Augmentation of progestin signaling rescues testis organization and spermatogenesis in zebrafish with the depletion of androgen signaling

Androgens and estrogens exert considerable influence on the formation of primary sex characteristic (PSC) and secondary sex characteristic (SSC) in fish. In the zebrafish model, the steroidogenic pathway in reproduction has been extensively investigated as zebrafish possess several advantages for research, including the ease of germ cells observation, gonadal dissection, fertility capacity assessment, knockout strategy (Crowder et al., 2018; Lau et al., 2016; Li et al., 2020; Lu et al., 2017; Tang et al., 2018; Tang et al., 2016; Yin et al., 2017; Yu et al., 2018; Zhai et al., 2018; Zhu et al., 2015), as well as chemical reagent administration, including estradiol (E2), testosterone (T), 11-ketotestosterone (11-KT), fadrozole, flutamide, and 17α,20β-dihydroxy-4-pregnen-3-one (DHP) (Chen et al., 2013; Fenske and Segner, 2004; Shu et al., 2020; Zhai et al., 2018; Zhai et al., 2017). Based on observations of ar- or npgr-deficient zebrafish, androgen signaling is reported to be essential for sex determination, testis organization, male-typical SSCs, and fertility (Crowder et al., 2018; Li et al., 2020; Tang et al., 2018; Yu et al., 2018; Zhai et al., 2018), while progestin signaling has been suggested to be important for female ovulation (Tang et al., 2016; Wu and Zhu, 2020).

In humans, the CYP17A1 mutation causes pseudohermaphroditism, delay in sexual maturation, and absence of masculinization (Kater and Biglieri, 1994; Marsh and Auchus, 2014; Yanase, 1995; Yanase et al., 1991). In mice, Cyp17a1 knockout leads to infertility and loss of sexual behaviors (Liu et al., 2005). In zebrafish, the knockout of ar, cyp11c1 (encoding 11β-hydroxylase) or cyp11a2 (encoding cytochrome P450 side-chain cleavage enzyme, the first and rate-limiting enzyme for steroid hormone biosynthesis) results in impaired spermatogenesis and disorganized testes, which is accompanied by androgen signaling insufficiency (Crowder et al., 2018; Li et al., 2020; Oakes et al., 2020; Tang et al., 2018; Yu et al., 2018; Zhang et al., 2020). However, as evidenced by a recent study reported from our laboratory, a phenotype of all-male cyp17a1-deficient fish that exhibited reductions of T and 11-KT in plasma and brain samples and loss of male-typical SSCs and mating behaviors with enhanced testicular development and spermatogenesis was observed (Shu et al., 2020; Zhai et al., 2018; Zhai et al., 2017). In another well-known freshwater fish model with an XX/XY genetic sex determination system, medaka, the scl mutant strain carrying a loss-of-function mutation in the cyp17a1 gene also results in the loss of male-typical SSCs while spermatozoa develop normally (Sato et al., 2008). Similarly, results were also observed in another cyprinid fish, the common carp (Cyprinus carpio L.), with cyp17a1 deletion (the cyp17a1-/- XX fish) developed normal testis structure with normal spermatogenesis and sperm capacity, and lost male-typical SSCs. Using artificial fertilization, the neomale common carp were mated with wild-type (WT) females (cyp17a1+/+ XX genotype). We confirmed that all offspring from the neomale-WT female mating have the cyp17a1±XX genotype, and 100.00% normal development of gonads to ovaries (n > 500) (Zhai et al., 2022). However, other than impaired male-typical SSCs, the disorganized testicular development and impaired spermatogenesis were not observed in the cyp17a1-deficient cyprinid fish (zebrafish and common carp) and scl mutant medaka (Sato et al., 2008; Zhai et al., 2022; Zhai et al., 2018), but in tilapia (Yang et al., 2021). Our initial hypothesis was that, compared with the male-typical SSCs and mating behaviors, testicular development and spermatogenesis may not be susceptible to androgen signaling deficiency. Nevertheless, this hypothesis is not supported by the phenotypes of the ar, cyp11a2, and cyp11c1 knockout zebrafish that show disorganized testes and impaired spermatogenesis. Therefore, an alternative pathway might exist in the cyp17a1-/- fish, facilitating testicular development and spermatogenesis when androgen signaling deteriorates.

Progestin signaling is important in mediating spermatogenesis, sperm maturation, and spermiation (Baynes and Scott, 1985; Miura et al., 1992; Tubbs and Thomas, 2008; Vizziano et al., 1996b). However, the npgr loss-of-function models diverged in phenotypes both in male mammals and fish. In mice, homozygous progesterone receptor (Pr) knockout males were fertile as WT and heterozygous male siblings (Lydon et al., 1995). Consistently, no evidence of fertility effects in the npgr knockout male zebrafish has been reported (Tang et al., 2016; Wu and Zhu, 2020). In male tilapia, npgr knockout resulted in disorganized spermatogenic cysts, decreased sperm number, motility, spermatocytes, and spermatozoa, showing the essentiality for the fertility of XY tilapia (Fang et al., 2018). The underlying mechanism of this discrepancy has been only scarcely investigated. We hypothesize that the different requirements for progestin receptors and the alternative between progestin signaling and other pathways (or factors) may contribute to it.

Although an alternative pathway might exist in the cyp17a1-/- fish to facilitate testicular development and spermatogenesis when androgen signaling deteriorates, it had not yet been proposed. The cyp17a1-/- zebrafish generated in our laboratory shows stimulated testicular development and spermatogenesis with deteriorated androgen synthesis (Zhai et al., 2018). In the cyp17a1-/- fish, the accumulation of progestins, DHP, and P4, which are the upstream products of androstenedione and testosterone, was observed, possibly due to the loss of the critical 17α-hydroxylase and 17, 20-lyase activities of Cyp17a1 during gonadal steroidogenesis (Tokarz et al., 2013; Zhai et al., 2018). To dissect the roles of androgen signaling and progestin signaling in adult zebrafish, we established and analyzed a series of deficiency models of sex steroid signaling (cyp17a1-/-, ar-/-, cyp17a1-/-;ar-/-, cyp17a1-/-;ar-/-;npgr-/-, and cyp17a1-/-;ar-/-;fshβ-/-) and initiated a concurrent study of DHP administration in ar-/- males as well. Here, we report that an augmentation of progestin/nuclear progestin receptor (nPgr) signaling can sufficiently compensate for proper spermatogenesis and testis organization when androgen signaling deteriorates in the cyp17a1-/- and cyp17a1-/-;ar-/- zebrafish.

Spermatogenesis is a highly coordinated developmental process involving diploid spermatogonia proliferation and differentiation, during which diploid spermatogonial stem cells produce a large number of highly differentiated spermatozoa (Schulz et al., 2010). Progestin signaling, which mainly acts via the progestin receptor expressed in Sertoli cells, is closely related to spermatogenesis (Schulz et al., 2010). First, non-flagellated germ cells in rainbow trout testes can synthesize DHP in the early phase of spermatogenesis (Vizziano et al., 1996a). In salmonid fish, the plasma concentration of DHP peaks during the progression of spermatogonial proliferation (Scott and Sumpter, 1989). Second, DHP treatment induces spermiation in Salmonidae and Cyprinidae (Ueda et al., 1985), increases milt production (Baynes and Scott, 1985), and regulates meiosis in male eels (Miura et al., 2006). In the testis of Nile tilapia, npgr was expressed in Sertoli cells surrounding spermatogonia and spermatids. The treatment with RU486, a synthetic nPgr antagonist, inhibits progestin signaling and disrupts spermatogenesis in Nile tilapia. The expression levels of piwil1, dazl, and sycp3 genes were found to be downregulated in the testes of RU486-treated fish (Liu et al., 2014). The GSI and the expression levels of the germ cell markers piwil1, dazl, and the meiotic marker, sycp3, increased after 2-week E2 exposure in the presence of DHP (Chen et al., 2013). Third, npgr-knockout male tilapia exhibited disorganized spermatogenic cysts, decreased sperm number and motility, as well as spermatocytes and spermatozoa (Fang et al., 2018). Although progestin signaling may play important roles in spermatogenesis, the in vivo evidence and its relationship with androgen signaling are yet to be elucidated, especially in the species that the males with npgr mutation did not exhibit obvious phenotypes in reproduction.

To our knowledge, the cyp17a1-/- zebrafish generated by us is a unique model exhibiting deteriorated androgen signaling but enhanced testicular development and spermatogenesis (Li et al., 2020; Zhai et al., 2018). In our newly generated cyp17a1-/-;ar-/- fish, the phenotype with testicular development and spermatogenesis similar to cyp17a1-/- fish was observed. The introduction of cyp17a1 knockout successfully rescued the impaired testis organization and spermatogenesis in ar-/- males, clearly demonstrating the existence of an alternative androgen-independent signaling pathway promoting testis organization and spermatogenesis in the cyp17a1-/- fish (Figure 1). Zebrafish Ar showed an affinity for the non-androgenic steroid progesterone in the high nanomolar range (de Waal et al., 2008). The abnormalities in the ar-/- male zebrafish rescued by a compensatory mechanism demonstrated that the restorations of the testicular development and spermatogenesis after the introduction of the cyp17a1-deletion are not dependent on Ar (Figure 1). DHP and P4 are the androgen upstream products and accumulate in the cyp17a1-/- fish as measured using ELISA (Zhai et al., 2018) and UPLC-MS/MS (Figure 2). DHP is a fish-specific progestin that might play critical roles in spermatogenesis, sperm maturation, and spermiation (Fang et al., 2018). The administration of DHP effectively restored the phenotypes of GSI and the number of spermatozoa in ar-/- males (Figure 3). In addition, compared with the cyp17a1-/-;ar-/- fish that showed the hypertrophic testis and enhanced spermatogenesis, the cyp17a1-/-;ar-/-;npgr-/- fish exhibited phenotypes of defective spermatogenesis and testis organization, which are also seen in the ar-, cyp11a2-, or cyp11c1-deficient male zebrafish (Li et al., 2020; Oakes et al., 2020; Tang et al., 2018; Yu et al., 2018). These suggest that the accumulated progestin may play an alternative signaling role in promoting testis organization and spermatogenesis, which is independent of androgen signaling (in cyp17a1-/- fish and cyp17a1-/-;ar-/- fish) (Figures 4 and 5).

In mice with knockout of Pr, a member of the nuclear receptor superfamily of transcription factors, homozygous females displayed defects in reproductive tissues. These defects included the inability to ovulate, uterine hyperplasia and inflammation, severely limited mammary gland development, and loss of stereotypical sexual behavior (Lydon et al., 1996). In fact, larger testes and greater sperm production have been observed in male Pr-/- mice (Lue et al., 2013; Schneider et al., 2005). Combined with the observations in the cyp17a1-/- fish and Pr-/- male mice, we speculate that the stimulated effects on spermatogenesis in the testis are not caused by the depleted actions of progestin or androgen signaling. These effects are likely to stem from the highly coordinatively regulated actions of androgen and progestin signaling cascades. In teleosts, various effects of progestins, including oocyte maturation, ovulation, spermatogenesis initiation, and spermatogenesis stimulation, have been previously reported (Chen et al., 2013; Miura et al., 2006; Nagahama and Yamashita, 2008). However, most early studies on teleosts have been conducted through progestin administration without robust genetic studies. Considering this evidence, the unimpaired fertility observed in the npgr homozygous males may be attributed to the presence of androgen signaling (Tang et al., 2016; Zhu et al., 2015). Recently, subfertility has been observed in npgr-deficient male tilapia (Oreochromis niloticus), which provides the first genetic evidence of the functions of progestin signaling in teleost spermatogenesis (Fang et al., 2018). The decreased levels of pituitary follicle-stimulating hormone subunit β (fshβ) and testis follicle stimulating hormone receptor (fshr) in the npgr-deficient tilapia were attributed to impaired spermatogenesis, the decline of milting, and livability of offspring (Fang et al., 2018). Nevertheless, the npgr-deficient male tilapia can successfully sire offspring, which might be due to the presence of androgen signaling, as evidenced by the increased concentration of 11-KT (Fang et al., 2018). On the other hand, the CYP17A1 deficiency in humans and mice results in disorders of sex development and absence of masculinization (Kater and Biglieri, 1994; Liu et al., 2005; Marsh and Auchus, 2014; New, 2003; Yanase, 1995; Yanase et al., 1991), as well as accumulated P4, 11-deoxycorticosterone, and corticosterone (Auchus, 2017). We believe that the role of progestin signaling in facilitating spermatogenesis and testis organization to compensate for androgen signaling insufficiency is highly divergent between fish and mammals. Unfortunately, the DHP content in people with 17-hydroxylase/17,20-lyase deficiency was not measured and analyzed in that previous study (Auchus, 2017).

It has been reported that the activation of the gonadal-pituitary feedback axis was observed in cyp17a1 and cyp19a1a knockout fish, as evidenced by the significant upregulation of pituitary fshβ and lhβ (Li et al., 2020; Tang et al., 2017; Zhai et al., 2018). These mutants also displayed impaired sex steroid biosynthesis, as measured by sex steroid concentrations in homozygous fish (Li et al., 2020; Tang et al., 2017; Zhai et al., 2018). The upregulation of gonadotropins observed in these knockout zebrafish could be attributed to the absence of negative feedback due to androgen or estrogen deficiency (Chen et al., 2017; Tang et al., 2017; Zhai et al., 2018). In the testes of adult zebrafish, Fshβ stimulates the proliferation and differentiation of spermatogonia and its entry into meiosis (Holdcraft and Braun, 2004; Nobrega et al., 2015; Patiño et al., 2001; Ramaswamy and Weinbauer, 2014). This supports the observations in the cyp17a1-/- fish and cyp19a1a-/- fish, as well as our newly generated cyp17a1-/-;ar-/- fish, in which testicular development and spermatogenesis were stimulated (Figure 6—figure supplement 2D, F and J). Compared with that in the cyp17a1-/-;ar-/- fish, the normal GSI and spermatozoa number displayed in cyp17a1-/-;ar-/-; fshβ-/- fish (Figure 6—figure supplement 2E, F and K) also support that the upregulated Fshβ contributes to the hypertrophic testicular development and overactivated spermatogenesis in the cyp17a1-/-;ar-/- zebrafish. By monitoring the expression of pituitary fshβ in fish of the aforementioned eight genotypes, we found that the expression of fshβ was upregulated in the cyp17a1-/- fish and cyp17a1-/-;ar-/- fish. This was an expected result based on our previous study (Zhai et al., 2018). The npgr depletion did not affect pituitary fshβ expression as npgr-/- males and ar-/-;npgr-/- males exhibited comparable fshβ expression to the control males. However, the additional deletion of cyp17a1 significantly upregulated fshβ expression in the males of these genotypes (in the cyp17a1-/-;npgr-/- fish and cyp17a1-/-;ar-/-;npgr-/- fish) (Figure 6—figure supplement 3). These results demonstrated that cyp17a1-/-;ar-/-;npgr-/- fish have impaired spermatogenesis and testis organization, and upregulated pituitary fshβ.

The upregulations of gonadal steroidogenesis-related genes, star, hsd3β1, cyp11a2, and cyp11c1, in Leydig cells were observed in the ar-/- males, npgr-/- males, cyp17a1-/-;ar-/- fish, cyp17a1-/-;npgr-/- fish, ar-/-;npgr-/- males, and cyp17a1-/-;ar-/-;npgr-/- fish. The upregulated expression of steroidogenesis-related genes in the ar-/- males has been reported previously (Tang et al., 2018), which may be attributed to the positive feedback effect caused by androgen or progestin signaling insufficiency. Another marker of the Leydig cells, insl3, which has been reported to be essential for maintaining germ cell differentiation, was significantly decreased in the ar-/- males, ar-/-;npgr-/- males, and cyp17a1-/-;ar-/-;npgr-/- fish (the lowest), the only three genotype groups examined with impaired spermatogenesis and testis organization (Figure 6A). Considering the downregulation of insl3 expression shown in the Venn diagram (Figure 6—figure supplement 1A and B), and reported in both cyp11c1 and cyp11a2 mutant male zebrafish, which are associated with disorganized testicular structure and significantly decreased numbers of mature spermatozoa (Li et al., 2020; Zhang et al., 2020), it is reasonable to speculate that insl3 may be a target that is co-regulated by androgen signaling and high level of progestin signaling. Among the upstream signals of the insl3, the role of the accumulated progestin may be a compensatory signaling pathway that regulates testis organization and spermatogenesis in the absence of androgen signaling. The expression levels of the markers of Sertoli cells, sox9a, amh, dmrt1, and igf3 were decreased in the ar-/- males, cyp17a1-/- fish, npgr-/- males, cyp17a1-/-;ar-/- fish, cyp17a1-/-;npgr-/- fish, ar-/-;npgr-/- males, and cyp17a1-/-;ar-/-;npgr-/- fish (Figure 6B), indicating impaired functions of Sertoli cells in these fish. The divergent expression patterns of the germ cell markers, dazl, vasa, dnd, nanos1, nanos2, piwil1, and piwil2, were observed in the fish of different genotypes, except cyp17a1-/-;ar-/-;npgr-/- fish (Figure 6C). These results might indicate that the upregulated expression levels of germ cell markers are not critical for the restoration of the defective phenotypes of the testis organization and spermatogenesis caused by the lack of androgen signaling in these various mutant lines (Tang et al., 2018). The Insl3 receptors, rxfp2a and rxfp2b, expressed in type A spermatogonia and Sertoli cells/myoid cells, respectively, were also decreased in the cyp17a1-/-;ar-/-;npgr-/- fish compared with that in the control males (Figure 6B and C), suggesting compromised Insl3 signaling in cyp17a1-/-;ar-/-;npgr-/- fish. The upregulated expression of the retinoic acid-degrading enzyme, cyp26a1, decreased GSI and disrupted testis morphology in the cyp17a1-/-;ar-/-;npgr-/- fish (Figures 6D and 4H and I), correlating with the observations in the insl3-/- males (Crespo et al., 2021).

The sperm fertilities of the ar+/+ males and ar-/- males after treatment with DHP were also measured. Unfortunately, both experiments showed impaired fertility after DHP treatment via artificial fecundation assays with WT females (Figure 3—figure supplement 1). Based on our observations and several previous publications, the chemical reagents of sex steroids used in the animal administration would result in developmental abnormalities, such as the E2 usually leads to abnormal body growth, enlarged abdomen and liver, and a large amount of liquid in the peritoneal cavity induced by 10 nM E2 treatment from 20 dpf to 40 dpf (Chen et al., 2017), and 10 μg/L (36.71 nM) E2 treatment from 18 dpf to 90 dpf, as well as DHP treatment in the present study (data not shown). On the other hand, based on the results in Figure 2B, the dynamic levels of the elevated DHP seen in the cyp17a1-/- fish might suggest the failure of rescued fertility due to being unable to precisely match in vivo DHP dynamic levels or the potential side effects of the constant levels of exogenous DHP exposure. However, partial restoration of the testicular phenotypes of ar-/- males via DHP administration supports the mechanism by which an enhancement of progestin signaling pathway is an alternative to testis organization and spermatogenesis to androgen signaling (probably via Insl3) in cyp17a1-/- fish. Therefore, a high level of endogenous or exogenous DHP can compensate for androgen signaling insufficiency to facilitate testis organization and spermatogenesis (Figure 7C–E).

Figure 7

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In summary, our present study with a series of sex steroid signaling deficiency models and DHP administration provides compelling evidence demonstrating that an augmentation of progestin/nPgr signaling can sufficiently compensate for proper spermatogenesis and testis organization when androgen signaling is depleted in zebrafish.

The knockout fish and genes involved in this study have been cited and clearly listed in the references. The transcriptomics raw data files in this article are available in Sequence Read Archive (SRA) at NCBI, and the BioProject is PRJNA796639.

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   https://www.ncbi.nlm.nih.gov/sra/?term=PRJNA796639

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Author details

1. Gang Zhai

   1. State Key Laboratory of Freshwater Ecology and Biotechnology, Chinese Academy of Sciences, Wuhan, China
   2. College of Advanced Agricultural Sciences, University of Chinese Academy of Sciences, Beijing, China

   Contribution

   Data curation, Funding acquisition, Investigation, Methodology, Validation, Visualization, Writing – original draft

   Contributed equally with

   Tingting Shu

   Competing interests

   No competing interests declared

2. Tingting Shu

   1. State Key Laboratory of Freshwater Ecology and Biotechnology, Chinese Academy of Sciences, Wuhan, China
   2. College of Advanced Agricultural Sciences, University of Chinese Academy of Sciences, Beijing, China
   3. Chinese Sturgeon Research Institute, China Three Gorges Corporation, Hubei, China

   Contribution

   Investigation, Methodology, Validation, Visualization, Writing – review and editing

   Contributed equally with

   Gang Zhai

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-3020-9329

3. Guangqing Yu

   1. State Key Laboratory of Freshwater Ecology and Biotechnology, Chinese Academy of Sciences, Wuhan, China
   2. College of Advanced Agricultural Sciences, University of Chinese Academy of Sciences, Beijing, China

   Contribution

   Methodology, Resources

   Competing interests

   No competing interests declared

4. Haipei Tang

   5State Key Laboratory of Biocontrol, Institute of Aquatic Economic Animals and Guangdong Provincial Key Laboratory of Improved Variety Reproduction in Aquatic Economic Animals, School of Life Sciences, Sun Yat-Sen University, Guangzhou, China

   Contribution

   Resources

   Competing interests

   No competing interests declared

5. Chuang Shi

   1. State Key Laboratory of Freshwater Ecology and Biotechnology, Chinese Academy of Sciences, Wuhan, China
   2. College of Advanced Agricultural Sciences, University of Chinese Academy of Sciences, Beijing, China

   Contribution

   Investigation, Methodology, Visualization

   Competing interests

   No competing interests declared

6. Jingyi Jia

   College of Fisheries, Huazhong Agriculture University, Wuhan, China

   Contribution

   Investigation, Methodology, Software

   Competing interests

   No competing interests declared

7. Qiyong Lou

   State Key Laboratory of Freshwater Ecology and Biotechnology, Chinese Academy of Sciences, Wuhan, China

   Contribution

   Investigation

   Competing interests

   No competing interests declared

8. Xiangyan Dai

   Key Laboratory of Freshwater Fish Reproduction and Development and Key Laboratory of Aquatic Science of Chongqing, School of Life Science, Southwest University, Chongqing, China

   Contribution

   Validation, Visualization

   Competing interests

   No competing interests declared

9. Xia Jin

   State Key Laboratory of Freshwater Ecology and Biotechnology, Chinese Academy of Sciences, Wuhan, China

   Contribution

   Resources, Supervision

   Competing interests

   No competing interests declared

10. Jiangyan He

    State Key Laboratory of Freshwater Ecology and Biotechnology, Chinese Academy of Sciences, Wuhan, China

    Contribution

    Methodology, Project administration, Resources

    Competing interests

    No competing interests declared

11. Wuhan Xiao

    1. State Key Laboratory of Freshwater Ecology and Biotechnology, Chinese Academy of Sciences, Wuhan, China
    2. College of Advanced Agricultural Sciences, University of Chinese Academy of Sciences, Beijing, China
    3. The Innovative Academy of Seed Design, Chinese Academy of Sciences, Wuhan, China

    Contribution

    Resources

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0002-2978-0616

12. Xiaochun Liu

    5State Key Laboratory of Biocontrol, Institute of Aquatic Economic Animals and Guangdong Provincial Key Laboratory of Improved Variety Reproduction in Aquatic Economic Animals, School of Life Sciences, Sun Yat-Sen University, Guangzhou, China

    Contribution

    Resources

    Competing interests

    No competing interests declared

13. Zhan Yin

    1. State Key Laboratory of Freshwater Ecology and Biotechnology, Chinese Academy of Sciences, Wuhan, China
    2. College of Advanced Agricultural Sciences, University of Chinese Academy of Sciences, Beijing, China
    3. The Innovative Academy of Seed Design, Chinese Academy of Sciences, Wuhan, China

    Contribution

    Conceptualization, Funding acquisition, Methodology, Project administration, Resources, Supervision, Writing – review and editing

    For correspondence

    zyin@ihb.ac.cn

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0002-7969-3967

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