[Development of prospective diapause-germs (Bombyx mori L.) in vitro without dormancy : II. Medium with foreign proteins (LYS) and extraembryonic deposits from different phases of egg diapause].

When extraembryonic egg material is placed apart from prospective diapause germ anlagen or early germ bands, it will stimulate their development without dormancy via the medium. This method of a bipartial systemin vitro (21° C) helps to analyze the regulatory mechanisms involved in the egg diapause ofBombyx mori. 1. In preliminary experiments, a culture medium free of egg extracts but containing foreign proteins (LYS) proved useful, since 100% of the test germs reached dormancy in the absence of stored egg material. Mitoses decrease and morphogenesis decelerates until the stage of the fully segmented germ-band is reached, which means the end of the prediapausal period. 2. When eggs were opened they developedin vitro without egg diapause. One may assume that the access of free oxygen activates some regulatory mechanisms permitting development without dormancy (nondormancy=Nd). In addition, the separate deposit of chorion, serosa and yolk cells (CDS Depot) will in any case prevent dormancy. Thus, the factors responsible for egg diapause must be sought in the extraembryonic egg system. A direct contact between the extraembryonic action-system and the embryonic reactionsystem is not a prerequisite. TheLYS medium without deposits offers sufficient oxygen to the test germ. Therefore the prospective diapause germ possesses a tendency to dormancy, according to its reaction norm. The potency to stimulateNd was tested with various depotmaterials (C, D, S, CS, DS, CDS) removed from eggs during prediapause (21° C), diapause (3° C) and post-diapause (returned to 21° C for 4 days). Each material produced a specificNd rate.In vitro, the test germs can progress in their organogenesis optimally to the stage of a small larva. The means of a collective effect in development are determined and related to one of the nine possibledegrees of organogenesis. 3. In comparison to serosa and chorion, yolk material has the highest mean both inNd rate (68 %,n=219) and degree of organogenesis. Surprisingly, cell-free chorionic material prevents dormancy development in 55% (n=296). As compared to theD Depot, the combination ofDS elicits a higherNd rate (79%,n=234), which is only surpassed by theCDS combination (100%,n=76). In comparison toS Depot(44%,n=294) theNd rate of aCS Depot reaches only 37% (n=161), presumably due to a restriction of the experiments to young material only. Probes, tested separately according to germ anlage or germ band, showed that there was no influence of the operational age of the test germ on theNd rate. 4. However, theNd-stimulating potency ofC, D, S, CS andDS depends on the operational age of the donator egg. Yolk material starts out having a highNd effect, decreasing with pre-diapausal age and staying relatively high in diapausal age. Similar changes are observed in the combination of yolk and serosa. TheNd rate of chorion starts low, increases steeply with the operational age and remains rather uniform. TheNd rate of serosa increases steeply in the stage critical to the beginning of egg diapause (dish-like germ anlage), decreases after pre-diapause and increases again after the minimal period for diapause (3 months at 3° C). HigherNd rates are observed whenS, D, andDS Depot were returned to 21° C for 4 days.D Depot has the maximal potency favouring organogenesis at the dish-like germ anlage stage. 5. The following subjects are discussed: the results of Chino (1957, 1958) on glycogen metabolismin ovo, the findings of Okada (1971) on the development of de-chorionized eggs under paraffin oil and our ownin ovo observations on the ultrastructural changes in the chorion, the mitotic activity before and after diapause and the distribution of glycogen in germ, yolk cells and serosa. These facts can be utilized to formulate a concept of the physiological phases of egg diapausein ovo: Egg diapause begins during a critical stage of the germ anlage with a reaction between serosa and chorionic material, which reduces the rate of oxygen consumption. Under these conditions, glycogen is metabolized into sorbitol and glyoerol. The physiologicalprophase of egg diapause is terminated, when the germ-band reaches dormancy. Diapause begins (e.g. at 3° C) with themesophase, during which the metabolism of glycogen continues decreasingly. Now glycogen is found only in the germ.Metaphase may begin with the re-uptake of oxygen, which starts the re-synthesis of glycogen from sorbitol and glycerol via oxydation and phosphorylation. However, the exposure to cold (3° C) will inhibit mitosis in the dormant germ band. In thetelophase of egg diapause, after terminated resynthesis, the dormant germ can remain in quiescence. When exposed to 21° C during the embryonic post-diapause period, it consumes the stored glycogen. If the high temperature starts prematurely during the mesophase, no embryo will hatch. However, when high temperatures set in during the metaphase, glycogen resynthesis and glycogen-breakdown in embryogenesis will compete and thus the hatching rate will be low. 6. Assuming that in the depot experimentsin vitro at 21° C and with free access of oxygen, glycogen metabolism can be considered one parameter of theNd rate, a satisfactory explanation of our experiments can be offered. With aCDS Depot,Nd stimulatory mechanism will always work satisfactorily, assuming a considerable resynthesis of glycogen of previously cold-exposed depot material.Nd rate ofD Depot will first follow the glycogen parameterin ovo; when removed from diapause, it may be capable of the resynthesis of glycogen. This will also explain the correspondingNd rates of theDS Depots. There is no correlation between theNd rates ofC Depots and the glycogen parameterin ovo. S Depots acquire a dependency on the glycogen parameter, which is independent of exposure to high temperature and oxygenin vitro. Further investigations on the glycogen metabolim of the depot materialin vitro are necessary to clarify these hypotheses. 7. The observations on the physiological phases inBombyx may also hold true for egg diapause of other insects. Various experiments with eggs of other strains ofBombyx with different reaction norms may substantiate our present conclusions. The enzymatic basis of the regulatory mechanisms with special regard to chorion should receive further clarification.