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Effects of Section of Utero-Ovarian Vascular Connections on the Duration of Pseudopregnancy in the Rat

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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.
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