Effects of Section of Utero-Ovarian Vascular Connections on the Duration of Pseudopregnancy in the Rat
EFFECTS OF SECTION OF UTERO-OVARIAN VASCULAR CONNECTIONS ON THE DURATION OF PSEUDOPREGNANCY IN THE RAT
J. D. O'SHEA and C. S. LEE Department of Veterinary Preclinical Sciences, University of Melbourne, Parkville, Victoria 3052, Australia (Received 20th March 1972)
Summary. Two experiments were performed on female Hooded Wistar rats to determine the effects ofsection of utero-ovarian connections on the duration of pseudopregnancy. Ligation and section of the anterior uterine blood vessels on Day 6 of pseudopregnancy caused a prolongation of the pseudopregnancy during which the operation was performed, and also of the subsequent pseudopregnancy (P<0\m=.\01).Neither section of the Fallopian tube nor that of the mesosalpinx and broad ligament significantly affected the duration of pseudopregnancy. When the anterior uterine vessels were sectioned separately, it was shown that severing the artery led to a prolongation of pseudopregnancy (P<0\m=.\001).Section of the vein did not significantly affect the duration of pseudopregnancy. These results suggest that the effects on pseudopregnancy of section of utero-ovarian connections depend primarily on interruption of the anterior uterine artery.
INTRODUCTION There is considerable evidence that in many species of mammals the life-span of the corpus luteum is controlled, at least in part, by the release of a luteolytic substance or substances from the uterus (Schomberg, 1969; Rowson, 1970). The concept that uterine luteolytic activity involves a local, unilateral com¬ ponent, whereby each uterine horn is able to exert a preferential effect on the ipsilateral ovary, has also been supported by data from several species (Fischer, 1967; Ginther, 1967). Evidence of local uterine luteolytic activity in the rat derives principally from the demonstration by Barley, Butcher & Inskeep (1966) that unilateral hysterectomy accompanied by contralateral ovariectomy leads to a prolonga¬ tion of pseudopregnancy, whereas unilateral hysterectomy with ipsilateral ovariectomy is without effect. Ligation and cutting of the oviduct and meso¬ salpinx also led to a prolongation of pseudopregnancy, and Barley et al. (1966) concluded that the ability of the uterus to limit the life-span of the corpora lutea 245 Downloaded from Bioscientifica.com at 09/26/2021 11:10:10PM via free access 246 J. D. 0'Shea and C. S. Lee depended on the proximity of the uterus to the ovary. However, subsequent experiments involving reapposition of the uterus to the contralateral (Butcher, Barley & Inskeep, 1969) or ipsilateral (O'Shea & Lee, 1972a) ovary have failed to restore the normal duration of pseudopregnancy, suggesting that spatial separation is not an essential feature. Clemens, Minaguchi & Meites (1968) were able to prolong pseudopregnancy in the rat simply by ligating blood vessels 'joining the uterus to the ovary', and similar effects have been reported in the guinea-pig (Bland & Donovan, 1969) and sheep (Dobrowolski & Hafez, 1970). In view of recent theories on the rôle of connections between the uterine and ovarian blood vessels in the mediation of local uterine luteolytic activity (Phar- riss, 1970; McCracken, Baird & Goding, 1971), further clarification of the basis for prolongation of luteal life-span following interruption of utero-ovarian connections is desirable. This paper describes experiments concerned with defining more precisely the effects on the duration of pseudopregnancy of section of the various structures connecting the ovary and uterus in the rat.
MATERIALS AND METHODS General Female Hooded Wistar rats aged 8 weeks were used in two experiments. Pseudopregnancy was induced by sterile mating with vasectomized males, which remained caged with the females throughout the experiments. Vaginal smears were taken daily for 9 days before the introduction of males to select females showing normal oestrous cyclical activity. Only rats showing an oestrous cycle length of 4 to 6 days before the first pseudopregnancy were included in the experiments. Vaginal smears were continued daily throughout the experi¬ ments. All surgery was performed bilaterally under ether anaesthesia through a ventral mid-line incision. Operations were performed on Day 6 of pseudo¬ pregnancy in all rats, Day 0 being designated the first day of vaginal oestrus. The termination of pseudopregnancy was recognized by the return of vaginal oestrus. Experiment 1 The effects of section of the uterine tube (Text-fig. 1, a), the mesosalpinx and broad ligament (Text-fig. 1, b) and the anterior uterine artery and vein (Text-fig. 1, c) on the duration of pseudopregnancy were studied in a factorial experiment. Sixty-four rats, randomly allocated to eight groups each of eight rats (Table 1), were maintained throughout two consecutive pseudopreg- nancies. Rats in the control group (Group I) were subjected to laparotomy and handling of both uterine horns. Section of the uterine tube involved prior ligation with 4/0 monofilament nylon immediately above and below the site of section, which was performed close to the uterotubal junction (Text-fig. 1, a). Free ends of the two ligatures were then tied to one another to restore as far as possible the normal positional relationship between the uterus and ovary.
Downloaded from Bioscientifica.com at 09/26/2021 11:10:10PM via free access Section of rat utero-ovarian vascular connections 247 Section of the anterior uterine artery and vein, between the point of origin of these vessels from the utero-ovarian vessels and the point at which the first uterine branches arose (Text-fig. 1, c), was performed after ligation on either side of the point of section. The mesosalpinx and broad ligament were cut without ligation. On the first day of dioestrus following the end of the second pseudopregnancy, four rats from each group were anaesthetized and 0-5 to 10 ml of Indian ink (Pelikan) was injected into the left saphenous vein. After 2 to 5 min, these rats were killed with an overdose of chloroform. The uterine horns were pinned out to expose the blood vessels in the mesovarium and broad ligament. The ab¬ dominal contents were then fixed in situ in 10% formalin. After fixation, the entire genital tracts of these rats were dissected out, dehydrated in alcohol and cleared in xylene to permit examination of their blood vessels.
Text-fig. 1. Diagramatic representation of one side of the female genital tract of the rat, together with its accompanying blood vessels. The sites of section of the Fallopian tube (a), the mesosalpinx and broad ligament (b), and the anterior uterine vessels (c) in Exp. 1 are indicated. Ov == ovary; Ut = uterine horn.
Experiment 2 Four groups each of twenty-one rats (Table 3) were used in an experiment to differentiate the effects of section of the anterior uterine artery from those of section of the anterior uterine vein. Section of these vessels, individually or in combination, was performed at the same site as in the previous experiment, following ligation on both sides of the point of section. Rats in the control group (Group I) were subjected to laparotomy, handling of the uterus, and puncture of the mesosalpinx on both sides of the anterior uterine vessels. Puncture was performed in the same manner as that used to pass ligatures around these vessels in the experimental groups. All rats in the experimental groups, and fourteen control rats, were injected with Indian ink on the first day of dioestrus following the end of the second pseudopregnancy, using the technique described previously. In addition to the groups described above, a further six rats were subjected to section of the anterior uterine artery and six to section of the anterior uterine vein. Four rats from each group of six were injected with Indian ink 7 days
Downloaded from Bioscientifica.com at 09/26/2021 11:10:10PM via free access 248 J. D. 0'Shea and C. S. Lee after section to study the condition of the blood vessels. The remaining two rats from each group were killed 5 days after operation (on Day 11 of pseudo¬ pregnancy) and their uteri examined grossly and histologically for evidence of deciduoma formation. Material for histological examination was fixed in 10% buffered neutral formalin, and embedded in paraffin wax. Sections were stained with haematoxylin and eosin (H and E). Complete cross sections from the anterior, middle and posterior thirds of each uterine horn were examined. Histological examination of the anterior uterine vessels and mesosalpinx at the site where section would normally have been performed was carried out on a final additional group of five normal female rats. Tissues from these rats were fixed in Bouin's fluid and embedded in paraffin wax, and sections were stained with H and E. Statistical analysis of data Data were analysed for statistical significance using Student's t test and analysis of variance (Sokal & Rohlf, 1969).
RESULTS Experiment 1 The mean durations of two pseudopregnancies in each of the eight groups are shown in Table 1. Factorial analysis of variance (Table 2) showed that the only single factor to produce a significant lengthening of pseudopregnancy was section of the anterior uterine vessels (P<0-0\ in both pseudopregnancies).
Table 1. Duration of pseudopregnancy in rats following section of utero- ovarian connections
Structures sectioned Mean duration ofpseudopregnancy No. (days±S.E.M.) Group of Fallopian Mesosalpinx Anterior rats tube and broad uterine First Second ligament vessels pseudopregnancy pseudopregnancy I 13-5±0-42 13-5 + 0-57 II 13-8 ±0-49 12-6 + 0-18 III + 14-0 ±0-60 12-8±0-25 IV + 15-6 + 0-50 14-6 ±0-46 V + + 14-2 ±0-70 14-5 + 0-57 VI + + 14-9 + 0-52 14-1 ±0-35 VII + 15-0 ±1-00 14-4 ±0-46 VIII + + 15-8 ±0-45 14-6 + 0-65
Neither section of the uterine tube nor section of the mesosalpinx and broad ligament produced a significant effect. However, there was a significant inter¬ action between these two factors in the second pseudopregnancy only ( <0·05). In six of the eight groups, the mean duration of the second pseudopregnancy was less than that of the first. When the results of the two pseudopregnancies were combined in a single factorial analysis of variance, this shortening was significant (F = 8-79; P<0-01).
Downloaded from Bioscientifica.com at 09/26/2021 11:10:10PM via free access Section of rat utero-ovarian vascular connections 249 Examination of the specimens injected with Indian ink confirmed that section of the anterior uterine vessels had been successfully accomplished in the appropriate groups (Groups IV, VI, VII and VIII—Table 1). The result shown in PI. 1, Fig. 4 (from Exp. 2) indicates the type of appearance seen after the second pseudopregnancy in Group IV. Compared with normal control rats (PI. 1, Fig. 1), the vessels were widely separated at the point of section, as indicated by the distance between the two ligatures, and any regrowth of vessels across the gap was minimal. However, in some rats, small tubai vessels arising close to the bifurcation of the utero-ovarian vessels remained attached to the latter after section of the anterior uterine vessels. Small connections were sometimes present between these tubai vessels and the vessels supplying the anterior tip of the uterine horn. Table 2. Factorial analysis of variance of effects of section of Fallopian tube, mesosalpinx and broad ligament, and anterior uterine vessels on the duration of pseudopregnancy in rats
First pseudopreg\ancy Second pseudopregnancy Factor
Individual factors A Section of Fallopian tube 008 N.S. 0-23 N.S. Section of mesosalpinx and 0-52 N.S. 1-10 N.S. broad ligament C Section of anterior 11-06 <0-01 11-24 <0-01 uterine vessels Interactions AxB 0-75 N.S. 6-65 <0-05 AxC 0-08 N.S. 0-68 N.S. BxC 0-19 N.S. 0-39 N.S. AxBxC 0-75 N.S. 2-11 N.S.
N.S. = Not significant. > 005.
In those groups (Groups I, II, III and V—Table 1) where the vessels were not cut, the vascular arrangement appeared normal in the specimens injected with ink. Ligation and section of the uterine tubes led to the accumulation of watery fluid in the ovarian bursae, and distension of those parts of the tube anterior to the site of section in most, but not all, rats. Experiment 2 The mean durations of the two pseudopregnancy cycles in the four groups in this experiment are shown in Table 3. Both the first and second pseudopreg¬ nancies were longer in Group II (P<0-02) and Group IV ( <0·01) than in Group I. The only other significant differences were between Groups IV and III in both pseudopregnancies ( <0·01) and between Groups II and III in the second pseudopregnancy only ( <0 5). Analysis of variance (Table 4) showed a significant lengthening of both pseudopregnancies associated with
Downloaded from Bioscientifica.com at 09/26/2021 11:10:10PM via free access 250 J. D. 0'Shea and C. S. Lee section of the anterior uterine artery, but no significant effect from section of the anterior uterine vein. Nor was there any significant interaction between the effects of section of the artery and vein. In all groups, the second pseudopregnancy was significantly shorter than the first, by approximately 1 day (Table 3). Table 3. Duration of pseudopregnancy in rats following section of anterior uterine artery and anterior uterine vein
Structures sectioned Duration ofpseudopregnancy No. (days±S.E.M.) Group of Anterior Anterior (difference between rats uterine uterine First Second first and second artery vein pseudopregnancy pseudopregnancy pseudopregnancies) I 21 13-6 + 0-20 12-9 + 0-14 <001 II 21 + 14-7 + 0-36 13-7 + 0-29 <005 III 21 + 14-0 + 0-21 12-9 + 0-17 < 0-001 IV 21 + + 14-9±0-26 14-0 + 0-33 <0-05
Examination of specimens injected with ink (PI. 1, Figs 1 to 4) confirmed the efficacy of section of the vessels, both at 7 days after operation and at the end of the second pseudopregnancy. However, as in the previous experiment, tubai vessels joining the utero-ovarian vessels remained in many rats. Anasto- motic connections between these and vessels supplying the anterior tip of the uterine horn were also present in several rats. Table 4. Analysis of variance of effects of section of anterior uterine artery and anterior uterine vein in rats
First pseudopregnancy Second pseudopregnancy Factor
Individual factors A Section of anterior 13-36 <0001 15-70 <0001 uterine artery Section of anterior 1-37 N.S. 0-76 N.S. uterine vein Interaction AxB 0-07 N.S. 0-46 N.S.
N.S. = Not significant. f>005.
No evidence of deciduoma formation was found, either grossly or histo- logically, in any of the four rats studied for this purpose. Histological examination of the anterior uterine vessels and mesosalpinx revealed the features illustrated in PI. 2, Figs 5 and 6. The anterior uterine artery and vein were separated at, and near to, their points of origin from the utero-ovarian vessels (PI. 2, Fig. 5), but converged before the point of origin of the first uterine branches (PI. 2, Fig. 6). This arrangement generally pro-
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Fig. 1. Photograph of Indian ink-injected, cleared specimen of the left ovary (Ov) and anterior part of the uterine horn (Ut) from a normal control rat. The utero-ovarian (VUO), ovarian (VO), anterior uterine (VAU) and uterine (VU) blood vessels are indicated. The artery is the smaller vessel in each case. Between the upper limbs of the Y-shape formed by the bifurcation of the utero-ovarian vein can be seen the smaller Y-shaped branching of the utero-ovarian artery. Fig. 2. Specimen similar to Fig. 1, from a rat in which the anterior uterine artery had been sectioned. Ligatures are seen on the cut ends of this vessel. Fig. 3. Similar specimen, following section of the anterior uterine vein. Ligatures are seen on the cut ends of the vein, and the Y-shaped branching of the anterior uterine artery is clearly visible. Fig. 4. Similar specimen, following section of both anterior uterine artery and vein. The two ligatures are again visible and the considerable separation of the cut ends of the vessels is apparent by comparison with Fig. 1.
(Facing p. 250)
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Fig. 5. Rat anterior uterine artery (top) and vein at approximately the position where section was normally performed. At this site near their origins, these vessels are separated from one another by adipose tissue. Small nerves (n) and a small vessel identified as a lymphatic vessel (1) are seen. H and . 120. Fig. 6. Rat anterior uterine artery (top left) and vein, slightly further from their origins, lying in close apposition to one another. Two small nerves (n) and a probable lymphatic vessel (1) are present. and . 120.
(Facing p. 251)
Downloaded from Bioscientifica.com at 09/26/2021 11:10:10PM via free access Section of rat utero-ovarian vascular connections 251 duced a small triangular space between the two vessels, as is seen in PI. 1, Fig. 1. In addition to the anterior uterine artery and vein, the mesosalpinx contained smaller blood vessels, considerable amounts of fat, small nerves and occasional small thin-walled vascular channels presumed to be lymphatic vessels (PI. 2, Figs 5 and 6).
DISCUSSION The present demonstration that section of the anterior uterine vessels prolongs pseudopregnancy in the rat confirms the observations of Clemens et al. ( 1968), and accords with observations on the guinea-pig (Bland & Donovan, 1969) and sheep (Dobrowolski & Hafez, 1970). Neither section of the uterine tube nor section of the mesosalpinx and broad ligament produced any effect on the duration of pseudopregnancy, although a significant interaction between these two procedures was found in the second pseudopregnancy. The reason for this interaction, assuming that it represented a real effect, is not obvious. It seems possible that cutting these structures in combination might have placed an extra physical strain on the anterior uterine vessels, and thereby partially restricted blood flow, although no evidence of abnormal stretch was apparent from those specimens from this group which had been injected with ink. In some rats, persistent connections between small vessels at the anterior tip of the uterine horns and tubai vessels attached to the utero-ovarian artery and vein were observed. These may have permitted a small amount of vascular 'bypass' around the site of section of the anterior uterine vessels. It is possible that this could have lessened to some degree the extent of the effect on pseudo¬ pregnancy which resulted from section of these vessels. Significant effects were nonetheless produced by the sectioning procedures used, and there is no doubt that continuity between the uterine and utero-ovarian vessels was substantially interrupted throughout the duration of these experiments. Two pseudopregnancies were studied in all rats to minimize the danger of 'non-specific' effects (Nalbandov & Cook, 1968) on luteal life-span due to surgery performed during pseudopregnancy. In particular, the induction of deciduoma formation, with a resultant prolongation of luteal life-span (Velardo, Olsen, Hisaw & Dawson, 1953), poses a hazard in experiments involving surgery during pseudopregnancy (Shelesnyak & Marcus, 1967). It is not likely that deciduoma formation was a factor in the effect produced in the present study. All operations were performed on Day 6 of pseudopregnancy to avoid the period of maximal uterine sensitivity (De Feo, 1963), and no evidence of deciduomata was found in any of four animals examined 5 days after operation. The fact that the significant effects of section of the anterior uterine vessels in Exp. 1, and of the anterior uterine artery in Exp. 2, were observed in the second as well as the first pseudopregnancy adds support to the interpretation that these effects were specific. The second pseudopregnancy was shorter than the first in both experiments. This could have been partially due to a 'non-specific' effect of surgery during the first pseudopregnancy. More probably, however, this shortening was largely or entirely due to the phenomenon of shortening of vaginal oestrus and pseudo-
Downloaded from Bioscientifica.com at 09/26/2021 11:10:10PM via free access 252 J. D. 0'Shea and C. S. Lee pregnancy which normally follows a previous pseudopregnancy in the rat (O'Shea & Lee, 1972b). The possibility that incidental damage to structures other than the anterior uterine blood vessels may have contributed to, or produced, the observed effects on pseudopregnancy cannot be entirely dismissed. It is likely that both nerves and small lymphatic vessels were interrupted in the process of sectioning the blood vessels. However, it is unlikely that these structures could have been consistently interrupted in section of the artery, while remaining intact when the adjacent vein was cut. Two theories have recently been advanced to explain local uterine luteolytic activity in terms of release of prostaglandin F2c( (PGF2a) from the uterus. Pharriss (1970) proposed that PGF2„ released by the uterus could pass down the common utero-ovarian venous pathway (utero-ovarian vein in the rat) and exert a venoconstrictor action, thereby restricting ovarian blood flow and indirectly precipitating luteolysis. An alternative hypothesis in relation to the sheep (McCracken et al., 1971 ; Goding, Baird, Cumming & McCracken, 1971) suggested that PGF2ct may pass down the uterine vein and be transferred by a countercurrent mechanism to the adjacent ovarian artery. Thence, it would be carried to the ovary and bring about luteolysis by a mechanism as yet undefined. Both of these hypotheses involve the passage of luteolysin from the uterus down a common utero-ovarian venous pathway. From both, it could be pre¬ dicted that, in the case of the rat, section of the anterior uterine vein would interfere with the local luteolytic mechanism. The present observation that section of the anterior uterine vein was without significant effect on the duration of pseudopregnancy does not therefore support either hypothesis in relation to this species. It is not clear why section of the anterior uterine artery led to prolongation of pseudopregnancy. An important factor in interpretation of this result is the direction of blood flow in this artery. Clemens et al. (1968) appear to have assumed that this vessel carries blood from the uterine artery to the ovary. However, uterine branches of utero-ovarian arteries are more conventionally regarded as carrying blood in the opposite direction, and supplying the anterior part of the uterus. A definitive answer on this point is important since arterial flow towards the ovary could fulfil the essential conditions of the veno-arterial transfer hypothesis (McCracken et al., 1971), if it be assumed that transfer of luteolysin could take place between the uterine vein and the adjacent uterine artery. Another possibility, if the anterior uterine artery is a significant source of uterine blood supply, is that severance of this vessel might reduce uterine blood flow sufficiently to interfere with the production or release of luteolysin. Such an effect could result in a prolongation of pseudopregnancy whether the luteo¬ lytic mechanism operated locally or systemically. Experiments involving partial hysterectomy in the rat (Anderson, Melampy & Chen, 1966) have shown that a quantitative relationship exists between the duration of pseudopregnancy and the amount of uterine tissue removed. Uterine luteolytic activity is therefore not an all-or-none phenomenon, and a proportionate degree of lengthening of pseudopregnancy could be expected to follow any interference with the pro-
Downloaded from Bioscientifica.com at 09/26/2021 11:10:10PM via free access Section of rat utero-ovarian vascular connections 253 duction or release of luteolysin. Further studies are needed to determine whether uterine blood supply is reduced following section of the anterior uterine vessels, and if so whether such reduction of uterine blood flow is, in fact, sufficient to reduce uterine luteolytic activity. Finally, it is also possible that, if blood flow in the anterior uterine artery is towards the uterus, section of this vessel could result in an increased supply of blood to the ovary. It is conceivable that an improved blood supply to the corpora lutea could lengthen their life-span by conferring a partial protection against luteolytic influences operating at the end of pseudopregnancy. This possibility could be better evaluated if the mechanism of action of the uterine luteolysin, and the haemodynamic effects of interruption of the anterior uterine artery, were known. ACKNOWLEDGMENTS This work was supported by the Melbourne University Veterinary Research Fund. Rats were kindly donated by the C.S.I.R.O. Division of Animal Health. We are grateful to Dr R. G. Beilharz for advice and help with statistical analysis of the data. Skilled technical assistance was provided by Miss I. E. Kerry and Mr M. T. L. Greig. REFERENCES Anderson, L. L., Melampy, R. M. & Chen, C. L. (1966) The uterus and ovarian activity in the pseudopregnant rat. Anat. Ree. 154, 309. Barley, D. ., Butcher, R. L. & Inskeep, E. K. (1966) Local nature of utero-ovarian relationships in the pseudopregnant rat. Endocrinology, 79, 119. Bland, K. P. & Donovan, . T. (1969) Observations on the time of action and the pathway of the uterine luteolytic effect of the guinea-pig. J. Endocr. 43, 259. Butcher, R. L., Barley, D. A. & Inskeep, E. K. (1969) Local relationship between the ovary and uterus of rats and guinea pigs. Endocrinology, 84, 476. Clemens, J. ., Minaguchi, H. & Meites, J. (1968) Relation of local circulation between ovaries and uterus to lifespan of corpora lutea in rats. Proc. Soc. exp. Biol. Med. 127, 1248. De Feo, V. J. (1963) Temporal aspect of uterine sensitivity in the pseudopregnant or pregnant rat. Endocrinology, 72, 305. Dobrowolski, W. & Hafez, E. S. E. (1970) The effect of destruction of utero-ovarian vascular con¬ nections on the life-span of the corpus luteum in sheep. J. Reprod. Fert. 23, 165. Fischer, T. V. (1967) Local uterine regulation of the corpus luteum. Am.J. Anat. 121,425. Ginther, O. J. (1967) Local utero-ovarian relationships. J. Anim. Sci. 26, 578. Goding, J. R., Baird, D. T., Cumming, I. A. & McCracken, J. A. (1971) Functional assessment of auto- transplanted endocrine organs. In : Perfusion Techniques, 4th Karolinska Symposium on Research Methods in Reproductive Endocrinology, p. 169. Ed. E. Diczfalusy. Karolinska Institutet, Stockholm. Acta endocr., Copenh. Suppl. 158. McCracken, J. ., Baird, D. T. & Coding, J. R. (1971) The study of ovarian function in the ewe by autotransplantation to the neck. Recent Prog. Horm. Res. 27, 537. Nalbandov, A. V. & Cook, B. (1968) Reproduction. A. Rev. Physiol. 30, 245. O'Shea, J. D. & Lee, C. S. (1972a) Local uterine luteolysis in the rat. J. Reprod. Fert. 28, 155. O'Shea, J. D. & Lee, C. S. (1972b) Shortening of pseudopregnancy and the oestrous cycle following a previous pseudopregnancy in the rat. J. Reprod. Fert. 28, 281. Pharriss, . B. (1970) The possible vascular regulation of luteal function. Perspect. Biol. Med. 13,434. Rowson, L. E. A. (1970) The evidence for luteolysin. Br. med. Bull. 26, 14. Schomberg, D. W. (1969) The concept ofa uterine luteolytic hormone. In: The Gonads. Ed. K. W. McKerns. North-Holland Publishing Co., Amsterdam. Shelesnyak, M. C. & Marcus, G. J. (1967) On the incidence of spontaneous decidualization in the rat. J. Reprod. Fert. 14, 497. Sokal, R. R. & Rohlf, F. J. (1969) Biometry: the principles and practice of statistics in biological research. W. H. Freeman, San Francisco. Velardo, J. T., Olsen, A. G., Hisaw, F. L. & Dawson, A. G. (1953) The influence of decidual tissue upon pseudopregnancy. Endocrinology, 53, 216.
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