HYPOTHALAMIC LUTEINIZING HORMONE RELEASING FACTOR AND CORTICOTROPHIN RELEASING ACTIVITY IN RELATION TO PITUITARY AND PLASMA HORMONE LEVELS IN MALE AND FEMALE RATS
SHARON A. CHIAPPA AND G. FINK Department of Human Anatomy, South Parks Road, Oxford, 0X1 3QX
(Received
June
1976)
SUMMARY
Hypothalamic corticotrophin releasing (CR) activity and LH-releasing factor (RF) content, and pituitary and plasma LH, FSH and ACTH were measured in adult male and female Wistar rats maintained under 14 h light per day. Hypothalamic LH-RF and pituitary and plasma hormones were estimated by radioimmunoassay while CR-activity was assessed by the amount of ACTH released from hemipituitaries in vitro. Two experiments were carried out on male animals. In the first, some of the animals were kept in a room, distant from the animal house, in which the lighting was reversed with respect to the external environment. In animals exposed to the reversed lighting r\l=e'\gime,hypothalamic LH-RF content and pituitary gonadotrophin concentrations were significantly lower than the values in male rats kept in the animal house where they were in close proximity to female rats. In the second experiment, which was carried out on animals which had all been kept in the animal house, there were no significant differences between the LH-RF contents measured at 3\p=n-\4h intervals throughout the day. Pituitary LH and FSH contents, but not concentrations, were significantly increased at 12.00 h. There was little difference between the experiments in CR-activity, plasma ACTH concentrations and profiles of pituitary ACTH content and concentration. As expected there was a diurnal rhythm in plasma corticosterone concentrations (determined by competitive protein-binding assay) with the peak occurring between 15.00 and 18.00 h. The profiles of plasma and pituitary ACTH were similar to that of plasma corticosterone. Corticotrophin releasing activity dropped significantly between 12.00 and 16.00 h, but remained steady at the other times. In female rats there were no significant differences between hypothalamic LH-RF con¬ tent throughout the 4-day cycle. During pro-oestrus the mean LH-RF content rose to reach a high level at 18.00 h at which time plasma LH concentration had risen sharply to a level consistent with the peak of the preovulatory surge. Plasma FSH concentration also rose significantly between 15.00 and 18.00 h of pro-oestrus. At metoestrus and dioestrus, plasma FSH levels were lower in the morning than in the evening. These results suggest that (1) there is no diurnal rhythm in hypothalamic LH-RF, (2) there may be a diurnal rhythm in pituitary gonadotrophin content in the male and in plasma FSH concentration on the days of metoestrus and dioestrus in the female, (3) if a surge of LHRF does occur on the afternoon of pro-oestrus, the rate of LH-RF synthesis exceeds its release, and (4) the mechanism which regulates gonadotrophin secretion in the male may be affected by factors in the environment other than daylength. The results provide further evidence for the view that the diurnal rhythm of corticosterone secretion is under hypothalamo-hypophysial control.
INTRODUCTION
Several workers have studied hypothalamic corticotrophin releasing activity (CR-activity) at various times during the day in male animals (Cheifetz, Gaffud & Dingman, 1969; David-Nelson & Brodish, 1969; Hiroshige, Sakakura & Itoh, 1969; Hiroshige & Sakakura, 1971; Seiden & Brodish, 1972; Takebe, Sakakura & Mashimo, 1972) and hypothalamic Iuteinizing hormone releasing factor (LH-RF) during the oestrous cycle (Chowers & McCann, 1965; Ramirez & Sawyer, 1965; Kalra, Krulich & McCann, 1973; Araki, Ferin, Zimmerman & Vande Wiele, 1975; Kalra, 1976). No detailed study appears to have been published on hypothalamic LH-RF levels during the day in male rats, and, apart from data on plasma corticosterone concentrations which were included in most of the studies on CRactivity, there is no information on the relationship between other relevant plasma or pituitary hormone levels and hypothalamic releasing factor activity measured at the same times and in the same animals. The purpose of the present work was to determine whether (I) there are variations in CR-activity of the hypothalamus and immunoreactive corticotrophin (ACTH) in the pituitary gland and in plasma which can be linked with the well-known diurnal rhythm of plasma corticosterone in male rats, (2) there is a diurnal rhythm in the gonadotrophin regulatory system in male rats, and (3) the levels of immunoreactive LH-RF in the hypothalamus of female rats can be correlated with changes in pituitary and plasma gonadotrophin levels. MATERIALS AND METHODS
Animals and experimental procedures All the animals were rats of the Wistar strain supplied by Charles River (U.K.) Ltd, Mar¬ gate. They were maintained under controlled lighting and temperature (22 °C) and given free access to Diet 41 (E. Dixon and Sons Ltd, Ware) and tap-water. Male rats Two experiments were carried out on male animals. In Expt. 1, some of the animals were kept under a lighting régime in which the lights were on between 05.00-19.00 h ('ordinary light') while others were kept in a room, distant from the animal house, in which lighting was reversed with respect to that of the external environment (lights on 20.30-10.30 h; 'reversed light'). Food, water and cages were usually changed between 10.30 and 12.00 h in the room with ordinary lighting and between 09.30 and 10.00 h in the room with reversed lighting. The latter corresponded to 18-18-5 h after the animals' midnight. In Expt 2, all the animals were kept under ordinary light. In each experiment the animals were of the same age, arrived in the Department at the same time, and weighed about 400 g in Expt 1 and 300 g in Expt. 2. They were allowed 5 (Expt 1) or 3 (Expt 2) weeks to become accustomed to the conditions of the animal house or the reversed light room. All the animals were handled daily for at least a week before experimentation. Collection of samples was completed in 14 (Expt 1) and 24 h (Expt 2). At each time of day studied, a cage of animals (5-6 rats/cage) was removed from the housing to a nearby room. The animals were killed by rapid decapitation, and trunk blood was collected in ice-cooled plastic tubes containing 100/d heparin (500 i.u./tube; Pularin, Evans Medical Ltd, Liverpool) and 50 /*! Trasylol (1000 kallikrein inactivator units (K.I.U.)/tube; Bayer, U.K. Ltd., Pharmaceutical Division, Haywards Heath). No more than 3 min elapsed be¬ tween removal of the animals from their housing room and decapitation of the last animal in the group. The blood was immediately centrifuged and the plasma samples were each divided into two samples, the larger of which was reserved for ACTH extraction. The brains
removed from the heads (which had been placed on ice immediately after decapita¬ and a block extending from the optic chiasma to just caudal to the mammillary body, tion) as wide as the median eminence and about 2 mm deep, was dissected out. The block of tissue comprised the pituitary stalk, median eminence, the arcuate, ventromedial, dorsomedial, posterior hypothalamic, premammillary, mammillary, caudal part of the suprachiasmatic, and ventral part of the paraventricular nuclei of the hypothalamus, and the anterior hypothalamic area. Blocks were weighed (11-14 mg) and placed in an homogenizer tube kept on ice and containing 1-5 ml 0-1 M-HC1. Cerebral cortical tissue, corresponding in weight to that of the total weight of hypothalamic fragments in each group, was also re¬ moved and treated similarly. The extracts of tissues obtained at each time were pooled after each extract had been assayed for LH-RF. The anterior pituitary of each animal was dissected out, blotted, weighed and placed in 1-5 ml 0-9 % saline solution in an homogenizer tube kept on ice. The tissue samples were homogenized, starting within 15 min of decapita¬ tion, with the aid of a Potter-Elvehjem type homogenizer (15 strokes). The homogenates and plasma samples were stored at -20 °C. were
Female rats The female rats were all maintained under ordinary light and in the same room as the males used for Expt. 2. The animals weighed 200-320 g, and had all shown regular 4-day oestrous cycles (as assessed by the daily inspection of vaginal smears). At various times of the cycle, groups of animals were anaesthetized with 2-5 % Avertin (tribromoethanol plus amylene hydrate), and a 2 ml blood sample was taken from the external jugular vein. Hypothalamic, cortical and anterior pituitary tissues were removed and treated as for the males except that the pituitary and hypothalamic tissues were homogenized in 2-0 ml 0-9 % saline and 3-0 ml 0-1 M-HC1, respectively.
Assay methods Plasma and pituitary Iuteinizing hormone (LH) was measured by the method of Niswender, Midgley, Monroe & Reichert (1968) as used by Aiyer, Fink & Greig (1974). The standard was NIH-LH-S18. Plasma and pituitary follicle-stimulating hormone (FSH) was determined with the aid of the NlAMDD-rat-FSH kit using a method similar to that of Daane & Parlow (1971) as described by Aiyer et al. (1974). The standard was NIAMDD rat FSHRP-1. Estimations of plasma LH and FSH in the male rats were made on 200/d aliquots. Estimations of FSH in the female rats were also made on 200 µ\ plasma, but the volume used to assay LH concentrations ranged from 20 to 50 µ\. For a 200 µ\ sample the lower limit of sensitivity (90 % intercept) of the LH assay ranged from 0-29 to 0-40 ng/ml, and that of the FSH assay was 95 ng/ml. ACTH (males only) was measured using a double-antibody radioimmunoassay tech¬ nique, lodination of the ACTH (III IWS; Mill Hill, London) with 125I was similar to that described by Rees, Cook, Kendall, Allen, Kramer, Ratcliffe & Knight (1971). The III IWS (1 µg 100 mu.) was also used as a standard. The antiserum, purchased from Wellcome Reagents Ltd, Beckenham, Kent, was raised in rabbits against human ACTH and was used at a working dilution of 1:5000. To reduce damage to the ACTH molecule, which, com¬ pared with many other peptide hormones, is particularly sensitive to protease degradation, 2-mercaptoethanol (Sigma, Missouri) and Trasylol at concentrations of 0-5 ml/100 ml and 1000 K.I.U./ml respectively were added to the assay buffer which also contained human serum albumin (Fraction V, Sigma, 250 mg/100 ml). The antiserum, buffer and either standard or unknown were added and mixed in plastic tubes on Day 1. Three days later, 100/*l of buffer containing 10 pg 125I-labelled ACTH were added to the tubes which were then mixed. Separation of free and bound fractions was achieved by the addition, 24 h later, =
of sufficient anti-rabbit gamma globulin (Wellcome Reagents Ltd) diluted in 0-01 mphosphate-buffered saline (usually 1:15), followed by a further period of incubation for 24 h after which the tubes were centrifuged. The tubes were kept at 4 °C throughout the period of the assay. 3r MSH and
4-10 ACTH
2
Arginine-vasopressin
1 m
0
hypothalamic
fraemcnt
o
III IWS
KRB + cortical tissue
-3
_L
10 10 ng 111 IWS ACTH/ml ng ACTH/ml KRB medium 01 ng ACTH/pituitary 100 ng ACTH fragment/ml 10 mu. Pitressin or vasopressin/'ml
100
Fig. 1. ACTH radioimmunoassay showing similarity in slopes between standard (III IWS), rat pituitary extract, Krebs-Ringer bicarbonate (KRB) medium from flasks in in-vitro assay for cortico¬ trophin releasing activity to which saline, cortical or hypothalamic extract had been added, and fragments ( -24 and 1-24) of ACTH. Pitressin (Parke Davis & Co. Ltd), but not synthetic arginine-vasopressin, inhibited binding; however, the slope of the Pitressin curve was not parallel to that of III IWS. The slopes are based on at least three points with four replicates per point. To determine plasma ACTH it was necessary to extract the plasma samples with Vycor glass (Corning Glass International, London). The standards (III IWS ACTH) were made up in plasma obtained from hypophysectomized male rats and similarly extracted. The extraction method was as described in the details received with the antiserum (Wellcome Reagents Ltd), and was similar to that used by Ratcliffe & Edwards (1971) and Rees et al. (1971). The recovery of the standard ACTH extracted by this method was 37-0 ±1-6% (four standards in duplicate). The characteristics of the Wellcome anti-ACTH serum have been examined by Croughs, Tops & de Jong (1973). We checked the antiserum against similar fragments and obtained similar results (Fig. 1), with the exception that the slope of the percentage bound v. log-dose curve of a25-39 ACTH was much flatter than that found by Croughs et al. (1973). The slopes of serial dilutions of rat pituitary extracts and incubation medium from flasks containing rat pituitaries (in-vitro assay for CR-activity; below) were similar to that of the standard (Fig. 1). The intra-assay and inter-assay coefficients of variation were 5-1 and 11-0% (six assays; 4-6 replicates of pooled sample/assay). The sensitivity of the assay for unextracted samples was 158 ± 11 pg/ml (90 % intercept). The LH-RF content of the hypothalamic and cortical fragments was determined using the radioimmunoassay of Nett, Akbar, Niswender, Hedlund & White (1973) with the aid of an antiserum (R-42-anti-GnRH) provided by Dr G. D. Niswender. Synthetic LH-RF
decapeptide (I.C.I. Pharmaceuticals) was used as standard and also as tracer. The character¬ istics of the assay in our Laboratory have been described in detail by Fink, Chiappa & Aiyer (1976) and Fink & Jamieson (1976). This assay is sensitive to 2-3 pg LH-RF/tube using 90 % intercept; measurements were made on the equivalent of 01 mg hypothalamic tissue. The CR-activities of hypothalamic and cortical fragments (males only) were determined by an in-vitro technique. Male Wistar rats weighing 300-400 g were anaesthetized with sodium pentobarbitone (Expirai; Abbott Laboratories, Kent) and decapitated. The an¬ terior pituitary glands were removed, bisected and placed into separate 25 ml conical flasks which contained 2 ml Krebs-Ringer bicarbonate buffer (pH 7-4) with added glucose (2 g/litre). The flasks, containing two pituitary halves, each from a different rat, were preincubated at 37 CC in a shaking incubator under 95 % 02:5 % C02 for 2 h. The medium was changed at the end of each hour. During pre-incubation, the hypothalamic and cortical extracts were thawed and centrifuged at 10 000 g and 4 °C for 30 min. The supernatants were neutralized with 5 M-NaOH, diluted in saline to obtain the desired dose, and assayed at 3-5 flasks/dose. The amount of extract added (in a volume of 0-5 ml) was equivalent to 1 hypothalamus or a similar weight of cerebral cortical tissue. The flasks were assigned in advance for each treatment using Fisher's Tables of Random Numbers. Incubation was carried out for 15 min at 37 °C after which the flasks were removed and placed on ice. The medium was aspirated, divided into two samples and stored frozen until assayed by radio¬ immunoassay for ACTH content. The CR-activity of the hypothalamic and cortical extracts (males only) was also assayed in vivo using the method of Arimura, Saito & Schally (1967), as used by Fink, Smith & Tibballs (1971) except that plasma corticosterone was determined by a competitive proteinbinding method. The latter, based on that of Corker, Naftolin & Richards (1971), was also used to estimate plasma corticosterone concentrations in blood samples obtained from male animals in Expt 2. The results are presented as group means ± s.e.m. except where one or more values in the group was below the limits of detection, in which case only the group mean is given. The CR-activity is based on the amount of ACTH released/ml medium minus the amount of ACTH present in the tissue extracts (after correcting for dilution in the medium). The times stated in the text and tables are all related to the animals' midnight (0 h). The significance of the difference between two means was determined by the unpaired r-test, while the signifi¬ cance of differences between more than two means was determined by analysis of variance and multiple range test (Duncan, 1955; Harter, 1960). RESULTS
Male animals LH-RF and gonadotrophins
The results are presented in Tables 1 (Expt 1) and 2 (Expt 2). In Expt 1, the mean LH-RF contents of the three groups of animals kept under reversed light were significantly lower than those of the three groups maintained under ordinary light. This, together with the absence of a significant between-treatment (ordinary or reversed light) difference in data related to the hypothalamic-pituitary adrenal system (Table 3) suggested that the lower LH-RF content during the night may have been an artifact produced by the different animal housing conditions. Therefore we carried out Expt 2 in which it was found that although LH-RF levels changed, being low at 5 and 18 h and high at 1 and 12 h, there were no sig¬ nificant differences between the group means. At all times the levels of LH-RF in cerebral cortical extracts were undetectable (< 14pg/15-18mg tissue). In Expt 1, pituitary
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SHARON A. CHIAPPA AND G. FINK Department of Human Anatomy, South Parks Road, Oxford, 0X1 3QX
(Received
June
1976)
SUMMARY
Hypothalamic corticotrophin releasing (CR) activity and LH-releasing factor (RF) content, and pituitary and plasma LH, FSH and ACTH were measured in adult male and female Wistar rats maintained under 14 h light per day. Hypothalamic LH-RF and pituitary and plasma hormones were estimated by radioimmunoassay while CR-activity was assessed by the amount of ACTH released from hemipituitaries in vitro. Two experiments were carried out on male animals. In the first, some of the animals were kept in a room, distant from the animal house, in which the lighting was reversed with respect to the external environment. In animals exposed to the reversed lighting r\l=e'\gime,hypothalamic LH-RF content and pituitary gonadotrophin concentrations were significantly lower than the values in male rats kept in the animal house where they were in close proximity to female rats. In the second experiment, which was carried out on animals which had all been kept in the animal house, there were no significant differences between the LH-RF contents measured at 3\p=n-\4h intervals throughout the day. Pituitary LH and FSH contents, but not concentrations, were significantly increased at 12.00 h. There was little difference between the experiments in CR-activity, plasma ACTH concentrations and profiles of pituitary ACTH content and concentration. As expected there was a diurnal rhythm in plasma corticosterone concentrations (determined by competitive protein-binding assay) with the peak occurring between 15.00 and 18.00 h. The profiles of plasma and pituitary ACTH were similar to that of plasma corticosterone. Corticotrophin releasing activity dropped significantly between 12.00 and 16.00 h, but remained steady at the other times. In female rats there were no significant differences between hypothalamic LH-RF con¬ tent throughout the 4-day cycle. During pro-oestrus the mean LH-RF content rose to reach a high level at 18.00 h at which time plasma LH concentration had risen sharply to a level consistent with the peak of the preovulatory surge. Plasma FSH concentration also rose significantly between 15.00 and 18.00 h of pro-oestrus. At metoestrus and dioestrus, plasma FSH levels were lower in the morning than in the evening. These results suggest that (1) there is no diurnal rhythm in hypothalamic LH-RF, (2) there may be a diurnal rhythm in pituitary gonadotrophin content in the male and in plasma FSH concentration on the days of metoestrus and dioestrus in the female, (3) if a surge of LHRF does occur on the afternoon of pro-oestrus, the rate of LH-RF synthesis exceeds its release, and (4) the mechanism which regulates gonadotrophin secretion in the male may be affected by factors in the environment other than daylength. The results provide further evidence for the view that the diurnal rhythm of corticosterone secretion is under hypothalamo-hypophysial control.
INTRODUCTION
Several workers have studied hypothalamic corticotrophin releasing activity (CR-activity) at various times during the day in male animals (Cheifetz, Gaffud & Dingman, 1969; David-Nelson & Brodish, 1969; Hiroshige, Sakakura & Itoh, 1969; Hiroshige & Sakakura, 1971; Seiden & Brodish, 1972; Takebe, Sakakura & Mashimo, 1972) and hypothalamic Iuteinizing hormone releasing factor (LH-RF) during the oestrous cycle (Chowers & McCann, 1965; Ramirez & Sawyer, 1965; Kalra, Krulich & McCann, 1973; Araki, Ferin, Zimmerman & Vande Wiele, 1975; Kalra, 1976). No detailed study appears to have been published on hypothalamic LH-RF levels during the day in male rats, and, apart from data on plasma corticosterone concentrations which were included in most of the studies on CRactivity, there is no information on the relationship between other relevant plasma or pituitary hormone levels and hypothalamic releasing factor activity measured at the same times and in the same animals. The purpose of the present work was to determine whether (I) there are variations in CR-activity of the hypothalamus and immunoreactive corticotrophin (ACTH) in the pituitary gland and in plasma which can be linked with the well-known diurnal rhythm of plasma corticosterone in male rats, (2) there is a diurnal rhythm in the gonadotrophin regulatory system in male rats, and (3) the levels of immunoreactive LH-RF in the hypothalamus of female rats can be correlated with changes in pituitary and plasma gonadotrophin levels. MATERIALS AND METHODS
Animals and experimental procedures All the animals were rats of the Wistar strain supplied by Charles River (U.K.) Ltd, Mar¬ gate. They were maintained under controlled lighting and temperature (22 °C) and given free access to Diet 41 (E. Dixon and Sons Ltd, Ware) and tap-water. Male rats Two experiments were carried out on male animals. In Expt. 1, some of the animals were kept under a lighting régime in which the lights were on between 05.00-19.00 h ('ordinary light') while others were kept in a room, distant from the animal house, in which lighting was reversed with respect to that of the external environment (lights on 20.30-10.30 h; 'reversed light'). Food, water and cages were usually changed between 10.30 and 12.00 h in the room with ordinary lighting and between 09.30 and 10.00 h in the room with reversed lighting. The latter corresponded to 18-18-5 h after the animals' midnight. In Expt 2, all the animals were kept under ordinary light. In each experiment the animals were of the same age, arrived in the Department at the same time, and weighed about 400 g in Expt 1 and 300 g in Expt. 2. They were allowed 5 (Expt 1) or 3 (Expt 2) weeks to become accustomed to the conditions of the animal house or the reversed light room. All the animals were handled daily for at least a week before experimentation. Collection of samples was completed in 14 (Expt 1) and 24 h (Expt 2). At each time of day studied, a cage of animals (5-6 rats/cage) was removed from the housing to a nearby room. The animals were killed by rapid decapitation, and trunk blood was collected in ice-cooled plastic tubes containing 100/d heparin (500 i.u./tube; Pularin, Evans Medical Ltd, Liverpool) and 50 /*! Trasylol (1000 kallikrein inactivator units (K.I.U.)/tube; Bayer, U.K. Ltd., Pharmaceutical Division, Haywards Heath). No more than 3 min elapsed be¬ tween removal of the animals from their housing room and decapitation of the last animal in the group. The blood was immediately centrifuged and the plasma samples were each divided into two samples, the larger of which was reserved for ACTH extraction. The brains
removed from the heads (which had been placed on ice immediately after decapita¬ and a block extending from the optic chiasma to just caudal to the mammillary body, tion) as wide as the median eminence and about 2 mm deep, was dissected out. The block of tissue comprised the pituitary stalk, median eminence, the arcuate, ventromedial, dorsomedial, posterior hypothalamic, premammillary, mammillary, caudal part of the suprachiasmatic, and ventral part of the paraventricular nuclei of the hypothalamus, and the anterior hypothalamic area. Blocks were weighed (11-14 mg) and placed in an homogenizer tube kept on ice and containing 1-5 ml 0-1 M-HC1. Cerebral cortical tissue, corresponding in weight to that of the total weight of hypothalamic fragments in each group, was also re¬ moved and treated similarly. The extracts of tissues obtained at each time were pooled after each extract had been assayed for LH-RF. The anterior pituitary of each animal was dissected out, blotted, weighed and placed in 1-5 ml 0-9 % saline solution in an homogenizer tube kept on ice. The tissue samples were homogenized, starting within 15 min of decapita¬ tion, with the aid of a Potter-Elvehjem type homogenizer (15 strokes). The homogenates and plasma samples were stored at -20 °C. were
Female rats The female rats were all maintained under ordinary light and in the same room as the males used for Expt. 2. The animals weighed 200-320 g, and had all shown regular 4-day oestrous cycles (as assessed by the daily inspection of vaginal smears). At various times of the cycle, groups of animals were anaesthetized with 2-5 % Avertin (tribromoethanol plus amylene hydrate), and a 2 ml blood sample was taken from the external jugular vein. Hypothalamic, cortical and anterior pituitary tissues were removed and treated as for the males except that the pituitary and hypothalamic tissues were homogenized in 2-0 ml 0-9 % saline and 3-0 ml 0-1 M-HC1, respectively.
Assay methods Plasma and pituitary Iuteinizing hormone (LH) was measured by the method of Niswender, Midgley, Monroe & Reichert (1968) as used by Aiyer, Fink & Greig (1974). The standard was NIH-LH-S18. Plasma and pituitary follicle-stimulating hormone (FSH) was determined with the aid of the NlAMDD-rat-FSH kit using a method similar to that of Daane & Parlow (1971) as described by Aiyer et al. (1974). The standard was NIAMDD rat FSHRP-1. Estimations of plasma LH and FSH in the male rats were made on 200/d aliquots. Estimations of FSH in the female rats were also made on 200 µ\ plasma, but the volume used to assay LH concentrations ranged from 20 to 50 µ\. For a 200 µ\ sample the lower limit of sensitivity (90 % intercept) of the LH assay ranged from 0-29 to 0-40 ng/ml, and that of the FSH assay was 95 ng/ml. ACTH (males only) was measured using a double-antibody radioimmunoassay tech¬ nique, lodination of the ACTH (III IWS; Mill Hill, London) with 125I was similar to that described by Rees, Cook, Kendall, Allen, Kramer, Ratcliffe & Knight (1971). The III IWS (1 µg 100 mu.) was also used as a standard. The antiserum, purchased from Wellcome Reagents Ltd, Beckenham, Kent, was raised in rabbits against human ACTH and was used at a working dilution of 1:5000. To reduce damage to the ACTH molecule, which, com¬ pared with many other peptide hormones, is particularly sensitive to protease degradation, 2-mercaptoethanol (Sigma, Missouri) and Trasylol at concentrations of 0-5 ml/100 ml and 1000 K.I.U./ml respectively were added to the assay buffer which also contained human serum albumin (Fraction V, Sigma, 250 mg/100 ml). The antiserum, buffer and either standard or unknown were added and mixed in plastic tubes on Day 1. Three days later, 100/*l of buffer containing 10 pg 125I-labelled ACTH were added to the tubes which were then mixed. Separation of free and bound fractions was achieved by the addition, 24 h later, =
of sufficient anti-rabbit gamma globulin (Wellcome Reagents Ltd) diluted in 0-01 mphosphate-buffered saline (usually 1:15), followed by a further period of incubation for 24 h after which the tubes were centrifuged. The tubes were kept at 4 °C throughout the period of the assay. 3r MSH and
4-10 ACTH
2
Arginine-vasopressin
1 m
0
hypothalamic
fraemcnt
o
III IWS
KRB + cortical tissue
-3
_L
10 10 ng 111 IWS ACTH/ml ng ACTH/ml KRB medium 01 ng ACTH/pituitary 100 ng ACTH fragment/ml 10 mu. Pitressin or vasopressin/'ml
100
Fig. 1. ACTH radioimmunoassay showing similarity in slopes between standard (III IWS), rat pituitary extract, Krebs-Ringer bicarbonate (KRB) medium from flasks in in-vitro assay for cortico¬ trophin releasing activity to which saline, cortical or hypothalamic extract had been added, and fragments ( -24 and 1-24) of ACTH. Pitressin (Parke Davis & Co. Ltd), but not synthetic arginine-vasopressin, inhibited binding; however, the slope of the Pitressin curve was not parallel to that of III IWS. The slopes are based on at least three points with four replicates per point. To determine plasma ACTH it was necessary to extract the plasma samples with Vycor glass (Corning Glass International, London). The standards (III IWS ACTH) were made up in plasma obtained from hypophysectomized male rats and similarly extracted. The extraction method was as described in the details received with the antiserum (Wellcome Reagents Ltd), and was similar to that used by Ratcliffe & Edwards (1971) and Rees et al. (1971). The recovery of the standard ACTH extracted by this method was 37-0 ±1-6% (four standards in duplicate). The characteristics of the Wellcome anti-ACTH serum have been examined by Croughs, Tops & de Jong (1973). We checked the antiserum against similar fragments and obtained similar results (Fig. 1), with the exception that the slope of the percentage bound v. log-dose curve of a25-39 ACTH was much flatter than that found by Croughs et al. (1973). The slopes of serial dilutions of rat pituitary extracts and incubation medium from flasks containing rat pituitaries (in-vitro assay for CR-activity; below) were similar to that of the standard (Fig. 1). The intra-assay and inter-assay coefficients of variation were 5-1 and 11-0% (six assays; 4-6 replicates of pooled sample/assay). The sensitivity of the assay for unextracted samples was 158 ± 11 pg/ml (90 % intercept). The LH-RF content of the hypothalamic and cortical fragments was determined using the radioimmunoassay of Nett, Akbar, Niswender, Hedlund & White (1973) with the aid of an antiserum (R-42-anti-GnRH) provided by Dr G. D. Niswender. Synthetic LH-RF
decapeptide (I.C.I. Pharmaceuticals) was used as standard and also as tracer. The character¬ istics of the assay in our Laboratory have been described in detail by Fink, Chiappa & Aiyer (1976) and Fink & Jamieson (1976). This assay is sensitive to 2-3 pg LH-RF/tube using 90 % intercept; measurements were made on the equivalent of 01 mg hypothalamic tissue. The CR-activities of hypothalamic and cortical fragments (males only) were determined by an in-vitro technique. Male Wistar rats weighing 300-400 g were anaesthetized with sodium pentobarbitone (Expirai; Abbott Laboratories, Kent) and decapitated. The an¬ terior pituitary glands were removed, bisected and placed into separate 25 ml conical flasks which contained 2 ml Krebs-Ringer bicarbonate buffer (pH 7-4) with added glucose (2 g/litre). The flasks, containing two pituitary halves, each from a different rat, were preincubated at 37 CC in a shaking incubator under 95 % 02:5 % C02 for 2 h. The medium was changed at the end of each hour. During pre-incubation, the hypothalamic and cortical extracts were thawed and centrifuged at 10 000 g and 4 °C for 30 min. The supernatants were neutralized with 5 M-NaOH, diluted in saline to obtain the desired dose, and assayed at 3-5 flasks/dose. The amount of extract added (in a volume of 0-5 ml) was equivalent to 1 hypothalamus or a similar weight of cerebral cortical tissue. The flasks were assigned in advance for each treatment using Fisher's Tables of Random Numbers. Incubation was carried out for 15 min at 37 °C after which the flasks were removed and placed on ice. The medium was aspirated, divided into two samples and stored frozen until assayed by radio¬ immunoassay for ACTH content. The CR-activity of the hypothalamic and cortical extracts (males only) was also assayed in vivo using the method of Arimura, Saito & Schally (1967), as used by Fink, Smith & Tibballs (1971) except that plasma corticosterone was determined by a competitive proteinbinding method. The latter, based on that of Corker, Naftolin & Richards (1971), was also used to estimate plasma corticosterone concentrations in blood samples obtained from male animals in Expt 2. The results are presented as group means ± s.e.m. except where one or more values in the group was below the limits of detection, in which case only the group mean is given. The CR-activity is based on the amount of ACTH released/ml medium minus the amount of ACTH present in the tissue extracts (after correcting for dilution in the medium). The times stated in the text and tables are all related to the animals' midnight (0 h). The significance of the difference between two means was determined by the unpaired r-test, while the signifi¬ cance of differences between more than two means was determined by analysis of variance and multiple range test (Duncan, 1955; Harter, 1960). RESULTS
Male animals LH-RF and gonadotrophins
The results are presented in Tables 1 (Expt 1) and 2 (Expt 2). In Expt 1, the mean LH-RF contents of the three groups of animals kept under reversed light were significantly lower than those of the three groups maintained under ordinary light. This, together with the absence of a significant between-treatment (ordinary or reversed light) difference in data related to the hypothalamic-pituitary adrenal system (Table 3) suggested that the lower LH-RF content during the night may have been an artifact produced by the different animal housing conditions. Therefore we carried out Expt 2 in which it was found that although LH-RF levels changed, being low at 5 and 18 h and high at 1 and 12 h, there were no sig¬ nificant differences between the group means. At all times the levels of LH-RF in cerebral cortical extracts were undetectable (< 14pg/15-18mg tissue). In Expt 1, pituitary
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