The Eggs of Mammals (1936) 5

From Embryology

Chapter V Methods Employed in the Experimental Manipulation of Mammalian Ova

The Eggs of Mammals (1936): Introduction | The Origin of the Definitive Ova | The Growth of the Ovum | The Development and Atresia of Full-Grown Ova and the Problem of Ovarian Parthenogenesis | Methods Employed in the Experimental Manipulation of Mammalian Ova | The Tubal History of Unfertilized Eggs | Fertilization and Cleavage | The Activation of Unfertilized Eggs | The Growth and Implantation of the Blastodermic Vesicle | Summary and Recapitulation | Bibliography | Figures | Historic Disclaimer
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The first investigators of living tubal eggs (Barry, Cruikshank, Bischoff, Spee, et al.) used rather laborious methods of dissecting the tubes (see Squier, 1932, for an interesting historical discussion). The modern technique of securing eggs from the fallopian tubes of most mammals is a fairly simple one. Nonetheless, certain surprising differences in the behavior of the obtained ova arise when exactly the same methods are applied to two different species. Among the laboratory mammals the rabbit is by far superior, and for one very simple reason, namely, rabbit ova seem to withstand the process of handling better than other ova. Mouse, rat and guinea pig ova, for example, begin to fragment very soon after removal from the tubes (Lewis, 1931; Gilchrist and Pincus, 1932; Squier, 1932; and Defrise, 1933) and to date it has been possible to observe at most one or two cleavages in culture, whereas rabbit ova will go through the whole course of cleavage and blastulation in vitro.


The long, fairly straight tubes of most mammals can easily be washed through by a Ringer-Locke or similar balanced salt solution. The writer has found that a RingerLocke solution to which has been added an equal amount of homologous blood serum is most useful. It is necessary only to free the tubes of their mesenteric connections, and if the tubes only are to be employed to cut them away from the uterus. It is ordinarily best to cut off the uterus at about one-half inch from the ampulla so that if washing backward toward the fimbria is desired a certain length of uterine lumen will be available for the guidance of the washing pipette. When ova are to be washed downward from the fimbriated end of the tubes a rather broad bored capillary pipette is used; washing upward from the uterine end requires a very fine pipette. The ova are washed into Syracuse watch glasses and are easily observed under low magnification of a dissecting microscope.


In animals like the rat, mouse and guinea pig with coiled tubes a different procedure is followed. Here the coiled tubes are cut into several fairly straight portions and are squeezed with a pair of fine iris forceps or stroked gently with blunt needles. The contents of the tubal lumen are extruded and the ova are found among the cellular debris.


Ova from the uterus are obtained simply by flushing the uterine lumen with the washing fluid.


Allen, Pratt, Newell and Bland (1930a) describe a method for obtaining human tubal ova without removing the tubes or uterus. ^'The ovaries were examined as soon as possible after the abdominal cavity was opened. In some instances the findings at operation necessitated removal of the most recently ovulating ovary and its tube was not justified, the tube was flushed in situ and the corpus luteum alone removed from the ovary. This method consisted of clamping the cervix with a special clamp and injecting isotonic saline solution directly into the uterine cavity from above by hypodermic syringe while first one and then the other uterine tube was gently pinched by the assistant. The injected solution in most cases flowed back freely through the tube and was collected in a series of watch-glasses held beneath the fimbriated end. Apparently the development of valveHke folds of mucosa at the tubo-uterine junction as described for several mammals by Lee (1928) is not appreciable in woman. Usually from 10 to 30 c.c. was flushed through each tube. The most recent corpus was carefully excised from the ovary, and since it is a transitory structure, without sacrificing any considerable amount of ovarian tissue.


"It is believed that this method of flushing the tubes in situ is harmless to uterus and tubes and opens up new possibilities, not only for the recovery of human ova, but also for checking the patency of tubes at operation.
The tubes which could be removed were washed by direct injection through either the uterine or the fibriated ends after first trimming the tube carefully along the attachment of the mesosalpinx. The trimming seemed advisable, for otherwise when the tube was distended with injected fluid it would often kink badly.
A search for human tubal ova is sometimes complicated by the follicle cells of the cumulus still surrounding the specimens which make difficult clear observation and certain identification. Although while fresh such specimens are fairly transparent, it is often difficult to observe or measure them accurately. Since it is probable that ova may remain in the tubes for three or more days, degenerative changes may be expected in a certain number of unfertilized tubal ova. Also small masses or balls of cells are often encountered in the tubes. These may originate in the peritoneal ca\'it.y, be pinched ofT from the fimbria of the tube, or (in cases where injected fluid is forced back through the tubes from the uterine cavity) derived from cast-off endometrium. Sometimes such cell balls contain structures which before sectioning can easily be mistaken for ova. For this reason unless an ovum is free from follicle cells or the cells of cumulus are partly dispersed, it w^ould seem necessary that it be sectioned before certain identification is possible. Further check should also be made by histologic study of the most recent corpus luteum."


In obtaining both unfertilized and fertihzed ova for culture in litro the use of a warm washing solution is preferable. This is often practically difficult and rabbit ova at least are not materially affected by handling at room temperature over a period of several hours.


The usual methods of tissue culture have been employed in the cultivation of mammalian ova. These include the hanging drop with the ovum held in a plasma clot on a coverslip over a fluid-free cavity; a plasma clot occupying


Mirrifons for tiik ma\ipi:latio\ of o\'a go

the total area under a raised coverslip; the CJarrf^I flask; and the watch-glass teehnirjue in which the sterile watch glass containing the culture rnediun^i is contained in a nnoist chamber, iilood plasma or serum ordinarily form the basis of the most successful culture media. I'he longest perirjd of rfigular development of normally fertilized rabbit ova has bef*n obtained by IxnvLs and Gregory (1929j who photographed the development of rabbit ova from the initial cleavage stages through late blastocyst stages. They placed the ova in homologous plasma upon glass slides. Pincus (unpublished data) has obtained similar development by this technique and also with ova grown in Carrel flasks. IxwLs and Ilartman (\iy.V4) observed the development of a Macacus rhesus ov^um from the 2-cell to the 8-cell stage using the Ixwis and Gregory technifjue. The ova of the rat, moase, and guinea pig have failed to de\'elop beyond one or two cleavages with the use of a \'ariety of culture media. I'hus Defrise 0933; used the following media for culturing rat ova: (\) Ringer's solution, bufferr.'d or not with sodium bicarbone; (2) Tyrode's solution, (\) Isotonic with NaCl, milimol: fa) 120, (h) VM), ((■) 151, (B) K+ - Ca^+ - Mg"+ equilibrium on the basis of the triangular diagram of Loewe, milimol: Ta; 2.05 KCL, 1.90 CaCU, 2.20 MgCF,, ^b; 5.63 KCl, 3.60 CaCU, 0.52 MgCla: the above solutions were used at pll 6.8, 7.2, 7.6; f3j Tyrode's solution rXaCl: milimol 136 - KCl: 5.6 - CaCU: 2.16 - MgCU: 0.52 - XaHC03: 8.6 - CJI 1206:5.5) with the addition of gelatine 0.5 per cent; (4) Tyrode's solution (as above) with the addition of blood serum: faj 1/1, fb) 3/1; (o) Tyrode's solution (a.s above) with the addition of plasma (heparin): (a) 1/1, (b) 3/1; (6) pure blood serum of (a) pregnant female, (b) male, (c) newborn; (7) plasma (secured from the heart and mixed with heparin): (a) pregnant female, (b) male; (8) spinal fluid (secured by suboccipital puncture) ; (9) foetal hystolymph (secured by Mart.ino\itch's technique); HO) uterine fluid (II oestral period): (a) pure, (h) with the addition of blood serum.


In a few cases, in some of the above media, and especially inthis:NaClmilimol 130 - KCl 2.65 - CaCU 174 - MgCls 1.18 - NaHCOa 8.6 - at pH 7.2 - drops III = bloodserum drops II, one or two mitoses were obtained. The addition of small quantities of embryonic extract, of rat foUiculine, of extract of the anterior lobe of the hypophysis to the medium (either solid or liquid, natural or artificial) has not noticeably modified the culture results.

Squier (1932) using a less extensive variety of media was similarly unsuccessful with guinea pig ova (see also Lewis, 1931).

The limitations of the ordinary methods of tissue culture are discussed further in Chapter IX in connection with the investigation of the normal physiological environment of developing ova.


Nicholas and Rudnick (1933) have cultivated rat embryos upon the chorioallantois of the chick, but ovum development has not been studied. The embryos survive and differentiate over a considerable period of time in the foreign environment.


Cinematography of developing ova has been undertaken in a number of recent investigations. Standard motion picture cameras adapted for microphotography are employed.


For a study of the comparative behavior of ova in vivo and in vitro the writer has transplanted cultured ova into the fallopian tubes of rabbit does (see Pincus and Enzmann, 1934). The operative technique requires the use of a light anaesthesia, e.g., either ether preceded by atropine sulphate injection to inhibit excessive mucous secretion or simple urethane anaesthesia. The exposure of both tubes and ovaries is had by a simple laparotomy. The ova are held in a special pipette with an opening in the tube above the capillary. This type of pipette permits one to take up a minimum amount of fluid with the eggs, and also prevents the ova from being drawn into the wide-bored portion of the pipette. The capillary portion is inserted into the upper 3^ of the tubes and the ova expelled by gentle pressure on the bulb when the opening in the tube is closed over. No amount of pressure on the bulb will expel the ova if the opening is not closed. Extreme care should be taken to expel only the ova and the fluid containing them. If air is also pumped into the tubes it often blows the eggs down too far into the tubes or even into the uterus. Excessive fluid acts in the same way.


The writer (in collaboration with Dr. E. V. Enzmann) has also transplanted mouse ova into the fallopian tubes. Here it is necessary to slit the capsule and expose the tubal opening, which is slightly wider than at the ampulla, but not as wide as the rabbit's fimbriated opening. The tubes are observed under a dissecting microscope and the opening exposed by manipulation with iris or watchmaker's forceps. The delicate mouse ova are best handled in warm RingerLocke solution plus serum.


Nicholas (1933a) has transplanted rat ova from the fallopian tubes into the uterus. In this case the tubes are excised at the isthmus and the ova expelled from a capillary pipette into the uterine lumen.



Historic Disclaimer - information about historic embryology pages 
Mark Hill.jpg
Pages where the terms "Historic Textbook" and "Historic Embryology" appear on this site, and sections within pages where this disclaimer appears, indicate that the content and scientific understanding are specific to the time of publication. This means that while some scientific descriptions are still accurate, the terminology and interpretation of the developmental mechanisms reflect the understanding at the time of original publication and those of the preceding periods, these terms and interpretations may not reflect our current scientific understanding.     (More? Embryology History | Historic Embryology Papers)
The Eggs of Mammals (1936): Introduction | The Origin of the Definitive Ova | The Growth of the Ovum | The Development and Atresia of Full-Grown Ova and the Problem of Ovarian Parthenogenesis | Methods Employed in the Experimental Manipulation of Mammalian Ova | The Tubal History of Unfertilized Eggs | Fertilization and Cleavage | The Activation of Unfertilized Eggs | The Growth and Implantation of the Blastodermic Vesicle | Summary and Recapitulation | Bibliography | Figures | Historic Disclaimer

Cite this page: Hill, M.A. 2017 Embryology The Eggs of Mammals (1936) 5. Retrieved October 22, 2017, from https://embryology.med.unsw.edu.au/embryology/index.php/The_Eggs_of_Mammals_(1936)_5

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