a positive feedback mechanism in zebrafish, consistent with previous studies [68–70]. It is well known that LHβ is an essential regulator of reproduction in stimulating spermiation [33]. This reduction of LHβ probably accounts for the reduced sperm release in the male mutants. The generation and characterization of total and conditional AR knockout male mice from different laboratories revealed different impacts on spermatogenesis, suggesting that AR signaling is critical in maintaining male fertility [18–27]. The similarities and differences in phenotypes between mice and zebrafish are summarized in Table 1. The overall fertility of the ar mutant zebrafish is similar to the T-AR−/y, S-AR−/y, and L-AR−/y mice, with infertility in all of them. Meanwhile, no sperm could be found in the T-AR−/y, S-AR−/y, and L-AR−/y mice [19–22,24]. In the PM-AR−/y mice, there is a reduction by 57% in sperm count, and fertility is normal [27]. However, a very small number of sperms with a low fertilization rate could still be found in the ar mutant zebrafish, and this is the main difference between AR mutant mice and ar mutant zebrafish. In addition, the expression levels of the spermatogenesis-related genes are abnormal in the ar mutants, suggesting defective spermatogenesis. Moreover, the levels of estradiol and 11-KT are significantly decreased and the protein level of LHβ in serum is significantly decreased in the mutant males, again suggesting defective steroidogenesis. In summary, we have demonstrated that lacking AR not only results in impaired spermatogenesis but also defective steroidogenesis, thus causing fertility impairment eventually, and illustrating the essentiality of ar for male fertility in zebrafish. Supplementary data Supplementary data are available at BIOLRE online. Supplemental Figure 1. The mRNA levels of genes in the brain from ar+/+ and ar−/− fish. (a–g) The relative expression of kiss1 (a), kiss2 (b), gnrh2 (c), gnrh3 (d), gnih (e), th1 (f) and th2 (g) in the brain of ar+/+ and ar−/- fish. (h–j) The relative expression of esr1 (h), esr2a (i), and esr2b (j) in the brain of ar+/+ and ar−/- fish. Each value represents the mean value ± SEM. (*, P < 0.05). Supplemental Table 1. Primers used in the present study. Acknowledgments We thank Ms Mi Yao and Ms Xi Wu for expert technical assistance. Footnotes † Grant Support: This research was supported by the National Natural Science Foundation of China (NSFC Grant No. 31701302, 31372512), Guangdong Provincial Natural Science Foundation (Grant No. 2017A030310312), Fundamental Research Funds for the Central Universities (Grant No. 17lgpy108) and the Research Grant Council of Hong Kong (Grant No. 463013). References 1. O’Hara L, Smith LB. Androgen receptor roles in spermatogenesis and infertility. Best Pract Res Clin Endocrinol Metab  2015; 29( 4): 345– 364. 2. Walters KA, Simanainen U, Handelsman DJ. Molecular insights into androgen actions in male and female reproductive function from androgen receptor knockout models. 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Published by Oxford University Press on behalf of Society for the Study of Reproduction. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Biology of Reproduction Oxford University Press

Fertility impairment with defective spermatogenesis and steroidogenesis in male zebrafish lacking androgen receptor

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© The Authors 2017. Published by Oxford University Press on behalf of Society for the Study of Reproduction.
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Abstract

Abstract The pivotal role of androgen receptor (AR) in regulating male fertility has attracted much research attention in the past two decades. Previous studies have shown that total AR knockout would lead to incomplete spermatogenesis and lowered serum testosterone levels in mice, resulting in azoospermia and infertility. However, the precise physiological role of ar in controlling fertility of male fish is still poorly understood. In this study, we have established an ar knockout zebrafish line by transcription activator-like effectors nucleases. Homozygous ar mutant male fish with smaller testis size were found to be infertile when tested by natural mating. Intriguingly, a small amount of mature spermatozoa was observed in the ar mutant fish. These mature spermatozoa could fertilize healthy oocytes, albeit with a lower fertilization rate, by in vitro fertilization. Moreover, the expression levels of most steroidogenic genes in the testes were significantly elevated in the ar mutants. In contrast, the levels of estradiol and 11-ketotestosterone (11-KT) were significantly decreased in the ar mutants, indicating that steroidogenesis was defective in the mutants. Furthermore, the protein level of LHβ in the serum decreased markedly in the ar mutants when compared with wild-type fish, probably due to the positive feedback from the diminished steroid hormone levels. Introduction Androgens are among the main hormones involved in normal male reproduction in mammals [1–3]. Extensive evidence suggests the roles of androgens in spermatogenesis, in the development of secondary sex characters, and in the expression of reproductive behaviors. Testosterone alone could maintain spermatogenesis in vitro [4]. Disruption of androgen production by hypophysectomy [5], Leydig cell ablation [6], and knockout of luteinizing hormone receptor (LHR) [7] demonstrated that spermatogenesis cannot proceed to completion under these circumstances. These studies provide strong evidence that androgens are critical in maintaining the normal function of the male reproductive system [1,8–10]. Nearly all the biological actions of androgens are mediated by the specific androgen receptor (AR, NR3C4), a member of the nuclear receptor gene superfamily [11–14]. Androgen and AR have been shown to play important roles in normal male fertility [10,15]. In males, AR has been detected in Sertoli cells, Leydig cells, peritubular myoid cells and vascular smooth cells in mature testis [16,17]. It is widely accepted that spermatogenesis depends highly on autocrine and paracrine communication among these different cell types. For instance, the action of testosterone on spermatogenesis is mediated via AR on these cell types. The development of the reproductive tract and the gonad as well as sexual behavior is severely impaired in ARKO male mice. The fact that spermatogenesis in ARKO male mice is arrested at different stages [18–27] suggests a role of ar for the maintenance of a normal male reproduction in mammals. Androgens also play pivotal roles in controlling reproduction in teleosts. Two AR subtypes (arα and arβ) have been identified in fish and they are all predominantly expressed in the gonad, suggesting their functional roles on reproduction [28–30]. In addition, the plasma levels of androgens (testosterone, T; 11-ketotestosterone, 11-KT) increase gradually during the later stages of spermatogenesis in most teleost species [31–33]. Moreover, androgen treatment induces spermatogenesis in a number of teleost species. Treatment with 11-KT induced precocious spermatogonial proliferation and meiosis as well as development of secondary sexual characteristics in African catfish [34]. Using a long-term testis tissue culture system, it was shown that 11-KT could promote full spermatogenesis in the Japanese eel [35]. In prepubertal trout, androgen treatment affects the expression of a series of genes involved in spermatogenesis and steroidogenesis. Various transcription factors important for testis development were regulated by androgen treatment [33]. Although the important role of androgen and ar in male fertility of teleosts has been recognized, the precise roles of ar in male fertility are still not fully defined. To address this issue, we undertook this study to analyze the function of ar in zebrafish using transcription activator-like effectors nucleases (TALENs). Our in vivo genetic data provide valuable information for a more thorough understanding of the functional role of ar in vertebrates. Materials and methods Zebrafish husbandry All zebrafish (Danio rerio) were reared and kept in a circulated water system with a 14 h light and 10 h dark cycle at 28°C. The larval and adult zebrafish were fed with brine shrimp (hatched from 20 g eggs in 4 L salt water) twice daily. All animal experiments were conducted in accordance with the guidelines and approval of the respective Animal Research and Ethics Committees of Sun Yat-Sen University. Generation of ar gene mutant zebrafish using TALENs To obtain the zebrafish mutant lines, specific TALEN target sites were designed in the first exon of ar. Paired TALENs were constructed using the Golden Gate TALEN Kit (Addgene, Cambridge, MA, USA) as described previously [36,37]. Approximately 200–500 pg TALEN mRNAs were microinjected into one-cell stage zebrafish embryos, which were then incubated at 28.5°C. Two days after injection, 8–10 embryos were collected for DNA extraction to check whether the targeted genomic fragment was deleted. The target genomic regions were amplified by PCR and subcloned into the pTZ57R/T vector (Fermentas). Single colonies were genotyped by sequencing. To obtain germline mutations, the TALEN-injected embryos were raised to adulthood and the P0 founders were outcrossed with the wild-type fish. The F1 progeny were genotyped by sequencing. To obtain homozygous mutants, heterozygous mutant with the same mutation were obtained and self-crossed. The primers used in this study are listed in Supplemental Table 1. PCR genotyping To assess TALEN-induced mutations of the ar target region, PCR genotyping was performed. Genomic DNA extracted by phenol-chloroform method from the embryo or tail fin of F1 or F2 littermates of a heterozygote cross was employed for PCR. The PCR was conducted with specific primers as listed in Supplemental Table 1. PCR products were sequenced to resolve and identify the mutations. Morphological and histological analyses of the zebrafish mutant lines Morphological and histological analyses were performed as described before [38,39]. Briefly, the gross morphology of adult fish was analyzed at 90 days post fertilization (dpf). Fish were first anesthetized by submersion in 250 mg/L MS-222, and images were taken using a digital camera. Total body length and body weight were measured for each fish, respectively. Then, the testis was isolated from the body cavity for histological examination. The gonadsomatic index (GSI) was calculated as (gonad weight/body weight) × 100%. For gonad histology, the testis samples were fixed in Bouin's solution overnight at room temperature and then immediately dehydrated in a graded series of ethanol, immersed in xylol and finally embedded in paraffin wax. The samples were serially cut into 2–3 μm sections on a Leica microtome. After rehydration, the sections were stained with hematoxylin and eosin and mounted with Canada balsam (Sigma-Aldrich) for microscopic examination. Fertility assessment The fertility of the mutant fish was assessed by natural mating with wild-type females in a spawning tray. At 1 h before the end of the light period, adult male zebrafish (wild-type or homozygotes) were transferred to a breeding aquarium and mate with one wild-type female fish. One hour after light on in the next morning, the embryos were collected by siphoning the bottom of the tank. The number of spawned eggs produced by each female fish was recorded. Individuals that failed to spawn after at least 12 trials were considered infertile [40]. Sperm quality assessment Intact testis from adult zebrafish were carefully dissected out after anesthetization and decapitation, then placed in a tube containing 20 μL zebrafish sperm immobilizing solution (140 mM NaCl, 10 mM KCl, 2 mM CaCl2, and 20 mM HEPES titrated to pH 8.5 with 1.0 M NaOH) on ice, and homogenized by gently grinding the tube with a pipette tip. Samples were kept on ice and used within 1 h. To activate the sperms, 1 μL of semen suspension in zebrafish sperm immobilizing solution was mixed with 20 μL of aged tap water and quickly applied into a single well of a 12-well multitest slide (MP Biomedicals). The slides and coverslips were coated with 1% (wt/vol) polyvinyl alcohol to reduce sticking of sperms. Sperm quality was assessed using CASA (computer-assisted sperm analysis). Fertilization assay by in vitro fertilization Female zebrafish was selected for egg collection using a net to place it gently in a dish after anesthetization. The eggs were squeezed out from the abdomen by stroking the belly gently with fingers. Eggs were scooped out with a fine probe from the belly surface. The fish was then gently lifted and returned to the tank. Ten microliters of the semen suspension were added to the clutch of eggs, and then mixed with 50 μL of aged tap water. The mixture was then placed at 28°C for subsequent incubation. The number of fertilized/unfertilized eggs was discerned under a dissecting microscope at 4 h post fertilization. At least 50 embryos from each replicate were scored. For each fish line, the fertilization rate was accessed from five matured males and five wild-type females in three independent crosses. RNA isolation and real-time PCR Total RNA was extracted from the testis of zebrafish using TRIzol reagent (Invitrogen). The cDNA was produced from 500 ng total RNA using ReverTra Ace α-first strand cDNA Synthesis Kit (TOYOBO, Japan), and used as template in the subsequent real-time PCR analyses. The specific primers used in this study are listed in Supplemental Table 1. The transcription levels of the target genes were measured using the SYBR Green PCR Master Mix Kit (ABI, USA) carried out on an ABI Real-Time PCR Fast System (ABI, USA). The quantitative RT-PCR conditions were as follows: denaturation at 95°C for 5 min, followed by 40 cycles of 95°C for 15 s, 58°C for 15 s, 72°C for 15 s, and then 84°C for 10 s (fluorescent data collection). All mRNA quantification data were normalized to ef1a and expressed as fold differences of target gene expression relative to the control. Whole-body steroid determination Whole-body steroid determination was performed as described previously [41]. Whole-body extractions of steroids were performed to measure the amount of estradiol in each fish. First, the male fish (n = 6) were anesthetized by submersion in 250 mg/L MS-222, weighed on an analytical balance, and then homogenized in 3 mL phosphate-buffer saline (PBS) in glass tubes using a Polytron homogenizer. Steroids were extracted by adding 5 mL diethyl ether to each tube followed by vortexing for 1 min, then centrifuging at 3000 g for 2 min. Thereafter, the tubes were frozen in a methanol/dry ice bath at –30°C, and the ether layer containing the steroids was poured off into another set of tubes. The diethyl ether was then evaporated off under a flow of air. The extraction procedure was repeated twice to enhance the extraction efficiency and the extract was dissolved in PBS. The amount of each steroid (estradiol and 11-KT) was determined using Enzyme-linked immunosorbent assay (ELISA) according to the manufacturer's protocol (Cayman Chemical Company, Ann Arbor, MI, USA). All standards and samples were run in triplicates. Western blot analyses Western blot analysis was performed as previously described with slight modifications [42]. Briefly, the pituitary samples were collected from male fish (90 dpf). The samples were lysed in 60 μl cell lysis reagent (Sigma, USA) with proteinase inhibitor cocktail (1:100; Sigma, USA), mixed with 15 μL of 5× DualColor Protein Loading Buffer (Fudebio-tech) and heated at 100°C for 10 min. For the serum samples, the fish were first anesthetized by submersion in 250 mg/L MS-222, then blood was gently aspired from the heart using a 10-μL tip and centrifuged (10 000 g, 30 min, 4°C) after being held in a 4°C environment for half an hour. The supernatant was transferred to a new tube, and then diluted in PBS to stated concentrations. Thereafter, the solution was mixed with 5× DualColor Protein Loading Buffer and heated at 100°C for 10 min. The lysates or serum samples were first separated on a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel. The separated proteins were then transferred onto poly vinylidene fluoride (PVDF) membranes (Pall, USA). The membranes were blocked with 5% bovine serum albumin (BSA) in Tris-buffered saline with 0.05% Tween 20 (TBST) and then cut into two halves according to the visible marker bands (25 and 35 KD). The membranes containing the larger Mw proteins were incubated with GAPDH mouse monoclonal primary antibodies (1:4000, Proteintech, RRID: AB_2107436), and the membranes containing smaller Mw proteins were incubated with FSHβ (follicle-stimulating hormones, 1:3000, RRID: AB_2651068) or LHβ (luteinizing hormones, 1:6000, RRID: AB_2651067) rabbit polyclonal primary antibodies, as previously reported [42]. The membranes were blocked for 30 min in 5% BSA in TBST prior to the addition of the primary antibody and then incubated with secondary antibodies conjugated to horseradish peroxidase, the protein bands were visualized using enhanced chemiluminescence reagents (ThermoFisher, USA). The bands were analyzed semi-quantitively by densitometry using the ImageJ software (grayscale analysis) and normalized to their loading controls. All western blot analyses were performed twice. Statistical analyses All data were expressed as mean values ± SEM, and analyzed by Student's t-test using the GraphPad Instat software (GraphPad Software, USA). P < 0.05 was considered statistically significant. All experiments were performed at least two times to confirm the results. Results Generation of ar gene knockout zebrafish line To study the role of androgens in male fertility, the ar mutant line was generated using TALENs. The target sequences were designed based on the sequence of ar provided in NCBI. A pair of TALEN-binding sites was chosen within the first exon (Figure 1A). The mutated genotype with a 13-bp deletion was further used to establish the homozygous mutant line in this study (Figure 1A). Successful ar deletion was confirmed by sequencing (Figure 1B). This mutation could induce an open reading frame (ORF) shift, generating a truncated AR protein lacking the DNA-binding domain, hinge domain, and the ligand-binding domain (Figure 1C) [43]. Figure 1. View largeDownload slide Targeted disruption of the ar locus in zebrafish using TALENs. (A) TALEN target site and the mutated genotype. The TALEN-binding site was chosen within the first exon. A genotype with an 8-bp deletion was used to establish the ar mutant line. (B) Sanger sequencing analysis of the 8-bp deletion genotype. (C) Schematic representation of the protein functional domains of AR from the WT and the mutant. TAD: transactivation domain; DBD: DNA-binding domain; HD: hinge domain; LBD: ligand binding domain. Figure 1. View largeDownload slide Targeted disruption of the ar locus in zebrafish using TALENs. (A) TALEN target site and the mutated genotype. The TALEN-binding site was chosen within the first exon. A genotype with an 8-bp deletion was used to establish the ar mutant line. (B) Sanger sequencing analysis of the 8-bp deletion genotype. (C) Schematic representation of the protein functional domains of AR from the WT and the mutant. TAD: transactivation domain; DBD: DNA-binding domain; HD: hinge domain; LBD: ligand binding domain. Phenotypic characterization of ar −/− male zebrafish The ar mutant line was viable and developed normally. The gross morphology of adult male fish was analyzed at 90 dpf. As showed in Figure 2A, male ar mutant fish exhibited female-like appearance. The testis size was significantly decreased in the homozygotes (Figure 2A), while the body length and overall body weight were not affected (Figure 2B and C). Accordingly, a significant reduction in the GSI was found in the homozygous mutants (Figure 2D). Mating between homozygous ar mutant males and wild-type females failed to induce spawning, indicating that ar-deficient males were infertile (Figure 2E). These results suggested that both testis development and fertility were impaired in male zebrafish lacking ar. Figure 2. View largeDownload slide Phenotypic characterization of the ar−/− male zebrafish. (A) Gross and gonad morphology of the ar+/+ and ar−/–fish. The testis size was significantly decreased in the homozygotes. (B–D) Body length (B), body weight (C), and GSI (D) of the ar+/+ and ar−/–fish. (E) The reproductive capacity of males from the indicated genotypes crossed with wild-type females (n = 12). Each value represents the mean value ± SEM (n = 6) (***, P < 0.001 vs control). Figure 2. View largeDownload slide Phenotypic characterization of the ar−/− male zebrafish. (A) Gross and gonad morphology of the ar+/+ and ar−/–fish. The testis size was significantly decreased in the homozygotes. (B–D) Body length (B), body weight (C), and GSI (D) of the ar+/+ and ar−/–fish. (E) The reproductive capacity of males from the indicated genotypes crossed with wild-type females (n = 12). Each value represents the mean value ± SEM (n = 6) (***, P < 0.001 vs control). Testicular histology, sperm morphology, and sperm quality in the ar mutant fish To further analyze the causation of fertility defects observed in the male ar mutants, histological and sperm morphology assessment was performed on the testis sections from male fish at 90 dpf. The overall testicular size was reduced in the ar mutant males when compared with wild-type males (Figure 3A). Histological analysis from hematoxylin and eosin (H&E) staining revealed that germ cell development was severely disrupted. A pronounced reduction in the number of germ cells was observed in the ar mutant fish and most of the germ cells were blocked at the early development stages. Interestingly, a small amount mature spermatozoon could still be found in the ar mutants. In addition, these spermatozoa from the ar mutants were viable and their morphology was also normal (Figure 3A). The sperm quality was assessed and no significant difference in sperm motility (Figure 3B), average curvilinear velocity (VCL) (Figure 3C) and average straightness (STR) (Figure 3D) were observed in both genotypes. Finally, fertilization assays were performed by in vitro fertilization. Our data showed that sperm obtained from the ar mutants could fertilize healthy oocytes, albeit with a low fertilization rate (Figure 3E). Overall, it implies that ar plays a pivotal role in normal spermatogenesis in zebrafish. Figure 3. View largeDownload slide Testicular histology, sperm morphology, and sperm quality of the ar +/+ and ar −/− male zebrafish at 90 dpf. (A) Testicular histology and sperm morphology of adult male zebrafish of ar +/+ and ar −/–genotypes. A pronounced reduction in germ cell number was observed in the ar mutant fish, while a very small number of mature spermatozoa of normal morphology could still be found in the ar mutant fish. (B–D) Sperm motility (B), VCL (C), SRT (D) in adult male zebrafish of the indicated genotypes. (E) Fertilization rate of sperm obtained from the ar mutant fish performed by in vitro fertilization. sz: Spermatozoa; VCL: average curvilinear velocity; STR: average straightness. Each value represents the mean value ± SEM (n = 6) (***, P < 0.001 vs control). Figure 3. View largeDownload slide Testicular histology, sperm morphology, and sperm quality of the ar +/+ and ar −/− male zebrafish at 90 dpf. (A) Testicular histology and sperm morphology of adult male zebrafish of ar +/+ and ar −/–genotypes. A pronounced reduction in germ cell number was observed in the ar mutant fish, while a very small number of mature spermatozoa of normal morphology could still be found in the ar mutant fish. (B–D) Sperm motility (B), VCL (C), SRT (D) in adult male zebrafish of the indicated genotypes. (E) Fertilization rate of sperm obtained from the ar mutant fish performed by in vitro fertilization. sz: Spermatozoa; VCL: average curvilinear velocity; STR: average straightness. Each value represents the mean value ± SEM (n = 6) (***, P < 0.001 vs control). The mRNA level of selected testicular genes in the double mutants To understand the mechanism of spermatogenetic defects caused by ar deficiency, we have selected a number of spermatogenesis-related genes as candidates for real-time PCR analyses. We found that the genes expressed in the type Aund spermatogonia, spermatogonia, and spermatocyte such as nanos2, piwil1, piwil2, dazl, and sycp3l were elevated in the mutant testis relative to the wild-type (Figure 4A–E). In contrast, the expression of odf3b, a gene expressed in spermatid, was significantly decreased. The genes expressed in Sertoli cells such as amh and gsdf were significantly upregulated (Figure 4G and H), while the expression of igf3 was significantly downregulated in the ar mutants (Figure 4I). The gene expressed in Leydig cells, such as insl3, significantly decreased in the mutants (Figure 4J). We also analyzed the mRNA level of cx43 (an important gap junction protein that participates in the control of cell proliferation) and found that its expression was significantly downregulated in the mutants (Figure 4K). Taken together with the histological analyses, the number of type Aund spermatogonia, spermatogonia, spermatocyte increased, whereas the number of spermatid and proliferative cells decreased in the ar mutants when compared with wild-type zebrafish, suggesting that spermatogenesis was defective in the ar mutants. Figure 4. View largeDownload slide The mRNA level of selected testicular genes in the wild-type and mutant fish at 90 dpf. (A–K) The relative expression of spermatogenesis-related genes in the testis of wild-type and mutant lines. The mRNA levels were normalized to that of ef1a, with the level in the ar−/– fish expression as the fold change of the level in the ar+/+ fish. Each value represents the mean value ± SEM. (*, P < 0.05; **, P < 0.01; ***, P < 0.001 vs control). Figure 4. View largeDownload slide The mRNA level of selected testicular genes in the wild-type and mutant fish at 90 dpf. (A–K) The relative expression of spermatogenesis-related genes in the testis of wild-type and mutant lines. The mRNA levels were normalized to that of ef1a, with the level in the ar−/– fish expression as the fold change of the level in the ar+/+ fish. Each value represents the mean value ± SEM. (*, P < 0.05; **, P < 0.01; ***, P < 0.001 vs control). Defective steroidogenesis in ar mutant males In order to determine whether endocrine abnormalities might underlie or contribute to the ar mutant fish phenotype, the expression profile of the steroidogenic-related genes was analyzed. Real-time PCR quantification revealed that testicular star, cyp11a1, cyp11a2, cyp17a1, hsd3β1, hsd3β2, hsd17β3, and hsd11β1 mRNA levels were increased (Figure 5A–D, F, G, I and J), whereas hsd17β1 mRNA level was decreased (Figure 5H) in the ar mutants when compared with the age-matched wild-type fish. But no significant alteration in testicular mRNA levels of cyp17a1, hsd11β2, and cyp19a1a was observed in the ar mutant males when compared with wild-type males (Figure 5E, K and l). Hormone assays showed that estradiol and 11-KT levels were lower in the ar mutants (Figure 5M and N). These data indicated that the steroidogenesis pathway was affected in the ar mutant males (Figure 5O). Figure 5. View largeDownload slide Defective steroidogenesis in the ar mutant males. (A–L) The relative expression of steroidogenic pathway genes in the testis of ar+/+ and ar−/– fish. The mRNA levels were normalized to that of ef1a, with the level in the ar−/– fish expression as the fold change of the level in the ar+/+ fish. (M and N) Whole-body estradiol (M) and 11-KT (N) levels. (O) Summing of the expression profile of steroidogenic pathway genes in the ar deficient zebrafish. Each value represents the mean value ± SEM. (*, P < 0.05; **, P < 0.01; ***, P < 0.001 vs control). Figure 5. View largeDownload slide Defective steroidogenesis in the ar mutant males. (A–L) The relative expression of steroidogenic pathway genes in the testis of ar+/+ and ar−/– fish. The mRNA levels were normalized to that of ef1a, with the level in the ar−/– fish expression as the fold change of the level in the ar+/+ fish. (M and N) Whole-body estradiol (M) and 11-KT (N) levels. (O) Summing of the expression profile of steroidogenic pathway genes in the ar deficient zebrafish. Each value represents the mean value ± SEM. (*, P < 0.05; **, P < 0.01; ***, P < 0.001 vs control). The protein levels of LHβ and FSHβ in the pituitary and serum The protein levels of LHβ and FSHβ in the pituitary gland and serum of male zebrafish were analyzed using western blot. It was found that the expression levels of LHβ and FSHβ in the pituitary gland were not significantly altered in the ar mutant male fish (Figure 6A–C). The expression levels of LHβ and FSHβ in the serum were subsequently analyzed (Figure 6D and E). No change was observed in serum FSHβ, whereas serum LHβ decreased markedly in the ar mutants when compared with the age-matched wild-type fish (Figure 6F and G). Thereafter, the mRNA levels of lhr, fshr, and ers in the testis were analyzed by real-time PCR. It was found that the mRNA level of lhr was significantly downregulated in the ar mutants, while fshr did not vary markedly (Figure 6H and I). In addition, of the three er genes in zebrafish, the esr2b gene but not esr1 and esr2a exhibited significant upregulation in the testis of ar mutants (Figure 6j–L). Finally, we analyzed the expression of several neuroendocrine factors including kiss1, kiss2, gnrh2, gnrh3, gnih, th1, and th2, in the brain by real-time PCR. As showed in Supplemental Figure S1, th2 was significantly decreased in the ar mutants (Supplemental Figure S1a–g). The expression levels of the three esr genes in the brain were further analyzed and on significant changes were detected (Supplemental Figure S1h–j). Figure 6. View largeDownload slide Western blot of LHβ and FSHβ in the pituitary gland and serum from ar+/+ and ar−/− fish. (A) Western blot for LHβ and FSHβ in the pituitary gland from ar+/+ and ar−/− fish. (B and C) Densitometric analysis of blot on expression of LHβ (B) and FSHβ (C) in the pituitary gland from ar+/+ and ar−/− fish. (D and E) Western blot for LHβ (D) and FSHβ (E) in the serum from the ar+/+ and ar−/− fish. Ponceau protein stain of the transfer membrane indicating approximately equal loading across the gel. (F and G) Densitometric analysis of blot on expression of LHβ (F) and FSHβ (G) in the serum from ar+/+ and ar−/− fish. (H and I) The relative expression of lhr (H) and fshr (I) in the testis of ar+/+ and ar−/- fish. (j–l) The relative expression of esr1 (J), esr2a (K), and esr2b (L) in the testis of ar+/+ and ar−/- fish. Western blot was performed twice. Each value represents the mean value ± SEM. (*, P < 0.05; **, P < 0.01). Figure 6. View largeDownload slide Western blot of LHβ and FSHβ in the pituitary gland and serum from ar+/+ and ar−/− fish. (A) Western blot for LHβ and FSHβ in the pituitary gland from ar+/+ and ar−/− fish. (B and C) Densitometric analysis of blot on expression of LHβ (B) and FSHβ (C) in the pituitary gland from ar+/+ and ar−/− fish. (D and E) Western blot for LHβ (D) and FSHβ (E) in the serum from the ar+/+ and ar−/− fish. Ponceau protein stain of the transfer membrane indicating approximately equal loading across the gel. (F and G) Densitometric analysis of blot on expression of LHβ (F) and FSHβ (G) in the serum from ar+/+ and ar−/− fish. (H and I) The relative expression of lhr (H) and fshr (I) in the testis of ar+/+ and ar−/- fish. (j–l) The relative expression of esr1 (J), esr2a (K), and esr2b (L) in the testis of ar+/+ and ar−/- fish. Western blot was performed twice. Each value represents the mean value ± SEM. (*, P < 0.05; **, P < 0.01). Table 1. Comparison of reproductive phenotypes in T-AR−/y, G-AR−/y, S-AR−/y, L-AR−/y, PM-AR−/y mice and ar−/− zebrafish.   Overall fertility  Testis size  Mutant testis size/WT testis size(%)  Sperm count  Testosterone/11-KT level  LH (Serum)  FSH (Serum)  References    T-AR−/y  Infertile  Decreased  7  No epididymis  Decreased  Elevated  Elevated  [21, 22, 23, 26]    AR−/y  Infertile  Decreased  23.40  No sperm  Decreased  Elevated  Normal  [19, 20, 22, 23, 25, 26]  Mice  L-AR−/y  Infertile  Decreased  31.10  No sperm  Decreased  Elevated  Elevated  [23, 24]    PM-AR−/y  Normal fertility  Decreased  79.00  Decreased by 57% of WT sperm count  Normal  Normal  Normal  [27]    G-AR−/y  Normal fertility  Normal  Normal  Within normal range  Normal  Normal  Normal  [23]  Zebrafish  ar−/−  Infertile  Decreased  20.88  Very few sperm with a low fertilization rate  Decreased  Decreased  Normal  Present study    Overall fertility  Testis size  Mutant testis size/WT testis size(%)  Sperm count  Testosterone/11-KT level  LH (Serum)  FSH (Serum)  References    T-AR−/y  Infertile  Decreased  7  No epididymis  Decreased  Elevated  Elevated  [21, 22, 23, 26]    AR−/y  Infertile  Decreased  23.40  No sperm  Decreased  Elevated  Normal  [19, 20, 22, 23, 25, 26]  Mice  L-AR−/y  Infertile  Decreased  31.10  No sperm  Decreased  Elevated  Elevated  [23, 24]    PM-AR−/y  Normal fertility  Decreased  79.00  Decreased by 57% of WT sperm count  Normal  Normal  Normal  [27]    G-AR−/y  Normal fertility  Normal  Normal  Within normal range  Normal  Normal  Normal  [23]  Zebrafish  ar−/−  Infertile  Decreased  20.88  Very few sperm with a low fertilization rate  Decreased  Decreased  Normal  Present study  T-AR−/y, Total AR knockout; S-AR−/y, Sertoli cell-specific AR knockout; L-AR−/y, Leydig cell-specific AR knockout; PM-AR−/y, PMcell-specific AR knockout; G-AR−/y, Germ cell-specific AR knockout View Large Discussion Unlike the mammalian counterparts, the functional roles of ar in teleosts are not fully understood. Using TALENs, we have undertaken the present study to evaluate the function and importance of ar in zebrafish. Our data demonstrate that fertility is impaired with defective spermatogenesis and steroidogenesis in male zebrafish lacking AR. Naturally occurring androgens as well as synthetic androgens are potential ligands for ARs [44]. In fish, the principal androgens are testosterone and 11-KT, which had been shown to bind and activate fish ar in vitro [44–46]. Different from the single copy of mammalian nuclear AR gene, many teleost species possess two ar genes (arα and arβ) [28–30], arising from a teleost-specific genome duplication that occurred in the teleost lineage [47–49]. However, only a single copy of functional ar gene is found in zebrafish [45]. Actually, species such as zebrafish that possess only a single copy ar gene cannot help to elucidate the potential role complete role of ars in teleosts since many species possess two ars. As a first step, we have utilized the convenient zebrafish model because of the ease of genome-editing. As demonstrated in this study, the ar mutant males possess testes of diminished size. The testis mass was reduced by approximately 80% in the ar mutant males when compared with wild-type fish, and all male mutants were infertile. These findings are in agreement with ARKO models in mice [18–24]. These phenotypes indicate that the function of ar is well conserved from fish to mammals. To further examine whether spermatogenesis proceeded normally, histological examination of the testis was performed. It was found that loss of the ar has resulted in reduction in sperm count in the ar mutant zebrafish. Despite the presence of some sperms in the small testis, the mutant fish were infertile. This is very different from previous studies in mice that a reduced sperm count is associated with normal fertility [27,50–53]. Furthermore, ar mutant fish did produce some viable spermatozoa of normal motility, and these sperms were able to fertilize healthy oocytes although with a lower fertilization rate. It has been reported previously that androgens could mediate its action through estrogen receptors [54]. Indeed, we have detected the expression of esr genes in the testis and found that esr2b was significantly upregulated in the ar mutants, suggesting that compensation of androgen by cross-activating ER may exist in the male mutants. As a matter of fact, ar is not the only AR in the zebrafish. Membrane AR (mAR, ZIP9) [55,56] may have a compensation effect as a result of ar disruption. Moreover, the G protein-coupled receptor family C group 6 subtype A (GPRC6A) is functionally important in regulating the nongenomic actions of androgens in mice [57], indicating that androgens might exert their function by binding to and activating other receptors or factors. The finding of the present study in zebrafish is different from the phenotype in AR null mice in which spermatogenesis was arrested in the pachytene spermatocyte stage [21,58]. We have analyzed a series of genes expressed in the germ cells, Sertoli cells and Leydig cells. In concordance with the testicular histology, the genes expressed in spermatogonia and spermatocytes were upregulated, while the genes expressed in the spermatids were downregulated in the ar mutant fish. Moreover, the transcript level of amh, a gene known to inhibit the differentiation of spermatogonia, was upregulated. In contrast, the transcript level of igf3, a gene known to stimulate spermatogenesis, was downregulated. Taken together, spermatogenesis is severely impaired in the ar mutant zebrafish, in spite of the presence of a small number of viable spermatozoa. Spermatogenesis (the production of haploid germ cells) and steroidogenesis (the production of steroid hormones that support male reproductive development and function) are the major functions of the testes. Both of them are regulated in a highly coordinated manner [10]. The expression profile of the steroidogenic genes was also investigated. It was found that the expression levels of most steroidogenic genes were upregulated, concordant with what was reported in ARKO mice [26]. However, the levels of steroid hormones such as estradiol and 11-KT were significantly decreased in the ar mutants, probably explaining dramatically the small size of the testes in the mutants. In addition, 11-KT is one of the factors involved in the initiation of spermatogonial proliferation [59,60]. The low level of 11-KT also caused the pronounced reduction in the number of germ cells in the testes of the ar mutants. Furthermore, AR antagonists (flutamide and vinclozolin) also impact the reproductive process via multiple pathways related to steroidogenesis, spermatogenesis, and fertilization [61]. Therefore, the mutant fish line generated in this study is a valuable resource for investigating compounds with endocrine disrupting potential. We further observed that the protein levels of LHβ and FSHβ in the pituitary gland were not significantly affected in the ar mutant males, despite the expression of th2, code for rate-limiting enzyme in dopamine biosynthesis [62], was significantly decreased in the ar mutant males. Interestingly, both the protein level of LHβ in the serum and mRNA level of lhr in the testis were reduced in the ar mutant males, probably due to the strong positive feedback from the diminished steroid hormones levels. In fact, previous work has demonstrated that positive feedback control of LHβ (GTH-II) release is a common feature in adult teleost [63,64]. Moreover, we found that the mRNA levels of kiss1, kiss2, gnrh2, and gnrh3 were not markedly altered in the ar mutants when compared with wild-type fish. Additionally, our previous studies have demonstrated that double knockout of kiss1; kiss2 and triple knockout of kiss1; kiss2; gnrh3 exhibited no defect in the reproduction of zebrafish [38,65]. Likewise, ontogeny and reproductive activity were not altered upon disruption of gnrh2; gnrh3 in zebrafish [66]. Furthermore, a very recent study from our team revealed that E2 could directly act on the pituitary level to stimulate LHβ expression during puberty in zebrafish [67]. Taken together, it can be concluded that steroid hormones can directly regulate LHβ of the pituitary level in a kiss; gnrh system independent manner via a positive feedback mechanism in zebrafish, consistent with previous studies [68–70]. It is well known that LHβ is an essential regulator of reproduction in stimulating spermiation [33]. This reduction of LHβ probably accounts for the reduced sperm release in the male mutants. The generation and characterization of total and conditional AR knockout male mice from different laboratories revealed different impacts on spermatogenesis, suggesting that AR signaling is critical in maintaining male fertility [18–27]. The similarities and differences in phenotypes between mice and zebrafish are summarized in Table 1. The overall fertility of the ar mutant zebrafish is similar to the T-AR−/y, S-AR−/y, and L-AR−/y mice, with infertility in all of them. Meanwhile, no sperm could be found in the T-AR−/y, S-AR−/y, and L-AR−/y mice [19–22,24]. In the PM-AR−/y mice, there is a reduction by 57% in sperm count, and fertility is normal [27]. However, a very small number of sperms with a low fertilization rate could still be found in the ar mutant zebrafish, and this is the main difference between AR mutant mice and ar mutant zebrafish. In addition, the expression levels of the spermatogenesis-related genes are abnormal in the ar mutants, suggesting defective spermatogenesis. Moreover, the levels of estradiol and 11-KT are significantly decreased and the protein level of LHβ in serum is significantly decreased in the mutant males, again suggesting defective steroidogenesis. In summary, we have demonstrated that lacking AR not only results in impaired spermatogenesis but also defective steroidogenesis, thus causing fertility impairment eventually, and illustrating the essentiality of ar for male fertility in zebrafish. Supplementary data Supplementary data are available at BIOLRE online. Supplemental Figure 1. The mRNA levels of genes in the brain from ar+/+ and ar−/− fish. (a–g) The relative expression of kiss1 (a), kiss2 (b), gnrh2 (c), gnrh3 (d), gnih (e), th1 (f) and th2 (g) in the brain of ar+/+ and ar−/- fish. 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Published by Oxford University Press on behalf of Society for the Study of Reproduction. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

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Biology of ReproductionOxford University Press

Published: Feb 1, 2018

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