of embryology

CHAPTER 1 History of embryology

Embryology, the study of development from fertilization to birth, has always intrigued philosophers and scientists. Universal fascination with the way in which ‘life’ unfolds has led to spirited discussion of the complexities of the process amongst embryologists over the years. In 1899, Ernst Haeckel (1834–1919) considered that ‘Ontogeny is a short recapitulation of phylogeny’. In other words, ontogeny (the development of an organism from the fertilized egg to its mature form) reflects, in a matter of days or months, the origin and evolution of a species (phylogeny), a continuing process that is measured in millions of years. Although this is not entirely true, one has only to look at a 19-day-old sheep embryo to understand Haeckel’s viewpoint; the gill-like pharyngeal arches and the somites, for example (Fig. 1-1) are common to embryos of all chordates. Although trained as a physician, Haeckel abandoned his practice after reading The Origin of Species by Charles Robert Darwin (1809–1882) published in 1859 (and available at that time for fifteen shillings!). Always suspicious of teleological and mystical explanations of life, Haeckel used Darwin’s theories as ammunition for attacking entrenched religious dogma on the one hand and elaborating his own views on the other. However, in projecting phylogeny into ontogeny, Haeckel made one mistake: his mechanism of change required that formation of new characters, diagnostic of new species, occur through their addition to a basic developmental scheme. For example, because most metazoans pass through a developmental stage called a gastrula (a ball of cells with an infolding that later forms the gut) Haeckel thought that at one time an organism called a ‘gastraea’ must have existed, looking much like the gastrula stage of ontogeny. This hypothesized ancestral metazoan, he thought, gave rise to all multi-celled animals. Such a ‘single straight line’ conception of phylogeny, with all creatures standing on the shoulders of predecessors in one trajectory, has now been abandoned; phylogeny divides into a multitude of lines.

Fig. 1-1: Sheep embryo at Day 19 of development.

Many of the ancient Greek philosophers were interested in embryology. According to Democritus (ca. 455–370 BC), the sex of an individual is determined by the origin of the sperm: males arising from the right testicle (of course!) and females from the left. This hypothesis was somewhat modified by Pythagoras, Hippocrates and Galen. However, gender bias was always evident, for science was the privilege of men; philosophers mostly positioned females between man and animals, so males were supposed to originate from the stronger sperm of the right testicle.

The first real embryologist that we know about was the Greek philosopher Aristotle (384–322 BC). In The Generation of Animals (ca. 350 BC) he described the different ways that animals are born: from eggs (oviparity, as in birds, frogs and most invertebrates), by live birth (viviparity, as in placental animals and some fish) or by production of an egg that hatches inside the body (ovoviviparity, which occurs in certain reptiles and sharks). It was Aristotle who also noted the two major patterns of cell division in early development: holoblastic cleavage in which the entire egg is divided into progressively smaller cells (as in frogs and mammals) and the meroblastic pattern in which only that part of the egg destined to become the embryo proper divides, with the remainder serving nutritive purposes (as in birds). The fetal membranes and the umbilical cord in cattle were described by Aristotle and he recognized their importance for fetal nutrition. By sequential studies of fertilized chick eggs, Aristotle made the very important observation that the embryo develops its organ systems gradually – they are not preformed. This concept of de novo formation of embryonic structures, which is referred to as epigenesis, remained enormously controversial for more than 2000 years before becoming fully accepted; Aristotle was way ahead of his time. The enigma of sexual reproduction also intrigued Aristotle. He realized that both sexes are needed for conception but felt that the male’s semen did not contribute to conception physically, but by providing an unknown form-giving force that interacts with menstrual blood in the womb of the female to materialize as an embryo. In this case Aristotle was wrong. Ironically, in contrast to the non-acceptance of his correct views on epigenesis, his error about conception prevailed for about 2000 years, and it took a battle of almost a hundred years to correct it!

During those 2000 years, the science of embryology developed very slowly along descriptive anatomical lines. One of the first anatomical descriptions of the pregnant uterus of the pig was by Kopho (years of birth and death unknown) in the early 13^th century in his work Anatomio Porci. Kopho worked at the famous medical school of Salerno using pigs as models because human cadavers could not be dissected for religious reasons. During the early Renaissance, the famous and incomparably artistic anatomical studies of Leonardo da Vinci (1452–1519) included investigation of the pregnant uterus of a cow. His drawings of a pregnant bicornuate uterus and of the fetus and fetal membranes released from the uterus are reproduced in Fig. 1-2. In an accompanying drawing, Leonardo depicted a human uterus cut open to ‘reveal’, not a human placenta but a multiplex, villous, ruminant placenta (see Chapter 9)! Clearly it had not been possible for him to actually study a human pregnancy. The structures are described in the characteristic mirrored writing of Leonardo, who worked mostly in Florence.

Fig. 1-2: Drawings of Leonardo da Vinci. A: Top: A pregnant bicornuate uterus of the cow. Bottom: Fetus and fetal membranes showing the cotyledons of the placenta (see Chapter 9). B: Opened simplex uterus of human showing the fetus with the umbilical cord. Note that the placenta is of the ruminant multiplex type with several placentomes drawn on the cut wall of the uterus and, at the right top, a single cotyledon drawn at a higher magnification displaying its villous surface.

The Renaissance, from the 13^th to the 15^th century, was a great time for anatomical studies and, happily, coincided with the invention of book printing by Johann Gutenberg. Thus, the first major publication on comparative embryology was De Formato Foetu in 1600 by the Italian anatomist Hieronymus Fabricius of Acquapendente (1533–1619). Fabricius described and illustrated the gross anatomy of embryos and their membranes in that book, but was not actually the first to do so; another Italian anatomist, Bartolomeo Eustachius (1514–1574), had previously published illustrations of dog and sheep embryos in 1552. We now recognize the names of Fabricius, in the term bursa Fabricii (the immunologically competent portion of the bird gut), and of Eustachius in the Eustachian tube.

The work of Eustachius, Fabricius and others gave insight into how organs develop from their immature to mature forms, but left unanswered the basic enigma of how and where the mammalian embryo originates. However, the development of the microscope by Zacharias Janssen, a Dutch eyeglass maker, in 1590 ushered in a new era of embryological science to tackle that 2000-year-old question. The Dutch dominance in the optical field at that time may not be just a coincidence; the naval ambitions of their new empire required excellent telescopes and lens systems. Janssen’s microscope in its original form, however, was not really appropriate for cell and tissue research; it was approximately 2 metres long, achieved only 10 to 20 times magnification, and its principal use was to attract an audience at country fairs! In 1672, the Italian medical doctor Marcello Malpighi (1628–1694) published the first microscopic account of chick development, identifying the neural groove, the somites, and circulation of blood in the arteries and veins to and from the yolk. Malpighi also observed that even the unincubated chick egg is considerably structured, leading him to think that a preformed version of the chicken resided in the egg. Later (in 1722), the French ophthalmologist Antoine Maître-Jan (1650–1730) pointed out that although the egg examined by Malpighi was technically ‘unincubated’, it had been left sitting in the Bolognese sun in August and so was certainly not ‘unheated’. Nevertheless, Malpighi’s notion of a preformed chicken initiated one of the great debates in embryology that was to last throughout the 17^th and 18^th centuries. The question was: are the organs of the embryo formed de novo (epigenesis), or they are already present in a miniature form in the egg (or the sperm when this cell was discovered), a concept referred to as preformation. We will return to this debate in a moment.

The new microscopic techniques also prompted a vigorous search for the mammalian gametes. The chicken egg and its initial transformation into a chick were obvious, as Aristotle had described, but what mediated the formation of the embryo in mammals? Where was the mammalian egg to be found?

One of the earliest and most influential names in the fascinating story of the discovery of the mammalian egg was that of William Harvey (1578–1657), personal physician to the English kings James I and Charles I, and famous for his description of the circulation of the blood. In 1651, Harvey published De Generatione Animalium (Disputations touching the Generation of Animals) with a famous frontispiece showing Zeus freeing all creation from an egg bearing the inscription Ex ovo omnia (All things come from the egg). However, it should be realized that, far from advancing 17^th century knowledge of reproduction and embryology, Harvey’s observations in some ways impeded progress. From having studied with Fabricius, Harvey was imbued with Aristotle’s view that the semen provided a force that interacted with the menstrual blood to materialize as an embryo. Harvey set out to understand this process by looking for the earliest products of conception in female deer killed during the breeding season in the course of King Charles I’s hunts in his Royal forests and parks over a 12-year period. In the red and fallow deer that he studied, the male’s rut begins in mid-September and so Harvey dissected uteri throughout the months of September to December. Believing, wrongly, that copulation coincides with the onset of the rut, Harvey was mystified to find nothing that he recognized as an embryo until mid-November, some two months later. This forced him to the erroneous, but entirely logical conclusion that ‘nothing after coition is to be found in [the] uterus for many days together’. When he did find a conceptus, that, for Harvey, was the egg: ‘Aristotle’s definition of an egg applies to it, namely, an egg is that out of a part of which an animal is begotten and the remainder is the food for that which is begotten’.

Three factors of veterinary interest had led this brilliant man astray. First, he did not appreciate that the females did not come into oestrus until early October and so his estimates of breeding dates were wrong. Second, he dismissed the ovaries (‘female testicles’) as making no contribution to conception because they failed to swell up as the testes of males do during rut. Third, expecting to find an egg-shaped conceptus, he failed to recognize the ‘purulent matter … friable … and inclining to yellow’, which he observed much earlier after mating and describes quite vividly, as being the filamentous blastocyst so characteristic of ruminants. Had Harvey used a lens, or conducted his studies on species (like rabbits or horses) with spherical early conceptuses, discovery of the real egg might have been advanced considerably!

It is easy to be critical with hindsight of course, and we should remember that Harvey made important contributions to embryology: his descriptions of early development were impeccable; he was the first to observe the blastoderm of the chick embryo (the small region of the egg containing the yolk-free cytoplasm that gives rise to the embryo proper) and to indicate that blood islands form before the heart does; and he was aware of the gradual development of the embryo, subscribing to the school of epigenesis as did Aristotle.

The observations by Harvey and the search for the mammalian egg were extended by Regnier de Graaf (1641–1673) who performed detailed studies of the female reproductive organs, especially the ovary. De Graaf, like his friend Leeuwenhoek, worked in Delft. From a comparison of mammalian ovaries with those of chicken, de Graaf considered mammalian antral ovarian follicles to be the eggs; an assumption he confirmed by tasting! His contribution to science was later acknowledged by the German medical doctor Theodor Ludwig Wilhelm Bischoff (1807–1882) who introduced the nomenclature ‘Graafian follicle’. De Graaf also noted some connection between follicular maturation and the development of oocytes but, without an appropriate microscope, he could not substantiate this and his observations were for a long time forgotten. Had he lived a little longer (he died tragically early, at the age of 32), the discovery of the mammalian egg could probably have avoided a delay of about 150 years!

As it was, the first scientist to actually see the mammalian egg (which everyone believed to exist, but no one had seen) was the Estonian medical doctor Karl Ernst von Baer (1792–1876). He opened the ‘Graafian egg’, as the follicle was known at that time, and saw with his naked eye a small yellow point which he released and examined under the microscope (Baer, 1827). There, upon a first glance, Baer was stunned and could hardly believe that he had found what so many famous scientists including Harvey, de Graaf, Purkinje and others had failed to find. He was so overcome that he had to work up courage to look into the microscope a second time. The mammalian egg had been identified.

How about the spermatozoa? Anton van Leeuwenhoek (1632–1723), a Dutch tradesman and scientist from Delft, was the first to report having seen moving spermatozoa. He constructed a single lens microscope that magnified up to about 300 times. Technically, this microscope was an amazing achievement – no bigger than a small postage stamp and resembling a primitive micromanipulator. It was used close to the eye like a magnifying glass. Using this, Leeuwenhoek drew spermatozoa from different species. Initially, Leeuwenhoek was reluctant to study sperm and he questioned the propriety of writing about semen and intercourse. When he first focused his microscope on semen, Leeuwenhoek discovered what he then took to be globules. However, he so disliked the prospect of having to discuss his findings that he quickly turned to other matters. Three or four years later, however, in 1677, a student from the medical school at Leiden brought him a specimen of semen in which he had found small animals with tails, which Leeuwenhoek now observed as well. Consequently, Leeuwenhoek resumed his own observations and, in his own semen (acquired, he stressed, not by sinfully defiling himself but as a natural consequence of conjugal coitus), observed a multitude of ‘animalcules,’ less than a millionth the size of a coarse grain of sand and with thin, undulating, transparent tails. A month later, Leeuwenhoek described these observations in a brief letter to Lord Brouncker, president of the Royal Society in London. Still uneasy about the subject matter, he begged Brouncker not to publish it if he thought it would give offence. Leeuwenhoek’s observations prompted vivid discussions and controversies on the significance of these living objects. At first, it was commonly held that the tadpole-like creatures were parasites. Leeuwenhoek may have been biased towards that view as he was actually engaged in parallel studies of parasites and was the first to see Giardia, a protozoan parasite that infects the gastrointestinal tract. Giardia are flagellated and may, at poor microscopical resolution, resemble spermatozoa. Other scientists considered that the whirling action of the objects was meant to prevent solidification of the semen. In the preformation school, however, Leeuwenhoek’s observations encouraged the thought that the sperm head contained preformed miniatures of babies, foals, calves etc. and so these scientists became known as the spermists.

The two mammalian gametes had been identified. Instead of forming a common platform for further endeavours, however, this knowledge prompted a bitter dispute, as to whether the embryo arose from the egg or from the sperm!

In parallel with the search for the mammalian gametes, the combat between the schools of epigenesis and preformation became more and more intense. The latter had the backing of 18^th century science, religion and philosophy for several reasons. First, if the body is prefigured and just needs to be unrolled, no extra mysterious force is needed to initiate embryonic development. This was a religiously convenient point of view, paying proper respect to God’s creation of mankind. Second, if the body is prefigured in the germ cells, a further generation will already exist prefigured in the germ cells of the next, rather like Russian nested Matryoshka dolls. This concept was also convenient, ensuring that the forms of species would remain constant. The fact that at a certain point Matryoshka dolls cannot get any smaller would seem like an obvious objection to the concept today. However, there was no biological size scale at the time of these arguments because the cell theory of Theodor Schwann (1810–1882) was not proposed until 1847. Thus preformationists could claim, as formulated in 1764 by the Swiss naturalist and philosophical writer Charles Bonnet (1720–1793), that ‘Nature works as small as it wishes’. Basically, preformation was a conservative theory, and it was unable to answer some of the questions raised by the limited knowledge of genetic variation at that time. It was, for example, known that matings between black and white parents resulted in babies of intermediate colour, an outcome incompatible with preformation in either gamete.

In the late 18^th century, Caspar Friedrich Wolff (1734–1794), a German embryologist working in St. Petersburg, made detailed observations on chick embryos that resulted in the first strong case for epigenesis. He demonstrated how the gut arises from the folding of an originally indifferent flat tissue and interpreted his findings as evidence of epigenesis when, in 1767, he wrote that ‘When the formation of the intestine in this manner has been duly weighed, almost no doubt can remain, I believe, of the truth of epigenesis’. Wolff’s name lives on in the term ‘Wolffian duct’ for the mesonephric duct.