Retinoic acid induces differentiation of buffalo (Bubalus bubalis) embryonic stem cells into germ cells

Gene 631 (2017) 54–67

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Erratum

Retinoic acid induces differentiation of buffalo (Bubalus bubalis) embryonic stem cells into germ cells

MARK

Syed Mohmad Shah⁎, Suresh Kumar Singla, Prabhat Palta, Radhey Sham Manik, Manmohan Singh Chauhan Animal Biotechnology Centre, National Dairy Research Institute, Karnal, 132001, Haryana, India

A R T I C L E I N F O

A B S T R A C T

Keywords: Embryonic stem cells Differentiation Germ cells Retinoic acid Buffalo FACS Methylation erasure

Development of precise and reproducible culture system for in vitro differentiation of embryonic stem (ES) cells into germ cells counts as a major leap forward for understanding not only the remarkable process of gametogenesis, otherwise obscured by limited availability of precursor primordial germ cells (PGCs), but in finally treating the catastrophic infertility. Taking into account the significant role of retinoic acid (RA) during in vivo gametogenesis, we designed the present study to investigate the effects of its stimulation on directing the differentiation of ES cells into germ cells. The effects of RA were analyzed across dose-and-time upon various stages of gametogenesis like PGC induction, meiosis initiation and completion, haploid cell formation and development of the final gamete (oocyte and spermatozoa). Out of the series of RA doses (2, 4, 8, 16, 20 and 30 μM), 16 μM RA for 8 day culture interval was found to induce highest expression of PGC- and meiosis-associated genes like DAZL, VASA, SYCP3, MLH1, TNP1/2 and PRM2, while mature germ cell genes like BOULE and TEKT1 (Spermatocyte markers), GDF9 and ZP2 (Oocyte markers) showed higher expression at 2 μM RA dose, suggesting functional concentration-gradient of RA activity. Immunocytochemistry revealed expression of germ lineagespecific markers like: c-KIT, DAZL and VASA (PGC-markers); SYCP3, MLH1 and PROTAMINE1 (Meiotic-markers); ACROSIN and HAPRIN (Spermatocyte-markers); and GDF9 and ZP4 (Oocyte-markers) in optimally differentiated embryoid bodies (EBs) and adherent cultures. We observed significantly reduced (p < 0.05) concentration of 5-methyl-2-deoxycytidine in RA-differentiated EBs which is suggestive of the occurrence of methylation erasure. FACS analysis of optimally differentiated cultures detected 3.07% haploid cell population, indicating completion of meiosis. Oocyte-like structures (OLS) were obtained in adherent differentiated cultures. They had a big nucleus and a zona pellucida (ZP4) coat. They showed progression through 2-cell, 4-cell, 8-cell, morula and blastocyst-like structures upon extended culture beyond 14 days.

1. Introduction

study of these progenitor cells, known as PGCs, is rather difficult owing to their limited number, embedded nature within the embryo and paucity of the primary tissue, first trimester fetal gonads (Aflatoonian et al., 2009). In view of these limitations, embryonic stem cells could provide a direct system for experimental examination of the landmark events and genetic requirements in germ-cell formation, epigenetic reprogramming, meiosis and post-meiotic progression to gametogenesis (Kee et al., 2009). A number of studies have been conducted to screen the potential inducers of PGC formation from ES cells; most of them identifying bone morphogenetic protein (BMP) family members like BMP4, BMP2, BMP8b (Panula et al., 2011; Aflatoonian et al., 2009; Wei et al., 2008), retinoic acid (Cai et al., 2013; Zhu et al., 2012;

The origin of germ cells (sperm and oocytes) can be traced back to primordial germ cells (PGCs) which undergo a long and eventful journey till their differentiation into functional gametes. To fulfill their role as carriers of genetic information from generation to generation, germ cells undergo a unique process of meiosis to reduce their genetic material to half. The origin, properties and unique capabilities of germ cells are a special focus to reproductive biologists interested in in vitro generation of germ cells (artificial gametes). To elucidate molecular mechanisms and signaling pathways of germ cell formation, the ideal starting material would be the progenitor cells of germ lineage. The

DOI of original article: http://dx.doi.org/10.1016/j.gene.2017.05.037 Abbreviations: IVF, In vitro fertilization; ESC, Embryonic stem cells; AFP, Alpha fetoprotein; BMP4, Bone morphogenetic protein4; EB, Embryoid body; HNF4, Hepatocyte Nuclear Factor 4; GATA4, Global Transcription Factor Alpha 4; MSX1, Msh Homeobox 1; NF-68, Neurofilament Light peptide 68; RA, Retinoic acid; 5mC, 5-methylcytosine; RAR, Retinoic acid receptors ⁎ Corresponding author at: Animal Biotechnology, Embryo Biotechnology Lab, Animal Biotechnology Centre, National Dairy Research Institute, Karnal 132001, India. E-mail address: [email protected] (S.M. Shah). http://dx.doi.org/10.1016/j.gene.2017.07.041

Available online 16 August 2017 0378-1119/ © 2017 Elsevier B.V. All rights reserved.

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Fig. 1. qPCR analysis of normalized expression of key genes involved in ES cell differentiation to germ cell lineage upon RA induction over 14 days of culture period. Bars represent 2− ΔΔcT values ± S.E. of mean, and are calibrated against the corresponding normalized values of undifferentiated ES cell colonies. Bars with different superscripts differ significantly (p < 0.05).

cultures (Aflatoonian et al., 2009). The differentiation was assayed for protein markers and gene expression profile consistent with germ lineage cells. For example, immunocytochemical analysis for PGC-specific, meiosis-specific, spermatocyte-specific as well as oocyte-specific markers was performed in both EB and monolayer adherent cultures, differentiated at the optimum dose- and time-period. Methylation erasure was assayed by Global DNA methylation analysis for quantification of 5-methyl-2-deoxycytidine at optimum EB differentiation conditions. The monolayer differentiation cultures were subjected to FACS analysis for determination of haploid cell population. This study is, as per our knowledge, the first of its kind in farm animals, especially bubaline species. It would provide for understanding genetics, epigenetics and biochemistry of gametogenesis, especially of the bubaline species. In addition to its future applications in transgenesis, elite animal conservation and propagation as well as in development of designer gametes, it would provide a higher mammalian model for understanding human gametogenesis in vitro.

Aflatoonian et al., 2009), testicular cell conditioned media (LachamKaplan et al., 2006), cumulus cell conditioned media (Shah et al., 2015b) and other growth factors like bFGF, KIT-ligand and Wnt proteins etc. (Park et al., 2009; Wei et al., 2008; West et al., 2008; Tilgner et al., 2008). Most of these studies were carried in murine ES cells, and hence, warrant a caution in their extrapolation to human and higher mammalian species, because inferences from murine studies may not always translate to advances in higher mammalian germ cell development. This highlights the need to investigate germ cell signaling factors and their role in PGC specification, meiosis and mature germ cell formation either directly in the species of interest or in an ontogenetically closer one. On the basis of some preliminary reports that retinoic acid stimulation induces ES cell differentiation to germ cell lineage; we designed this study to investigate the effects of all-trans retinoic acid (RA), in different dose- and time- combinations, on ES cell differentiation to germ cell lineage. RA has been demonstrated to be produced in both male and female mesonephroi and has been implicated in in vivo gametogenesis (Bowles et al., 2006). A source/sink system of RA has also been demonstrated in developing gonads which directs meiotic progression or inhibition, depending upon sex of the embryo (McCaffery et al., 1999; Romand et al., 2006). We employed the two most commonly used strategies: i) differentiation in floating cultures via embryoid body (EB) formation; and ii) differentiation in monolayer adherent cultures in absence of feeder cells. We initially adopted EB differentiation protocol based on the understanding that 3D culture would more closely resemble to in situ conditions than the monolayer

2. Materials and methods 2.1. Chemicals Chemicals and plastic ware were purchased from Sigma Aldrich (St. Louis, MO) and Falcon (Paignton, UK), respectively, unless stated otherwise. 55

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Fig. 2. Comparison of normalized expression of key genes involved in ES cell differentiation to germ cell lineage under embryoid body and monolayer adherent cultures, upon RA induction over 14 days of culture period. Bars represent 2− ΔΔcT values ± S.E. of mean and are calibrated against the corresponding normalized values of undifferentiated ES cell colonies. Bars with different superscripts differ significantly (p < 0.05).

(ES cell medium supplemented with RA). RA was exogenously added to the medium at 2, 4, 8, 16, 20 and 30 μM concentrations. The cultures were replenished with half of the medium every alternate day. EBs so formed were collected at day 4, 8 and 14 for analysis of germ lineage differentiation.

2.2. Ethical approval The study was approved by National Dairy Research Institute Animal Ethics Committee as well as by Department of Biotechnology, New Delhi, for use of animal tissues as well as for development of the embryonic stem cell lines.

2.4.2. Differentiation in adherent cultures Embryonic stem cells were also differentiated in adherent cultures as MaxGel adherent monolayers for 14 day culture period in presence of exogenous RA. For establishment of monolayer cultures, the colonies were cut into as small clumps as possible, using a Microblade, and washed thrice in sterile DPBS−−. The colonies were treated with trypsin-EDTA (0.25%) for 5 min in a 35 mm Petri dish so as to remove the attached feeder cells (if any). This was followed by two DPBS−− washes and enzymatic digestion for which colonies were transferred into 2 ml Eppendorf tubes containing 1 ml Accutase. After a brief incubation of 5 min, colonies were disaggregated by repetitive pipetting into single cells. The single cell suspension was subjected to centrifugation at 1500 rpm for 10 min. The supernatant was discarded; pellet resuspended in differentiation medium (2, 4, 8, 16, 20 and 30 μM RA) and seeded for establishment of monolayer cultures in MaxGel (1:200) coated 4-well plates. The cultures were replenished with half of the culture medium every alternate day. The differentiated cultures were subjected to qPCR analysis after 4, 8 and 14 day culture intervals so as to identify the optimum RA dose-and-time period in monolayer differentiation. The optimally differentiated cultures were subsequently subjected to qPCR germ lineage gene profiling, immunodetection of germ lineage proteins, methylation analysis and FACS analysis for determination of haploid cell population.

2.3. Establishment, characterization and culture of buffalo ES cells Three buffalo ES cell lines (48 + XX) were developed and characterized for pluripotency and self-renewal markers, differentiation capability (ectoderm, mesoderm and endoderm) and stable karyotype, as described previously (Shah et al., 2015a). The cell lines were developed from in vitro fertilization (IVF) derived blastocysts and propagated on buffalo fetal fibroblast feeders in ES cell culture medium. The ES cell culture medium was composed of KoDMEM and 15% Knock out serum replacer (KoSR), supplemented with 5 ng/ml basic fibroblast growth factor (bFGF), 2 mM L-glutamine, 1000 U/ml recombinant murine lukaemia inhibitory factor (rmLIF), 1× nonessential amino acids and 50 μg/ml gentamicin sulphate. The cultures were propagated at 38 °C in a 5% CO2 incubator. The cell lines were subcultured for > 100 passages and were used for the present study at around 25–40 passages. 2.4. Differentiation into germ lineage cells upon RA stimulation 2.4.1. Differentiation in floating cultures ES cell colonies were subjected to differentiation in low attachment 35 mm culture dishes, as floating cultures, in differentiation medium

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Fig. 3. Immunocytochemical analysis for Primordial germ cell (PGCs) markers (c-KIT, DAZL and VASA) induced by RA stimulation (16 μM) in 8 day old EBs developed in static suspension cultures. BF - Bright field image; DAPI - Nucleus stained image; 2°Ab - PE/FITC- labeled secondary antibody image; M - Merged images of DAPI and 2°Ab (Magnification 200 ×, Scale bar - 100 μm).

threshold cycle (Ct) values were calculated for each reaction. All the PCR reactions were performed in triplicates. The PCR efficiency and target gene expression were calculated as described previously (Shah et al., 2015a, 2015b).

2.4.3. qPCR analysis for germ lineage markers 60 EBs were collected separately from each of the media formulations on day 4, 8 and 14. Total RNA was extracted using RNeasy RNA extraction kit (Qiagen, Germany) and used for cDNA synthesis (SuperScript III first strand cDNA synthesis kit; Invitrogen, USA). qPCR was performed for quantification of such genes associated with: i) PGC like NANOG, OCT4, DAZL and VASA; ii) Meiosis, like MLH1, SYCP3, TNP1/2 and PRM2; iii) Spermatocytes, like BOULE and TEKT1; and iv) Oocytes, like GDF9 and ZP2 and 3. The primers, their sequences as well as annealing temperatures have been previously mentioned (Shah et al., 2015b). The optimized qRT-PCR reaction mixtures contained 10 μl SYBR Green PCR Master Mix Buffer (2 ×), 2 μl (100 ng) cDNA template, and 10 pmol each of forward and reverse primers in a total volume of 20 μl. GAPDH and β-ACTIN were used as the endogenous controls for each sample. The PCR parameters were: initial denaturation at 95 °C for 10 min; followed by 40 PCR cycles (denaturation: 95 °C for 30 s, annealing at X °C (respective for each gene as mentioned previously) for 30s, and extension at 72 °C for 30 s. This was followed by a dissociation protocol to provide evidence for a single reaction product. A no-template control (NTC) of nuclease-free water was included in each run and

2.4.4. Immunocytochemical examination for germ lineage protein expression Day 8 EBs collected from the optimum differentiation cultures were analyzed by immunocytochemistry for expression of proteins like c-KIT, DAZL, VASA (PGC-specific proteins); MLH1, SYCP3, PROTAMINE1 (Meiotic proteins); ACROSIN and HAPRIN (Spermatocyte-specific proteins); and GDF9 and ZP4 (Oocyte-specific proteins). All the primary antibodies (Shah et al., 2015b) were diluted 1:100 in blocking solution (4% normal goat serum in DPBS−−) except for DAZL which was diluted at 1:50 ratio. Undifferentiated buffalo ES cells were used as negative controls, while testicular and/or ovarian tissue sections were used as positive controls so as to confirm antibody specificity. Immunocytochemical analysis was performed as per the already discussed protocol (Shah et al., 2015b, 2015c). Briefly, the EBs were washed twice in DPBS−−, and fixed for 20 min in 4% paraformaldehyde (PFA).

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Fig. 4. Immunocytochemical analysis for meiotic markers (SYCP3, PROTAMINE1 and MLH1) induced by RA stimulation (16 μM) in 8 day old EBs developed in static suspension cultures. BF - Bright field image; DAPI - Nucleus stained image; 2°Ab - PE/FITC labeled secondary antibody image; M - Merged images of DAPI and 2°Ab (Magnification - 200 ×, Scale bar 100 μm).

(gDNA) extraction. gDNA samples were digested to nucleosides by treatment with nuclease P1 and alkaline phosphatase, as described (Shah et al., 2015b). Centrifugation was performed at 6000g for 5 min and supernatant, containing individual nucleosides, was used for 5MedCyd ELISA assay. This was followed by addition of 50 μl sample DNA or 5MedCyd standard to the wells of the 5MedCyd-DNA conjugate coated plate and incubation at room temperature for 10 min on an orbital shaker. Pre-diluted primary antibody was added and incubation was performed for 2 h on an orbital shaker. After 1 h incubation in blocking buffer, secondary antibody enzyme conjugate was added to each well followed by three washings and addition of substrate solution. 100 μl stop solution was added as soon as the color started to appear and the results were read immediately on spectrophotometer using 450 nm as primary wavelength. The standard curve was prepared using standard 5-methyl-2-deoxycytidine concentrations versus corresponding absorbance values. The concentration of 5-methyl-2-deoxycytidine was calculated from the respective mean absorbance values of three independent measurements, each taken in duplicate.

This was followed by treatment with 0.1% Triton X-100 for 15 min, and incubation for 1 h in blocking solution (4% normal goat serum in DPBS−−). The colonies were exposed to primary antibodies at 4 °C overnight, followed by incubation with fluorescein isothiocyanate (FITC) or phycoerythrin (PE) labeled class specific secondary antibody (Shah et al., 2015b). The EBs were put individually on a clean grease free microscopic glass slide and the nuclei were stained with DAPI (4–6diamidino-2-phenylindole) (Life Technologies, USA). This was followed by visualization under a fluorescence microscope (Diaphot; Nikon, Tokyo, Japan) for examination of protein expression. Immunocytochemical analysis was also performed on buffalo ES cell monolayer cultures differentiated at optimum RA dose, following the same protocol. 2.4.5. Analysis of methylation erasure Global DNA methylation analysis of EBs collected from the optimum RA differentiation cultures, spontaneously differentiated EBs and undifferentiated (control) ES cells was performed by employing Global DNA Methylation ELISA Kit (Cell Biolabs, USA), following the previously discussed protocol (Shah et al., 2015b). NucleoSpin Tissue DNA purification kit (Macherey-Nagel, Germany) was used for genomic DNA

2.4.6. Analysis for haploid cell population Monolayers of ES cells differentiated under optimum RA

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Fig. 5. Immunocytochemical analysis for spermatocyte specific (ACROSIN and HAPRIN) and oocyte specific (GDF9 and ZP4) markers induced by RA stimulation (16 μM) in 8 day old EBs developed in static suspension cultures. BF - Bright field image; DAPI - Nucleus stained image; 2°Ab - PE/FITC-labeled secondary antibody image; M - Merged images of DAPI and 2°Ab (Magnification - 200 ×, Scale bar - 100 μm).

The results are expressed as mean ± S.E. of mean and the statistical significance was accepted at p < 0.05.

concentration for 14 day culture period were washed twice with DPBS−− and dissociated into single cells by Accutase treatment and repetitive pipetting. Centrifugation at 2000 rpm for 10 min pelleted the cells. 1 ml of 70% alcohol was added drop wise to the pellet, while vortexing. This was followed by a brief vortex, pipetting and incubation at room temperature for 2 h, so as to fix the cells. The fixed cells were washed thrice with DPBS−− to remove last traces of alcohol. This was followed by addition of 0.5 ml staining solution (0.5% Triton X-100, 0.2 mg ml− 1 RNase A and 0.05 mg ml− 1 Propidium iodide in DPBS−−) and incubation at 37 °C for 30 min. The cell suspension was then subjected to FACS analysis (MoFlo XDP, Beckman Coulter). DNA content was analyzed by determination of PI intensity, employing the FACS parameters for haploid DNA content examination by using both PI stained as well as non-PI stained frozen thawed buffalo semen as positive controls, while undifferentiated buffalo embryonic stem cells were used as the negative control. The cell doublets were excluded by pulse processing using pulse area versus pulse width and the single cells were identified by Forward Scatter (FS) and Side Scatter (SS).

3. Results 3.1. Transcriptional profiling of germ lineage genes Most of the genes associated with PGC formation, meiosis and mature gametes started expressing from day 4 but their expression was highest in day 8 EBs. The optimum concentration of RA that induced highest expression of genes like DAZL (6–7 fold), PLZF (4–5 fold), VASA (250–300 fold), TNP1 (5–6 fold), TNP2 (16 fold), SYCP3 (400–500 fold), MLH1 (25–30 fold), TEKT1 (30 fold), was 16 μM and the corresponding culture interval was 8 days. BOULE, however, showed highest expression (30 fold) at 2 μM RA dose in 8 day old EBs, followed by 16 μM RA concentration for the same time period (10 fold). GDF9 (150–200 fold) and ZP2 (100–120 fold) also exhibited the highest expression at 2 μM RA dose for 8 day culture period, unlike ZP3 (10–12 fold) whose expression was highest at 20 μM RA dose for the same culture period (Fig. 1). Similar pattern of gene expression was observed in monolayer adherent cultures differentiated under the same dose-andtime regimen (Supplementary Fig. 1), but was significantly lesser as compared to the corresponding floating cultures (Fig. 2) Thus, from the foregoing results it could be concluded that the optimum dose of RA for inducing PGC differentiation and progression through meiosis is 16 μM

2.4.7. Statistical analysis One way ANOVA followed by Duncan multiple range test, using a statistical software program (SPSS 11.5, 2004, IBM, USA), was used for qPCR data analysis. GraphPad PRISM version 4 (GraphPad Software Inc., San Diego CA) was used for calculation of 5-methyl-2-deoxycytidine concentrations from the corresponding absorbance values.

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Fig. 6. Immunocytochemical analysis for Primordial germ cell (PGCs) markers (c-KIT, DAZL and VASA) induced by RA stimulation (16 μM) in monolayer adherent cultures differentiated for 8 days. BF - Bright field image; DAPI - Nucleus stained image; 2°Ab - PE/FITC labeled secondary antibody image; M - Merged images of DAPI and 2°Ab (Magnification - 200 ×, Scale bar - 100 μm).

spherical shape and bigger size than undifferentiated ES cells. These structures also stained positive for ZP4 protein (Fig. 8). These findings together indicate that RA does function to induce differentiation of ES cells into PGCs which subsequently develop into germ cells (spermatocytes and oocytes). None of these markers were expressed in undifferentiated ES cell colonies and monolayer cultures, when analyzed by immunocytochemistry (Supplementary Figs. 2 and 3). Out of the total 300 EBs which were analyzed for germ lineage marker expression, more number of EBs collected randomly from optimum RA differentiation media exhibited germ lineage marker expression as compared to spontaneously differentiated EBs (Table 1).

for 8 day culture period, while genes associated with mature germ cells like BOULE (spermatocytes), GDF9 and ZP2 (oocytes) get induced by comparatively lower levels of RA, indicating a functional concentration-gradient of RA activity. In view of these observations, we used 16 μM RA dose for further differentiation studies.

3.2. Immunocytochemical examination for germ lineage protein expression Based on mRNA quantification results, examination for PGC-specific proteins was performed on EBs collected from cultures containing 16 μM RA dose, at day 8 of the culture period. Upon immunocytochemistry, expression of PGC-specific proteins (c-KIT, DAZL and VASA) (Fig. 3) as well as meiotic proteins (PROTAMINE1, SYCP3 and MLH1) (Fig. 4) was observed. EBs also exhibited expression of spermatocyte-specific proteins (ACROSIN and HAPRIN) (Fig. 5) and oocyte-specific proteins (GDF9 and ZP4) (Fig. 5). Monolayer adherent cultures also showed expression of PGC- (Fig. 6), meiotic- (Fig. 7), spermatocyte- (Fig. 7) and oocyte-specific proteins (Fig. 8). We observed oocyte-like structures (OLS) in the cultures which had a

3.3. Analysis of methylation erasure Day 8 EBs differentiated under optimum RA stimulation had significantly (p < 0.05) lower concentration of 5-methyl-2-deoxycytidine (3.16 ± 0.10 ng μl− 1) as compared to control ES cell colonies (5.33 ± 0.15 ng μl− 1) and spontaneously differentiated EBs for 14 day culture interval (4.9 ± 0.22 ngμl− 1) (Fig. 9).

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Fig. 7. Immunocytochemical analysis for meiotic markers (PROTAMINE1 and MLH1) and spermatocyte markers (ACROSIN and HAPRIN) induced by RA stimulation (16 μM) in monolayer adherent cultures differentiated for 8 days. BF - Bright field image; DAPI - Nucleus stained image; 2°Ab - PE/FITC-labeled secondary antibody image; M - Merged images of DAPI and 2°Ab (Magnification - 200 ×, Scale bar - 100 μm).

activation without fertilization (Hubner et al., 2003). Upon extended culture, these OLS showed two distinct developmental patterns: i) Attached development - The semi-attached OLS got completely attached to the culture substrate after a couple of days. At days 14–22, these OLS developed into 2-cell embryo-like structures. After 2–3 days, 4-cell embryo like structures developed which progressed further to 8-cell and morula-like embryonic stages, within another 3–7 days (Fig. 11). In order to confirm the cell number in these structures and elucidate their embryonic nature, we analyzed them for nucleus staining (DAPI) and immunocytochemistry for ZP4 protein, presuming that all the embryonic stages are enclosed within zona pellucida, if the zona pellucida coat has not been removed by enzymatic treatment. These structures did stain for DAPI as well as for ZP4, indicating their embryonic origin as well as the actual cell number. The number of cells (nuclei) in presumed morula-stage counted to 70–80 (Fig. 12). The development did not proceed beyond this stage up to another week of the culture (total culture interval was 40 days). ii) Semi-attached development - OLS did not attach to the flask substrate after 14 day culture progression. On day 16–18, a polar body like structure seemed to be extruded from them, indicating in vitro maturation (Fig. 13). On days 19–22, a putative matured oocyte (PMO) like structure was evident in the differentiation cultures. Most of these OLS disrupted in the culture flask. However, compaction and blastocyst hatching -like process was observed in some developing embryos which was followed by formation of hatched blastocyst-like structures (Fig. 13).

3.4. Haploid cell population analysis FACS parameters of haploid cells (1N) were set by using buffalo semen sample, both PI stained and unstained, procured from Animal Breeding and Research Centre (ABRC) of the Institute. We observed a small percentage of haploid cell population (3.07%) in our differentiation cultures (Fig. 10), indicating completion of meiosis in a fraction of cell population, while undifferentiated ES cells did not reveal presence of any haploid cell population (Suppl. Fig. 4).

3.5. Development into embryo-like structures The oocyte-like structures had a bigger size (~ 30–60 μm) as compared to an individual undifferentiated ES cell (10–20 μm), stained positive for ZP4 and harboured a big nucleus. A distinction with them was that most, if not all, of them grew as spheroid semi-attached structures. IVF of these structures was hindered due to: i) small size (compared to an average buffalo oocyte, ~120–160 μm) making it difficult to separate them from rest of the cells; and ii) detachment of the adhered cells upon addition of capacitated sperm to culture, and subsequent loss upon washings, deemed necessary for normal embryo production under in vitro culture (IVC). So we decided to examine the developmental competence of these OLS in extended cultures, probably, by parthenogenetic activation as parthenogenesis leads to

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Fig. 8. Immunocytochemical analysis for oocyte markers (GDF9 and ZP4) induced by RA stimulation (16 μM) in monolayer adherent cultures differentiated for 8 days. BF - Bright field image; DAP I - Nucleus stained image; 2°Ab - PE/FITC-labeled secondary antibody image; M - Merged images of DAPI and 2°Ab. Also shown is enlarged section of oocyte-like structures with a big nucleus and ZP4 coat (Magnification - 200 ×, Scale bar - 100 μm).

Table 1 Immunocytochemical analysis results for germ lineage gene induction in embryoid bodies developed under RA induction (16 μM for 8 days) and spontaneous differentiation. Germ lineage protein

c-KIT

DAZL

VASA

MLH1

SYCP3

PROTAMINE1

ACROSIN

HAPRIN

GDF9

ZP4

Spontaneous differentiation Total EBs analyzed EBs expressing the protein

24 6 (25%)

24 7 (34.3%)

24 8 (30%)

30 4 (13.3%)

30 3 (10%)

30 3 (10%)

35 3 (8.5%)

35 4 (11.4%)

34 5 (14.7%)

34 4 (11.4%)

RA induced differentiation Total EBs analyzed EBs expressing the protein

24 18 (75%)

24 18 (75%)

24 20 (83.3%)

30 17 (56.7%)

30 20 (66.7%)

30 17 (56.7%)

35 15 (42.8%)

35 19 (54.3%)

34 21 (61.7%)

34 23 (67.6%)

4. Discussion

across germ cell lineage in other species (Aflatoonian et al., 2009; Kee et al., 2009; Okita et al., 2007; Clark et al., 2004; Geijsen et al., 2004). The present study, as per our knowledge, is the first draft reporting RAinduced differentiation of bubaline embryonic stem cells into germ lineage cells. A battery of germ lineage markers in association with embryonic stem cell markers was analyzed to model the in vitro differentiation to germ lineage. A number of evidences support the validity of this approach for assaying germ cell development (Hubner et al., 2003; Clark et al., 2004; Toyooka et al., 2003). A belief, though untrue, is held that ES cells express all the genes promiscuously, thus

4.1. Assaying differentiation to germ lineage A fairly good number of reports exist which demonstrate that ES cells, irrespective of the species of origin, are pluripotent and capable of differentiating into all cell types under suitable culture conditions (Odorico et al., 2001; Clark et al., 2004). Apart from some preliminary studies in mice and humans, little information is available regarding the differentiation of embryonic and/or induced pluripotent stem cells

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Taking this role of RA into consideration, we exposed buffalo ES cells to different concentrations of exogenous RA and for different time periods in order to investigate its effect on differentiation to germ lineage cells. Based on the qPCR data, we observed that germ lineage gene induction is subject to RA concentration as well as culture duration. This probably reflects to a functional concentration-gradient which expectedly might be a case, based on the observation of RA source/sink system under in vivo conditions (McCaffery et al., 1999; Romand et al., 2006). The beginning of meiosis in germ cells in a developing ovary occurs in an anterior-posterior wave, rather than simultaneously or stochastically, which further supports the existence of a concentration gradient in the gonad (Bullejos and Koopman, 2004; Yao et al., 2003; Menke et al., 2003) This corroborates with the gene expression patterns, we obtained in our study, where PGC-specific and meiotic genes showed highest expression at higher concentration of RA (16 μM) in contrast to mature germ cell markers whose expression peaked at lower concentration (2 μM), for the same culture period. The germ lineage gene expression followed the trend similar to that observed during EB differentiation when analyzed for the optimum dose-and time- period, and thus 16 μM RA was selected as the optimum induction dose. The expression level of all genes, under monolayer differentiation was, nevertheless, significantly (p < 0.05) lesser as compared to that in EB culture for the corresponding RA concentrations. This could probably be due to enhanced differentiation induction in embryoid bodies which resemble three-dimensional aggregates and thus get exposed to an inducer over a larger surface area in contrast to adherent monolayers where exposed area is comparatively lesser. An appreciable apoptosis was also observed in ES cell monolayers differentiated at higher RA concentrations (20 and 30 μM doses); probably reflecting deleterious effects of RA, while no such cell death was observed at lower doses (2–8 μM). Little cell death was also observed at 16 μM RA dose but it was still employed as the optimum differentiation dose for it induced significantly (p < 0.05) higher germ lineage gene expression than both the lower and higher doses. Our results are in agreement with Aflatoonian et al. (2009) who also observed higher expression of germ lineage cell-markers like DAZL, VASA, TNP1, PRM2 etc. at day 7 of culture interval in presence of 2 μM RA. The authors however, did not optimize the RA concentration for maximizing germ lineage induction but arbitrarily employed the 2 μM dose. Detection of PGC-specific (c-KIT, DAZL and VASA); Meiotic (SYCP3, PROTAMINE1 and MLH1) Spermatocyte-

Fig. 9. Global DNA methylation analysis for 5-methyl-2-deoxycytidine concentration of undifferentiated ES cell colonies and EBs collected from spontaneous and RA differentiation cultures. Bars with different superscripts differ significantly (p < 0.05).

annulling cell-specific marker analysis as inappropriate means to monitor their differentiation into any given cell type. However, a number of reports have demonstrated that such promiscuous gene expression does not occur in ES cells, thereby validating marker based analysis approach (Tanaka et al., 2005; Ivanova et al., 2002; RamalhoSantos et al., 2002; Hsiao et al., 2001; Warrington et al., 2000). A nonrandom pattern of gene expression, beginning with expression of early markers (PGC-specific) at lesser culture intervals, and late markers (meiotic, spermatocyte- and oocyte specific) at later stages of culture interval was also observed. We further studied transcription as well as translation of germ cell-specific genes. The reports that only germ line cells express proteins like DAZL, VASA, SYCP3, GDF9, TEKT1, TNPs, PRMs, ACROSIN, BOULE, ZP, HAPRIN etc. (Aflatoonian et al., 2009; Tilgner et al., 2008; Clark et al., 2004) further support our model. We used three IVF-derived ES cell lines for the study in order to nullify and/ or reduce the bias that could arise from a single cell line.

4.2. RA signaling induces ES cell differentiation to germ lineage RA has been shown to play a key role in early gametogenesis and meiosis induction in vivo (Bowles et al., 2006; Koubova et al., 2006).

Fig. 10. Analysis of DNA content for haploid cell population determination in ES cell monolayer cultures differentiated under optimum RA conditions (16 μM for 8 days).

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Fig. 11. Developmental competence of the oocyte like structures (OLS) in extended cultures in attached mode of embryonic development. d14, d16, etc. represent the total culture duration from day 1. (Magnification - 200 ×, Scale bar - 100 μm).

previous studies have reported the detection of haploid cell population upon FACS analysis. Kee et al. (2009) for example, reported the detection of ~2% haploid cell population on day 14, after over expression of DAZL, BOULE and DAZ genes in human ES cells. It is also noteworthy to mention that we observed no haploid cell population in differentiated buffalo embryonic stem cell monolayer cultures using cumulus cell conditioned medium as well as testicular cell conditioned medium upon FACS analysis (Shah et al., 2015b, 2016). Our results also demonstrate methylation erasure event, as inferred from significantly reduced levels of 5-methyl-2-deoxycytidine in day 8 EBs derived from optimum RA differentiation cultures as compared to EBs derived from spontaneous differentiation as well as undifferentiated ES cells. The epigenetic reprogramming, primarily by global demethylation, has been ascribed to be an essential event in gametogenesis bestowing pluripotency to germ cells, across all the mammalian species (Maatouk et al., 2006). The increased reprogramming, proportional to increased demethylation (and so decreased 5mCytosine concentration) in RA inducing conditions could be attributed to enhanced RA signaling, in presence of exogenous RA (16 μM in our experiments). It has been reported that signaling through RARs (retinoic acid receptors) plays critical role in cellular reprogramming of somatic cells into iPSCs (Wang et al., 2011) and might also perform a somewhat similar function in ES cell reprogramming to germ cells. RARs activate OCT4 expression via binding of RAR: RXR heterodimers to RAREoct (Barnea and Bergman, 2000). These heterodimers subsequently recruit coactivators (CBP/ p300, P/CAF and SRC1/TIF2) to the OCT4 locus to facilitate further chromatin remodeling, binding as well as OCT4 expression (Niederreither and Dolle, 2008). They further promote reprogramming

specific (ACROSIN and HAPRIN); and Oocyte-specific (GDF9 and ZP4) proteins by immunocytochemical analysis of both EBs and adherent cultures further demonstrates differentiation across germ lineage like PGC formation, meiosis and development to spermatocyte- and oocytelike cells, in our study. The expression of meiotic proteins together with spermatocyte- and oocyte- markers is an indicator of post-meiotic gametogenesis. This observation is in agreement with the previous findings (Lacham-Kaplan et al., 2006; Clark et al., 2004) that ES cells upon differentiation in vitro, under optimum culture conditions, may express genetic programmes for both the genders, regardless of the sex karyotype (48 +XX, in our case). Some of the previous studies (Aflatoonian et al., 2009; Lacham-Kaplan et al., 2006; Clark et al., 2004) reported detection of SYCP3 protein in day 14 EBs differentiated from human ES cells either spontaneously or in presence of germ line inducers. These studies, however, did not report detection of MLH1 and/or PROTAMINE1, other meiotic markers which are suggestive of undoubtful meiosis. West et al. (2008) however, reported detection of MLH1 and SYCP3 in continuous monolayer cultures of human ES cells differentiated in presence of bFGF and mouse feeder cells for 16 days. In contrast to these studies, we detected the presence of meiotic proteins (SYCP3, MLH1 and PROTAMINE1) as well as mature germ cell proteins (ACROSIN, HAPRIN, GDF9 and ZP4) at reduced culture interval of 8 days only. This could probably be due to the presence of optimum concentration of RA used in our study. We also report presence of haploid cell population (3.07%) in monolayer differentiation cultures, which suggests completion of meiosis. This, as per our knowledge, is the first study reporting the detection of haploid cell population upon RA induction of germ lineage differentiation in any species. A number of

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Fig. 12. Immunofluorescence staining for oocyte-specific protein (ZP4) and nuclei (DAPI) to elucidate developmental competence of OLS in extended cultures in attached mode of development. Also shown are the enlarged versions of merged image of morula-stage like structures (Magnification - 200 ×, Scale bar - 100 μm).

preimplantation embryos, representing parthenotes. Such structures were also observed by Hubner et al. (2003) upon differentiation of mouse ES cells into oocytes. We demonstrated an optimal culture system for differentiation of ES cells into germ lineage cells upon stimulation by optimal RA dose. We showed that upon optimal induction, ES cells express germ lineage associated genes in several hundred-fold higher quantities as compared to undifferentiated ES cells. The detection of germ lineage specific proteins as well as methylation erasure indicates critical events of gametogenesis occurring in vitro. The detection of haploid cell population is suggestive of completion of meiosis and development of haploid gametes. The progression of oocyte-like structures through various embryonic stages to blastocyst-like structures is an indicator of their developmental competence. This, as per our knowledge, is the first study in higher mammalian species, especially farm animals, that investigates the key events of gametogenesis right from PGC-specification, meiosis, methylation erasure, haploid cell formation and developmental competence of the OLS, so formed. It could be concluded that buffalo ES cells are capable of differentiating into oocytes as well as spermatocytes under suitable in vitro culture conditions. However, more studies are required to determine the fertilizability of these OLS and spermatocyte-like cells and whether the resulting zygotes are able to develop into a complete blastocyst with an outer trophectoderm and inner cell mass.

in a ligand-independent manner by binding to additional genomic loci (Hua et al., 2009; Ross-Innes et al., 2010). This probably explains for the highest hypomethylation status observed under RA induced differentiation. This reprogramming mechanism of RA via up-regulation of key pluripotency genes finds support from the observation that RA stimulation was coupled with increased expression of OCT4 and NANOG genes in day 4 and day 8 EBs, as observed in our study. 4.3. Development into embryonic-like structures We observed oocyte-like structures (OLS) in monolayer differentiation cultures at day 8–14 of the culture interval. These OLS had a distinct nucleus and showed ZP4 expression. This is in contrast to previous reports where immunolocalization of ZP4 in the OLS was not detected (Aflatoonian et al., 2009). The probable reason could be less optimal differentiation conditions or species-to-species variation between the studies. These OLS when put for extended cultures probably got parthenogenetically activated and progressed through embryonic development. In completely attached mode of development, these OLS developed upto morulae-stage only, indicating lesser competence. However, blastocyst-like structures were observed in semi-attached mode of development which progressed through such stages like compaction, zona-disintegration and hatching. We found a number of structures that resembled blastocysts and are likely to be

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