Reagents and antibodies

Follicle-stimulating hormone (FSH) was purchased from Bioniche (Canada); Dulbecco's phosphate-buffered saline (DPBS) and DMEM/F12 medium were purchased from Gibco (USA); penicillin–astreptomycin and fetal bovine serum were obtained from Gibco Products; Luteinizing hormone (LH) was purchased from Vetoquinol (France); Estradiol (E2), Bovine serum albumin (BSA), and polyvinyl alcohol were all purchased from Sigma Company (Germany); TRIzol, PrimeScript™ RT reagent kit, and SYBR^® Premix Ex Taq^™ reagents were purchased from Takara Company (Japan); Revert Aid First Strand cDNA Synthesis Kit was purchased from Thermo Scientific (USA); Polymerase chain reaction (PCR) product purification kit were purchased from TaKaRa Company (Japan); qRT-PCR primers were synthesized by Shenggong Bioengineering Co., Ltd. (Shanghai, China); pMD19-T vector and RNA extraction reagent were purchased from RNAiso Plus; terminal transferase and dATP were purchased from Dalian TaKaRa; and rabbit polyclonal POSTN antibody (19899-1-AP) was purchased from Wuhan Sanying (China). The recovery kit was purchased from Tiangen Company (China). RIPA lysate (P0013B), PMSF (ST506), and BCA protein concentration determination kit (P0010) were purchased from Biyuntian (China).

Experimental animal model and sample preparations

The study was performed in accordance with the Principles of the Care and Use of Laboratory Animals and China Council on Animal Care. All experiments were approved by the Institutional Animal Care and Use Committee of the University. Unmated adult Merino sheep (1.5–2.5 years old) were euthanized (n = 10). The ovaries, heart, liver, spleen, lungs, kidneys, and muscles were collected and rinsed with 37 °C normal saline and sent back to the laboratory for follow-up experiments.

Cloning of POSTN gene

Total RNA was extracted from follicle tissue by the TRIzol method, and its OD₂₃₀, OD₂₀₆, and OD₂₀₈ were detected by UV spectrophotometer and then reverse transcribed to synthesize the first strand after electrophoresis detection. One pair of primers was designed with reference to the sheep cDNA sequence (GenBank accession number: XM_004012111.5)—upstream primer: 5′-TACCTTCAAAGAAATCCCCAT-3′; downstream primer: 5′-GGTGAAACGGTAACTGAAG-3′. PCR amplification was performed using the first strand of cDNA synthesized by reverse transcription as a template. The PCR product was purified, cloned by TA, identified by double restriction digestion, and then sent to Shanghai Sangon Technology Co., Ltd. for sequencing.

Bioinformatics analysis of POSTN gene cDNA

Sheep POSTN nucleic acid and amino acid sequences were analyzed using NCBI online BLAST ( http://blast.ncbi.nlm.nic.gov/blast.cgi). The sequences of Homo sapiens (accession: AAI06711), Rattus norvegicus (accession: AIS73061), Bos taurus (accession: AAS02052), Gallus gallus (accession: AAT72403), Callithrix jacchus (accession: JAB23422), Danio rerio (accession: AAI62274), Pan troglodytes (accession: JAA40911), Bos mutus (accession: ELR48035), Ailuropoda melanoleuca (accession: EFB16759), Salmo trutta (accession: XP_029626198), Chiroxinphia lanceolata (accession: XP_032536357), Bos indicus × Bos taurus (accession: XP_027413176), and Theropithecus gelada (accession: XP_025198) were analyzed using the MEGA software. The phylogenetic tree was constructed using the neighbor-joining method.

Follicles with different diameters and corpus luteum isolation

In the ultra-clean workbench, sheep ovaries were randomly collected, the connective tissue was removed to clean the area to isolate follicles, and they were rinsed at least three times with sterile PBS with DEPC water. The follicles were divided into large follicles (>5.5 mm), medium follicles (3.5–5.4 mm), and small follicles (2–3.4 mm) by detecting the diameter of the follicles, and the follicular fluid of different follicles was collected (). The corpus luteum of the ovary was cut into small pieces about 2 mm in size with sterilized scissors, and they were placed in sterile PBS prepared with diethypyrocarbonate (DEPC) water, washed three times, put in liquid nitrogen, and frozen for subsequent experiments.

Collection of GCs, TCs, COC, and oocyte in follicles of different diameters

COCs and follicular membrane cells (theca cells (TCs)) from follicular fluid of different diameters were isolated. After culturing COCs in vitro in mature fluid for 22–24 h, COCs were treated with 100 IU/mL hyaluronidase for 1–2 min and pipetted several times to get Oo and cumulus cells (CCs). There are three replicates in each group, and each replicate contains about 50–120 Oo cells and the collected CCs were mixed into a pool, and 1 mL RNAiso Plus was added separately and stored at −80 °C for later use. TCs were rinsed with PBS prepared with DEPC water for three times to wash away the granulosa cells (GCs) on the inner wall of the follicular membrane, and 1000 rmp centrifuge to remove the supernatant. There are three replicates in each group (large, medium, and small follicles), and each replicate contains 10 membranes separated from follicles.

Immunohistochemistry

The ovarian tissue was soaked in 4% formaldehyde, and paraffin-embedded after different concentrations of alcohol (70%, 80%, 90%, 95%, 100%, and 100%) and dimethylbenzene. The paraffin-embedded tissue was cut into 5 µm sections. The slices were then placed in an oven at 70 °C overnight. The sections were deparaffinized twice in xylene (5 min each time) and then immersed in 100%, 100%, 95%, 90%, 80%, and 70% alcohol for 5 min. The slices were placed in sodium citrate buffer (0.021% citric acid + 0.029% sodium citrate) for 5 min to boil for antigen retrieval. The paraffin sections were then stained using the immunohistochemistry kit (MXB Biotechnologies) according to the instructions. POSTN antibody (Protentech, China, 19899-1-AP) (1:50) was used for section staining. The section was developed using DAB color development kit (MXB Biotechnologies), and then the results were observed using a fluorescence microscope (Olympus, BX53FL).

Immunofluorescence

The cells were washed with PBS, fixed with an immunostaining fixative for 1 h, and then incubated with 0.1% Triton X-100 for 30 min. Then the cells were blocked with 5% donkey serum for 2 h. The cells were then incubated overnight at 4 °C with POSTN (Protentech, China, 19899-1-AP) (1:50) primary antibody. The cells were washed with PBS and stained with donkey anti-rabbit 488. The results were observed using an immunofluorescence microscope.

Western blot

Tissues and cells were lysed with NP40, and then the protein concentration was detected with the BCA protein concentration detection kit and used for aliquoting. Protein samples were aliquoted into 30 µg/15 µL and boiled for 5 min. Then 4% stacking gel and 12% separating gel were used to separate the proteins in the samples. The protein was then transferred to the polyvinylidene fluoride (PVDF) membrane and blocked with 5% skimmed milk, followed by blocking with the following primary antibody overnight. Subsequently, the PVDF membrane was washed with TBS + Tween (TBST) and incubated with the murine monoclonal primary antibody POSTN (Protentech, China, 19899-1-AP) (1:1000) and secondary antibody (Boster, BA1055/BA1051, USA) for 1 h, and then visualized with enhanced chemiluminescence (ECL) hypersensitive luminescent solution.

Real-time quantitative PCR

Total RNA was extracted from sheep follicles, corpus luteum, and cells in each follicle using TRIzol reagent according to the manufacturer's instructions. Total RNA was reverse transcribed into cDNA. Real-time quantitative PCR was performed on a CFX96 system using SYBR Green Supermix. β-actin was used as a reference gene, and the amount of mRNA was calculated with the 2^–△△CT method. All real-time quantitative PCR analyses were repeated three times. POSTN primers are shown in Table 1.

Table 1.

Sequences of primers for POSTN.

Statistical analysis

The experiments were repeated at least three times, as detailed in the figure legends. Experimental data were presented as mean ± standard, and statistics were calculated for one-way repeated measure ANOVA using the Prism 5.0 statistical software (GraphPad Software, San Diego, CA, USA). Significant differences were determined by comparing means using the Bonferroni post-test. Values of p  < 0.05 were considered to be statistically significant. Follicles are defined as large follicles (>5.5 mm), medium follicles (3.5–5.4 mm), and small follicles (2–3.4 mm) according to their diameters.

Cloning and bioinformatics analysis of POSTN cDNA sequence

To analyze the characteristics of the POSTN gene, we first analyzed the cluster of differentiation (CD) region of the POSTN gene. The results showed that the sequence of the cloned PCR product was about 2000 bp (  Fig. S1 (cjas-2021-0036_suppla.docx)). Using ORF Finder analysis, it was found that the CDs were 1557 bp in size ( Fig. S2 (cjas-2021-0036_suppla.docx)), encoding a protein consisting of 518 amino acids ( Fig. S3 (cjas-2021-0036_suppla.docx)), and the protein encoded by the cloned CDs was sheep deletion POSTN isoform X3 ( Fig. S4 (cjas-2021-0036_suppla.docx)). Molecular phylogenetic tree analysis found that the cloned POSTN protein has close homology with B. mutus and B. taurus (Fig. 1).

Fig. 1.

The phylogenetic tree of POSTN in ovine.

POSTN gene expression analysis in sheep tissues

The qRT-PCR method was used to detect the relative expression of POSTN gene in eight tissues, including heart, liver, spleen, lung, kidney, muscle, uterus, and ovary. Figure 2 shows that the POSTN gene is expressed in uterus, ovarian large, medium, and small follicles, heart, liver, spleen, lung, kidney, muscle, and especially in the ovary (p < 0.05).

Fig. 2.

The expression mRNA of POSTN in different tissues of ovine. Note: Different letters indicate significant differences between groups (p < 0.05).

Expression of POSTN in follicles and follicular cells of different diameters

To study the expression pattern of POSTN in the development of sheep follicles, qRT-PCR, Western blot, and immunohistochemical staining were used to analyze the expression of POSTN mRNA and protein in antral follicles, corpus luteum, and follicles of different sizes. The results showed that the expression of POSTN gene and protein gradually increased with the increase in follicle diameter (p < 0.05). Meanwhile, the mRNA expression of POSTN in medium and large follicles was significantly higher than that of the corpus luteum (p < 0.05) (Figs. 3A–3C). To further study the expression of POSTN in the development of sheep follicles, qRT-PCR was used to detect POSTN mRNA in GCs, CCs, TCs, Oo, and membrane cells in antral follicles of different sizes. The results showed that POSTN gene is higher in GCs in large follicles, followed by Oo and CCs, while TC expression is relatively low (p < 0.05). The expression of POSTN in CCs and TCs of large, medium, and small follicles had no significant difference (p > 0.05) (Fig. 3D). Moreover, immunohistochemistry was used to analyze the expression of POSTN protein in follicular cells. The results showed that POSTN protein was highly expressed in corpus luteum tissue and primary follicles, secondary follicles, and Oo, CCs, GCs, and TCs with antral follicles (Fig. 3E).

Fig. 3.

The expression and distribution of POSTN in cells of different antrum follicles. (A) Western blot was used to detect the expression of POSTN in follicles of different sizes. Different lowercase letters indicate significant differences between groups (p < 0.05). (B) ImageJ was used to quantify the immunoblot bands of POSTN, and β-actin was used as an internal reference and the ratio was calculated. Different letters indicate significant differences between groups (p < 0.05). (C) Real-time PCR analysis showed the expression of POSTN in small follicle (2–3.4 mm), medium follicle (3.5–5.4 mm), large follicle (>5.5 mm), and corpus follicle. Different capital letters indicate extremely significant differences between groups (p < 0.01). (D) Real-time PCR analysis showed the expression of POSTN in GCs, TCs, CCs, and Oo. Lowercase letters indicate the comparison of the same type of cells in different follicles. A indicates that there is no difference between MGCs, CCs, Oo, and TCs in small follicles. B indicates that there is no difference between MGCs, CCs, Oo, and TCs in the medium follicle. C and D indicate comparisons between MGCs, CCs, Oo, and TCs in large follicles. Different letters indicate significant differences (p < 0.05). (E) Protein in the ovine ovary illustrates (a) primordial follicle, (b) primary follicle, (c) secondary follicle, (d) small antral follicle, (e) a large antral follicle, (f) COC of a large antral follicle, (g) corpus luteum, and (h) negative control.

Position and expression of POSTN in GCs

To further study the role of POSTN in GCs, we then examined the expression of POSTN in GCs using immunofluorescence assays. The results showed that POSTN was expressed in the nucleus and cytoplasm of GCs (Fig. 4).

Fig. 4.

The distribution and expression of POSTN of ovine GCs. Note: GCs were cultured under complete medium. (A) GCs were observed under a microscope. (B) The cell nucleus was restained with DAPI (blue). (C) The POSTN antibody (green) was used for immunofluorescence staining of GCs. (D) DAPI, POSTN, and GC cell images are merged. Scale, 50 µm.