Expression patterns and immunohistochemical analysis of ERK, HRAS and MEK1 proteins during ovarian prehierarchical follicular development in Zi geese (Anser cygnoides)

Ethical statement
 
The Goose Industry Research and Development Centre of Jilin Agricultural University, enforcing the Regulations for the Administration of Affairs Concerning Experimental Animals, approved all the experimental procedures to ensure animal welfare (Permission number: GR(J)18-011. Date: 22 May 2018).
 
Animals and sample collection
 
Six egg-laying geese (35 to 37 weeks old) were randomly selected from Jilin Agricultural University Goose Farm Research Center. The geese were reared under semi-intensive deep litter system, with ad libitum access to feed and water and exposed to natural photoperiod. The geese were anaesthetized with ether before exsanguination to collect the prehierarchical follicles sampled according to their sizes in diameter and colour as follows; the primary follicles (PF, < 1 mm), small white follicles (SWF, 1 to 4 mm), middle white follicles (MWF, 4 to 6 mm), large white follicles (LWF, 6 to 10 mm) and small yellow follicles (SYF, 10 to 15 mm). The vascular membranes of the outermost layer of the follicles were stripped and ruptured to remove the follicular liquid and the oocyte, thoroughly rinsed with Phosphate-buffered saline (PBS) buffer and then immediately liquid nitrogen and stored in -80°C refrigerator for experimental analysis. Other intact samples of prehierarchical follicles were immediately fixed in 4% neutral buffered formalin at 4°C and then transferred to 70% ethanol for subsequent embedding into paraffin wax.
 
Total RNA extraction and complementary DNA (cDNA) synthesis
 
Total RNA was extracted from the geese prehierarchical follicles using the Trizol reagent method according to the manufacture’s protocol (Invitrogen, USA). The total RNA for each sample was treated with DNAse 1 (Ambion/Life Technologies) to genomic DNA contamination. The RNA quality and concentration were assessed with Agilent 2100 Bio analyser (Agilent Technologies, USA); the purity and degradations were also assessed on 1.2% agarose gels before proceeding to the next experimental procedures. The first strand cDNA was synthesized using a cDNA synthesis Kit (TOYOBO, Japan) according to the operation procedures in the instruction manual.  Concisely, the reverse transcription reaction consisted of 2 µg total RNA, 4 µL of 5XRT buffer, 1 µL of RT Enzyme Mix, 1 µL of Primer Mix and finally Nuclease-Free Water added to a total volume of 20 µL; the reaction conditions of the reverse transcription reaction were 37°C, 15 min and the enzyme deactivation was 98°C, 5 min. The final products were kept at 4°C for daily experimental usage and at -20°C for longer storage.
 
Quantitative real-time PCR
 
The Bio-Rad CFX Real-time PCR Detection System and software (BIO-RAD, California, USA) was used to assess the mRNA quantitative expression of the target genes (ERK, MEK1 and HRAS) in prehierarchical follicles and specific sets of primer pairs were designed using the Primer Premier 5.0 program (Primer-E Ltd., Plymouth, UK) (Table 1).The PCR reaction (20 μl) consisted of 2 μl of cDNA, 0.6 µl of each of the forward and reverse primers (10 μmol), 0.4 µl of ROX Reference Dye, 10 μl of SYBR Green Master Mix and 6.4 µl of nuclease-free water. Amplification conditions were as follows: pre-denaturation at 95°C for 5 min, 45 cycles of amplification (95°C for 15 s and 59°C for 60 s). A melt curve analysis was performed from 60°C to 95°C by taking readings every 0.3°C. The relative quantification of gene expression was detected in triplicate per sample. Gene expression levels were calculated by the 2^-ΔΔCT method using β-actin as an internal control.
 



Western blot analysis
 
The prehierarchical follicles sample tissues were homogenized in RIPA buffer (10 μl of phosphatase inhibitor, 1 μl of protease inhibitor and 5 μl of 100 mM PMSF per 1 mL) followed by centrifugation at 14 000 rpm, at 4°C for 15 min and the supernatant was separated into a whole protein extract. The concentration of protein samples was determined by using the BCA protein content detection kit (Biotechnology Development, Kaiji, Nanjing, China). For every sample, 10 μg total protein was pipetted onto a 12% SDS-PAGE Gel Electrophoresis Kit (Biotechnology Development, Kaiji, Nanjing, China) initially set at 60 V and the voltage was increased to 90 V for 90 min when the protein samples enter the separation gel. The electro blotting was carried out in a transfer liquid at 200 mA for 60-120 min. The membranes were incubated in 5% dry milk (EASYBIO, Inc. Beijing, China. Cat# No.BE6250) for 1.5 to 2 h at 25°C room temperature and after wards the membranes were washed with Tris- buffered saline/Tween 20 (TBST) 3 times for 5 min then incubated overnight at 4°C immersed with the following primary antibody: an anti-ERK antibody, an anti-HRAS and an anti-MEK1 (LifeSpan BioSciences, Inc. Shanghai, China). Finally, the membranes were rinsed with 1×PBST (phosphate-buffered saline with 0.1% Tween-20), then incubated with an HRP Affinipure conjugated goat anti-mouse IgG (H+L) secondary antibody (Wuhan, Hubei, China) for 1 h at room temperature. The membranes were washed three times with 1×PBST at room temperature for a total of 15 min. The membranes were visualized with an ECL Test Kit (Millopore WBKLS0100, Germany). The chemiluminescence of each protein band was quantified using the ImageJ software and protein levels were normalized by β-actin as internal control.

Immunohistochemistry
 
The prehierarchical follicles were fixed for 48 h with 4% paraformaldehyde in PBS, pH 7.4, at 4°C. The follicles were then cut and immersed in different concentrations of ethanol (75%, 85%, 90% and 95%) and immersed in 100% xylene twice for 10 min each time. The treated samples were embedded in paraffin wax and then the tissues were sliced at 5μm thickness. The sliced tissues were stretched and baked at 65°C for 3 h and finally soaked twice in xylenes. The treated tissue sections were washed 3 times with PBS solution for 5 min each time. The slides were then subjected to antigen repair by heating in citrate buffer solution (pH 6) two times for 15 min. Tissue sections were washed 3 times with PBS solution for 5 min each time. Following deparaffinization, rehydration and enzyme digestion, slides were blocked with 3% goat serum at room temperature for 20 min then incubated with 50 μl of the target gene primary antibody at 4°C overnight, and the sections subjected to the incubation were rewarmed for 45 min. The slides were washed 3 times in PBS for 2 min each time and then incubated at room temperature for 60 min with 20 μl secondary antibody. After the incubation with the secondary antibody, the tissues were washed 3 times with PBS solution for 5 min each time followed by DAB staining (5 to 10 min). The stained sections were then rinsed with tap water for 10 min and then counterstained with hematoxylin for 2 min. Then, the sections were decolourized in 1% hydrochloric acid-ethanol for 10 to 15 s, quickly taken out and placed in distilled water and then placed in PBST about pH 7.4 for 5 to 10 min. The stained sections were rinsed with tap water for 15 min, then dehydrated and translucent using different concentrations of ethanol and xylene. The film was then sealed with a neutral resin. The slides were examined with a BX53 electric fluorescent illuminator microscope (Olympus, Tokyo).

Statistical analysis
 
The relative expression levels of target genes in different grades of prehierarchical follicles were analyzed by SPSS23.0 statistical software. Single factor analysis of variance (One-way ANOVA) was used among groups and least square significant difference (LSD) test was used to compare the means. The significant difference of the data was considered as p<0.05.

mRNA expression levels of ERK, HRAS and MEK1 genes in the prehierarchical follicles
 
As illustrated in (Fig 1), the RT-qPCR method was used to determine the differential expression levels of ERK, HRAS, and MEK1 in five stages of prehierarchical follicles in geese. All the candidate genes had varying expression patterns during the prehierarchical follicular development. The ERK mRNA transcripts were predominantly expressed in PF. However, ERK exhibited no significant difference between the SYF, MWF and LWF (p>0.05). These results indicate that ERK may play a significant role during the initial stage of prehierarchical follicle establishment. Lee et al., (2017) found that ERK was highly expressed in oviduct cells and cumulus cells of canine during the estrus stage. HRAS showed a significant noticeable trend of expression levels by decreasing exponentially from PF to SYF (p<0.05), which suggest that HRAS may have dynamic functional activities during the prehierarchical follicle development. Previous studies reported the spatial and temporal HRAS mRNA transcripts during mice development expressed in a stage development and tissue specific manner with the highest expression in the adult brain (Newlaczyl et al., 2017). Moreover, higher expression of MEK1 was observed in SWF suggesting that MEK1 may have an active role by either suppressing or promoting the development of prehierarchical follicle. Studies have been conducted on various types of disease conditions in which the highest MEK1 mRNAs were detected in cancer cells (Gong et al., 2015). Interestingly, undetectable mRNA quantities of ERK and MEK1 were observed in SYF and MWF respectively. These results may be associated with the restricted contributory effect of ERK and MEK1 at these stages of prehierarchical follicular growth and maturation.


 
Expression levels of ERK, HRAS and MEK1 proteins in prehierarchical follicle
 
This study explored the protein expression levels of the three selected genes in the prehierarchical follicles using western blot. The results showed that all the target genes were ubiquitously expressed in all the follicle tissue samples as shown in (Fig 2).The expression of ERK was predominant in both PF and SWF with the least expression in SYF (P<0.05). The highest protein expression in HRAS was detected in SWF (p<0.05) and had the lowest expression in LWF. Additionally, the expression of MEK1 was highest in PF, followed by LWF, SYF, SWF and MWF. Herein, the data demonstrated that the ERK/HRAS/MEK1 proteins might be required in a context-dependent manner to maintain the prehierarchical biological development processes before transiting into hierarchical maturity stage, but the potential roles of this differential expression remain unknown. To support our contention on the expression pattern discrepancy of our target proteins, the highest expression of ERK proteins was found in rats’ kidneys during development than their adult counterparts (Omori et al., 2000). Liang et al., (2005) found that MEK1 proteins level increased in metastatic lymph nodes more than adjacent normal mucosa lymph nodes. Park et al., (2016) found that HRAS protein levels were significantly increased in the neural stem cells during mouse brain development.


 
Immunolocalization of ERK, HRAS and MEK1 proteins in prehierarchical follicles
 
The paracrine communications between the granulosa cells and the adjacent theca cells are critical in regulating survival and fate of ovarian follicles, more precisely the prehierarchical follicles that are known to be susceptible to undergoing atresia (Johnson 2003). This study reported for the first time the localization of goose ERK, HRAS and MEK1 protein kinases in the ovarian follicular walls during the prehierarchical follicular growth and development using immunohistochemical staining procedure as shown in (Fig 3).It was found that ERK proteins were predominantly expressed in the granulosa cells of MWF and LWF. Constant weak expression of ERK protein was observed in PF and SWF theca and granulosa cells. Chen et al., (2014) found that ERK was mainly localized in the cytoplasm of epithelial cells in the renal tubules of Cyprinus carpio. The strong immunostaining signal of HRAS was comparatively detected in MWF and less intensive in SYF. Vairaktaris et al., (2008) detected the highest expression levels of HRAS proteins in normal oral mucosa and precancerous lesions, compared to well-differentiated carcinomas. The MEK staining was most profound in PF and LWF followed by MWF, SWF and less immunoreaction in small SYF. Bi et al., (2017) found the highest expression of MEK1 in gastric tissues in rats with spleen deficiency syndrome treated with herbal cake-partitioned moxibustion. The immunohistochemical analysis results suggest the efficient biological participation of ERK, HRAS and MEK1 proteins during the growth and development of the undifferentiated granulosa and the theca cell layers.



In this study, quantitative expression and the localization of ERK, HRAS and MEK1 genes at mRNA and protein levels were conducted using RT-qPCR, western blot and immunohistochemistry respectively in different stages of geese ovarian prehierarchical follicles. At mRNA level, all the target genes showed inconsistent expression pattern in all prehierarchical follicles stages with the least expression level small yellow follicles (ERK and HRAS) and middle white follicles for MEK1. The relative high expression levels of these genes were observed at initial stage of prehierarchical follicular development with greater expression in PF for both ERK and HRAS followed by SWF for MEK1. These findings suggest that ERK, HRAS, MEK1 might have a less functional biogenetic effects towards the recruitment of the prehierarchical follicles into hierarchical stages. At protein level, the expression of the candidate genes unveiled different expression patterns from mRNA level tendencies. ERK showed a significant coexpression level between PF and SWF and HRAS proteins were more predominant in SWF whereas MEK1 was also highly expressed in PF. This may be due to multiple importance processes beyond transcript concentration that contribute to establishing the expression level of a proteins such as translation rates and modulation of a protein’s half-life (Liu et al., 2016). Immunostaining revealed the positive detection of the three proteins of interest in both theca cells and the granulosa cells in the five stages prehierarchical follicles. The results of this current study imply that ERK, HRAS and MEK1 might be involved in differentiation and proliferation of prehierachical follicular walls. Taken together these results showed that all the target genes were differentially expressed at mRNA and protein level during the geese prehierarchical ovarian follicles growth and maturation.

This study established that the presence of ERK, HRAS, and MEK1 during the growth and development of prehierarchical follicles maybe regulated in a stage-dependent manner. However, further studies on molecular interactions and functional roles of these protein kinases in ovarian follicular cell fate or survival need to be elucidated.