The Arabidopsis thaliana-Alternaria brassicicola pathosystem: A model interaction for investigating seed transmission of necrotrophic fungi

Stephanie Pochon
Emmanuel Terrasson
Thomas Guillemette
Beatrice Iacomi-Vasilescu
Sonia Georgeault
Marjorie Juchaux
Romain Berruyer
Isabelle Debeaujon
Philippe Simoneau
Claire Campion
The Arabidopsis thaliana-Alternaria brassicicola pathosystem: A model interaction for investigating seed transmission of necrotrophic fungi
M E T H O D O L O G Y Open Access
Background: Seed transmission constitutes a major component of the parasitic cycle for several fungal pathogens. However, very little is known concerning fungal or plant genetic factors that impact seed transmission and mechanisms underlying this key biological trait have yet to be clarified. Such lack of available data could be probably explained by the absence of suitable model pathosystem to study plant-fungus interactions during the plant reproductive phase. Results: Here we report on setting up a new pathosystem that could facilitate the study of fungal seed transmission. Reproductive organs of Arabidopsis thaliana were inoculated with Alternaria brassicicola conidia. Parameters (floral vs fruit route, seed collection date, plant and silique developmental stages) that could influence the seed transmission efficiency were tested to define optimal seed infection conditions. Microscopic observations revealed that the fungus penetrates siliques through cellular junctions, replum and stomata, and into seed coats either directly or through cracks. The ability of the osmosensitive fungal mutant nik1Δ3 to transmit to A. thaliana seeds was analyzed. A significant decrease in seed transmission rate was observed compared to the wild-type parental strain, confirming that a functional osmoregulation pathway is required for efficient seed transmission of the fungus. Similarly, to test the role of flavonoids in seed coat protection against pathogens, a transparent testa Arabidopsis mutant (tt4-1) not producing any flavonoid was used as host plant. Unexpectedly, tt4-1 seeds were infected to a significantly lower extent than wild-type seeds, possibly due to over-accumulation of other antimicrobial metabolites. Conclusions: The Arabidopsis thaliana-Alternaria brassicicola pathosystem, that have been widely used to study plant-pathogen interactions during the vegetative phase, also proved to constitute a suitable model pathosystem for detailed analysis of plant-pathogen interactions during the reproductive phase. We demonstrated that it provides an excellent system for investigating the impact of different fungal or plant mutations on the seed transmission process and therefore paves the way towards future high-throughput screening of both Arabidopsis and fungal mutant.

Background

Transmission from infected to healthy hosts is a key component of pathogen fitness, as pathogens strongly depend on this process for their long-term survival [1]. For several plant-parasite interactions, transmission is strictly "horizontal" with a pathogen spreading between related and unrelated individuals through mechanisms ranging from direct contact to wind-dispersal. In other systems, transmission may also be "vertical", that is to say the pathogen infects the host's offspring [2]. Although many pathogens spread by a combination of horizontal and vertical transmission, these two transmission modes require specific adaptations of the pathogen that can be mutually exclusive, resulting in a trade-off between horizontal and vertical transmission [1]. While an inverse relation between aggressiveness and vertical transmission has been shown for some pathogens [3], the ability to transmit by seed may have significant advantages for pathogens, such as long-term survival, maximum opportunity for progeny infection and long distance dissemination [4,5].

From an epidemiological standpoint, seed transmission represents a possible mode of primary infection from which dangerous polycyclic microorganisms can start epidemic attacks. The early presence of a small quantity of infected plants (i.e. 0.05% of a crop) is sometimes sufficient for the development of destructive attacks [6]. The use of seeds infected with pathogenic fungi thus represents a major economic threat and prevention of plant infection by seedborne pathogens in seed crops is essential. Although the chain of events leading to seed transfer through the plant has been extensively documented [4], almost nothing is currently known concerning the factors that mediate seed transmission by fungal pathogens. Iacomi-Vasilescu et al. [7] recently reported that Alternaria brassicicola strains deficient in a group III osmosensor histidine kinase were highly jeopardized in their ability to infect radish seeds. This was interpreted as a consequence of their failure to overcome severe osmotic stress conditions consecutive to the gradual decrease in the water potential in maturing seeds. Besides this pioneer study, no other data are available for fungal pathogenic factors that impact seed transmission. This could be probably explained by the difficulty to carry out such experiments under field conditions, particularly due to the reduced number of reproduction cycles per year and the lack of available model pathosystem to study plant-fungus interactions during the plant reproductive phase.

Arabidopsis thaliana has become an important model host for studying plant-pathogen interactions during the vegetative phase due to the development of numerous genetic and genomic tools. However, to our knowledge, this plant species has never been used as host to explore plant-pathogen interactions during the reproductive phase although it has several attributes such as small size and rapid life cycle, which make it well suited for this kind of study under carefully controlled environmental conditions. Among the numerous Arabidopsispathogen systems that have been developed to investigate the molecular mechanisms of virulence and plant resistance [8], the interaction of Arabidopsis with Alternaria brassicicola has been extensively used as a model for diseases caused by fungal necrotrophs [9]. Alternaria brassicicola causes dark spot disease, one of the most common and destructive fungal diseases of Brassicaceae worldwide. This fungus can be seedborne via mycelium within the seed and is the dominating Alternaria spp. in Brassica seed crops and may be responsible for important yield losses [10][11][12][13]. In such crops, a vertical disease gradient can be observed on fruits, with the lowest siliques becoming infected first and infection spreading slowly upwards [14]. Concerning seeds, hilum was described to be the more contaminated seed area, as a consequence of mycelial growth through the suture of siliques and the funicle [15,16]. To a lesser extent, A. brassicicola can infect seeds in an internal manner. In this instance, the pathogen is mainly found in seed coats. Both external and internal inoculum can survive for several years but the longevity of internal inoculum is higher than that of external inoculum [10].

The main objective of the present study was to develop, in controlled conditions, a model pathosystem suitable for detailed analysis of the molecular mechanisms underlying the seed transmission stage in both the host plant and the fungal pathogen. Reproductive organs of A. thaliana Landsberg erecta (Ler), an ecotype susceptible to A. brassicicola, were thus inoculated with fungal conidia, and transmission levels of this pathogen to seeds were evaluated. The influence of different parameters on the seed transmission "probability" was established in order to design a robust and easy to handle protocol. This model pathosystem was then used to confirm that an A. brassicicola strain deficient in the osmosensing histidine kinase AbNIK1 was impaired in its capacity to efficiently colonize seeds. In addition, transmission of A. brassicicola to seeds of an A. thaliana transparent testa 4 (tt4-1) mutant deficient with respect to flavonoid synthesis was also analyzed.

Results

Seed transmission route and influence of the seed collection date on seed transmission

As seed infection through the vascular supply has been described for wilt pathogens [17], only floral and fruit routes of seed infection were explored with Alternaria brassicicola. The floral infection route for seed transmission was studied in plants at the bolting stage by inoculating floral buds with an A. brassicicola (Abra43, WT) conidial suspension adjusted to either 1 x 10 4 or 1 x 10 5 conidia mL -1 . For inoculations, 5 μl drops of an A. brassicicola conidial suspension were deposited on intact host tissue. Six days after inoculation, necrotic lesions were observed on flower buds and 8 days later, flower buds were covered with mycelium and conidia, irrespective of the inoculum charge. As a consequence, flower formation was impeded (data not shown).

For examination of the fruit route for seed transmission, plants with 10 siliques on the main flowering stem, were inoculated by application of two distinct 2.5 μL drops of an A. brassicicola (Abra43) conidial suspension adjusted to 1 x 10 5 conidia mL -1 on the outer surface of the 10 oldest siliques (one drop at the base of silique and one in the middle). Inoculations resulted in mycelium development, conidia formation and the appearance of necrotic lesions typical of black spot on siliques within a few days after inoculation ( Figure 1a, 1b and 1c). Inoculated siliques were collected 10 or 20 days after inoculation, and seeds were individually harvested. Seeds showed mycelium development and conidia formation ( Figure 1d). They were separately plated on PDA medium, to estimate the seed contamination level. Transmission of A. brassicicola to seeds was similar (32% of contaminated seeds) at the two collection dates ( Figure 2). Siliques and seeds were thus harvested 10 days after inoculation for further experiments, since longer incubation did not induce a higher contamination level. Seeds harvested in control plants that were integrated to all experiments, and whose siliques were treated with two 2.5 μL drops of a 0.01% Tween 20 solution, never showed any A. brassicicola development.

Influence of the plant and silique developmental stage on seed transmission

The influence of the plant developmental stage on the incidence of A. brassicicola on seeds was examined by inoculating all siliques of plants with either 10 or 20 siliques on the main flowering stem. For inoculations, two distinct 2.5 μL drops of a conidial suspension adjusted to 1 x 10 5 conidia mL -1 were applied. The results indicated that the contamination level per plant was significantly higher (35%) when siliques were inoculated at the 10-silique stage rather than later at the 20-silique stage (8%) ( Figure 3). Further experiments were thus conducted by inoculating plants at the 10-silique stage.

The impact of the silique developmental stage on A. brassicicola seed transmission was assessed through the inoculation of 10 siliques numbered from 1 (the oldest silique, at the base of the bolting stem) to 10 (the youngest silique) (Figure 4a), and the seeds were identified according to their corresponding silique. The global probability of seed infection by A. brassicicola was similar to previous b a c d results (32%, data not shown), but the seed infection probability differed depending on the silique: 74% for seeds from the oldest silique at the base of the bolting stem (n°1 ), only 7% for seeds from a younger silique near the apical meristem (n°10) (Figure 4b). The incidence on seeds was thus significantly different according to the silique developmental stage. As the A. brassicicola incidence on seeds of the youngest siliques was very low, in further experiments, evaluation of A. brassicicola seed transmission capacity took only the results concerning the five oldest siliques (n°1 to n°5) into account.

Microscopic assessment of seed and silique colonization by A. brassicicola

Siliques and seeds harvested 10 days after inoculation of siliques with an A. brassicicola conidial suspension adjusted to 1 x 10 5 conidia mL -1 were assessed by scanning electron microscopy (SEM) and confocal laser scanning microscopy. These studies focused on the histopathology of infected seeds and siliques, while observing the extent and amount of mycelium and conidia.

SEM observations of seeds provided evidence of A. brassicicola mycelium superficial growth and conidia formation on seeds harvested 10 days after inoculation (Figure 5a and 5b). Penetration of hyphae into seed coat tissues, directly (Figure 5c) or through cracks in the seed coat, was observed by SEM. Penetration and fungal development within the seed tissues were also revealed by staining hyphae with solophenyl flavin and observing the tissues under a confocal laser scanning microscope ( Figure 5d). These findings indicated that the fungus actively colonized the seed coat and was not only a passive contaminant of the seed surface. After fungal colonization, infected seeds frequently showed alteration of the seed coat appearance (Figure 5e) by comparison with seeds harvested on control plants ( Figure 5f): infected seeds seemed shrivelled as if embryo aborted early during development.

On the outer surface of siliques, mycelium was found to be growing in many directions from germinated conidia in the inoculation zone (Figure 6a and 6b). Hyphae penetrated into the fruit occasionally via stomata present on the outer surface of siliques (Figure 6c) or, more often directly, in particular at the cellular junctions ( Figure 6d). Stomata also allowed mycelium reemergence (Figure 6e). Conidia formation was evident on the outer surface of siliques ( Figure 6f ). Intense mycelium development and conidia formation were also visible on the inner surface of siliques (Figure 6g). Replum seemed to be a preferential zone for penetration into siliques (Figure 6h). The silique results indicated that the fungal pathogen spread into the seed from the fruit.

Influence of fungal and host genotypes on seed transmission

In order to confirm that the above-described model pathosystem is a useful tool for analyzing the molecular factors involved in the seed transmission process, the seed transmission capacity of an osmosensitive fungal mutant nik1Δ3 [18] and its wild-type parental strain (Abra43) were assessed on an A. thaliana Ler ecotype. This was performed as described above by inoculating the five oldest siliques on plants on which 10 siliques were present on the main flowering stem, and individually harvesting seeds 10 days after inoculation. Parallel leaf inoculation experiments were conducted to ensure that the nik1Δ3 mutant strain was still virulent on A. thaliana vegetative organs (Additional file 1).

In comparison to the wild-type strain Abra43, the nik1Δ3 mutant strain, defective in AbNIK1, encoding a group III osmosensor histidine kinase, was significantly affected with respect to the transmission capacity to Ler ecotype seeds ( Figure 7). Indeed, infection probability was 40% after inoculation with Abra43, and 12% after inoculation with the nik1Δ3 mutant strain (Figure 7a). This difference was statistically significant overall (p < 2 × 10 -16 ), and for each silique (p ≤ 1.14 × 10 -8 ) (Figure 7b). The gradient of fungal incidence from the oldest silique (n°1) to the youngest inoculated one (n°5) observed after inoculation with Abra43 was also noted for the nik1Δ3 mutant strain.

Seed transmission efficiencies of A. brassicicola (Abra43) on two A. thaliana genotypes, the Ler ecotype and the tt4-1 mutant, inactivated in flavonoid biosynthesis, were also compared. Unexpectedly, the infection probability of A. brassicicola to tt4-1 seeds was significantly lower (15%) than to seeds of the parental wild-type Ler ecotype (27%) (Figure 8a). The difference in the infection probability was significant overall (p < 2 × 10 -16 ), and for each inoculated silique (p ≤ 12 × 10 -4 ) (Figure 8b). Parallel leaf inoculation assays did not reveal any obvious difference in the capacity of A. brassicicola to develop symptoms on vegetative organs from either A. thaliana genotypes (Additional file 1).

Discussion

The main purpose of this study was to set up a new pathosystem that could be used to analyze molecular factors that mediate the transmission of fungal pathogens to seeds. Arabidopsis thaliana reproductive organs were inoculated with the seedborne pathogen A. brassicicola. Some general conclusions can be drawn from the findings of experiments designed to define the optimal conditions for studying fungal transmission to seeds of A. thaliana. First, concerning the seed transmission routes, our results did not reveal whether A. brassicicola was able to cause seed infection via flowers, since floral development was interrupted due to inoculation with a virulent isolate. Oliver et al. [2] demonstrated, via glasshouse inoculation trials and examination of naturally infected Cakile seeds, that seed infection could occur through the flowers, though less efficiently than the fruit route. Indeed, the floral route seems to be more specific to opportunistic pathogenic strains of Alternaria alternata, with this phenomenon being explained by the weakness of flower defence mechanisms. By contrast, we found that the fruit route was efficient and the seed contamination levels (30%) thus observed in A. thaliana under our experimental conditions were comparable to those obtained by Oliver et al. [2] for Cakile maritima seeds in field and laboratory conditions, with the frequency of infection positively correlated with the lesion density on the fruit surface.  Our results also confirmed data obtained on Brassica seed crops in field conditions, indicating a vertical disease gradient on fruits, with the infection first affecting the lowest siliques and then spreading slowly upwards [14]. This was explained by the observation that conidia are often disseminated short distances and retained within the canopy. As in our laboratory conditions, inoculum was distributed equally on all siliques, the more efficient transmission to seeds from the lower siliques could be mainly due to silique maturity differences, as Alternaria development is favored on senescent tissues [19].

The mode of seed infection by A. brassicicola and its localization on the reproductive organ tissues was already described several years ago [15]. We used our model pathosystem to perform a microscopic analysis of contaminated seeds, but also of contaminated siliques. Concerning seeds, surface colonization via conidia and hyphae was observed and resulted in desquamation of the seed surface, in accordance with previously published data [15]. Such intensive fungal development could be favored by the release of carbohydrates at the surface of damaged seeds as proposed by Knox-Davies [15]. Conidiogenesis has frequently been observed on contaminated seeds, with spores preferentially located in seed coat folds and cracks, which may provide protection against harsh environments [20]. Superficial hyphae also penetrated within the seed, as confirmed by SEM and confocal laser scanning microscopy. Our observations did not allow us to determine if A. brassicicola was limited to the seed coat or also present inside the endosperm and embryo. However, according to Singh & Mathur [17], whereas biotrophic fungi are often located in the embryo, necrotrophic fungi like A. brassicicola are generally located on seed and fruit coats, and deeper penetration is uncommon. In line with this, A. brassicicola was found to be confined to the testa of naturally infected Brussels sprout seeds while being mainly on the surface cabbage seeds [15]. The extent of testa colonization could not be accurately determined and would require transmission electronic microscopy analysis of ultra-thin seed cuts or confocal laser scanning microscopy analysis of seed cuts. Nevertheless, hyphal penetration of A. thaliana seed coats was evidently accompanied by seed surface damage. This phenomenon could be due to the production of plant tissue degradation enzymes, for example cutinases, as already described for A. brassicicola, and it could potentially accompany the penetration of cabbage tissues [21,22]. The same hypothesis was put forward by Vaughan et al. [23] with respect to soybean seed colonization by several Alternaria sp.. To our knowledge, no detailed microscopic analysis has ever been reported concerning silique colonization. A few days before seed harvest, typical black-spot symptoms developed on fruits at inoculation sites where intensive development of the fungus was observed on the outer surface of siliques. Penetration inside the fruit was either via stomata, intercellular spaces or replum, with occasional differentiation of appressorialike structures. The model pathosystem thus defined was then used to specify the molecular factors impacting the seed colonization process by using mutant genotypes of the pathogen and host plant. In maturing seeds, the water content dramatically decreases, leading to a reduction in the metabolism necessary for seed conservation. During seed colonization, fungi could thus be exposed to severe water and osmotic stresses that they have to overcome to be efficiently transmitted to seeds and complete their infection cycle. Then the ability to cope with water and osmotic stress could be a factor that determines their transmission to seeds. Arabidopsis thaliana silique inoculation in controlled conditions allowed us to repeatedly show a lower seed transmission capacity for nik1Δ3, a disruption mutant deficient in a group III histidine kinase involved in the osmotic stress response [18], by comparison with its wild-type parental strain Abra43. The contamination level was significantly lower at every silique stage, with a gradient from the   oldest to the youngest silique, as observed after Abra43 inoculation. This confirms previous findings indicating that A. brassicicola null mutants in the AbNIK1 gene, although their virulence on host vegetative tissues remained intact, were affected in their transmission to radish seeds after artificial inoculation in field conditions [7].

Using our model pathosystem, we took advantage of the availability of A. thaliana transparent testa (tt) mutants, to assess the influence of flavonoids present in seed testa on their susceptibility to fungal infection. The selected mutant line tt4 is altered in the chalcone synthase gene [24] and therefore disrupted in the first step of the flavonoid biosynthesis pathway. It produces pale yellow seeds and does not produce any of the flavonoid compounds present in WT seeds, i.e. proanthocyanidins and flavonols [25]. Based on the well-documented plant protection functions of flavonoids against abiotic and biotic stresses [26], and previously published data showing that peas with dark seed coats were less susceptible to fungal infections than those with light-colored coats [27], we anticipated that A. brassicicola would colonize seeds of the tt4-1 allele more efficiently than the wild type. Unexpectedly, although a downward vertical gradient of seed infection was still observed for both wild-type and mutant genotypes, significantly lower A. brassicicola infection probabilities were obtained with the flavonoid deficient mutant. A recent metabolomic study characterized several compounds over-accumulating in seeds of tt4-1 plants compared to those of wild-type Ler, as phenolic choline esters, sinapate-derived metabolites and glucosinolate breakdown products [28]. Although the antimicrobial properties of the former compounds have yet to be characterized, glucosinolate derivatives have been well documented as potentially toxic for pathogens, including A. brassicicola [29], and such metabolomic changes in tt4-1 seeds might explain our observations. These hypotheses can be tested by investigating the transmission behaviour of Arabidopsis mutants disrupted in sinapate and glucosinolate metabolisms. Complementary investigations using different tt mutants and different tt4 alleles are required to reliably conclude on the role of flavonoids in the seed transmission process of A. brassicicola to A. thaliana.

Conclusions

We have established a reliable Arabidopsis-based pathosystem allowing investigations of fungal transmission to seeds. The results obtained by comparing the seed transmission ability of two fungal genotypes (wild-type vs. nik1Δ3) on the Ler ecotype and of the wild-type isolate Abra43 on two plant genotypes (Ler vs. tt4-1) confirmed that the described assay was suitable for assessing the impact of different fungal or plant mutations on the seed transmission process. This new pathosystem therefore paves the way towards future studies involving other fungal genotypes, e.g. hypersensitive to osmotic and/or water stresses, and other plant genotypes, e.g. mutants producing abnormal seed coats. Moreover, fungusinfected seeds can now be reproducibly obtained and used as starting material to study the ability of the pathogen to survive in seeds during storage and to transmit from seeds to seedlings.

Methods

Fungal strains and growth conditions

The A. brassicicola strains Abra43 (wild-type) and nik1Δ3 (mutant inactivated in the AbNIK1 gene) used in this study have previously been described [18,29]. For routine culture, Abra43 and nik1Δ3 were grown and maintained on potato dextrose agar (PDA) and PDA supplemented with 12 μg/ mL hygromycin B, respectively.

Arabidopsis thaliana genotypes and growth conditions

The capacity of Alternaria brassicicola to transmit to seeds was studied in plants obtained by sowing seeds of Arabidopsis thaliana individually in pots (7 x 7 x 7 cm) containing prewetted compost (NEUHAUS HUMINSUBSTRAT *N4*, NFU 44-551). The Landsberg erecta (Ler) ecotype and its tt4-1 mutant allele (NASC reference N85), inactivated in the chalcone synthase gene and totally defective in flavonoids [24,30], were used. The plants were grown in a controlled climatic room with day/night temperatures of 22°C/19°C and a 16 h photoperiod. ARACON base and tube (ARASYSTEM, BETATECH bvba, Gent, Belgium) were placed as soon as the floral stem appeared.

Infection assays and seed contamination assessment

The floral infection route for seed transmission was tested by inoculating A. thaliana floral buds with 5 μL drops of an A. brassicicola conidial suspension. Two parallel experiments were performed with conidial suspension adjusted to either 1 x 10 4 or 1 x 10 5 conidia mL -1 . The plants were maintained under saturating humidity for 2 days in the dark and were monitored 6 and 14 days after inoculation.

Most of the seed transmission assays used 1-monthold A. thaliana plants with 10 (or 20 for determination of the influence of the plant developmental stage on seed transmission) siliques. The position of a silique at a precise stage of development is determined by the number of flowers produced per inflorescence per day, which is in general three or four. Two 2,5 μL drops of an A. brassicicola conidial suspension (1 x 10 5 conidia mL -1 in water) supplemented with 0,01% (v/v) Tween 20 were placed on the 10 (or 20 for determination of the influence of the plant developmental stage on seed transmission) youngest siliques (one drop at the silique base and one in the middle). The oldest silique (at the base of the bolting stem) was numbered 1 and the youngest 10 (or 20 for determination of the influence of the plant developmental stage on seed transmission). At least three plants per fungal or plant genotype were inoculated and the experiment was repeated twice. As a control for all experiments, two 2,5 μL drops of a 0,01% (v/v) Tween 20 solution were placed on 10 siliques of one plant. The plants were then maintained under saturating humidity for 2 days in the dark.

Contaminated siliques were harvested 10 days after inoculation (or 20 days after inoculation for determination of the influence of the harvest date on seed transmission). Inoculated or control siliques were dissected with sterile forceps and seeds were carefully harvested to avoid contact with the fungus potentially present on the outer surface of siliques. Seeds were incubated separately on PDA medium for 2 days. A seed was considered to be contaminated when incubation resulted in typical colony development. As the first results showed that contamination levels were very low for siliques n°6 to n°10, only results for siliques n°1 to n°5 were taken into account for further experiments.

Statistical analysis

Because of their dichotomous nature (i.e. each seed is either contaminated or not), seed contamination data were analyzed using logistic (logit) generalized linear models (for an introduction, see [31]). Statistical analyses were performed with the R software package (version 2.12.0, [32]), using the glm function with "family" set to "binomial (link = "logit")". IC 95 (95% confidence interval) values were extracted from such models. For ease of reading, log odd values predicted by the models were converted to infection probabilities, thus resulting in slightly asymmetric IC 95 s. Repetitions were treated as a factor, and were found not to have a significant effect.

Scanning electronic microscopy (SEM)

For SEM observations, specimens (siliques valves and seeds harvested 10 days after inoculation) were fixed under vacuum in 4% (v/v) glutaraldehyde in 0.1 M phosphate buffer (pH 7.2) for 4 h at 4°C and stored overnight at 4°C in the same fixative buffer. Specimens were rinsed once in 0.1 M phosphate buffer (pH 7.2) and post-fixed in 2% (w/v) osmium tetroxide in 0.1 M phosphate buffer (pH 7.2) in the dark for 2 h. Samples were rinsed three times with distilled water, dehydrated in a graded ethanol series (50, 70, 95 and 100% for 10 min each). The last 100% ethanol bath was repeated three times. The dehydration process was completed by treating samples in ethanol/acetone (1:1, v/v) for 15 min and then twice in acetone for 15 min. Specimens were dried by the critical-point method. Silique walls and seeds were then sputter-coated with a thin carbon layer and examined using a JSM 6301-F (JEOL, Tokyo, Japan) scanning electron microscope operating at 3 kV.

Figure 1
1Mycelium development, conidia formation on siliques and seeds, and necrotic lesions on siliques 10 days after inoculation with Alternaria brassicicola. (a) and (b), mycelium development and conidia formation (arrows) on siliques. (c), necrotic lesions (arrowhead) on siliques. (d), mycelium development and conidia formation (arrow) on seeds. Bar = 1 mm.

Figure 2
2Influence of incubation duration on transmission capacity of Alternaria brassicicola (Abra43) to seeds of Arabidopsis thaliana plants (Ler ecotype). Harvesting was done 10 days and 20 days after inoculation (dai). Values represent means with IC 95.

Figure 3
3Influence of plant developmental stage on transmission capacity of Alternaria brassicicola (Abra43) to seeds of Arabidopsis thaliana plants (Ler ecotype). Siliques were inoculated when 10 or 20 siliques were present on the main flowering stem. Harvesting was done 10 days after inoculation. Values represent means with IC 95.

Figure 4
4Influence of silique stage on transmission capacity of Alternaria brassicicola (Abra43) to seeds of Arabidopsis thaliana plants (Ler ecotype). (a) Representation of the silique nomenclature. (b) Seed transmission level according to silique stage. Harvesting was done 10 days after inoculation. Values represent means with IC 95.

Figure 5 A
5. brassicicola localization on A. thaliana seeds. Scanning electron microscopy a, b, c, e and f) and confocal laser scanning microscopy (d) images of Arabidopsis thaliana seeds (Ler ecotype) 10 days after inoculation with Alternaria brassicicola (Abra43). (a) Global colonization of a seed by A. brassicicola. (b) conidia formation on a seed. (c) direct hyphal penetration into the seed coat (white arrow). (d) mycelium development on a seed. Hyphae were stained with solophenyl flavin, and seed coat with propidium iodide. Hyphae grew on the seed surface, then penetrated into the seed coat (white arrows): hyphal brightness was lower when hyphae were growing within the seed coat. (e) and (f), alteration of the seed surface after fungal colonization: seed seemed shrivelled (e) by comparison with a seed harvested on a control plant (f).

Figure 6 A
6. brassicicola localization on A. thaliana siliques. Scanning electron microscopy images of Arabidopsis thaliana (Ler ecotype) siliques 10 days after inoculation with Alternaria brassicicola (Abra43). (a), mycelium development on the outer surface of a silique in many directions from the inoculation point. (b), conidia germination (white arrow) on the outer surface of a silique. (c), hyphal penetration into silique tissues via a stomatum (St). (d), direct hyphal penetration into silique tissues at cellular junctions (white arrowhead). (e), hyphal reemergence from silique tissues via a stomatum (St). (f), conidia formation (double white arrowheads) on the outer surface of a silique. (g), mycelium development and conidia formation (double white arrowheads) on the inner surface of a silique. (h), abundant fungal colonization of the replum.

Figure 7
7Influence of A. brassicicola genotypes on seed transmission capacity. Transmission capacity of two Alternaria brassicicola genotypes, Abra43 and nik1Δ3, to Arabidopsis thaliana seeds (Ler ecotype). (a), global seed transmission capacity. (b), seed transmission capacity according to the silique stage. Hatched bars: Abra43, white bars: nik1Δ3. Harvesting was done 10 days after inoculation. Values represent means with IC 95.

Figure 8
8Influence of A. thaliana genotypes on seed transmission capacity. Transmission capacity of Alternaria brassicicola (Abra43) to seeds of two Arabidopsis thaliana genotypes: Ler ecotype and tt4-1 mutant. (a), global seed transmission capacity. (b), seed transmission capacity according to silique stage. Hatched bars: Ler ecotype, white bars: tt4-1 mutant. Harvesting was done 10 days after inoculation. Values represent means with IC 95.
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Pochon et al. Plant Methods 2012, 8:16
AcknowledgementsThis work was supported by the French Région Pays de la Loire (QUALISEM research program). We are grateful to Sylvain Hanteville and Bruno Hamon for technical assistance. We thank David Manley for editing the manuscript.Confocal laser scanning microscopyFor confocal laser scanning microscopy observations, seeds harvested 10 days after inoculation were observed after double staining: seed cell walls were stained with propidium iodide[33]and fungal cell walls, with solophenyl flavin[34].Seeds were immersed for 2 min in a 1% (w/v) propidium iodide solution and rinsed twice in 0.1 M phosphate buffer (pH 7.2). Then the seeds were incubated in a 0.1% (w/v) solophenyl flavin solution for 5 min and rinsed twice in ultrapure water, and then stored in 50% (v/v) glycerol at 4°C until analysis under a Nikon (Nikon Instruments, Melville, NY) A1S1 confocal laser microscope equipped with argon-ion (488 nm) and diode (561 nm) lasers. Solophenyl flavin staining was detected with the emission light set at 520 nm, and propidium iodide staining with the emission light set at 617 nm. Several images were overlapped to obtain a tridimensional picture.Additional fileAdditional file 1: Effects of the disruption of AbNIK1 on the virulence of A. brassicicola on A. thaliana and of the tt4 mutation on the susceptibility of A. thaliana to A. brassicicola. Leaves of A. thaliana Ler ecotype or its tt4 mutant were inoculated with conidia suspensions of Abra43 and nik1Δ3 strains. For each plant-pathogen combination, five leaves are shown that correspond to representative symptoms obtained at 6 dpi.Competing interestsThe authors declare that they have no competing interests.Authors' contributions SP developed the seed transmission pathosystem, performed the experimental work and wrote a draft of the manuscript. ET prepared most of the plant samples for microscopy. BI carried out initial experiments aimed at studying the seed transmission routes. TG and IB supervised experiments involving A. brassicicola and A. thaliana mutants, respectively. SG and MJ provided technical assistance with scanning electronic and confocal laser scanning microscopy, respectively. RB performed the statistical analyses. PS and CC designed and coordinated the project and wrote the manuscript. All authors performed a critical reading and approved the final version of the manuscript.
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Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution. Pochon, Plant Methods. 816The Arabidopsis thaliana-Alternaria brassicicola pathosystem: A model interaction for investigating seed transmission of necrotrophic fungi. Submit your manuscript at www.biomedcentral.com/submitCite this article as: Pochon et al.: The Arabidopsis thaliana-Alternaria brassicicola pathosystem: A model interaction for investigating seed transmission of necrotrophic fungi. Plant Methods 2012 8:16. Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit


Injurious Effects of Emodin on Maturation of Mouse Oocytes, Fertilization and Fetal Development via Apoptosis
2012

Mei-Hui Chang
Department of Bioscience Technology and Center for Nanotechnology
Chung Yuan Christian University
Chung Li 32023Taiwan

Shao-Chung Chang
Department of Bioscience Technology and Center for Nanotechnology
Chung Yuan Christian University
Chung Li 32023Taiwan

Wen-Hsiung Chan firstname.lastname@example.com
Department of Bioscience Technology and Center for Nanotechnology
Chung Yuan Christian University
Chung Li 32023Taiwan

Injurious Effects of Emodin on Maturation of Mouse Oocytes, Fertilization and Fetal Development via Apoptosis

Int. J. Mol. Sci
13201210.3390/ijms131113911Received: 24 September 2012; in revised form: 25 October 2012 / Accepted: 25 October 2012 /International Journal of Molecular Sciences * Author to whom correspondence should be addressed; E-Mail: OPEN ACCESS Int. J. Mol. Sci. 2012, 13 13912emodinapoptosisoocyte maturationembryonic development
Emodin (1,3,8-trihydroxy-6-methylanthraquinone), a major constituent of rhubarb, has a wide range of therapeutic applications. Previous studies have established that emodin induces apoptosis in the inner cell mass and trophectoderm of mouse blastocysts and leads to decreased embryonic development and viability, indicating a role as an injury risk factor for normal embryonic development. However, the mechanisms underlying its hazardous effects have yet to be characterized. In the current study, we further investigated the effects of emodin on oocyte maturation and subsequent pre-and post-implantation development, both in vitro and in vivo. Notably, emodin induced a significant reduction in the rates of oocyte maturation, fertilization, and in vitro embryonic development. Treatment of oocytes with emodin during in vitro maturation (IVM) led to increased resorption of postimplantation embryos and decreased fetal weight. Experiments using an in vivo mouse model disclosed that consumption of drinking water containing 20-40 μM emodin led to decreased oocyte maturation and in vitro fertilization, as well as early embryonic developmental injury. Notably, pretreatment with a caspase-3-specific inhibitor effectively prevented emodin-triggered injury effects, suggesting that impairment of embryo development occurs via a caspase-dependent apoptotic process.

Introduction

Emodin (1,3,8-trihydroxy-6-methylanthraquinone), one of the major chemical constituents of the root of rhubarb (Rheum palmatum L.), is widely used in the Orient [1], and exerts immunosuppressive, anticancer, antiinflammatory, antiatherosclerotic, and vasorelaxant effects [2][3][4][5]. Emodin inhibits cell proliferation in different cancer cell lines, including HER-2/neu-overexpressing breast cancer [6], hepatoma [7], leukemia [8], and lung cancer [9]. An earlier study reported that emodin-stimulated apoptosis is mediated via reactive oxygen species (ROS) and mitochondria-dependent pathways in human tongue squamous cancer SCC-4 cells [10]. Interestingly, emodin exerts both cytotoxic and protective effects in rat C6 glioma cells [11]. Moreover, recent experiments by our group showed that emodin induces a decrease in mouse embryonic development and viability in vitro and in vivo [12]. Interestingly, the hazardous effects of emodin on embryonic development were effectively prevented by caspase-9 and -3 inhibitors [12], implying that emodin triggers apoptosis of mouse blastocysts, leading to impairment of embryo development through intrinsic apoptotic pathways. Accordingly, we propose that emodin is an injury risk factor for normal embryonic development in mouse blastocysts.

Oocyte viability is affected by the microenvironment during growth and maturation. Heat stress, oxygen concentration, and glucose content are key determinants of oocyte viability [13][14][15]. A number of researchers have investigated the influence of environmental biological toxins on oocyte maturation in vivo and in vitro. During normal embryogenesis, apoptosis (a unique morphological pattern of cell death) functions to remove abnormal or redundant cells in preimplantation embryos [16,17]. However, apoptotic processes do not occur prior to the blastocyst stage during normal mouse embryonic development [18], and induction of cell death during oocyte maturation and early stages of embryogenesis (i.e., via exposure to a teratogen) leads to developmental injury [14,[19][20][21][22]. While we have established that emodin promotes cell apoptosis and developmental injury in blastocyst-stage embryos [12], the influence of this compound on early-stage embryogenesis processes, such as oocyte maturation, fertilization, and sequential embryo development from zygotes, is currently unclear. Here, we focused on ascertaining whether emodin has a hazardous effect on oocyte development. Briefly, oocytes were incubated with emodin for 24 h and sequential development compared with that of oocytes under emodin-free conditions, with the aim of determining whether short-term exposure to the compound at the oocyte stage has a long-term injurious impact on embryo development. Our results clearly demonstrate that emodin exposure during the oocyte stage not only inhibits oocyte maturation but also promotes injurious effects on in vitro fertilization and embryonic development.

Results

Effects of Emodin on Oocyte Maturation Status, Fertilization Rate, and in Vitro Embryo Development

While emodin evidently induces apoptosis and developmental injury in mouse blastocysts [12], its effects on oocyte maturation have not been clarified to date. Oocyte nuclear maturation status was measured using eight independent experimental replicates, with ~250 oocytes per group. The number of oocytes that reached the metaphase II (MII) stage of maturation after in vitro maturation (IVM) ranged to about 97%. A lower maturation rate was observed in the emodin-treated oocyte group, which was dose-dependent ( Figure 1). Male pronucleus formation was assessed for the detection of fertilization. Our data showed that the ability of oocytes to be fertilized by fresh sperm was significantly decreased upon pretreatment with emodin, prior to IVM (Figure 1).

We further analyzed in vitro embryo development to the two-cell and blastocyst stages. Emodin pretreatment led to a significant decrease in cleavage of oocytes to the two-cell stage, indicative of an injurious effect (Figure 1). In addition, the number of embryos cleaved to form blastocysts in the emodin-treated groups was markedly lower than that in untreated control groups ( Figure 1).

Figure 1. Effects of emodin on mouse oocyte maturation and embryo development in vitro.

Oocytes were collected from 21 day-old mice, cultured for 24 h in in vitro maturation (IVM) medium containing emodin (5, 10 or 20 μM), fertilized in vitro, and transferred to in vitro culture (IVC) medium. Oocyte maturation, in vitro fertilization, cleavage and blastocyst development were analyzed. Values are presented as means ± SD of eight determinations. Data are based on 250-280 samples per group. *** p < 0.001 versus the untreated control group.

Effects of Emodin on Cell Proliferation and Apoptosis of Embryos during Oocyte Maturation in Vitro

Total blastocyst cell numbers were determined following emodin treatment during IVM of oocytes, with a view to establishing its effects on cell proliferation, assessed using differential staining followed by cell counting. Significantly lower blastocyst cell numbers were derived from emodin-pretreated oocytes, compared to control oocytes ( Figure 2A). Additionally, the numbers of inner cell mass (ICM) cells in blastocysts during IVM were decreased upon emodin pretreatment, while, in contrast, trophectoderm (TE) cell numbers were not affected ( Figure 2A).

Apoptosis of blastocysts derived from emodin-pretreated oocytes was additionally evaluated. TUNEL staining revealed a dose-dependent increase in apoptosis of blastocysts from the emodin-pretreated oocyte group ( Figure 2B). Further quantitative analysis showed a 7-to 10-fold increase in apoptotic blastocysts derived from emodin-pretreated oocytes, compared to the control group ( Figure 2C).

Developmental Potential of Blastocysts from Oocytes Treated with Emodin and in Vivo Effects of Emodin Intake on Oocyte Development

Embryos were transferred to 45 recipients per group (8 per horn). A total of 40 recipients were pregnant in at least one horn at day 18. The implantation ratio of blastocysts derived from the oocyte group treated with 20 μM emodin during IVM was ~27%, which was significantly lower than that observed for control blastocysts (~79%) ( Figure 3A). Embryos that implanted but failed to develop were subsequently resorbed in the uterus. The proportion of implanted embryos that failed to develop normally was significantly higher in the 20 μM emodin-treated group (~70%), compared to the control group (~36%). Moreover, the emodin-pretreated group displayed a higher resorption rate than the untreated control group ( Figure 3A). In terms of embryo survival rate (surviving fetuses), 64% of the control group survived to 14 days post-transfer (18-day fetuses), compared to only 30% of the 20 μM emodin-treated group ( Figure 3A). Interestingly, however, the placental weights of blastocysts derived from emodin-treated oocytes in IVM were not significantly different from those of the control group ( Figure 3B). Importantly, fetal weights were lower in the groups treated with 10-20 μM emodin, relative to the untreated control group. Furthermore, only 9% of fetuses in the 20 μM emodin-pretreated group weighed over 600 mg, whereas 37% of control fetuses exceeded this threshold, an important indicator of successful embryonic and fetal development ( Figure 3C). Our findings collectively indicate that exposure of oocytes to emodin during IVM reduces the potential of postimplantation development.

In view of the injurious effects of emodin on oocyte maturation and embryo development in vitro, we analyzed emodin activity in vivo via intake in an animal model. Female mice were fed a standard diet and drinking water supplemented with emodin (10-40 μM) for 4 days, or left untreated, prior to COC collection. Oocyte maturation status, fertilization rate, and in vitro embryo development were evaluated. Dietary emodin induced a significant decrease in oocyte maturation and fertilization, resulting in inhibition of embryonic development from the zygote to blastocyst stage ( Figure 3D).

Effects of Emodin Intake on the Developmental Potential of Blastocyst-Stage Embryos

To further determine the effects of emodin on embryo implantation and post-implantation development, we analyzed its in vivo activity via intake and transfer of blastocyst-stage embryos to the uterus horn using the embryo transfer assay in an animal model. Female mice were fed a standard diet and drinking water continuously supplemented with emodin (10-40 μM) or left untreated for 4 days before embryo transfer to the uterus during the experimental period. Embryos were transferred to 50 recipients per group (8 per horn). A total of 40 recipients were pregnant at day 18. Notably, the implantation ratio of blastocysts in the emodin intake group was significantly lower than that of control blastocysts ( Figure 4A). Moreover, the emodin intake group displayed a higher overall resorption rate than the emodin-free group ( Figure 4A). The embryo survival rate of the emodin intake group was additionally markedly lower than that of emodin-free group ( Figure 4A). However, the placental weights of blastocysts derived from both groups were comparable (data not shown). Finally, fetal weights were lower in the emodin intake (40 μM) than the untreated control group ( Figure 4B). Our results collectively demonstrate that exposure of embryos to emodin reduces the potential of implantation and postimplantation development.

Apoptotic Effects of Emodin on Oocyte Maturation Status, Fertilization Rate, and Embryo Development during IVM

To further clarify the regulatory mechanisms of emodin, oocytes were pretreated with 100 μM Ac-DEVD-cho, a caspase-3 specific inhibitor, with the aim of preventing emodin-triggered embryo cell apoptosis during IVM of oocytes. Pretreatment with the caspase-3 inhibitor effectively prevented apoptosis of blastocysts derived from the oocyte group pretreated with 20 μM emodin ( Figure 5A). Moreover, the caspase-3 inhibitor blocked emodin-triggered hazardous effects on oocyte maturation, fertilization rate, and sequential embryo development during IVM ( Figure 5B), and additionally rescued reduction in postimplantation development potential following embryo transfer, leading to improvements in embryo implantation rate, fetal survival rate, and fetal development status (determined on the basis of fetal weight) ( Figure 5C). These results strongly indicate that emodin regulation of oocyte development during IVM involves apoptotic processes.

Discussion

During the complex and precisely orchestrated process of oocyte maturation and early-stage embryonic development, chemical or physical injury can affect normal progression and lead to malformation or miscarriage of the embryo. Thus, it is crucial to ascertain the possible teratogenic effects of various agents on oocyte maturation and early-stage embryonic development. Emodin is a natural chemical compound in the root of rhubarb widely used in Chinese medicine. Recently, Wei and colleagues (2011) demonstrated that emodin not only induces apoptosis of cancer cells directly but also enhances the anticancer effects of gemcitabine in pancreatic cancer. Specifically, treatment with gemcitabine in combination with emodin efficiently inhibited tumor growth in mice inoculated with pancreatic tumor cells. The combination therapy induced a reduction in Akt and NF-κB activation and the Bcl-2/Bax ratio, and increase in caspase-9 and -3 activation, as well as cytochrome C release from the mitochondria to cytosol [23]. Thus, emodin appears to function as a chemopreventive and/or chemotherapeutic agent in different cancer types by decreasing cell viability, inhibiting cell proliferation and increasing apoptosis. A previous investigation by our group showed that co-incubation of embryos with emodin for 24 h triggers apoptosis in mouse blastocysts [12], in turn, suppressing cell viability. Measurement of embryo cell numbers of blastocysts using dual differential staining revealed emodin-induced cell loss and apoptosis in both ICM and TE cell populations [12]. In this report, we have examined the possible cytotoxic effects of emodin on oocyte maturation, fertilization, and sequential embryo development.

Oocyte maturation, fertilization, and embryonic development are complex processes during which chemical injury can lead to developmental problems or embryonic malformation. Previously, we reported that emodin induces apoptosis, impairment of blastocyst development from the morula and promotion of early-stage mouse blastocyst death [12]. In view of these findings, it is important to establish whether emodin has possible teratogenic effects. Here, we have shown for the first time that emodin inhibits mouse oocyte maturation, fertilization, and sequential embryonic development ( Figure 1). Importantly, the emodin-pretreated oocyte group displayed a significant decrease in cell number and increased apoptosis (Figure 2A,B). In our experiments, cumulus-oocyte complexes (COCs) were isolated from female hybrid ICR mice (21 days old) injected with 5 IU human chorionic gonadotropin (hCG), 44 h prior to oocyte collection. Our coworkers additionally collected COCs from 42 day-old ICR mice. Importantly, no differences were observed in emodin-induced hazardous effects on mouse oocyte maturation and sequent embryonic development between oocytes from 21 and 42 day-old female mice (data not shown). The results collectively indicate that emodin treatment at the oocyte stage triggers both oocyte maturation injury and abnormal apoptosis of cells at the blastocyst stage, an important step in embryo implantation.

The TE arises from the trophoblast at the blastocyst stage, and develops into a sphere of epithelial cells surrounding the ICM and blastocoel. These cells contribute to the placenta, and are required for mammalian conceptus development [24]. Reduction of cells from the TE and/or ICM lineage leads to suppressed implantation and lower embryonic viability [25,26]. ICM and total blastocyst cell numbers are positively correlated with embryonic development during the embryo transfer assay [27]. In our experiments, application of emodin during oocyte maturation had no effect on the TE cell number of blastocysts, but induced a dramatic decrease in ICM and total (TE plus ICM) cell numbers ( Figure 2). Our results imply that emodin treatment during IVM causes mortality and/or developmental delay in postimplantation mouse embryos via ICM cell death or decreased proliferation (Figures 2 and 3). Interestingly, blastocysts derived from emodin-treated oocytes appeared to undergo decreased implantation, and exhibited increased embryo resorption and lower fetal survival rate ( Figure 3A), but comparable placental weights to the control group. TE cells of embryos play important roles in implantation and placental development. Our data showed that emodin does not exert a hazardous effect on TE cells of blastocysts, and consequently has no effect on placental development. Based on these findings, we propose that the decrease in ICM cell number induced by emodin during oocyte maturation is the major injurious factor leading to inhibition of embryonic development.

Emodin-pretreated oocytes displayed significantly decreased fertilization rates and cleavage to two-cell and blastocyst stages, compared to untreated control groups (Figure 1), indicating that emodin induces loss of fertilization and sequent embryonic development. Moreover, in an embryo transfer study, mouse blastocyst stage embryos derived from emodin-pretreated oocytes displayed lower implantation and higher resorption rates than control blastocyst-derived untreated oocytes ( Figure 3A). Further experiments demonstrated significantly lower embryo implantation rates and fetal weights and higher resorption rate of blastocyst stage embryo transfer to mouse uterus in the emodin intake group, compared to those of the emodin-free control group ( Figure 4A,B). Emodin has been shown to inhibit proliferation and induce apoptosis with an IC 50 value of 20 μM in Jurkat cells [28]. Preliminary experiments by our group further revealed that emodin triggers mouse embryonic stem cell (ESC) apoptosis in a dose-dependent manner with an IC 50 value of 12 μM, as determined from the MTT assay after 24 h of exposure (data not shown). In addition, our initial HPLC results disclosed serum emodin levels of about 8.7 μM in mice drinking water supplemented with 40 μM emodin over 4 days (data not shown). Our data showed that 10-20 μM emodin induces significant cytotoxic effects, i.e., reduction in the rates of oocyte maturation, fertilization, and in vitro embryonic development. Accordingly, we propose that emodin suppresses oocyte maturation and in vitro fertilization and promotes early embryonic developmental injury at concentrations that may be attained via dietary intake. Clearly, emodin has potential hazardous effects on early-stage oocyte maturation and fertilization.

Emodin has been reported to trigger apoptosis of SCC-4 cells via mitochondria-dependent signaling pathways, and mitochondrial dysfunction as a result of Bcl-2 and Bax modulation, mitochondrial cytochrome c release and caspase activation [10]. Recent research in our laboratory demonstrated that pretreatment of cells with specific inhibitors against caspase-9 (Z-LEHD-FMK) and caspase-3 (Z-DEVD-FMK) effectively blocks apoptosis in mouse blastocysts [12]. In the current investigation, we showed that pretreatment of oocytes with emodin leads to decreased cell number, apoptosis, and delay in postimplantation development of blastocysts, compared to the control group. These injurious effects were prevented by pretreatment of oocytes with a caspase-3 inhibitor to suppress blastocyst apoptosis ( Figure 5). Treatment with the caspase-3 inhibitor additionally rescued emodin-induced reduction in postimplantation development potential following embryo transfer, leading to improvements in embryo implantation rate, fetal survival rate, and fetal development status. Our results indicate that emodin triggers improper cell apoptotic processes in early-stage embryos, leading to loss of embryo cell numbers and suppression of post-implantation development, further supporting its role as a teratogen through apoptosis induction in early-stage cells. Accordingly, we conclude that developmental injury by emodin occurs via induction of apoptosis processes in oocyte maturation and early-stage embryos.

Experimental Section

Chemicals and Reagents

Dulbecco's modified Eagle's medium (DMEM), emodin, and pregnant mare serum gonadotropin (PMSG) were obtained from Sigma (St. Louis, MO, USA). Human chorionic gonadotropin (hCG) was purchased from Serono (NV Organon, Oss, The Netherlands). TUNEL in situ cell death detection kits were acquired from Roche (Mannheim, Germany), and CMRL-1066 medium from Gibco Life Technologies (Grand Island, NY, USA). Acetyl Asp-Glu-Val-Asp aldehyde (Ac-DEVD-cho) was from Calbiochem.

COC Collection and in Vitro Maturation (IVM)

ICR mice were acquired from the National Laboratory Animal Center (Taiwan). This research was approved by the Animal Research Ethics Board of Chung Yuan Christian University (Taiwan). All animals received humane care following Principles of Laboratory Animal Care (National Institutes of Health publication 85-23, revised 1996 [29]). Mice were maintained on breeder chow (Harlan Teklad chow) with food and water available ad libitum. Housing was provided in standard 28 cm × 16 cm × 11 cm (height) polypropylene cages with wire-grid tops, and maintained under a 12 h day/12 h night regimen. Cumulus-oocyte complexes (COCs) were obtained according to a previous protocol [14]. Briefly, COCs were isolated from female hybrid ICR mice (21 days old) injected with 5 IU human chorionic gonadotrophin (hCG) 44 h prior to oocyte collection. COCs were collected in HEPES-buffered α minimum essential medium (MEM) (containing 50 μg/mL Streptomycin sulfate, 75 μg/mL Penicillin G, and 5% fetal bovine serum) by gently puncturing visible antral follicles present on the ovary surface. Germinal vesicle stage oocytes containing an intact vestment of cumulus cells were collected and pooled in at least 10 animals. For oocyte maturation, one drop (~100 μL) of buffer (αMEM supplemented with 50 μg/mL Streptomycin, 75 μg/mL Penicillin G, 5% FBS and 50 mIU/mL recombinant human FSH) containing 10 COCs was added under oil in 35 mm culture dishes. COC maturation was analyzed following treatment with or without various concentrations of emodin (5, 10 or 20 μM) for 24 h under an atmosphere of 5% O 2 , 6% CO 2 and balance of N 2 at 37 °C.

Maturation Status Assessment

After in vitro maturation (IVM), COCs of each group were treated with 50 U/mL ovine hyaluronidase and gently pipetted for the removal of all cumulus cells. Denuded oocytes were collected, and washed with fresh medium, followed by phosphate-buffered saline (PBS). Oocytes were fixed in ethanol:glacial acetic acid (1:3) for 48 h, and stained with 1% aceto-orcein solution. Nuclear structures were visualized using phase-contrast microscopy.

In Vivo Maturation

For obtaining in vivo matured oocytes, 21 day-old mice were injected with 5 IU equine chorionic gonadotrophin (eCG) and 5 IU hCG, 61 and 13 h prior to fertilization, respectively. Mature ova were collected from the oviduct into HEPES-buffered in MEM medium.

Effects of Emodin Intake on Oocyte Maturation in an Animal Model

The effects of emodin on oocytes were analyzed in 21 day-old ICR virgin albino mice. Female mice were randomly divided into two groups of 20 animals each, and administered a standard diet with or without 10-40 μM emodin in drinking water for 4 days. COCs were collected by pre-treatment with 5 IU human chorionic gonadotrophin (hCG) for 44 h prior to oocyte collection, and analyzed for oocyte maturation, in vitro fertilization, and embryonic development.

In Vitro Fertilization

For in vitro fertilization, ova were washed twice in bicarbonate-buffered α-MEM medium (containing 50 mg/mL Streptomycin, 75 mg/mL Penicillin G and 3 mg/mL fatty acid free bovine serum albumin), and fertilized in the same medium with fresh sperm (obtained from a CBAB6F1 male donor). After incubation with sperm for 4.5 h, eggs were washed three times in potassium simplex optimized medium (KSOM) without amino acids in the presence of L-alanyl-L-glutamine (1.0 mM). Next, eggs were placed in 20 mL drops of KSOM under oil, and cultured overnight. During cleavage to the 2-cell stage, embryos were transferred to a fresh drop of KSOM under oil, and cultured for another 72 h. All fertilization steps and embryo culture were additionally carried out under 5% O 2 , 6% CO 2 and balance of N 2 at 37 °C.

Fertilization Assessment

For the examination of fertilization, ova were incubated with sperm for 4.5 h, followed by 3 h of culture in fresh medium. Zygotes were assessed for the presence of the male pronucleus with orcein staining, as described previously [14].

Cell Proliferation

Cell proliferation was analyzed by dual differential staining, which facilitated the counting of cell numbers in inner cell mass (ICM) and trophectoderm (TE) [25,30,31]. Blastocysts were incubated with 0.4% pronase in M 2 -BSA medium (M 2 medium containing 0.1% bovine serum albumin) for the removal of zona pellucida. Denuded blastocysts were exposed to 1 mM trinitrobenzenesulfonic acid (TNBS) in BSA-free M 2 medium containing 0.1% polyvinylpyrrolidone (PVP) at 4 °C for 30 min, and washed with M 2 [32]. Blastocysts were further treated with 30 μg/mL anti-dinitrophenol-BSA complex antibody in M 2 -BSA at 37 °C for 30 min, followed by M 2 supplemented with 10% whole guinea pig serum as a source of complement, along with 20 μg/mL bisbenzimide and 10 μg/mL propidium iodide (PI) at 37 °C for 30 min. The immunolysed blastocysts were gently transferred to slides, and protected from light before observation. Under UV light, ICM cells (which take up bisbenzimidine but exclude PI) appeared blue, whereas TE cells (which take up both fluorochromes) appeared orange-red. Since multinucleated cells are not common in preimplantation embryos [33], the number of nuclei represent an accurate measurement of cell number.

TUNEL Assay of Blastocysts

For TUNEL staining, embryos were washed in emodin-free medium, fixed, permeabilized, and subjected to labeling using an in situ cell death detection kit (Roche Molecular Biochemicals, Mannheim, Germany), according to the manufacturer's protocol. Photographic images were obtained with a fluorescence microscope under bright-field illumination.

Blastocyst Development Following Embryo Transfer

To determine the ability of expanded blastocysts to implant and develop in vivo, embryos generated were transferred to recipient mice. ICR females (6-8 week-old, white skin) were mated with vasectomized males (C57BL/6J; black skin; National Laboratory Animal Center, Taiwan) to produce pseudopregnant dams as recipients for embryo transfer. To ensure that all fetuses in pseudopregnant mice were derived from embryo transfer (white color) and not fertilization by C57BL/6J (black color), we examined skin color at day 18 post-coitus. To assess the impact of emodin on postimplantation growth in vivo, COCs were exposed to 0-20 μM emodin for 24 h, followed by fertilization and in vitro maturation to the blastocyst stage. Subsequently, 8 untreated control embryos were transferred to the left uterine horn, and 8 emodin-treated embryos to the right uterine horn in day 4 pseudopregnant mice. Forty surrogate mice were analyzed and killed on day 18 post-coitus, and the frequency of implantation calculated as the number of implantation sites per number of embryos transferred. The incidence rates of resorbed and surviving fetuses were calculated as number of fetuses per number of implantations, respectively. The weights of the surviving fetuses and placenta were measured immediately after dissection.

Statistical Analysis

Data were analyzed using one-way ANOVA and t-tests, and presented as means ± SD. Data were considered statistically significant at p <0.05.

Conclusions

Here, we have shown for the first time that emodin exerts injurious effects on oocyte maturation, fertilization, and embryonic development, which clearly suggesting that emodin is a risk factor for normal embryonic development that may reduce oocyte maturation in infertile couples. Further studies are required to determine the possible teratogenic actions of emodin on human oocyte maturation and embryogenesis.

Conflicts of Interest

The authors declare no conflicts of interest.

Figure 2 .
2Effects of emodin on cell number and apoptosis in embryos during IVM of oocytes. Oocytes were cultured for 24 h in IVM medium containing emodin (5, 10 or 20 μM), fertilized in vitro, and transferred to in vitro culture (IVC) medium for in vitro development. (A) Cell numbers of total, trophectoderm (TE) lineages and inner cell mass (ICM) were counted in blastocysts. (B) Apoptotic cells were examined at the blastocyst stage using TUNEL staining, followed by light microscopy. Positive cells are depicted in black. (C) The mean number of apoptotic (TUNEL-positive) cells per blastocyst was calculated. Values are presented as means ± SD of six determinations. Data are based on at least 200 samples in each group. *** p < 0.001 versus the untreated control group.

Figure 3 .
3Effects of emodin treatment or dietary emodin intake on embryo development during oocyte IVM. Oocytes were cultured for 24 h in IVM medium containing emodin (5, 10 or 20 μM), fertilized in vitro, and transferred to in vitro culture medium for development. (A) Implantation, resorption and surviving fetuses were analyzed, as described in Materials and Methods. The implantation percentage represents the number of implantations per number of transferred embryos × 100. The percentage of resorption or surviving fetuses represents the number of resorptions or surviving fetuses per number of implantations × 100. (B) Placental weights of 40 recipient mice were measured. (C) Weight distribution of surviving fetuses at 14 days post-transfer (18-day fetuses). Surviving fetuses were obtained via embryo transfer of control and emodin-pretreated groups, as described in Materials and Methods (320 total blastocysts across 40 recipients). (D) Random female mice (21 days old) were fed a standard diet and drinking water supplemented with emodin (10-40 μM) for 5 days or left untreated. Oocytes were collected for in vitro maturation, in vitro fertilization, cleavage, and blastocyst development analyses. Data are based on at least 300 samples in each group. * p < 0.05, ** p < 0.01 and *** p < 0.001 versus the emodin-free group.

Figure 4 .Figure 5 .
45Effects of dietary emodin on embryo development in mouse blastocysts. Random female mice (21 days old) were fed a standard diet and drinking water continuously supplemented with emodin (10-40 μM) or left untreated for 4 days before embryo transfer to the uterus during the experimental period. (A) Implantation, resorption and surviving fetuses were analyzed, as described in Materials and Methods. The implantation percentage represents the number of implantations per number of transferred embryos × 100. The percentage of resorption or surviving fetuses represents the number of resorptions or surviving fetuses per number of implantations × 100. (B) Weight distribution of surviving fetuses at day 18 post-coitus. Surviving fetuses were obtained by embryo transfer of control and emodin intake groups, as described in Materials and Methods (320 total blastocysts across 40 recipients). * p < 0.05, ** p < 0.01 and *** p < 0.001 versus the emodin-free group. Effects of inhibition of apoptosis on embryo development in emodin-treated oocytes during IVM. Oocytes were collected from 21 day-old mice, cultured for 24 h in IVM medium alone or that containing 100 μM Ac-DEVD-cho and 20 μM emodin, fertilized in vitro, and transferred to in vitro culture (IVC) medium for in vitro development. (A) Apoptotic cells were examined at the blastocyst stage using TUNEL staining. (B) Oocyte maturation, in vitro fertilization, cleavage and blastocyst development rates were analyzed. (C) Implantation, resorption, and surviving fetuses were analyzed with the embryo transfer assay, as described forFigure 3. *** p < 0.001 versus the emodin-free group. # p < 0.001 versus the emodin-treated only group.

Figure 5 .
5Cont
© 2012 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).

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