J. Physiol. (1976), 256, pp. 167-174 With 4 text-fitgure8
167
Printed in Great Britain
PLASMA KININOGEN DURING THE OESTROUS CYCLE, EARLY PREGNANCY AND PSEUDOPREGNANCY IN THE RAT
BY JUDITH SENIOR AND E. T. WHALLEY From the School of Studies in Pharmacology, University of Bradford, Bradford BD7 1DP
(Received 11 August 1975) SUMMARY
1. Changes in the plasma kinin precursor, kininogen, occur following treatment with oestrogens and progestogens. This work was undertaken to attempt to relate plasma kininogen values to hormonal changes in various reproductive states. 2. During the oestrous cycle kininogen concentrations follow a nychthemeral rhythm, kininogen depletion occurring during the increase in general activity of the rat. 3. Superimposed on the nychthemeral changes in kininogen concentration are changes which may be related to the hormonal events of the oestrous cycle. 4. During early pregnancy and pseudopregnancy the kininogen level rises; this increase may be due to changes in the circulating levels of oestrogens and progestogens. 5. It is suggested that the changes in kininogen concentration may result from utilization of kinin which could be of functional significance. INTRODUCTION
The components of the kinin forming system have been shown to alter during the later stages of pregnancy in several species. Because of the short half-life and low plasma levels of circulating free kinin, changes in the kinin forming system have been estimated by measuring the plasma kinin precursor, kininogen, and the activity of the kininase enzymes. Methods for the measurement of kininase activity tend to be unsatisfactory because the enzymes which cleave the peptide chain of kinins have widely differing properties. Using the technique of measuring the plasma kinin precursor it has been shown in the human that kininogen values increase during pregnancy and
JUDITH SENIOR AND E. T. WHALLEY there is some evidence that kininogen may be mobilized during foetal delivery (Periti & Gasparri, 1966; Porter, Shennan & Smith, 1972). In the rabbit plasma kininogen also increases as gestation advances and depletion of kininogen occurs at parturition (Weigerhausen, Klausch, Hennighausen & Sosat, 1968). A similar pattern of plasma kininogen changes occurs during late pregnancy in the rat, although the depletion of kininogen at parturition is only small (McCormick & Senior, 1974a). Changes in plasma kininogen concentrations also occur during prolonged pseudopregnancy in the rat; the pattern is similar to that seen in late pregnancy but the increase in kininogen is less (Senior & Whalley, 1974a). Changes in the plasma kininogen concentration in the rat can be induced by the administration of reproductive hormones and the levels of kininogen can be moderated by changes in the ratio of oestrogen to progesterone (Senior & Whalley, 1974 b). To examine further the hypothesis that plasma kininogen levels are under hormonal control in the rat, measurements have been made during the oestrous cycle, early pregnancy and pseudopregnancy. Any changes in plasma kininogen seen in these conditions can thus be related to known hormonal changes. 168
METHODS
Virgin female and male rats of a CFE derived strain and weighing between 200 and 250 g were used. They were housed in plastic cages with not more than six animals per cage and were allowed free access to food (modified Diet 41B, Oxoid Ltd) and water. The rooms were temperature and light controlled (light 07.0019.00 hr). The stage of the oestrous cycle was determined by vaginal smear each morning, using the criteria of Long & Evans (1922) to designate the various stages of the cycle. Only females exhibiting regular 4 day oestrous cycles were used in the experiments. Pregnant animals were obtained by caging a pro-oestrous female overnight with a male rat of proven fertility. Females showing a vaginal plug or the presence of spermatozoa in the vaginal smear were designated pregnant and were housed in separate cages until taken for kininogen determinations. Pseudopregnant animals were obtained likewise, but a vasectomized male was used for mating. Day 1 of pregnancy or pseudopregnancy refers to the day of finding the vaginal plug. The concentrations of plasma kininogen were determined using the method of Diniz & Carvalho (1963), which involves the conversion of kininogen to kinin in the presence of excess trypsin (Tryptar, Armour Ltd). Blood samples were collected by cardiac puncture from rats lightly anaesthetized with ether. Rats were sampled once only. To ensure that all trypsin was inactivated at the end of the conversion, 10 ml. boiling 95 % ethanol was added to the digest and the mixture was immersed in a water-bath at 750 C for 15 min. After drying under reduced pressure the kinin was dissolved in physiological saline and assayed against synthetic bradykinin (BRS 640, Sandoz Products Ltd). A four point assay was performed using the isolated rat uterus or guinea-pig ileum preparations. The plasma kininogen concentration is expressed in ,Ag/ml., 1 jug referring to the amount of liberated kinin which is
KININOGENS IN REPRODUCTION
169
equivalent to 1 jug synthetic bradykinin. Statistical analysis of the results was performed using the Student's t test. RESULTS
Oestrous cycle Plasma kininogen determinations were performed at 6 hourly intervals in each 24 hr period. The results for the 4 days of the cycle are shown in Fig. 1; for comparison groups of male rats were also studied at the same 3
()()(5)
(6) (5)
E
(S)
(5)
(5) (5)
) ()(5)(5
(5
(5)~~~~~~5)(6 (5)~~~~~~~~~~~~~~~~~~5
(5)
2:
(5)
(5)
2
0
39 1521 3 91521 3 91521 3 9
1521
Pro-oestrous
D II
Oestrous
DI
.3 91521 Male
Fig. 1. Changes in the plasma kininogen concentration during the oestrous cycle in the rat. The numbers indicate the times at which samples were taken during pro-oestrous, oestrous and dioestrous (day 1, day 11) stages of the cycle. Values for male rats are shown for comparison. The dark bars indicate the period of darkness (19.00-07.00 hr). Standard errors and numbers of observation are indicated.
time intervals. Male and female rats show a diurnal rhythm in the plasma kininogen concentrations. This nychthemeral rhythm is characterized by high kininogen concentrations at 09.00 and 15.00 hr during the period of illumination and low values at 03.00 hr, coinciding with the eighth hour of the darkness phase. Comparison of the plasma kininogen values during the oestrous cycle reveals that for all days the results at 09.00 and 15.00 hr show no
JUDITH SENIOR AND E. T. WHALLEY 170 significant differences. The values obtained during the dark periods (at 03.00 and 21.00 hr) show a quantitative difference on various days of the cycle. At 03.00 hr on the first (DI) and second (Dli) dioestrous days the levels of kininogen (0.95 + 041 and 0-96 + 0 1 ug/ml.) are significantly lower (P
167
Printed in Great Britain
PLASMA KININOGEN DURING THE OESTROUS CYCLE, EARLY PREGNANCY AND PSEUDOPREGNANCY IN THE RAT
BY JUDITH SENIOR AND E. T. WHALLEY From the School of Studies in Pharmacology, University of Bradford, Bradford BD7 1DP
(Received 11 August 1975) SUMMARY
1. Changes in the plasma kinin precursor, kininogen, occur following treatment with oestrogens and progestogens. This work was undertaken to attempt to relate plasma kininogen values to hormonal changes in various reproductive states. 2. During the oestrous cycle kininogen concentrations follow a nychthemeral rhythm, kininogen depletion occurring during the increase in general activity of the rat. 3. Superimposed on the nychthemeral changes in kininogen concentration are changes which may be related to the hormonal events of the oestrous cycle. 4. During early pregnancy and pseudopregnancy the kininogen level rises; this increase may be due to changes in the circulating levels of oestrogens and progestogens. 5. It is suggested that the changes in kininogen concentration may result from utilization of kinin which could be of functional significance. INTRODUCTION
The components of the kinin forming system have been shown to alter during the later stages of pregnancy in several species. Because of the short half-life and low plasma levels of circulating free kinin, changes in the kinin forming system have been estimated by measuring the plasma kinin precursor, kininogen, and the activity of the kininase enzymes. Methods for the measurement of kininase activity tend to be unsatisfactory because the enzymes which cleave the peptide chain of kinins have widely differing properties. Using the technique of measuring the plasma kinin precursor it has been shown in the human that kininogen values increase during pregnancy and
JUDITH SENIOR AND E. T. WHALLEY there is some evidence that kininogen may be mobilized during foetal delivery (Periti & Gasparri, 1966; Porter, Shennan & Smith, 1972). In the rabbit plasma kininogen also increases as gestation advances and depletion of kininogen occurs at parturition (Weigerhausen, Klausch, Hennighausen & Sosat, 1968). A similar pattern of plasma kininogen changes occurs during late pregnancy in the rat, although the depletion of kininogen at parturition is only small (McCormick & Senior, 1974a). Changes in plasma kininogen concentrations also occur during prolonged pseudopregnancy in the rat; the pattern is similar to that seen in late pregnancy but the increase in kininogen is less (Senior & Whalley, 1974a). Changes in the plasma kininogen concentration in the rat can be induced by the administration of reproductive hormones and the levels of kininogen can be moderated by changes in the ratio of oestrogen to progesterone (Senior & Whalley, 1974 b). To examine further the hypothesis that plasma kininogen levels are under hormonal control in the rat, measurements have been made during the oestrous cycle, early pregnancy and pseudopregnancy. Any changes in plasma kininogen seen in these conditions can thus be related to known hormonal changes. 168
METHODS
Virgin female and male rats of a CFE derived strain and weighing between 200 and 250 g were used. They were housed in plastic cages with not more than six animals per cage and were allowed free access to food (modified Diet 41B, Oxoid Ltd) and water. The rooms were temperature and light controlled (light 07.0019.00 hr). The stage of the oestrous cycle was determined by vaginal smear each morning, using the criteria of Long & Evans (1922) to designate the various stages of the cycle. Only females exhibiting regular 4 day oestrous cycles were used in the experiments. Pregnant animals were obtained by caging a pro-oestrous female overnight with a male rat of proven fertility. Females showing a vaginal plug or the presence of spermatozoa in the vaginal smear were designated pregnant and were housed in separate cages until taken for kininogen determinations. Pseudopregnant animals were obtained likewise, but a vasectomized male was used for mating. Day 1 of pregnancy or pseudopregnancy refers to the day of finding the vaginal plug. The concentrations of plasma kininogen were determined using the method of Diniz & Carvalho (1963), which involves the conversion of kininogen to kinin in the presence of excess trypsin (Tryptar, Armour Ltd). Blood samples were collected by cardiac puncture from rats lightly anaesthetized with ether. Rats were sampled once only. To ensure that all trypsin was inactivated at the end of the conversion, 10 ml. boiling 95 % ethanol was added to the digest and the mixture was immersed in a water-bath at 750 C for 15 min. After drying under reduced pressure the kinin was dissolved in physiological saline and assayed against synthetic bradykinin (BRS 640, Sandoz Products Ltd). A four point assay was performed using the isolated rat uterus or guinea-pig ileum preparations. The plasma kininogen concentration is expressed in ,Ag/ml., 1 jug referring to the amount of liberated kinin which is
KININOGENS IN REPRODUCTION
169
equivalent to 1 jug synthetic bradykinin. Statistical analysis of the results was performed using the Student's t test. RESULTS
Oestrous cycle Plasma kininogen determinations were performed at 6 hourly intervals in each 24 hr period. The results for the 4 days of the cycle are shown in Fig. 1; for comparison groups of male rats were also studied at the same 3
()()(5)
(6) (5)
E
(S)
(5)
(5) (5)
) ()(5)(5
(5
(5)~~~~~~5)(6 (5)~~~~~~~~~~~~~~~~~~5
(5)
2:
(5)
(5)
2
0
39 1521 3 91521 3 91521 3 9
1521
Pro-oestrous
D II
Oestrous
DI
.3 91521 Male
Fig. 1. Changes in the plasma kininogen concentration during the oestrous cycle in the rat. The numbers indicate the times at which samples were taken during pro-oestrous, oestrous and dioestrous (day 1, day 11) stages of the cycle. Values for male rats are shown for comparison. The dark bars indicate the period of darkness (19.00-07.00 hr). Standard errors and numbers of observation are indicated.
time intervals. Male and female rats show a diurnal rhythm in the plasma kininogen concentrations. This nychthemeral rhythm is characterized by high kininogen concentrations at 09.00 and 15.00 hr during the period of illumination and low values at 03.00 hr, coinciding with the eighth hour of the darkness phase. Comparison of the plasma kininogen values during the oestrous cycle reveals that for all days the results at 09.00 and 15.00 hr show no
JUDITH SENIOR AND E. T. WHALLEY 170 significant differences. The values obtained during the dark periods (at 03.00 and 21.00 hr) show a quantitative difference on various days of the cycle. At 03.00 hr on the first (DI) and second (Dli) dioestrous days the levels of kininogen (0.95 + 041 and 0-96 + 0 1 ug/ml.) are significantly lower (P
