Tenovus Institute for Cancer Research, Welsh National School of Medicine, Heath, Cardiff, CF4 4XX, UK
ISOLATION OF CANINE PROLACTIN BY POLYACRYLAMIDE GEL ELECTROPHORESIS
By G. E.
Jones,
A. D. Brownstone
and A. R.
Boyns
ABSTRACT
electrophoretic mobility
of prolactin obtained from canine pituitary studied with the aid of polyacrylamide disc electrophoresis. Using a preparative gel electrophoretic system the immunoreactive material was purified on a quantitative scale which was then used to develop a homologous radioimmunoassay for canine prolactin. The radioimmunoassay system was able to detect prolactin in the plasma of dogs after the administration of agents which would be expected to affect prolactin secretion. The
extract
was
Recent work from these laboratories has shown that canine pituitary extracts immunological characteristics of prolactin (Jones et al.
contain material with the
1976). A heterologous radioimmunoassay (using antiserum raised against canine prolactin and labelled rat prolactin) was then developed which was capable of detecting and measuring this material (Jones et al. 1976). This system was used to characterise canine prolactin by analytical polyacrylamide gel electrophoresis whereby it was shown that canine pituitaries contained reasonable quantities of prolactin. Therefore a preparative polyacrylamide gel electrophoresis system was em¬ ployed to purify canine prolactin from a relatively small number of pituitaries ]) National Institute for Medical
Research, The Ridgeway, Mill Hill, London
N. W. 7.
691 44s'
purified canine prolactin obtained was then used develop homologous radioimmunoassay, capable of measuring prolactin various physiological states of the dog. which
were
collected. The
a
to
in
MATERIALS AND METHODS
Canine
pituitary extracts Pituitaries were removed from dogs within 30 min of death. Each gland was blotted gently on moist paper, weighed, washed once with 2 ml of cold bicarbonate buffer, pH 9.5 and then homogenised in 2 ml of buffer using a glass homogeniser with a Teflon pestle cooled in ice. The homogenate was then centrifuged at 100 000 g at 4°C for 40 min in a Beckman L2-65B ultracentrifuge. The supernatant was removed, and either analysed immediately or stored at -20°C (Extract B). Purified canine growth hormone D 1001A and canine pituitary extract D 864C were prepared by Dr. A. E. Wilhelmi, Emory University, Georgia, USA. D 864C contained 4 IU/mg of prolactin. Other hormones Rat prolactin (rat prolactin 1-1 potency 30 IU/mg) ovine prolactin NIH-P-S8 and bovine prolactin NIH-P-Bl were obtained from the National Institute of Health, Bethesda, USA.
Other materials
2-bromo-a-ergocryptine-methane-sulphonate (CB154) was obtained from Sandoz Ltd., (Basle, Switzerland); Chlorpromazine from May & Baker Ltd., (Dagenham, Essex) and human serum albumin (HSA) from Blood Products Ltd., (Lister Institute, Hertfordshire). Plasma from a hypophysectomised male dog was a gift from Dr. Ramon Alvarez-Buylla, Istituto Politécnico Nacional, Mexico and Dr. Anna Sirek, Toronto University, Canada. Antisera Rabbit anti-canine prolactin (ACPF9S-R.3) and antiserum to rabbit serum globulin were raised at our laboratory (Jones et al. 1976). In some cases antiserum to canine prolactin was processed in an attempt to reduce plasma interference subse¬ quently observed in the radioimmunoassay. Anti-canine prolactin (100 fil) was mixed with 2 ml plasma from a hypophysectomised male dog and centrifuged at 1500 g for 40 min. The precipitate was discarded and the supernatant re-centrifuged. Any further precipitate was again discarded and the supernatant diluted to 1:1000 in EDTA buffer (0.1 m phosphate buffer pH 7.5 containing 0.15 m NaCl, 0.05 M EDTA and 1:400 nonimmune rabbit serum).
(ARGS-S)
Radioimmunoassay for prolactin Canine prolactin was estimated in rabbit canine prolactin, and labelled rat negligible activity in this assay.
a heterologous radioimmunoassay using antiprolactin (Jones et al. 1976). Canine GH showed
Analytical polyacrylamide electrophoresis This
was
carried out
by the method
of Ornstein
692
(1964)
and Davis
(1964).
Localisation of
protein in gels after polyacrylamide gel electrophoresis (PGE)
a) Staining. The gels were placed in the stain (0.05 % Coomassie brilliant Blue in methanol, water and acetic acid (5:5:1) (BDH Chemicals, Poole, Dorset) for 12 h. They were destained with a glacial acetic acid-methanol-water mixture (5:1:17.5). b) Trichloroacetic acid precipitation (TCA). The gels were placed in 12 % (w/v) TCA solution for 1 h. The protein bands in the gel were then visible as white opaque -
-
precipitates. c) Radioimmunological
Gels were removed from the glass tubing, placed on assay. perspex board, and sliced into discs approximately 2 mm in length using a razor blade. Each disc was placed in a LP3 tube (Luckhams Ltd., Burgess Hill, Sussex) con¬ taining 400 ¡A 0.1 M phosphate buffer pH 7.5 and left overnight at 4°C. Samples of each eluate were removed (100 ¡il) and assayed for prolactin. -
a
Isolation
of specific bands
As staining with Coomassie Blue resulted in shrinkage of gels, accurate localisation of bands in an unstained gel by comparing with a duplicate stained gel was not possible. TCA treatment did not produce gel shrinkage and this technique was used to isolate specific bands. Samples were run in duplicate. One of each gel pair was treated with TCA, placed alongside its untreated duplicate and the latter bisected longitudinally to produce two hemicylinders. One hemicylinder was cut transversely so as to isolate regions in the untreated gel corresponding to selected bands in the TCA treated gel. The isolated segments were placed in LP3 tubes containing 0.1 M phosphate buffer pH 7.5, left overnight at 4°C and the supernatant assayed for prolactin content. The remaining hemicylinder and the TCA treated gel were then stained with Coomassie Blue.
Quantitative preparation of canine prolactin/'Sephadex chromatography Pituitary extract B (15 ml) was mixed with 40% (w/v) solution of sucrose and passed down a Sephadex G-150 column (50 x 2.2 cm) equilibrated with 0.05 m NH4HC03/ NH4OH buffer pH 10.0. The eluate was collected in 3 ml fractions and the absorbance at 280 nm of each fraction read on a Zeiss spectrometer (Lewis Se Cheever 1967). Fractions 1-24 and fractions 25-80 were pooled, and each pool concentrated to 3 ml by pressure using a Bio-fibre device (Bio-rad Laboratories, California, USA). The solutions were then dialysed against 0.05 M NH4HCO3/NH4OH buffer.
Preparative gel electrophoresis Pools 1 and 2 were made up to 10 ml with 0.015 M Tris and 0.05 m Glycine buffer 8.7 and sufficient sucrose added to give a concentration of 5 % (w/v). The solution was layered onto the gel and electrophoresis carried out at 120 V for 1 h. The voltage was raised to 175 V and the buffer re-circulated. Fractions ranging between 0.5 and 1 ml volume were collected as soon as the absorbance of the eluate started to increase (Brownstone 1969). The fractions were then assayed for prolactin activity. Those con¬ taining the highest concentrations of prolactin were pooled, divided into 1 ml portions (containing approximately 60 fig protein) and lyophilised. This material was designated
pH
PPGE-1.
assay using PPGE-1 This was performed as for canine LH (Boyns et al. 1972). PPGE-1 (5 ug) in 25 fil 0.05 M phosphate buffer pH 7.5 was iodinated by a modification of the method of Greenwood et al. (1963) previously described (Boyns et al. 1972).
Radioimmunological
693
Dose-response curves were prepared for PPGE-1 in the presence of hypophysectomised canine plasma using the system 'treated' ACPF9S-R-3 and 131I-labelled PPGE-1. Pro¬ lactin levels were measured in canine plasma obtained from dogs in various physiological states.
Physiological studies a) Chforpromazine (150 mg) was administered iv to two anoestrous bitches at time 0. samples (10 ml) were collected at intervals, mixed with 1 mg EDTA/ml and placed on ice. At the end of the experiment, samples were centrifuged, and the plasma collected and stored at -20°C until assayed. b) Perphenazine (2 mg) was administered iv to one male dog at time 0. Blood samples (10 ml) were collected at intervals, mixed with 1 mg EDTA/ml and placed on ice. At the end of the experiment, samples were centrifuged and the plasma was collected and stored at -20°C until assayed. c) CB154 (2 mg/day) was administered im for 1 week to one male dog. Plasma samples were taken between 10 a.m. and 11 a.m. each day before the injection of CB154 was given. Samples were collected and stored as above. d) Blood samples (10 ml) were collected from the cephalic vein into heparinised tubes from beagle bitches at regular intervals throughout pregnancy. The plasma was sepa¬ rated by centrifugation and stored at -20°C until assayed. Blood
Fig. L Analysis of canine pituitary extracts by polyacrylamide gel electrophoresis. The gels were stained with Coomassie Blue after the electrophoresis of 40 ¡ul samples of a mixture of canine growth hormone and ovine prolactin (tube 1), canine pituitary extract B (tube 3) and canine plasma (tube 5). A fourth gel which contained fraction D 864C is not shown as the bands were too faint to be photographed. GH Growth hor¬ =
mone.
Pr
=
Prolactin. Al
=
Albumin.
694
CANINE GH AND OVINE PROLACTIN SLICE 13 (TUBE 1)
D864 C (TUBE ; SLICE 15
CANINE PITUITARY EXTRACT B
(TUBE 3) SLICE
5
10
15
20
25
14
30
35
SLICES
Fig. 2. immunologically active material in canine pituitary extracts by poly¬ acrylamide gel electrophoresis. Duplicate gels were divided into approximately 2 mm slices without staining and the slices eluted into buffer. Samples of each eluate were radioimmunoassayed for prolactin activity using the homologous antibody system (Jones et al. 1976). GH Growth hormone. Distribution of
=
e) Plasma samples were collected from one intact beagle bitch at regular during the oestrous cycle. Samples were collected and stored as stated above. f) Amniotic fluid was taken from three foetuses during Caesarian section.
intervals
RESULTS
Analytical polyacrylamide gel electrophoresis of canine pituitary extracts Fig. 1 shows the appearance of gels stained with Coomassie Blue after the electrophoresis of 40 fil samples of a mixture of canine growth hormone and ovine prolactin (tube 1) canine pituitary extract B (tube 3) and canine plasma 695
which contained fraction D 864C is not shown as the bands were too faint to be photographed. Tentative identification of some of the bands was made on the basis of the findings of Saluja et al. (1973). Duplicate gels were divided into 2 mm slices without staining, the slices eluted into buffer, and samples of each eluate radioimmunoassayed for prolactin. The distribution of immunologically active material in each gel is shown in Fig. 2. The activity was concentrated in the slices nearest the cathode except in the case of canine plasma which was devoid of activity. The sharpness of the peaks suggested that prolactin activity resided in narrow bands near that of albumin. In further experiments, an attempt was made to more accurately localise the distribution of prolactin activity and assign it to one or other of the stained
(tube 5).
A fourth
gel
Fig.
3.
specific bands by analytical polyacrylamide disc electrophoresis. Gels were run in duplicate. One of each gel pair was treated with trichloroacetic acid, placed alongside its untreated duplicate and bissected longitudinally to give hemicylinders. One hemicylinder was cut transversely so as to isolate regions in the untreated gel corresponding to selected bands in the TCA treated gel. The isolated segments were assayed for prolactin content. The remaining hemicylinder and the TCA treated gel were then stained with Coomassie Blue. The transverse interrupted lines indicate the sites of transection of the bisected gels. Immunoreactive material was present in the gels containing D 864C (gel b), female canine pituitary extract B (gel c) and male canine pituitary extract B (gel d). The amount of prolactin present in each segment is stated alongside gels b, c and d. Isolation of
696
20
30
40
60
50
TUBE No.
70
(60 drops)
Fig.
4.
Fractionation of canine pituitary extract B, on Canine pituitary extract B (5 ml) fractionated NH4OH buffer pH 10.0.
Sephadex G-150. Sephadex G-150
on
in 0.05
M
NH4HCO3/
Table 1.
Pigeon-crop
assay
on
prolactin purified
the
preparative gel electrophoresis (PPGE-1)
IU/mg) injection
Ovine
prolactin (NIH-P-S-9
0.3 ml
0.4 fig
2.0 fig
10.0 m
6.0 6.3 0.9 5.1 2.5
22.2 11.3 7.4 19.7 11.4 8.9
21.8 18.5 35.3 21.7 31.5 37.2
55.1 33.8 26.0 51.1 32.0 43.0
3.88
13.48
27.66
40.17
Saline
injection
2.5 Mean
on
Canine Prolactin
Mean
30
(PPGE-1) injection
2.0 fig
10.0 ms
24.7 30.2 24.2 23.2
30.6 29.7 40.5 30.4
25.57
32.:
Responses are wet weight (mg) of a 3 cm diameter disc of crop sac mucosal epithelium. The PPGE-1 was not absolutely parallel to the ovine prolactin standard curve, but the deviation from parallelism was not significant and may have been due to the birds completing a moult (Forsyth, personal communication). 697
HYPOPHYSECTOMIZED MALE DOG PLASMA
ng ml
Fig.
5.
Standard curve for canine prolactin (PPGE-1). Each point is the mean of two replicates expressed as a percentage of the mean radio¬ activity bound in the tubes with no added standard. On one curve 200 fit hypophysecto¬ mised canine plasma replaced 200 ¡A of HSA buffer. Probability log scale is shown.
bands. Thus the specific bands were isolated (see Methods) and assayed for prolactin. Immunoreactive material was present in the gels containing female pituitary extract B, male canine pituitary extract B, and D 864C. Maximum concentrations appeared to be present in those gel segments containing the pro¬ minent albumin band (Fig. 3). Isolation of canine pituitary prolactin by preparative polyacrylamide gel electrophoresis Canine pituitary extract (3x5 ml) was fractionated on Sephadex G150. Pool 1 (fractions 1-24) contained little prolactin when assayed while the pro¬ lactin content of pool 2 (fractions 25-80) was 880 //g/ml (Fig. 4). Pool 2 was subjected to preparative PGE and the eluates from the column collected in approximately 1 ml fractions. The fractions were assayed for prolactin activity, the highest concentrations were found in tubes 36-46. The contents of these tubes were then pooled, divided into 1 ml portions containing 60 fig protein and lyophilised. 698
Bioassay of isolated material The lyophilised material (designated PPGE) was assayed for prolactin activity by Isobel Forsyth (National Institute for Research in Dairying, Reading Univer¬ sity) (Table 1). Although the dose response curve for PPGE-1 was not absolutely parallel to the ovine prolactin standard, the deviation from parallelism was not significant and was probably due to variation in the physiological state of the birds completing a moult (Forsyth, personal communication). A rough graph¬ ical estimate gave prolactin content as 16 IU/mg but as the response at the 2 and 10 fig doses were within the range of the responses to the corresponding doses of the ovine prolactin, the prolactin content may have been even higher i. e. similar to that of the purified ovine standard.
700
600
500
Ë c
400
300
200
100
i-1-1-1-1-1-1-1-1-1
0
20
40 TIME (min)
60
80
Fig. 6 a. chlorpromazine on plasma prolactin levels in one anoestrous bitch. Chlorpromazine was injected iv at time 0. Blood samples were taken at regular
Effect of
699
intervals.
2 mg PERPHENAZINE 60
§
O)
40
20-^
0
60
30
TIME
90
120
(min)
Fig. 6 b. Effect of perphenazine on plasma prolactin levels in an intact male dog. Perphenazine was injected iv at time 0. Blood samples were then taken.
60
40 4
20
0J
DAYS
Fig.
6
c.
Effect of CB154 on plasma prolactin levels in an intact male dog. Two mg CB154 was injected im for 7 days into an intact male dog.
700
Iodination
of PPGE-1
phosphate buffer pH 7.5 was added to the contents of a tube ¡A removed for iodination. The reaction mixture was fraction¬ ated on Biogel P.60. The specific activity of the iodinated protein was 17 fiCi/fig. Replicate dose-response curves were prepared with PPGE-1 using the system 'treated' ACPF9S-R-3 and labelled PPGE-1. In one set of dose-response curves for PPGE-1, 200 fd of plasma from a hypophysectomised male dog replaced 200 pd of HSA buffer. The addition of hypophysectomised canine plasma in¬ creased the counts bound of the zero tube (Fig. 5). Sixty fa
0.05
M
of PPGE-1 and 5
BITCH 202
300
WHELPED 260
220
180
140
100 4
60 A
20
A 20 30 AUG.
10
20 30 SEPT
10
30 OCT.
20
Fig. 6 d. Plasma prolactin levels during pregnancy in one Beagle bitch. Bitch 202. Pro-oestrus was from 21.8 to 22.8. Oestrus was from 23.8 to 1.9. Mated 27.8 and 29.8. Whelped on 30.10. 701
APRIL
MAY
Fig. e. cycle 6
Plasma prolactin levels during the oestrous Pro-oestrus was from 27.4 to 30.4. Oestrus
was
of an adult intact from 1.5 to 11.5.
Beagle
bitch
Physiological studies Fig. 6 a shows the plasma prolactin levels from one bitch before and after the administration of 200 mg chlorpromazine. Plasma prolactin was 200 ng/ml at time 0 and maximum levels (675 ng/ml) were obtained after 75 min. Fig. 6 b shows the plasma prolactin levels in one male dog after 2 mg perphenazine was administered iv at time 0. The levels increased from 19 ng/ml at 30 min to 70 ng/ml at 60 min. Fig. 6 c shows the plasma prolactin levels in one male dog during treatment withCBl54. One im injection of CB154 (2 mg) was given daily for 1 week. Plasma samples were taken between 10 a.m. and 11 a.m. each day before the injection of CB154. On day 0, plasma prolactin concentration was 77 ng/ml. After one injection of CB154, plasma prolactin levels fell to 17 ng/ml and was undectable thereafter. Fig. 6 d shows the plasma prolactin levels measured during pregnancy in one beagle bitch. Although prolactin levels fluctuated a great deal, at parturition they increased. 702
Fig. 6 e shows the plasma prolactin levels during the oestrous cycle beagle bitch. Levels were increased on the last day of pro-oestrus.
in
one
Prolactin levels were measured in amniotic fluid obtained from three foetuses taken during Caesarean section. The prolactin levels were 2097 ng/ml, 1297 ng/ml and 2411 ng/ml respectively.
DISCUSSION
analytical polyacrylamide disc electrophoresis system, radioimmunoassayable canine prolactin had a mobility slightly less than that of albumin. In this respect, it resembled bovine prolactin (Kwa et al. 1965) and differed from rat prolactin which had a mobility slightly greater than that of serum albumin at pH 8.3 (Nicoll et al. 1969). Canine growth hormone had a com¬ pletely different mobility. These results were essentially similar to those ob¬ tained by Saluja et al. (1973) who used a pigeon crop assay for prolactin de¬ termination. The above studies suggest that canine pituitary contains a material which had the biological and immunological properties of prolactin. An attempt was made to use a preparative gel electrophoresis system (Lewis et al. 1968; Groves 8c Sells 1968; Cheever et al. 1969) to prepare canine pro¬ lactin on a quantitative scale. Canine prolactin obtained from this preparative gel electrophoresis system has a biological activity similar to that obtained for mouse prolactin (Cheever et al. 1969) bovine prolactin (Lewis et al. 1968) and rat prolactin (Groves 8c Sells 1968). Using this purified preparation a radio¬ immunoassay was developed for canine prolactin. In the work described here, chlorpromazine and perphenazine increased plasma prolactin levels in dogs. Similar results have been obtained by other investigators e. g. the rat (Lu et al. 1970) and man (Frantz et al. 1972). CB154, a synthetic ergot alkaloid inhibits prolactin secretion in many species including rat (Wuttke et al. 1971) and sheep (Niswender 1974). In the male dog CB154 (2 mg/day) lowered prolactin secretion to undetectable levels within one day of the first injection. High levels of prolactin were found at the end of pro-oestrus, 2 days before the LH peak, in the bitch. At this time, oestradiol-17/5 levels are at their maximum (Jones et al. 1973a). In the rat, maximum levels of serum prolactin are seen at pro-oestrus, levels declining on the day of oestrus (Niswender et al. 1969). Serum levels of prolactin were also reported to be elevated during prooestrus or oestrus in the sheep (Bryant 8c Greenwood 1968) and cow (Raud et al. 1971), although no change in plasma prolactin values during ovulation in women have been observed (McNeilly 8c Chard 1974). Plasma prolactin levels rose on the day before parturition in the bitch. In pregnant rats prolactin is low until the day before parturition (Amenomori et al. In the
703
1970). Low levels of prolactin have been found during most of pregnancy in the sheep (McNeilly 1971) cow (Karg 8c Schams 1970) and goat (Bryant 8c Green¬ wood 1968). In women during the first trimester, mean prolactin levels were 30 ng/ml, at the second trimester they were 60 ng/ml and at term 200 ng/ml (Friesen 8c Hwang 1973). All the above species show a rise in prolactin levels at the end of pregnancy and on the day of parturition. Low prolactin values during most of pregnancy in the bitch may be associated with low oestradiol-17/5 secretion (Jones et al. \973b). Prolactin concentrations in amniotic fluid in the bitch were very high. Similar findings have been reported in the human and female Rhesus monkey (Friesen et al. 1972). It would appear that when serum prolactin levels were barely elevated in the first trimester amniotic fluid prolactin was at its highest (mean 2130 ng/ml in women) but at term levels had fallen to a mean of 1120 ng/ml. Amniotic fluid prolactin immunologically, electrophoretically and chromatographically, appears identical with pituitary prolactin (Friesen 8c Hwang 1973). These preliminary studies have demonstrated that canine pituitaries contain material with the immunological activity of prolactin. Attempts to isolate pro¬ lactin seemed to result in some degree of purification of the active material, and although more work needs to be done on the radioimmunoassay, it was able to detect prolactin in plasma samples and amniotic fluid. The results in the present stage are too incomplete to make concrete conclusions but they suggest that prolactin secretion in the dog has certain similarities with the patterns seen in other species.
ACKNOWLEDGMENTS We are grateful to the Tenovus Organisation for generous financial assistance, Dr. A. E. Wilhelmi, Emory University, Atlanta Georgia, USA for the gifts of canine growth hormone and canine pituitary fraction D 864C. We thank Pedigree Petfoods Limited and Schering Research Laboratories, Germany, for the gifts of canine plasma and amniotic fluid. We are grateful to Professor K. Griffiths for his advice and encourage¬ ment.
REFERENCES Amenomori Y'., Chen C. L. Se Mettes J.: Endocrinology 86 (1970) 506. G. E., Bell E. T., Christie D. W. Se Parkes M. F.: J. Endocr. 55
Boyns A. R., Jones (1972) 279.
Brownstone A. D.: Analyt. Biochem. 27 (Í969) 24. Bryant G. D. & Greenwood F. C: Biochem. J. 709 (1968) 831. Cheever E. V'., Seavey B. K. & Lewis U. J.: Endocrinology 85 (1969) 698. Davis B. J.: Ann. N. Y. Acad. Sei. 727 (1964) 404. Frantz A. G., Kleinberg D. L. Se Noel G. L.: Recent Progr. Hormone Res. 28
704
(1972)
539.
Friesen H. Se Hwang P.: Ann. Rev. Med. 24 (1973) 251. Friesen H., Hwang P., Guyda H., Tolis G., Tyson J. Se Myers R. P. In: Boyns A. R. and Griffiths K., Eds. Fourth Tenovus Workshop, Prolactin & Carcinogenesis, vol. 64, Cardiff (1972). Greenwood F. C, Hunter W. M. Se Glover J. S.: Biochem. J. 89 (1963) 114. Groves W. E. Se Sells B. H.: Biochim. biophys. Acta (Amst.) 168 (1968) 113. Jones G. E., Boyns A. R., Cameron E. H. D., Bell E. T., Christie D. W. Se Parkes M. F.: J. Endocr. 57 (1973a) 331. Jones G. E., Boyns A. R., Cameron E. H. D., Bell E. T., Christie D. W. Se Parkes M. F.: J. Reprod. Fértil. 35 (19735) 187. Jones G. E.. Stocked Hartree A. Se Boyns A. R.: Acta endocr. 82 (1976) 475. Karg H. Se Schams D. In: Falconer I. R., Ed. Lactation. Butterworth, London (1970) p. 141.
Kwa H. G., Van Der Bent E. M., Feltkamp C. A., Rumke P. Se Bloemendal H.: Biochim. biophys. Acta (Amst.) 777 (1965) 447. Lewis U. J. Se Cheever E. V.: Endocrinology 81 (1967) 1338. Lewis U. J., Cheever E. V. Se Seavey B. K.: Analyt. Biochem. 24 (1968) 162. Lu K. H., Amenomori Y., Chen C. L. Se Mettes J.: Endocrinology 87 (1970) 667. McNeilly J.: J. Endocr. 49 (1971) 141. McNeilly A. S. Se Chard T.: Clin. Endocr. 3 (1974) 105. Nicoll C. S., Parsons J. A., Fiorindo R. P. Se Nichols C. W.: J. Endocr. 45 (1969) 183. Niswender G. D.: Endocrinology 94 (1974) 612. Niswender G. D., Chen C. L., Midgley A. R., Jr., Meites J. Se Ellis S.: Proc. Soc. exp. Biol. (N. Y.) 730 (1969) 793. Ornstein L.: Ann. N. Y. Acad. Sei. 727 (1964) 321. Raud H. R., Kiddy C. A. Se Odell W. D.: Proc. Soc. exp. Biol. (N. Y.) 736 (1971) 689. Saluja P. G., Gronow M. Se Hamilton J. M.: J. Endocr. 56 (1973) 245. Wuttke W., Cassell E. Se Meites J.: Endocrinology 88 (1971) 737. Received
on
September 23rd,
1975.
705 Acta endocr. 82, 4
ISOLATION OF CANINE PROLACTIN BY POLYACRYLAMIDE GEL ELECTROPHORESIS
By G. E.
Jones,
A. D. Brownstone
and A. R.
Boyns
ABSTRACT
electrophoretic mobility
of prolactin obtained from canine pituitary studied with the aid of polyacrylamide disc electrophoresis. Using a preparative gel electrophoretic system the immunoreactive material was purified on a quantitative scale which was then used to develop a homologous radioimmunoassay for canine prolactin. The radioimmunoassay system was able to detect prolactin in the plasma of dogs after the administration of agents which would be expected to affect prolactin secretion. The
extract
was
Recent work from these laboratories has shown that canine pituitary extracts immunological characteristics of prolactin (Jones et al.
contain material with the
1976). A heterologous radioimmunoassay (using antiserum raised against canine prolactin and labelled rat prolactin) was then developed which was capable of detecting and measuring this material (Jones et al. 1976). This system was used to characterise canine prolactin by analytical polyacrylamide gel electrophoresis whereby it was shown that canine pituitaries contained reasonable quantities of prolactin. Therefore a preparative polyacrylamide gel electrophoresis system was em¬ ployed to purify canine prolactin from a relatively small number of pituitaries ]) National Institute for Medical
Research, The Ridgeway, Mill Hill, London
N. W. 7.
691 44s'
purified canine prolactin obtained was then used develop homologous radioimmunoassay, capable of measuring prolactin various physiological states of the dog. which
were
collected. The
a
to
in
MATERIALS AND METHODS
Canine
pituitary extracts Pituitaries were removed from dogs within 30 min of death. Each gland was blotted gently on moist paper, weighed, washed once with 2 ml of cold bicarbonate buffer, pH 9.5 and then homogenised in 2 ml of buffer using a glass homogeniser with a Teflon pestle cooled in ice. The homogenate was then centrifuged at 100 000 g at 4°C for 40 min in a Beckman L2-65B ultracentrifuge. The supernatant was removed, and either analysed immediately or stored at -20°C (Extract B). Purified canine growth hormone D 1001A and canine pituitary extract D 864C were prepared by Dr. A. E. Wilhelmi, Emory University, Georgia, USA. D 864C contained 4 IU/mg of prolactin. Other hormones Rat prolactin (rat prolactin 1-1 potency 30 IU/mg) ovine prolactin NIH-P-S8 and bovine prolactin NIH-P-Bl were obtained from the National Institute of Health, Bethesda, USA.
Other materials
2-bromo-a-ergocryptine-methane-sulphonate (CB154) was obtained from Sandoz Ltd., (Basle, Switzerland); Chlorpromazine from May & Baker Ltd., (Dagenham, Essex) and human serum albumin (HSA) from Blood Products Ltd., (Lister Institute, Hertfordshire). Plasma from a hypophysectomised male dog was a gift from Dr. Ramon Alvarez-Buylla, Istituto Politécnico Nacional, Mexico and Dr. Anna Sirek, Toronto University, Canada. Antisera Rabbit anti-canine prolactin (ACPF9S-R.3) and antiserum to rabbit serum globulin were raised at our laboratory (Jones et al. 1976). In some cases antiserum to canine prolactin was processed in an attempt to reduce plasma interference subse¬ quently observed in the radioimmunoassay. Anti-canine prolactin (100 fil) was mixed with 2 ml plasma from a hypophysectomised male dog and centrifuged at 1500 g for 40 min. The precipitate was discarded and the supernatant re-centrifuged. Any further precipitate was again discarded and the supernatant diluted to 1:1000 in EDTA buffer (0.1 m phosphate buffer pH 7.5 containing 0.15 m NaCl, 0.05 M EDTA and 1:400 nonimmune rabbit serum).
(ARGS-S)
Radioimmunoassay for prolactin Canine prolactin was estimated in rabbit canine prolactin, and labelled rat negligible activity in this assay.
a heterologous radioimmunoassay using antiprolactin (Jones et al. 1976). Canine GH showed
Analytical polyacrylamide electrophoresis This
was
carried out
by the method
of Ornstein
692
(1964)
and Davis
(1964).
Localisation of
protein in gels after polyacrylamide gel electrophoresis (PGE)
a) Staining. The gels were placed in the stain (0.05 % Coomassie brilliant Blue in methanol, water and acetic acid (5:5:1) (BDH Chemicals, Poole, Dorset) for 12 h. They were destained with a glacial acetic acid-methanol-water mixture (5:1:17.5). b) Trichloroacetic acid precipitation (TCA). The gels were placed in 12 % (w/v) TCA solution for 1 h. The protein bands in the gel were then visible as white opaque -
-
precipitates. c) Radioimmunological
Gels were removed from the glass tubing, placed on assay. perspex board, and sliced into discs approximately 2 mm in length using a razor blade. Each disc was placed in a LP3 tube (Luckhams Ltd., Burgess Hill, Sussex) con¬ taining 400 ¡A 0.1 M phosphate buffer pH 7.5 and left overnight at 4°C. Samples of each eluate were removed (100 ¡il) and assayed for prolactin. -
a
Isolation
of specific bands
As staining with Coomassie Blue resulted in shrinkage of gels, accurate localisation of bands in an unstained gel by comparing with a duplicate stained gel was not possible. TCA treatment did not produce gel shrinkage and this technique was used to isolate specific bands. Samples were run in duplicate. One of each gel pair was treated with TCA, placed alongside its untreated duplicate and the latter bisected longitudinally to produce two hemicylinders. One hemicylinder was cut transversely so as to isolate regions in the untreated gel corresponding to selected bands in the TCA treated gel. The isolated segments were placed in LP3 tubes containing 0.1 M phosphate buffer pH 7.5, left overnight at 4°C and the supernatant assayed for prolactin content. The remaining hemicylinder and the TCA treated gel were then stained with Coomassie Blue.
Quantitative preparation of canine prolactin/'Sephadex chromatography Pituitary extract B (15 ml) was mixed with 40% (w/v) solution of sucrose and passed down a Sephadex G-150 column (50 x 2.2 cm) equilibrated with 0.05 m NH4HC03/ NH4OH buffer pH 10.0. The eluate was collected in 3 ml fractions and the absorbance at 280 nm of each fraction read on a Zeiss spectrometer (Lewis Se Cheever 1967). Fractions 1-24 and fractions 25-80 were pooled, and each pool concentrated to 3 ml by pressure using a Bio-fibre device (Bio-rad Laboratories, California, USA). The solutions were then dialysed against 0.05 M NH4HCO3/NH4OH buffer.
Preparative gel electrophoresis Pools 1 and 2 were made up to 10 ml with 0.015 M Tris and 0.05 m Glycine buffer 8.7 and sufficient sucrose added to give a concentration of 5 % (w/v). The solution was layered onto the gel and electrophoresis carried out at 120 V for 1 h. The voltage was raised to 175 V and the buffer re-circulated. Fractions ranging between 0.5 and 1 ml volume were collected as soon as the absorbance of the eluate started to increase (Brownstone 1969). The fractions were then assayed for prolactin activity. Those con¬ taining the highest concentrations of prolactin were pooled, divided into 1 ml portions (containing approximately 60 fig protein) and lyophilised. This material was designated
pH
PPGE-1.
assay using PPGE-1 This was performed as for canine LH (Boyns et al. 1972). PPGE-1 (5 ug) in 25 fil 0.05 M phosphate buffer pH 7.5 was iodinated by a modification of the method of Greenwood et al. (1963) previously described (Boyns et al. 1972).
Radioimmunological
693
Dose-response curves were prepared for PPGE-1 in the presence of hypophysectomised canine plasma using the system 'treated' ACPF9S-R-3 and 131I-labelled PPGE-1. Pro¬ lactin levels were measured in canine plasma obtained from dogs in various physiological states.
Physiological studies a) Chforpromazine (150 mg) was administered iv to two anoestrous bitches at time 0. samples (10 ml) were collected at intervals, mixed with 1 mg EDTA/ml and placed on ice. At the end of the experiment, samples were centrifuged, and the plasma collected and stored at -20°C until assayed. b) Perphenazine (2 mg) was administered iv to one male dog at time 0. Blood samples (10 ml) were collected at intervals, mixed with 1 mg EDTA/ml and placed on ice. At the end of the experiment, samples were centrifuged and the plasma was collected and stored at -20°C until assayed. c) CB154 (2 mg/day) was administered im for 1 week to one male dog. Plasma samples were taken between 10 a.m. and 11 a.m. each day before the injection of CB154 was given. Samples were collected and stored as above. d) Blood samples (10 ml) were collected from the cephalic vein into heparinised tubes from beagle bitches at regular intervals throughout pregnancy. The plasma was sepa¬ rated by centrifugation and stored at -20°C until assayed. Blood
Fig. L Analysis of canine pituitary extracts by polyacrylamide gel electrophoresis. The gels were stained with Coomassie Blue after the electrophoresis of 40 ¡ul samples of a mixture of canine growth hormone and ovine prolactin (tube 1), canine pituitary extract B (tube 3) and canine plasma (tube 5). A fourth gel which contained fraction D 864C is not shown as the bands were too faint to be photographed. GH Growth hor¬ =
mone.
Pr
=
Prolactin. Al
=
Albumin.
694
CANINE GH AND OVINE PROLACTIN SLICE 13 (TUBE 1)
D864 C (TUBE ; SLICE 15
CANINE PITUITARY EXTRACT B
(TUBE 3) SLICE
5
10
15
20
25
14
30
35
SLICES
Fig. 2. immunologically active material in canine pituitary extracts by poly¬ acrylamide gel electrophoresis. Duplicate gels were divided into approximately 2 mm slices without staining and the slices eluted into buffer. Samples of each eluate were radioimmunoassayed for prolactin activity using the homologous antibody system (Jones et al. 1976). GH Growth hormone. Distribution of
=
e) Plasma samples were collected from one intact beagle bitch at regular during the oestrous cycle. Samples were collected and stored as stated above. f) Amniotic fluid was taken from three foetuses during Caesarian section.
intervals
RESULTS
Analytical polyacrylamide gel electrophoresis of canine pituitary extracts Fig. 1 shows the appearance of gels stained with Coomassie Blue after the electrophoresis of 40 fil samples of a mixture of canine growth hormone and ovine prolactin (tube 1) canine pituitary extract B (tube 3) and canine plasma 695
which contained fraction D 864C is not shown as the bands were too faint to be photographed. Tentative identification of some of the bands was made on the basis of the findings of Saluja et al. (1973). Duplicate gels were divided into 2 mm slices without staining, the slices eluted into buffer, and samples of each eluate radioimmunoassayed for prolactin. The distribution of immunologically active material in each gel is shown in Fig. 2. The activity was concentrated in the slices nearest the cathode except in the case of canine plasma which was devoid of activity. The sharpness of the peaks suggested that prolactin activity resided in narrow bands near that of albumin. In further experiments, an attempt was made to more accurately localise the distribution of prolactin activity and assign it to one or other of the stained
(tube 5).
A fourth
gel
Fig.
3.
specific bands by analytical polyacrylamide disc electrophoresis. Gels were run in duplicate. One of each gel pair was treated with trichloroacetic acid, placed alongside its untreated duplicate and bissected longitudinally to give hemicylinders. One hemicylinder was cut transversely so as to isolate regions in the untreated gel corresponding to selected bands in the TCA treated gel. The isolated segments were assayed for prolactin content. The remaining hemicylinder and the TCA treated gel were then stained with Coomassie Blue. The transverse interrupted lines indicate the sites of transection of the bisected gels. Immunoreactive material was present in the gels containing D 864C (gel b), female canine pituitary extract B (gel c) and male canine pituitary extract B (gel d). The amount of prolactin present in each segment is stated alongside gels b, c and d. Isolation of
696
20
30
40
60
50
TUBE No.
70
(60 drops)
Fig.
4.
Fractionation of canine pituitary extract B, on Canine pituitary extract B (5 ml) fractionated NH4OH buffer pH 10.0.
Sephadex G-150. Sephadex G-150
on
in 0.05
M
NH4HCO3/
Table 1.
Pigeon-crop
assay
on
prolactin purified
the
preparative gel electrophoresis (PPGE-1)
IU/mg) injection
Ovine
prolactin (NIH-P-S-9
0.3 ml
0.4 fig
2.0 fig
10.0 m
6.0 6.3 0.9 5.1 2.5
22.2 11.3 7.4 19.7 11.4 8.9
21.8 18.5 35.3 21.7 31.5 37.2
55.1 33.8 26.0 51.1 32.0 43.0
3.88
13.48
27.66
40.17
Saline
injection
2.5 Mean
on
Canine Prolactin
Mean
30
(PPGE-1) injection
2.0 fig
10.0 ms
24.7 30.2 24.2 23.2
30.6 29.7 40.5 30.4
25.57
32.:
Responses are wet weight (mg) of a 3 cm diameter disc of crop sac mucosal epithelium. The PPGE-1 was not absolutely parallel to the ovine prolactin standard curve, but the deviation from parallelism was not significant and may have been due to the birds completing a moult (Forsyth, personal communication). 697
HYPOPHYSECTOMIZED MALE DOG PLASMA
ng ml
Fig.
5.
Standard curve for canine prolactin (PPGE-1). Each point is the mean of two replicates expressed as a percentage of the mean radio¬ activity bound in the tubes with no added standard. On one curve 200 fit hypophysecto¬ mised canine plasma replaced 200 ¡A of HSA buffer. Probability log scale is shown.
bands. Thus the specific bands were isolated (see Methods) and assayed for prolactin. Immunoreactive material was present in the gels containing female pituitary extract B, male canine pituitary extract B, and D 864C. Maximum concentrations appeared to be present in those gel segments containing the pro¬ minent albumin band (Fig. 3). Isolation of canine pituitary prolactin by preparative polyacrylamide gel electrophoresis Canine pituitary extract (3x5 ml) was fractionated on Sephadex G150. Pool 1 (fractions 1-24) contained little prolactin when assayed while the pro¬ lactin content of pool 2 (fractions 25-80) was 880 //g/ml (Fig. 4). Pool 2 was subjected to preparative PGE and the eluates from the column collected in approximately 1 ml fractions. The fractions were assayed for prolactin activity, the highest concentrations were found in tubes 36-46. The contents of these tubes were then pooled, divided into 1 ml portions containing 60 fig protein and lyophilised. 698
Bioassay of isolated material The lyophilised material (designated PPGE) was assayed for prolactin activity by Isobel Forsyth (National Institute for Research in Dairying, Reading Univer¬ sity) (Table 1). Although the dose response curve for PPGE-1 was not absolutely parallel to the ovine prolactin standard, the deviation from parallelism was not significant and was probably due to variation in the physiological state of the birds completing a moult (Forsyth, personal communication). A rough graph¬ ical estimate gave prolactin content as 16 IU/mg but as the response at the 2 and 10 fig doses were within the range of the responses to the corresponding doses of the ovine prolactin, the prolactin content may have been even higher i. e. similar to that of the purified ovine standard.
700
600
500
Ë c
400
300
200
100
i-1-1-1-1-1-1-1-1-1
0
20
40 TIME (min)
60
80
Fig. 6 a. chlorpromazine on plasma prolactin levels in one anoestrous bitch. Chlorpromazine was injected iv at time 0. Blood samples were taken at regular
Effect of
699
intervals.
2 mg PERPHENAZINE 60
§
O)
40
20-^
0
60
30
TIME
90
120
(min)
Fig. 6 b. Effect of perphenazine on plasma prolactin levels in an intact male dog. Perphenazine was injected iv at time 0. Blood samples were then taken.
60
40 4
20
0J
DAYS
Fig.
6
c.
Effect of CB154 on plasma prolactin levels in an intact male dog. Two mg CB154 was injected im for 7 days into an intact male dog.
700
Iodination
of PPGE-1
phosphate buffer pH 7.5 was added to the contents of a tube ¡A removed for iodination. The reaction mixture was fraction¬ ated on Biogel P.60. The specific activity of the iodinated protein was 17 fiCi/fig. Replicate dose-response curves were prepared with PPGE-1 using the system 'treated' ACPF9S-R-3 and labelled PPGE-1. In one set of dose-response curves for PPGE-1, 200 fd of plasma from a hypophysectomised male dog replaced 200 pd of HSA buffer. The addition of hypophysectomised canine plasma in¬ creased the counts bound of the zero tube (Fig. 5). Sixty fa
0.05
M
of PPGE-1 and 5
BITCH 202
300
WHELPED 260
220
180
140
100 4
60 A
20
A 20 30 AUG.
10
20 30 SEPT
10
30 OCT.
20
Fig. 6 d. Plasma prolactin levels during pregnancy in one Beagle bitch. Bitch 202. Pro-oestrus was from 21.8 to 22.8. Oestrus was from 23.8 to 1.9. Mated 27.8 and 29.8. Whelped on 30.10. 701
APRIL
MAY
Fig. e. cycle 6
Plasma prolactin levels during the oestrous Pro-oestrus was from 27.4 to 30.4. Oestrus
was
of an adult intact from 1.5 to 11.5.
Beagle
bitch
Physiological studies Fig. 6 a shows the plasma prolactin levels from one bitch before and after the administration of 200 mg chlorpromazine. Plasma prolactin was 200 ng/ml at time 0 and maximum levels (675 ng/ml) were obtained after 75 min. Fig. 6 b shows the plasma prolactin levels in one male dog after 2 mg perphenazine was administered iv at time 0. The levels increased from 19 ng/ml at 30 min to 70 ng/ml at 60 min. Fig. 6 c shows the plasma prolactin levels in one male dog during treatment withCBl54. One im injection of CB154 (2 mg) was given daily for 1 week. Plasma samples were taken between 10 a.m. and 11 a.m. each day before the injection of CB154. On day 0, plasma prolactin concentration was 77 ng/ml. After one injection of CB154, plasma prolactin levels fell to 17 ng/ml and was undectable thereafter. Fig. 6 d shows the plasma prolactin levels measured during pregnancy in one beagle bitch. Although prolactin levels fluctuated a great deal, at parturition they increased. 702
Fig. 6 e shows the plasma prolactin levels during the oestrous cycle beagle bitch. Levels were increased on the last day of pro-oestrus.
in
one
Prolactin levels were measured in amniotic fluid obtained from three foetuses taken during Caesarean section. The prolactin levels were 2097 ng/ml, 1297 ng/ml and 2411 ng/ml respectively.
DISCUSSION
analytical polyacrylamide disc electrophoresis system, radioimmunoassayable canine prolactin had a mobility slightly less than that of albumin. In this respect, it resembled bovine prolactin (Kwa et al. 1965) and differed from rat prolactin which had a mobility slightly greater than that of serum albumin at pH 8.3 (Nicoll et al. 1969). Canine growth hormone had a com¬ pletely different mobility. These results were essentially similar to those ob¬ tained by Saluja et al. (1973) who used a pigeon crop assay for prolactin de¬ termination. The above studies suggest that canine pituitary contains a material which had the biological and immunological properties of prolactin. An attempt was made to use a preparative gel electrophoresis system (Lewis et al. 1968; Groves 8c Sells 1968; Cheever et al. 1969) to prepare canine pro¬ lactin on a quantitative scale. Canine prolactin obtained from this preparative gel electrophoresis system has a biological activity similar to that obtained for mouse prolactin (Cheever et al. 1969) bovine prolactin (Lewis et al. 1968) and rat prolactin (Groves 8c Sells 1968). Using this purified preparation a radio¬ immunoassay was developed for canine prolactin. In the work described here, chlorpromazine and perphenazine increased plasma prolactin levels in dogs. Similar results have been obtained by other investigators e. g. the rat (Lu et al. 1970) and man (Frantz et al. 1972). CB154, a synthetic ergot alkaloid inhibits prolactin secretion in many species including rat (Wuttke et al. 1971) and sheep (Niswender 1974). In the male dog CB154 (2 mg/day) lowered prolactin secretion to undetectable levels within one day of the first injection. High levels of prolactin were found at the end of pro-oestrus, 2 days before the LH peak, in the bitch. At this time, oestradiol-17/5 levels are at their maximum (Jones et al. 1973a). In the rat, maximum levels of serum prolactin are seen at pro-oestrus, levels declining on the day of oestrus (Niswender et al. 1969). Serum levels of prolactin were also reported to be elevated during prooestrus or oestrus in the sheep (Bryant 8c Greenwood 1968) and cow (Raud et al. 1971), although no change in plasma prolactin values during ovulation in women have been observed (McNeilly 8c Chard 1974). Plasma prolactin levels rose on the day before parturition in the bitch. In pregnant rats prolactin is low until the day before parturition (Amenomori et al. In the
703
1970). Low levels of prolactin have been found during most of pregnancy in the sheep (McNeilly 1971) cow (Karg 8c Schams 1970) and goat (Bryant 8c Green¬ wood 1968). In women during the first trimester, mean prolactin levels were 30 ng/ml, at the second trimester they were 60 ng/ml and at term 200 ng/ml (Friesen 8c Hwang 1973). All the above species show a rise in prolactin levels at the end of pregnancy and on the day of parturition. Low prolactin values during most of pregnancy in the bitch may be associated with low oestradiol-17/5 secretion (Jones et al. \973b). Prolactin concentrations in amniotic fluid in the bitch were very high. Similar findings have been reported in the human and female Rhesus monkey (Friesen et al. 1972). It would appear that when serum prolactin levels were barely elevated in the first trimester amniotic fluid prolactin was at its highest (mean 2130 ng/ml in women) but at term levels had fallen to a mean of 1120 ng/ml. Amniotic fluid prolactin immunologically, electrophoretically and chromatographically, appears identical with pituitary prolactin (Friesen 8c Hwang 1973). These preliminary studies have demonstrated that canine pituitaries contain material with the immunological activity of prolactin. Attempts to isolate pro¬ lactin seemed to result in some degree of purification of the active material, and although more work needs to be done on the radioimmunoassay, it was able to detect prolactin in plasma samples and amniotic fluid. The results in the present stage are too incomplete to make concrete conclusions but they suggest that prolactin secretion in the dog has certain similarities with the patterns seen in other species.
ACKNOWLEDGMENTS We are grateful to the Tenovus Organisation for generous financial assistance, Dr. A. E. Wilhelmi, Emory University, Atlanta Georgia, USA for the gifts of canine growth hormone and canine pituitary fraction D 864C. We thank Pedigree Petfoods Limited and Schering Research Laboratories, Germany, for the gifts of canine plasma and amniotic fluid. We are grateful to Professor K. Griffiths for his advice and encourage¬ ment.
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on
September 23rd,
1975.
705 Acta endocr. 82, 4
