History of cryobiology, with special emphasis in evolution of mouse sperm cryopreservation
Introduction
Living in the computing era obviously changed the way we act, the way we think and the way we analyze the past. Today computers, plays the role of a kind of modern oracle that is able to answer all our questions and give us access to an incredible amount of digitalized historical documents now in the public domain. This matter helps us to verify data that not long ago was transmitted orally, but not verified. Taking advantage of the internet technical benefit, this review was done digging in the past searching for the original documents.
The main question is where to start? Which fact is important enough to consider it as a historic milestone? In general, most of the reviews suggest that the glycerol discovery is the starting point, in fact, as J.K Sherman [49] pointed out, there is a pre and post 1949, the year of the glycerol discovery.
Greeks and Romans used ice brought from the mountains to primarily chill their drinks and not their food. Lowering the temperature for meal conservation using ice cold was an important step that occurs much later. The primitive method of chilling was achieved using natural ice or compacted snow, which can be considered as the precursor of our refrigeration system. The use of ice was kept for a long time, until 1842 when a patent was issued in Britain for freezing food by immersion in an ice and salt brine [9], describing that the use of cold temperature could maintain the freshness of food. Nevertheless, all those issues had happened a long time before the possibility of applying the use of low temperature for maintaining live cells in an inanimate state was even thought about.
One of the most relevant inventions that brought the opportunity to many other scientific discoveries, like in this case the cryopreservation of embryos and gametes, was the creation of the microscope. Antonie Philips van Leeuwenhoek (October 1632 –August 1723) invented a tool that was essential for understanding biology at the cellular level. With his device, Leeuwenhoek was able to identify what he called animalcules, like worms or parasites living in human and other animal seminal fluid. From his letters, describing an observation of a dog sperm sample he wrote: “At the beginning of October in the year 1684, however, I again took male seed from the same dog, a very strong and vigorous animal, and found that a few animalcules were still alive at the end of seven days and seven nights, a very small number of them still swimming about as actively as if they had only just come from the dog” [18]. He was supporting the theory of the fluids where the male semen will fuel the egg growing in the mother's womb.
In 1776 Lazzaro Spallanzani (1729–1799), an Italian priest and scientist, devoted to investigating and understanding all biological processes, using Leeuwenhoek's microscope described the animalcules he observed in the semen of different animals: horse, bull, dog, frog, salamander and human [55], although he also saw animalcules in almost any liquid, fluid and material in putrefaction. In a personal opinion, the reason that this reference has been cited many times as one of the starting points of cryobiology history is because Spallanzani described for the first time the reaction of those animalcules exposed to different ambient temperatures, and he scores the survival from hot to ice cold. He also observed that some of these animalcules survived and continues swimming after being exposed at low temperatures. At that time, the temperature was measured with the Réaumur scale, which is similar to the Celsius scale where 0° is equivalent to ice formation. Spallanzani also collected samples from human cadavers and found out that some movement was still happening after death. He also was interested in the fecundation (the generation, as it was called at that time). Spallanzani challenged his colleagues and thereby contradicted the argument of the fertility theory of that time, supporting that semen was able to carry the “seed”. He proved with a series of experiments that the sperm “aura” does not fertilize; instead, he postulated that both sexes are needed to create an embryo. In an effort to prove his theory about generation, he documented the first artificial insemination with a nice and methodic study using a female dog housed in a separated room where was impossible that the sperm aura can reach her. He collected sperm “from spontaneous ejaculations” of a male dog (he need to be discreet, he was a priest) and deeply introduced it into the vagina in two separate days, and the female got pregnant delivering live pups [5].
On the other hand, thanks to the Spallanzani's premonitory studies demonstrating that heat kills the “animalcules”, gave Louis Pasteur the key to discover the sterilization (Pasteurization) method we use today.
Something quite important also happened during those years. In 1779, Carl W. Scheele, a Swedish chemist, was overjoyed when he discovered a new transparent, syrupy liquid by heating olive oil and litharge (an oxide of lead, PbO) [8]. This “sweet” discovery named Glycerol (in Greek means sweet) was not as important as it is today, not just for its relevance in cryopreservation, but for what it really means for the pharmaceutical and food industry.
Going back to the animalcules or parasites described in the seminal fluid in the 1700, are still today using the parasite-like name: spermatozoa. Nevertheless, at that time, the theories about the sperm function and fertility were contradictory among research groups. A group of scientists named the sperm as Homunculus because they thought that a tiny human being was laying in the sperm head (the seed) leaving the female only as an “incubator”. Others supported the importance of the seminal fluid transporting the essentials “food”, thus nurturing the growth of the egg (the Fluidist theory). Another group supported the ovist theory, where the egg was the only element for the proper generation [20].
It was in 1840 when the Swiss physician J.L Prevost and the French scientist J.B Dumas, working in different laboratories and located in different places, discover simultaneously that the sperm is created in the testis, contradicting one of the most supported theories that those animanculus were parasites. On the other hand, these scientists discovered the real function of both the spermatozoa and the egg, describing and obtaining a real fertilization by showing that both gametes are needed to fertilize a frog egg [49].
Paolo Mantegazza (1831–1910), an Italian physician, scientist, anthropologist, politician and visionary, who travelled around the globe studying human sexual behavior, it is commonly cited in cryobiology publications for a series of observations he did when cooling human and frog sperm to −14 and −17° Celsius. His prediction in 1866 was extremely imaginative about the future existence of sperm banks:
“If the human sperm can remain unchanged for more than four days at the melting ice temperature, it is certain that the science of the future will improve the breeds of horses and oxen, without forcing the enormous expense of transporting stallions and bulls, and they will be able to make artificial fertilization with the frozen sperm, shipped at great speed from one country to another. It may also be that a husband dead on the battlefields can fertilize his wife from his cadaver, and have legitimate children even after he died.” [22].
Indeed, Mantegazza was a visionary. Almost 100 years later the use of frozen/thawed sperm from a person who was dead at the time of inseminating his wife was reported for the first time in 1954 by Sherman and Bunge and was front-page news [4,56].
In 1878, the Viennese embryologist Samuel Leopold Schenk performed the first attempt of an IVF with mammalian eggs. Working with rabbit's and guinea pig's ova, Schenk noted that complex cumulus cells–egg was disintegrated with the introduction of sperm in the dish [6]. Then after hours of culture he observed 2-cell embryos; however, other scientists critically suggested that the cell division he described could as well be parthenogenic cell division [1,72].
Before advancing too far in time, it is important for this historical review to describe the work of Robert Boyle, an Irish philosopher and scientist who in 1662 published what we know today as the Boyle's law. His study shows that if a gas in a proper container receives an amount of pressure, the volume of the gas decreases proportionally to the pressure applied [2,3]. Boyle described this phenomenon analyzing the gas (air) behavior; however, the same observation was described in cells during the process of cryopreservation [19].
We know today that a penetrant cryoprotectant additive or agent (CPA), chemically a polyalcohol at 1 or 1.5 M concentration in solution has the property of protecting a cell during the cooling process by displacing the inner water contents to avoid intra-cellular ice formation [25,26]. But how could this interchange between cryoprotectant from outside the cell and the water from inside the cell happen? It is because of the physics of the osmotic pressure. The CPA has an osmolarity of approximately 1500 mOsm (milliosmoles) and the cell has an osmolarity of 300 mOsm; therefore to respond to the outside pressure the cell eliminates water and incorporates CPA, equilibrating the osmotic pressure. As consequence of that pressure, the cell volume reduces (or contracts). Following Boyle's law, Jocobus Van't Hoff, a Dutch physical chemist, described this phenomenon in liquid solutions, showing that the water under pressure can pass through a membrane concentrating the solute. Cells exposed to a strong osmotic pressure (hypertonic) will shrink until most of the water comes out and only the residual solute is left. With the aid of the Van't Hoff's factor plot it was shown that 80% of the cell is water. For this magnificent work Jacobus Van't Hoff received the first chemistry Nobel Prize in 1901.
Returning to our historical route, in 1930 two scientists working in collaboration, but in different laboratories, published studies on rabbit sperm chilled to 0 °C or maintained at 10 °C. Stanley Leibo in his chapter in the book “Life in the frozen state”, described the work of Walton and Hammond [19]. John Hammond [13] studied ejaculated semen and Arthur Walton [75] collected the sperm from the vas deferens; both studies were published together in the same journal. The results showed that the sperm maintained at 10 °C kept its fertility for more than 100 h. It is remarkable what Hammond wrote in the introduction of his paper: “A knowledge of the factors affecting the vitality of the sperm outside the body is essential to the success of this method of breeding, which may in the future play an important part in the improvement of livestock, for with the development of rapid aeroplane transport long distances can be covered in a short time and the transportation of semen would be far less expensive than the transport of breeding animals”. Today we can compare the futuristic view of that study with the studies published 80 years later, in 2010 and 2014 by Toru Takeo et al., on cold transportation of embryos and sperm. [63,64].
Basile Luyet was a Roman Catholic Franciscan priest, having earned at age of 28 doctorates on both Natural Sciences and Physics; later he became the first president of the Society for Cryobiology. He was also the first one to use and describe the vitrification process. In 1938 he published along with Eugene Hodapp the results of their experiments on frog sperm vitrification in liquid air [21]. The authors noted that when they treated spermatozoa with a hypertonic sucrose solution the survival increased in relation to the sucrose concentration. Therefore, they showed the need of using an osmotic buffer to dehydrate the cell before exposing it to low temperatures.
In 1942 Hudson Hoagland and Gregory Pinkus followed Luyet's technique to vitrify human sperm. In the introduction of their paper the authors elaborate on a comment about a frozen organism maintained under liquid helium, that “with subsequent warming and recovery, would, if it were possible to effect, be precisely equivalent to projecting the organism forward a century into the future - a sort of Wellsian time machine idea”. The result of the study after storage in liquid nitrogen was better with human sperm where 65% recover motility than with rabbit sperm where only 0.5% survived [15].
In 1945 Alan Parkes, later named Sir for his outstanding scientific contributions, showed that large volumes of human sperm frozen in ampoules or tubes have better freezability than smaller quantities [50]. His studies demonstrated also that a slow freezing curve was less harmful to the cells than a rapid one. But most important was that in 1949 Christopher Polge, Audrey Smith and Alan Parkes published in the journal Nature the letter “Revival of Spermatozoa after Vitrification and Dehydration at Low Temperatures” [46] where for the first time glycerol is described as a cryoprotectant. As many other great discoveries, the glycerol protective property was discovered by mistake. To everyone's surprise a bottle kept in the fridge labeled as Laevulose (Fructose) suddenly preserved the fowl (cock) sperm motility after being exposed to −79 °C. A freshly made solution did not have that property. Then the “original” bottle content was sent for analysis and the report described that the solution contained 10% glycerol and 1% albumen. In other words, the sperm sample was treated with the Meyer's histological albumen solution originally used for fixing the smears before staining [50] which was, obviously, wrongly labeled. The glycerol discovery marks an important milestone in cryopreservation and the beginning of a new era. Thanks to the addition of glycerol to the cryopreservation solution most mammalian sperm, from an immense variety of species, have been able to be cryopreserved; except … ….mouse and rat.
Section snippets
Other important milestones in cryobiology
The first calf born from frozen thawed sperm by artificial insemination was reported by C. Polge and L Rowson in 1952 [47]. Sperm was frozen in ampoules and kept at −79 °C. For the study they inseminated 38 cows from which 30 become pregnant. From this experiment, 27 healthy calves were born, and the first one was named Frosty. Many years later, in 1973, Ian Wilmut working in Chris Polge's lab who was collaborating with L Rowson produced the first calf born from a frozen embryo and it was named
Milestones on mouse sperm freezing
In December 1981, Gordon and Ruddle published in Science the creation of the first transgenic mouse [11]. Thanks to this new transgenic technology and the prolific creation of transgenic animals most animal facilities were overcrowded in no time. The colony expansion as consequence of the breeding of the transgenic founders and their progeny, who needed to be screened for the gene insertion, resulted in not enough physical space to house animals. At that time, the embryo freezing technique was
Acknowledgements
The authors especially thank to Dr. Larry Mobraaten for review and comments on this manuscript and to Shuuji Tsuchiyama (CARD, Kumamoto University) for his great help with the graphic work.
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