25th ANNIVERSARY OF CLONING BY SOMATIC-CELL NUCLEAR TRANSFER: Scientific and technological approaches to improve SCNT efficiency in farm animals and pets

The birth of Dolly through somatic cell nuclear transfer (SCNT) was a major scientific breakthrough of the last century. Yet, while significant progress has been achieved across the technics required to reconstruct and in vitro culture nuclear transfer embryos, SCNT outcomes in terms of offspring production rates are still limited. Here, we provide a snapshot of the practical application of SCNT in farm animals and pets. Moreover, we suggest a path to improve SCNT through alternative strategies inspired by the physiological reprogramming in male and female gametes in preparation for the totipotency required after fertilization. Almost all papers on SCNT focused on nuclear reprogramming in the somatic cells after nuclear transfer. We believe that this is misleading, and even if it works sometimes, it does so in an uncontrolled way. Physiologically, the oocyte cytoplasm deploys nuclear reprogramming machinery specifically designed to address the male chromosome, the maternal alleles are prepared for totipotency earlier, during oocyte nuclear maturation. Significant advances have been made in remodeling somatic nuclei in vitro through the expression of protamines, thanks to a plethora of data available on spermatozoa epigenetic modifications. Missing are the data on large-scale nuclear reprogramming of the oocyte chromosomes. The main message our article conveys is that the next generation nuclear reprogramming strategies should be guided by insights from in-depth studies on epigenetic modifications in the gametes in preparation for fertilization

The fibre type composition of the striated muscle of the oesophagus in ruminants and carnivores

The fibre type composition of the striated muscle layer of the oesophagus of the cow, sheep, donkey, dog and cat was examined with standard histochemical methods and immunohistochemical staining using type-specific antimyosin sera. The heavy chain and light chain composition of oesophageal myosin was also examined using electrophoretic peptide mapping and 2-dimensional gel electrophoresis respectively. In the ruminants and donkey the oesophagus was composed of fibre types I, IIA and IIC with immunohistochemical characteristics identical to those of the same fibre types found in control skeletal muscle. In the ruminants there was a gradient in the proportion of type I fibres from 1% (at the cervical end) to about 30% (at the caudal end). In the carnivores the oesophageal muscle was composed of a very small percentage of type I and IIC fibres, but the predominant type was very different hisotchemically and immunohistochemically from all the fibre types (I, IIA, IIB, IIC) present in the control muscles. This oesophageal fibre type (IIoes) had an acid- and alkaline-stable m-ATP-ase activity, a moderate histochemical Ca-Mg actomyosin ATPase activity, and reacted weakly with anti-IIA and antiIIB myosin sera. Although the light chains of the IIoes myosin were the same as the light chains of a mixture of IIA and IIB myosins, their respective heavy chains gave different peptide maps. Greater differences were obtained between the heavy chains of IIoes and other striated muscle myosins. These observations lead us to conclude that this predominant fibre type of the carnivore oesophageal striated muscle is of the fast type, and contains a distinct isoform of myosin similr but not identical to the other fast type myosins

Histochemical and immunohistochemical profile of pink muscle fibres in some teleosts.

The pink muscle of several Teleosts was examined immunohistochemically using antisera specific for the myosins of red and white muscle, and histochemically using various methods for demonstrating myosin ATPase (mATPase) activity. In the catfish the pink muscle consists of 2 different layers of fibres. The superficial layer has a low mATPase activity after both acid and alkali pre-incubation, whereas the deeper layer has a high mATPase activity after acid and alkali pre-incubation, being more resistent to these conditions even than is the white muscle. In the trout the pink muscle is composed of fibres with the same mATPase activity as in the superficial pink muscle of the catfish, whereas in the rock goby, goldfish, mullet and guppy the pink muscle is like the deep pink layer of the catfish. Immunohistochemically the fibres of the pink muscle behave like the white muscle fibres except in the guppy and rock goby in which at the level of the lateral line there occurs a transition zone between red and pink fibres. The fibres of this region react with both anti-fast and (to a lesser extent) anti-slow myosin antisera, and have a mATPase activity which, going from the superficial to the deeper fibres, gradually loses the red muscle characteristics to acquire those of the main pink muscle layer

Hyperplastic and hypertrophic growth of lateral muscle in Dicentrarchus labrax (L.)

In this EM study of lateral muscle in Dicentrarchus labrax, we observed that during the larval period, growth of the presumptive red and white muscle layers occurs both by hypertrophy (as fibres already present at hatching complete their maturation) and by production of new fibres in germinal zones specific to the two muscle layers. In the first half of larval life the presumptive white muscle increases in thickness by the addition, superficially, of new fibres derived from a germinal zone of presumptive myoblasts lying beneath the red muscle layer. In the second half of larval life new fibres produced in this same zone form the intermediate (or pink) muscle layer. Dorsoventrally the myotome grows throughout larval life, largely by addition of new fibres from germinal zones at the hypo- and epi-axial extremities. Towards the end of larval life all these germinal zones are becoming exhausted, but another source of fibres arises as satellite cells, associated with large-diameter presumptive white muscle fibres, are activated to produce new fibres. The addition of small, new fibres gives the white muscle its mosaic appearance. Morphometric analysis of fibre diameters in the white muscle confirms that whereas these hyperplastic processes are important during the larval and juvenile periods, when growth is very rapid, they have ceased by the time the adult stage is attained. By contrast, fibre hypertrophy continues through into adult life. The presumptive red muscle consists initially of a monolayer of fibres present only near the lateral line, and during larval life it grows hypo- and epi-axially by addition of fibres derived from myoblasts already present in these areas at hatching. Lying superficially to the presumptive red muscle monolayer there is a near-continuous layer of external cells with a ldquoflattenedrdquo profile. During the second half of larval life, differentiation of these external cells into myoblasts provides the source of new fibres which are added to the red muscle layer. This process, which occurs initially in the region around the lateral line and later spreads outwards, is responsible for the increase in thickness of the red muscle. Key words Fish - Muscle growth - Hyperplasia - Hypertrophy - Ultrastructur

Developmental transitions of myosin isoforms and organisation of the lateral muscle in the teleostDicentrarchus labrax (L.)

InDicentrarchus labrax (the sea bass) the differentiation of lateral muscle fibres occurs at different stages and in different ways in the superficial (red), intermediate (pink) and deep (white) regions of the myotome. At hatching the myotomes are composed of presumptive white and red fibres, the latter forming a superficial monolayer present only near the transverse septum. At this stage, differences between the fibre types are mainly ultrastructural. From their different reactions with isoform-specific antibodies to mullet myosin, and the appearance of histochemical mATPase activity, it appears that in both red and white muscle fibres there is a transition in myosin composition from an early larval form (L1R and L1W respectively) to a late larval form (L2R and L2W) and then to the isoforms typical of adult red and white muscle. The transition from L1W to L2W in the deep muscle occurs very rapidly and early in larval life (between 10 and 28 days), whereas the equivalent transition in the superficial muscle (from L1R to L2R) is a gradual process beginning in fibres near the trasverse septum and spreading hypo- and epi-axially as this layer grows around the deep muscle. The definitive adult forms (AR and AW), distinguishable by the appearance of characteristic histochemical myosin ATPase activity, are present in the superficial red muscle by 80 days, but later in the deep white muscle (by 20 months), respectively. Compared to the superficial red and deep white muscle, the intermediate (pink) muscle layer first appears relatively late (80 days), but then acquires the histo- and immunohistochemical profile characteristic of the adult form much more rapidly. The mosaic appearance of the deep white muscle is first seen during larval life. However, at this stage the smaller fibres of the mosaic have a different histo- and immunohistochemical profile from that seen in the adult small white fibres, which may indicate different mechanisms of histogenesis of new fibres in the deep muscle layer in the early and adult stages. Key words Fish - Muscle development - Myosin - Immunohistochemistry - Histochemistr

Post-hatching development of motor innervation of lateral muscle in the seabream Sparus aurata

In many fish, both production of new muscle fibres and neurogenesis continue into juvenile life. To test the hypothesis that new motoneurons are produced to supply the expanding muscle target we used the seabream (Sparus aurata), which shows a many-fold increase in the number of fibres in lateral muscle during posthatching juvenile development. A motor nerve branch innervating a segment of epaxial lateral white muscle was identified, and the type and number of its axons were measured in fish of several larval and post-larval ages. Contrary to expectation, total axon number was greatest in the larval fish (114.3\ub122.6); unmyelinated axons were found only in the larval nerves, and the number of myelinated axons increased only modestly over the ages examined, from 58.5\ub112.4 in larval fish to 77.5\ub17.3 in post-larval juveniles. We conclude that in seabream the larval nerve still includes axons of motoneurons destined to die during the normal developmental phase of targetdependence in addition to those axons which will survive into juvenile life, and that the definitive number of motoneurons is already present in the larval fish before the main increase in muscle fibre number occurs

Comparative study of myosins present in the lateral muscle of some fish: species variations in myosin isoforms and their distribution in red, pink and white muscle

Myosin isoforms and their distribution in the various fibre types of the lateral muscle of eight teleost fish (representing a wide range of taxonomic groups and lifestyles) were investigated electrophoretically, histochemically and immunohistochemically. Polyclonal antisera were raised against slow (red muscle) and fast (white muscle) myosins of the mullet, and used to stain sections of lateral muscle. Antisera specific for fast and slow myosin heavy chains only (anti-FHC and anti-SHC respectively) and for whole fast and slow myosins (anti-F and anti-S respectively) were obtained, and their specificity was confirmed by immunoblotting against electrophoretically separated myofibrillar proteins. The ATPase activity of myosin isoforms was examined histochemically using methods to demonstrate their acid- and alkali-lability and their Ca-Mg dependent actomyosin ATPase. As expected, the predominant myosin (and fibre) type in the red muscle showed an alkali-labile ATPase activity, reacted with the anti-S and anti-SHC sera (but not anti-F or anti-FHC) and contained two slow light chains, whereas the predominant myosin (and fibre) type in the white muscle showed an alkali-stable ATPase activity, reacted with anti-F and anti-FHC sera (but not anti-S or anti-SHC) and contained three fast light chains. However, superimposed upon this basic pattern were a number of variations, many of them species-related. On analysis by two-dimensional gel electrophoresis fish myosin light chains LC1s, LC2s and LC2f migrated like the corresponding light chains of mammalian myosins, but fish LC1f consistently had a more acidic pI value than mammalian LC1f. Fish LC3f varied markedly inM r in a species-related manner: in some fish (e.g. eel and mullet) theM r value of LC3f was less than that for the other two light chains (as in mammalian myosin), whereas in others it was similar to that of LC2f (e.g. cat-fish) or even greater (e.g. goldfish). Species differences were also seen in the relative intensity of LC1f and LC3f spots given by the fish fast myosins. In most of the fish examined the red muscle layer showed some micro-heterogeneity, containing (in addition to the typical slow fibres) small numbers of fibres with a histo- and immunohistochemical profile typical of white muscle (fast) fibres. However, other immunohistochemically distinct minority fibres were found in the red muscle of the goldfish. Three types of pink muscle were distinguished: (1) a mosaic of immunohistochemically typical red and white fibres (e.g. grey mullet); (2) a transition zone with properties intermediate between those of red and white muscle (e.g. guppy); and (3) a layer of fibres which appeared on the basis of their myosin and actomyosin ATPase activities to contain a distinct myosin type, although this could not be distinguished from the white muscle fast myosin by any of the antisera used (e.g. goldfish, cat-fish, carp). Four of the fish examined had a mosaic white muscle consisting of fibres of a very wide range of diameters. The larger sized fibres always had the histo- and immunohistochemical profile of fast (white) fibres, but the characteristics of the small fibres varied according to the species. In the trout no histo- or immunohistochemical difference between large and small fibres could be detected, and in the mullet there was a histochemical difference only. In the eel some of the smallest fibres reacted with the anti-S serum as well as with anti-fast sera, and in the carp the small fibres reacted with both anti-S and anti-SHC in addition to the anti-fast sera, but in neither species could any trace of slow light chains be found in this muscle. In these two cases the small fibres may contain a cross-reacting form of myosin analogous to mammalian embryonic myosin. The significance of the small fibres of mosaic white muscles in the context of postlarval hyperplastic growth mechanisms in fish muscle is discussed