Paper - In vitro fertilization and cleavage of human ovarian eggs: Difference between revisions
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==The Two-Cell Stage of the Human Egg== | ==The Two-Cell Stage of the Human Egg== | ||
The first specimen was obtained from Mrs. D. D. (No. 20,768), a 38-year old Para IV, who underwent laparotomy on the 10th day of her menstrual cycle, at a time when the endometrium was in the early pro- liferative stage. When first observed in the fluid drained from a 2.3-cm bluish follicle, the egg was | The first specimen was obtained from Mrs. D. D. (No. 20,768), a 38-year old Para IV, who underwent laparotomy on the 10th day of her menstrual cycle, at a time when the endometrium was in the early pro- liferative stage. When first observed in the fluid drained from a 2.3-cm bluish follicle, the egg was enclosed within a moderate investment of granulosa cells. It was washed in Locke’s solution and incubated for 27 hours in the serum of the same patient. Then it was exposed for one hour at room temperature to a washed sperm suspension in Locke’s solution. The watch-glass containing the ovum and spermatozoa was left on the stage of the dissecting microscope and the egg was kept in constant view (at a magnification of X35)“ The spermatozoa showed great activity throughout the period of observation; they were clearly seen to travel through the interstices of the loose cellular formation surrounding the egg, and many were noted in active motion just outside the ovular boundary. At the end of one hour, the ovum was transferred to fresh serum from a post-meno- pausal patient. As the egg was pipetted into the culture flask, the cellular investment suddenly dropped off and it appeared as a single round cell with a fuzzy border. When it was again observed after 40% hours’ culture, it was found to consist of two blastomeres, eachimeasuring 8611. in diameter, and was enclosed within a zona pellucida of uniform width, measuring 14 u. A sketch of' the specimen was made and it was fixed in Bouin’s solution, but in the lengthy process of dehydration, it was, unfortunately, lost. | ||
A stained section of the follicle from which this egg was obtained showed a typical preovulatory phase. | A stained section of the follicle from which this egg was obtained showed a typical preovulatory phase. | ||
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Thirteen eggs in all were recovered from the follicles of this patient and were cultured in three batches. The egg to be described was one of a set ‘of four, of which three, when first seen, were covered by a thick granulosal cell investment, and one by only a few rows of cells. The eggs were incubated in | Thirteen eggs in all were recovered from the follicles of this patient and were cultured in three batches. The egg to be described was one of a set ‘of four, of which three, when first seen, were covered by a thick granulosal cell investment, and one by only a few rows of cells. The eggs were incubated in serum<ref>In all three experiments reported here, the serum used for culture of the eggs prior to exposure to spermatozoa was taken from the patient who had furnished the eggs, while subsequent culture (following contact with spermatozoa) was carried out in serum of a post~menopausal patient. </ref> for 22.5 hours, being washed in salt solution before and after incubation, and then exposed to a Washed sperm sus- pension in Locke’s solution for two hours at room temperature. They were again washed in Locke’s solution and cultured in fresh serum for 45 hours. When examined at the end of the, incubation period, one egg was found to be in the two-cell stage; it re- sembled very closely the first specimen described. Two blastomeres of fairly uniform size and appearance, and containing granular cytoplasm, were en- closed within a zona pellucida along the border of which were numerous spermatozoa. At least one of them was clearly seen within the zone. The entire egg (including the zona pellucida) measured 153 11. x155 u; the cellular portion was 100 u x 113 u, and the average width’ of the zona pellucida was 23 M. The blastomeres measured 88 u x 58 u, and 105 u x 58 M, respectively. | ||
The egg was fixed in a plasma clot according to the method described by Pincus<ref>G. Pincus, Jour. Exp. Zool., 82: 85, 1939.</ref> and the clot was carried through the double embedding celloidin-parafiin method, serially sectioned at 8M, and stained with hematoxylin and eosin. The specimen is included in 8 sections; hence the total thickness of the egg is ap- proximately 64 it. In a section through the middle of ‘the specimen, the blastomeres (designated for con- venience as “A” and “B”) measure 63p.x39u and 66 it x 36 M, respectively. The cytoplasm appears uni- formly granular, with the exception of the polar re- gions, where there is beginning vacuolization, as had been noted in the fresh specimen just prior to fixa- tion. In the approximate center of each cell there is a round, vesicular nucleus measuring 18 P. x 13 p., and l6p.x15p. (blastomeres “A” and ‘.‘B,” respectively), and containing a chromatin meshwork. The zona pellucida surrounds the egg over about two thirds of its circumference. The failure to retain the entire zona pellucida in section was doubtless due to the method of fixation, as it had been intact in the fresh specimen. In its widest portion, the zona measures 7 to 8 p. in width. At least 4 sperm heads may be identified; one of them appears to be just within the cell body of blastomere “B.” In a section adjacent to the one just described, a polar body measuring 18y.x10p. is seen beside blastomere “A.” It contains what appears at first as a more or less definite number of chromatin units discrete enough to be counted. However, upon further magnification, the chromatin is seen to be in the form of a lobulated body, only two clumps being definitely separated from the general mass. Opposite blastomere “B” at the outer boundary of the zona, two sperm heads may be identified. Other sections of the egg, devoid of nuclear material, show 1, 5, 7 and 9 spermatozoa, re- spectively, in the neighborhood of the zona pellucida or of the cytoplasm itself. | |||
==The Three-Cell Stage== | ==The Three-Cell Stage== | ||
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Rock J. and Menkin MF. In vitro fertilization and cleavage of human ovarian eggs. (1944) Science 100 (2588): 105-107. PMID 17788930
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In Vitro Fertilization and Cleavage of Human Ovarian Eggs
John Rock and Miriam F. Menkin
From the Department of Gynecology, Harvard Medical School, Boston, and the Fertility Clinic Laboratory, Free Hospital for Women, Brookline, Mass.
- Aided by grants from the William F. Milton Fund of Harvard University, the Committee for Research in Problems of Sex, National, Research Council, and the Carnegie Corporation of New York.
Introduction
First stages in the cleavage of the fertilized human egg have, as far as we know, never been reported, and while in oitro fertilization of tubal eggs of the rabbit has been described[1] we have found no record of such experiments in higher mammals. A monkey egg fertilized in vivo has been cultured in vitro from the two- to the eight-cell stage.[2]
Utilizing the surgical material available at the Free Hospital for Women, we have, during the past six years, made numerous attempts to achieve in oitm fertilization and cleavage of human eggs obtained from ovarian tissue removed just prior to the expected time of ovulation. Throughout this period of investigation,[3] several factors have been varied; e.g., the conditions of culture of the eggs, both before and after exposure to spermatozoa, the duration of contact of egg and spermatozoa, and the concentration of the sperm suspensions employe.[4] As a result of recent modifications of our method, we believe we have succeeded in three experiments, which constitute the subject-matter of the present report.
In two of these cases (D. D. and R. P.), the egg, after being subjected to certain procedures (to be described later), was found to be in the two-cell stage.
In the third case (J. D.), two eggs divided. One of these, when, first seen in cleavage, consisted of one large blastomere and two smaller ones, each of the three containing a round, vesicular nucleus. The second egg from this same patient was in a similar stage, but part of the cytoplasm appeared fragmented, and soon proceeded to undergo rapid degenerative changes. In this first report, we will confine our discussion, therefore, to the two eggs in the two-cell stage and the more normal of the two eggs in the three-cell stage.
The Two-Cell Stage of the Human Egg
The first specimen was obtained from Mrs. D. D. (No. 20,768), a 38-year old Para IV, who underwent laparotomy on the 10th day of her menstrual cycle, at a time when the endometrium was in the early pro- liferative stage. When first observed in the fluid drained from a 2.3-cm bluish follicle, the egg was enclosed within a moderate investment of granulosa cells. It was washed in Locke’s solution and incubated for 27 hours in the serum of the same patient. Then it was exposed for one hour at room temperature to a washed sperm suspension in Locke’s solution. The watch-glass containing the ovum and spermatozoa was left on the stage of the dissecting microscope and the egg was kept in constant view (at a magnification of X35)“ The spermatozoa showed great activity throughout the period of observation; they were clearly seen to travel through the interstices of the loose cellular formation surrounding the egg, and many were noted in active motion just outside the ovular boundary. At the end of one hour, the ovum was transferred to fresh serum from a post-meno- pausal patient. As the egg was pipetted into the culture flask, the cellular investment suddenly dropped off and it appeared as a single round cell with a fuzzy border. When it was again observed after 40% hours’ culture, it was found to consist of two blastomeres, eachimeasuring 8611. in diameter, and was enclosed within a zona pellucida of uniform width, measuring 14 u. A sketch of' the specimen was made and it was fixed in Bouin’s solution, but in the lengthy process of dehydration, it was, unfortunately, lost.
A stained section of the follicle from which this egg was obtained showed a typical preovulatory phase.
Of the second egg in the two-cell stage we have a complete series of stained sections. Essentially the same procedure, as described above, was carried out on an ovum, washed out of a follicle of Mrs. R. P. (No. 14,518), a 31-year old Para VI, Gravida VIII, who was operated upon on the 11th day of her cycle. The endometrium at this time was in the early to mid-proliferative phase of its development.
Thirteen eggs in all were recovered from the follicles of this patient and were cultured in three batches. The egg to be described was one of a set ‘of four, of which three, when first seen, were covered by a thick granulosal cell investment, and one by only a few rows of cells. The eggs were incubated in serum[5] for 22.5 hours, being washed in salt solution before and after incubation, and then exposed to a Washed sperm sus- pension in Locke’s solution for two hours at room temperature. They were again washed in Locke’s solution and cultured in fresh serum for 45 hours. When examined at the end of the, incubation period, one egg was found to be in the two-cell stage; it re- sembled very closely the first specimen described. Two blastomeres of fairly uniform size and appearance, and containing granular cytoplasm, were en- closed within a zona pellucida along the border of which were numerous spermatozoa. At least one of them was clearly seen within the zone. The entire egg (including the zona pellucida) measured 153 11. x155 u; the cellular portion was 100 u x 113 u, and the average width’ of the zona pellucida was 23 M. The blastomeres measured 88 u x 58 u, and 105 u x 58 M, respectively.
The egg was fixed in a plasma clot according to the method described by Pincus[6] and the clot was carried through the double embedding celloidin-parafiin method, serially sectioned at 8M, and stained with hematoxylin and eosin. The specimen is included in 8 sections; hence the total thickness of the egg is ap- proximately 64 it. In a section through the middle of ‘the specimen, the blastomeres (designated for con- venience as “A” and “B”) measure 63p.x39u and 66 it x 36 M, respectively. The cytoplasm appears uni- formly granular, with the exception of the polar re- gions, where there is beginning vacuolization, as had been noted in the fresh specimen just prior to fixa- tion. In the approximate center of each cell there is a round, vesicular nucleus measuring 18 P. x 13 p., and l6p.x15p. (blastomeres “A” and ‘.‘B,” respectively), and containing a chromatin meshwork. The zona pellucida surrounds the egg over about two thirds of its circumference. The failure to retain the entire zona pellucida in section was doubtless due to the method of fixation, as it had been intact in the fresh specimen. In its widest portion, the zona measures 7 to 8 p. in width. At least 4 sperm heads may be identified; one of them appears to be just within the cell body of blastomere “B.” In a section adjacent to the one just described, a polar body measuring 18y.x10p. is seen beside blastomere “A.” It contains what appears at first as a more or less definite number of chromatin units discrete enough to be counted. However, upon further magnification, the chromatin is seen to be in the form of a lobulated body, only two clumps being definitely separated from the general mass. Opposite blastomere “B” at the outer boundary of the zona, two sperm heads may be identified. Other sections of the egg, devoid of nuclear material, show 1, 5, 7 and 9 spermatozoa, re- spectively, in the neighborhood of the zona pellucida or of the cytoplasm itself.
The Three-Cell Stage
The third experiment to be reported was performed on ova of Mrs. J. D. (No. 21,012), a 38-year old sterility patient, in whom the diagnosis of tuberculous endometritis had been made after routine biopsy taken in the course of an investigation for sterility. Opera- tion on the 12th day of her cycle, at a time when the endometrium was in the late proliferative stage, re- vealed extensive tuberculous involvement of the uterus and tubes. Both tubes were sealed and the fimbriae were inverted.
Two out of four eggs, recovered in washings of incised follicles and subjected to- essentially the same procedures as outlined above, were found to be in cleavage. These ova had been cultured in serum for 27 hours prior to contact with spermatozoa, exposed to the latter for one hour and ten minutes, and rein- cubated for 46 hours. At the end of the incubation period, the more normal of the two specimens con- sisted of three well-defined, round, regular blasto- meres, each of which contained a round, vesicular nucleus. A photograph taken two hours later already shows beginning signs of degeneration; 1I.e., shrinkage and vacuolization. Within the zona pellucida, which is of even width, at least 5 spermatozoa are seen. The entire egg (including the zona pellucida) measured 170 u X 183 u; the cellular portion was 103 :1 X 127 M, and the zona pellucida averaged 21 u in Width. The largest blastomere measured 97 u x 73 u, and the two smaller ones 62 11. x 62 u, and 50 11. X 63 M, respectively.
The ovum was fixed, serially sectioned, and stained in the same manner as the second egg, described above.
Since it includes 10 sections, cut at 8 M, the specimen, is approximately 80 u. thick. A section through the middle measures 50 pix 86 M. The largest blastomere is here seen to be 66 u. X 49 M, and the two smaller ones, 35 M x 38 M and 33 M x 44 u., respectively. In addition to vacuolization of the cytoplasm, the presence of multiple nuclei in the individual cells is evidence that the egg had undergone definite degenerative changes since it was first observed in cleavage that afternoon and sketched.
In one section there is a structure measuring 14 p. X 9. p., which, is strongly suggestive of a polar body. Nowhere throughout the preparation is there any sign of the zona pellucida; this had evidently been dissolved by the fixative.
In regard to the duration of early cleavage stages, it‘ is pertinent to citethe report of Lewis and Hartmann on the culture in vitro of the monkey egg fertilized in vivo. They state that in their experiment, in which fertilization was believed to occur soon after ovulation, the one- and two-cell stages lasted at least 36 hours. We observed two eggs in the two-cell stage 40% and 45 hours, respectively, following contact with spermatozoa. Lewis and Hartman considered that the three- and four-cell stages in their egg extended to the 48th hour following fertilization. Our two eggs were seen in the three-cell stage 46 hours after exposure to spermatozoa. Hence, our findings, in this respect, are in general agreement with those reported for the monkey egg.
These experiments will be described in greater detail elsewhere, and photographs of the fresh and fixed specimens will be included.
References
- ↑ G. Pincus and E. V. Enzmann, Proc. Nat. Acad. Sci, 20: 121, 1934.
- ↑ W. H. Lewis and C. G. Hartman, Carnegie Inst. Wash. Pub. 443, Contrib. to Embryol., 24: 187, 1933.
- ↑ Nearly 800 human follicular eggs have been isolated and studied during the course of this investigation; of these, 138 have been observed after exposure to spermatozoa.
- ↑ We very gratefully acknowledge the invaluable advice and encouragement generously given us by Dr. Gregory Pincus, as well as the helpful assistance furnished at various stages by Dr. Nicholas T. Werthessen, Miss Lotte Lee Sichel, Miss Eleanor C. Adams, James M. Snodgrass and Dr. Harold Brown. We are also deeply indebted to Dr. Austin M. Brues for his help in the early part of this investigation, and to Dr. Arthur T. Hertig for his constant encouragement, advice and material aid through his grant from the Carnegie Corporation of New York.
- ↑ In all three experiments reported here, the serum used for culture of the eggs prior to exposure to spermatozoa was taken from the patient who had furnished the eggs, while subsequent culture (following contact with spermatozoa) was carried out in serum of a post~menopausal patient.
- ↑ G. Pincus, Jour. Exp. Zool., 82: 85, 1939.
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