TERMINATION OF PREGNANCY IN THE RABBIT
BY INTRAVENOUS INJECTION OF ANTERIOR
LOBE PITUITARY EXTRACT
BY C. W. BELLERBY.
(From the Department of Social Biology in the University of London.)
(Received 31st January, 1935.)
I. INTRODUCTION.
MUCH of the work carried out on the physiological properties of various preparations of the anterior lobe of the pituitary has led to contradictory results. No exception is provided by the investigations that have been made on the effects of injection of extracts or implantation of tissue during pregnancy. In 1926 Teel showed that the subcutaneous injection of extracts prepared by Evans and Long's method into pregnant rats resulted in disturbance of the parturition mechanism. Birth of litters was delayed for several days and the young were subsequently born in a more advanced stage of development than usual. It appeared that the fresh luteal tissue produced in the ovaries by the extract extended the function of the existing corpora lutea of pregnancy, with the result that the ovarian changes which are believed to be responsible for the induction of parturition did not take place at the normal time. Similar results were reported later by Evans and Simpson (1929),x but meanwhile totally different results had been obtained by Engle and Mermod (1928). In both rats and mice serial implantation of anterior lobe tissue during the middle stages of pregnancy was always followed by abortion or resorption of the embryos. No effect was produced if the treatment was carried out during the latter stages of gestation and normal litters were born. They concluded that termination of pregnancy always followed if sufficient glandular substance was given, and they stressed the fact that ovulation only seemed to occur after pregnancy had been ended. In the same year Zondek and Ascheim published similar results. In their experiments single im-plantations of anterior lobe tissue resulted in ovulation but not in termination of pregnancy. Larger amounts, however, produced abortion as well as ovulation. Results of the same kind were also obtained by Fels (1928). In all these experi-ments the contrast in results was possibly due to differences in the amounts of active substance administered.
Further work on the rabbit showed that injection of saline suspensions was fol-lowed by ovulation but not by any disturbance of the foetuses or placentae (Wolfe, 1931). A number of investigations have been carried out on the effects produced during pregnancy by the injection of the ovary-stimulating substance which can be
1
prepared from the urine of pregnant women. In the normal and hypophysectomised doe rabbit ovulation could be induced with ease. As a rule resorption of the embryos was caused but ovulation was also compatible with their further development, although some subsequent disturbance of the mechanism of parturition occurred (Hill and Parkes, 1931, 1932). Martius and Fabiao (1930), Snyder and Wislocki (1931) also produced ovulation in the rabbit by injection of the same substance and some of their experiments showed that termination of pregnancy did not necessarily result, and that it was possible to obtain fertilisation of a second batch of ova in the presence of the existing blastocysts (Wislocki and Snyder, 1931). The present communication also deals with the effects of injection during pregnancy in the rabbit of extracts prepared from the anterior lobe of the pituitary.
II. METHODS AND MATERIALS.
Animals. The doe rabbit was selected for experiment for several reasons. The
large size of the ear vein permits the intravenous administration of extracts without difficulty. Changes in the ovary can be detected without having to resort to histological methods. The duration of pregnancy is very constant (30-32 days), so that variation in length of any particular stage of gestation is reduced to a minimum. All does were mated twice and as far as could be ascertained only those which were definitely pregnant used for experiment. No special breed of rabbit was used and each batch of experimental animals consisted of several different kinds of doe. They were fed on a diet consisting of hay, bread, pressed dry clover, and sugar-beet pulp soaked in warm water.
Extracts. Two methods of preparation were used. In the first and simpler
method dried gland was employed. A requisite amount of powder was first ex-tracted for 24 hours at 370 C. with twice its weight of 1 per cent, acetic acid. The extract was then separated from the residue by Biichner filtration, neutralised with 50 per cent. NaOH (phenol red as indicator) and filtered a second time. The clear yellow filtrate was used for injection without further treatment. In most cases 2 ml. was equivalent to about 1 gm. of original tissue. This assumes that 1 gm. of powder corresponded to 5-5 gm. of fresh gland. In the second method of extraction fresh pituitaries were used. These were obtained from the slaughter-houses in London and were mainly from male oxen. They were removed from the head and placed in the refrigerator at — 150 C. within 15 min. of the death of the animal. The frozen glands were taken to the laboratory in Thermos flasks and the anterior lobes dis-sected out, extraction starting within 4 hours from the time of collection of all material. The extract was prepared according to the following method.
The anterior lobes are first weighed and ground to a fine paste with silver sand. After thoroughly mixing with twice its weight of iV/10 NaOH the mass is allowed to stand at room temperature for 10 min. 1 ml. of glacial acetic acid is next added for every 100 gm. of mash, and extraction allowed to proceed for a further 10 min. The extract is then filtered off under pressure and the residue well stirred up with distilled water and filtered again. After combining the water and acid extracts, a few
drops of phenol red are added and the fluid neutralised with 50 per cent. NaOH. The extract is then mixed with an equal volume of absolute alcohol, the heavy precipitate filtered off and acetone and ether added to the filtrate until maximum precipitation occurs. After collecting the precipitate on a Biichner filter, it is finally worked up with a small volume of distilled water and filtered a second time. Most of the precipitate remains undissolved. The volume of water used should be ad-justed so that the final volume of extract is such that 1 ml. is equivalent to 4 gm.
of original tissue, o-i ml. of this type of extract has consistently produced ovula-tion in the normal doe rabbit. In its final form, it contains about 4-5 mg. of water-soluble substance per ml.
Technique. Single injections of extract were made intravenously at definite
times after mating. Five does were used in each series of experiments. The volume of extract injected in every series (excepting the first) was such that every doe re-ceived the same equivalent of original tissue per kg. body weight. Rabbits injected with extracts were killed 3 days later for the reasons given below. Controls were injected with saline and were allowed to proceed to full term.
III. EXPERIMENTAL.
Two groups of experiments are described. The first was carried out during 1929-30 as part of a general investigation on the physiology of the anterior lobe of the pituitary. The second and larger group was performed in the spring of 1934.
Group 1.
Four series consisting of two does each were injected with extract made ac-cording to the first method described above. They were all killed on the third day, and each received the equivalent of 5-5 gm. of original tissue (i.e. 1 gm. of dried gland). The results are given in Table I.
Table I.
No. of d o e
A i A 2 A3 A4
A 5 A 6 Ay A 8
Duration of pregnancy in days at time of injection
18 18 19 19
2 2 2 2
27 27
Number of structures in when killed
Old white corpora lutea of pregnancy
1 2 1 2 1 2
9
1 0 1 1
9 8
corpora lutea
2 1
' 4 14
2 0 1 2 1 1
16 17
ovaries
rhagic follicles
8 6
1 1
9 13
1 1 1 2
16
Contents and con-dition of uterus
6 resorptions Empty, flaccid Empty, flaccid g resorptions 9 resorptions 4 placentae only 8 resorptions 3 placentae only
The data exhibited in this table may be summarised thus:
(a) Ovulation was induced in all cases. From the appearance of the young
ap-peared to have reached the same stage of development and most of them showed the characteristic pimple exhibited by recently formed corpora lutea.
(b) The functional life of the corpora lutea of pregnancy had presumably been
terminated, all these bodies being white and non-vascularised and apparently in an early stage of atrophy.
(c) Ovulation did not occur exclusively, varying numbers of haemorrhagic
follicles being produced in both ovaries. A considerable difference existed in the response of each individual doe, and the proportions of young corpora lutea or haemorrhagic follicles formed showed a wide variation. For this reason subse-quent experiments are considered in groups of not less than four does.
(d) In every case abortion or resorption of the foetuses took place.
(e) In five out of the eight does, a fur nest was made during the third night after
injection.
The last observation led to the decision to kill does on the third day in subse-quent experiments because the occurrence of this behaviour clearly showed that pregnancy ended within this interval. In fact nesting was the only obvious sign that the pregnancy had been terminated. Although the cages were examined carefully every day, in no case was it possible to ascertain from inspection of the bedding alone whether abortion or resorption of the young had occurred. It appears that the doe is meticulous in removing any signs which might indicate that pregnancy had come to an end. She eats immediately not only the aborted embryos and placentae but also any straw which may bear traces of blood. It was often possible, however, to determine whether resorption of embryos had taken place by pressing the lower part of the abdomen when a turbid brown or yellow fluid could be expressed from the vulva.
Group 2.
In this group six series of experiments were undertaken. Each series consisted of five does. Extracts were prepared from the fresh gland. Each doe received a dose which was equivalent to 0-5 gm. (0-125 m'-) °f fresh tissue per kg. body weight. The results of the first two series are given in Table II, and are grouped together because they have certain features in common.
All these animals made a nest during the third night after injection. Pregnancy was terminated in every case and was associated with the same features observed in the previous series.
Two further series are summarised in Table III. One doe in each series was found to have been non-pregnant on post-mortem (Nos. 11 and 19). Nests were made by only two does in each series (Nos. 14, 15, 17, 19), but as before they were constructed during the third night after injection.
In three cases ovulation without the accompanying formation of haemorrhagic follicles occurred (Nos. 12, 13, 15).
The last two series are given in Table IV. None of these does made a nest. One new feature was observed. Examination showed that in six cases the ovaries
contained two types of corpora lutea of pregnancy. In addition to the old white non-vascularised corpora lutea previously observed, there were others which were orange and highly vascularised. These were apparently normal young corpora lutea which had not undergone atrophy.
Table II. No. of doe 2 1 2 2 23 24 25 26 27 28 2 9 3 0 No of doe 11 1 2 13 1 4 i s 1 6 1 7 18 ' 9 2 0 Weight of doe in kg.
2 8 0
2-22 2-50 2-69 2-29 2-54 2-70 2-60 2-57 2 6 2
Weieht of doe in kg
2 1 2
2-82 2-30 2-67 2-24 2-12 2-53 1-79
2 6 2 2 - I O
Number of structures when killed
__
Old white corpora lutea of pregnancy
Series injected 9 1 1 9 8 8 Series injected 6 1 2 8 9 1 0 Young corpora lutea
on the 23rd day
1 3 1 0
13 11 1 1
on the 26th day
2 5 13 2 2 1 9 Table III.
Number of structures when killed
Old white corpora lutea of pregnancy
Series injected 7 9 7 6 7 Series injected 7 6 9 8 0 Young corpora lutea
on the 16th day
7 8 4
11 1 4
on the 19th day
8 11 8 8 6 in ovaries Haemor-rhagic follicles of pregnancy 16 18 8 7 1 3 of pregnancy 1 4 4 11 8 4 in ovaries Haemor-rhagic follicles of pregnancy 0 0 0 4 0 of pregnancy 5 7 4 1 1 3
Contents and con-dition of uterus
Empty, flaccid Empty, flaccid
2 resorptions 1 resorption
5 resorptions and
2 placentae
2 resorptions and 4 placentae 6 resorptions 2 resorptions
Empty, flaccid
1 resorption
dition of uterus
Pseudo-pregnant 9 resorptions 7 resorptions 4 resorptions 1 resorption and
6 placentae 2 placentae 6 resorptions 9 resorptions 4 resorpttons Pseudo-pregnant
instances (Nos. 9 and 10) only ruptured follicles were found in association with the atrophic corpora lutea of pregnancy.
A summary of the results of the control experiments is given in Table V. The data pertaining to the condition of the ovaries in the six series of does injected with anterior lobe extract is summarised in Table VI.
Table IV. No. of doe 1 2 3 4 S 6 8 9 10 Weight of doe in kg.
2-45 2'12 2 1 1 I-87 1-97 2 34 240 2-30 i-95 2 2 4
Number of structures in ovaries when killed
Corpora lutea of pregnancy
Atrophic Normal
Series injected on the 5th day of pregnancy Young corpora lutea Haemor-rhagic follicles
Contents and condition of uterus 6 7 9 7 4 6 2 1 0 5 2 O O 2 O IO 14 9 8 11
SerieB injected on the 12th day of pregnancy
No traces of implantation 9 blastocysts
No traces of implantation No traces of implantation No traces of implantation
IO o 6 8 6 6 7 10 placentae 9 placentae
4 placentae, 2 reaorptions 3 placentae, 2 resorptions 7 placentae Table V. No. of series 1 2 3 4 5 6 Totals No. producing litters 4 4 5 5 5 4 27
No. making pseudo-pregnant nests 1 0 0 0 0 1 2
No. failing to ovulate after mating 0 M 0 0 0 0 1 Table VI. No. of series Duration of pregnancy in
days at time of injection 5 12 16 19 23 26
Average number of structures in both ovaries when killed
Corpora lutea of pregnancy 94 8-2 7-2 7'5 90 90 Young corpora lutea o-8 6-2 9'2 87 116 12-6 Haemor-rhagic follicles 10-4 i-4 i-o 67 12-4 8-2
Two main conclusions can be drawn from the data presented in this table. Firstly, there is a definite increase in the total number of haemorrhagic follicles and young corpora lutea which are produced in the second half of pregnancy. Nearly double the number are found after 19 days. It is clear that there is a great increase in the number of follicles which will respond to injection in the later stages. Secondly, considerable difference exists in the nature of the response occurring at various stages of pregnancy. From 19 days onward approximately equal propor-tions of haemorrhagic follicles and young corpora lutea are produced. Between 12 and 16 days ovulation mainly occurs, but at 5 days a preponderance of haemorrhagic follicles results from injection.
Details concerning the fate of the developing embryos is summarised in Table VII. From the previous tables it will be seen that several different phe-nomena followed injection. In some cases (Nos. A2, A3, 29, 21, 22) the uterine contents were completely expelled, so that one cannot say whether the foetuses had died before abortion. In other does (Nos. A 6, A 8, 16, 6, 7, 10) only placentae were found. These were attached to the wall of the uterus, and as far as could be seen were not autolytic. In these cases again one cannot say whether abortion of live embryos had occurred or whether death was followed by complete resorption. In the majority of cases the uterus only contained foetuses and placentae in an ad-vanced stage of resorption. Frequently autolysis had progressed so far that it was difficult to distinguish between the two (Nos. A i , A4, A5, A7, 27, 28, 30, 23, 24, 12, 13, 14, 17, 18, 19). In three other does (Nos. 15, 25, 26) normal placentae at-tached to the uterine wall were found side by side with resorbing foetuses.
No. of series
1 2 3 4 5 6
Duration of pregnancy in
days at time of injection
5 1 2
16 19 23 26
No. of does pregnant
when injected
5 5 4 4 5 5
Table VII
Contents
Retained placentae
0
2 4
6 2 2 4
of uterus
Resorptions
0 4 2 0 1 9 8
9
No. of corpora lutea of pregnancy
3« 4 1 2 9 3° 45 45
No. of embryos unaccounted
for
3« 13 3 9 35 32
On the other hand the contractile strength of the uterus in the early stages of pregnancy may be insufficient to expel the more firmly attached placentae. The large number of embryos unaccounted for in Series 1 is probably due to the fact that at such an early stage it was practically impossible to detect any signs that the blastocysts had undergone resorption.
Before dealing with the conclusions reached from the present experiments it is necessary to consider one point of importance. The results are similar to those ob-tained by Knaus (1926) with injection of pituitrin. In view of the fact that some evidence is available which indicates that rapid post-mortem diffusion of the auto-coids of the pituitary gland can occur from one lobe to the other, one must exclude the possibility of the results being due to the action of the oxytocic substance or of the possible action upon the uterus of non-specific substances formed during preparation of the extracts. This applies particularly to the crude acid extracts prepared from the dried gland. Specimens of extract used in the second group of experiments were tested for oxytocic activity and for histamine and tyramine. It was found that 1 ml. of extract contained 0-05 unit of pitocin. The greatest volume of extract ever administered to a doe (0-375 m^-) therefore only contained 0-018 unit: an amount which according to Knaus's figures would not have any action. The presence of histamine or tyramine could not be detected in 2 ml. of extract. In the present experiments any effects due to these substances can be therefore excluded.
It would not be out of place to deal briefly with the occurrence of nest making The data shows that when this occurred it did so with unfailing regularity 36-48 hours after ovulation had been induced. A summary of the data is given in Table VIII. It is apparent that pregnancy must be of at least 12-16 days' duration before nesting behaviour can arise. If some relationship exists between the cessation or function of corpora lutea and the occurrence of the nest formation—as is believed from the fact that the latter occurs after pseudo-pregnancy—it is clear that there is a time factor involved. That is to say, the induction of this behaviour depends not only upon the fact that corpora lutea have been present in the ovaries but also upon the time they have been active.
Table VIII.
No. of series No. of does pregnantwhen injected
5 S 4 4 5 5
Duration of pregnancy in days when injected
5
12 16 19 23
z6
IV. CONCLUSIONS.
The main point that emerges from the present communication is that pregnancy can be readily terminated in the rabbit after intravenous injection with single small doses of extract prepared from the anterior lobe of the pituitary. In the first group of experiments twenty-seven out of a possible twenty-eight does failed to produce their litters, whilst in the controls all twenty-seven pregnant animals did so. This was associated with atrophy of the existing corpora lutea of pregnancy, ovulation and the formation of haemorrhagic follicles. There are several alternative explana-tions as to how termination of pregnancy may be brought about. In some cases it took place in the absence of ovulation, so it does not mean that the latter process must take place before pregnancy can end. This is rendered feasible when it is remembered that secretion of oestrin by the ovary could possibly be stimulated by anterior lobe pituitary extracts in the absence of detectable morphological changes, and that the production of oestrin is incompatible with the continuance of preg-nancy (Smith, 1926; Parkes and Bellerby, 1926). In this event termination results solely from the cessation of function of the corpora lutea of pregnancy, and the problem reduces to finding the reason why resorption or abortion should occur in the absence of secretory activity of these bodies. That is to say, the sequence of events is the same as when double ovariectomy is carried out during pregnancy. Another alternative is that the extract has some direct or indirect action upon the uterus. This is not supported by the present experiments, although Reynolds (1932) has found some such action in that the contracting uterus of the rabbit becomes fully quiescent after a single intravenous injection of extract prepared from the anterior lobe of the pituitary. A third alternative is that the extract has a direct toxic effect upon the foetuses themselves. Before any final conclusions can be drawn, further work must be carried out to exclude some of these possibilities.
V. SUMMARY.
1. Ovulation can be induced in the pregnant rabbit at different stages of gesta-tion by a single intravenous injecgesta-tion of extract prepared from the anterior lobe of the pituitary.
2. It is almost invariably associated with either resorption or abortion of the embryos.
3. In most cases, atrophy of all the existing corpora lutea of pregnancy is pro-duced, but in the early stages both atrophic and non-atrophic structures can be found together.
4. Ovulation does not occur exclusively; haemorrhagic follicles being also produced as a result of the action of the extract.
5. The proportions in which ruptured and haemorrhagic follicles are formed varies according to the stage of pregnancy at which injection is made.
available, in this as in other experiments, a supply of the dried gland. The investi-gation was carried out during the tenure of a grant from the Birth Control Investigation Committee.
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