Antibody Preparation, Protein Expression, and Function Analysis of Cyp19a1b in Ovarian Differentiation in a Natural Triploid Teleost Qi River Crucian Carp (Carassius auratus)

Abstract

Estrogen is an essential sex steroid that functions in numerous biological systems including female reproduction, neuroendocrine, vascular, skeletal, and immune systems. The synthesis of estrogen is controlled by the rate-limiting enzyme, which has been confirmed to exist in two different forms, named brain aromatase and ovary aromatase, and encoded by cyp19a1a and cyp19a1b respectively in teleosts. However, existing studies have primarily focused on the expression and function of cyp19a1b in the brain and cyp19a1a in the gonad, the roles of cyp19a1b in the female gonad of teleosts are largely unknown. In our previous study, we cloned the full length of the cyp19a1b gene from a natural triploid teleost Qi River crucian carp (Carassius auratus), andthe spatial and temporal expression patterns of cyp19a1b mRNA were detected. To further clarify the roles of cyp19a1b in the ovarian differentiation of Qi River crucian carp, we produced a polyclonal antibody of Cyp19a1b in this study. Western blotting results showed that Cyp19a1b was mainly expressed in the brain and then in the ovary, heart, liver, and muscle. During embryogenesis, Cyp19a1b was abundantly expressed in the neurula stage. Immunohistochemistry demonstrated that Cyp19a1b was expressed in the radioactive glial cells (RGCs) of the brain from 20 days after hatching (dah) and the somatic cells of the ovaries from 30 dah, the critical period of ovarian differentiation in Qi River crucian carp. With the treatment of letrozole, an inhibitor of the aromatase, the expression of Cyp19a1b was downregulated both in the brain and gonad. Our results suggested that Cyp19a1b might be involved in the development of the nervous system and also participate in the ovarian differentiation of Qi River crucian carp.

1. Introduction

The developing gonads secrete steroid hormones testosterone (T) and estradiol (E2), which play essential roles in differentiating into either testicles or ovaries. Likewise, it had been proposed that the brain’s neuronal networks are influenced by T and E2, developing in a way that is specific to either males or females [1]. Similar to other vertebrates, the brain of teleosts also serves as a target organ of steroids. The effect of these steroids has primarily been investigated in relation to the neuroendocrine control of reproduction [2]. Among the various sex steroids, estrogen is crucial for numerous physiological and biochemical processes, including sex determination and differentiation, the development and maintenance of gonads, and the regulation of the reproductive cycle. Aromatase, the rate-limiting enzyme in estrogen synthesis, catalyzes the demethylation of carbon 19 in androgens, resulting in the production of phenolic 18-carbon estrogens. In teleosts, this enzyme is encoded by two genes: cyp19a1a (also known as P450aromA, cyp19a, or cyp19a1) and cyp19a1b (also known as P450aromB, cyp19b, or cyp19a2). The cyp19a1a gene encodes the ovarian or gonadal type of aromatase, while the cyp19a1b gene encodes the neural or brain type of aromatase. These isoforms are predominantly expressed in the gonads and the brain, respectively [3,4]. Cyp19a1a is known for its pivotal role in the process of sex differentiation in fishes [5]. The upregulation of cyp19a1a is crucial for ovarian differentiation, while downregulation is needed for testicular differentiation [6]. The occurrence of high aromatase activity in the brain was found to correspond to the expression of the brain aromatase (cyp19a1b) [7,8,9,10,11,12], but the roles of cyp19a1b in the ovary are largely unknown.

Increasing evidence has identified important roles for brain-derived estrogens in reproduction, neuroendocrine function, and neurogenesis. In the initial phases of gonadal differentiation and reproductive development in fish, the expression level of cyp19a1b is elevated both in the brain and gonads [13]. Function analysis indicates that the complete inhibition of aromatase activity leads to a decrease in sexual behavior and deficits in social recognition in both mice and teleosts [14,15], suggesting the significant role of cyp19a1b in the regulation of sex differentiation and reproductive behaviors. In contrast to mammals, fish are born with a fully developed brain. The adult fish brain is distinct from that of mammals due to its ongoing and widespread neurogenesis, which is facilitated by the presence of persistent radial glial cells (RGCs) [16]. The RGCs have been identified as the precursors of neuronal cells [17,18], and are proposed to play a critical role in facilitating the newly generated neurons by providing a scaffold for cell migration [19]. Furthermore, expression analysis has revealed that cyp19a1b, together with the estrogen receptor, reaches its peak expression during the neurula stage [20]. This finding indicates the essential role of Cyp19a1b in neurogenesis through regulating estrogen synthesis in the brain. Moreover, Cyp19a1b exhibits high expression levels, specifically in RGCs within the brain, accompanied by the co-expression of multiple steroid synthase genes, including CYP11a1 (cytochrome P450 side chain cleavage), CYP17 (P450 17α-hydroxylase/c17,20 lyase), 3β-HSD (3β-hydroxysteroid dehydrogenase), 17β-HSD (17β-hydroxysteroid dehydrogenase), and 5α-reductase, which, together, regulate reproduction and neuroendocrine function in the brain [21]. Therefore, clarifying the essential role of brain-derived estrogens in teleost sex differentiation is most important and urgently needed.

The Qi River crucian carp (Carassius auratus) is a remarkable polyploid cyprinid species that has evolved from gibel carp (Carassius gibelio) through long-term ecological adaptation [22]. Studies found that the gibel carp possess both gynogenesis and sexual reproduction, which makes it as an excellent model to study ovarian development in vertebrates [23]. During the gonadal development in Qi River crucian carp, we found that the oogonia first appeared in female gonads at 15 days after hatching (dah), and underwent mitosis from 20 to 25 dah, followed by meiosis from 25 to 30 dah (unpublished data), indicating that the crucial time of sex determination in Qi River crucian carp is from 20 dah to 30 dah. However, genes and pathways that participated in the sex determination and differentiation of Qi River crucian carp are largely unknown. Estrogen, which is frequently thought of as a natural inducer of ovarian differentiation, is essential for the maintenance and differentiation of fish ovaries. Nonetheless, the majority of pertinent research focuses on the role of ovarian aromatase in the ovary, with relatively few studies examining the function of brain aromatase in fish sex differentiation and determination. Thus, in this study, we used Qi River crucian carp as an experiment model to examine the functions of Cyp19a1b in female sex differentiation in teleosts. Through polyclonal antibody preparation, protein expression dictation, and inhibitor treatment, the role of Cyp19a1b in the development of the brain and ovary in Qi River crucian carp was systematically elucidated, offering a theoretical framework for the further exploration of the role of steroid hormones in the regulation of sex differentiation in teleosts.

2. Materials and Methods

2.1. Construction of pCold I-Cyp19a1b Recombinant Plasmid

For the construction of pCold I-Cyp19a1b recombinant plasmid, the coding sequence of cyp19a1b was cloned in our previous study [24]. The antigenicity of the Cyp19a1b amino acid sequence from Qi River crucian carp was analyzed using DNASTAR Lasergene 7.1 to identify suitable target fragments. The cyp19a1b-pGEMT-easy plasmid was used as the template for PCR amplification, utilizing a forward primer that included an NdeI recognition site (5ʹ-attggatccAGTCTGTTCTTCATGCTTCT-3ʹ) and a reverse primer that included an XbaI recognition site (5ʹ-agcaagcttTTCCTGTGTTTTTTCGCCCA-3ʹ). The PCR conditions were established as follows: an initial denaturation at 94 °C for 3 min, followed by 37 cycles of denaturation at 94 °C for 30 s, annealing at 59 °C for 45 s, and extension at 72 °C for 1 min, concluding with a final extension at 72 °C for 5 min. The resulting PCR products were analyzed via electrophoresis on a 1.2% agarose gel. The purified PCR fragment with a size of 597 bp was ligated into pCold I vector and transformed into E. coli BL21(DE3). Positive clones were chosen and sequenced at Life Technologies Corporation (Shanghai, China) following an overnight incubation on an LB solid medium at 37 °C. The following experiment was conducted after the sequence’s accuracy was confirmed.

2.2. Expression and Purification of Recombinant Cyp19a1b Protein

The LB medium supplemented with ampicillin was utilized for the cultivation of bacteria containing the recombinant plasmid, maintained at 150 rpm and 37 °C. Upon reaching an optical density of 0.6 to 0.8 at 600 nm, 1 mM IPTG was introduced, and the culture was incubated for an additional six hours at 200 rpm. Following this incubation, centrifugation was employed to harvest the cells, which were subsequently analyzed for the target protein using Western blotting and SDS-PAGE electrophoresis. The bacteria expressing the recombinant protein were resuspended in phosphate-buffered saline (PBS) and subjected to ultrasonication at 100% power for 30 min, incorporating intervals of 3 s of sonication followed by 6 s of rest. The insoluble fractions were then separated through centrifugation at 4 °C for 30 min at 13,000 rpm. The protein elution and purification of inclusion bodies were performed using Ni Sepharose Fast Flow with imidazole eluates of varying concentration gradients. The purity of the protein was assessed through SDS-PAGE analysis. The purified protein was subsequently collected and stored at −80 °C.

2.3. Production of Cyp19a1b Polyclonal Antibody

The purified recombinant protein was utilized for the generation of antibodies in rats. Initially, the rats were immunized subcutaneously with 200 μg of the protein in Freund’s complete adjuvant. Subsequently, two booster injections of 100 μg of the protein were administered at three-week intervals using incomplete Freund’s adjuvant. The antiserum was harvested seven days following the final booster injection, and was subsequently stored at −80 °C for future use.

2.4. Antiserum Titer Determination by ELISA

Antibody titer was measured using an indirect enzyme linked immunosorbent analysis (ELISA) as below. The purified antigen was diluted to a concentration of 2 µL in 50 mM carbonate salt buffer at pH 9.6 and subsequently coated onto the wells of the plates at a volume of 100 µL per well, followed by incubation at 4 °C overnight. The wells were then subjected to three washes with PBS-Tween buffer (0.05% Tween 20 in PBS). Following this, the coated wells were blocked with 3% bovine serum albumin (BSA) for one hour at 37 °C, after which they were incubated with 100 µL of polyclonal antibodies targeting the Cyp19a1b protein at various dilutions ranging from 1:1000 to 1:51,200. After a two-hour incubation at 37 °C, the wells were washed thoroughly and then incubated with 100 µL of horseradish peroxidase-conjugated goat anti-rabbit IgG (dilution 1:5000, Sigma-Aldrich, St. Louis, MO, USA) for one hour at 37 °C. Peroxidase activity on the immunoblot was detected using o-phenylenediamine and H₂O₂ as enzyme substrates. Color development was stopped with 2 mol/L of H₂SO₄ and the absorbance was measured at 490 nm using SYNERGY H1 Microplate Reader (BioTek, Burlington, VT, USA).

2.5. Sample Collation

For tissues distribution, three adults were selected and the brain, heart, intestine, liver, spleen, kidney, ovary, and muscle were dissected, frozen in liquid nitrogen, and then placed at −80 °C for use.

For exploding the expression patterns of Cyp19a1b during embryonic development and ontogenesis, the fertilized eggs of Qi River crucian carp obtained by gynogenesis were obtained by artificial insemination during the breeding season, and materials from various stages of embryo development (unfertilized, fertilized, blastocyst, gastrula, neurula, tail sprout, heartbeat, hatching) and ontogenesis (10, 20, 25, 30, 40, 60, 90, 150, 210, 720 dah) were collected and placed in liquid nitrogen for quick freezing and set aside at −80 °C for further study. It is noteworthy that all animal experiments conducted within this study strictly adhered to the ethical guidelines stipulated by the Ethics Committee of Henan Normal University (HNSD-2019-03-02). To minimize any possible distress, the fish were immersed in an MS-222 solution as a means of euthanasia.

2.6. Western Blotting

Proteins (20 μg) from each tissue were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinyl difluoride membranes (Millipore, Burlington, MA, USA). The membranes were blocked with 5% skim milk dissolved in TBST buffer overnight at 4 °C. After that, the membranes were washed three times with TBST, then incubated with primary antibody (anti-Cyp19a1b, 1:2000) in TBST for one hour at 37 °C. Horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (Beyotime, A0208, Shinghai, China) or horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (Beyotime, A0216) diluted 1:1000 in TBST were incubated on membranes for 30 min at 37 °C following five rounds of TBST washing. As a control, a mouse monoclonal antibody against a-tubulin (abcam, ab7291, Cambridge, UK) was diluted 1:2000 in TBST. Protein expression was detected using enhanced chemiluminescence detection reagents (Vazyme, E411, Nanjing, China) and visualized using the GE Image Quant LAS4000mini system (GE Healthcare, Buckinghamshire, UK).

2.7. Immunohistochemistry Analysis

The protein localization of Cyp19a1b was determined by dissecting the brain and female gonads of Qi River crucian carp at 10, 20, 30, 50, 90, 150, 210, and 720 dah, and then fixing them in Bouin’s solution at room temperature for a duration of 12 h. Afterwards, the tissues were dehydrated, embedded in paraffin, and then sectioned at a 5 μm thickness. After that, these parts underwent a number of procedures, including blocking, hydration, and deparaffinization. After that, the sections were incubated with a 1:1000 diluted primary polyclonal antibody for an entire night at 4 °C. They were then rinsed three times for five minutes each using 1× PBS. The sections were then treated to a 30 min incubation period with a secondary antibody coupled with horseradish peroxidase (diluted 1:2000; Bio-Rad, Hercules, CA, USA). Subsequently, they were cleaned with PBS and visualized using 3, 3′-diaminobenzidine (Sigma-Aldrich, St. Louis, MO, USA). Ultimately, hematoxylin was used as a counterstain on the components. The primary antibody was swapped out for normal rabbit serum as a negative control. All immunohistochemistry (IHC) images in this study were captured using a Zeiss Axio Scope A1 microscope (Carl Zeiss AG, Oberkochen, Germany), which was equipped with an AxioCam MRc5 digital camera (Carl Zeiss AG, Oberkochen, Germany).

2.8. Letrozole Treatment

For inhibitor treatment, letrozole powder was dissolved in alcohol with a concentration of 200 μg/mL. Then, 200 Qi River crucian carp at 5 dah were divide into two groups according our previous study [25]; one group was fed with the food mixed with letrozole liquor with 200 mg/kg, and the other group was fed with normal food mixed with alcohol. The animals were treated for 60 days, and then the fish were normally reared until sampling at 210 dah. Finally, the brain and gonad tissues of Qi River crucian carp with letrozole treatment were fixed in Bouin’s solution for observation of histological structure, and the tissues were also frozen in liquid nitrogen and then placed at −80 °C for use.

3. Results

3.1. Protein Purification and Antibody Preparation

SDS-PAGE analysis was used to detect the purification of the recombinant protein. The induced pure recombinant protein, as demonstrated by the results, had a single band at roughly 44 kDa, indicating effective purification (Figure 1A). The greatest serum A490 nm ratio dilution that surpassed 2.1—that is, the ratio of post-immunization serum to negative control serum at A490 nm—was used to calculate the antibody titer. According to the ELISA results, the Cyp19a1b antibody showed a great specificity (

Table 1. Antibody titer test.

Dilution Ratio	OD	Positive/Negative
1:1000	3.213	36.9
1:2000	2.879	33.1
1:4000	2.507	28.8
1:8000	2.108	24.2
1:16,000	1.538	17.6
1:32,000	1.076	12.3
1:64,000	0.663	7.6
1:128,000	0.358	4.1
Negative	0.087	1

). Additionally, a single band measuring roughly 44 kDa was detected by Western blotting in the whole protein of Qi River crucian carp brain tissue, indicating that the Cyp19a1b polyclonal antibody had good specificity (Figure 1B).

3.2. Tissue Distribution of Cyp19a1b in Qi River Crucian Carp

The results showed that the Cyp19a1b protein exists at the highest level in the brain, while at a relatively lower level in the heart, liver, ovary, and muscle (Figure 2), which was consistent with cyp19a1b mRNA expression patterns detected by qRT-PCR in our previous study [24].

3.3. Expression of Cyp19a1b During Embryonic Development in Qi River Crucian Carp

As the results showed, the expression of Cyp19a1b increased with embryonic development, and reached the highest level in the neural embryo and then decreased (Figure 3), which was basically consistent with cyp19a1b mRNA expression patterns detected by qRT-PCR in our previous study [24].

3.4. Cellar Location of Cyp19a1b in the Brain

The RGCs were identified as the main cells responsible for Cyp19a1b expression. As the IHC results showed, the positive signal of Cyp19a1b protein was first detected at 20 dah (Figure 4B). With the development of the brain, the signal of the Cyp19a1b protein was solely detected at the putative RGCs of the forebrain, close to the brain ventricles (Figure 4).

3.5. Cellar Location of Cyp19a1b in the Ovary

In the ovary, a positive signal was observed in somatic cells starting from 30 days after hatching (dah), which marks a critical period for ovarian differentiation in Qi River crucian carp (Figure 5C). As ovarian development progresses, the expression of Cyp19a1b exhibits a modal increase, peaking at 90 dah when the gonads are filled with phase II oocytes (Figure 5F). However, at 150 dah, when the ovary is filled with phase III oocytes, the expression of Cyp19a1b is downregulated. By 720 dah, Cyp19a1b expression is detected in both somatic cells and phase V oocytes (Figure 5H).

3.6. Expression of Cyp19a1b with Letrozole Treatment

With the treatment of letrozole, Qi River crucian carp were sex-reversed. The IHC result revealed the presence of Cyp19a1b in the somatic cells of the ovary and spermatogonia and spermatocytes of the testis (Figure 6). Western blotting results showed that the expression level of Cyp19a1b in the brain and ovary of the female control group was higher than that in the brain and testis of the letrozole-induced sex reversal group (Figure 7).

4. Discussion

The adult fish brain is well known for its high aromatase activity. Although the functional significance for this unique feature is not fully understood, the mechanisms underlying the high expression of Cyp19a1b are starting to be well deciphered. In the current study, a polyclonal antibody against Cyp19a1b was developed to examine the expression patterns and cellular localization of brain aromatase in Qi River crucian carp. Additionally, letrozole treatment was employed to further investigate the role of Cyp19a1b in sex differentiation within this species, thereby providing insights for future research on the influence of steroid hormones in the brain on gonadal development in teleost fish.

Cyp19a1 is a duplicated gene in all investigated teleost fish, except in eels [26]. These two genes were identified in a variety of species such as Oncorhynchus mykiss, C. auratus, Danio rerio, Oreochromis niloticus, Dicentrarchus labrax L., Oryzias latipes, Cyprinus carpio L., and Clarias gariepinus [5,27,28,29,30]. The expression of Cyp19a1b in Qi River crucian carp exhibited a certain degree of tissue specificity, being particularly abundant in the brain and gonads, a pattern that is consistent with findings in rhubarb fish (Larimichthys crocea) [31] and Monopterus albus [32]. Furthermore, Cyp19a1b was also detected in the liver, suggesting its potential role in the conversion of androgens to estrogens during circulation, thereby facilitating estradiol-related functions such as the stimulation of yolk protein synthesis in the liver and the promotion of germ cell development [33]. The relatively low expression of Cyp19a1b was also observed in the muscle of Qi River crucian carp, which might be linked to the effects of estrogen on growth, metabolism, and other physiological processes, warranting further investigation [34].

Cyp19a1b has been previously detected in unfertilized eggs from various fish species, including zebrafish (Danio rerio) and rainbow fish [35,36,37]. Our Western blotting analysis indicated that the expression of Cyp19a1b exhibited a progressive increase during embryonic development, peaking at the neurula stage, which suggested the important role of Cyp19a1b in neurogenesis. In zebrafish, immunohistochemical studies revealed strong Cyp19a1b expression in the RGCs of the telencephalon, preoptic area, and hypothalamus [38,39,40]. Similarly, in Qi River crucian carp, Cyp19a1b expression was exclusively detected in RGCs and showed an increase corresponding to brain development. This expression of Cyp19a1b in RGCs also suggests the existence of a high local production of estradiol [39,41]. In mammals, RGCs are known to diminish following embryonic neurogenesis; however, in teleosts, RGCs persist in the adult brain, which may be associated with their potential as progenitor cells [42,43], contributing to the high neurogenic capacity observed in fish brains. The specific role of aromatase in RGCs remains inadequately understood; yet, there is growing evidence to suggest that estrogens may play a critical role in embryonic, adult, or reparative neurogenesis [44,45,46].

The ontogenetic expression of ovarian aromatase is associated with sex differentiation in fish [3,47,48]; however, the expression and location of brain aromatase within the ovary have only been documented in a few species [10,13,37]. During the early stages of gonad differentiation and reproduction in fish, cyp19a1b exhibits high expression levels in both the brain and gonads, indicating its involvement in regulating sex differentiation and reproductive behavior [49]. In Micropterus salmoides, the upregulation of cyp19a1b expression was observed to coincide with the occurrence of germ cells. This is comparable to our study in Qi river crucian carp [50], suggesting the potential involvement of Cyp19a1b in promoting germ cell differentiation. Moreover, recent studies indicate that developing brains exhibit sex-specific differences prior to the influence of gonadal steroids, indicating that the brain may play a pivotal role in determining the trajectory of gonadal development. In zebrafish, depending on the expression of brain-specific aromatase (cyp19a1b), larvae can be segregated into high- and low-expression groups prior to gonadal differentiation [51,52]. Fish aromatase significantly influences gonad development through the brain–pituitary–gonadal axis [53]. In Qi River crucian carp, we found that the expression of Cyp19a1b in the brain is detectable earlier than that in the ovary, which indicated that brain-specific aromatase might influence ovary differentiation through the brain–pituitary–gonadal axis, which requires further confirmation.

Sex steroids, including androgen and estrogen, play a crucial role as endocrine factors in the process of sex differentiation. Endogenous estrogens are critical for directing initial ovarian differentiation in fish. The enzyme cytochrome P450 aromatase is integral to the hormonal pathway that facilitates the conversion of androgens to estrogens, thereby significantly influencing the phenomenon of sex change. The administration of exogenous estrogen or aromatase inhibitors has been shown to induce sex reversal in fish [3]. Moreover, the cyp19a1b gene has been demonstrated to be highly sensitive to estrogens. In both Labeo rohita and Micropterus salmoides, the expression of cyp19a1b in the brain and ovary was all induced by exogenous E2 [9,35]. In our previous study, letrozole treatment induced sex reversal in Qi River crucian carp. Following aromatase inhibitor treatment, the expression of the Cyp19a1b gene was suppressed in both the brain and gonads, exhibiting notable sexual dimorphism between females and individuals undergoing sex reversal. This finding was consistent with the study in Micropterus salmoides [50], Oncorhynchus mykiss [54], Oryzias latipes [55], and goldfish (Carassius auratus) [56], suggesting that the high level of Cyp19a1b is essential for ovary development in fish. Moreover, the expression of Cyp19a1b also was detected in the spermatogonia and spermatocytes of testicles, with a comparatively lower level than that in ovary. As we all know, estrogen in testis also plays an important role in spermatogenesis and testis development. In zebrafish, cyp19a1-deficient XY individuals induce spermatocyte apoptosis, resulting in male infertility [57]. The expression of Cyp19a1b in the testes of Qi River crucian carp may also indicate that this gene plays an essential role in male gonadal development, which requires further investigation. In zebrafish, promoter analysis has revealed that the proximal promoter of the cyp19a1b gene contains an estrogen-responsive element (ERE), along with an upstream regulatory sequence, which contributes to the gene’s heightened sensitivity to estrogen. However, this effect is highly cell-specific, being observed exclusively in RGCs or astrocytes [38,39,41,42]. This observation is consistent with the restricted expression of Cyp19a1b in the brains of both developing and adult fish, which is localized to RGCs. With the treatment of letrozole, the expression of cyp19a1b in the brain was also inhibited with sex reversal, indicating a critical role of estrogens in RGCs concerning ovarian development and sex differentiation in Qi River crucian carp.

5. Conclusions

In this study, we successfully generated a polyclonal antibody against Cyp19a1b in Qi River crucian carp, utilizing the full-length cDNA sequence of the cyp19a1b gene obtained from our previous study. The spatio-temporal expression pattern was detected, providing a solid foundation for investigating the role of this gene in ovarian differentiation. Treatment with letrozole resulted in the conversion of gonads that were destined to develop into testes into ovaries, concurrently inhibiting the expression of Cyp19a1b. Our study firstly identified the expression of Cyp19a1b in the ovary and brain, and preliminarily elucidated the roles of Cyp19a1b in the ovarian differentiation and development of the nervous system in the brain of Qi River crucian carp.