Renato C Juac ¸aba Neto

Rossana M C Andrade
email: rossana@ufc.br

Reinaldo B Braga
email: reinaldo.braga@ifce.edu.br

Fabrice Theoleyre
email: theoleyre@unistra.fr

Carina T Oliveira
email: carina.oliveira@ifce.edu.br

Performance Issues with Routing in Multi-Channel Multi-Interface IEEE 802.11s Networks

Keywords: IEEE 802.11s, Multi-Interface, Routing, Performance Evaluation

Wireless Mesh Networks have the potential to serve as an all-wireless backbone. They represent a key building block of the new Internet of Things to collect all the traffic. However, IEEE 802.11 performs very poorly in multi-hop topologies leading to unfairness and a poor throughput. Thus, the standard IEEE 802.11s proposes to re-use the IEEE 802.11 medium access in a mesh multi-hop topology while using a routing protocol to create routes at the layer 2. IEEE 802.11s may exploit multichannel multi-interface wireless routers to increase the throughput. In particular, the routing protocol must select carefully the forwarders to limit inter-and intra-flow interference. In this paper, we propose to fully characterize the performance of IEEE 802.11s when using multi-interface nodes, and to isolate some key performance issues. We also propose some key mechanisms that must be implemented to solve these pathological cases and to improve the throughput.

I. INTRODUCTION

Wireless Mesh Networks (WMN) have attracted increasing attention in recent years because of their low-cost, ease of deployment, and robustness [START_REF] Campista | Routing Metrics and Protocols for Wireless Mesh Networks[END_REF]. WMN are composed of interconnected wireless routers, called mesh stations, that are dynamically self-organized, self-configured and self-healing without any centralized control [START_REF]IEEE Standard for Information Technology -Telecommunications and Information Exchange Between Systems -Local and Metropolitan Area Networks -Specific Requirements -Part 11: Wireless LAN Medium Access Control (MAC) and Physical Layer (PHY) Specifications -Amendment 10: Mesh Networking[END_REF]. In this way, they provide broadband wireless coverage to large areas without infrastructure requirements. WMN have been used in numerous application scenarios such as broadband home networking, community networks, and intelligent transportation systems.

The popularity of WMN has lead to the IEEE 802.11 standardization of a mesh networking solution for broadcast and unicast packet delivery over a multi-hop topology. The standard is called IEEE 802.11s Mesh Networking [START_REF]IEEE Standard for Information Technology -Telecommunications and Information Exchange Between Systems -Local and Metropolitan Area Networks -Specific Requirements -Part 11: Wireless LAN Medium Access Control (MAC) and Physical Layer (PHY) Specifications -Amendment 10: Mesh Networking[END_REF].

In the initial design of WMN, mesh stations were typically equipped with a single IEEE 802.11 wireless network interface card that operates over only a small portion of the available spectrum (a channel). However, it is well-known that the performance of IEEE 802.11 quickly degrades in multi-hop topologies [START_REF] Chaudet | Performance Issues with IEEE 802.11 in Ad Hoc Networking[END_REF]. In particular, a group of emitters may not sense the same medium activity because they are spatially far from each other. This asymmetrical view leads to unfairness. Even when we consider one single flow relayed over multiple hops (i.e., intra-flow interference), collisions impact very negatively the throughput [START_REF] Campista | Routing Metrics and Protocols for Wireless Mesh Networks[END_REF].

One way of improving the performance is to use multiple orthogonal channels so that different mesh stations may simultaneously operate on different channels. For instance, IEEE 802.11a provides 12 orthogonal channels in the 5 GHz spectrum [START_REF] Crichigno | Protocols and Architectures for Channel Assignment in Wireless Mesh Metworks[END_REF]. The main goal of multi-channel singleinterface networks is to distribute the load across the available orthogonal channels. Consequently, while transmissions are multiplexed over different channels, interference and channel congestion problems are minimized [START_REF] Li | How to Effectively Use Multiple Channels in Wireless Mesh Networks[END_REF]. However, the solution has to be carefully designed to avoid the deafness problem [START_REF] Crichigno | Protocols and Architectures for Channel Assignment in Wireless Mesh Metworks[END_REF]: the emitter must know a priori the channel used by the receiver when transmitting a packet.

To fully take advantage of multiple orthogonal channels, nodes may have multiple interfaces so that different mesh stations may operate on different channels at the same time. A pair of neighboring stations may even use several interfaces to increase the throughput. In other words, full-duplex operation is possible at each node. Equipping mesh stations with multiple interfaces then leads to efficient spectrum utilization and increases the bandwidth available to nodes in the network. Such networks are often designated as Multi-Channel Multi-Interface (MCMI) WMN [START_REF] Si | An Overview of Channel Assignment Methods for Multi-Radio Multi-Channel Wireless Mesh Networks[END_REF].

Although there has been significant work in MCMI WMN [START_REF] Draves | Routing in Multi-Radio, Multi-Hop Wireless Mesh Networks[END_REF], [START_REF] Pirzada | Performance Analysis of Multi-Radio AODV in Hybrid Wireless Mesh Networks[END_REF], [START_REF] De Oliveira | Connectivity in Multi-Channel Multi-Interface Wireless Mesh Networks[END_REF], understanding finely the limits of IEEE 802.11s is still an open problem. We propose here to focus on the performance issues of the Hybrid Wireless Mesh Protocol (HWMP) [START_REF]IEEE Standard for Information Technology -Telecommunications and Information Exchange Between Systems -Local and Metropolitan Area Networks -Specific Requirements -Part 11: Wireless LAN Medium Access Control (MAC) and Physical Layer (PHY) Specifications -Amendment 10: Mesh Networking[END_REF], which is the routing protocol recommended by the IEEE 802.11s standard. To the best of our knowledge, the performance evaluation of HWMP in MCMI WMN has not been addressed prior to this work. In fact, there is only few studies on the performance evaluation of HWMP in singlechannel single-interface WMN [START_REF] Bari | Performance study of Hybrid Wireless Mesh Protocol (HWMP) for IEEE 802.11s WLAN mesh networks[END_REF], [START_REF] Guesmia | Performance Evaluation of the HWMP Proactive Tree Mode for IEEE 802.11s based Wireless Mesh Networks[END_REF].

In this paper, we thoroughly evaluated the behavior of IEEE 802.11s in multi-hop topologies. In particular, we highlighted the limits of HWMP to deal with multi-interface nodes: the replication of control messages leads to a misbehavior, impacting negatively the performance.

Our contribution is threefold:

• we thoroughly evaluate the behavior of IEEE 802.11s with multi-interface nodes;

• we provide a summary of the different performance issues extracted from simulations;

• we present a set of mechanisms which should be implemented to solve these performance issues.

II. IEEE 802.11S

In this section, we present the most important concepts of IEEE 802.11s [START_REF]IEEE Standard for Information Technology -Telecommunications and Information Exchange Between Systems -Local and Metropolitan Area Networks -Specific Requirements -Part 11: Wireless LAN Medium Access Control (MAC) and Physical Layer (PHY) Specifications -Amendment 10: Mesh Networking[END_REF], which is the IEEE 802.11 (Wi-Fi) amendment for WLAN mesh networks.

A. Mesh Network Architecture

The architecture of a WMN consists of a backbone of autonomous nodes that provides large coverage, connectivity and robustness to the network. Figure 1 -Mesh Stations (Mesh STA) are wireless routers that implement mesh facilities such as topology construction, path selection and data forwarding. They can establish mesh peering with multiple neighbor Mesh STA, but they do not offer support to STA. They are usually stationary and, consequently, they can be permanently power-supplied and benefit from resources such as memory, energy, and computation power.

-Mesh Gates are Mesh STA with Access Point (AP) functionalities that enable the integration of the WMN with STA as well as with different wired and wireless networks like Ethernet, cellular, Wi-Fi, and sensors.

-Mesh Portals are Mesh STA integrated with gateway functions to interoperate with external networks such as the Internet.

It can be seen that IEEE 802.11s mesh networks can be incrementally deployed, one node at a time, according to the demand. Through this integrated WMN, the users of wired or wireless networks (e.g., Ethernet, cellular, Wi-Fi) can benefit from otherwise impossible services of these networks.

In this work, we focus on the issues of Mesh STA and Mesh Portal, leaving the Internet and the last hop issues (e.g., stations mobility, communication between Mesh Gates and stations, etc) to other areas of research. Moreover, we focus on IEEE 802.11s-based backbone wireless mesh networks.

B. Hybrid Wireless Mesh Protocol (HWMP)

HWMP is the path selection protocol recommended by IEEE 802.11s [START_REF] Carrano | IEEE 802.11s Multihop MAC: A Tutorial[END_REF]. In particular, it uses MAC addresses to route the packets based on an IEEE 802.11 link metric. It combines the flexibility of a reactive path selection with a proactive tree-based approach. These two modes of operation provide different levels of functionalities.

The HWMP On-demand Mode allows mesh stations to communicate with each other using peer-to-peer paths (i.e. Any2Any traffic). This mode is based upon the Ad hoc On-Demand Distance Vector (AODV) protocol [START_REF] Perkins | Ad hoc On-Demand Distance Vector (AODV) Routing[END_REF]. Specifically, a source node that does not have a valid path to the destination can use the Path REQuest (PREQ) broadcasting mechanism to discover link metric information to destination. Upon receiving a PREQ, the destination sends a unicast Path REPly (PREP) message back to the source. Intermediate nodes which have forwarded PREQs and PREPs messages create or update their own routing table to relay data packets between the source and the destination. The HWMP Tree-based Proactive Mode maintains a tree path from the Mesh Portal to all other nodes. Indeed, forwarding the packets to the Mesh Portal is common when interconnecting the mesh network to the Internet. The IEEE 802.11s standard defines two mechanisms for proactively disseminating path selection information for reaching the Mesh Portal.

In the first mechanism, the root announces its presence by broadcasting a proactive PREQ. Each receiving Mesh STA updates the PREQ (TTL and path metric) and rebroadcasts it to its neighbors. As the proactive PREQs are disseminated in the entire network, a Mesh STA may receive multiple copies of a proactive PREQ. Thus, a Mesh STA updates its current path to the Mesh Portal if and only if: (i) the new PREQ contains a greater HWMP sequence number, or (ii) the HWMP sequence number is the same as the current path, but the new PREQ offers a better metric than the current path. If the proactive PREQ is sent with the Proactive PREP subfield set to 0 (respectively 1), every Mesh STA may (respectively must) respond to the received PREQ by sending a PREP back to the Mesh Portal. The overhead may consequently be significant.

As an alternative, the root may flood a Root ANNouncement (RANN). Only the Mesh STA that aims at constructing a route may reply with a PREQ forwarded to the neighbor from which the RANN is received. The root then reply with a PREP to create explicitly the route. This mode is particularly relevant when only a small subset of the Mesh STA may establish a route with the Mesh Portal.

Path ERRor (PERR) messages can be used by any of the available HWMP modes for announcing one or more unreachable destinations. The announcement is sent to all traffic sources that have an active path to the destination(s). Finally, the PERR messages reach the corresponding sources, which initiate a new path discovery cycle in order to find an alternative path.

In this work, we focus on the HWMP On-demand Mode and the first mechanism of HWMP Tree-based Proactive Mode. We have chosen this proactive mode because we evaluate the impact of HWMP in scenarios where different number of Mesh STA may establish a path with the Mesh Portal. We highlight that on-demand and tree-based proactive modes are not exclusive. In fact, they are used concurrently, since the proactive mode is an extension of the on-demand mode.

In this context, we show that HWMP is unsuitable for MCMI WMN because it does not take into consideration the high overhead incurred by the replication of control messages on multiple interfaces. Specifically, when multiple interfaces are used, control messages such as PREQ and PREP are retransmitted by every interface. As a result, the amount of transmitted control messages increases with the number of interfaces, which causes severe performance degradation due to heavy contention and collisions. We discuss the impact of this misbehavior of the HWMP in Section IV and present possible solutions in Section V.

C. Airtime Link Metric (ALM)

ALM is the default link metric of IEEE 802.11s, which is computed for each pairwise link within the mesh network. The path selection protocol accumulates all the link metric values included in the selected multi-hop path to obtain the overall cost of the path. This metric estimates the amount of channel bandwidth consumed when transmitting a frame over a link l. It is defined in terms of overhead, data rate, and transmission errors:

ALM l = O ca + O p + S r * 1 1-e f ,
where O ca is the channel access overhead, O p is the protocol overhead, and S is the number of bits in the test frame. The parameter r is the data rate in megabits per second. The frame error rate e f is the probability that when a frame of standard size S is transmitted at the current transmission bit rate r, the frame is corrupted due to transmission error. A closer look at the ALM reveals that this metric is similar to the well-know Expected Transmission Time (ETT) metric [START_REF] Draves | Routing in Multi-Radio, Multi-Hop Wireless Mesh Networks[END_REF].

D. Channel and Interface Assignment

Although the importance of the channel and interface assignment in WMN [START_REF] De Oliveira | Connectivity in Multi-Channel Multi-Interface Wireless Mesh Networks[END_REF], the current IEEE 802.11s mesh standard lets this part unspecified. The previous drafts advocated the use of a common assignment to preserve connectivity [START_REF] Camp | The IEEE 802.11s Extended Service Set Mesh Networking Standard[END_REF]. In this way, the same channel is used for all i th interfaces. Fig. 2 shows an example of the common channel assignment. Each node has two interfaces. Four orthogonal channels are available. Bold lines represent links. The channel assigned to each interface is shown inside the square brackets nearby the nodes. Note that any two neighbors have always multiple independent links to communicate with each other, resulting in a stable network topology without partitions.

III. SIMULATION SETUP

We have evaluated HWMP in MCMI WMN through extensive simulations in NS-3.19. This simulator was chosen because it already contains protocols and components of IEEE 802.11s implemented as a module of its own. We have developed extensions to detail the HWMP information captured by each interface. Besides, new features were developed to restrict simulation to fully connected scenarios (i.e., there is always a path between any two nodes). The simulation characteristics are summarized in Table I.

We have performed the evaluation in different disk scenarios with 1 Mesh Portal and 49 static Mesh STA. To represent a typical WMN traffic pattern, several flows are originated from different Mesh STA (transmitters) to the Mesh Portal. Each node has on average six neighbors in the disk scenario. Mesh STA are equipped with IEEE 802.11a wireless interfaces and therefore 12 orthogonal channels are available. On each node, interface i is assigned to the orthogonal channel C i .

Each data point in the graphical results is computed as being a result of 64 different simulations. For each simulation Fig. 2 run, a node is randomly designated as the Mesh Portal to receive the Poisson traffic generated by the transmitters. The network traffic is equally distributed among the flows.

The variations in the obtained results mainly occur due to (1) the randomness of the topology, (2) the gateway and transmitters positions, and (3) the flows components. An initial period of 10 seconds of the simulation is undertaken before the flows start ("warm-up period"). We have considered a total simulation time of 80 seconds and a packet size of 1024 bytes.

IV. RESULTS

In this section, we present the results of the performance evaluation of HWMP. We focus on the most interesting results that show the misbehavior of HWMP in MCMI WMN. We present the results with confidence intervals of 95%.

A. Impact of Traffic Load

We first investigate the impact of the traffic load on the performance of HWMP, as shown in Fig. 3. The objective is to understand the behavior of the routing protocol under different number of interfaces and network traffic.

As illustrated in Fig. 3(a), 3(b) and 3(c), in scenarios where Mesh STA have a larger number of interfaces the network has a better performance in terms of delivery rate, aggregate throughput and average end-to-end delay, respectively. This behavior is expected as the increase of interfaces also increases the number of paths using different channels. Consequently, inter-flow and intra-flow interference can be reduced. Also, note that the increase of the traffic load tends to reduce the gap between the curves in terms of delivery rate and delay. In part, this is because Mesh STA are more susceptible to suffer from quickly buffer overflow with the increase of the traffic load, especially when they are close to the Mesh Portal, which creates an unfair situation for the traffic originated by the Mesh STA close to the Mesh Portal. In this specific case, we are convinced that a solution to address this issue is to give channel and interface assignment priority to links closer to the Mesh Portal based on the number of available channels and interfaces per node (see Section V-4).

When multiple interfaces are used, HWMP broadcasts many control messages such as PREQ (Fig. 3(d)) and PREP (Fig. 3(e)) on each of its interfaces. As a result, the amount of transmitted control messages increases with the number of interfaces, which causes severe performance degradation due to heavy contention and collisions. We refer to these problems associated with flooding as the broadcast storm problem [START_REF] De Oliveira | Connectivity in Multi-Channel Multi-Interface Wireless Mesh Networks[END_REF]. As a result, a considerable part of transmission opportunities is used to send control packets instead of data packets.

Besides, Fig. 3(d) presents a clear dominance of PREQ over the other HWMP control packets: PREP (Fig. 3(e)) and PERR (Fig. 3(f)). Another important issue seen on all scenarios is the dominance of PREQ forwarded (Fig. 3(g)) over initiated PREQ (Fig. 3(h)) and initiated proactive PREQ (Fig. 3(i)). Similarly, the number of PREP forwarded (Fig. 3(j)) is superior to the number of initiated PREP (Fig. 3(k)).

B. Impact of the Number of Flows

Next, we investigate the impact of the number of flows (cf. Fig. 4). The objective is to evaluate the behavior of the MCMI WMN when the traffic is distributed through the network.

Similarly to the previous results, we observe that networks composed of Mesh STA with more interfaces perform better concerning the delivery rate (Fig. 4(a)), the aggregate throughput (Fig. 4(b)) and the end-to-end delay (Fig. 4(c)). Also, note that the delay slightly increases with the number of flows whatever the number of interfaces is. The main reason is that HWMP does not perform load-balancing and then does not seek to distribute the various flows through different paths in order to prevent channel congestions (i.e., bottleneck links). More specifically, HWMP tends to forward new flows through already established paths, which means that all flows are led to the same congested areas. Consequently, it results in poor quality paths that spend more time to transmit data because of the high level of interference and contention. In this context, we consider that HWMP performance can be improved if dynamic characteristics of the network are considered to perform routing, such as the link quality and the traffic load (see Sections V-1 and V-3).

Figures 4(d),4(e) and 4(f) show that the amount of transmitted control messages increases considerably with the number of flows. As the previous results, the number of transmitted PREQ is superior to other HWMP control packets (PREP and PERR). Besides, it is clear the dominance of PREQ forwarded (Fig. 4(g)) over initiated PREQ (Fig. 4(h)) and initiated proactive PREQ (Fig. 4(i)).

V. POSSIBLE SOLUTIONS

The problems highlighted in the previous section are mainly due to the exploitation of multiple interfaces. HWMP was not designed originally to create a route through different interfaces. We present here four mechanisms that may improve the IEEE 802.11s efficiency.

1) Metrics Quantification: Many routing metrics are not able to depict all the factors that impact the performance of MCMI WMN: the exhaustive identification of all these factors is not trivial. Among the well-known factors, the inter-and intra-flow interference, the traffic load and the link quality can be regarded as the most recent trend to depict the behavioral aspects of the network. For example, interference can be measured to favor paths with channel diversity, while routing solutions aware of the traffic load may prevent congested areas. Also, link quality measures may be used to avoid links with high packet losses. However, most routing metrics do not consider the combination of these factors when judging the goodness of a path. Besides, they use different information gathering methods to obtain the measurements. In consequence, each metric has an appropriate use case, which generally is not extensible to different network scenarios (e.g., mesh networks with variations of traffic and interference).

2) Route Discovery through Multiple Interfaces: When forwarding a PREQ, a node should select the channel to use based on the channels which will be selected further in the route: channel selection is not independent among the different hops. In particular, the same channel should not be used by two interfering hops. Consequently, a node cannot filter the PREQ received through one interface just because the same PREQ has already been relayed through another interface. Thus, PREQ should be independently considered through all the interfaces. However, multiple radio links between each pair of Mesh STA may be exploited to reduce the complexity of HWMP. We are convinced that each node should be able to piggyback in its PREQ the information of all the links with the previous hop. Else, the interdependency would create a PREQ storm: to explore all the possible combinations of channels, each node would have to forward several PREQ per interface. Since the overhead would grow with the path length, an additional mechanism should filter this information, selecting the best choice for the beginning of the path.

3) Bandwidth Estimation: Different flows may interfere with each other locally. Since IEEE 802.11 does not permit to share fairly and linearly the bandwidth among the competing flows, we should be able to estimate the available residual bandwidth.

We may exploit the mechanism described in [START_REF] Nguyen | Retransmission-based Available Bandwidth Estimation in IEEE 802.11based Multihop Wireless Networks[END_REF] to modify the Airtime Link Metric. A model of the medium access should translate the number of retransmissions into a channel occupancy. Coupled with a call admission, we should be able to select the path minimizing the ALM while respecting the requested bandwidth for the flow.

4) Channel and Interface Assignment:

A key challenge in MCMI WMN consists in achieving an effective channel and interface assignment (mapping) to decide "when to switch interfaces" as well as "which channel to assign". There is an inherent trade-off between connectivity and interference [START_REF] De Oliveira | Connectivity in Multi-Channel Multi-Interface Wireless Mesh Networks[END_REF]. The more interfaces are assigned to the same channels, the better connectivity. However, interference and contention have oppositely a negative impact on network capacity.

In this way, we are convinced that a promising approach for improvement of HWMP consists in combining routing with assignment according to the network conditions (e.g., number of available channels and interfaces, traffic behavior, neighborhood, interference, QoS requirements, etc). Surprisingly, to the best of our knowledge, the impact of channel and interface assignment strategies on the HWMP performance has never been studied, although it is a major component of network operation.

VI. CONCLUSIONS

In this paper, we summarize the performance issues that arise with HWMP when used in MCMI WMN. The results and discussions provide guidelines for academic researchers, research engineers, and practitioners in planning MCMI IEEE 802.11s, as well as to improve HWMP.

The contributions presented in this paper bring up interesting perspectives for future research. For instance, we plan to evaluate the HWMP performance when different channel and interface assignment strategies are adopted. Also, we plan the development of a cross-layer routing solution for MCMI WMN with a precise characterization of the wireless links in order to increase the overall network performance.

VII. ACKNOWLEDGEMENTS

  gives an example of WMN, where dashed and solid lines indicate wireless and wired links, respectively. According to the IEEE 802.11s, nodes fall into one of the following categories: -Stations (STA) are the basic entities in an IEEE 802.11 network. They correspond to nodes that request services but do not forward frames, nor participate in path discovery mechanisms. Laptops, cell phones, smartphones, and tablets are examples of STA.

Fig. 1 .

 1 Fig. 1. IEEE 802.11s Mesh Network Architecture.

Fig. 3 .

 3 Fig. 3. Impact of Traffic Load -50 nodes (1 Mesh Portal), 6 flows.

Fig. 4 .

 4 Fig. 4. Impact of the Number of Flows -50 nodes (1 Mesh Portal), 500 pps.

  . Example of Common Channel Assignment in MCMI WMN.

	Parameter	Default Value
	Simulation Time	80s
	Number of Runs	64 for each data point
	Network Size	50 nodes-1 gateway
	HWMP Routing	On-demand Mode and Tree-based Proactive Mode
	Assignment Strategy	Common channel assignment
	Topology Type	Disk (radius = 300)
	Traffic Type	Poisson
	Traffic Load	500 packets per second (pps)
	Number of Flows	6
	Packet Size (bytes)	1024
	PHY specification	IEEE 802.11a

TABLE I

 I

	.	SIMULATION PARAMETERS.

The authors thank CNPq, which provided financial support through the Notice MCT/CNPq 14/2013-Universal n o 486287/2013-0 (Project Cross-Layer Optimization in Multi-Interface Wireless Mesh Networks). The authors would also like to thank CENAPAD-UFC for computational facilities.


Cai-Xia C.-X Yang

Zichuan Z Liu

Renaud R Fleurot

Pierre Adenot

Véronique Duranthon

Xavier Vignon

Qi Q Zhou

Jean Paul

Jean-Paul P Renard

Nathalie Beaujean
email: nathalie.beaujean@jouy.inra.fr

Cai-Xia Yang

Ve ´ronique Duranthon

C-X Yang

Heterochromatin reprogramming in rabbit embryos after fertilization, intra-, and inter-species SCNT correlates with preimplantation development

come   L'archive ouverte pluridisciplinaire

Introduction

Somatic cell nuclear transfer (SCNT) has obtained success in many kinds of mammals. However, the low efficiency associated with development both in intraand inter-species SCNT is still the major obstacle to its widespread application [START_REF] Dominko | Bovine oocyte cytoplasm supports development of embryos produced by nuclear transfer of somatic cell nuclei from various mammalian species[END_REF][START_REF] Sansinena | Nuclear transfer embryos from different equine cell lines as donor karyoplasts using the bovine oocyte as recipient cytoplast[END_REF][START_REF] Arat | Gene expression and in vitro development of inter-species nuclear transfer embryos[END_REF][START_REF] Chang | Blastocyst formation, karyotype, and mitochondrial DNA of interspecies Heterochromatin reprogramming in rabbit embryo embryos derived from nuclear transfer of human cord fibroblasts into enucleated bovine oocytes[END_REF], 2004[START_REF] Ikumi | Interspecies somatic cell nuclear transfer for in vitro production of Antarctic minke whale (Balaenoptera bonaerensis) embryos[END_REF][START_REF] Lorthongpanich | Development of interspecies cloned monkey embryos reconstructed with bovine enucleated oocytes[END_REF][START_REF] Song | Nucleologenesis and embryonic genome activation are defective in interspecies cloned embryos between bovine ooplasm and rhesus monkey somatic cells[END_REF][START_REF] Hong | Production of transgenic canine embryos using interspecies somatic cell nuclear transfer[END_REF][START_REF] Kwon | Blastocysts derived from adult fibroblasts of a rhesus monkey (Macaca mulatta) using interspecies somatic cell nuclear transfer[END_REF]. SCNT involves fundamental questions about differentiation and its reversibility. After being introduced into recipient oocytes, differentiated somatic nuclei must be completely remodeled to initiate another round of embryonic development. Although the abnormal chromatin remodeling has been implicated as primary reasons for the low efficiency of the SCNT procedure [START_REF] Arat | Gene expression and in vitro development of inter-species nuclear transfer embryos[END_REF][START_REF] Park | Bovine oocyte cytoplasm supports nuclear remodeling but not reprogramming of murine fibroblast cells[END_REF][START_REF] Wang | Bovine ooplasm partially remodels primate somatic nuclei following somatic cell nuclear transfer[END_REF], the associated mechanisms have not been completely elucidated. In order to obtain a deeper insight into these events, we focused on centromeric and pericentric heterochromatin, known to form higher-order chromatin structures within nuclei in the interphase.

Indeed, numerous studies have addressed the nuclear organization of centromeric and pericentric chromosome regions in somatic cycling cells [START_REF] Haaf | Centromeric association and non-random distribution of centromeres in human tumor cells[END_REF][START_REF] Haaf | Chromosome topology in mammalian interphase nuclei[END_REF][START_REF] Weierich | Three-dimensional arrangements of centromeres and telomeres in nuclei of human and murine lymphocytes[END_REF][START_REF] Solovei | Differences in centromere positioning of cycling and postmitotic human cell types[END_REF]). In mouse interphase somatic nuclei, pericentric heterochromatin from several different chromosomes tends to aggregate into clusters called chromocenters, which are surrounded by the centromeres from the chromosomes involved in their formation [START_REF] Hsu | Arrangement of centromeres in mouse cells[END_REF][START_REF] Haaf | Chromosome topology in mammalian interphase nuclei[END_REF][START_REF] Cerda | Organisation of complex nuclear domains in somatic mouse cells[END_REF][START_REF] Guenatri | Mouse centric and pericentric satellite repeats form distinct functional heterochromatin[END_REF]. Such chromocenters can also be observed in bovine fibroblasts (Martin et al. 2006a[START_REF] Pichugin | Dynamics of constitutive heterochromatin: two contrasted kinetics of genome restructuring in early cloned bovine embryos[END_REF], but not in human or rabbit cells. In human cells, pericentromeric heterochromatin is predominantly found in specific nuclear domains associated with centromeric regions called pericentromeres [START_REF] Hayakawa | Cell cycle behavior of human HP1 subtypes: distinct molecular domains of HP1 are required for their centromeric localization during interphase and metaphase[END_REF]. However, these domains do not seem to cluster, which may be because of the varieties of repeat sequences comprised in human pericentromeric regions [START_REF] Lee | Human centromeric DNAs[END_REF]. Similarly, computational approaches in rabbit 8-cell and blastocyst nuclei revealed that centromeres form regularly spaced patterns, thereby suggesting the presence of isolated pericentromeres in this species as in human cells [START_REF] Andrey | Statistical analysis of 3D images detects regular spatial distributions of centromeres and chromocenters in animal and plant nuclei[END_REF].

In mouse cells, chromocenters represent the major heterochromatin regions and play important roles in the regulation of gene expression [START_REF] Brown | Dynamic repositioning of genes in the nucleus of lymphocytes preparing for cell division[END_REF][START_REF] Francastel | A functional enhancer suppresses silencing of a transgene and prevents its localization close to centrometric heterochromatin[END_REF], Schu ¨beler et al. 2000[START_REF] Skok | Nonequivalent nuclear location of immunoglobulin alleles in B lymphocytes[END_REF][START_REF] Guenatri | Mouse centric and pericentric satellite repeats form distinct functional heterochromatin[END_REF]. Remarkably, in mouse embryos, we observed that these regions adopt a peculiar 'cartwheel' organization after fertilization characterized by the positioning of pericentric regions to the periphery of the nucleolus precursor bodies (NPBs; Martin et al. 2006a). This configuration has been suggested to maintain transcriptional silencing during parental genome maturation [START_REF] Probst | Structural differences in centromeric heterochromatin are spatially reconciled on fertilisation in the mouse zygote[END_REF]). Reorganization of these heterochromatin regions into 'chromocenters' occurs during the second cell stage, concomitantly with the major phase of major embryonic genome activation (EGA) in this species (Martin et al. 2006a[START_REF] Probst | Structural differences in centromeric heterochromatin are spatially reconciled on fertilisation in the mouse zygote[END_REF]. Remarkably, interference with pericentromeric transcription disturbs chromocenter formation and results in developmental arrest [START_REF] Probst | A strand-specific burst in transcription of pericentric satellites is required for chromocenter formation and early mouse development[END_REF][START_REF] Santenard | Heterochromatin formation in the mouse embryo requires critical residues of the histone variant H3.3[END_REF].

In bovine, we observed a switch of pericentric heterochromatin distribution in fertilized embryos only at the 8-cell stage, at the time of EGA in this species (Martin et al. 2006a[START_REF] Pichugin | Dynamics of constitutive heterochromatin: two contrasted kinetics of genome restructuring in early cloned bovine embryos[END_REF]. Similarly, in rabbit, major EGA also takes place at the 8-to 16-cell stage [START_REF] Manes | The participation of the embryonic genome during early cleavage in the rabbit[END_REF]. However, as rabbit 8-cell and blastocyst nuclei do not present chromocenter-like structures but only isolated pericentromeres [START_REF] Andrey | Statistical analysis of 3D images detects regular spatial distributions of centromeres and chromocenters in animal and plant nuclei[END_REF], we questioned whether early rabbit embryos would present an embryonic-specific nuclear architecture (as in the two other species studied so far) and whether pericentromeres would form only at EGA. We therefore tracked HP1b and CENP, two wellcharacterized protein markers of the pericentric and centromeric compartments respectively.

Interestingly, in mouse and bovine cloned embryos obtained by nuclear transfer (NT), pericentric heterochromatin is rapidly reorganized to adopt an embryonic-like nuclear architecture (Martin et al. 2006b[START_REF] Merico | Epigenomic differentiation in mouse preimplantation nuclei of biparental, parthenote and cloned embryos[END_REF][START_REF] Maalouf | Trichostatin A treatment of cloned mouse embryos improves constitutive heterochromatin remodeling as well as developmental potential to term[END_REF][START_REF] Pichugin | Dynamics of constitutive heterochromatin: two contrasted kinetics of genome restructuring in early cloned bovine embryos[END_REF]. However, reprogramming after NT is not perfect and aberrations are quite frequent: remains of somatic-like chromocenters, for example, are often observed (Martin et al. 2006b[START_REF] Maalouf | Trichostatin A treatment of cloned mouse embryos improves constitutive heterochromatin remodeling as well as developmental potential to term[END_REF][START_REF] Pichugin | Dynamics of constitutive heterochromatin: two contrasted kinetics of genome restructuring in early cloned bovine embryos[END_REF]. In bovine NT embryos obtained from fibroblasts, we could also observe a premature compaction of pericentric heterochromatin at the 2-to 4-cell stages, i.e. much earlier than in the fertilized embryos [START_REF] Pichugin | Dynamics of constitutive heterochromatin: two contrasted kinetics of genome restructuring in early cloned bovine embryos[END_REF].

We therefore also analyzed the dynamics of HP1b and CENP in early embryos reconstructed by intra-species SCNT. Then, we tested the reprogramming ability of rabbit oocytes on bovine donor cells characterized by the presence of large chromocenters (inter-species SCNT). Finally, we addressed whether heterochromatin remodeling was associated with the onset of transcriptional activity in these embryos.

Results

Nuclear distribution of HP1b and CENP differs in mouse, rabbit, and bovine somatic cells

To clarify centromeric and pericentric heterochromatin organization in rabbit and bovine fibroblasts (used as donor cells in the present study), we first analyzed the nuclear distributions of HP1b and centromere-specific CENP proteins in these two kinds of primary cultured fibroblasts in comparison with mouse NIH-3T3 established cell line. As expected, in mouse interphase nuclei, HP1b-enriched pericentric heterochromatin was clustered, forming chromocenters, and each chromocenter was surrounded by several centromeres (Fig. 1, upper panel). We observed a similar organization in bovine fibroblasts (Fig. 1, lower panel); conversely, no chromocenters were observed in rabbit fibroblasts (Fig. 1, middle panel). In rabbit interphase nuclei, HP1b was diffusely distributed in the nucleoplasm with discrete accumulations that were quite small compared with mouse and bovine chromocenters. These HP1b foci were often associated with single centromeres (1:1 ratio), and occasionally single centromeres without HP1b association were observed. Furthermore, 40.2G2.9 CENP dots on average were observed in interphase rabbit fibroblasts (nZ10), which correlated with the number of chromosomes in rabbits (44). This distribution pattern of pericentric heterochromatin and centromeres in rabbit cells is clearly similar to the pericentromeres observed in human cells [START_REF] Hayakawa | Cell cycle behavior of human HP1 subtypes: distinct molecular domains of HP1 are required for their centromeric localization during interphase and metaphase[END_REF].

Compaction of pericentric and centromeric compartments in rabbit fertilized embryos and parthenotes is associated with major EGA Next, we assessed the spatio-temporal distribution of HP1b and centromeres in early rabbit embryos obtained after natural fertilization to investigate when the somatic-like organization would be established during preimplantation development (Fig. 2). In 1-cell rabbit embryos, the female pronucleus (fPN) is usually smaller than the male one (mPN), whatever the time point analyzed (Reis [START_REF] Silva | Dynamics of DNA methylation levels in maternal and paternal rabbit genomes after fertilization[END_REF]. At 19 hphCG (w7 hpi), we observed that HP1b was diffused in the nucleoplasm of both mPN and fPN, with no enrichment at the peripheries of any NPB (Flechon & Kopecny 1998). Only a more brightly stained nucleoplasmic region adjoining the NPB was observed in the fPN (42 of 50 embryos; Fig. 2). After quantification, we found around 40 bright CENP dots in both PNs (nZ27), of which 27.5% were associated with NPBs, while the other ones were dispersed in the nucleoplasm (Table 1). At 22 hphCG (w10 hpi, late 1-cell; Fig. 2), the number of centromeres associated with NPBs decreased (in mPN only) as well as the total number of NPBs (Table 1). As a result, the total percentage of centromeres associated with NPBs dropped dramatically from 27.5% at 19 hphCG to 17% at 22 hphCG. These results in rabbit 1-cell embryos are very different from mouse in which NPBs seem to play a major role in attracting both pericentromeric heterochromatin and centromeres to form a 'cartwheel' (Martin et al. 2006a).

Upon cleavage to 2-cell (Fig. 2), HP1b was still quite diffused in the nucleoplasm, also few HP1b foci appeared usually not associated with any CENP dot (15 of 21 embryos; Table 2). Nearly all CENP dots were clearly partitioned on one side of the nuclei as in mouse and bovine embryos (Martin et al. 2006a). At the 4-cell stage, more HP1b foci were observed in all embryos (nZ18; Figs 2 and3) and CENP dots started to associate with them (6 of 18 embryos; Table 2). Subsequently, more prominent accumulations of HP1b appeared in the nucleoplasm, most probably by the clustering of smaller foci (Fig. 3). These accumulations were mostly associated with one centromere and the number of single isolated centromeres clearly decreased at the early 8/16-cell stage (Fig. 2). The typical somatic-like organization into pericentromeres was completely established at the late 8/16-cell stage (Figs 2 and 3), which correlates with major EGA in this species [START_REF] Manes | The participation of the embryonic genome during early cleavage in the rabbit[END_REF].

In parallel, we analyzed the distribution of these two markers, HP1b and CENP, in rabbit diploid embryos derived from parthenogenesis to evaluate the impact of the activation protocol later used for NT (Fig. 4, left column). At the 1-cell stage, the organization of pericentric and centromeric heterochromatin was similar to the one described above in fertilized 1-cell embryos. The foci of HP1b that are associated with centromeres appeared at the 4-cell stage (Fig. 4, left column). In addition, centromeres tend to distribute in one part of the nucleus during the 2-and 4-cell stages. The typical somatic-like organization was established at the 8/16-cell stage, although HP1b foci remained quite smaller (Fig. 3) compared with fertilized embryos. Taken together, these data show that a similar spatio-temporal reorganization of pericentric and centromeric regions is recapitulated at the early stages of rabbit parthenotes and fertilized embryos.

After rabbit intra-species SCNT, somatic nuclear organization is disrupted but compact foci of pericentric heterochromatin reappear before 8-cell

We next wondered whether rabbit NT embryos derived from somatic nuclei would adopt the somatic or embryonic type of nuclear organization. To address this question, the distribution of HP1b and CENP was observed in rabbit-to-rabbit SCNT embryos.

In 1-cell SCNT embryos fixed at 8 hpf (nZ35), the pronucleus-like structure formed contained uniform HP1b labeling in the nucleoplasm in 80% of embryos (Fig. 4, middle column), whereas the other 20% displayed some condensed HP1b accumulations (image not In contrast, interphase rabbit nuclei showed diffusely distributed HP1b in the nucleoplasm with discrete accumulations that were quite small compared with mouse and bovine chromocenters, and HP1b foci were often associated with single centromeres.

shown, Table 2). In these embryos, we counted 37.5G3.9 CENP dots, of which 11.1% were organized at the NPB periphery (Fig. 4 and Table 1). These data show that after SCNT, the different components of somatic pericentromeres were rapidly reorganized into an embryonic type in the majority of 1-cell embryos.

In 2-cell SCNT embryos (15 hpf, nZ32), HP1b was still diffusely distributed in the nucleoplasm in 59% of embryos (image not shown) but 41% displayed discrete accumulation of HP1b dots (Fig. 4), which were sometimes already linked with centromeres (13% of embryos; Table 2).

Upon cleavage to 4-cell (nZ35; Fig. 4), most embryos showed somatic-like pericentromeres with compact pericentric and centromeric heterochromatin, i.e. 1-cell cycle earlier than in rabbit fertilized embryos (Table 2). In total, 85% of the 4-cell embryos (30 of 35; Fig. 4) displayed pericentromeres: in each of the four nuclei (31%), in three nuclei (34%), or in two nuclei (20%). The remaining nuclei (and embryos) had a similar nuclear organization to fertilized embryos (image not shown). HP1b foci were also more numerous in SCNT embryos than in fertilized ones (Fig. 3). We also observed that in vitro development of these rabbit SCNT embryos was not disturbed until the 4-cell stage and dramatically decreased thereafter (Table 3). Altogether these observations suggest that, although rabbit recipient oocytes can trigger nuclear remodeling of rabbit somatic donor cells, this process is insufficient.

Bovine nuclei can be remodeled by rabbit oocytes after SCNT to some extent but typical bovine chromocenters are then rapidly re-established

We then addressed the problem of epigenetic memory in inter-species SCNT and questioned whether pericentromeric heterochromatin and centromeres in embryos derived from bovine fibroblasts would adopt the donor species-type (bovine) or recipient species-type (rabbit) nuclear organization.

At the 1-cell stage in bovine-to-rabbit SCNT embryos (Fig. 4, last column), 100% of the embryos (nZ32; Table 2) presented partially condensed HP1b foci mainly in the nucleoplasm and, in a few cases, on NPBs. Furthermore, the number of NPBs (w6.4G3.5) was significantly lower than that in rabbit-to-rabbit 1-cell Statistical comparisons were performed i) within the fertilized 1-cell stage for mPN and fPN separately, ii) between the early and late 1-cell (mPNC fPN), and iii) between SCNT embryos. Values with different superscript letters within the same column are significantly different (P!0.05). Each value corresponds to the meanGS.E.M.

SCNT (w11.0G4.3, nZ35; Table 1). In bovine-to-rabbit 1-cell embryos, 52.8G4.3 CENP dots on average were observed, which correlated with the number of chromosomes in the bovine donor cells (2nZ60); 9.7% of these CENP were located at the NPB periphery as in rabbitto-rabbit SCNT (Table 1). At 2-cell, nucleoplasmic accumulations of HP1b associated with CENP dots already appeared in 12% of the embryos (3 of 25; Fig. 4; Table 2), as in intra-species SCNT. Surprisingly, 88% of the embryos (22 of 25; Fig. 4) had HP1b rims on NPBs, a phenomenon that we did not observe in the other types of embryos analyzed so far in this study.

In the 4-cell embryos (nZ26; Fig. 4), these peri-NPB rims of HP1b disappeared while large nucleoplasmic HP1b foci appeared (Figs 3 and4). The decrease in HP1b association with NPBs was not correlated with a decrease in NPB numbers, suggesting that HP1b actually dissociated from NPBs. Remarkably, 100% of the embryos displayed bovine-like chromocenters (nZ26; Table 2): in all four nuclei (65%), in three nuclei (27%), or in two nuclei (8%). Even in bovine-to-bovine SCNT, we never observed such a high percentage of abnormal heterochromatin distribution [START_REF] Pichugin | Dynamics of constitutive heterochromatin: two contrasted kinetics of genome restructuring in early cloned bovine embryos[END_REF]). This may be related to the fact that all reconstructed bovine-to-rabbit SCNT embryos arrested before the 8-cell stage (Table 3).

The fluorescence intensity profile of HP1b staining (Fig. 4, last panel) suggested that the size of HP1b foci in bovine-to-rabbit SCNT embryos was different from that of rabbit parthenotes and rabbit-to-rabbit SCNT embryos. Quantification confirmed that HP1b foci in bovineto-rabbit SCNT were 3.5 times larger at the 4-cell than in the 2-cell embryos (Fig. 3).

Altogether it suggests that donor cells maintain an epigenetic memory of their original nuclear status that determines pericentric and centromeric reorganization after NT.

Global transcriptional activation is delayed in early 1-cell embryos derived from bovine-to-rabbit SCNT Next, we questioned whether incomplete remodeling in bovine-to-rabbit SCNT embryos could be associated with abnormal transcriptional activity. Although we did not observe differences at the time of major EGA (all embryos were transcriptionally active, data not shown), we noticed differences at the 1-cell stage when transcriptional activity starts, i.e. when minor EGA takes place (Memili & First 1999). Very weak signals of BrUTP incorporation were observed only in 9.2% of bovine-to-rabbit SCNT embryos at 5 hpf, which was significantly lower than in rabbit-to-rabbit SCNT embryos (36.7%; Table 4). At 10 hpf, the percentage significantly increased to 71.4% and reached 85 and 97.8% at the 2-cell stage, at 13 and 17 hpf respectively. However, it was not statistically different The first columns present a summary of the different patterns observed in fertilized and SNCT embryos after HP1b immunodetection (uniform vs foci).

The presence of somatic-like structures (pericentromeres or chromocenters) is given in the last column. Heterochromatin reprogramming in rabbit embryo from rabbit-to-rabbit SCNT embryos anymore (Table 4). This shows a delayed transcriptional activation in bovine-to-rabbit SCNT embryos at the early 1-cell stage, compared with rabbit intra-species SCNTembryos.

Discussion

In the present study, we show that the assembly of pericentric heterochromatin and centromeres in rabbit fibroblasts is different from that in mouse and bovine.

In interphase mouse and bovine somatic nuclei, pericentric regions from different chromosomes are organized into chromocenters enriched in HP1b and each chromocenter is surrounded with several centromeres [START_REF] Hsu | Arrangement of centromeres in mouse cells[END_REF][START_REF] Haaf | Chromosome topology in mammalian interphase nuclei[END_REF][START_REF] Cerda | Organisation of complex nuclear domains in somatic mouse cells[END_REF][START_REF] Guenatri | Mouse centric and pericentric satellite repeats form distinct functional heterochromatin[END_REF], Martin et al. 2006a[START_REF] Pichugin | Dynamics of constitutive heterochromatin: two contrasted kinetics of genome restructuring in early cloned bovine embryos[END_REF]. Differently, in rabbit somatic nuclei, individualized HP1b dots are associated with single centromere (1:1 ratio) as in human somatic nuclei [START_REF] Hayakawa | Cell cycle behavior of human HP1 subtypes: distinct molecular domains of HP1 are required for their centromeric localization during interphase and metaphase[END_REF]. The differences in genome organization among species probably result from the characteristic feature of satellite DNA repeats at Values with different superscript letters within the same column are significantly different (P!0.05).

pericentric and centromeric regions. Three different families of human satellite sequences are indeed found in pericentromeres of different chromosomes [START_REF] Miller | Human Chromosomes[END_REF], whereas mouse major satellites are quite conserved [START_REF] Vissel | Mouse major (gamma) satellite DNA is highly conserved and organized into extremely long tandem arrays: implications for recombination between nonhomologous chromosomes[END_REF]. In rabbit, [START_REF] Ekes | Isolation, cloning and characterization of two major satellite DNA families of rabbit (Oryctolagus cuniculus)[END_REF] identified two families of DNA repeat sequences: Rsat I and Rsat II that do not hybridize to every chromosome but are both localized in the centromeric regions of the chromosomes. It would therefore be interesting to perform fluorescent in situ hybridization with DNA probes directed against Rsat I and Rsat II both to evaluate i) whether these two families occupy distinct nuclear regions in somatic nuclei and ii) whether different chromosomes have similar dynamics or not during nuclear embryonic reorganization.

In rabbit embryos following fertilization, our results show that centromeres and associated pericentric heterochromatin are quite dispersed and the somaticlike organization is progressively established and completed only by the 8/16-cell stage, a stage that corresponds to major EGA in this species. This is in agreement with previous studies showing striking coincidences between these two events in mouse and bovine embryos (Martin et al. 2006a[START_REF] Pichugin | Dynamics of constitutive heterochromatin: two contrasted kinetics of genome restructuring in early cloned bovine embryos[END_REF]. We also observed that this precise spatio-temporal reorganization of pericentromeric heterochromatin is disturbed in SCNT embryos. After the introduction of somatic cells, either from rabbit or bovine origin, into rabbit enucleated oocyte cytoplasm, the formation of pronucleus-like structures with the disruption of somatic heterochromatin clusters (pericentromeres and chromocenters respectively) was very rapid in both types of embryos. However, decondensation of HP1b-labeled heterochromatin was incomplete in 20% rabbit-to-rabbit embryos and 100% bovine-to-rabbit embryos at the 1-cell stage. Also, compared with rabbit intra-species SCNT embryos, delayed transcriptional activation was observed at the early 1-cell stage in bovine-to-rabbit SCNT embryos. Furthermore, the reorganization of somatic-like/donor species-specific heterochromatin clusters took place more quickly than in fertilized embryos, at least 1-cell cycle earlier. We believe that these abnormalities may correlate with lower embryonic developmental potential. This hypothesis is supported by the fact that these abnormalities are observed just before major EGA and that most embryos get blocked at major EGA.

Remarkably, pericentromeric heterochromatin organization in inter-species SCNT embryos was mainly dependent on the donor cell organization, showing the presence of an 'epigenetic memory'. In contrast to rabbit-to-rabbit SCNT, we observed a lower proportion of embryos with transcriptional activity and a higher proportion of embryos with incompletely decondensed HP1b at the 1-cell stage. We also found that the re-establishment of compact heterochromatin at the 4-cell stage leads to the formation of large foci that resembled somatic chromocenters, suggesting that the nuclear remodeling of bovine nuclei in rabbit cytoplasm is less sufficient. One possible explanation is that inter-species nucleo-cytoplasmic compatibility between the introduced bovine genome and maternal rabbit factors present in the recipient oocytes is lower than that in the case of rabbit intra-species SNCT, probably because some remodeling factors are species-specific.

The mechanisms involved in heterochromatin formation are still subject to discussion. Recently, it has been shown in mouse embryos that, similarly to fission yeast and plants, transcripts generated by pericentromeric repeats would be processed to small RNAs that in turn guide heterochromatin formation [START_REF] Probst | A strand-specific burst in transcription of pericentric satellites is required for chromocenter formation and early mouse development[END_REF][START_REF] Santenard | Heterochromatin formation in the mouse embryo requires critical residues of the histone variant H3.3[END_REF]. It was also shown that the presence of histone variant H3.3 and low levels or absence of H3K27 methylation would provide a chromatin environment permissive for the transcription of pericentromeric chromatin, leading to heterochromatin formation at pericentromeric repeats [START_REF] Santenard | Heterochromatin formation in the mouse embryo requires critical residues of the histone variant H3.3[END_REF]. Such data are not available yet in other species and further studies are required. It would be of particular interest to see whether the epigenetic memory of heterochromatin organization in the donor genome is based on one of these mechanisms.

In the present study, immunodetection of HP1b and CENP also reveals centromeres/chromocenters partitioning in one part of the nuclear volume in 2-cell fertilized rabbit, rabbit-to-rabbit SCNT, and bovine-to-rabbit SCNT embryos. This is in agreement with the description of nuclear partitioning in mouse and bovine early-stage embryos [START_REF] Mayer | Spatial separation of parental genomes in preimplantation mouse embryos[END_REF], Martin et al. 2006b[START_REF] Pichugin | Dynamics of constitutive heterochromatin: two contrasted kinetics of genome restructuring in early cloned bovine embryos[END_REF]; a Rabl polarization of chromosomes that was first described in plant cells [START_REF] Shaw | The architecture of interphase chromosomes and nucleolar transcription sites in plants[END_REF]. It suggests that in reconstructed SCNT embryos, the movements of chromosome territories (CTs) could occur as in fertilized ones. The first study dealing with CT distribution during major genome activation has recently been published in fertilized bovine embryos [START_REF] Koehler | Changes of higher order chromatin arrangements during major genome activation in bovine preimplantation embryos[END_REF]). The authors have found that during Values with different superscript letters within a column are significantly different (P!0.05).

Heterochromatin reprogramming in rabbit embryo the preimplantation period, a non-random radial CT arrangement is established but they do not have proof that this nuclear reorganization is a consequence or a cause of EGA. Looking at these CTs, large-scale movements in fertilized vs SCNT embryos would bring new insights to this question. As bovine-to-rabbit embryos do not develop beyond the 4-cell stage, it might indeed be that the large-scale movements leading to the Rabl-like orientation are not sufficient after SCNT.

Materials and Methods

Animals

Animals were handled and used in accordance with the French legal requirements of animal care. et al. 2003). Female rabbits were then mated with vasectomized or normal males at 12 h after the last FSH injection and 30 IU of human chorionic gonadotropin (hCG, Choluron; Intervet, Angers, France) were injected a few minutes after mating. MII oocytes or 1-cell embryos were recovered 16 h post-hCG injection [START_REF] Christians | Sequential acquisition of transcriptional control during early embryonic development in the rabbit[END_REF][START_REF] Challah-Jacques | Production of cloned rabbits by somatic nuclear transfer[END_REF].

Collection of rabbit oocytes and embryos

The cumulus of MII oocytes was removed by exposure to 0.5% hyaluronidase (Sigma) for a few minutes followed by gentle pipetting, and then denuded oocytes were subjected to NT experiments. In vivo-derived zygotes, after the assessment of fertilization by the presence of two polar bodies, were in vitro cultured up to different experimental stages in B2 medium with 2.5% FCS (Sigma) in 5% CO 2 at 38.5 8C. In rabbits, insemination normally occurs at w12 h post-hCG (tZ0 postinsemination, pi).

Cell culture

Mouse fibroblasts (3T3) were cultured in DMEM supplemented with L-glutamine and 10% newborn calf serum, incubated in 5% CO 2 at 37 8C. Bovine ear and rabbit fetal skin fibroblasts were cultured in DMEM with 15% FCS in 5% CO 2 at 38.5 8C.

For immunofluorescence detection, cells were grown on coverslips to sub-confluence for about 24 h.

Nuclear transfer

Rabbit oocytes without cumulus cells were enucleated according to our laboratory's routine protocol [START_REF] Chesne | Cloned rabbits produced by nuclear transfer from adult somatic cells[END_REF][START_REF] Challah-Jacques | Production of cloned rabbits by somatic nuclear transfer[END_REF]. Briefly, after 20 min culture in M199 medium containing 10% FCS (M199-FCS) and 0.5 mg/ml Hoechst 33342, oocytes were placed into a micromanipulation chamber containing 0.5 mg/ml Hoechst 33342 and 7 mg/ml cytochalasin B (CB) in M199-FCS buffered with 20 mM HEPES. Under the control of a video-enhanced camera at a low ultraviolet light level, metaphase II plate together with the first polar body (PB) was removed. Bovine ear and rabbit fetal skin fibroblasts at three to ten passages were induced into the G 0 /G 1 stage by serum starvation culture (0.5% FCS) for 2-6 days and then used as nuclear donors. Cells were introduced into the subzonal space of enucleated oocytes. Cell-oocyte pairs were electrostimulated (3 DC of 3.2 kV/cm for 20 ms each) with a BTX stimulator (Biotechnologies & Experimental Research, Inc., San Diego, CA, USA) to induce fusion in 0.3 M mannitol in distilled water containing 0.1 mM CaCl 2 and 0.1 mM MgCl 2 . One hour later, fused embryos were activated by a second set of pulses (same parameters as fusion) followed by 1 h incubation with 5 mg/ml cycloheximide and 2 mM 6-dimethylaminopyridine in M199-FCS, and then were cultured in B2 medium supplemented with 2.5% FCS in a humidified atmosphere of 5% CO 2 at 38.5 8C up to different experimental stages.

For parthenogenesis, rabbit oocytes underwent a similar activation protocol except that they were then cultured 4 h in B2 medium supplemented with 7 mg/ml CB in order to inhibit the extrusion of the second PB after electrostimulation.

Immunofluorescence

Rabbit fertilized embryos were collected at 7 and 10 hpi (1-cell), 15 hpi (2-cell), 22 hpi (4-cell), 33 hpi (early 8/16-cell), and 40 hpi (late 8/16-cell). The collection of rabbit parthenogenetic embryos was performed at 8 h post-activation (hpa; 1-cell), 15 hpa (2-cell), and 22 hpa (4-cell). Rabbit-to-rabbit and bovine-to-rabbit SCNT embryos were collected at 8 hpf (1-cell), 15 hpf (2-cell), and 22 hpf (4-cell) respectively. Two to four replicates were performed at each time point.

Collected embryos were processed for HP1b and CENP immunostaining as described previously [START_REF] Pichugin | Dynamics of constitutive heterochromatin: two contrasted kinetics of genome restructuring in early cloned bovine embryos[END_REF]. Briefly, after 20 min fixation at room temperature (RT) using 2% paraformaldehyde and 30 min permeabilization at RT using 0.5% Triton X-100, embryos were blocked for 45 min with 3% BSA in PBS. Incubation with primary antibodies was performed overnight at 4 8C before three washes with 0.05% Tween-20 in PBS (15 min each) followed by incubation with secondary antibodies (1 h, RT). Embryos were washed again and postfixed 10 min using 2% paraformaldehyde. For microscopic observation, embryos were mounted onto glass slides in Vectashield (Vector Laboratories, Les Ulis, France). HP1b was detected with a mouse monoclonal anti-HP1b antibody (1:250 in 3% BSA/PBS, clone 1 MOD 1A9; Euromedex, Souffelweyersheim, France), using a lissamine-rhodamine-conjugated antimouse secondary antibody (1:150 in 2% BSA/PBS; Jackson ImmunoResearch, Interchim, France). The centromeres were labeled with a human CREST antibody that recognizes mostly CENPA (1:250 in 3% BSA/PBS; Immunovision, Cellon Sarl, Luxembourg), using a FITC-conjugated anti-human secondary antibody (1:150 in 2% BSA/PBS; Jackson ImmunoResearch). Rabbit, bovine, and mouse fibroblasts were grown on glass coverslips and then processed similarly.

Assessment of global transcriptional activity in embryos

Rabbit-to-rabbit and bovine-to-rabbit SCNT embryos were collected at 5 hpf (early 1-cell), 10 hpf (late 1-cell), 13 hpf

Figure 1

 1 Figure 1 Immunostaining of HP1b (red) and CENP (green) in mouse, rabbit, and bovine somatic cells. Scale bars: 5 mm. (Upper panel) Mouse NIH-3T3 cell. (Middle panel) Rabbit fibroblast. (Lower panel) Bovine fibroblast. The left column represents maximum projections of confocal Z-stacks. The right column represents confocal single sections. In mouse and bovine interphase nuclei, HP1b-enriched pericentric heterochromatin was clustered into chromocenters, and each chromocenter was surrounded by several centromeres (green).In contrast, interphase rabbit nuclei showed diffusely distributed HP1b in the nucleoplasm with discrete accumulations that were quite small compared with mouse and bovine chromocenters, and HP1b foci were often associated with single centromeres.

Figure 2

 2 Figure 2 Immunostaining of HP1b (red) and CENP (green) in rabbit naturally fertilized embryos (maximum projections of confocal Z-stacks) at the 1-cell (19 and 22 hphCG), 2-cell, 4-cell, early 8/16-cell, and late 8/16-cell stages. Scale bars: 5 mm. Only one nucleus per stage is shown but all blastomeres had similar staining within the embryos.

Figure 3

 3 Figure3Graphs showing computational quantification of HP1b foci number and mean size within the nuclei of parthenotes, fertilized embryos, rabbit-to-rabbit (RR) SCNT, and bovine-to-rabbit (BR) SCNT, from 2-to 8-cell (except for BR-SCNT embryos that did not develop beyond the 4-cell stage). Errors bars correspond to S.E.M.

Figure 4

 4 Figure 4 Maximum projections of confocal Z-stacks presenting HP1b (red) and CENP (green) distributions in rabbit parthenogenetically activated embryos, rabbit-to-rabbit SCNT embryos, and bovine-to-rabbit SCNT embryos. The most representative images are shown. Scale bars: 5 mm. 1-, 2-, and 4-cellstage embryos are presented on the top, second, and third row of panels respectively. The bottom row of panels fluorescence intensity profile of HP1b in the 4-cell nuclei.

Table 1

 1 Distribution of NPBs and CENP in 1-cell rabbit in vivo fertilized embryos, rabbit-to-rabbit SCNTembryos, and bovine-to-rabbit SCNTembryos.

					Percentage of
		Timing	CENP dots		CENP dots	No. of		Percentage
		(no. of	associated	Total	associated with	NPBs with	Total no.	of NPBs with
	Stage	embryos)	with NPBs	CENP dots	NPBs	CENP dots	of NPBs	CENP dots
	Fertilized 1-cell mPN only Fertilized 1-cell fPN only Fertilized 1-cell mPNCfPN Rabbit-to-rabbit SCNT 1-cell Bovine-to-rabbit SCNT 1-cell	19 hphCG (27) 22 hphCG (23) 19 hphCG (27) 22 hphCG (23) 19 hphCG (27) 22 hphCG (23) 8 hpf (35) 8 hpf (32)	7.1G3.0 a 2.3G1.8 b 3.8G1.8 a 4.2G2.0 a 10.9G3.7 a 6.6G3.0 b 4.3G3.1 c 4.9G3.9 c	18.7G1.7 a 18.1G2.3 a 20.7G1.5 a 19.9G1.6 a 39.5G2.3 a 38.0G2.5 a 37.5G3.9 d 52.8G4.3 e	37.7G16.4 a 12.4G9.8 b 18.4G8.3 a 21.0G9.9 a 27.5G9.1 a 17.2G7.7 b 11.1G8.3 d 9.7G4.7 d	5.6G2.3 a 2.1G1.6 b 2.9G1.3 a 3.4G1.5 a 8.5G2.5 a 5.5G2.2 b 3.3G2.4 d 2.8G1.9 d	9.3G1.7 a 7.2G2.9 b 7.4G1.8 a 6.4G1.5 b 16.7G2.5 a 13.7G3.5 b 11.0G4.3 e 6.4G3.5 f	61.5G26.2 a 27.9G19.1 b 40.7G16.9 a 54.1G22.2 b 51.5G15.1 a 41.0G15.7 b 29.0G16.6 e 52.8G27.1 f

Table 2

 2 HP1b distribution in early rabbit in vivo fertilized embryos, rabbit-to-rabbit SCNT embryos, and bovine-to-rabbit SCNT embryos.

			Total no.	No. of embryos with uniform	No. of embryos	No. of embryos with somatic-
	Type of embryo	Stage	of embryos	HP1b distribution (%)	with HP1b foci (%)	like structures (%)
	Fertilized	1-cell	50	50 (100)	0	0
		2-cell	21	6 (29)	15 (71)	0
		4-cell	18	0	18 (100)	6 (33)
	Rabbit-to-rabbit SCNT	1-cell	35	28 (80)	7 (20)	0
		2-cell	32	19 (59)	13 (41)	4 (13)
		4-cell	35	0	35 (100)	26 (75)
	Bovine-to-rabbit SCNT	1-cell	32	0	32 (100)	0
		2-cell	25	0	25 (100)	3 (12)
		4-cell	26	0	26 (100)	26 (100)

Table 3

 3 In vitro developmental potential of fertilized, intra-, and inter-species SCNT embryos.

		No. of reconstructed
	Type of embryo	embryos	Fused (%)	2-to 4-cell (%)	Morula (%)	Blastocyst (%)
	Bovine-to-rabbit SCNT Rabbit-to-rabbit SCNT Rabbit fertilized in vivo,	356 173 165	252 (70.8) a 127 (73.4) a -	213 (84.5) a 108 (85.0) a 153 (92.7) a	0 a 49 (38.6) b 145 (87.9) c	0 a 37 (29.1) b 142 (86.1) c
	cultured in vitro

Table 4

 4 Global transcriptional activity at 1-cell in rabbit-to-rabbit and bovine-to-rabbit SCNT embryos.BrUTP incorporation: timing and number of positive embryos/total number (%)

	Type of embryo	5 h BrUTPC/n (%)	10 h BrUTPC/n (%)	13 h BrUTPC/n (%)	17 h BrUTPC/n (%)
	Rabbit-to-rabbit embryos Bovine-to-rabbit embryos	13/34 (36.7G16.9) a 3/31 (9.2G8.0) b	21/23 (92.5G6.6) a 27/38 (71.4G15.3) a	27/29 (92.9G1.7) a 34/41 (85.0G17.5) a	26/27 (96.2G5.4) a 32/33 (97.8G3.9) a

Reproduction (2013) 145 149-159www.reproduction-online.org

www.reproduction-online.org Reproduction (2013) 145 149-159

Acknowledgements

We are grateful to the platform MIMA2 (Microscopie et Imagerie des Microorganismes, Animaux et Elements) for confocal microscopy and UCEA for animal care. We would like to thank Sandrine Peron for the culture of bovine fibroblasts, Laurent Boulanger and Nathalie Daniel for the culture of rabbit fibroblasts, as well as Linda Maulny for the collection of rabbit oviducts.

Funding

The present work was supported by INRA Jeune Equipe funding, the French embassy in Beijing, and the MRI at INRA.

(early 2-cell), and 17 hpf (late 4-cell) to detect transcriptional activity. Three replicates were done at each time point. Immunofluorescent detection of BrU incorporation into nascent RNA transcripts was performed, using BrUTP (Sigma) as a precursor. Embryos using the plasma permeabilized method were treated according to a previous report in mice [START_REF] Aoki | Regulation of transcriptional activity during the first and second cell cycles in the preimplantation mouse embryo[END_REF], except that: i) the plasma membrane of embryos was permeabilized for 3-5 min with 0.1% Triton X-100 in 'physiological buffer' (PB); ii) BrUTP incorporation (working concentration, 1.6 mM) was performed for 30 min at 33 8C; and iii) the nuclear membrane was permeabilized by a treatment using 0.4% Triton X-100 in PB for 3 min. After fixation with 3.7% paraformaldehyde in PB overnight at 4 8C, embryos were washed completely using PBS with 0.3% BSA and blocked 30 min in the same medium at RT. Incubation with the primary antibody (1:100 dilution, mouse monoclonal anti-BrUTP antibody; Caltag Laboratories, Burlingame, CA, USA) was allowed to proceed overnight at 4 8C followed by three washes. Incubation with a FITC-conjugated anti-mouse secondary antibody (1:400 dilution; Sigma) was performed at RT for 1 h. Nucleic acids were counterstained with PI (50 mg/ml) for 15 min at 37 8C. Embryos were mounted onto glass slides in Vectashield.

Confocal microscopy

All samples were scanned on a Zeiss LSM 510 confocal microscope (MIMA2 Platform; INRA) using a Plan-Apochromat 63!/1.4 oil objective. Imaging was performed with sequential multi-track scanning using 488 and 543 nm wavelength lasers separately. Complete Z-stacks were acquired using a frame size of 512!512 with a pixel depth of 8 bits and 0.37 mm Z-step. Images were prepared using Adobe Photoshop CS version.

Image analysis

Analysis of the fluorescence intensity profiles for HP1b was performed with the software LSM510 3.2 version. Manual counting of CENP dots and NPBs was performed with ImageJ software.

To determine HP1b foci size and number, automated 3D image processing and analysis were performed with the help of the ITK library (http://www.itk.org) interfaced with the Python script language [START_REF] Lehmann | Kappa Sigma Clipping[END_REF]. Briefly, images were first automatically resized to a constant voxel size (0.284!0.284! 0.36 mm). Granules were then detected using an automatic threshold segmentation procedure: i) the regions of interest (ROI) of nuclei were determined by a threshold method largely used in astronomy which analyzed only the intensities of background and computed the intensity value (meanCsigma) as the lower threshold [START_REF] Lehmann | Wrap ITK: Enhanced languages support for the Insight Toolkit[END_REF]; ii) images were processed according to two different methods: the most intense granules were segmented following subtraction of a background image generated by a 3D median filtering, whereas a 2D top-hat filtering was applied to detect small and/or faint granules; and iii) these two segmented images were then combined and granule parameters in the nuclear ROI were determined.

To qualify the BrUTP incorporation signal, the embryo showing at least three clear staining dots in each interphase nucleus was referred to as positive transcription nucleus.

Statistical analysis

Comparison of the number of CENP dots and NPBs (nuclear precursor bodies) among 1-cell-stage embryos was done with the Wilcoxon-Mann-Whitney test. The cleavage rates as well as the percentage of embryos with positive transcription were compared using the c 2 -test. Statistical significance was determined when the P value was !0.05.

Declaration of interest

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.