BLOOD LEVELS OF PROGESTERONE IN THE EWE

Transcription

1 BLOOD LEVELS OF PROGESTERONE IN THE EWE By D. G. EDGAR and J. W. RONALDSON From the Ruakura Animal Research Station, Department of Agriculture, Hamilton, New Zealand {Received 12 August 1957) SUMMARY Progesterone levels in the blood of ewes were assayed chemically. No progesterone was detected in blood from the jugular vein of any ewe. It was found in blood from the vein draining the active ovary during the oestrous cycle. Detectable amounts appeared on the 3rd day, and the mean concentration increased to about 1\m=.\8\g=m\g/ml. on the 7th day. This level was maintained until the 16th day and fell to <0\m=.\15 \g=m\g/ml. on the 17th or last day of the cycle. Blood from the vein draining the active ovary during pregnancy showed a mean level similar to that reached during the oestrous cycle until about the 17th week when it gradually fell to, and remained at, <0\m=.\15\g=m\g/ml. a few days before parturition. In blood from the vein draining the pregnant horn of the uterus, progesterone was detected in low concentrations between the 9th and 18th weeks of pregnancy in only six out of 143 cases. Considerable variation between ewes in progesterone concentration was found. The measurement of individual hormones at known physiological states of the organs they influence is probably an essential step towards an understanding of endocrine balances and their relationship to bodily functions. This paper describes an attempt to assay blood levels of progesterone at known stages throughout the oestrous cycle and pregnancy in the ewe. MATERIALS AND METHODS The oestrous cycles of 113 Romney ewes were defined by means of raddled vasec tomized rams, and the commencement of pregnancy in 108 Romney ewes was indi cated by raddled rams which marked the ewes at mating. Forty-six of the nonpregnant and forty of the pregnant ewes were yearlings, the remainder being 3- and 4-year-old sheep. Blood samples were obtained at known stages after mating from the jugular vein, the ovarian vein draining the active ovary, and the uterine vein draining the pregnant horn of the uterus. The ovarian and uterine venous samples were obtained at laparotomy under Nembutal anaesthesia, and, for this reason, most ewes were sampled only once or twice. All samples were withdrawn into a hypodermic syringe, ovarian venous blood being obtained from one of the tributaries of the ovarian vein at about 1 cm from the ovary. The results, in non-pregnant ewes, were analysed for an average oestrous cycle of 17 days, day 1 being the day of onset of oestrus. This was done by relating the day of blood sampling to the nearer mating date. In the pregnant ewes the analysis was made for weekly intervals, to the nearest whole week, throughout pregnancy. The assay method consisted of extraction of 20 ml. blood by, and partition between,

2 observed BLOOD LEVEL OF PROGESTERONE 379 organic solvents (see Fig. 1), final separation by Chromatographie partition on filter paper (system A, without water, of Bush [1952]), and subsequent estimation of the hormone by ultraviolet absorption spectroscopy [Edgar, 1953]. The ultraviolet 20 ml. oxalated blood Lake with equal vol. of water Extract with ethyl acetate, 3 60 ml. Add 5 ml. ethyl alcohol acetic acid Discard aqueous phase Add 2 drops Evaporate to dryness in vacuo I I Dissolve with warming in petroleum ether, b.p C, 3 10 ml. Discard solids Partition with 70 % ethyl alcohol, 3x8 ml. Discard petroleum ether Add 24 ml. water Partition with chloroform, 3 X 60 ml. Evaporate to dryness Discard alcohol Fig. 1. Transfer to chromatogram with ethyl ether, 3 2 ml. Scheme for extraction of progesterone from blood. absorption measurements were made in 90% ethyl alcohol at wavelengths and 270 µ, and to these was applied the formula: of 242 where Dt µg progesterone = j --Dx -D2 J, optical density at 242 µ, = D2 observed optical density at 270 µ, œ the ratio of the optical densities at 270 mfi and 242 µ for = pure pro gesterone, b the = average of the same ratios for fifty extracts from blood containing no detectable progesterone. This formula is a modification of that of Gibson & Evelyn [1938]. Five samples of ovarian venous blood containing naturally occurring progesterone were divided equally and the two halves were assayed. The average percentage difference in progesterone concentration of the divided samples was 4-2 % (range 0-10%). In fifty-two recovery experiments, known amounts of progesterone ranging from 5 to 60 µg were added in every case to 20 ml. samples of blood. The mean percentage

3 . 380 D. G. EDGAR AND J. W. RONALDSON added was recovery was (s.d.). The mean percentage recovery from 5 µg (s.d.); from 10 µg 60-1 ± 8-5; from 20 µg 61-0± 12-6; from 40 µg 59-1 ± 10-2 and from 60 µg 65 2 ± 8-3. There was thus no evidence of an association between the percentage recovery, or variation in it, and the amount added. The limit of sensitivity was about 0-15 µg progesterone/ml. blood. On the basis of statistical analysis of the data, the best predictor (using inverse regression methods) of the amount of pro gesterone present was obtained by scaling up the observed recovery by the factor The approximate standard error of this factor was 0-035, and that of the resulting estimate was 5-65, i.e. 18-8% of the mean dosage represented (30 µg). In this study, therefore, the observed concentrations have been scaled up by the fac tor 1 64 to yield estimates ofthe naturally occurring progesterone in the blood samples. Table 1. Ewe Progesterone concentration in ovarian blood of ewes at intervals on the same day of the oestrous cycle Day of oestrous cycle st Progesterone concentration (fig/ml.). Collections 2nd rd Interval between collections (min) and 20 RESULTS No progesterone was detected in blood from the jugular vein of any ewe. The mean concentrations of progesterone in the ovarian venous blood of ewes during the 17 days of the oestrous cycle are shown in Fig. 2. During the first 2 days no progesterone was detected, except in one apparently anomalous case on the 2nd day. From the 3rd to the 7th day, the mean concentration increased to about 1-8 µg al., that level being maintained until the 16th day, with a sudden fall to < 015 /tg/ml. on the 17th day. The concentrations for yearling ewes fell within the range for older sheep. On those days of the cycle when progesterone was detectable, there was considerable variation between sheep. Two or three samples taken from the ovarian vein of each of eight ewes at intervals of less than 1 hr (Table 1), and three or four samples taken at daily intervals from the ovarian vein of each of two ewes (Table 2) showed fluctuations in concentration in a given ewe which were much less than the variations between different ewes (Fig. 2). Five ewes, each sampled once between the 7th and 16th days of two different oestrous cycles (Table 3), showed variations in progesterone concentration between cycles as wide as those between different ewes (Fig. 2). The progesterone concentrations in the ovarian venous blood of three ewes with one corpus luteum in each ovary are recorded in Table 4. Where two corpora lutea were present in one ovary (Table 5), the concentration of progesterone fell in the same range as for ewes with one corpus luteum; in ewe 20,

4 BLOOD LEVEL OF PROGESTERONE No. of observations Ewes under 2 years Ewes over 2 years J L _L _L _! L J L -L -L Days of cycle Fig. 2. Concentration of progesterone in ovarian venous blood of ewes during oestrous cycle. Table 2. Table 3. Table 4. Progesterone concentration in ovarian venous blood of ewes on consecutive days of the oestrous cycle Progesterone concentration (pg/ml.). Day of cycle Ewe Progesterone concentration in ovarian venous blood between 1th and 16th days of two different oestrous cycles in same ewes Progesterone concentration Progesterone concentration Ewe Day (/ig/ml.) Day (/ig/ml.) Progesterone concentration in ovarian venous blood of one corpus luteum in each ovary Progesterone concentration (pg/ml.) Ewe Day of cycle Left ovary Right ovary ewes with

5 382 D. G. EDGAR AND J. W. RONALDSON which had two corpora lutea in one ovary and one in the other, the concentrations in the blood from the two ovarian veins were almost the same. In pregnant ewes (Fig. 3), the mean concentration of progesterone in the ovarian venous blood rose no higher than in non-pregnant ewes. The maximum level reached during the oestrous cycle was maintained, when pregnancy supervened, until within about 4 weeks of lambing. The latest stage of pregnancy when progesterone was detected was 15 days before lambing. In eleven cases, <0-15 /ng/ml. progesterone was present at a mean period of 8 days (range: 1-14 days) before lambing. As in the non-pregnant ewes, wide variations existed between ewes, and the con centrations for yearling ewes fell within the range for older sheep. In only six out of 143 uterine venous samples was progesterone detected. The mean value was 0-30 µg al. blood, the range being from 0-24 to These instances were found between the 9th and 18th weeks of pregnancy. Table 5. Progesterone concentration in ovarian venous blood of ewes with two corpora lutea in one ovary Progesterone concentration Ewe Day of cycle (/xg/ml.) (100 in blood from other ovary with one c.l.) th week of 1-30 pregnancy T of observations " r Ewes under 2 years Ewes over 2 years E 3 XX xxxxxxx 0 1 J_L J_I_I_U J_L J-L Weeks of pregnancy Fig. 3. Concentration of progesterone in ovarian venous blood of ewes during pregnancy.

6 BLOOD LEVEL OF PROGESTERONE 383 DISCUSSION In this work the assumption has been made that progesterone secretion in anaes thetized laparotomized ewes is at normal levels. In the peripheral blood of ewes, Neher & Zarrow [1954], using the bioassay of Hooker & Forbes [1947], found µg 'progestin'/ml. plasma at oestrus, and up to 6 /xg/ml. plasma at the 8th-12th day of the oestrous cycle. Because the validity of the Hooker-Forbes bioassay has been questioned, Neher & Zarrow postulated that they were measuring another circulating progestational hormone, not progesterone. Zander & Simmer [1954] have shown, by infra-red spectroscopy, that the sub stance extracted from human blood and other unspecified biological materials, by methods essentially, but not exactly the same as those used in this paper, is pro gesterone. They extracted, with ethyl acetate, the residue from evaporation of the mixture of alcohol and ether used in the initial extraction, whereas in the work reported here the initial extraction was made with ethyl acetate. The chromatography in both cases followed the ligroin and methanol system of Bush [1952]. The results reported here show that progesterone is secreted by the ovary. The hormone is probably physiologically active in the peripheral blood in concentrations < 0-15 µg ai. Concentrations of this order have been reported in human peripheral blood during pregnancy [Salhanick, Jones, Merrill & Neal, 1954; Zander, 1954]. Short [personal communication] has recently found much lower concentrations of progesterone and of its metabolites in the peripheral blood of the ewe. Cytological studies [Warbritton, 1934] have suggested that the progesterone output of the corpus luteum may fall during about the last 3 days of the oestrous cycle, but it appears that the reduction occurs within about the last 24 hr. This sudden withdrawal of progesterone possibly influences the occurrence of the next heat and ovulation. Dutt [1953] and Robinson [1954] have shown that, during anoestrus, some ewes will exhibit oestrus and ovulation following the end of a course of progesterone injections. The concentration of progesterone was similar in blood from ovaries with one or with two corpora lutea. This is of interest in view of the observation of Wallace [personal communication] that individual corpora lutea tend to be smaller when more than one is present in the ovary. In contrast to that of the dioestrous corpus luteum, the mean progesterone output of the corpus luteum of pregnancy falls off gradually. Progesterone was never de tected during the last 15 days of pregnancy, and in some cases it was absent as early as 23 days before lambing. This suggests that parturition in the ewe does not im mediately follow the disappearance of progesterone from the blood. The finding of low concentrations in the uterine vein in only a few instances sug gests that the uterine contents in the pregnant ewe are not an important source of progesterone. The blood flow through this vessel greatly increases, however, during pregnancy, and a low concentration might represent a relatively large secretion of progesterone. There is no apparent tendency during either the oestrous cycle or pregnancy for yearling sheep to have lower progesterone concentrations than older sheep, which might have been postulated as a cause of the lower fertility of the younger ewe.

7 384 D. G. EDGAR AND J. W. RONALDSON The wide variation in estimated progesterone concentrations between sheep is difficult to interpret. It is not known if rate of blood flow through the ovary plays any part, and this might be difficult to measure. In spite of the evidence to the con trary obtained by sampling at intervals, it is possible that variations as great may occur throughout 1 day in individual ewes. In that case, the amount of progesterone present at any moment may be less important than the daily total. Although we are uncertain about the constancy of the level of concentration during 24 hr from one corpus luteum, we do know that the levels resulting from corpora lutea present during different oestrous cycles in one ewe can differ markedly. The absolute amount of progesterone secreted may be less important than a minimal amount. Those ewes secreting less than this minimal amount may be unable to maintain pregnancy, while those secreting more may simply have varying sur pluses of little significance. The responsiveness of the target organs cannot be ignored. The same progesterone level may be adequate in one ewe but inadequate in another. Progesterone concentration may be significant only in relation to that of other progestational substances, oestrogens and/or perhaps other hormones. Thanks are due to Dr A. H. Carter for the statistical analysis of the preliminary recovery experiments. REFERENCES Bush, I.E. [1952]. Biochem. J. 50, 370. Dutt, R. H. [1953]. J. Anim. Sci. 12, 515. Edgar, D. G. [ Biochem. J. 54, 50. Gibson, J. G. & Evelyn, K. A. [1938]. J. din. Invest. 17, 153. Hooker, C. W. & Forbes, T. R. [1947]. Endocrinology, 41, 158. Neher, G. M. & Zarrow, M. X. [1954]. J. Endocrin. 11, 323. Robinson, T. J. [1954]. J. Endocrin. 10, 117. Salhanick, H., Jones, J., Merrill, N. & Neal, L. [1954]. Fed. Proc. 13, 401. Warbritton, V. [1934]. J. Morph. 56, 187. Zander, J. [1954]. Nature, Lond., 174, 406. Zander, J. & Simmer, H. [1954]. Klin. Wschr. 32, 529.