Author: Ibon Cancio, UPV/EHU Associated Professor in Cell Biology; researcher in the ‘Cell Biology & Environmental Toxicology’ (CBET) research group of the Plentzia Marine Station (PiE-UPV/EHU); Spanish scientific representative in the EMBRC Committee of Nodes

Sexual reproduction and fertilisation are a ‘tale of two cells’. Their discovery, the egg and the sperm, took place during a thrilling decade of research between 1660 and 1670 that occupied some of the most amazing scientists at the time: van Leeuwenhoek, de Graaf, Swammerdam, Steno. Eggs are not only a thing of hens and ducks, and the little things swarming animal testes (spermato…ZOA) are not parasites! These discoveries make for a thrilling story of competition and rivalry among scientists that has been published in a riveting text: ‘An Amazing 10 Years: The Discovery of Egg and Sperm in the 17th Century’ (Cobb, M. 2012). It’s a must-read! In any case, this short decade finished abruptly leading to 200 years of confusion… with tales of storks transporting children and little bees touching flowers! So we had an actress and an actor, but no movie! For that, two exceptional scientists would have to be born, Herman Fol (born in 1845) and Oscar Hertwig (1849), and they would have to turn to marine creatures (they studied ctenophores, echinoderms, cnidarians, molluscs, etc) to uncover the real story of the ‘birds and the bees’.

Picture of Herman Fol and Oscar Hertwig
Portraits of Oscar Hertwig (left) and Herman Fol (right), public domain.

Fol, although born in France, was a Swissman from Geneva and while he graduated in Berlin he initiated his studies in medicine and zoology at the University of Jena, in Germany. This was also the alma matter of the Hertwig brothers, Oscar and Richard, who both did most of their research in ‘brotherly’ collaboration. They all received the same influence, that of Ernst Haeckel. That is like being mentored in the business of scoring goals by Cristiano Ronaldo. For Haeckle, the explanation of all things in biology resided in the oceans. The professor took his young student Fol with him on a sampling expedition to the north and west coasts of Africa and the Canary islands in the winter of 1866-1867. Curiously enough, such a trip imitated the winter migration of the storks from central Europe to Africa. Later, in 1871, Haeckle took the Hertwig brothers to another sampling expedition to the Dalmatian coast (Austria, now Croatia). These trips awoke the appetite of the young students for zoological research by the sea, and marked the beginning of the movie that Fol and Hertwig filmed rather independently to demonstrate sperm entry into the egg and the union of their nuclei. For the Hertwig brothers, this turn to marine creatures was always done without losing their standpoint as doctors in medicine, with human-related problems in the horizon. Not in vain did both brothers participate in the Franco Prussian war (1870-1871), Oscar as surgeon major and Richard as an auxiliary in a field hospital. What was at stake?

Did the sperm establish any contact with the oocyte? Was the sperm contributing anything to the egg to initiate embryo development, or was it just waking up the ‘sleeping beauty’? The nucleus of the egg was seen to disappear with the onset of the first mitosis, so was the first nucleus of the newly created organism generated from accretion of chemical material in the egg cytosol? What was the function of mitosis and the mitotic spindle?

While on the Dalmatian expedition, Hertwig spent his nights in the campaign laboratory that Haeckel arranged in a nearby monastery. It was there that Hertwig discovered an organism suitable for detailed microscopic observations, the Mediterranean sea urchin Toxopneustes lividus (today, Paracentrutus lividus). Common in coastal ponds and sexually mature throughout most of the year, its transparent eggs are easily accessible in large numbers. It took a little bit of ‘in vitro love’, mixing sperm and eggs together, to observe the sperm cell entering the egg. Waiting 10 minutes allowed the observation of the union of both nuclei (pronuclei). Then, all the nuclei of the growing embryo derived from the fused nucleus resulted in the fertilisation process. Hertwig published his observations in 1876. Fol made similar observations in four sea urchin species (in addition to P. lividus, he used the species now-called Sphaerechinus granularis, Echinus esculentus, Arbacia lixula) and in the sea star Asterias glacialis (now called Marthasterias glacialis). His observations, published in 1877 and 1879, most clearly showed the entry of a single sperm cell into an egg, finally recognising fertilisation as the union of sperm and egg.

Picture of sea urchins
Echinoderms. Sea urchins (Paracentrotus lividus) and a sea star (Marthasterias glacialis) in the intertidal ponds.* Photos by Ibon Cancio.

Hertwig recognised the role of the cell nucleus during inheritance and chromosome reduction during meiosis. In 1885, he published that the substance present in the nucleus, which he named nuclein (DNA), was responsible upon fertilisation for the transmission of hereditary characteristics. This suggestion did not receive experimental proof until 60 years later with the publication of the experiment of Avery, MacLeod and McCarthy of 1944.

Fol, as a professor at the University of Geneva, studied a wide variety of zoological problems spending winters in Villefranche-sur-mer, near Nice, France, to collect samples to study the embryology of invertebrates. Fol had been born in a rich family and, with his own money, in 1880 he established a marine station in an abandoned lazaret building constructed so sailors arriving at Villefranche with malaria and cholera could observe their quarantines. Later, the Russian Marine Station, the present-day Observatoire Oceanologique de Villefranche, would be founded nearby. Fol, somewhat a problematic character, resigned in 1886 from his position in the University and retired permanently to continue his research at Villefranche.

Image of book cover in French: Research on Fertilization
The front page and the plate showing the entry of sperm into the egg in Marthasterias glacialis as published by Hermann Fol, “Research on Fertilization and the Beginning of Henogenesis in Various Animals”, in 1879.

Fol’s last important microscopic research, published in 1891, was centred on the function of the role and characteristics of the centrosome during fertilisation. There, he argued that in sea urchins, each gamete contributed two centrosomes, or one centrosome that divided immediately after fertilisation. Fol was of course wrong this time. Shortly after, he initiated a sampling trip in the Mediterranean to study the distribution of sponges in the Tunisian coast. Himself, and his crew of two men, initiated a voyage in a new yacht, the Aster (an aster is the structure with a characteristic star shape, formed in an animal cell by a centrosome and its associated microtubules during early mitosis). It is like initiating an expedition on board of your last piece of research. The three men and the Aster disappeared at sea in March 1892, and nothing else was known about them. Mind you, storks always avoid the Mediterranean in their migration from Europe to Africa!

Picture of mitotic asters
Mitotic asters and spindle during anaphase and metaphase in the embryo cells during blastodisc stage in a starfish. Photos by Ibon Cancio.

*Additional information regarding sea urchins (pictured above): The discovery by Oscar Hertwig of the sea urchin as a model organism in developmental biology is not just an anecdotal fact. The sea urchin has really been a marine guinea pig, an important model in many discoveries in biology. Think of the seminal work of Theodor Boveri with sea urchins and the chromosome theory. Boveri was a student of Richard Hertwig.

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