Expression pattern and regulation of head-to-head genes Vps36 and Ckap2 during chicken follicle development

Vacuolar protein sorting 36 (VPS36), a protein primarily known for its role in the Endosomal Sorting Complex Required for Transport pathway, has recently been shown to be linked to chicken reproduction. Previous research showed that Vps36 is significantly downregulated in sexually mature chicken ovaries compared to immature ones. In this study, using real-time quantitative RT-PCR, we investigated the expression pattern of Vps36 and its head-to-head gene Ckap2 mRNA in chicken follicles. Small white follicles were found to have significantly higher expression of Vps36 and Ckap2 mRNA than any other sized follicles (P< 0.05). The expression of Vps36 and Ckap2 mRNA were detected in both granulosa and theca layers of pre-ovulatory follicles, the expression of Ckap2 in theca layers was slightly higher than in granulosa cells. Treatment of small yellow follicles with folliclestimulating hormone and estradiol resulted in a marked decrease of both Vps36 and Ckap2 mRNA (P< 0.05); however, progesterone, transforming growth factor-β 1 and luteinizing hormone induced no significant changes in Vps36 and Ckap2 mRNA expression in these follicles. These results indicate that the head-to-head genes of Vps36 and Ckap2 exhibit similar expression in chicken follicles and are involved in chicken follicle development.

Genetic analysis of class E vps genes has mainly been performed in yeast [2,10]. In Drosophila, the Vps36 mutant increases apoptotic resistance and shows neoplastic characteristics [11]. Mutants in all subunits of the ESCRT-II complex (Vps22, Vps25 and Vps36) abolish the final Staufen-dependent step in bcd RNA localization [12]. Ckap2 (cytoskeleton associated protein 2) is the head-to-head gene of Vps36 and shares a 247 bp promoter with the latter on chicken chromosome 1. CKAP2 is an important mitotic regulator [13][14][15], and has been shown to be upregulated in various human malignancies [16][17][18].
It is widely accepted that follicle growth and differentiation are mediated by endocrine, paracrine and autocrine factors, among which the most important are the gonadotropins and growth factors [19]. Follicle-stimulating hormone (FSH) is responsible for follicular recruitment and growth of the smaller follicles. Subsequent to selection, a follicle undergoes a transition from largely FSH-dependence to luteinizing hormone (LH)-dependence [20][21][22]. LH is the primary gonadotropin responsible for promoting progesterone production in pre-ovulatory follicles [19,23]. Our previous study showed that Vps36 is significantly downregulated in sexually mature ovaries compared to immature ones in chicken ovary [24], suggesting that Vps36 is likely to be involved in the process of ovarian follicular development. The objective of this study is to investigate the mRNA expression pattern of Vps36 and Ckap2 genes and the effect of gonadotrophins, steroid hormones and transforming growth factor-β1 (TGF-β1) on their expression in chicken follicles. We demonstrated that the expression of the head-to-head genes Vps36 and Ckap2 is regulated by FSH and estradiol in chicken follicles.

Materials and methods
2.1 Birds, follicle collection, and separation of granulosa and theca layer cells The Hy-Line Brown laying hens were housed under standard conditions with food and water. Sexually mature hens (23 weeks old, n = 4) were slaughtered to collect follicles of various sizes, including pre-ovulatory follicles (F1, F2, F3 and F5) and pre-hierarchical follicles (small white follicles, SWF, 2-4 mm; and small yellow follicles, SYF, 4-8 mm). Then the yolk in follicles was removed and the separated follicles were immediately frozen in liquid nitrogen. All the tissues were stored at -80°C until processed. The granulosa and theca cell layers from preovulatory follicle (F1, F3 and F6) were separated using a dissection microscope following the method described previously [25].

Follicle culture and treatment
The SYF of the ovaries of egg-laying hens (35 -48 weeks old) were collected, then individual follicles were washed with three dishes of 1 Â PBS (pH 7.2) and placed in M199 medium (1 mL) (HyClone, Logan, UT, USA) with 1% ITS (Sigma, ST. Louis, MO, USA) and seeded in 24-well culture plates at a density of one per well according to the reference [26]. The SYF were divided into four groups: one control group and three treatment groups. The cultured SYF in treatment groups were treated with 10, 50 and 100 ng$mL -1 of porcine FSH (Sioux Biochemical, Sisoux center, IA, USA), β-estradiol (E2; Sigma), progesterone (P4; Sigma) and equine LH (Sigma), and the SYF that were treated with human TGF-β1 (Prospecbio, Rehovot, Israel) were divided into three groups (control, 2.5 and 5 ng$mL -1 ). Each group had four repeats. Then all SYF were cultured at 39°C in a water-saturated atmosphere of 95% air and 5% CO 2 for 16 h [27]. After 16 h, the yolk in SYF was removed and the remaining SYF were immediately frozen in liquid nitrogen for RNA isolation. Each treatment was repeated at least three times.

Total RNA isolation and cDNA synthesis
Total RNA from follicles of different levels was extracted using Trizol Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions and treated with DNase I (Qiagen, Beijing, China) to remove DNA contamination. Total RNA from SYF cultured and treated as above was isolated using MicroElute TM Total RNA Kit (Omegabiotek, Norcross, GA, USA) according to the manufacturer's instructions. One microgram of total RNA was reverse transcribed using oligo-d(T) primer (10 µmol$L -1 ) and 2 U reverse transcriptase (Roche Applied Science, Mannheim, Germany) according to manufacture's protocol. The amount and integrity of isolated total RNA were measured using a spectrophotometer (Eppendorf, Hamburg, Germany) and checked by loading total RNA onto a 1% agarose gel that was stained with ethidium bromide.

Real-time quantitative RT-PCR
Chicken Vps36 and Ckap2 mRNA were quantified using 3 μL of the reverse transcription reaction product (equivalent of 150 ng of single-stranded cDNA) as template in real-time quantitative RT-PCR (qRT-PCR) with β-actin as internal control. The qRT-PCR was performed in a 20 μL reaction volume consisting of SYBR® Premix Ex Taq TM (Takara Biotechnology, Dalian, China) and 0.2 μmol$L -1 of forward and reverse primers ( confirm the presence of a single amplification product. Each sample was run in duplicate to obtain average loglinear threshold (CT) values for Vps36 and Ckap2 mRNA and β-actin mRNA. The efficiencies were close to 100%, allowing the use of the 2 -ΔΔCT method for calculation of relative gene expression [28]. All qRT-PCR were performed at least in triplicate and with a negative control. The expression of chicken Vps36 and Ckap2 mRNA in different sized follicles and in cultured SYF was examined individually, except that for SWF, due to their small sizes, three to four were pooled for analysis.

Statistical analysis
For the quantitative measurement of the mRNA levels of Vps36 and Ckap2 genes in follicles, values are expressed as the meansAESEM. Data from the experiments were analyzed by one-way analysis of variance (ANOVA) followed by the Duncan's multiple range test. When P < 0.05, the difference was considered as significant.

Expression of Vps36 and Ckap2 mRNA in follicles
The mRNA expression pattern of chicken Vps36 and Ckap2 genes were investigated in chicken follicles of various sizes, i.e. SWF, SYF and from F5 to F1. The mRNA expression of chicken Vps36 gene was markedly reduced from SWF to SYF (P < 0.05), then remains stable from F5 to F1 follicles (Fig. 1a). The expression of chicken Ckap2 mRNA was progressively reduced from SWF to F2 follicles (P < 0.05) and then slightly increased in F1 follicles (Fig. 1b). Both Vps36 and Ckap2 exhibited the highest mRNA expression levels in chicken SWF. The expression of Vps36 and Ckap2 mRNA were detected in both granulosa and theca layers of chicken pre-ovulatory follicles, and the expression of Ckap2 in theca layers was slightly higher than in granulosa cells (Fig. 2).

Effect of FSH and LH on the expression of Vps36 and
Ckap2 mRNA in SYF The expression of chicken Vps36 and Ckap2 mRNA in SYF was significantly decreased in response to FSH treatment (P < 0.05, Fig. 3a). The effect of FSH on chicken Vps36 and Ckap2 mRNA expression was not significant by different concentrations (10, 50 and 100 ng$mL -1 ). LH treatment in chicken SYF produced no significant effect on mRNA expression of Vps36 and Ckap2 genes (Fig. 3b).

Effect of E2 and P4 on Vps36 and Ckap2 mRNA expression in SYF
In SYF, E2 treatment for 16 h resulted in a significant decrease in the mRNA expression of chicken Vps36 and Ckap2 genes (P < 0.05, Fig. 4a). With the increasing concentration of E2, a gradual decrease in mRNA expression level of chicken Vps36 was observed. Treatment of SYF with 10 ng$mL -1 E2 did not affect chicken Ckap2 mRNA expression, however, when the concentration of E2 was increased to 50 and 100 ng$mL -1 , Ckap2 mRNA expression was remarkably decreased (P < 0.05, Fig. 4a). By contrast, P4 treatment produced no significant effect on chicken Vps36 mRNA expression (P > 0.05, Fig. 4b). The same situation occurred with chicken Ckap2 (Fig. 4b) except that Ckap2 mRNA expression was significantly decreased after treatment with higher concentration of P4 (100 ng$mL -1 ) compared to any other concentrations (Fig. 4b).

Effect of TGFβ1 on the expression of Vps36 and Ckap2 mRNA in SYF
In chicken SYF, the expression of Vps36 mRNA levels was not significantly affected by TGFβ1 at the concentrations of 2.5 and 5.0 ng$mL -1 (Fig. 5). When the concentration of TGFβ1 was increased, the chicken Ckap2 mRNA expression level was gradually decreased, but the effect was not significant (Fig. 5).

Discussion
The process of ovarian follicle development in vertebrates is closely associated with the functional differentiation of granulosa cells. In the process of chicken follicular development, through selection from the pool of SYF, follicles successively become dominant. The functional differentiation of granulosa cell regulated by hormone and many paracrine/autocrine factors is coupled with changes in the expression level of many related genes. By cDNA-   AFLP, Vps36 was indentified to be related to sexual maturity in chicken and its expression was related to the ovary function [24]. Ckap2 is the head-to-head gene to Vps36 on the same chicken chromosome. However, the role of Vps36 and Ckap2 in animal follicle growth and ovulation remains unknown. Therefore, in this study, the expression pattern and regulation of these two genes in different sized follicles, as well as in cultured SYF, were investigated. The result indicated that Vps36 was expressed in chicken pre-ovulatory and pre-hierarchical follicles, and was much higher in SWF than any others. In addition, both granulosa and theca layers of chicken pre-ovulatory follicles expressed Vps36 mRNA. These results indicate that Vps36 is involved in follicle development. It is known that activated growth factors, hormones and cytokine receptors are brought inside the cell by endocytosis and delivered to lysosomes by multivesicular endosomes of which Vps36 is one important component [29]. Thus it can be postulated that the cellular process, mentioned above, is more active in SWF than any other sized follicles.
To investigate whether the head-to-head gene of Vps36 is also closely associated with the functional differentiation of follicles, the mRNA expression of Ckap2 was analyzed in follicles of different sizes. The result showed that there was a similar expression pattern between Vps36 and Ckap2 mRNA in follicles sized from SWF to F1, but there were also slight differences in which the mRNA expression of Ckap2 gradually reduced from SWF to F2, implying that, like Vps36, Ckap2 is also involved in follicle development and the related cellular process.
The decrease in Vps36 and Ckap2 mRNA levels in SYF compared to SWY follicles indicates their possible involvement in follicle recruitment, which is likely to be also regulated by sex hormones and gonadotropins. Therefore, the regulatory mechanism of Vps36 and Ckap2 in the process of follicle recruitment was investigated by treating cultured chicken SYF with FSH, β-estradiol, progesterone, TGF-β1 and LH. The results indicated that FSH and β-estradiol inhibited the mRNA expression of both Vps36 and Ckap2, but progesterone, TGF-β1 and LH produced no effect on the mRNA expression of Vps36 and Ckap2. These results are consistent with a previous study that the regulatory mechanism on the transcription of head-to-head genes is similar [30].
Follicle-stimulating hormone promotes the development of granulosa cells, and causes them to proliferate, subsequently differentiate and finally to become steroidogenic via de novo synthesis of steroidogenic factors, steroidogenic enzymes, and transcription factors [31][32][33][34][35]. Transcription factors like AP-1, CBP, Egr-1, SF-1, and SP1 were suggested to be regulated through the ERK signaling pathway that is stimulated by FSH [36]. It is also reported that FSH promotes rapid activation of protein kinase A (PKA) [37]. The cAMP-response-element binding-protein (CREB) is the best-known transcription factor regulated by PKA [38,39] and was initially predicted to regulate expression of most, if not all, PKA-regulated target genes in granulosa cells. In this study, bioinformatics analysis revealed two CREB binding sites in Vps36 gene (data not shown), indicating a PKA-CREB pathway that FSH regulates Vps36 transcription. The biological actions of estrogens are mediated by estrogen binding to one of two specific estrogen receptors, ERα and ERβ, which belong to the nuclear receptor superfamily [40]. Bioinformatics analysis shows that both Vps36 and Ckap2 genes contain ERα binding sites. We postulate β-estradiol inhibits the mRNA expression of Vps36 and Ckap2 mainly through the ERα pathway, but this idea requires investigation.

Conclusions
In this study, we found that Vps36 and Ckap2 were highly expressed in pre-hierarchical follicles. The mRNA expression of Vps36 and Ckap2 in chicken SYF was strongly inhibited by FSH and estrogen treatment. In addition, the head-to-head genes of Vps36/Ckap2 exhibit similar expression and regulatory modes in chicken follicles. These results indicate that Vps36 and Ckap2 are important in follicle development.