1. Introduction
In the poultry industry, the sex of animals is directly associated with economic traits [
1,
2]. For instance, in the egg-producing industry, a large number of male chickens are culled after hatching [
3], whereas in the broiler industry, male chickens are preferred because of their higher growth rate and feed conversion ratio [
4]. Therefore, controlling the poultry sex ratio is essential for improving the efficiency of poultry production. Overall, we have a preliminary understanding of male sex-determining genes in poultry, such as the
Z chromosome gene
doublesex and
mab-3 related transcription factor 1 (
DMRT1), which is known to function as a testis determinant; however, the molecular mechanisms underlying female ovarian development have not been fully elucidated.
The
FOXL2/CYP19A1/ERα signaling pathway, which is only activated in females (suppressed in males), is a well-known pathway that plays a key role in ovarian development. However, a recent study showed that although a male (ZZ) chicken with a single functional copy of
DMRT1 (generated by CRISPR-Cas9) developed ovaries in place of testes, which expressed
FOXL2/CYP19A1/ERα as in the wild-type female, the male-to-female sex-reversed gonads could not further develop into functional ovaries [
5,
6]. This indicates that more factors may be involved in chicken ovary development. The
RSPO1/WNT4/β-catenin pathway, which has been shown to be associated with female ovarian development in mammals, is likely to be one of the candidates [
7,
8].
R-spondin 1 (
RSPO1) is a member of the R-spondin family and encodes a protein containing a type 1 thrombospondin repeat sequence (TSR-1) [
9], and it regulates the Wnt/β-catenin signaling pathway. Studies in humans have found that the loss-of-function mutation of
RSPO1 led to sex reversal in 46 XX women, indicating that it plays an important role in ovarian development [
10]. In mice,
RSPO1 is expressed in embryonic gonads, and mutations in mouse
RSPO1 lead to masculinization, dysregulation of
WNT4 expression, and ectopic testosterone production in female mice [
11]. Smith et al. found that
RSPO1 showed a conserved female-biased expression in gonads of chicken, mouse, and red-eared slider turtle [
12]. Based on these findings,
RSPO1 is considered to be a regulator of vertebrate ovarian development [
13]. However, the function of this gene, especially its interaction with the
FOXL2/CYP19A1/ERα pathway, has not been well studied in poultry.
Therefore, we performed a systematic study to determine the spatiotemporal expression of RSPO1 and to explore its relation with the FOXL2/CYP19A1/ERα pathway during chicken ovarian development. In this study, we determined the expression of RSPO1 in both gonadal tissues and cells of E12 (12 days of incubation) male and female embryos by qPCR, detected the expression of the RSPO1 protein in gonads of E12 male and female embryos by Western blotting (WB), and explored the interaction between RSPO1 and the estrogen pathway, another important pathway of ovarian development, at in vivo and in vitro levels using drug treatment and gene overexpression, respectively.
Our study showed that RSPO1 exhibited a female-biased expression in chicken embryonic gonads, which was not inherently present in gonadal cells but was dependent on regulation by the estrogen pathway.
2. Materials and Methods
2.1. Egg Incubation and Sample Collection
Fertilized eggs of HY-LINE variety white chicken (domestic chicken) from a poultry farm (Yangzhou, China) were hatched in an intelligent incubator at 37.5 °C and 60% humidity. (The hens were raised according to the commercial management guides of Hy-line W-36,
https://www.hyline.com/literature/W-36 (accessed on 11 May 2022).) For the spatiotemporal expression experiment, 100 eggs were incubated. For FAD and E2 injection groups and the control group, 60 eggs each were used. The blunt end was rotated upwards every 30 min until the required embryonic stage. On day 6.5 (E6.5), E9, E12.5, or E18.5, eggs were removed from the incubator, and the embryos were carefully dissected to expose the gonads. The gonadal tissues of E6.5, E9, E12.5, and E18.5 embryos were collected into 1.5 mL centrifuge tubes, quickly frozen in liquid nitrogen, and stored at −80 °C for subsequent RNA extraction. Tissues of liver, gonads, brain, spleen, intestine, kidney, stomach, heart, and muscle obtained from E12 (4 female and 4 male) were harvested for the organ-specific expression analysis. The gonads of E12 embryos were collected into 100 μL RIPA lysate (Cat No. C1053, APPLYGEN, Beijing, China) for protein extraction. For E6.5 and fadrozole (FAD)-injected chicken embryos, a small piece of tissue (wings or toes) was collected from each embryo to determine the genetic sex.
2.2. RNA Isolation, cDNA Synthesis, and Quantitative (Real-Time) Polymerase Chain Reaction (q-PCR)
Total RNA was extracted from gonads, cells, and other tissues using TRNzol Universal (TIANGEN, Beijing, China) reagent according to the manufacturer’s instructions. Briefly, gonads were collected into a 1.5 mL RNA enzyme-free centrifuge tube containing 1 mL of TRNzol reagent and homogenized with beads for 2 min. Then, 200 μL of chloroform was added to the lysate, the mix was shaken vigorously for 15 s, and then it was left at room temperature for 5 min. Subsequently, 1 mL of 75% ethanol was added to wash the total RNA, and then RNA was dried and dissolved with ddH
2O. For E6.5 embryos, five pairs of gonads for each gender were pooled (4 pools generated for each sex). For E9, E12.5, and E18.5 embryos, total RNA was extracted from single pairs of gonads (
n = 4 individuals for each sex). For sex reversal studies, total RNA was extracted from pairs of gonads from different groups (control male, control female, FAD-treated male, and FAD-treated female,
n = 5 per group). First-strand cDNA was synthesized using a commercial kit (Cat No. R123-01, Vazyme Biotech Co., Ltd., Nanjing, China), according to the manufacturer’s instructions. Briefly, genomic DNA was removed by mixing 4 × gDNA wiper mix into total RNA and heating at 42 °C for 2 min, and then cDNA was amplified with 5 × qRT SuperMix II at 50 °C for 15 min, followed by a step at 85 °C for 2 min to inactivate the enzyme. Primers were designed according to NCBI, optimized for qPCR, and the most effective primer pair (efficiency > 95% and <105%) was selected. qRT-PCR was performed using an RT-PCR kit (Cat No. Q111-02/03, Vazyme Biotech Co., Ltd., Nanjing, China), and the level of mRNA expression was detected using the QuantStudio3 real-time PCR detection system (Thermo Fisher Scientific, Waltham, MA, USA). Relative expression was measured based on the expression of a housekeeping gene (
β-actin) and was quantitatively analyzed using the 2
−ΔΔCq method. The primer sequences used for qRT-PCR are listed in
Table 1.
2.3. Protein Extraction and Protein Imprinting
Total protein in gonads was extracted using the RIPA buffer according to the manufacturer’s instructions (Cat No. C1053, APPLYGEN, Beijing, China). Briefly, gonads were collected into a 1.5 mL centrifuge tube containing 300 μL RIPA buffer and 3 μL protease inhibitor and then blown to pieces by pipetting. After lysing on ice for 10 min, the lysate was centrifuged at 4 °C at 12,000× g for 10 min, and the supernatant, which contained the total protein, was moved to another tube until later use. For E12 embryos, total protein was extracted from the left gonads of each individual (n = 5 per sex). For FAD-treated embryos, total protein was extracted from a single pair of gonads in each treatment group (n = 3 per group). The relative level of RSPO1 protein in a single sample was estimated by analyzing the value of the target protein band in Western blots using the ImageJ V1.8.0.112 software (National Institutes of Health, Bethesda, MD, USA). The RSPO1 antibody (AF3474) was purchased from R&D Systems (Minneapolis, MN, USA), and staining with a tubulin protein antibody (#2144, CST, Peachtree, GA, USA) was used as an internal reference. The working concentrations of the RSPO1 and tubulin antibodies were 1:1500 and 1:1000, respectively.
2.4. Paraffin Section and Immunostaining
For histological analysis, the complex renal tissues were placed in 4% paraformaldehyde for 24 h. Next, the tissue shape was adjusted under a stereomicroscope. Subsequently, the tissue and corresponding labels were placed in a dehydration cassette, dehydrated with low to high ethanol concentrations, treated with xylene, and cleared for paraffin embedding. The embedded tissues were serially sliced to a thickness of 3 μm. Next, the slices were floated on a spreader of 40 °C warm water to spread the tissues. Then, the tissues were fished up with slides, baked in an oven at 60 °C, dried, and removed for storage at room temperature. Immunohistochemistry was performed as previously described [
14]. Briefly, the slides were washed in PBS at 37 °C for 30 min and immersed in PBS containing 10% donkey serum, 1% BSA (Bovine Serum Albumin), and 0.3% Triton X-100 at 24 °C for 2 h. The slides were incubated with primary antibody overnight at 4 °C, washed in PBS containing 0.3% Triton X-100 before incubation, and then incubated with secondary antibody at room temperature for 2 h. Lastly, the slides were washed in PBS containing 0.3% Triton X-100, and the sections were treated with Hoechst solution (10 mg/mL) for 5 min to stain cell nuclei. The working concentrations of the antibodies used were as follows: goat anti-mouse R-Spondin 1 antigen affinity-purified polyclonal antibody (AF3474, R&D Systems, Minneapolis, MN, USA) was diluted 1:200 for immunostaining, and donkey anti-goat secondary antibody (GB21404, Servicebio, Wuhan, China) was diluted at 1:300 for immunofluorescence.
2.5. Construction of Overexpression Vector
According to the coding region of chicken RSPO1 gene (NCBI ID: 419613, accession number: NM_001318444.2), the RSPO1 overexpression vector was designed and synthesized by Shanghai Gima Gene Company (Shanghai, China) using a pcDNA3.1 vector. The empty vector was a pcDNA3.1 vector containing the CMV promoter. The same sequence of the RSPO1 open reading frame that was cloned and tested in vitro was PCR amplified from cDNA using forward primer 5′-GCTTGGTACCGAGCTCGGATCC-3′ and reverse primer 5′-TGCTGGATATCTGCAGAATTCCTATTGGGCAGGGCTGG-3′ with an included BamHI and EcoRI site. The resultant 783 bp fragment was subcloned into the NcoI and BamHI sites of pcDNA3.1 vector.
2.6. Acquisition, Culture, and Treatment of Chicken Embryo Gonad Cells
Chicken gonadal cells were isolated from the gonads of E12 HY-LINE variety white chicken embryos by 0.25% trypsin-EDTA (Gibco, Grand Island, NY, USA) digestion. First, the gonads were collected, washed three times with PBS (Solarbio, Beijing, China), and then trypsinized. Following the termination of digestion with 10% FBS-DMEM (Gibco, Hongkong, China), cells were washed with PBS, the supernatant was discarded, and cells were resuspended in 1 mL PBS. Centrifugation was performed at 700× g at 25 °C for 5 min, the supernatant was discarded, and 1 mL FBS-DMEM was added. A 70 µm nylon mesh was used to filter the resuspended cells into 50 mL conical tubes, to which complete medium was added, mixed well, and plated for culturing. Cells were seeded at 1 × 106 cells/well in 12-well plates and cultured overnight in complete medium (DMEM medium with 10% fetal bovine serum, 1% penicillin streptomycin solution (100 IU/mL), and 10 μL EGF (PEPROTECH, Cranbury, NJ, USA) (20 ng/mL)). DMEM complete medium was prepared in advance. Cells reaching confluence were treated with β-estradiol. Before estradiol treatment, complete medium and working estradiol solutions were prepared at final concentrations of 0, 100, 150, 200, and 300 μmol/L. After treatment, cells were cultured for 24 h; five replicates were performed for each treatment. After washing with PBS, cells were collected and RNA was extracted using TRNzol Universal reagent. The transfection of the RSPO1 overexpressing and empty plasmids in the control group was carried out according to the instructions for the jetPRIME® in vitro DNA and siRNA transfection reagent (No. 101000046, Polyplus, Shanghai, China). The amount of overexpressing plasmid vectors and empty plasmid bodies was 0.8 μg per well, and cells were collected as described above.
2.7. Sexual Reversal and Genetic Sex Determination
To construct a model of sex reversal from female to male, 0.2 mg fadrozole (experimental group) or 100 μL PBS (control group) was injected into E2.5 eggs [
15]. For the model of sex reversal from male to female, 17β-estradiol (E2; Sigma-Aldrich, St. Louis, MI, USA) was resuspended in 100% ethanol (10 mg/mL) and diluted to 1 mg/mL in sesame oil. Subsequently, 100 μL of 1 mg/mL solution (0.1 mg E2) (experimental group) or 10% ethanol sesame oil solution (control group) was injected into E2.5 eggs [
16]. Briefly, a small hole (0.5 cm in diameter) was made in the blunt end of the egg, and then E2 or FAD was injected into the inner shell membrane above the chick embryo. A very small hole was then made in the membrane using sterilized tweezers, allowing the E2 and FAD fluid to diffuse into the embryo area. Then, the hole in the eggshell was sealed with a breathable medical tape, and the eggs were returned to incubation. The FAD injection group was incubated to E12, whereas the E2 injection group was incubated to E10.5. Follow-up experiments were conducted.
For genetic sex determination, a small piece of wing tissue was collected and used to extract DNA using a commercial kit (Cat No. DC102-01; Novozyme Biotechnology Co., Ltd., Nanjing, China). Subsequently, PCR was performed to amplify the CHD gene sequence located on both sex chromosomes. The CHD-forward/reverse primer sequences were as follows: F: AGTGCATTGCAGAAGCAATATT; R: GCCTCCTGTTTATTATAGAATTCAT. The female (ZW) had two bands at 506bp and 351bp, while the male (ZZ) had only one band at 506 bp [
15].
2.8. Data Analysis
All statistical analyses were performed using the SPSS 22.0 software (IBM, Armon, NY, USA). Following qPCR, the relative gene expression was calculated using the 2−ΔΔCq method. All experiments were repeated twice, and the data are expressed as the mean ± SEM. t-test was used for the significance analysis of data between two groups; Duncan test of one-way ANOVA analysis (SPSS 22.0) was used for data from E2 treatment of gonadal cells. Statistical significance was set at p < 0.05. GraphPad Prism V7 software (GraphPad, San Diego, CA, USA) was used for visualizing data (generating graphs and plots).
4. Discussion
Although sexual differentiation in birds has been studied for decades, the specific molecular mechanisms remain unclear. Recent studies have revealed that
RSPO1 is closely associated with ovarian development in mammalian and scleractinian females and suggested that it might have a similar role in birds [
12,
17,
18].
Our study aimed to investigate the spatiotemporal expression pattern of RSPO1 in various tissues, especially the gonads, of male and female chicken embryos, and delineate the relationship between the expression of RSPO1 and that of other key genes in the estrogen pathway, another important pathway of ovarian development, using drug treatment or gene overexpression both in vivo and in vitro. Our results confirm the female bias of expression of RSPO1 in chicken embryonic gonad tissues; however, this bias was not inherent to gonadal cells but depended on estrogen stimulation. More specifically, we revealed a partial feedback loop regulation of the estrogen pathway by RSPO1, realized mainly through the modulation of the downstream target gene ERα. This study provided new evidence for the function and mechanism of RSPO1 in chicken ovarian development.
A previous study found that
RSPO1 was expressed in a female-biased manner in vertebrate gonads, suggesting its functional conservation in ovarian development [
12]. In addition, our comparison of the amino acid sequences of RSPO1 homologous proteins from different species supported the existence of similar structural RSPO1 domains among different species (
Figure 1). However, the amino acid sequences of RSPO1 exhibited different degrees of variation among species. Previous studies have found differences in the function of RSPO1 among different species [
19,
20,
21,
22,
23,
24]. Therefore, although RSPO1 appears to have conserved functions in ovarian development among different species, its specific mechanisms of action or associated target genes might differ.
Smith et al. [
12] examined the expression of
RSPO1 mRNA in E6.5, E8.5, E10.5, and E12 male and female embryonic gonads and found that
RSPO1 was expressed in a female-biased manner at all developmental stages. Specifically,
RSPO1 was consistently expressed at low levels in male gonads, whereas in females, its expression was gradually increased with gonadal development. This was consistent in part with the results of the present study (
Figure 2B), namely, the female-biased expression of
RSPO1 and its consistently low expression in males. However, in our study, the expression of
RSPO1 in females did not gradually increase in the four periods examined (E6.5, E9, E12.5, and E18.5) but was relatively low at E6.5 (initiation of gonadal differentiation) and E9 (stage of gonadal differentiation), increased sharply at E12.5 (completion of gonadal differentiation, stage of ovarian development), and was maintained at that level until E18.5. This implied that
RSPO1 might be more strongly associated with ovarian development than gonadal differentiation. This notion was supported by the
RSPO1 expression in the left gonad of E12 females being much higher than that of the degenerating right gonad (
Figure 2D), suggesting that the high expression of
RSPO1 in the left gonad at this stage was required for ovarian development. This is consistent with Smith’s whole in situ hybridization results, which showed
RSPO1 expression in the left and right gonads of female chicken embryos [
12].
Furthermore, we verified the involvement of
RSPO1 in chicken ovarian development by its female-biased RSPO1 protein expression (
Figure 2C). Our study confirmed that
RSPO1 was mainly distributed in the cortical layer (
Figure 2E), which agreed with the results of Smith et al. [
12]. Considering that the estrogen receptor ERα is also expressed in the ovarian cortex [
25,
26], it is likely that RSPO1 interacts with the
FOXL2/CYP19A1/ERα axis, another important pathway for ovarian development in chicken embryos. Smith et al. [
12] demonstrated that FAD-induced inhibition of estrogen synthesis reduced the expression of
RSPO1 in chicken embryos; we confirmed this in the present study (
Figure 3C). However, it is not clear whether the effect of FAD treatment on
RSPO1 is mediated by estrogen, as FAD treatment apart from blocking estrogen synthesis also leads to changes in the expression of
AROM and that of other genes in the pathway (such as
FOXL2,
Figure 3B). Therefore, we constructed a male-to-female sex reversal model by injecting estradiol into E2.5 chicken embryos and directly confirmed that estradiol stimulation induced the expression of
RSPO1 in chicken embryonic gonadal tissues (
Figure 4).
These findings confirm that the estrogen pathway regulates the expression of
RSPO1 in chicken embryonic gonadal tissues, causing its female-biased high expression. Cheng et al. [
27] treated Chinese soft-shelled turtle embryos before sex differentiation using different concentrations of estradiol and found that it resulted in significantly increased expression of
FOXL2,
CYP19A1,
WNT4, and
RSPO1. In addition, overexpression of the key enzyme CYP19A1, responsible for estrogen synthesis in male gonads, can upregulate the expression of
RSPO1 [
28]. However, whether this female-biased expression of
RSPO1 was estrogen-dependent remains unclear. To answer this, we isolated gonadal somatic cells from differentiated male and female gonads (E12) and examined the expression of
RSPO1 mRNA and that of other genes after 2 d of culture. Of note, Smith et al. [
12] demonstrated that
RSPO1 was mainly expressed in mouse gonadal somatic cells. However, the expression of
RSPO1 in chicken embryonic gonadal cells was not detected. Here we found that, unlike
FOXL2/CYP19A1/ERα, there was no cell-intrinsic difference in the expression of
RSPO1 between male and female gonadal cells; this pattern was different from the female-biased expression of
RSPO1 in gonadal tissues. We hypothesized that the high expression of
RSPO1 in female gonads might be dependent on estrogen stimulation, as the in vitro culture estrogen synthesis was inhibited despite the high expression of
FOXL2/CYP19A1/ERα in female gonadal cells, probably due to lack of required substrates. Therefore, we treated gonadal cells with estradiol and found that stimulation with estradiol significantly upregulated the expression of
RSPO1, indicating that the female-biased expression of
RSPO1 was estrogen-dependent. The expression of
ERα,
CYP19A1, and
FOXL2, key genes for female pathway development, were also upregulated in estrogen-treated male gonadal cells, further demonstrating the effectiveness of estrogen treatment. Cai et al. [
29] also reported enhanced expression of
RSPO1 during estrus, a phase with high estrogenic signaling activity, suggesting that
RSPO1 is a hormone-mediated local factor whose expression could be upregulated by estrogen and progesterone [
30].
Regarding the mechanism of function of
RSPO1, studies in mammals have suggested that
WNT4 and
β-catenin might be downstream target genes [
10,
11,
31,
32], with
RSPO1 activating the β-catenin pathway together with
WNT4 to participate in ovarian development [
11]. In the present study, we found a similar regulatory relationship in chickens. Overexpression of
RSPO1 in chicken embryonic gonadal cells induced the mRNA expression of
WNT4 and
CTNNB1 (
Figure 6). Interestingly, we identified a partial feedback loop regulation of the
FOXL2/CYP19A1/ERα pathway by
RSPO1, as the levels of
ERα mRNA were upregulated after overexpression of
RSPO1, whereas the expression of the other 2 genes,
FOXL2 and
CYP19A1, did not show significant changes, nor did the expression of the key gene for masculinization,
DMRT1. This indicated that despite the feedback loop regulation of the downstream factors of the
FOXL2/CYP19A1/ERα pathway to a certain extent,
RSPO1 does not directly affect the upstream sex-determining genes. In line with our results, Geng et al. [
33] also found that
RSPO1 promoted
ERα expression in mouse mammary duct luminal cells. This is consistent with our finding that the expression of
ERα was significantly upregulated after overexpression of
RSPO1 in chicken embryonic male gonadal cells. However, Zhang et al. [
24] observed a significant decrease in the expression of
FOXL2 after knocking down the expression of
RSPO1 in the gonads of female Chinese soft-shelled turtles in vivo, which demonstrates the feedback regulation of
RSPO1 on the
FOXL2/CYP19A1/ERα pathway and also highlights the differences in the degree of this feedback among different species. The above studies demonstrate the importance of
RSPO1 in ovarian development and suggest that there is indeed an interaction between
RSPO1 and the estrogen pathway, but the exact mechanisms need to be further investigated.