Since we show that type of culture medium affects multiple individual morphokinetic parameters, our findings may have consequences for the discriminatory value and applicability of other (de)selection tools. Therefore, it is recommended that IVF clinics should either validate morphokinetic-based selection tools prior to implementation or develop their own clinic-specific selection tool, since the ideal developmental kinetics of human embryos under different culture conditions remains largely unknown.
A number of studies in the 1960s and 1970s reported biochemical evidence suggesting that myosins isolated from mammalian embryonic or fetal skeletal muscle differ from adult muscle myosins (see references in [2, 3]). However, Whalen et al. [2] were the first to provide unambiguous evidence for the existence of distinct developmental myosins. They identified two specific MyHCs, called embryonic and neonatal (also called perinatal) MyHCs, hereafter referred to as MyHC-emb and MyHC-neo, which precede the appearance of adult fast myosins in the developing rat skeletal muscle [2]. The corresponding MYH genes were identified [4, 5] and found to be located in the same chromosomal locus as gene coding for adult fast myosin heavy chains on chromosome 11 (mouse) or 17 (human) [6]. The gene coding for MyHC-neo (MYH8) shows considerable sequence similarity with adult fast MYH genes, whereas the gene coding for MyHC-emb (MYH3) is quite different (see [7] for a comparative sequence analysis of MYH genes). Embryonic skeletal muscles also contain a unique type of essential MLC, MLC-1emb, encoded by the MYL4 gene, which is also expressed in the developing heart and in adult atrial myocardium but not in adult skeletal muscle [8, 9].
download human embryology and developmental 17
The developmental pattern of myosin isoform expression in the human embryonic and fetal skeletal muscle has been comparatively less investigated. At week 8 of gestation, primary generation fibers with central nuclei are present in the human skeletal muscle, whereas secondary generation fibers are formed after week 10 and become the predominant fiber population by week 21 [42]. MyHC-emb, MyHC-slow, and MyHC-neo transcripts are detectable in the developing skeletal muscle at week 9 (Fig. 1). At the protein level, all primary myofibers express MyHC-emb and MyHC-slow [43, 44], with MyHC-emb being detectable before MyHC-slow in the initial myotubes [45]. The proportion of fibers staining for MyHC-slow decreases from 75 % at week 10 to 3 % at week 21 of gestation, due to the dramatic increase in secondary fibers that initially do not contain MyHC-slow [45]. Secondary generation fibers express only MyHC-emb at week 12, MyHC-neo protein being detected at later stages [45]. Quantitative RNA analysis indicates that MYH3 transcripts account for about 81 % of all MYH transcripts in the human fetal skeletal muscle at week 15 of gestation [46]. At week 16 to 17, a tertiary fiber population has been identified, initially composed of very small myofibers stained by an anti-myosin antibody reactive with adult fast but not with neonatal MyHC [44, 47]. In situ hybridization indicates that MyHC-2A transcripts are weakly expressed at week 19 and more strongly at birth, whereas MyHC-2X transcripts are barely present at birth and are clearly expressed at 30 days after birth (Fig. 1). After week 27, a proportion of secondary fibers starts to express MyHC-slow, and by week 30, about 50 % of all muscle fibers express MyHC-slow, like in adult muscle [45, 44]. In the developing human muscles, both developmental MyHC isoforms are downregulated toward the end of gestation, the corresponding MyHC transcripts are expressed at low levels at birth, and in a 1-month-old infant, MyHC-neo persists only in a few fibers [48] (Fig. 1). In conclusion, most human skeletal muscle fibers, probably more than 95 %, appear to derive from secondary and tertiary waves of myogenesis and their diversification into the fast type 2A or slow type 1 lineage occurs before birth, during the third trimester of gestation, whereas the differentiation of type 2X fibers takes place in the first week after birth.
The view that, in mammals, neonatal myosin has kinetics similar to 2A myosin but slower than 2X and 2B myosins has received support from the experiments on recombinant human myosin S1 motor domain expressed in C2C12 myotubes [100]. An additional interesting feature emerging from Resnicow et al. [100] data is that K m values for actin are much greater for developmental than for adult myosins. This suggests even lower values of ATP hydrolysis rate for immature myofibrils in conditions other than maximal actin activation.
Some preliminary experiments have already been performed, such as injection of human ESC into a mouse blastocyst [16]. This first experiment failed; however, more recent attempts using human iPSC led to the production of mice with a significant percentage of chimerism. These mouse embryos were sacrificed at early developmental stages for ethical reasons [17]. Injection of macaque rhesus ESC into mice blastocysts also led to a significant proportion of chimerism [18].
Understanding the molecular basis of embryonic development is of basic importance, in and of itself, but this pursuit is also key to understanding mechanisms that underlie disease and birth defects in humans. Many of the regulatory mechanisms that govern embryonic development are similar if not identical to those that control the normal function of adult cells and tissues. Furthermore, many advances in biotechnology are fueled by new methods that stem from the study of embryonic development (e.g. the recent rise of RNAi through studies of C. elegans). Closer to our interests, the study of frog embryos (by many groups) has led to discovery and insight into the function of developmental regulators that have made their way into clinical testing, such as BMP, FGF and Wnt growth factors and their various inhibitors.
G.H. Thomsen (2006). A new century of amphibian developmental Biology. Seminars in Cell and Developmental Biology 17:78-79. Note: This issue highlights Xenopus research and was edited by G.H. Thomsen (see below). [ download PDF] 2ff7e9595c
Comments