List of most probable exam, interview and viva questions on Embryology!
Embryology Question # Q.1. What are the main stages of frog and chick embryos?
Ans. Frog Embryo after Completion of Neurulation, Stage 17:
The neural system is in the form of a closed tube, broadened anteriorly where the brain will develop. The parts of the brain begin to be indicated by thickenings and constrictions of the neural tube, and the eye rudiments bulge laterally from the fore-brain. The notochord stretches underneath the neural tube from the midbrain level to the posterior end of the body, where it fuses with other tissues which are, as yet, in an undifferentiated state.
Under the notochord in the posterior part of the body there lies a strand of endodermal cells – the sub-chorda, a structure which exists for a short time and soon disappears. Lateral to the notochord the mesoderm is subdivided into segments, the somites. The endoderm surrounds the gut cavity, which is broad anteriorly and narrow in the posterior part of the body.
In the anterior part of the gut, lateral out-pushing’s represent the pharyngeal pouches. At the posterior end, an anal opening has been formed, but the mouth is not perforated, although the endoderm and ectoderm are in contact at the site of the future mouth. Above the place where the mouth will be, an ingrowth of the ectodermal layer represents the rudiment of the hypophysis.
Lateral to the foregut, masses of mesectoderm can be seen which give rise to the visceral skeleton. Underneath the foregut, groups of mesodermal cells represent the rudiment of the heart. The posterior end of the body above the anus has elongated somewhat in the form of a tail-bud, still in an early stage of development; hence this stage may be referred to as an early tail-bud stage.
4 mm. Frog (Rana Pipiens) Embryo, Stage 19:
The tail-bud has elongated and has developed into a clearly recognizable tail with an axis consisting of neural tube, notochord, and segmented mesoderm, and a fin fold all around its edge. The neural system has developed further; the eye vesicles have been constricted at the base and thus sub-divided from the brain.
The parts of the brain are more distinctly indicated. Ear vesicles have been formed to the right and left of the hindbrain. The nose rudiments are in the form of placodes, thickenings of the epidermis. The oropharyngeal membrane separating the stomodeum from the endodermal foregut has become thin and will soon be perforated.
Ventral to the mouth, the adhesive organs have become well developed. The foregut has become sub-divided into an anterior part, the pharynx, and a posterior part which will give rise to the gastric and duodenal section of the alimentary canal. The pharynx has produced pharyngeal pouches laterally.
Underneath the pharynx the heart rudiment is developing; in cross section one can see the thin-walled endocardial tube and the thicker parts of lateral plate mesoderm which will give rise to the myocardium and pericardium. The pronephric tubules are in the process of formation. The posterior part of the endodermal gut is still much in the same condition as in the preceding stages.
10 mm. Frog (Rana Pipiens) Embryo, Stage 23:
The embryo has changed its shape and has become a tadpole. The head and trunk are bloated and together form a more or less egg-shaped body. The tail has elongated and has become a powerful swimming organ, with segmented muscle laterally and broad fin folds dorsally and ventrally.
The brain is rapidly differentiating, and the forebrain has produced the two hemispheres of the telencephalon. The roof of the medulla has become membranous. The nose rudiments have invaginated and are in the form of sacs connected to the exterior by nares. The eye cup is clearly differentiated into a neural retina and a pigment layer. A lens has been formed.
The ear vesicles are in the process of producing the labyrinths; endolymphatic ducts can be seen on the dorsomedial aspect of each labyrinth. The pronephros consists of several (three) convoluted tubules which form thickenings on the sides of the body anteriorly, and the mesonephric ducts have been developed. The mouth is open and is surrounded by horny jaws, teeth, and soft circumoral papillae.
The branchial clefts are perforated, and finger-like external gills project from the sides of the head in the branchial region. The endodermal gut is differentiated into its various parts. In sections one can distinguish pharynx, esophagus, lung rudiments, stomach, liver, gallbladder, pancreas, and intestine. The intestine has already become coiled spirally and largely contributes to the bloating of the body of the tadpole.
Chick Embryo, Stage 10 (29 to 30 Hours):
The most conspicuous parts of the chick embryo in this stage are the brain and spinal cord rudiments and somites, of which there are 9 or 10 pairs. The brain shows a subdivision into primary brain vesicles, and eye vesicles are already prominent. Nevertheless, the closing of the neural tube is not quite completed – anteriorly the cavity of the neural tube is still open to the exterior by the neuropore; at the posterior end the neural folds have not come together, and there is still an open neural plate present.
Still farther back, the remnants of the primitive streak have not yet disappeared. Underneath the neural tube lies the notochord which at its posterior end is continuous with the primitive streak material. Posterior to the somites lies the still un-segmented mesoderm. The body folds have undercut the anterior end of the embryo, so that the foremost part of the head is lifted above the surface of the yolk, but farther back the separation of the body from the yolk sac has not yet begun.
The anterior part of the gut is already closed and separated from the yolk sac, but from the anterior trunk region onward the gut is open ventrally. Pronephroi have started developing. The blood vessel system is represented by separate vessels on the right and left sides of the body, and only in the heart the rudiments of the right and the left sides are fusing together. There is one pair of aortic arches (the first) at the anterior end of the body. The embryo as a whole is symmetrical.
Chick Embryo, Stage 15 (50 to 55 Hours):
The anterior end of the embryo is twisted to the right, so that the left side of this part of the embryo lies flat on the surface of the yolk, largely because the brain of the embryo has become bent at an angle at the midbrain level. The parts of the brain are clearly recognizable. The eye is already in the eye cup stage with a lens lying in the pupil.
The lens is, however, not yet separated from the epidermis and is in the form of a sac with an opening to the exterior. The ear rudiments are also in the form of pockets open to the exterior. The nose rudiment is still only a thickening of the epidermis. Masses of cells lateral to the brain represent the rudiments of the large cranial ganglia.
The number of pairs of somites has greatly increased, but at the posterior end of the body there is still some un-segmented mesoderm present. The neural tube is not completely closed at the posterior end, and even a remnant of the primitive streak is to be found, though immigration of cells from the streak has ceased.
The first three pairs of pharyngeal pouches have been formed. The blood vessel system of the embryo has undergone a marked development. The heart is a large and conspicuous organ just underneath the head. A paired ventral aorta conveys blood into the first pair of aortic arches, which are still the only ones to carry blood.
The dorsal aortae are paired anteriorly and posteriorly but are fusing in the median region. Anterior cardinal veins are well developed, but the posterior cardinal veins are very small. The yolk sac circulation is carried on by means of large vitelline veins joining the sinus venosus arid the vitelline arteries, which at this stage appear to be direct continuations of the dorsal aortae.
Embryology Question # Q.2. Explain the process of induction and differentiation of animal cell.
Ans. The physicochemical nature of the processes of induction concerns the way in which the inducing substances control the differentiation of cells. In principle it may be assumed that the inducing substances penetrate into the cells and, by interfering in the metabolic mechanisms in the interior of the cells, change their physicochemical composition. Although this mechanism of action seems to be fairly plausible, it is not self-evident.
Radioactive tracers have been used to learn whether, in the process of embryonic induction, substances pass from the inducing tissue into the reacting tissue. Labeled amino acids, methionine 35S and glycine 14C, and a nucleic acid precursor, orotic acid 14C, were used in some experiments. These compounds are taken up into the tissues of the animal (embryo).
Next, inducing parts—roof of the archenteron or parts of brain rudiments—are excised from the embryo containing radioactive substances and are transplanted into a normal animal (embryo) where the graft may induce neural plates or other structures from the host tissue. The animal (embryo) is then fixed and cut into sections, and autoradiographs are prepared.
It has been found that the radioactive atoms do not remain restricted to the cells of the grafts, but that they become fairly widely dispersed in the host embryo. A high radioactivity is shown by induced neural plates, which may be the result of the passage of inducing substances from the graft into the host tissue.
However, the animal (host) neural plates and tubes are also strongly radioactive, and radioactivity can also be discovered in the mesodermal and endodermal tissues of the host. These results are consistent with the assumption that the radioactive atoms are carried around in the host tissue by simple diffusion.
Furthermore, it cannot be claimed with certainty that the diffusing substance was actually the macromolecular material which can reasonably be assumed to be the inducing agent. It could well be that the radioactive atoms were carried around as components of small molecules (single amino acids, mononucleotides, and even smaller organic and inorganic molecules).
The preceding criticism does not apply to another set of experiments in which use was made of immunological methods to trace the passage of substances from the inductor into the reacting tissue. Antibodies were prepared against malignant tumor tissue used in one experiment and against a purified protein preparation isolated from guinea pig bone marrow in another experiment.
The antibodies in the antiserum were later coupled to a fluorescent dye, rhodamine B 200. Triturus ectoderm was exposed to the action of the inductor (tumor tissue in one case, protein preparation in the other) for a few hours, then fixed, and cut into sections. The sections were then treated with the antiserum coupled with the fluorescent dye.
Owing to antigen-antibody binding, the antibody molecules with the attached dye became localized in the section in positions which indicated the distribution of the antigen molecules (the molecules of the inductor). The fluorescent dye made these locations easily discernible. Distinct fluorescence was found in the reacting tissue, thus showing that antigen molecules had actually penetrated into it.
Since the specificity of the antibody-antigen reaction would have disappeared if the antigen molecules were degraded to any great extent, it is evident that the macromolecules of the materials used as inductors penetrated into the reacting cells without being split up into small components.
As a final proof, a radioactively labeled “mesodermalizing factor” was prepared, and it was found to actually enter the cells exposed to this factor.
An attempt was made to see whether the purified “mesodermalizing factor” binds to DNA. The results were inconclusive. The possibility remains, therefore, that there exists in the cells of the gastrula some kind of “receptor” molecules which interact with the inducing substance and mediate its influence on the genome of the reacting cells.
Some evidence on the mechanism of induction has been provided by the use of metabolic poisons coupled with induction experiments. If the induction is mediated by the diffusion of some specific proteins (or nucleoproteins) from the inducing to the reacting tissue, then it may be postulated that a specific protein has been manufactured in the inductor at some stage and that a corresponding mRNA had been transcribed from a locus on the DNA.
Secondly, if the reacting tissue acquires new properties, this, in all probability, means that some new substances have been produced in the reacting cells or that existing substances have been modified. In both cases, it would be suspected that new proteins are involved, a circumstance which again presupposes transcription of a new kind of mRNA and translation into a previously absent new protein.
That the change is caused simply by the presence in the reacting cells of the inducing substance, which had passed into them from the inductor, is highly improbable, especially in view of the possibility of neural induction by way of sublethal cytolysis.
The synthetic mechanisms of the reacting cells are obviously involved in the transformation. Consequently, the question is, what would happen to the process of induction if transcription or translation were prevented in the inductor, or in the reacting tissue, or in both?
In some experiments the dorsal blastopore lips of young Triturus gastrulae were cultivated for up to 20 hours in a medium containing actinomycin D or puromycin, and then the explant was confronted with reacting ectoderm. Mesodermal induction or both mesodermal and neural inductions were obtained.
These results prove that neither transcription of mRNA nor synthesis of new proteins occurs in the inductor at the time of its action; that is, the inducing substance is already present at the beginning of gastrulation (which could be expected, since a killed inductor can induce).
If, on the other hand, the dorsal blastopore lip was explanted with the adjacent ectoderm and cultivated in a medium containing sufficient quantities of actionomycin D to inhibit RNA synthesis completely, induction could not take place. After two to seven days of cultivation it was found that while some differentiation of muscle and notochord occurred, there was no differentiation of nervous tissue.
Since no new mRNA could be produced under the circumstances, it follows that the mRNA for notochord and muscle differentiation was already present at the time of explantation— at the beginning of gastrulation. For the ectoderm to start neural differentiation, however, it was necessary for the reacting cells to produce new mRNA by transcription from the DNA in the reacting cells. Such transcription was made impossible by the presence of actinomycin D, and the result was that no neural induction could be detected.
Lastly, Triturus gastrula ectoderm, which was exposed to an inductor in a “sandwich” experiment, was separated from the inductor and treated with actinomycin D for six hours, after having been disaggregated to allow better penetration of the poison into the cells.
In controls, the dis-aggregated cells fused together again and differentiated into neural and mesodermal tissues, as would be expected in view of the inductor which had been used – Actinomycin-treated explants also re-aggregated and remained viable for days, but only atypical epidermal differentiation took place.
This experiment shows that even though an “inducing substance” may have penetrated into reactive cells, their transformation into differentiated neural and mesodermal cells cannot occur without active participation of the nuclei of reacting cells, and particularly without an RNA (presumably mRNA) being produced as a crucial link in the process of induction.
Embryology Question # Q.3. Explain the Variation in Spermatozoon and Egg Cytoplasm.
Ans. There is some variation in different animals as to how much of the spermatozoon is taken into the interior of the egg. In many animals, notably in the mammals, all the internal parts of the spermatozoon, nucleus, and components of the middle piece and the axial filaments of the tail penetrate into the cytoplasm and for a short time may be seen lying intact in the interior of the egg.
In some animals (echinoderms), however, the tail of the spermatozoon breaks off and is left outside the vitelline membrane, and even the middle piece of the spermatozoon may be left without, so that only the nucleus and the centrosome enter the egg (Nereis). That the tail of the spermatozoon often does not enter the egg gives additional proof that its functions are purely locomotive.
The information concerning the middle piece of the spermatozoon is not unequivocal. Although components of the middle piece appear to enter the egg cytoplasm in most cases, there is no definite proof that any constituents of the spermatozoon except for the nucleus and the centrosome play an active part in subsequent development. The mitochondria contained in the middle piece have been observed in some cases to scatter in the cytoplasm of the egg, but it is not known how long, they maintain their existence there.
The subsequent behavior of the spermatozoon nucleus is dependent on the stage of maturation (reduction divisions) which the egg has reached at the time of fertilization. In the sea urchins the eggs are shed and become fertilized after the reduction divisions have been completed and both polar bodies have been extruded from the egg. This is, however, by no means a general occurrence.
In vertebrates the rule is that the egg completes its first reduction division in the ovary and reaches the metaphase stage of the second meiotic division. At this stage all further progress is arrested, ovulation takes place, and the egg may become fertilized. The second reduction division is completed and the second polar body extruded only if the egg is fertilized by a spermatozoon or activated in some other way.
In ascidians, some molluscs, and annelids, the egg reaches only the metaphase of the first meiotic division when it becomes ripe and is fertilized; only then does the egg complete the first reduction division and carry out the second. Lastly, in some annelids, in nematodes, and in chaetognaths, the eggs are fertilized even before the beginning of meiotic division, while the oocyte nucleus is still intact.
It follows that although in animals like the sea urchins the spermatozoon nucleus may immediately proceed to join the egg nucleus, in other cases the immediate effect of the fertilization, as far as the nuclear apparatus is concerned, is the completion of the meiotic divisions, only after which the fusion of the male and female pronuclei may take place.
When the spermatozoon first fuses with the egg cytoplasm, it moves with the acrosome (or acrosomal filament) at its front. The nucleus and the centrosome, in that order, are arrayed behind the acrosome. After fusion of the two gametes, however, a rotation of the nucleus and the centrosome can be observed in many, though not in all, animals, the centrosome coming ahead of the nucleus and the nucleus turning 180 degrees so that its original posterior end turns forward.
The other parts of the spermatozoon, if still discernible by this stage, lose connection with the nucleus and the centrosome. Both the nucleus and the centrosome change in appearance. The nucleus, which is now referred to as the male pronucleus, starts swelling, and the chromatin, which is very closely packed in the spermatozoon, again becomes finely granular.
By imbibition of fluid from the surrounding cytoplasm the pronucleus becomes vesicular. The centrosome, at the same time, becomes surrounded by an aster, similar to that of the centrosome in the early stages of an ordinary mitosis. While these changes are occurring, the sperm nucleus, together with the centrosome, moves through the egg cytoplasm toward the area where fusion with the egg nucleus, the female pronucleus, is to take place.
This area is generally near the center in holoblastic eggs having a fairly small amount of yolk, but in telolecithal eggs it is in the center of the active cytoplasm at the animal pole of the egg. As the sperm head moves inward it may be accompanied by some cortical and subcortical cytoplasm. If the latter is heavily pigmented, as in amphibian eggs, the trajectory of the sperm head may be marked by pigment granules trailing along its path. This is sometimes referred to as the penetration path.
The female pronucleus also has to traverse a greater or lesser way before it reaches the male pronucleus. At the beginning of its migration the female pronucleus is invariably at the surface of the egg, where the second meiotic division has been taking place, since it is only after the completion of the meiotic division that the nucleus of the egg may fuse with the nucleus of the spermatozoon.
The haploid nucleus of the egg, after the completion of the second meiotic division, is often in the form of several vesicles known as the karyomeres. This fuse together to form the female pronucleus, which swells and increases in volume as it approaches the male pronucleus. In the last stage before they meet, the male and female pronuclei may become indistinguishable.
The actual fusion of the male and female pronuclei to form a single zygote nucleus may differ in detail among different animals. In some animals the two pronuclei actually fuse together; that is, the nuclear membranes become broken at the point of contact, and the contents of the nuclei unite into one mass surrounded by a common nuclear membrane – the zygote nucleus.
At the approach of the first cleavage of fertilized eggs of sea urchins and vertebrates, the nuclear membrane dissolves, and the chromosomes of maternal and paternal origin become arranged on the equator of the achromatic spindle. In other cases, however, the male and female pronuclei do not fuse as such, but the nuclear membranes in both dissolve and the chromosomes become released.
In the meantime, the centrosome of the spermatozoon has divided in two and a spindle has been formed to which the chromosomes derived from the male and the female pronuclei become attached. (It is important to note that in normal fertilization the achromatic figure of the first and subsequent cell divisions is produced by the centrosome off the spermatozoon.)
Only after completion of the first division of the fertilized egg do the paternal and maternal chromosomes become enclosed by common nuclear membranes in the nuclei of the two daughter cells into which the egg has become divided (Ascaris, some molluscs, and annelids). In both types of fusion the chromosomes of the maternal and paternal sets retain, of course, their individuality.
Lastly, in some animals, of which the copepod Cyclops is a well-known example, the paternal and maternal nuclear components remain separate for some time, even after cleavage has started, so that each blastomere has a double nucleus consisting of two parts lying side by side, but each surrounded by its own nuclear membrane.
A closer union of the homologous chromosomes takes place much later in preparation for meiosis in the gonads of the new individual and also in cases of somatic conjugation of chromosomes, as in the salivary gland chromosomes of Drosophila.
In addition to activating the egg and providing an opportunity for amphimixis, the fusion of the spermatozoon with the egg (or the parthenogenetic activation of the egg) causes in many animals, perhaps in all, far-reaching displacements of the cytoplasmic constituents of the egg.
As a result of this, the distribution of various cytoplasmic substances and inclusions in the egg at the beginning of cleavage may be considerably different from that in the unfertilized egg, and even qualitatively new areas may sometimes appear. It will be evident later that these changes in the organization of the egg at fertilization may be profoundly important for further development of the fertilized egg.
One result of the extrusion of the cortical granules is that a large part of the original outer egg cell surface becomes replaced by the inner surfaces which surrounded the cortical granules and now are everted onto the exterior.
Embryology Question # Q.4. Parthenogenesis Approach of Fertilization?
Ans. A different approach to the solution of the problem of fertilization lies in attempting to imitate the action of the spermatozoon by some known agent. It has long been known that in some animals an egg can develop without fertilization, as in the aphids, phyllopods, and rotifers at some times of the year, or as in bees and wasps in which a fertilized egg produces a female individual and an unfertilized egg develops into a male.
These are cases of natural parthenogenesis (virginal reproduction). In other animals, such as most of the echinoderms and many others, the eggs under natural conditions do not develop unless they are fertilized. It has been found, however, that certain treatments of the ripe eggs may incite them to develop, and this phenomenon is known as artificial parthenogenesis.
A great amount of work on artificial parthenogenesis has been done with sea urchin eggs. O. Hertwig and R. Hertwig discovered that ripe sea urchin eggs may be caused to start developing by treatment with chloroform or strychnine.
Later, it was found that the same and even better results may be obtained by treating the eggs with a variety of substances – hypertonic or hypotonic seawater; various salts, such as the chlorides of potassium, sodium, calcium, magnesium, etc.; weak organic acids—butyric acid, lactic acid, oleic and other fatty acids; fat solvents—toluene, ether, alcohol, benzene, and acetone; and urea and sucrose.
Similar results are obtained by temperature shocks—that is, by transferring the eggs for a short time to warm (32°C.) or cold (0° to 10°C.) water; by electric induction shocks; by ultraviolet light; and even by shaking the eggs in ordinary seawater.
This long list of agents (which is by no means exhaustive) clearly shows that there is no one agent which can be recognized as the specific cause of the activation of the egg, the cause which determines the nature of the processes that are to take place.
Obviously, factors determining the nature of the reaction of the egg are contained in the egg itself. The agents causing the artificial parthenogenesis of the egg are instrumental only as factors triggering off this reaction of the egg. This becomes particularly clear due to experiments in which an ionophore was used as a parthenogenetic agent.
Ionophores are a group of chemicals which make cellular membranes permeable to certain ions—and not only the outer cell membrane, the plasmalemma, but internal membranes as well. An ionophore which is specific for bivalent positive ions—of which the Ca ion is one—have been found to be extremely efficient in causing parthenogenetic development of eggs in a wide group of animals including sea urchins, tunicates, molluscs, amphibians, and mammals.
It is obvious that in these experiments the ionophore caused the upsurge in the level of free Ca ions in the egg cytoplasm, which is such a characteristic, and possibly the key reaction of the egg to fertilization by the spermatozoon. The Ca ions must have been released from some internal source, as the ionophore can cause parthenogenetic activation in the absence of Ca in the medium surrounding the eggs.
The other agents used for parthenogenetic activation of the egg may either have the same effect, or possibly cause one of the other changes that take part in activation of development. Ammonia as a parthenogenetic agent may be causing the alkalinization of the egg cytoplasm—a later process, bypassing the Ca ion release.
We will conclude this section by adding some information on natural and artificial parthenogenesis in some vertebrates. In frogs, artificial parthenogenesis may be achieved by some of the methods used on echinoderm eggs, such as the use of hypertonic and hypotonic solutions, some poisons (corrosive sublimate), and electric shock.
The activation achieved by these methods is incomplete, however, and the development does not go beyond abortive cleavage. A more efficient method is pricking the eggs with a fine glass needle (a method also used successfully with echinoderm eggs); for full success with this method, however, it is necessary that the needle be smeared with blood or be contaminated by cells or cellular particles from other tissues.
If particles from foreign cells are thus introduced into the ripe egg cell, the cleavage is greatly improved, and a small percentage of the treated eggs may go through the whole development apparently quite normally. Instead of pricking the eggs with a contaminated needle, constituent parts of cells may also be introduced into the egg with a micropipette.
In this way it has been possible to investigate what fractions of cell constituents are most active in causing parthenogenesis, and it has been found that “large granules” of a centrifuged tissue homogenate (this fraction includes the mitochondria) have the strongest effects, while the liquid supernatant had no more effect than did the pricking with a clean needle or a needle wetted with a buffer saline solution.
No successful experiments have been reported in producing artificial parthenogenesis in birds, but in connection with other work it may be of interest to note that spontaneous parthogenetic development has been observed in two representatives of the class, the domestic turkey and the common domestic hen.
Eggs laid by female turkeys which had been isolated from males start developing in a fairly high percentage of cases (up to 41.7 per cent in some recent experiments); although there is still a high percentage of abnormal development, a small number of embryos reach the hatching stage.
All the poults, the sex of which could be determined, were males. A few of these reached maturity and one was used, with success, to fertilize eggs of normal females. More recently, similar results have been obtained with hens.
A very peculiar case of spontaneous parthenogenesis is presented by some lizards, in particular the lizard Lacerta saxicola armeniaca which is found in the Caucasus. In this subspecies no males exist in nature, and the population is propagated exclusively by parthenogenetic females. Some embryos start developing as males, but these all die in early stages.
In mammals the possibility of artificial parthenogenesis was discovered in connection with experiments on cultivating unfertilized eggs in vitro which had been collected from the fallopian tubes. Extensive investigations in this field were undertaken by Pincus and his collaborators. Most of the experiments were done with rabbits’ eggs.
It was found that if the eggs are simply kept for up to 48 hours in the ordinary tissue culture medium (blood plasma plus embryo extract), some of them become activated. The first sign of activation in this case is completion of the second meiotic division and extrusion of a second polar body.
Some of the eggs even go beyond that stage and start cleaving. Chemical treatment (with butyric acid, which has been used for activating sea urchin eggs) does not seem to yield any better results. However, a temperature shock, particularly a treatment with cold, is more effective.
To allow the activated eggs to develop further, in some experiments they have been injected into the fallopian tubes of rabbit does, which were made “pseudo-pregnant” by mating with a sterile buck or by injection of hormones (the luteinizing hormone).
In the body of the female the development progressed further, and quite a number of embryos reached the blastocyst stage (18 per cent in one experiment in which the eggs were activated by cooling for 24 hours at 10°C.). In two cases in which the fertilization of the eggs by spermatozoa seems to have been completely excluded, the parthenogenetic embryos completed intrauterine development, and one of the young was born alive.
In another case the eggs were given a cold shock in vivo by opening the body cavity of a rabbit doe which had unfertilized eggs in its fallopian tubes and cooling the fallopian tubes with cold water. One live young was born. So far, these are the only records of living mammalian young produced by parthenogenesis.
Embryology Question # Q.5. Explain the Chemical Analyses of Inducing Substances in animals.
Ans. it is not very difficult to obtain extracts from tissues showing inductive action when brought into contact with competent embryonic cells, and such extracts may be prepared in a variety of ways. Perhaps the simplest way is to homogenize the tissue and to sediment the larger particles by centrifugation.
However, chemical extraction by a variety of solvents has also been tried out. Adult tissues, as a source of inducing substances, enable the investigator to make use of a number of methods of purification which are normally applied in biochemical investigations, since one may start with sufficiently large quantities of material.
Two schools have been especially successful in purifying extracts from tissues capable of imitating the action of the natural “primary organizer” – namely, the school of Yamada in Japan and the school of Tiedemann in Germany. Yamada and his collaborators used guinea pig liver and guinea pig bone marrow as their main sources of inducing substance.
The tissues in their experiments were homogenized, and separation of fractions was achieved by ultracentrifugation, by sedimentation of nucleoproteins with streptomycin sulfate and of proteins with acidified solutions and with ammonium sulfate, and eventually by chromatographic separation on a diethylaminoethyl cellulose chromatographic column. Tiedemann and collaborators used 11-day-old chicken embryos as starting material.
The extracts prepared by workers of both schools contain macromolecular substances, some of which show all the chemical and spectrophotometric reactions of proteins; others are ribonucleoproteins. The inducing power of these preparations is no weaker than that of the natural organizer.
Another similarity is that substances obtained by certain modifications of the preparation procedures have more specific inducing properties, such as those of archencephalic, deuterencephalic, and spinocaudal inductors. A particular protein fraction, with spinocaudal inducing properties was prepared from 11-day-old chick embryos.
After precipitation of homogenates with ammonium sulfate, the supernatant was extracted with phenol and the protein chromatographed on carboxymethyl cellulose, and subsequently subjected to electrophoresis on Sephadex G 100. In the last stage the substance was purified by electro-focusing at pH 7-9.
Approximately 40µg. was obtained from 1 kg. of chicken trunks. The purified substance had a molecular weight of 35,000, and its activity was increased 10,000,000-fold compared with the initial material. 1 × 10-4 µq. of the purified product introduced into the blastocoele of a gastrula caused inductions in 50 per cent of the cases.
While tissues of the chick embryo are an “abnormal inductor” insofar as the amphibian gastrula is concerned, a preparation has also been made of a similar substance from Xenopus morulae and gastrulae, although the purification could not be performed to the same extent because of difficulty in providing sufficient initial material. Fractions containing only deoxyribonucleoproteins were completely inactive.
It seems somewhat disconcerting that inducing activity should be present in two different groups of chemical compounds such as proteins on the one hand and ribonucleoproteins on the other. Furthermore, seeing that synthetic processes in cells are controlled by different kinds of ribonucleic acids, it would be of great importance to know whether the participation of ribonucleoproteins in induction may mean that the ribonucleic acids involved are instrumental in bringing some kind of “information” from the inductor cells to the reacting cells.
Fortunately, some further experiments provide a clear and satisfactory answer to these problems. It has been convincingly shown by various investigators that the degradation of ribonucleic acid in inducing preparations by ribonuclease does not reduce the inducing ability of such preparations.
In one experiment, for instance, a very active preparation was obtained from a liver homogenate by ultracentrifugation. The microsome fraction was used, which contains most of the cytoplasmic ribonucleic acid. The experimental lot of this preparation was incubated for 3.5 hours with ribonuclease.
After ribonuclease treatment the substance was precipitated, and its inducing ability (as well as that of untreated controls) was tested by placing pieces of precipitate between two layers of gastrula presumptive ectoderm. The preparations were cultivated as explants in a saline solution.
Although the ribonucleic acid content of the inductor was reduced to less than 1 per cent of the original quantity, the inducing power of the preparation was the same as in controls, not only with respect to the total number of inductions but also with respect to the type of induction observed. This result is supported by many similar experiments, and it appears therefore that ribonucleic acid cannot be the essential part in the chemical composition of an inductor.
In contrast to the action of ribonuclease, treatment of tissues or tissue extracts with proteolytic enzymes—pepsin, trypsin, or chymotrypsin—destroys their ability to induce.
It was found that the preparation completely lost its ability to induce neural structures after treatment for 120 minutes and only caused weak atypical inductions. The induction in the control compared with the lack of neural induction after treatment of the liver extract with trypsin (120 minutes).
There seems to be no doubt that the integrity of the proteins is essential for inductions by tissues and tissue extracts. Exactly similar results have been obtained by workers of the Tiedemann School. Likewise, the “conditioned medium” in the experiments of Twitty and Niu is not substantially affected by ribonuclease treatment, but addition of trypsin or chymotrypsin prevents induction completely.
The general conclusion from the preceding experiments is that the inducing principle in both types of experiment is of a protein nature. The protein may or may not be coupled with ribonucleic acid, but the inducing action is due to protein alone.
There is hardly reason to believe that the treatment with purified extracts of inducing tissues, any more than the “conditioned medium,” acts in some roundabout way by “unmasking” an inducing substance present in the reacting cells. There is no trace of sublethal damage, and the specificity of the preparations and their activity in minute quantities are evidence in the same direction.
Rather, one feels that the proteins contained in the tissue of advanced embryos (nine-day-old chicken embryos) and adults have some properties in common with the natural inducing agents liberated from the roof of the archenteron, if they are not actually identical to these agents.
As a further corroboration of this view, we may mention that an extract obtained from gastrulae and neurulae of Triturus, and presumably containing the “natural” inducing substance, is inactivated by trypsin treatment.
Embryology Question # Q.6. How to develop an embryo?
Ans. In every stage of its development the embryo is a living organism, and like every living organism it requires foodstuffs for its vital processes. It assimilates its food and metabolizes organic substances to gain energy for its maintenance and for performing the processes which together constitute its development.
Furthermore, the new individual has to increase in size to a variable, but usually very considerable, degree until it approximates the structure of its parents. The growth process again involves the consumption, processing, and partial combustion of a vast quantity of foodstuffs.
Since the egg is a single cell and does not have any of the organs which an adult uses to procure and utilize its food supply, the supplying and utilization of food for the developing embryo have to be organized along lines that are quite special and specific for the process of embryonic development.
The primary source of nutrition in the eggs of most animals is the reserve material stored in the egg cell during its development in the ovary. This material is metabolized; it is partly broken down as a source of energy and partly transformed into the substances of which the various organs of the new individual are built in the course of development.
In addition to materials contained in the egg, various substances may be taken up from the environment. The nature and the amount of extraneous materials used by the embryo depend very largely on the environment in which the embryo develops. A vast number of animals rather early attain a stage in which the young individual can start feeding and thus can become self-supporting.
This is often greatly facilitated by the egg’s developing in the first instance into a larva, instead of directly into an adult. Larvae do not necessarily have the ability to take in food. In some animals, for instance in the tunicates, no food is taken during larval life.
In this latter case the advantage of a larval stage lies in the ability of the larva to move. The adult is sedentary. The larva thus serves to effect dispersal of the animals. In many parasites, especially internal parasites, larval stages serve for the infestation of new hosts.
The sea urchin egg may be taken as an example of a small egg with relatively little yolk. It develops in seawater and after a very short time (35 to 40 hours) produces a larva, called a pluteus, which has an alimentary canal of three parts- a foregut, opening to the exterior by a mouth, a dilated stomach, and a hindgut with anus.
The pluteus swims freely in water with the aid of a ciliary band drawn out into loops along the edges of arms supported by a complicated calcareous skeleton. It feeds on minute planktonic organisms, mainly algae, which are injected into the gut by the ciliary action of the stomodeum.
The developing embryo takes in some water from the environment and also quite a considerable quantity of mineral substances which are mainly utilized in building up the calcareous skeleton. The embryo loses a large part of its carbohydrates (including almost all of the glycogen) and some fat.
There is only a very slight decrease in nitrogen; according to some more recent investigations, the total nitrogen of the sea urchin egg does not change during the period up to the pluteus stage.
Water is absorbed as a general rule by embryos developing in an aquatic medium. The amount absorbed may be quite considerable, as can be seen in the case of an egg-laying dogfish, Scyllium canicula. Additional ash (salts) is also taken up from surrounding seawater by marine invertebrates (coelenterates, molluscs, crustaceans, and echinoderms).
Among fishes, the developing embryos of the elasmobrancns absorb salts, but the embryos of some freshwater fishes, such as the Salmonidae, do not absorb salts from the surrounding medium although they take in water. The same is true of the developing embryos of the Amphibia. The animals whose eggs develop in fresh water cannot depend on the surrounding medium for the intake of salts because fresh water does not contain the necessary salts (especially Na and K ions) in sufficient quantity.
The turnover of substances in the developing egg of a frog will serve as an example of an animal developing in fresh water. Very little increase in the amount of active cytoplasm during the early development of the embryo, that there is somewhat more of an increase in the amount of nucleic acid nitrogen (about 25 per cent), and that simultaneously there is a very considerable decrease in the total carbohydrates, due almost exclusively to the loss of glycogen, which is thus the main source of energy for the maintenance and development of the embryo.
The increase of water is considerable. According to some available data, it accounts for a 75 per cent increase in the total weight of the embryo during the corresponding stages. There is also some uptake of salts, particularly calcium, from the environment.
An entirely new situation is faced by animals that have abandoned the aquatic medium and have become completely terrestrial. Some of them have attempted a compromise by returning to the water for egg laying. This is true of most of the amphibians.
Other amphibians (many terrestrial frogs, some salamanders, and the Gymnophiona) lay their eggs on land but in damp places—in burrows underground, etc.—where the eggs can absorb the minimal quantities of water that are necessary for their development.
Even among the reptiles there are some whose eggs take up water from the environment. This is the case with turtles. For instance, the egg of the turtle, Malaclemys centrata, which weighs 10.58 gm., absorbs 3.07 gm. of water during its development. This is made possible because the eggs are laid in damp sand.
In the eggs of other reptiles and of birds, there is no longer a possibility for the absorption of water from the exterior. The egg membranes have become watertight. On the other hand, the loss of water from the egg by evaporation is reduced to a minimum. The only substance that is taken from without is the oxygen necessary for oxidative processes in the egg.
Otherwise, the egg has become a closed system, developing at the expense of the substances stored inside the egg itself. Such an egg, which has become self-sufficient (except for oxygen intake and CO2 outflow), is called a cleidoic egg. Cleidoic means boxlike.
Independently of the vertebrates, the terrestrial arthropods have also evolved cleidoic eggs. Some of the stages of this evolution can still be traced among contemporary insects. For instance, in the eggs of grasshoppers (Melanoplus and Locusta) there is an intake of water during development.
In Melanoplus the water content increases from 2.5 to 4.7 mg. In the eggs of other insects, however, no water can be taken in, and throughout the development of the eggs there is only a certain amount of water loss by evaporation. Such eggs are therefore as much cleidoic as the egg of a bird.
There is a fairly obvious advantage in the young individual’s being more advanced in development and growth when it emerges from the egg. This advancement may be achieved by the amassing of greater and greater supplies of foodstuffs in the egg. Increase of food supplies in the egg may be correlated with the elimination of a larval stage—as in cephalopods, among the molluscs; and in elasmobranchs, reptiles, and birds, in the vertebrate phylum.
The eggs of birds present an example of a very abundant supply of the egg with food material for the nourishment of the embryo. The chicken that hatches from the egg is already essentially a bird. The principal difference, besides the small size, lies in the development of the feathers. The body of a newly hatched chicken is covered with down, which is a simplified form of a feather. As soon as the down is replaced by typical feathers, the chicken acquires all the typical features of a bird.
A very special though widespread method of increasing the chances of survival of the offspring consists in retaining the eggs in the mother’s body and letting them develop there to a greater or lesser degree. The eggs are usually retained in the oviducts (which are then usually referred to as uteri), but in some cyprinodont fishes (Poecilia and Girardinus) the eggs begin their development while still in the ovaries. Sometimes, as in some salamanders, the young hatch from the eggs inside the mother’s body.
Occasionally, as in the adders, the eggs are laid intact, but the young begin to hatch as soon as the eggs are laid. The bearing of young developed from eggs which have been retained in the body of the mother, but without the maternal organism providing additional nourishment for the embryo, is known as ovoviviparity. Oviparity is, of course, the form of reproduction in which all or most of the development of the embryo in the egg occurs after the egg has been laid.
In typical cases of ovoviviparity the embryo is nourished by the food stored in the egg. A next step in evolution may be made when the embryo absorbs some substances present in the fluids filling the oviducts. The degree to which such oviductal fluids are used for the nourishment of the embryo is very variable.
In different species of the sharks and rays, all possible gradations may be found between such forms in which the embryo depends chiefly on the food supplied in the egg, and such forms in which the embryo depends chiefly on the food supplied by the mother.
The proportions of these two sources of nourishment may be best estimated by comparing the weight of organic substances in the egg and in the newly born offspring.
It will be seen that in the development of an egg-laying fish, the turnover of substances is very similar to that in the embryo of the sea urchin or frog – there is a decrease in organic substances, due to their combustion, and an increase in water content, which makes the young at hatching exceed the weight of the egg.
In the ovoviviparous fish, however, the uptake of organic substances from the maternal body not only compensates for their loss through combustion but brings about a total increase in the amount of organic material during development.
There is also a very considerable intake of water, so that the overall increase in weight of the embryo is many times that observed in a species in which the embryo depends on the egg reserves as the sole source of its organic materials. Ovoviviparity in several groups of animals seems to have been a transitional phase in the development of true viviparity.
True viviparity is achieved when the embryo establishes a direct connection with the maternal body, so that the nutrition can pass from the mother to the embryo without the intermediate state of being dissolved in the uterine fluid. The connection is established through a special organ, the placenta, which is an outgrowth of the embryo joined to parts of the maternal body especially modified for this purpose.
Viviparity and placentae have been developed in several groups of the animal kingdom independently of one another. Placentae are found in the protracheates (Peripatus), in the tunicates (Salpa), in several elasmobranchs, and in the placental mammals. The mode of origin and the structure of the placenta are different in each case mentioned.
The supply of nutrition to the embryo through a placenta appears to be a much more efficient method than the absorption of nourishment from the uterine fluids. The dogfish Mustelus laevis, in which a placenta is developed when the yolk sac of the embryo becomes connected to the walls of the uterus.
In this particular case the increase in organic materials during intrauterine life is more than tenfold as compared with the roughly fourfold increase of organic substances in Mustelus vulgaris, taken as an example of the ovoviviparous fish.
In mammals, which have developed the placenta to a degree of perfection not found in other animals, the increase in weight of the embryo is much greater, especially since the eggs of mammals are very small as compared with the eggs of viviparous fishes.