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| Jean Rostand |
In a previous article I described some of the work on frogs and toads done by the French biologist, Jean Rostand (1894-1977), who in later years has been described as ‘brilliant’ and ‘eccentric’. Rostand had another claim to fame that caused a flurry of excitement and embarrassment at the National Institute of Medical Research (NIMR) in London. In 1952 Rostand wrote, asking for offprints of papers written by members of the Institute concerning the use of glycerol as the first agent that protected spermatozoa and other cells from the effects of freezing to and thawing from very low temperatures. What happened is described by Sir Alan Parkes FRS (1900-1990) in his autobiography Off-beat Biologist:
Early in May 1952 a most interesting and friendly new connection opened up - Audrey [Smith] had a request for offprints from Jean Rostand, the distinguished French biologist, who at the same time sent us a copy of a paper published by him as early as 1946 in the Comptes-Rendus de l'Academie des Sciences entitled 'Glycérine et résistance du sperme aux basses températures'. His paper was entirely unknown to us and the mere title put us in a twitter. Had we perpetrated some fearful scientific blunder? However, on reading the paper it seemed that, although we ought to have known of it, the work was in no way similar to ours. Rostand had used glycerol to keep suspensions of frog sperm from solidifying at temperatures slightly below freezing. Under such conditions the spermatozoa regained mobility, but had lost fertilising power. In acknowledging Audrey's reprints Rostand said that he would try removing the glycerol slowly by dialysis, but we never heard whether fertilising power was thus restored.
Early in July 1955 Ruth Deanesly [Lady Parkes] and I were at a conference in Paris and took the opportunity of calling on Rostand. He received us most kindly at his combined home and laboratory and proved to be a delightful person. On leaving Paris on the Sunday morning we bought a newspaper which, to our surprise and pleasure, carried a detailed article about him, illustrated with sketches, from which we learnt that Jean was the son of Edmond Rostand, the playwright who had immortalised Cyrano de Bergerac, that on Wednesday of the previous week (the very day we had called on him) he had been awarded a prize of three million francs by the Singer-Polignac Foundation and that at one stage of his researches he had fallen foul of the gendarmes when he set free 70,000 toads in the woods of Saint-Cloud.
Thus Rostand is recorded as a pioneer in cryobiology. The work at NIMR became famous because, when applied to agriculture, it revolutionised artificial insemination and enabled the rapid genetic improvement of livestock. It was no accident that two of the triumvirate involved started off with degrees in agriculture, Parkes himself and Chris Polge, later FRS, (1926-2006). The other member was Audrey Ursula Smith (1915-1981) who was medically qualified with a degree in physiology.
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| Audrey Smith, Alan Parkes and Chris Polge see below for sources |
Accounts of how glycerol was found to be affective differ slightly. The Parkes team was following up work which suggested that some sugars might be protective against freezing and thawing. Fructose or laevulose, to use the terminology in use at the time, had been used to some effect in the early 1940s and this work was being checked. However, nothing of any great significance was found and the sugar solutions were put in the cold store for some months.
Parkes in 1956 wrote of what happened next:
Some months later work was resumed with the same material and negative results were again obtained with all of the solutions except one which almost completely preserved motility in fowl spermatozoa frozen to -79°C [the temperature of ‘dry ice’ solid carbon dioxide]. Further experiments confirmed this result and at this point, with some trepidation, the small amount (10 or 15 ml) of the miraculous solution remaining was handed over to our colleague Dr D. Elliott for chemical analysis. He reported that the solution contained glycerol, water, and a fair amount of protein! It was then realized that Meyer's albumen[*] - the glycerol and albumen of the histologist - had been used in the course of morphological work on the spermatozoa at the same time as the laevulose solutions were being tested, and with them had been put away in the cold-store. Tests with new material very soon showed that the albumen played no part in the protective effect.
Although we never discovered exactly what had happened it is very likely that during the long spell in the cold-store the labels had fallen off some of the bottles, a habit they have in cold-store, and had been replaced by some zealot on the wrong bottles, though there was no evidence that any of our own technicians had been involved. Be that as it may, there can have been few experiments in which blind chance stepped in more effectively.
Other accounts indicate that it was Audrey Smith who deduced that glycerol was a constituent of the ‘wrong bottle’ as the result of her accidentally dropping the bottle in the lab sink. A droplet of the contents flew out of the sink and landed on a hotplate which was indeed hot. She immediately recognised the smell from the puff of smoke as acrolein, the thermal degradation product of glycerol.
Labels falling off bottles was commonplace in laboratories of the time. Labelling glass was difficult. The usual method was a water-soluble gummed paper label for reagents. Sellotape, the UK equivalent of Scotch Tape may also have been used but at the time but was expensive and not widely available. It too suffered on bottles and the labels also often fell off especially in the cold. A mystery remains in who put the labels back the wrong—but fortuitously right—way round.
It would appear that in using glycerol to suspend frog spermatozoa, as Parkes indicated, Rostand also accidentally discovered its cryoprotective effects but only took his experiments to slightly below freezing point.
*The recipe I have is 50 ml white of egg, 50 ml glycerol, 1 g of sodium salicylate
Hunter RHF. 2008. Ernest John Christopher Polge. 16 August 1926 – 17 August 2006. Biographical Memoirs of Fellows of the Royal Society. 54, 275–296. doi:10.1098/rsbm.2008.0006
Parkes AS. 1985. Off-beat Biologist. Cambridge: Galton Foundation.
Polge C, Smith AU, Parkes AS. 1949. Revival of spermatozoa after vitrification and dehydration at low temperatures. Nature 164, 666.
Rostand J. 1946. Glycérine et résistance du sperme aux basses températures. Comptes-Rendus de l'Academie des Sciences 222, 1524-1525.
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