Objetivos gerais

1) Induzir a expressão de marcadores de pluripotência em fibroblastos bovinos e diferenciá-los em células germinativas primordiais e em oócitos.

2) Isolar células-tronco da linhagem germinativa a partir de ovários de bovinos adultos e diferenciá-las em células germinativas primordiais e em oócitos.

7.2Objetivos específicos

1) Estudar os efeitos da 5-aza-citidina na indução da aquisição pluripotência em fibroblastos bovinos, através da expressão de marcadores de pluripotência (OCT-4, NANOG, REX-1, SOX2).

2) Avaliar os efeitos da BMP-2, BMP-4 e do fluido folicular na diferenciação de fibroblastos tratados com 5-aza-citidina em células germinativas primordiais e em oócitos.

3) Identificar a capacidade de células-tronco isoladas de ovários de animais adultos se diferenciarem em células germinativas primordiais e em oócitos após estímulo com BMP-2, BMP-4 e fluido folicular.

4) Quantificar a expressão de marcadores de células germinativas (VASA e DAZL), de meiose (SCP3) e de oócitos (ZPA e GDF-9) após o cultivo in vitro de fibroblastos tratados com 5-aza-citidina e de células-tronco isoladas de ovários bovinos em meio suplementado com BMP-2, BMP-4 e fluido folicular.

8 ARTIGO II

Expression of markers for germ cells and oocytes in cow dermal fibroblast treated with 5- azacytidine and cultured in presence of BMP-2, BMP-4 or follicular fluid

(Expressão de marcadores de células germinativas e oócitos em fibroblastos da pele bovina tratados com 5-aza-citidina e cultivados na presença de BMP-2, BMP-4 ou fluido folicular)

Artigo submetido ao periódico Molecular Biology Reports (Qualis B1 - Biotecnologia)

Expression of markers for germ cells and oocytes in cow dermal fibroblast treated with 5- azacytidine and cultured in presence of BMP-2, BMP-4 and follicular fluid

José Jackson do Nascimento Costa1, Glaucinete Borges de Souza1,Joyla Maria Pires Bernardo1, Regislane Pinto Ribeiro1, José Renato de Souza Passos1, Francisco Taiã Gomes Bezerra1, Márcia

Viviane Alves Saraiva1_{, José Roberto Viana Silva}1*

1_{Biotechnology Nucleus of Sobral} – NUBIS, Federal University of Ceara, CEP 62042-280, Sobral, CE, Brazil.

*Corresponding address (J. R. V. Silva): Biotechnology Nucleus of Sobral - NUBIS, Federal University of Ceara, Av. Comandante Maurocélio Rocha Ponte 100, CEP: 62.041-040, Sobral, CE, Brazil. [roberto_viana@yahoo.com]

Abstract

I. Background: This study aims to investigate the effect 5-azacytidine during induction of pluripotency in bovine fibroblast skin, evaluate the effects of culture for 7 or 14 days in a medium containing BMP-2, BMP-4 or follicular fluid in the differentiation of reprogrammed fibroblasts in primordial germ cells and oocytes, as well as to analyze the mRNA levels for OCT- 4, NANOG, REX-1, SOX2, VASA, DAZL, c-KIT, SCP3, ZPA and GDF -9, after induction of pluripotency or differentiation in bovine fibroblasts.

II.Methods and Results: Dermal fibroblasts were cultured and exposed to 0.5, 1.0 or 2.0 μM of 5-Aza for 18 h, 36 h or 72 h. Then, cultured in DMEM/F12 supplemented with with 10 ng/mL BMP-2, or 10 ng/mL BMP-4 or 5% follicular fluid. After the cell culture, were evaluated

morphological characteristics, viability and gene expression by qPCR. Treatment of skin fibroblasts at 2.0 μM 5-Aza for 72 h results in changes in the morphology (oval or round shape) and cellular proliferation rate, and significantly increased expression of pluripotency factors. The culture in medium supplemented with BMP-2, BMP-4 or follicular fluid, for 7 or 14 days, altering cellular morphology, inducing formation of cell, and gene expression of germ cells and oocytes markers.

III. Conclusions: This study describes the possibility to convert bovine skin fibroblasts into another cell type, oocyte-likes cells, contribute to increase the development of therapies aimed at solving the problems of infertility in humans, as well as enhance the reproductive efficiency of animals.

Keywords: Gene expression. Fibroblast. 5-Azacytidine. BMP-2. BMP-4. Follicular fluid.

Introduction

The development of new regenerative therapies adapted to reproductive needs of female has been challenging in the last years [1]. Stem cell-based strategies for ovarian regeneration and oocyte production have been proposed as future clinical therapies for treating infertility in women [2]. Various studies have been conducted to identify, characterize, and differentiate cells from various sources [3-5], that can potentially be used for clinical studies, including embryonic stem cells (ESC) [6], stem cells isolated from adult tissues like the mesenchymal stem cells (MSC) [7], and induced pluripotent stem cells (iPS) which are adult somatic cells reprogrammed to pluripotency [8].

In 2006, Takahashi and Yamanaka [9] generated pluripotent cells by reprogramming somatic cells, this discovery it is now possible to convert differentiated somatic cells into pluripotent stem cells that have the capacity to generate all cell types of adult tissues [10]. iPS were generated by using a combination of 4 reprogramming factors, including OCT4 (Octamer binding transcription factor-4), SOX2 (Sex determining region Y)-box 2, KLF4 (Kruppel Like Factor-4), and c-MYC and were demonstrated both self-renewing and differentiating like ESCs [11]. Conventional reprogramming techniques depend on the stable integration of transgenes, using viral vectors, such as retroviruses [9] and lentiviruses [12], but it can introduce the current risk of insertional mutagenesis [13]. A chemical reprogramming is a promising strategy for safety and efficiency of iPS generation, many small molecules have been identified that can be used in place of exogenous transcription factors and significantly improve iPS reprogramming efficiency and quality [14]. Small molecules promote a chemical reprogramming, accompanied by remodeling of the epigenome, modulations of the epigenetic processes may facilitate such conversion of cell fate by making cells more permissive to these epigenomic changes [15].

To this purpose, 5-Azacytidine (5-Aza), a DNA methyltransferase inhibitor, can also improve reprogramming efficiency, by activating the expression of silent genes and altering the differentiation state of cells [16, 17]. Taylor and Jones, (1979) [18], used the 5-Aza in the conversion of a mesenchymal cell line into muscle cells, adipocytes and chondroblasts. Pennarossa et al. [19, 20] reprogrammed human and pig dermal fibroblast into insulin secreting cells by a brief exposure to 5-Aza. However, the use of 5-Aza to reprogram bovine skin fibroblast followed by a differentiation protocol, have not yet been reported.

The efficiency of differentiation of iPS cells into female germ cells has been considered low [21], since Eguizabal et al. (2011) [22] observed between 1.0%–2.0% haploid cells differentiated from a human female iPS cell line. The formation of iPS-derived germ cells

requires a strategy that involves differentiating iPS cells into primordial germ cells (PGCs), and subsequently directing the PGCs to undergo meiosis to form functional gametes [23, 21]. Several studies indicate that efficiency of differentiation to PGC can be increased with the addition of bone morphogenetic proteins (BMP-4, -7 and -8b) in human [24] and murine [25, 26] species. Günesdogan, et al. (2014) [27] indicated the BMP signals acts through a receptor complex including BMP receptor type II and ALK3/6, which results in SMAD1/5 phosphorylation, which form a complex with SMAD4 and translocate to the nucleus to control target gene expression. Mice carrying null mutations for BMP4, BMP8B, SMAD1 and SMAD5 showed impaired PGC development [28-30]. Ying et al. (2001) [31], Ying and Zhao (2001) [32] indicated mutation in BMP2 affects the size of the PGC founding population rather than PGC proliferation and/or survival, besides that, BMP2 produced by the endoderm (together with BMP4 and BMP8B) acts as a part of the first signal in PGC precursor generation. In human, Kee et al. (2006) [24] founded that addition of BMP-4 increased the expression of the germ cell-specific markers VASA and SYCP3 during differentiation of hES cells. These authors also showed that BMP-7 and BMP8b have additive effects on germ cell induction when added together with BMP4. West et al. (2010) [33] reported that BMP-4 increased the expression of pre-migratory (OCT-4, NANOG, c-KIT) and post-migration (DAZL, VASA) markers in PGC differentiated from ESC. Studies have also shown that follicular fluid contains many bioactives factors such as GDF9b, stem cell factor, basic fibroblast growth factor and oestrogen [34-36]. Dyce et al. (2006) [37] used follicular fluid in porcine skin-derived cells, and observed the induction of germ-cell formation and supported the expression of the markers, as OCT4, GDF9B, VASA and DAZL. In woman, the addition of follicular fluid, rich in substances for oocyte growth and maturation, to the culture medium triggers development of putative stem cells from the ovarian surface epithelium of women, and express several genes related to pluripotency and oocytes [38].

The aims of the present study were to investigate the expression of pluripotency markers (OCT-4, NANOG, REX-1, SOX2) in bovine fibroblast treated with different concentration of 5- Aza, as well as to evaluate the effects of BMP-2, BMP-4 or follicular fluid on mRNA levels for germ cell (VASA, DAZL, c-KIT), meiosis (SCP3) and oocytes (ZPA and GDF -9) markers in 5- Aza treated fibroblasts cultured for 7 and 14 days.

Materials and Methods

Skin Fibroblasts

All biological material were collected from animals at the local abattoir. Primary bovine skin fibroblast cultures were established from fresh biopsies from fetal ear skin. Fragments of tissues were washed twice in saline solution (0.9% NaCl) that contained antibiotics (100 IU/mL penicillin and 100 mg/mL streptomycin) (Sigma, St. Louis, MO, USA) and then, transported within 1 h to the laboratory in this same solution.

In the laboratory, fragments of tissues of approximately 2 mm3 were transferred into 24- well culture dishes (Corning, Lowell, MA, USA) that contained 1 mL of culture media. The basic culture medium consisted of D-MEM (pH 7.2–7.4) supplemented with 20% FBS (Sigma), 2 mM glutamine (Sigma) and antibiotics (100 IU/mL penicillin and 100 mg/mL streptomycin) (Sigma). Cells were cultured at 38.5 °C in 5% CO2 in a humidified incubator. After 7 days, primary fibroblast cultures started to grow out of the tissue fragments which were carefully removed and passaged twice a week in a 1:3 ratio. All experiments were performed on at least three lines.

Dermal fibroblasts (1.5×105 cells/well) were cultured in 0.1 % gelatin (Sigma) precoated 4-well multidish (Nunc) and exposed to 0.5, 1.0 or 2.0 μM of 5-Aza (Sigma) for 18 h, 36 h or 72 h. The basic culture medium consisted of D-MEM (pH 7.2–7.4) supplemented with 20% FBS (Sigma), 2 mM glutamine (Sigma) and antibiotics (100 IU/mL penicillin and 100 mg/mL streptomycin) (Sigma). Cells were cultured at 38.5 °C in 5% CO2 in a humidified incubator. At the end of the culture period, the proportion of living and dead cells was assessed with calcein AM (Molecular Probes) and ethidium homodimer-1 (Molecular Probes). Calcein AM (4 µM) and ethidium homodimer-1 (2 µM) was added to wells and incubated protected from light for a period of 15 min at room temperature. Determination of calcein and ethidium fluorescence at excitation/emission wave lengths of 488/568 nm was performed using an inverted microscopy (NIKON, Eclipse, TS100). Besides morphological evaluation, from each treatment, samples of cells were collected and stored at –80 ºC until RNA extraction to analyze the expression of markers for pluripotency (OCT4, NANOG, SOX2 and REX1).

Influence of follicular fluid and BMPs on the differentiation of germ cells

After treatment with 5-Aza (concentration and time of incubation determined previously), the bovine fibroblast cells were cultured in DMEM/F12 medium supplemented with 0.1 mM β- mercaptoetanol (Sigma), 2 mM glutamine (Sigma), 1 mM sodium pyruvate (Sigma), 1 mM Non- Essential Amino Acids (Sigma ), antibiotics (100 IU/mL penicillin and 100 mg/mL streptomycin) (Sigma), 20% Knockout Serum Replacement (KSR) (Life, Grand Sland, NY, USA). For the treatments, this medium was supplemented with 10 ng/mL BMP-2 (R&D systems, Minneapolis, MN, USA), or 10 ng/mL BMP-4 (R&D systems) or 5% follicular fluid [39]. Cells were cultured

at 38.5 °C in 5% CO2 in a humidified incubator. Every 2 days, the culture medium was replaced with fresh medium. After 7 and 14 days of culture, morphological analysis was performed and cellular viability was determined by immunofluorescence analysis (Calcein AM and ethidium homodimer-1) as described previously. Besides morphological evaluation, from each treatment, samples of cells were collected and stored at –80 ºC until RNA extraction to analyze the expression of markers for germ cells (VASA, DAZL and c-KIT), and oocytes (GDF -9, SCP3 and ZPA).

RNA extraction and cDNA synthesis

Isolation of total RNA was performed using the Trizol® Plus purification kit (Invitrogen, São Paulo, Brazil). According to the manufacturer’s instructions, 800 µL of Trizol solution was added to each frozen samples and the lysate was aspirated through a 20-gauge needle before centrifugation at 10,000 g for 3 min at room temperature. Thereafter, all lysates were diluted 1:1 with 70% ethanol and subjected to a mini-column. After binding of the RNA to the column, DNA digestion was performed using RNAse-free DNAse (340 Kunitz units/mL) for 15 min at room temperature. After washing the column three times, the RNA was eluted with 30 µL RNAse-free water. The RNA concentration was estimated by reading the absorbance at 260 nm and was checked for purity at 280 nm in a spectrophotometer (Amersham Biosciences, Cambridge, England). For each sample, RNA concentrations were adjusted and used to synthesize cDNA. Before the reverse transcription reaction, samples of RNA were incubated for 5 min at 70 ºC and then cooled in ice. The reverse transcription was performed in a total volume of 20 µ L composed of 10 µ L of sample RNA, 4 µ L reverse transcriptase buffer (Invitrogen, São Paulo, Brazil), 8 units RNase out, 150 units of reverse transcriptase Superscript III, 0036 U random primers, 10

mM DTT and 0.5mMof each dNTP (Invitrogen, São Paulo, Brazil). The mixture was incubated at 42 ºC for 1 h, subsequently at 80 ºC for 5 min, and finally stored at –20ºC. The negative control was prepared under the same conditions, but without the addition of reverse transcriptase.

qPCR

Quantification of mRNA was performed using GoTaq® qPCR Master Mix. PCR reactions were composed of 1μL cDNA as a template in 7.5 μL of GoTaq® qPCR Master Mix (Promega Corporation, Madison, WI, USA), 5.5 µ L of ultra-pure water, and 0.5 μM of each primer. The primers were designed by using the PrimerQuestSM program (http://www.idtdna.com). Primers used in this study are shown in table 1. The specificity of each primer pair was confirmed by melting curve analysis of PCR products. The thermal cycling profile for the first round of PCR was: initial denaturation and activation of the polymerase for 10 min at 95 o_{C, followed by 40 cycles of 15 sec at 95} o_{C, 30 sec at 58} o_{C, and 30 sec at 72} o_{C. The} final extension was for 10 min at 72 oC. All reactions were performed in StepOne Real-Time PCR (Applied Biosystems, Foster, CA, USA). Relative quantifications of mRNA were carried out using the comparative threshold (Ct) cycle method. The delta-delta-Ct method was used to transform the Ct values into normalized relative expression levels [40].

Statistical analysis

Levels of mRNA for pluripotency (SOX2, NANOG, OCT4, REX1), germ cells (VASA, DAZL, c-KIT) and oocytes (ZPA, GDF9, SCP3) genes were analyzed by using the non-parametric Kruskal–Wallis test and Dunn’s test for post hoc pair-wise comparisons (P<0.05). Data were expressed as mean ± s.e.m.

Results

Cellular morphology and viability after treating fibroblasts with 5-Aza

Fibroblasts obtained from biopsies from ear skin fetuses grew out of the original explants within 7 days and formed a monolayer, displaying a standard elongated morphology and a vigorous growth in culture typical of this cell population. After the exposure to 5-Aza, cell phenotype changed and fibroblast elongated morphology (Figure 1A) was replaced by an oval or round shape (Figure 1B), furthermore, there was a reduction in cell number.

During the exposure to 5-Aza, the total cell number remained substantially unstable, resulting from the effect of different concentrations of 5-Aza at different times, in manner time x concentration-dependent, it can be observed in immunofluorescence analysis (calcein AM and ethidium homodimer-1) (Figure 1C and Figure 1D). Cell proliferation rapidly decreased after exposure to 5-Aza, and can observed a high apoptotic index, especially with the treatment with 2.0 μM of 5-Aza for 72 h showed a greater reduction in the number of cells when compared to other treatments.

Expression of mRNA for markers of pluripotency after treating fibroblasts with 5-aza-cytidine

After treatment with 5-Aza for 18 h, real-time PCR analysis demonstrated that the levels of mRNA for SOX2 (Figure 2A) and REX (Figure 2D) did not significant changes in any of the treatments tested. However, NANOG expression was significantly higher after treatment with 2.0 μM of 5-Aza, when compared to 0.5 μM (Figure 2B). While the treatment with 1.0 μM of 5-Aza

significantly increased the levels of mRNA for OCT4, when compared to 0.5 μM of 5-Aza (Figure 2C).

Figure 3 shows the expression of pluripotency genes after treatment with different concentrations of 5-Aza for 36 h. The expression of mRNA for SOX2 (Figure 3A) did not significant change in any of the treatments tested. The expression of mRNA for NANOG (Figure 3B), was significantly increased after treatment with 1.0 μM of 5-Aza when compared to 0.5 μM of 5-Aza. The expression of mRNA for OCT4 (Figure 3C) was significantly increased after treatment with 2.0 μM of 5-Aza when compared to 0.5 μM of 5-Aza. In addition, expression of mRNA for REX (Figure 3D) was significantly increased after treatment with 1.0 or 2.0 μM of 5- Aza when compared to 0.5 μM of 5-Aza.

After culture for 72 h in different treatments with 5-Aza, the expression of SOX2 (Figure 4A), NANOG (Figure 4B), OCT4 (Figure 4C), and REX (Figure 4D) was significantly higher after supplementation of the medium with 2.0 μM of 5-Aza compared to 0.5 μM of 5-Aza.

Regarding the effects of incubation time on the expression of pluripotency genes, the culture with 0.5 (Figure 5Ai), 1.0 (Figure 5Aii) or 2.0 µM (Figure 5Aiii) 5-Aza for 72 h increased SOX2 mRNA levels, when compared to those seen after 18h or 36h. Fibroblasts cultured with 5- Aza (0.5 μM or 2.0 μM) for 72 h had higher levels of mRNA for NANOG than those cultured for 18 and 36 h (Figure 5Bi and 5Bii). However, no effect of incubation time on expression of NANOG was observed after culturing in presence of 1.0 μM of 5-Aza (Figure 5Bii).

After culturing cells with 0.5 μM and 1.0 μM of 5-Aza, a significant reduction in OCT4 expression was observed when the incubation time was increased from 18 to 72h (Figures 5Ci and 5Cii). On the other hand, fibroblasts cultured with 2.0 μM of 5-Aza for 72h had higher levels of OCT4 mRNA than those cultured for 18 and 36h a (Figure 5Ciii). The mRNA levels for REX1 in fibroblasts cultured either with 0.5 μM or 2.0 μM of 5-Aza for 72h were significantly higher

than those observed after 18 and 36h (Figures 5Di and 5Diii). When the cells were cultured with 1.0 μM of 5-Aza, a progressive and significant increase in REX1 expression was observed when the incubation time was increased from 18 to 36 and 72h (Figure 5Dii).

Cellular morphology and viability after culturing 5-Aza treated cells with BMP-2, BMP-4 or follicular fluid

After a series of preliminary experiments, we established that 2.0 μM of 5-Aza for 72 h represented the optimal combination for bovine skin fibroblasts. At the end of this treatment, cells were cultured in differentiation medium supplemented with BMP-2, or BMP-4 or follicular fluid, for 7 or 14 days.

Initially, growing in differentiation medium with BMP-2, BMP-4 or follicular fluid, induced the cells to present the ability to form colony. The culture in differentiation medium associated with 10 ng/mL of BMP-2 (Figure 6A, B, C, D) or BMP-4 (Figure 6E, F, G, H) for 14 days, resulted in a resumption of proliferative index, and morphological changes, gradually cells organized in clusters. The culture in BMP-2 or BMP-4 for 7 days did not affect the cell proliferation rate. The culture in 5% follicular fluid for 7 or 14 days (Figure 6I, J, K, L), promoted an large increase in cell proliferation rate, with vigorous growth and large rate of apoptosis.

Expression of mRNA for markers of germ cells and oocytes after culturing 5-Aza treated cells with BMP-2, BMP-4 or follicular fluid

Since exposure of fibroblasts to 2.0 µM of 5-Aza for 72h promoted higher expression of pluripotency genes, this concentration and incubation time were chosen to treat the cells before culture in differentiation medium containing BMP-2, BMP-4 or follicular fluid for 7 or 14 days.

After 7 days of culture, BMP-4 stimulated a significant increase in the expression of VASA, when compared to control medium. However, BMP-4 or follicular fluid had no effect on VASA expression (Figure 7A). While the culture for 14 days in medium containing follicular fluid, significantly increased the expression of VASA, compared to the culture in medium control (Figure 7B). DAZL expression was significantly increased after culture in medium containing BMP-2 for 7 or 14 days (Figure 7C, D), when compared to other treatments. The culture for 7 days in medium supplemented with BMP-2, stimulated mRNA expression of c-KIT (Figure 7E), when compared to the culture in to other treatments. The culture for 14 days, did not significantly modify the expression of c-KIT (Figure 7F).

Regarding the expression of oocyte markers, after 7 days of the culture, ZPA expression was significantly reduced when cells were cultured in medium containing BMP-2 (Figure 8A). Already, the culture in medium containing follicular fluid for 14 days, significantly increased ZPA expression, when compared to the culture in medium containing BMP-4 or control medium only (Figure 8B). Follicular fluid increased significantly mRNA levels for GDF -9 (Figure 8C), when compared to culture in control medium. After 14 days of culture in medium supplemented with BMP-2, increased significantly GDF -9 expression, when compared to other treatments (Figure 8D). An increase in SCP3 mRNA expression was observed after culture in presence of BMP-4 for 7 days, when compared to other treatments (Figure 8E). After 14 days culture in medium supplemented with BMP-2, promoted an increase in expression of SCP3 (Figure 8F), when compared to other treatments.

Figures 9, 10, 11 and 12, showed the expression of makers for germ cells and oocyte after 0 h, 7 or 14 days in the different treatments. For cells cultured in control medium, VASA expression was significantly increased after 7 days, when compared to the time 0 or after 14 days (Figure 9A). DAZL expression was not altered after 7 or 14 days culture (Figure 9B). An increase in the mRNA levels for c-KIT was observed after 7 or 14 days of culture, when compared to 0h (Figure 9C). Culture for 14 days significantly increased ZPA (Figure 9D) and GDF -9 (Figure 9E) expression, compared to culture for 0 h or 7 days. A progressive and significant increased in