EFFECTS OF ANDROGENS, OESTROGENS AND DEOXYCORTICOSTERONE ACETATE ON PLASMA CONCENTRATIONS OF LUTEINIZING HORMONE IN LAYING HENS SUSAN C. WILSON AND P. J. SHARP Council's Poultry Research Centre, King's Buildings, Research Agricultural West Mains Road, Edinburgh, EH9 3JS
(Received 19 June 1975) SUMMARY
Testosterone, androstenedione, oestrone, oestradiol-17\g=b\ or deoxycorticosterone acetate (DOCA) were injected intramuscularly at several dose-levels and at various stages of the
ovulatory cycle, and subsequent changes in plasma LH concentration were measured by radioimmunoassay. In 19 out of 24 hens, injection of 0\m=.\1,0\m=.\5or 1\m=.\0mg DOCA/kg resulted in a mean maximal increase in plasma LH concentration of between 0\m=.\47and 2\m=.\10ng/ml. The magnitude of this response was not related to either the dose or the stage of the cycle at which the DOCA was injected. In the remaining five hens DOCA failed to stimulate LH secretion. Injection of either androstenedione, oestrone or oestradiol did not result in any increase in LH level in the circulation. In contrast, injection of 0\m=.\5, 1\m=.\0or 2\m=.\0mg testosterone/kg between
22 and 26 h after the terminal ovulation of a sequence resulted in mean maximal incremental changes in plasma LH level of 1\m=.\98 \m0\=+-m=.\17,\2\m=.\17\m=+-\0\m=.\21and 2\m=.\41\m=+-\0\m=.\31(s.e.m.) ng/ml from pre-injection values of 1 \m=.\38\m=+-\0\m=.\16, 1\m=.\58\m=+-\0\m=.\30and 1 \m=.\43\m=+-\0\m=.\39ng/ml (n 7, 6 and 5, respectively). The interval between the injection and the resulting rise in LH level was inversely proportional to the dose. The same doses of testosterone injected between 0 and 8 h after ovulation failed to stimulate LH secretion. There was also no significant increase in LH levels after injection of 0\m=.\5and 1\m=.\0mg testosterone/kg between 8 and 9 h after ovulation. However, injection of 2 mg testosterone/kg at this time resulted in a small but significant (P < 0\m=.\05)increase in LH levels. Since the largest ovarian follicle is more mature at 22\p=n-\26h after ovulation than at 0\p=n-\9h after ovulation, the ability of testosterone to cause the release of LH therefore appears to depend upon the degree of maturation of the ovarian follicle next due to ovulate. =
INTRODUCTION
During the ovulatory cycle of the hen, plasma concentrations of the following hormones highest between 9 and 4 h before ovulation: androgens (Peterson, Henneberry & Common, 1973; Etches, 1974), oestrogens (Peterson & Common, 1972; Senior & Cunning¬ ham, 1974), progesterone (Peterson & Common, 1971; Kappauf & van Tienhoven, 1972; Furr, Bonney, England & Cunningham, 1973; Haynes, Cooper & Kay, 1973) and luteinizing hormone (LH) (Furr et al. 1973; Wilson & Sharp, 1973). Although the temporal relationships during this period between the secretion of oestrogen and LH (Senior & Cunningham, 1974) and progesterone and LH (Furr et al. 1973) have been examined, it is not clear which sex steroids are responsible for triggering the are
pre-ovulatory rise in plasma LH level. Progesterone may be important because appropriately timed injections of this steroid induce premature ovulation (Fraps & Dury, 1943; Neher & Fraps, 1950; Fraps, 1955a). Furthermore, injection of progesterone at any time during the ovulatory cycle, except during the 4 h preceding ovulation, results in an increase in plasma LH concentration (Wilson & Sharp, 1975). Of the remaining sex steroids, Fraps (1955Ò)
found testosterone to be about half as effective as progesterone in inducing premature ovulation, while large doses of oestrogen delayed ovulation (Fraps, 1954). The only other steroid shown to be effective in inducing ovulation was deoxycorticosterone acetate (Fraps,
19550). This
17/?,
study examines the effects of injections of testosterone, androstenedione, oestradioldeoxycorticosterone acetate on the secretion of LH in the laying hen.
oestrone and
MATERIALS AND METHODS
two-year-old hens derived from a commercial White Leghorn strain and laying of 4 to 10 eggs were maintained in individual cages on a lighting schedule sequences regular of 14 h light and 10 h darkness (lights on from 07.00 to 21.00 h) with food and water constantly available. Precise oviposition times were recorded using an automatic recording One- to
device. All steroids were dissolved in a solution of 1:1 propylene glycol and ethyl alcohol, acidified with 1 drop of 0-1 M-hydrochloric acid in each 4 ml of solution, and were injected intra¬ muscularly in a volume of between 0-3 and 0-4 ml. A blood sample of about 0-75 ml was taken by venepuncture from a wing vein at the time of the steroid injection, followed by four more samples at 15-min intervals and a further seven samples at 30-min intervals. The samples were centrifuged at 1800 g and the plasma was separated and stored at —20 °C until required for assay. Plasma LH concentrations were estimated using the radioimmunoassay described by Follett, Scanes & Cunningham (1972). The standard and 125l-labelled tracer were a prepara¬ tion of fowl LH (Fraction AE1) purified to minimize thyroid-stimulating hormone activity (Scanes & Follett, 1972). Each plasma sample was assayed in triplicate at one dilution and the LH concentrations, together with their 95 % confidence limits, were calculated using a modified version of a computer program described by Rodbard & Lewald (1970). In the 22 assays in this study the lowest detectable level of LH ranged from 0-01 to 0-29 ng/ml (mean 0-15 + 0-08 (s.d.) ng/ml) and the potency of a control plasma sample included in each assay ranged between 4-1 and 6-0 ng/ml (mean 5-2 ±0-54 (s.d.) ng/ml). The stages of the ovulatory cycle at which the steroids were injected were defined in relation to the time of the previous ovulation, which was estimated to occur within 14 to 75 min of oviposition (Warren & Scott, 1935). The first ovulation in a sequence which is not associated with a previous oviposition, was estimated to occur at approximately 05.00 h. This was based on observations that the interval between the peak of the pre-ovulatory LH surge and ovulation is about 4 h (Furr et al. 1973) and that in hens maintained under the same lighting conditions as were used in the present study, peak pre-ovulatory LH levels occur at about 01.00 h on the day of the first ovulation in a 4-10 egg sequence (S. C.Wilson, unpublished observation). All incremental changes in plasma LH concentration were expressed in relation to the pre-injection LH values. Student's r-test was used for statistical calculations.
RESULTS
Testosterone The effects of injected testosterone on plasma LH concentration varied according to the dose and the stage of the cycle at which the steroid was injected. Hens were injected between 0 and 8 h after any ovulation (C„), between 8 and 9 h after the first ovulation (Q) or between 22 and 26 h after the final (C,) ovulation of a sequence.
Injections 0 to 8 h after ovulation The mean change in plasma LH concentration after injection of 0-1, 0-5, 1-0 or 2-0 mg testosterone/kg between 0 and 8 h after ovulation is shown in Fig. 1. The mean change after injection of the carrier is shown in Fig. 2d. In all cases, the plasma LH concentration tended to fall during the first 90 min after injection. However, the plasma LH concentration at 90 min was only significantly lower than the pre-injection value after injection of 0-1 (P < 005) and 0-5 (P < 0-01) mg testosterone/kg. Data for the control injections include observations on four hens injected between 8 and 9 h after ovulation. Since there was no difference between changes in LH levels in hens injected at this time and those injected between 0 and 8 h after ovulation, the data were combined. No consistent change occurred in plasma LH concentration during the remaining 3-5 h of the sampling period. Injections 8 to 9 h after Cx ovulation A similar depression of LH secretion
to that described above was observed after injection of 0-1, 0-5 or 1-0 mg testosterone/kg between 8 and 9 h after a Ci ovulation (Fig. 1). How¬ ever, in no case was this depression found to be statistically significant. In contrast, after injection of 2-0 mg testosterone/kg the plasma LH level immediately began to rise and reached a maximum after 1 h. The LH concentration at this time was significantly (P < 0-05) higher than the pre-injection value. The incremental change in LH level was 0-86 + 0-25 (s.e.m.) ng/ml from the pre-injection value of 1-23 ±0-30 ng/ml. Data for control hens injected between 8 and 9 h after ovulation were combined with those from control hens injected between 0 and 8 h after ovulation as previously described (Fig. 2a"). There was no significant change in plasma LH concentration after an injection of carrier.
Injections 22 to 26 h after Ct ovulation The LH response to injection of testosterone during the period between 9 and 22 h after ovulation was not investigated. Injection of 0-5, 1-0 and 2-0 mg testosterone/kg between 22 and 26 h after ovulation resulted in a significant (P < 0-01 in all cases) mean maximal incremental change in LH levels of 1-98±017, 2-17 + 0-21 and 2-41 ±0-31 ng/ml respec¬ tively (Fig. lb, c and d). The dose of 0-1 mg/kg failed to stimulate LH secretion (Fig. la). The time taken for the LH level to start rising after injection was inversely related to the
dose of testosterone; for doses of 0-5, 1-0 and 2-0 mg testosterone/kg the interval was, 2-5 ±0-13, 1-4 + 0-28 and 0-7 ±0-10 h. The plasma LH level continued to rise 2 for between and 3 h in each case (Fig. 1). The plasma LH concentration showed no signifi¬ cant change from the pre-injection value after an injection of the carrier between 22 and 26 h after ovulation (Fig. 2 a*).
respectively,
Androstenedione Androstenedione was injected at dose levels of 0-1 (n 3), 0-5 (n 3) and 1-0 (n 3) after hens were h ovulation. Three 0 and 9 injected at each of the same dosemg/kg between levels between 22 and 26 h after the terminal ovulation of a sequence. In each case the =
=
=
0-8 h after C. ovulation
8-9 h after
22-26 h after C, ovulation
C, ovulation (a) 0· 1 mg testosterone/k
0-5 mg
(b) testosterone/kg
«=11
1 I
'_'
'
I_I_
1
_'
_I_
I
_ _1_L.
(c) 0 mg testosterone/kg
S 3 "SB ß
3 -
X -J
n
=
4
1 —
20 mg
'
'_ _ _ _
0
1
(d) testosterone/kg
0 3 4 12 Time after injection (h)
Fig. 1. Changes in plasma LH concentrations in laying hens after i.m. injections of (a) 01, (6) 0-5, (c) 1-0, (d) 20 mg testosterone/kg at 0 to 8 h after any ovulation of a sequence (Cn), 8 to 9 h after the first ovulation of a sequence (Q), 22 to 26 h after the terminal ovulation of a sequence (C(). Vertical lines indicate ± s.e.m.
plasma LH concentration tended to fall during the first 90 min after injection but was never significantly lower than the pre-injection value. A similar response was observed in the control injected hens (Fig. 2d); the extent to which the plasma LH level was depressed was not related to the dose of androstenedione. Changes in plasma LH levels after injection of 0-5 mg/kg are shown in Fig. 2c. Oestradiol
Oestrone
Time after
injection (h) Fig. 2. Changes in plasma LH concentrations in laying hens after i.m. injections of (a) 01 mg oestrone/kg, (6) 0-1 mg oestradiol-17/3/kg, (c) 0-5 mg androstenedione/kg either between 0 and 9 h after ovulation (solid line), or between 22 and 26 h after the terminal ovulation of a sequence (broken line), (d) Carrier-injected controls injected between 0 and 9 h after ovulation, including four hens injected between 8 and 9 h after ovulation (solid line), and carrier-injected controls injected between 22 and 26 h after the terminal ovulation of a sequence (broken line). The control hens also serve as controls for hens injected with testosterone and deoxycorticosterone acetate (Figs 1 and 3) since all steroids were injected in the same volume. Vertical lines indicate ± s.e.m. Oestrone and oestradiol-17ß Oestrone and oestradiol were injected into groups of three or four hens at dose levels of 0-01, 0-1 and 1-0 mg/kg between 0 and 9 h after ovulation. Either one or three hens were injected at the same dose-levels between 22 and 26 h after the terminal ovulation of a sequence. Neither oestrogen at any dose-level stimulated LH secretion during these two periods of the ovulatory cycle. The pattern of LH secretion after injection was similar to that in the control hens (Fig. 2o"). Plasma LH levels after injections of 0-1 mg oestrone or oestradiol/kg are shown in Fig. 2 a and b.
after
an
was a
Deoxycorticosterone acetate injected with 0-1, 0-5 or 1-0 mg DOCA/kg between 0
and 9 h ovulation or between 22 and 26 h after the terminal ovulation of a sequence, there rise in plasma LH level of between 0-47 and 2-10 ng/ml. The magnitude of the
In all but five out of 24 hens
incremental change in LH level was not related to either the dose-level or the phase of the ovulatory cycle at which the steroid was injected. The incremental changes observed were divided into three categories according to their magnitude: none, < 1-0 ng/ml and > 1-0 ng/ml; an example of each is shown in Fig. 3. Control-injected hens showed no signifi¬ cant change in LH secretion (Fig. 2 a*). (6)0-1 mgDOCA/kg
mg DOCA/kg 8 h 15 min after
(a) 01
8 h 45 min after C, ovulation
C, ovulation
I
3
JS
2
-
3
-
(c) 0-5 mg DOCA/kg 5 h 30 min after
C„ ovulation _L J_ _L _L J_ 01234
Time after injection
J_I_I
I_
01234
(h)
Fig. 3. Individual examples of three categories of LH response after i.m. injections of deoxy¬ corticosterone acetate (DOCA). (a) No response no significant rise in plasma LH levels, as deter¬ mined by 95 % confidence limits around each estimate. (6) Increase in plasma LH concentration of less than 1-0 ng/ml. (c) Increase in plasma LH concentration of greater than 10 ng/ml. Vertical lines indicate 95 % confidence limits. See text for explanation of Q and C„ ovulation. -
DISCUSSION
study shows that injection of testosterone or DOCA but not of oestrone or oestradiolstimulate secretion of LH in the laying hen. However, testosterone did not act in can 17/?, the same way as DOCA since the amount of LH released after an injection of testosterone seemed to be dependent on the degree of maturation of the ovarian follicle next due to ovulate. This did not apply to DOCA, which stimulated LH release when given at most stages of the ovulatory cycle in a manner similar to that previously demonstrated for progesterone (Wilson & Sharp, 1975). The response to DOCA was expected since Fraps (19556) found that ovulation was induced prematurely in 87 % of hens injected with 1-0 mg of the steroid about 14 h before the expected time of the first ovulation of a sequence. Similarly timed injections of 1-0 mg progesterone/hen were 95 % effective in inducing premature ovulation. Fraps (1955e) sug¬ gested that DOCA acts as a weak progestin since deoxycorticosterone is structurally similar to progesterone and possesses progesterone-like activity when administered systemically to mammals (Courrier, 1940; Gros, Benoit, Kehl & Paris, 1942; Hooker & Forbes, 1949). However, other studies have suggested that the progesterone-like activities of DOCA are the result of its conversion in the adrenal and kidney to a progesterone-like substance (Zarrow, Hisaw & Bryans, 1950; Lazo-Wasem & Zarrow, 1955). However, the conversion rate of less than 1 % (Lazo-Wasem & Zarrow, 1955) suggests that any increase in blood progesterone levels resulting from the injection of between 0-1 and 1-0 mg DOCA/kg would be insufficient to induce LH release. It is therefore more probable that DOCA itself was directly stimulating LH secretion. The LH response to DOCA was similar to the response to progesterone. After the latter steroid is injected at any stage of the ovulatory cycle, except during the 4 h period preceding This
ovulation, plasma LH level begins to rise within 50 min (Wilson & Sharp, 1975). Similarly, whenever injections of DOCA stimulated LH secretion, plasma LH levels started to rise
after the same time-interval. As in the case of progesterone, DOCA could stimulate LH secretion whether it was injected 0-8 h, 8-9 h or 22-26 h after ovulation. However, DOCA was not as uniformly effective as progesterone in causing the release of LH, since the mag¬ nitude of the LH response was variable and not related either to the dose of DOCA or to the stage of the cycle at which the steroid was injected. This variation could be due to differences among individual hens in the ability of the positive feedback receptor sites in the hypothalamus to distinguish between progesterone and deoxycorticosterone. The pattern of deoxycorticosterone secretion throughout the ovulatory cycle has not yet been determined, and it remains to be shown whether or not this steroid has any physiological role in triggering the pre-ovulatory LH surge. Plasma concentrations of both oestrone (Peterson & Common, 1972) and oestradiol (Peterson & Common, 1972; Senior & Cunningham, 1974), as measured by radioimmuno¬ assay, rise to a peak between 7 and 4 h before ovulation. When both oestradiol and LH were measured in the same plasma samples, the rise in oestradiol level was found to precede slightly that of LH (Senior & Cunningham, 1974), suggesting that a rising plasma level of oestrogen may be involved in causing the pre-ovulatory LH surge. This is not supported by earlier studies which showed that injection of large doses of oestradiol benzoate fail to cause premature ovulation (Fraps, 1954). Nor is it supported by the present study, which shows that injection of between 0-01 and 1-0 mg/kg of either oestrone or oestradiol-17/? did not increase plasma LH concentrations. These findings contrast with those in mammals, where appropriately timed injections of oestrogen can induce an LH surge (Schwartz, 1969; Harris & Naftolin, 1970; Brown-Grant, 1971; Knobil, 1974). In mammals the ability of injections of either oestrogen or progesterone to stimulate LH secretion is influenced by the pre-existing baseline plasma concentrations of sex steroids and, in some cases, in the length of time the hypothalamus and pituitary have been exposed to these concentrations. It is therefore possible that the inability of a single injection of oestrogen to cause a release of LH in the hen may be due to an inappropriate balance of gonadal steroids in the circulation. Alternatively, though oestrogen itself may not directly stimulate LH release, there is evidence that it may be needed to 'prime' the hypothalamus to facilitate the positive feedback effect of progesterone (S. C. Wilson, unpublished observation). Peak values of plasma testosterone have been observed between 9 and 8 h before ovulation (Etches, 1974). Since plasma LH concentration rises to a peak between 7 and 4 h before ovulation (Furr et al. 1973; Wilson & Sharp, 1973), it seems that the rise in plasma testo¬ sterone level precedes that of LH by 1-2 h. This implies that testosterone could be involved in triggering the preovulatory surge of LH. Earlier studies support this concept, since Fraps (1955Ò) observed premature ovulation in 41 % of hens injected with 1 mg testosterone/hen about 14 h before the first ovulation of a sequence. The interval between injection and ovulation was always greater than 9 h. In contrast, Fraps also observed that the interval between injection of 1 mg progesterone/hen and ovulation was always less than 8 h, suggest¬ ing that progesterone and testosterone were not acting in the same way. This observation led him to suggest that testosterone may have to be converted to an 'active substance' before ovulation could be induced (Fraps, 1955e). In the present study, after injection of testosterone between 22 and 26 h after the final ovulation of a sequence, plasma LH levels rose to a peak similar to the naturally occurring pre-ovulatory LH surge (Wilson & Sharp, 1973). No comparable LH surge resulted from testosterone injection between 0 and 9 h after ovulation. It therefore appears that the ability of testosterone to stimulate LH release depends on the functional state of the ovary. The concentration of progesterone in the largest ovarian follicle increases six- to sevenfold about
19 h after the previous ovulation (Shahabi, Norton & Nalbandov, 1975) and it is possible that testosterone injected after this time causes a release of ovarian progesterone, which then exerts a positive feedback effect on LH secretion. Since no increase in LH secretion was observed following testosterone injections between 0 and 8 h after ovulation, the ovarian follicle at this stage of the ovulatory cycle may not have been able to secrete sufficient progesterone to stimulate the release of LH. The mechanism by which testosterone could stimulate progesterone secretion is unclear. It could either act directly on the follicle to enable it to release progesterone, or exert a weak positive feedback effect on the hypothalamo-hypophysial complex and cause a minor increase in plasma LH levels. Increased plasma LH levels are known to stimulate progesterone secretion in the hen (Etches & Cunningham, 1975; Shahabi, Bahr & Nalbandov, 1975) and the ability of a small increase in LH levels in the circulation to trigger an initial release of progesterone from the ovary may depend on the amount of this steroid stored in the follicle next due to ovulate. If, in the present study, testosterone caused an initial rise in plasma LH level, it may not have been observed because it was too small or was masked by the larger rise in LH level presumably caused by the positive feedback action of progesterone released from the ovary. Another possibility is that testosterone differentially stimulated FSH secre¬ tion, which then caused the release of progesterone from the maturing ovarian follicle. If testosterone does cause a release of gonadotrophins via a central nervous positive feedback mechanism it may, as Fraps (1955¿>) suggested, have to be converted first into an 'active substance'. It has been shown that testosterone can be metabolized to androstene¬ dione in both the brain (Nakamura & Tanabe, 1974) and ovary (Nakamura, Tanabe & Katukawa, 1974) of the hen. However, as injections of between 0-1 and 1-0 mg androstenedione/kg did not increase plasma LH concentrations it is unlikely that androstenedione is the 'active substance'. There is no evidence to suggest that testosterone can be converted to progesterone in the hen, though it is converted to oestradiol in the hen's ovary (Nakamura et al. 1974). Since both oestradiol-17/? and oestrone were ineffective in stimulating LH secretion in the hen, it is unlikely that the positive feedback of testosterone suggested by the results of this study are mediated via oestrogens. However, it is possible that other conversion products such as dihydrotestosterone and androstanediol (Nakamura & Tanabe, 1974) are involved. In conclusion, this study shows that testosterone, like progesterone, will induce an LH surge in the laying hen. Since the pre-ovulatory increase in testosterone secretion may precede that of progesterone, it is possible that before a normal ovulation, rising levels of plasma testosterone facilitate the release of other gonadal steroids, including oestrogens and progesterone. As it has been shown that progesterone rapidly induces a release of LH when injected at most stages of the ovulatory cycle (Wilson & Sharp, 1975), increased secretion of this steroid is thought to be the immediate cause of the pre-ovulatory surge of LH. The authors are grateful to Dr . . Follett and Dr C. G. Scanes for the chicken LH preparations and to Mr W. R. Carr, Mr D. Maxwell and Mr D. T. Wilson of the A.R.C. Animal Breeding Research Organization for adapting Dr Rodbard's computer program. S.C.W. was supported by an A.R.C. Research studentship.
REFERENCES
Brown-Grant, . (1971). The role of steroid hormones in the control of gonadotrophin secretion in adult female mammals. In Steroid hormones and brain function, pp. 269-288. Eds C. H. Sawyer & R. A. Gorski. Los Angeles: University of California Press. Courrier, R. (1940). La désoxycorticostérone est capable de maintenir la grossesse ou de provoquer Pavortement. La Presse Medicale 48, 658. Etches, R. J. (1974). Plasma testosterone during the ovulation cycle of the chicken. In Abstract in XV World's Poultry Congress, New Orleans, August 1974, pp. 517-518. Washington: U.S.A. Branch World's Poultry Science Association.
Etches, R. J. & Cunningham, F. J. (1975). The role of the preovulatory release of progesterone in the chicken. Journal of Endocrinology 64, 47F-48F. Follett, B. K., Scanes, C. G. & Cunningham, F. J. (1972). A radioimmunoassay for avian luteinizing hormone. Journal of Endocrinology 52, 359-378. Fraps, R. M. (1954). Neural basis of diurnal periodicity in release of ovulation-inducing hormone in fowl. Proceedings of the National Academy of Sciences of the U.S.A. 40, 348-356. Fraps, R. . (1955 a). Egg production and fertility in poultry. In Progress in the physiology offarm animals, vol. 2, pp. 661-740. Ed. J. Hammond. London: Butterworths. Fraps, R. M. (19556). The varying effects of sex hormones in birds. Memoirs. Society for Endocrinology 4,
205-219. M. &Dury, A. (1943). Occurrence of premature ovulation in the domestic fowl following adminis¬ tration of progesterone. Proceedings of the Society for Experimental Biology and Medicine 52, 346-349. Furr, B. J. ., Bonney, R. C, England, R. J. & Cunningham, F. J. (1973). Luteinizing hormone and progesterone in peripheral blood during the ovulatory cycle of the hen Callus domesticus. Journal of Endocrinology 51, 159-169. Gros, G, Benoit, J., Kehl, R. & Paris, R. (1942). Action de l'acétate de désoxycorticostérone sur le maintien de la grossesse chez la Chatte gestante castrée. Comptes rendus des Séances de la Société de Biologie 136, 749-750. Harris, G. W. & Naftolin, F. (1970). The hypothalamus and control of ovulation. British Medical Bulletin 26, 3-9. Haynes, . ., Cooper, . J. & Kay, M. J. (1973). Plasma progesterone concentrations in the hen in relation to the ovulatory cycle. British Poultry Science 14, 349-357. Hooker, C. W. & Forbes, T. R. (1949). Specificity of the intrauterine test for progesterone. Endocrinology 45, 71-74. Kappauf, B. & van Tienhoven, A. (1972). Progesterone concentrations in peripheral plasma of laying hens in relation to the time of ovulation. Endocrinology 90, 1350-1355. Knobil, E. (1974). On the control of gonadotrophin secretion in the rhesus monkey. Recent Progress in Hormone Research 30, 1-46. Lazo-Wasem, E. A. & Zarrow, M. X. (1955). The conversion of désoxycorticostérone acetate to a pro¬ gesterone-like substance. Endocrinology 56, 511-515. Nakamura, T. & Tanabe, Y. (1974). In vitro metabolism of steroid hormones by chicken brain. Acta Endocrinologica 75, 410-416. Nakamura, T., Tanabe, Y. & Katukawa, H. (1974). Steroidogenesis in vitro by the ovarian tissue of the domestic fowl (Gallus domesticus). Journal of Endocrinology 63, 507-516. Neher, B. H. & Fraps, R. M. (1950). The addition of eggs to the hen's clutch by repeated injections of ovulation-inducing hormone. Endocrinology 46, 482^188. Peterson, A. J. & Common, R. H. (1971). Progesterone concentration in peripheral plasma of laying hens as determined by competitive protein-binding assay. Canadian Journal of Zoology 49, 599-604. Peterson, A. J. & Common, R. H. (1972). Estrone and estradiol concentrations in peripheral plasma of laying hens as determined by radioimmunoassay. Canadian Journal ofZoology 50, 395^104. Peterson, A. J., Henneberry, G. O. & Common, R. H. (1973). Androgen concentrations in the peripheral plasma of laying hens. Canadian Journal of Zoology 51, 753-758. Rodbard, D. & Lewald, J. E. (1970). Computer analysis of radioligand assay and radioimmunoassay data. In Karolinska Symposia on Research Methods in Reproductive Endocrinology, pp. 79-103. Ed. E. Diczfalusy. Stockholm: Karolinska Institutet. Scanes, C. G. & Follett, B. K. (1972). Fractionation and assay of chicken pituitary hormones. British Poultry Science 13, 603-610. Schwartz, N. B. (1969). A model for the regulation of ovulation in the rat. Recent Progress in Hormone Research 25, 1-53. Senior, B. E. & Cunningham, F. J. (1974). Oestradiol and luteinizing hormone during the ovulatory cycle of the hen. Journal of Endocrinology 60, 201-202. Shahabi, . ., Bahr, J. M. & Nalbandov, A. V. (1975). Effect of LH injection on plasma and follicular steroids in the chicken. Endocrinology 96, 969-972.
Fraps, R.
Shahabi, . ., Norton, . W. & Nalbandov, . V. (1975). Steroid levels in follicles and the plasma of hens during the ovulatory cycle. Endocrinology 96, 962-968. Warren, D. C. & Scott, . M. (1935). The time factor in egg formation. Poultry Science 14, 195-207. Wilson, S. C. & Sharp, P. J. (1973). Variations in plasma LH levels during the ovulatory cycle of the hen, Gallus domesticus. Journal of Reproduction and Fertility 35, 561-564. Wilson, S. C. & Sharp, P. J. (1975). Changes in plasma concentrations of luteinizing hormone after the injection of progesterone at various times during the ovulatory cycle of the domestic hen (Gallus domesticus). Journal of Endocrinology 67, 59-70. Zarrow, M. X., Hisaw, F. L. & Bryans, F. (1950). Conversion of désoxycorticostérone acetate to pro¬ gesterone in vivo. Endocrinology 46, 403-404.
(Received 19 June 1975) SUMMARY
Testosterone, androstenedione, oestrone, oestradiol-17\g=b\ or deoxycorticosterone acetate (DOCA) were injected intramuscularly at several dose-levels and at various stages of the
ovulatory cycle, and subsequent changes in plasma LH concentration were measured by radioimmunoassay. In 19 out of 24 hens, injection of 0\m=.\1,0\m=.\5or 1\m=.\0mg DOCA/kg resulted in a mean maximal increase in plasma LH concentration of between 0\m=.\47and 2\m=.\10ng/ml. The magnitude of this response was not related to either the dose or the stage of the cycle at which the DOCA was injected. In the remaining five hens DOCA failed to stimulate LH secretion. Injection of either androstenedione, oestrone or oestradiol did not result in any increase in LH level in the circulation. In contrast, injection of 0\m=.\5, 1\m=.\0or 2\m=.\0mg testosterone/kg between
22 and 26 h after the terminal ovulation of a sequence resulted in mean maximal incremental changes in plasma LH level of 1\m=.\98 \m0\=+-m=.\17,\2\m=.\17\m=+-\0\m=.\21and 2\m=.\41\m=+-\0\m=.\31(s.e.m.) ng/ml from pre-injection values of 1 \m=.\38\m=+-\0\m=.\16, 1\m=.\58\m=+-\0\m=.\30and 1 \m=.\43\m=+-\0\m=.\39ng/ml (n 7, 6 and 5, respectively). The interval between the injection and the resulting rise in LH level was inversely proportional to the dose. The same doses of testosterone injected between 0 and 8 h after ovulation failed to stimulate LH secretion. There was also no significant increase in LH levels after injection of 0\m=.\5and 1\m=.\0mg testosterone/kg between 8 and 9 h after ovulation. However, injection of 2 mg testosterone/kg at this time resulted in a small but significant (P < 0\m=.\05)increase in LH levels. Since the largest ovarian follicle is more mature at 22\p=n-\26h after ovulation than at 0\p=n-\9h after ovulation, the ability of testosterone to cause the release of LH therefore appears to depend upon the degree of maturation of the ovarian follicle next due to ovulate. =
INTRODUCTION
During the ovulatory cycle of the hen, plasma concentrations of the following hormones highest between 9 and 4 h before ovulation: androgens (Peterson, Henneberry & Common, 1973; Etches, 1974), oestrogens (Peterson & Common, 1972; Senior & Cunning¬ ham, 1974), progesterone (Peterson & Common, 1971; Kappauf & van Tienhoven, 1972; Furr, Bonney, England & Cunningham, 1973; Haynes, Cooper & Kay, 1973) and luteinizing hormone (LH) (Furr et al. 1973; Wilson & Sharp, 1973). Although the temporal relationships during this period between the secretion of oestrogen and LH (Senior & Cunningham, 1974) and progesterone and LH (Furr et al. 1973) have been examined, it is not clear which sex steroids are responsible for triggering the are
pre-ovulatory rise in plasma LH level. Progesterone may be important because appropriately timed injections of this steroid induce premature ovulation (Fraps & Dury, 1943; Neher & Fraps, 1950; Fraps, 1955a). Furthermore, injection of progesterone at any time during the ovulatory cycle, except during the 4 h preceding ovulation, results in an increase in plasma LH concentration (Wilson & Sharp, 1975). Of the remaining sex steroids, Fraps (1955Ò)
found testosterone to be about half as effective as progesterone in inducing premature ovulation, while large doses of oestrogen delayed ovulation (Fraps, 1954). The only other steroid shown to be effective in inducing ovulation was deoxycorticosterone acetate (Fraps,
19550). This
17/?,
study examines the effects of injections of testosterone, androstenedione, oestradioldeoxycorticosterone acetate on the secretion of LH in the laying hen.
oestrone and
MATERIALS AND METHODS
two-year-old hens derived from a commercial White Leghorn strain and laying of 4 to 10 eggs were maintained in individual cages on a lighting schedule sequences regular of 14 h light and 10 h darkness (lights on from 07.00 to 21.00 h) with food and water constantly available. Precise oviposition times were recorded using an automatic recording One- to
device. All steroids were dissolved in a solution of 1:1 propylene glycol and ethyl alcohol, acidified with 1 drop of 0-1 M-hydrochloric acid in each 4 ml of solution, and were injected intra¬ muscularly in a volume of between 0-3 and 0-4 ml. A blood sample of about 0-75 ml was taken by venepuncture from a wing vein at the time of the steroid injection, followed by four more samples at 15-min intervals and a further seven samples at 30-min intervals. The samples were centrifuged at 1800 g and the plasma was separated and stored at —20 °C until required for assay. Plasma LH concentrations were estimated using the radioimmunoassay described by Follett, Scanes & Cunningham (1972). The standard and 125l-labelled tracer were a prepara¬ tion of fowl LH (Fraction AE1) purified to minimize thyroid-stimulating hormone activity (Scanes & Follett, 1972). Each plasma sample was assayed in triplicate at one dilution and the LH concentrations, together with their 95 % confidence limits, were calculated using a modified version of a computer program described by Rodbard & Lewald (1970). In the 22 assays in this study the lowest detectable level of LH ranged from 0-01 to 0-29 ng/ml (mean 0-15 + 0-08 (s.d.) ng/ml) and the potency of a control plasma sample included in each assay ranged between 4-1 and 6-0 ng/ml (mean 5-2 ±0-54 (s.d.) ng/ml). The stages of the ovulatory cycle at which the steroids were injected were defined in relation to the time of the previous ovulation, which was estimated to occur within 14 to 75 min of oviposition (Warren & Scott, 1935). The first ovulation in a sequence which is not associated with a previous oviposition, was estimated to occur at approximately 05.00 h. This was based on observations that the interval between the peak of the pre-ovulatory LH surge and ovulation is about 4 h (Furr et al. 1973) and that in hens maintained under the same lighting conditions as were used in the present study, peak pre-ovulatory LH levels occur at about 01.00 h on the day of the first ovulation in a 4-10 egg sequence (S. C.Wilson, unpublished observation). All incremental changes in plasma LH concentration were expressed in relation to the pre-injection LH values. Student's r-test was used for statistical calculations.
RESULTS
Testosterone The effects of injected testosterone on plasma LH concentration varied according to the dose and the stage of the cycle at which the steroid was injected. Hens were injected between 0 and 8 h after any ovulation (C„), between 8 and 9 h after the first ovulation (Q) or between 22 and 26 h after the final (C,) ovulation of a sequence.
Injections 0 to 8 h after ovulation The mean change in plasma LH concentration after injection of 0-1, 0-5, 1-0 or 2-0 mg testosterone/kg between 0 and 8 h after ovulation is shown in Fig. 1. The mean change after injection of the carrier is shown in Fig. 2d. In all cases, the plasma LH concentration tended to fall during the first 90 min after injection. However, the plasma LH concentration at 90 min was only significantly lower than the pre-injection value after injection of 0-1 (P < 005) and 0-5 (P < 0-01) mg testosterone/kg. Data for the control injections include observations on four hens injected between 8 and 9 h after ovulation. Since there was no difference between changes in LH levels in hens injected at this time and those injected between 0 and 8 h after ovulation, the data were combined. No consistent change occurred in plasma LH concentration during the remaining 3-5 h of the sampling period. Injections 8 to 9 h after Cx ovulation A similar depression of LH secretion
to that described above was observed after injection of 0-1, 0-5 or 1-0 mg testosterone/kg between 8 and 9 h after a Ci ovulation (Fig. 1). How¬ ever, in no case was this depression found to be statistically significant. In contrast, after injection of 2-0 mg testosterone/kg the plasma LH level immediately began to rise and reached a maximum after 1 h. The LH concentration at this time was significantly (P < 0-05) higher than the pre-injection value. The incremental change in LH level was 0-86 + 0-25 (s.e.m.) ng/ml from the pre-injection value of 1-23 ±0-30 ng/ml. Data for control hens injected between 8 and 9 h after ovulation were combined with those from control hens injected between 0 and 8 h after ovulation as previously described (Fig. 2a"). There was no significant change in plasma LH concentration after an injection of carrier.
Injections 22 to 26 h after Ct ovulation The LH response to injection of testosterone during the period between 9 and 22 h after ovulation was not investigated. Injection of 0-5, 1-0 and 2-0 mg testosterone/kg between 22 and 26 h after ovulation resulted in a significant (P < 0-01 in all cases) mean maximal incremental change in LH levels of 1-98±017, 2-17 + 0-21 and 2-41 ±0-31 ng/ml respec¬ tively (Fig. lb, c and d). The dose of 0-1 mg/kg failed to stimulate LH secretion (Fig. la). The time taken for the LH level to start rising after injection was inversely related to the
dose of testosterone; for doses of 0-5, 1-0 and 2-0 mg testosterone/kg the interval was, 2-5 ±0-13, 1-4 + 0-28 and 0-7 ±0-10 h. The plasma LH level continued to rise 2 for between and 3 h in each case (Fig. 1). The plasma LH concentration showed no signifi¬ cant change from the pre-injection value after an injection of the carrier between 22 and 26 h after ovulation (Fig. 2 a*).
respectively,
Androstenedione Androstenedione was injected at dose levels of 0-1 (n 3), 0-5 (n 3) and 1-0 (n 3) after hens were h ovulation. Three 0 and 9 injected at each of the same dosemg/kg between levels between 22 and 26 h after the terminal ovulation of a sequence. In each case the =
=
=
0-8 h after C. ovulation
8-9 h after
22-26 h after C, ovulation
C, ovulation (a) 0· 1 mg testosterone/k
0-5 mg
(b) testosterone/kg
«=11
1 I
'_'
'
I_I_
1
_'
_I_
I
_ _1_L.
(c) 0 mg testosterone/kg
S 3 "SB ß
3 -
X -J
n
=
4
1 —
20 mg
'
'_ _ _ _
0
1
(d) testosterone/kg
0 3 4 12 Time after injection (h)
Fig. 1. Changes in plasma LH concentrations in laying hens after i.m. injections of (a) 01, (6) 0-5, (c) 1-0, (d) 20 mg testosterone/kg at 0 to 8 h after any ovulation of a sequence (Cn), 8 to 9 h after the first ovulation of a sequence (Q), 22 to 26 h after the terminal ovulation of a sequence (C(). Vertical lines indicate ± s.e.m.
plasma LH concentration tended to fall during the first 90 min after injection but was never significantly lower than the pre-injection value. A similar response was observed in the control injected hens (Fig. 2d); the extent to which the plasma LH level was depressed was not related to the dose of androstenedione. Changes in plasma LH levels after injection of 0-5 mg/kg are shown in Fig. 2c. Oestradiol
Oestrone
Time after
injection (h) Fig. 2. Changes in plasma LH concentrations in laying hens after i.m. injections of (a) 01 mg oestrone/kg, (6) 0-1 mg oestradiol-17/3/kg, (c) 0-5 mg androstenedione/kg either between 0 and 9 h after ovulation (solid line), or between 22 and 26 h after the terminal ovulation of a sequence (broken line), (d) Carrier-injected controls injected between 0 and 9 h after ovulation, including four hens injected between 8 and 9 h after ovulation (solid line), and carrier-injected controls injected between 22 and 26 h after the terminal ovulation of a sequence (broken line). The control hens also serve as controls for hens injected with testosterone and deoxycorticosterone acetate (Figs 1 and 3) since all steroids were injected in the same volume. Vertical lines indicate ± s.e.m. Oestrone and oestradiol-17ß Oestrone and oestradiol were injected into groups of three or four hens at dose levels of 0-01, 0-1 and 1-0 mg/kg between 0 and 9 h after ovulation. Either one or three hens were injected at the same dose-levels between 22 and 26 h after the terminal ovulation of a sequence. Neither oestrogen at any dose-level stimulated LH secretion during these two periods of the ovulatory cycle. The pattern of LH secretion after injection was similar to that in the control hens (Fig. 2o"). Plasma LH levels after injections of 0-1 mg oestrone or oestradiol/kg are shown in Fig. 2 a and b.
after
an
was a
Deoxycorticosterone acetate injected with 0-1, 0-5 or 1-0 mg DOCA/kg between 0
and 9 h ovulation or between 22 and 26 h after the terminal ovulation of a sequence, there rise in plasma LH level of between 0-47 and 2-10 ng/ml. The magnitude of the
In all but five out of 24 hens
incremental change in LH level was not related to either the dose-level or the phase of the ovulatory cycle at which the steroid was injected. The incremental changes observed were divided into three categories according to their magnitude: none, < 1-0 ng/ml and > 1-0 ng/ml; an example of each is shown in Fig. 3. Control-injected hens showed no signifi¬ cant change in LH secretion (Fig. 2 a*). (6)0-1 mgDOCA/kg
mg DOCA/kg 8 h 15 min after
(a) 01
8 h 45 min after C, ovulation
C, ovulation
I
3
JS
2
-
3
-
(c) 0-5 mg DOCA/kg 5 h 30 min after
C„ ovulation _L J_ _L _L J_ 01234
Time after injection
J_I_I
I_
01234
(h)
Fig. 3. Individual examples of three categories of LH response after i.m. injections of deoxy¬ corticosterone acetate (DOCA). (a) No response no significant rise in plasma LH levels, as deter¬ mined by 95 % confidence limits around each estimate. (6) Increase in plasma LH concentration of less than 1-0 ng/ml. (c) Increase in plasma LH concentration of greater than 10 ng/ml. Vertical lines indicate 95 % confidence limits. See text for explanation of Q and C„ ovulation. -
DISCUSSION
study shows that injection of testosterone or DOCA but not of oestrone or oestradiolstimulate secretion of LH in the laying hen. However, testosterone did not act in can 17/?, the same way as DOCA since the amount of LH released after an injection of testosterone seemed to be dependent on the degree of maturation of the ovarian follicle next due to ovulate. This did not apply to DOCA, which stimulated LH release when given at most stages of the ovulatory cycle in a manner similar to that previously demonstrated for progesterone (Wilson & Sharp, 1975). The response to DOCA was expected since Fraps (19556) found that ovulation was induced prematurely in 87 % of hens injected with 1-0 mg of the steroid about 14 h before the expected time of the first ovulation of a sequence. Similarly timed injections of 1-0 mg progesterone/hen were 95 % effective in inducing premature ovulation. Fraps (1955e) sug¬ gested that DOCA acts as a weak progestin since deoxycorticosterone is structurally similar to progesterone and possesses progesterone-like activity when administered systemically to mammals (Courrier, 1940; Gros, Benoit, Kehl & Paris, 1942; Hooker & Forbes, 1949). However, other studies have suggested that the progesterone-like activities of DOCA are the result of its conversion in the adrenal and kidney to a progesterone-like substance (Zarrow, Hisaw & Bryans, 1950; Lazo-Wasem & Zarrow, 1955). However, the conversion rate of less than 1 % (Lazo-Wasem & Zarrow, 1955) suggests that any increase in blood progesterone levels resulting from the injection of between 0-1 and 1-0 mg DOCA/kg would be insufficient to induce LH release. It is therefore more probable that DOCA itself was directly stimulating LH secretion. The LH response to DOCA was similar to the response to progesterone. After the latter steroid is injected at any stage of the ovulatory cycle, except during the 4 h period preceding This
ovulation, plasma LH level begins to rise within 50 min (Wilson & Sharp, 1975). Similarly, whenever injections of DOCA stimulated LH secretion, plasma LH levels started to rise
after the same time-interval. As in the case of progesterone, DOCA could stimulate LH secretion whether it was injected 0-8 h, 8-9 h or 22-26 h after ovulation. However, DOCA was not as uniformly effective as progesterone in causing the release of LH, since the mag¬ nitude of the LH response was variable and not related either to the dose of DOCA or to the stage of the cycle at which the steroid was injected. This variation could be due to differences among individual hens in the ability of the positive feedback receptor sites in the hypothalamus to distinguish between progesterone and deoxycorticosterone. The pattern of deoxycorticosterone secretion throughout the ovulatory cycle has not yet been determined, and it remains to be shown whether or not this steroid has any physiological role in triggering the pre-ovulatory LH surge. Plasma concentrations of both oestrone (Peterson & Common, 1972) and oestradiol (Peterson & Common, 1972; Senior & Cunningham, 1974), as measured by radioimmuno¬ assay, rise to a peak between 7 and 4 h before ovulation. When both oestradiol and LH were measured in the same plasma samples, the rise in oestradiol level was found to precede slightly that of LH (Senior & Cunningham, 1974), suggesting that a rising plasma level of oestrogen may be involved in causing the pre-ovulatory LH surge. This is not supported by earlier studies which showed that injection of large doses of oestradiol benzoate fail to cause premature ovulation (Fraps, 1954). Nor is it supported by the present study, which shows that injection of between 0-01 and 1-0 mg/kg of either oestrone or oestradiol-17/? did not increase plasma LH concentrations. These findings contrast with those in mammals, where appropriately timed injections of oestrogen can induce an LH surge (Schwartz, 1969; Harris & Naftolin, 1970; Brown-Grant, 1971; Knobil, 1974). In mammals the ability of injections of either oestrogen or progesterone to stimulate LH secretion is influenced by the pre-existing baseline plasma concentrations of sex steroids and, in some cases, in the length of time the hypothalamus and pituitary have been exposed to these concentrations. It is therefore possible that the inability of a single injection of oestrogen to cause a release of LH in the hen may be due to an inappropriate balance of gonadal steroids in the circulation. Alternatively, though oestrogen itself may not directly stimulate LH release, there is evidence that it may be needed to 'prime' the hypothalamus to facilitate the positive feedback effect of progesterone (S. C. Wilson, unpublished observation). Peak values of plasma testosterone have been observed between 9 and 8 h before ovulation (Etches, 1974). Since plasma LH concentration rises to a peak between 7 and 4 h before ovulation (Furr et al. 1973; Wilson & Sharp, 1973), it seems that the rise in plasma testo¬ sterone level precedes that of LH by 1-2 h. This implies that testosterone could be involved in triggering the preovulatory surge of LH. Earlier studies support this concept, since Fraps (1955Ò) observed premature ovulation in 41 % of hens injected with 1 mg testosterone/hen about 14 h before the first ovulation of a sequence. The interval between injection and ovulation was always greater than 9 h. In contrast, Fraps also observed that the interval between injection of 1 mg progesterone/hen and ovulation was always less than 8 h, suggest¬ ing that progesterone and testosterone were not acting in the same way. This observation led him to suggest that testosterone may have to be converted to an 'active substance' before ovulation could be induced (Fraps, 1955e). In the present study, after injection of testosterone between 22 and 26 h after the final ovulation of a sequence, plasma LH levels rose to a peak similar to the naturally occurring pre-ovulatory LH surge (Wilson & Sharp, 1973). No comparable LH surge resulted from testosterone injection between 0 and 9 h after ovulation. It therefore appears that the ability of testosterone to stimulate LH release depends on the functional state of the ovary. The concentration of progesterone in the largest ovarian follicle increases six- to sevenfold about
19 h after the previous ovulation (Shahabi, Norton & Nalbandov, 1975) and it is possible that testosterone injected after this time causes a release of ovarian progesterone, which then exerts a positive feedback effect on LH secretion. Since no increase in LH secretion was observed following testosterone injections between 0 and 8 h after ovulation, the ovarian follicle at this stage of the ovulatory cycle may not have been able to secrete sufficient progesterone to stimulate the release of LH. The mechanism by which testosterone could stimulate progesterone secretion is unclear. It could either act directly on the follicle to enable it to release progesterone, or exert a weak positive feedback effect on the hypothalamo-hypophysial complex and cause a minor increase in plasma LH levels. Increased plasma LH levels are known to stimulate progesterone secretion in the hen (Etches & Cunningham, 1975; Shahabi, Bahr & Nalbandov, 1975) and the ability of a small increase in LH levels in the circulation to trigger an initial release of progesterone from the ovary may depend on the amount of this steroid stored in the follicle next due to ovulate. If, in the present study, testosterone caused an initial rise in plasma LH level, it may not have been observed because it was too small or was masked by the larger rise in LH level presumably caused by the positive feedback action of progesterone released from the ovary. Another possibility is that testosterone differentially stimulated FSH secre¬ tion, which then caused the release of progesterone from the maturing ovarian follicle. If testosterone does cause a release of gonadotrophins via a central nervous positive feedback mechanism it may, as Fraps (1955¿>) suggested, have to be converted first into an 'active substance'. It has been shown that testosterone can be metabolized to androstene¬ dione in both the brain (Nakamura & Tanabe, 1974) and ovary (Nakamura, Tanabe & Katukawa, 1974) of the hen. However, as injections of between 0-1 and 1-0 mg androstenedione/kg did not increase plasma LH concentrations it is unlikely that androstenedione is the 'active substance'. There is no evidence to suggest that testosterone can be converted to progesterone in the hen, though it is converted to oestradiol in the hen's ovary (Nakamura et al. 1974). Since both oestradiol-17/? and oestrone were ineffective in stimulating LH secretion in the hen, it is unlikely that the positive feedback of testosterone suggested by the results of this study are mediated via oestrogens. However, it is possible that other conversion products such as dihydrotestosterone and androstanediol (Nakamura & Tanabe, 1974) are involved. In conclusion, this study shows that testosterone, like progesterone, will induce an LH surge in the laying hen. Since the pre-ovulatory increase in testosterone secretion may precede that of progesterone, it is possible that before a normal ovulation, rising levels of plasma testosterone facilitate the release of other gonadal steroids, including oestrogens and progesterone. As it has been shown that progesterone rapidly induces a release of LH when injected at most stages of the ovulatory cycle (Wilson & Sharp, 1975), increased secretion of this steroid is thought to be the immediate cause of the pre-ovulatory surge of LH. The authors are grateful to Dr . . Follett and Dr C. G. Scanes for the chicken LH preparations and to Mr W. R. Carr, Mr D. Maxwell and Mr D. T. Wilson of the A.R.C. Animal Breeding Research Organization for adapting Dr Rodbard's computer program. S.C.W. was supported by an A.R.C. Research studentship.
REFERENCES
Brown-Grant, . (1971). The role of steroid hormones in the control of gonadotrophin secretion in adult female mammals. In Steroid hormones and brain function, pp. 269-288. Eds C. H. Sawyer & R. A. Gorski. Los Angeles: University of California Press. Courrier, R. (1940). La désoxycorticostérone est capable de maintenir la grossesse ou de provoquer Pavortement. La Presse Medicale 48, 658. Etches, R. J. (1974). Plasma testosterone during the ovulation cycle of the chicken. In Abstract in XV World's Poultry Congress, New Orleans, August 1974, pp. 517-518. Washington: U.S.A. Branch World's Poultry Science Association.
Etches, R. J. & Cunningham, F. J. (1975). The role of the preovulatory release of progesterone in the chicken. Journal of Endocrinology 64, 47F-48F. Follett, B. K., Scanes, C. G. & Cunningham, F. J. (1972). A radioimmunoassay for avian luteinizing hormone. Journal of Endocrinology 52, 359-378. Fraps, R. M. (1954). Neural basis of diurnal periodicity in release of ovulation-inducing hormone in fowl. Proceedings of the National Academy of Sciences of the U.S.A. 40, 348-356. Fraps, R. . (1955 a). Egg production and fertility in poultry. In Progress in the physiology offarm animals, vol. 2, pp. 661-740. Ed. J. Hammond. London: Butterworths. Fraps, R. M. (19556). The varying effects of sex hormones in birds. Memoirs. Society for Endocrinology 4,
205-219. M. &Dury, A. (1943). Occurrence of premature ovulation in the domestic fowl following adminis¬ tration of progesterone. Proceedings of the Society for Experimental Biology and Medicine 52, 346-349. Furr, B. J. ., Bonney, R. C, England, R. J. & Cunningham, F. J. (1973). Luteinizing hormone and progesterone in peripheral blood during the ovulatory cycle of the hen Callus domesticus. Journal of Endocrinology 51, 159-169. Gros, G, Benoit, J., Kehl, R. & Paris, R. (1942). Action de l'acétate de désoxycorticostérone sur le maintien de la grossesse chez la Chatte gestante castrée. Comptes rendus des Séances de la Société de Biologie 136, 749-750. Harris, G. W. & Naftolin, F. (1970). The hypothalamus and control of ovulation. British Medical Bulletin 26, 3-9. Haynes, . ., Cooper, . J. & Kay, M. J. (1973). Plasma progesterone concentrations in the hen in relation to the ovulatory cycle. British Poultry Science 14, 349-357. Hooker, C. W. & Forbes, T. R. (1949). Specificity of the intrauterine test for progesterone. Endocrinology 45, 71-74. Kappauf, B. & van Tienhoven, A. (1972). Progesterone concentrations in peripheral plasma of laying hens in relation to the time of ovulation. Endocrinology 90, 1350-1355. Knobil, E. (1974). On the control of gonadotrophin secretion in the rhesus monkey. Recent Progress in Hormone Research 30, 1-46. Lazo-Wasem, E. A. & Zarrow, M. X. (1955). The conversion of désoxycorticostérone acetate to a pro¬ gesterone-like substance. Endocrinology 56, 511-515. Nakamura, T. & Tanabe, Y. (1974). In vitro metabolism of steroid hormones by chicken brain. Acta Endocrinologica 75, 410-416. Nakamura, T., Tanabe, Y. & Katukawa, H. (1974). Steroidogenesis in vitro by the ovarian tissue of the domestic fowl (Gallus domesticus). Journal of Endocrinology 63, 507-516. Neher, B. H. & Fraps, R. M. (1950). The addition of eggs to the hen's clutch by repeated injections of ovulation-inducing hormone. Endocrinology 46, 482^188. Peterson, A. J. & Common, R. H. (1971). Progesterone concentration in peripheral plasma of laying hens as determined by competitive protein-binding assay. Canadian Journal of Zoology 49, 599-604. Peterson, A. J. & Common, R. H. (1972). Estrone and estradiol concentrations in peripheral plasma of laying hens as determined by radioimmunoassay. Canadian Journal ofZoology 50, 395^104. Peterson, A. J., Henneberry, G. O. & Common, R. H. (1973). Androgen concentrations in the peripheral plasma of laying hens. Canadian Journal of Zoology 51, 753-758. Rodbard, D. & Lewald, J. E. (1970). Computer analysis of radioligand assay and radioimmunoassay data. In Karolinska Symposia on Research Methods in Reproductive Endocrinology, pp. 79-103. Ed. E. Diczfalusy. Stockholm: Karolinska Institutet. Scanes, C. G. & Follett, B. K. (1972). Fractionation and assay of chicken pituitary hormones. British Poultry Science 13, 603-610. Schwartz, N. B. (1969). A model for the regulation of ovulation in the rat. Recent Progress in Hormone Research 25, 1-53. Senior, B. E. & Cunningham, F. J. (1974). Oestradiol and luteinizing hormone during the ovulatory cycle of the hen. Journal of Endocrinology 60, 201-202. Shahabi, . ., Bahr, J. M. & Nalbandov, A. V. (1975). Effect of LH injection on plasma and follicular steroids in the chicken. Endocrinology 96, 969-972.
Fraps, R.
Shahabi, . ., Norton, . W. & Nalbandov, . V. (1975). Steroid levels in follicles and the plasma of hens during the ovulatory cycle. Endocrinology 96, 962-968. Warren, D. C. & Scott, . M. (1935). The time factor in egg formation. Poultry Science 14, 195-207. Wilson, S. C. & Sharp, P. J. (1973). Variations in plasma LH levels during the ovulatory cycle of the hen, Gallus domesticus. Journal of Reproduction and Fertility 35, 561-564. Wilson, S. C. & Sharp, P. J. (1975). Changes in plasma concentrations of luteinizing hormone after the injection of progesterone at various times during the ovulatory cycle of the domestic hen (Gallus domesticus). Journal of Endocrinology 67, 59-70. Zarrow, M. X., Hisaw, F. L. & Bryans, F. (1950). Conversion of désoxycorticostérone acetate to pro¬ gesterone in vivo. Endocrinology 46, 403-404.
