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Pituitary gonadotropins in the Japanese quail (Coturnix coturnix japonica) during photoperiodically induced gonadal growth
GENERAL
AND
COMPARATIVE
Pituitary
ENDOCRINOLOGY
7,
125-131
Gonadotropins cofurnix
in the
japonica) Induced
BRIAN
of
Gonadal AND
Quail
(Cofurnix
Photoperiodically Growth1 DONALD
Zoophysiology, State University,
Received The
Japanese
during
K. FOLLETT2
Laboratories Washington
(1966)
Department Pullman,
September
S. FARNER” of Zoology, Washington
1, 1965
content of the anterior pituitary in Japanese cpiail (Coturniz was studied in relation to photoperiodically induced gonadal development. The content was low in birds held on short daily photoperiods and in which there was no gonadal development. Long daily photoperiods caused complete gonadal development and a steady increase in gonadotropin content during the first 21 days with a slight decline thereafter. The anterior pituitary gland increased in weight under a long daily photoperiod. The results are discussed with respect to the balance between hormone synthesis and hormone release. cotumiz
gonadotropin japonica)
Although the occurrence of both FSHand LH-like activities in the adenohypophysis of birds has been known for more than three decades (for review see van Tienhoven, 1961), our information about them is still meager. In particular, the relation of gonadotropin content of the pituitary to photoperiodically induced gonadal growth has not been studied in a quantitative manner. Only Greeley and Meyer (1953)) in their investigations on Phasianus colchicus, have studied the annual cycle of gonadotropin activity in the anterior pituitary in relation to the annual testicular cycle. The results of their investigations indicate an increase in gonadotropin content, during vernal testicular growth, a relationship suggested also for the domestic mallard (Anas p&y~hynch.os) by t.he data of Benoit and his colleagues (Benoit, 1935; Benoit et al., 1950). ‘The investigations described ported by a grant (NB 01353) Institutes of Health. ‘Present address: Department University, Leeds, England. ” Present address: Department versity of Washington, Seattle.
The bioassay of gonadotropins of the aoian pituitary has been difficult. because of a paucity of knowledge of their number and nature, and also because of a lack of suitable assay techniques. Chemical separat,ion of the FSH- and LH-like act,ivit,iea has been achieved in extracts in chicken pituitaries (Fraps et a.Z., 1947; Fraps and Fevold, 1955), but further purification has not been reported. Indeed, Nalbandov (1959) and. van Tienhoven (1959) have postulated that the avian pars distalis produces a single gonadotropin complex wit’h both FSH- and LH-like activit,ies. Recently, however, Tixier-Vidal (TixierVidal et ,nZ., 1962 ; Tixier-Vidal, 1963) has presented evidence for two types of gonadotropic cells in the pars distalis of the domestic mallard and other species, suggesting t,hat t.wo types of gonadotropin are produced. Considerable physiological differences also exist between mammalian and avian gonadotropins; thus NIH-LN is more effective than NIB-FSH in promoting an FSH-like response in birds, namely testicular growth in chicks (Breneman et a.l.$ 1962) and Japanese quail (McFarland et al., 1964). Without definitive information
herein were supfrom the National of Zoology, of Zoology,
The Uni125
126
FOLLETT
AND
on the nature of avian gonadotropins it is difficult to assess the many techniques used for their assay. However, the results of Herrick ,et al. (1962) suggest that an avian end-point assay would provide the most sensitive technique. The most frequently used methods have been based on the increase in testicular weight of young chicks, which has the disadvantage of relative insensitivity and a poor index of precision (See, for example, Breneman et al., 1962). More recently, Breneman et al. (1962) have introduced an assay based on the discovery of Florsheim et al.. (1959) that gonadotropins increase the uptake of radio-phosphorus into the testes of day-old chicks. Because of the superiority of this method over previous techniques, we have adopted it, with some modifications, for our investigations. The Japanese quail (C~&.crnix co turnix japonioa) has a number of characteristics, such as a precise photoperiodic control of gonadal growth, that make it ideal for the study of adenohypophysial function. This communication is concerned with the relationship between photoperiodieally induced gonadal growth and the gonadotropin content of t.he adenohypophysis in this species. METHODS The anterior pituitaries used in this investigation were from quail used for the study of neurohypophysial hormones in a separate investigation (for details, see Follett and Famer, 19’66). In two separate experiments, newly hatched quail were subjected to either a short (6L/lSD) or a long (2OL/4D) daily photoperiod until 1 month of age (for details, see Follett and Farner, 1966). Birds were taken at intervals throughout this period; all were killed 1 hour after the lights came on (i.e., 1000 hours). The adenohypophysis was removed, blotted on wet filter paper, weighed to the nearest 0.001 mg on a Cahn electrobalance, and then placed in cold A.R. acetone which was changed twice over the next 4 days. Thereafter the glands were dried in air and stored i7z ~ncuo over phosphorus pentoxide at 0°C. All material was assayed within 60 days. Gonadotropin Assay Method. As noted above, the method of Breneman et al. (1962), with some modification, was used. This method, in our experience, compares favorably with the available techniques for assay of pituitary gonadotropin.
FARNER
Breneman et al. (1962) have reported the assay preparation to be approximately 5 times more sensitive to’ the LH from National Institutes of Health (an ovine preparation) than to NIH-FSH. We have confirmed this and have found also that synergism between mammalian LH and FSH is negligible. In routine assays we have employed NIH-LH (sample NIH-LH-S7) as the standard preparation; the results of the assays are normally expressed as pg equivalents of NIH-LH per gland. Male cros*sbred chicks (New Hampshire X Delaware) were obtained from the Department of Animal Science, Washington State University. The chicks were selected so that all had been hatched within a short period, They were held, without food and water, in a wooden box under constant illumination and high humidity at 35°C. Under these conditions four chicks died in a total of more than 5,000. Initial experiments showed that a more accurate assay was obtained if the chicks were used at 24-30 hours after hatching. In all assays the injections were made at the same time of day. The standard preparation of NIH-LH was used over a period of 18 months, since Breneman et al. (1962) had shown that the hormone does not lose potency within at least a year if stored in zlacz~o. Glands were homogenized thoroughly in saline solution and diluted to the desired concentration. Control groups of chicks were included with each assay and injected with saline solution. The phosphorus preparation was carrier-free 31P (H”PO,-) from the Atomic Energy Commission, Oak Ridge, Tennessee. The dose of isotope employed throughout was 1.0 or 1.2 ye per chick, which was found to give a better index of precision than doses of 4-6 pc (Florsheim et al., 1959; Breneman et al., 1962). The chicks were divided randomly into batches of 8-10. Gonadotropins were administered subcutaneously (nape of the neck) via a 26-gauge needle. In all cases the volume administered was 0.2 ml. Six hours thereafter the animals received isotope, again subcutaneously, in a volume of 0.2 ml. Twenty four hours after the injection of the hormone the chicks were killed. Since the time involved in preparing 860 chicks required l-2 hours, the chicks were killed in rotation, one from each treatment. At autopsy the testes were removed, blotted, and weighed to the nearest 0.1 mg on a torsion balance. They were placed on aluminum planchets and dried for 30 minutes under a heat lamp. Under these circumstances selfabsorption was negligible. Counts were taken to a precision of 3% by using a Nuclear-Chicago D47 gas-flow detector. After correction for back-
PITUITARY
GOKADOTROPINS
Ih-
THE
JAPANESE
12’7
QUAIL
that for NH-LB and cor,sequently the value of h for the slope was much greatel (0.307). The slope of the dose-response curves for both male and female quail cxtracts was very close to that, of NH-LH; in only 3 of 46 assays were the siopes significantly different’. The TVeight of the dde,nohypophysis (Table I). Photoperiodically induced gonadal growth was invariably accompanied by an increase in weight of the adenohyRESULTS pophysis. In Expt. 2 when the photoperiod Characterization of the Assay Technique. was changed from 20L/4D to 16L/18D on The linear relationship between log dose and day 40, testicular regression was r’apid. Alresponse (cpm/mg) was confirmed in a though the adenohypophysial weight. also preliminary series of experiments for NIHdecreased at, this time, the individual LIZ, NIH-FSH, and an extract of quail weights are not statistically different from pituitary glands. Since, in general, t,he dose- the weight at. clay 40. Gon,ad,otrop’n Content of the Ademresponse curve was linear between 1.0 and 4.0 ,p.g NIH-LH, standards of 1.5 and 3.0 hypopkysis. In Expt. 1 pituitary gonadol&g LH were employed. Breneman et al. tropins were measured when young quail (1.962) obtained indices of precision (A, had been exposed to either 61,/18D or Bliss, 1952) of 0.19-0.21 with 40-52 chicks 20L/4D for 28 days beginning with the per dose. In our assay, in which 7-10 chicks clay of hatching. At, this time the mean per dose were used, the mean index of pre- testicular weight for those exposed to 6Lj cision for all assayswas 0.154 + 0.044 (57). 18D was 9.04 i- 1.55 mg (131, and the This compares favorably with the published mean ovarian weight was 17.4 t 5.6 mg values for other assay techniques (Parlow, (46), whereas for t,hose held on 20L/4D 1964). The slope of the dose-responsecurve the gonadal weights were 876.4 i 460.3 for NIH-FSH was significantly lower than (22) and 84.9 * 58.4 mg (40)) respectively,
ground, the results were expressed as counts per minute per milligram of wet tissue. Statisticcll. AvKII~SLS of Asmy. Estimates were made with a four-point (2 + 2) balanced factorial method. Rarely, when gonadotropic potency was low, the assay design was modified to the (2 + 1) form. The results of each experiment were subjected to a full analysis of variance (Bliss, 1952) ; botency estimates are given together with their 95% confidence limits and the index of precision (X).
TABLE THE
WEIGHT
OF THE
~~DENOHYPOPHYSIS GROWTH
Sex and photoyeriodic
0” c? 0 9 0”
6L/18Db 20L/4D” 6L/lSD” 20L/4D” 20L/4D Day 0 Day 17 Day 40
Day 48 Day 70 Day 40 6L/18D controls
regime
IX
IN Fresh
NEWLY
1
RELATIOS
TO
HATCHED
m-t. of adenohypophysis
0.845 1.113 0.918 1.100
+ * + +
0.027 0.031 0.016 0.031
0.731 * 0.049 1.129 + 0.042 1.360 k 0.130 Photoperiod changed from 1.179 5 0.071 1.114 f 0.059 0.555 f 0.056
PHOTOPERIOUICALLY
JA~AXESE
INUUCEE
Gox\;an~~
QCAIL
(rnp)~
Gonadal
wt. (mg)”
(35) (35)c (37) (38)c
9.0 876.4 17.4 s4.9
* f f f
0.4 (13) 34.2 (Z$’ 0.8 (46) 9.2 (IlO,,:
(7) (7)d (4)d 20L/4D (4)d (lO)d (4)
8.1 1,683 3,000
f 1.2 (II) + 144 (14)d It 42 (12)d
2,420 36.3 13.2
2 61 (10)” + 2.9 (16)” f 1 .2 (40)
t’o 6L/18D
o Mean + standard error. Numbers of birds are shown in parentheses. b Birds exposed to photoperiodic regime for 28 days subsequent to hatching. C Significantly greater (p < 0.001) than 6L/18D group. d Significantly greater (p < 0.001) than day 0 sample.
--__
128
THE
FOLLETT
EFFECT
OF LONG
DAILY
FARNER
TABLE (20L/4D)
PHOTOPERIODS NEWLY
AND
HATCHED
2 ON
JAPANESE Expt.
PITUITARY
GONADOTROPIN
Group
6L/lSD
1 2 3
20L/4D
1 2 3 4 5
Female
MfX%Il potencya
95% limits~
Lambdad
<0.5 -0.5 -1.0
-
-
5.5 5.3 5.8 4.7 5.5
6.8 6.6 7.7
6.3 7.4
8.2 8.8 11.8 7.9 9.5
-
-
IN
1
Male Photoperiodic regime
POTENCY
QUAIL
MIZUI potencyb
95% limits
Lamb&d
-0.3 -0.5
0.130 0.123 0.165 0.145 0.167
3.8 2.7 3.5 3.7 3.6
2.2 2.1 2.7 3.0 2.7
0.086 0.165 0.145 0.140 0.161
4.4 3.7 4.4 - 4.7 - 4.6 -
a Begun on day of hatching; all birds were killed 28 days after hatching. b Potency expressed as pg equivalents NIH-LH per gland. c All assays for groups from 20L/4D were (2 + 2) with ten chicks per dose level. d Index of precision.
The birds from each treatment were divided into batches of 5-10; single assays were performed in each batch. The pituitaries of the quail exposed to 6L/lSD (Group 1s) contained so little gonadotropin that an accurate assay was impossible, whereas THE
EFFECT
OF LONG
DAILY
TABLE 3 (20L/4D)
PHOTOPERIODS NEWLY
those held on 2OL/4D (Group 1L) had considerable potency (Table 2). The variation among batches in Group 1L was sufficiently small so that Expt. 2 (Table 3) only a single assay was made for each killing time. The time course of changes in
HATCHED
ON PITUITARY
JAPANESE Expt.
GONADOTROPIN
1 is
146 21s 255 and saline 26s 71, 14L 21L 25L and saline 26L 36L
MeWI potencyb
<0.3 -0.6 1.0 0 .9 1.0 1.4 12 2.8 9.9
7.3 4.9 9.3
95%
Femnln Lambdac
limits
0 8 -
1.2
0.8 0.7
-
1.1
1.4
0.9
-
1.5
2.1
-
4.8
i.2 - 13.8 5 7 3.1 6.9
-
9.6
6.5 13 2
IN
2
Male Age and photoperiod*
POTENCY
QUAIL
0.091 0.126 0.126
-
0.097 0.107 0.174c 0.109e 0.151e 0.144e
Testicular wt.
1.2 2.4 7.7 8.3 8.9 4.4 23.8 149.6
306.1 464.7 1494
MeC&Il potency~
0.3 0.6 0.8 0.9 0.9
1.2 1.0 1.5 “6 ;:5 3 1 -
a Age given in days; photoperiod S is 6L/18D, L is 2OL/4D. Photoperiodic hatching. b Mean potency expressed as pg equivalents NIH-LH per gland. c Index of precision. d Mean in milligrams. e Assays of (2 + 2) form; all others modified to (2 + 1).
95% limits
0.4 0.6 0.7 0.7 0.7 1.2 2.7 2.3 2.4
Lambda0
-
0.9 1.0
0.091 0.098
-
1.0 1.1
0.114 0.126
-
1.3 1.7
0.097 0. 06gd
4.9
0.126 0. 207d 0.114d
-
5.0
3.8
Ovarian wt.d
1.7 6.4 11.7 16.1 25.3 6.2 15.4 42.7 37.0 51.0
-
regime
was begun
on day
of
PITUITARY
GONADOTROPIN?
IN
Days
Group
2L
1.2
Testicular 4.4
Grwp
2s
-
2.4
I
THE
Cmg 1 149.6
7.7
8.3
, IO
Days Ovarian
129
QUAIL
Of Experiment
Weight 23.8
I 0
JAPANESE
Of
Weight
464.7
1494.0
8.9
10.0
I
/
213
30
Experiment (mg)
51.0 6.2 15.4 42.7 Group 2s 6.4 Il.7 16.1 25.3 PIG. 1. Change in pituitary gonadotropins as a function of time in young Japanese quail exposd fkom hatching to a daily photoperiod of 20L/4D (0 -------.I or 6L/lSD (O------0). Gonadotropic potency is expressed as microgram equivalents NIR-LH per gland; the vertical bars represent the 95% confidence limits for the assay. The mean gonadnl weight of each sample is shown below the graph. Group
2L
1.7
130
FOJLET
AKD
pituitary gonadotropin potency for Expt. 2 is shown in Table 3 and Fig. 1. The pituitaries of. both male and female quail on 6L/lSD (Group 2s) contained small amounts of gonadotropin by day 14; the content did not change thereafter through day 26. A steady increase in pituitary gonadotropic potency was observed through day 21 in both male and female quail exposed to 20L/4D (Group 2L). There may have been a slight decrease in potency on day 25 and 26. Saline treatment, on days 20-25 appears not to have affected the gonadotropic potency of the anterior pituitary; this is consistent with our observation that this treatment had no effect on gonadal growth (Follett and Farner, 1965). The changes in weight of the anterior pituitary in this experiment were small although significant. The differences among groups were not altered by expression of gonadotropin in terms of weight of the anterior pituitary gland. In all cases (Table 2 and 3; Fig. 1) the pituitaries from males contained more gonadotropin than similarly treated females. This is consistent with the observations of Bacon et al. (1964) on the Japanese quail. The pars distalis of the Japanese quail is deeply furrowed and, at least visually, can be divided into cephalic and caudal lobes. Some preliminary experiments were undertaken to determine gonadotropic potency of the separate lobes. Activity was found in both cephalic and caudal portions ; the former is somewhat larger than the latter; e.g., in one experiment mean cephalic lobe weight was 0.744 * 0.033 mg (20)) whereas the weight of the caudal lobe was 0.399 + 0.022 mg (20). The gonadotropic activities for two groups of males were as follows: (1) 20L/4D, cephalic lobe, 5.7 (3.78.7) pg/lobe or 6.2 (4&9,.4)/mg wet wt.; caudal lobe, 3.0 (2.0-4.6) ,pg/lobe or 6.8 (4.6-10.4)/mg wet wt. (2) 20L/4D, cephalic lobe, 5.5 (4.2-7.8) ,pg/lobe or 7.4 (5.7-10.4)/mg wet wt.; caudal lobe, 3.8 (2.7-5.4) pg/lobe or 9.4 (6.6613.4)/mg wet, wt. These results suggest that gonadotropins may occur in about equal concentrations in both lobes; further analysis must await the
FARNER
outcome of current studies combining assays and cytological techniques.
bio-
DISCUSSION
The interpretation of data from the assay of pituitary gonadotropins is difficult, since they represent steady-state levels without yielding direct information on rates of synthesis or release of the hormone. It is possible, however, that the rate of photoperiodically induced gonadal growth in some species of birds may afford an approximation of the rate of hormone release; this is especially so because of the logarithmic relationship between gonadal weight and time (See review, Farner and Follett, 1966). Moreover, in all assay techniques based on increases in gonadal weight in birds there is a logarithmic relation between dose of hormone and the increase in weight. Theoretically, the rate of hormone release in photoperiodically induced growth may represent a constant output during the logarithmic phase of growth. In the present experiment only two photoperiodic treatments were used; no accumulation of gonadotropins occurred under the short daily photoperiod (6L/18D), suggesting that not only release was blocked but also that synthesis must have occurred at a minimal rate. In the quail subjected to 2OL/4D the rate of synthesis apparently exceeded slightly the rate of release during the first 21 days, whereas the reverse was true thereafter. Emphasis has been placed on the separation of the mechanisms of gonadotropin release and synthesis, since there is evidence (Farner and Follett, 1966) in the white-crowned sparrow (Zon#otrichia lseuc,ophrysga&eZii) that the two functions may be controlled partially independently of each other. The data presented in this communication offer a quantitative analysis related to a standard preparation, and thus confidence limits may be calculated for each potency determination and experiments may be compared directly with each other. The results are in general agreement with the semi-quantitative observations of pituitary gonadotropin content during gonadal
PITUITARY
GONADOTROPINS
IN
THE
JAPANESE
QUAIL
731
growth in other birds, including the domestic fowl (Breneman, 1945)) the ringnecked pheasant (Greeley and Meyer, 1953) and, very recently, on the Japanese quail (Tanaka et al., 1965).
neurosecretion in the hypothalamo-hypophysial system of the Japanese quail (Coturniz cotuwk japozica). Gen. Comp. Endocrinol. 6, 111-324. FRAPS, R. M., AKD FEVOLD, H. L. (1955). Delaying action of gonadotropins on ovulation in the hen.
ACKNOWLEDGMENTS We are indebted to Professor James McGinnis and Dr. Jean K. Lauber, Department of Animal Science, Washington State University, who hatched and reared the quail used in these experiments. Standard preparations of gonadotropin were kindly supplied by the Endocrinology Study Section, National Institutes of Health.
FRAPS,
REFERENCES B.~coN, W., CHERMS, F. L., AND MCSKQN, W. H. (1964). Gonadotropin assay of pituitaries from sexually mature male and laying female Cotur?aix quail. Endocrinology 74, 498-500. BENOIT, J. (1935). R81e de l’hypophyse dans l’action stimulante de la lumikre sur le dhveloppement testiculaire chez le Canard. Compt. Rend. Sot.
Biol.
11’8, 672-674.
J.: ASSEXMACBER, I., ASD WALTER, F. X. (1950). Activitk gonadotrope de l’hypophyse du canard domestique, au coure de la rkgreesion testiculaire saisonniere et de la p&pub&&. Compt. Rend. Xoc. Biol. 144, 1403-1407. BLISS, C. I. (1952). “The Statistics of Bioassay with Special Reference to the Vitamins.” Academic Press, New York. BRENEMAN, W. R. (1945). The gonadotropic activity of the anterior pituitary of cockerels. BENOIT,
Endoc%ology
36,
190-199.
W. R., ZELLER, F. J., AND CREEK, R. 0. (1962). Radioactive phosphorus uptake by chick testes as an end-point for gonadotropin assay.
BRENEMAN,
Endocrinology
71,
790-798.
D. S., AND FOLLETT, B. K. (1966). Light and other environmental factors affecting avian reproduction. VII. Biennial Symposium on Animal Reproduction, 1965. J. Animal Sci., (in press). FLOKSHEIM, W. H., VELCOFF, S. M., AKD BODFISH, R. E. (1959). Gonadotrophin assay based on augmentation of radiophosphate uptake by the chick testes. Actn Endockol. 30, 175-182. FOLLEY~, 13. K., AND FARNER, D. S. (1965). The effects of the daily photoperiod of gonadal growth, neurohypophysial hormone content and FARNER,
PTOC Sot.
Exptl.
Biol.
Med.
‘90, 440-446.
R. M., FEVOLD, H. L., ASD N~-ti~a, B. H. (1947) Ovulatory response of the hen to prcsumptive luteinizing and other fractions from fowl anterior pituitary tissue. Anat. Kecor11 ‘99:
571-572. GREELET,
.AND METER, R. Ii. (1953). Seasonal in testis-stimulating activity of male pituitary glands. AU& 70, 350-358. HERRICK, R. B., MCGIBBOS, W. H., AND MCSHAA. W. H. (1962). Gondotropic activit.y of chicken pituitary glands. Endocrinology 71, 487-491. MCFARLAKD, L. Z., MATHER, F. B., ASD W~LSOE: W. 0. (1964). Testicular response to gonadol.rophins and intermittent light in Coturnix reared under limited light. Am. J. Vet. Res. 25, 1531F.,
variation pheasant
1534.
A. V. (1959). Nemoendocrine reflex mechanisms: bird ovulation. I?L “Comparative Endocrinology” (A. Gorbman, ed.), pp. 161-173, Wiley, New York. PARLOW, A. I?. (1964). Differential action of small doses of e&radio1 on gonadotrophins in the rat. NaLBhxDOV,
Endocrinology
175, 1-8.
F. B., Wrmos, W. O., ANI: L. Z. (1965). Effect of photoperiods on early growth of gonads and on potency of gonadotropins of the anterior pituitary gland in
TANAIU,
K.,
MATHER,
MCFARLAKD,
Coturnix.
Poultry
Sci.
44,
662-665.
VAN TIENHOVEK, A. (1959). Reproduction in th<, domestic fowl: Physiology of the female. 11~ “Reproduction in Domestic Animals” (H. II. Cole, P. T. Cupps, eds.), Vol. II, pp. 305-342. Academic Press, New York. VAN TIENHCIVEK, A. (1961). Endocrinology of reproduction in birds. 117 “Sex and Int#ernal SoereLions” (W. C. Young, ed.)? 3rd Ed., pp. 198S1169. Williams and Wilkins, Baltimore. TIXIER-VIDAL, A. (1963). Histophgsio;ogie tic l’adknohypophyee des oiseaux. 1,~ “Cytologic de I’AdCnohypophyse” (J. Benoit and C. DaLage, eds.); Colloq. Intern. Cetltw Nntl. Rech. Sci. (Paris) 12’8, 255-273. TISIER-VID.~L, A., HERL.IXT: M., AYSD BENOIT, 3. (1962). La prkhypophyse du Canard P&kin mlilc au tours du cycle annuel. Arch. BioE. (Liege) 73, 318-368.
AND
COMPARATIVE
Pituitary
ENDOCRINOLOGY
7,
125-131
Gonadotropins cofurnix
in the
japonica) Induced
BRIAN
of
Gonadal AND
Quail
(Cofurnix
Photoperiodically Growth1 DONALD
Zoophysiology, State University,
Received The
Japanese
during
K. FOLLETT2
Laboratories Washington
(1966)
Department Pullman,
September
S. FARNER” of Zoology, Washington
1, 1965
content of the anterior pituitary in Japanese cpiail (Coturniz was studied in relation to photoperiodically induced gonadal development. The content was low in birds held on short daily photoperiods and in which there was no gonadal development. Long daily photoperiods caused complete gonadal development and a steady increase in gonadotropin content during the first 21 days with a slight decline thereafter. The anterior pituitary gland increased in weight under a long daily photoperiod. The results are discussed with respect to the balance between hormone synthesis and hormone release. cotumiz
gonadotropin japonica)
Although the occurrence of both FSHand LH-like activities in the adenohypophysis of birds has been known for more than three decades (for review see van Tienhoven, 1961), our information about them is still meager. In particular, the relation of gonadotropin content of the pituitary to photoperiodically induced gonadal growth has not been studied in a quantitative manner. Only Greeley and Meyer (1953)) in their investigations on Phasianus colchicus, have studied the annual cycle of gonadotropin activity in the anterior pituitary in relation to the annual testicular cycle. The results of their investigations indicate an increase in gonadotropin content, during vernal testicular growth, a relationship suggested also for the domestic mallard (Anas p&y~hynch.os) by t.he data of Benoit and his colleagues (Benoit, 1935; Benoit et al., 1950). ‘The investigations described ported by a grant (NB 01353) Institutes of Health. ‘Present address: Department University, Leeds, England. ” Present address: Department versity of Washington, Seattle.
The bioassay of gonadotropins of the aoian pituitary has been difficult. because of a paucity of knowledge of their number and nature, and also because of a lack of suitable assay techniques. Chemical separat,ion of the FSH- and LH-like act,ivit,iea has been achieved in extracts in chicken pituitaries (Fraps et a.Z., 1947; Fraps and Fevold, 1955), but further purification has not been reported. Indeed, Nalbandov (1959) and. van Tienhoven (1959) have postulated that the avian pars distalis produces a single gonadotropin complex wit’h both FSH- and LH-like activit,ies. Recently, however, Tixier-Vidal (TixierVidal et ,nZ., 1962 ; Tixier-Vidal, 1963) has presented evidence for two types of gonadotropic cells in the pars distalis of the domestic mallard and other species, suggesting t,hat t.wo types of gonadotropin are produced. Considerable physiological differences also exist between mammalian and avian gonadotropins; thus NIH-LN is more effective than NIB-FSH in promoting an FSH-like response in birds, namely testicular growth in chicks (Breneman et a.l.$ 1962) and Japanese quail (McFarland et al., 1964). Without definitive information
herein were supfrom the National of Zoology, of Zoology,
The Uni125
126
FOLLETT
AND
on the nature of avian gonadotropins it is difficult to assess the many techniques used for their assay. However, the results of Herrick ,et al. (1962) suggest that an avian end-point assay would provide the most sensitive technique. The most frequently used methods have been based on the increase in testicular weight of young chicks, which has the disadvantage of relative insensitivity and a poor index of precision (See, for example, Breneman et al., 1962). More recently, Breneman et al. (1962) have introduced an assay based on the discovery of Florsheim et al.. (1959) that gonadotropins increase the uptake of radio-phosphorus into the testes of day-old chicks. Because of the superiority of this method over previous techniques, we have adopted it, with some modifications, for our investigations. The Japanese quail (C~&.crnix co turnix japonioa) has a number of characteristics, such as a precise photoperiodic control of gonadal growth, that make it ideal for the study of adenohypophysial function. This communication is concerned with the relationship between photoperiodieally induced gonadal growth and the gonadotropin content of t.he adenohypophysis in this species. METHODS The anterior pituitaries used in this investigation were from quail used for the study of neurohypophysial hormones in a separate investigation (for details, see Follett and Famer, 19’66). In two separate experiments, newly hatched quail were subjected to either a short (6L/lSD) or a long (2OL/4D) daily photoperiod until 1 month of age (for details, see Follett and Farner, 1966). Birds were taken at intervals throughout this period; all were killed 1 hour after the lights came on (i.e., 1000 hours). The adenohypophysis was removed, blotted on wet filter paper, weighed to the nearest 0.001 mg on a Cahn electrobalance, and then placed in cold A.R. acetone which was changed twice over the next 4 days. Thereafter the glands were dried in air and stored i7z ~ncuo over phosphorus pentoxide at 0°C. All material was assayed within 60 days. Gonadotropin Assay Method. As noted above, the method of Breneman et al. (1962), with some modification, was used. This method, in our experience, compares favorably with the available techniques for assay of pituitary gonadotropin.
FARNER
Breneman et al. (1962) have reported the assay preparation to be approximately 5 times more sensitive to’ the LH from National Institutes of Health (an ovine preparation) than to NIH-FSH. We have confirmed this and have found also that synergism between mammalian LH and FSH is negligible. In routine assays we have employed NIH-LH (sample NIH-LH-S7) as the standard preparation; the results of the assays are normally expressed as pg equivalents of NIH-LH per gland. Male cros*sbred chicks (New Hampshire X Delaware) were obtained from the Department of Animal Science, Washington State University. The chicks were selected so that all had been hatched within a short period, They were held, without food and water, in a wooden box under constant illumination and high humidity at 35°C. Under these conditions four chicks died in a total of more than 5,000. Initial experiments showed that a more accurate assay was obtained if the chicks were used at 24-30 hours after hatching. In all assays the injections were made at the same time of day. The standard preparation of NIH-LH was used over a period of 18 months, since Breneman et al. (1962) had shown that the hormone does not lose potency within at least a year if stored in zlacz~o. Glands were homogenized thoroughly in saline solution and diluted to the desired concentration. Control groups of chicks were included with each assay and injected with saline solution. The phosphorus preparation was carrier-free 31P (H”PO,-) from the Atomic Energy Commission, Oak Ridge, Tennessee. The dose of isotope employed throughout was 1.0 or 1.2 ye per chick, which was found to give a better index of precision than doses of 4-6 pc (Florsheim et al., 1959; Breneman et al., 1962). The chicks were divided randomly into batches of 8-10. Gonadotropins were administered subcutaneously (nape of the neck) via a 26-gauge needle. In all cases the volume administered was 0.2 ml. Six hours thereafter the animals received isotope, again subcutaneously, in a volume of 0.2 ml. Twenty four hours after the injection of the hormone the chicks were killed. Since the time involved in preparing 860 chicks required l-2 hours, the chicks were killed in rotation, one from each treatment. At autopsy the testes were removed, blotted, and weighed to the nearest 0.1 mg on a torsion balance. They were placed on aluminum planchets and dried for 30 minutes under a heat lamp. Under these circumstances selfabsorption was negligible. Counts were taken to a precision of 3% by using a Nuclear-Chicago D47 gas-flow detector. After correction for back-
PITUITARY
GOKADOTROPINS
Ih-
THE
JAPANESE
12’7
QUAIL
that for NH-LB and cor,sequently the value of h for the slope was much greatel (0.307). The slope of the dose-response curves for both male and female quail cxtracts was very close to that, of NH-LH; in only 3 of 46 assays were the siopes significantly different’. The TVeight of the dde,nohypophysis (Table I). Photoperiodically induced gonadal growth was invariably accompanied by an increase in weight of the adenohyRESULTS pophysis. In Expt. 2 when the photoperiod Characterization of the Assay Technique. was changed from 20L/4D to 16L/18D on The linear relationship between log dose and day 40, testicular regression was r’apid. Alresponse (cpm/mg) was confirmed in a though the adenohypophysial weight. also preliminary series of experiments for NIHdecreased at, this time, the individual LIZ, NIH-FSH, and an extract of quail weights are not statistically different from pituitary glands. Since, in general, t,he dose- the weight at. clay 40. Gon,ad,otrop’n Content of the Ademresponse curve was linear between 1.0 and 4.0 ,p.g NIH-LH, standards of 1.5 and 3.0 hypopkysis. In Expt. 1 pituitary gonadol&g LH were employed. Breneman et al. tropins were measured when young quail (1.962) obtained indices of precision (A, had been exposed to either 61,/18D or Bliss, 1952) of 0.19-0.21 with 40-52 chicks 20L/4D for 28 days beginning with the per dose. In our assay, in which 7-10 chicks clay of hatching. At, this time the mean per dose were used, the mean index of pre- testicular weight for those exposed to 6Lj cision for all assayswas 0.154 + 0.044 (57). 18D was 9.04 i- 1.55 mg (131, and the This compares favorably with the published mean ovarian weight was 17.4 t 5.6 mg values for other assay techniques (Parlow, (46), whereas for t,hose held on 20L/4D 1964). The slope of the dose-responsecurve the gonadal weights were 876.4 i 460.3 for NIH-FSH was significantly lower than (22) and 84.9 * 58.4 mg (40)) respectively,
ground, the results were expressed as counts per minute per milligram of wet tissue. Statisticcll. AvKII~SLS of Asmy. Estimates were made with a four-point (2 + 2) balanced factorial method. Rarely, when gonadotropic potency was low, the assay design was modified to the (2 + 1) form. The results of each experiment were subjected to a full analysis of variance (Bliss, 1952) ; botency estimates are given together with their 95% confidence limits and the index of precision (X).
TABLE THE
WEIGHT
OF THE
~~DENOHYPOPHYSIS GROWTH
Sex and photoyeriodic
0” c? 0 9 0”
6L/18Db 20L/4D” 6L/lSD” 20L/4D” 20L/4D Day 0 Day 17 Day 40
Day 48 Day 70 Day 40 6L/18D controls
regime
IX
IN Fresh
NEWLY
1
RELATIOS
TO
HATCHED
m-t. of adenohypophysis
0.845 1.113 0.918 1.100
+ * + +
0.027 0.031 0.016 0.031
0.731 * 0.049 1.129 + 0.042 1.360 k 0.130 Photoperiod changed from 1.179 5 0.071 1.114 f 0.059 0.555 f 0.056
PHOTOPERIOUICALLY
JA~AXESE
INUUCEE
Gox\;an~~
QCAIL
(rnp)~
Gonadal
wt. (mg)”
(35) (35)c (37) (38)c
9.0 876.4 17.4 s4.9
* f f f
0.4 (13) 34.2 (Z$’ 0.8 (46) 9.2 (IlO,,:
(7) (7)d (4)d 20L/4D (4)d (lO)d (4)
8.1 1,683 3,000
f 1.2 (II) + 144 (14)d It 42 (12)d
2,420 36.3 13.2
2 61 (10)” + 2.9 (16)” f 1 .2 (40)
t’o 6L/18D
o Mean + standard error. Numbers of birds are shown in parentheses. b Birds exposed to photoperiodic regime for 28 days subsequent to hatching. C Significantly greater (p < 0.001) than 6L/18D group. d Significantly greater (p < 0.001) than day 0 sample.
--__
128
THE
FOLLETT
EFFECT
OF LONG
DAILY
FARNER
TABLE (20L/4D)
PHOTOPERIODS NEWLY
AND
HATCHED
2 ON
JAPANESE Expt.
PITUITARY
GONADOTROPIN
Group
6L/lSD
1 2 3
20L/4D
1 2 3 4 5
Female
MfX%Il potencya
95% limits~
Lambdad
<0.5 -0.5 -1.0
-
-
5.5 5.3 5.8 4.7 5.5
6.8 6.6 7.7
6.3 7.4
8.2 8.8 11.8 7.9 9.5
-
-
IN
1
Male Photoperiodic regime
POTENCY
QUAIL
MIZUI potencyb
95% limits
Lamb&d
-0.3 -0.5
0.130 0.123 0.165 0.145 0.167
3.8 2.7 3.5 3.7 3.6
2.2 2.1 2.7 3.0 2.7
0.086 0.165 0.145 0.140 0.161
4.4 3.7 4.4 - 4.7 - 4.6 -
a Begun on day of hatching; all birds were killed 28 days after hatching. b Potency expressed as pg equivalents NIH-LH per gland. c All assays for groups from 20L/4D were (2 + 2) with ten chicks per dose level. d Index of precision.
The birds from each treatment were divided into batches of 5-10; single assays were performed in each batch. The pituitaries of the quail exposed to 6L/lSD (Group 1s) contained so little gonadotropin that an accurate assay was impossible, whereas THE
EFFECT
OF LONG
DAILY
TABLE 3 (20L/4D)
PHOTOPERIODS NEWLY
those held on 2OL/4D (Group 1L) had considerable potency (Table 2). The variation among batches in Group 1L was sufficiently small so that Expt. 2 (Table 3) only a single assay was made for each killing time. The time course of changes in
HATCHED
ON PITUITARY
JAPANESE Expt.
GONADOTROPIN
1 is
146 21s 255 and saline 26s 71, 14L 21L 25L and saline 26L 36L
MeWI potencyb
<0.3 -0.6 1.0 0 .9 1.0 1.4 12 2.8 9.9
7.3 4.9 9.3
95%
Femnln Lambdac
limits
0 8 -
1.2
0.8 0.7
-
1.1
1.4
0.9
-
1.5
2.1
-
4.8
i.2 - 13.8 5 7 3.1 6.9
-
9.6
6.5 13 2
IN
2
Male Age and photoperiod*
POTENCY
QUAIL
0.091 0.126 0.126
-
0.097 0.107 0.174c 0.109e 0.151e 0.144e
Testicular wt.
1.2 2.4 7.7 8.3 8.9 4.4 23.8 149.6
306.1 464.7 1494
MeC&Il potency~
0.3 0.6 0.8 0.9 0.9
1.2 1.0 1.5 “6 ;:5 3 1 -
a Age given in days; photoperiod S is 6L/18D, L is 2OL/4D. Photoperiodic hatching. b Mean potency expressed as pg equivalents NIH-LH per gland. c Index of precision. d Mean in milligrams. e Assays of (2 + 2) form; all others modified to (2 + 1).
95% limits
0.4 0.6 0.7 0.7 0.7 1.2 2.7 2.3 2.4
Lambda0
-
0.9 1.0
0.091 0.098
-
1.0 1.1
0.114 0.126
-
1.3 1.7
0.097 0. 06gd
4.9
0.126 0. 207d 0.114d
-
5.0
3.8
Ovarian wt.d
1.7 6.4 11.7 16.1 25.3 6.2 15.4 42.7 37.0 51.0
-
regime
was begun
on day
of
PITUITARY
GONADOTROPIN?
IN
Days
Group
2L
1.2
Testicular 4.4
Grwp
2s
-
2.4
I
THE
Cmg 1 149.6
7.7
8.3
, IO
Days Ovarian
129
QUAIL
Of Experiment
Weight 23.8
I 0
JAPANESE
Of
Weight
464.7
1494.0
8.9
10.0
I
/
213
30
Experiment (mg)
51.0 6.2 15.4 42.7 Group 2s 6.4 Il.7 16.1 25.3 PIG. 1. Change in pituitary gonadotropins as a function of time in young Japanese quail exposd fkom hatching to a daily photoperiod of 20L/4D (0 -------.I or 6L/lSD (O------0). Gonadotropic potency is expressed as microgram equivalents NIR-LH per gland; the vertical bars represent the 95% confidence limits for the assay. The mean gonadnl weight of each sample is shown below the graph. Group
2L
1.7
130
FOJLET
AKD
pituitary gonadotropin potency for Expt. 2 is shown in Table 3 and Fig. 1. The pituitaries of. both male and female quail on 6L/lSD (Group 2s) contained small amounts of gonadotropin by day 14; the content did not change thereafter through day 26. A steady increase in pituitary gonadotropic potency was observed through day 21 in both male and female quail exposed to 20L/4D (Group 2L). There may have been a slight decrease in potency on day 25 and 26. Saline treatment, on days 20-25 appears not to have affected the gonadotropic potency of the anterior pituitary; this is consistent with our observation that this treatment had no effect on gonadal growth (Follett and Farner, 1965). The changes in weight of the anterior pituitary in this experiment were small although significant. The differences among groups were not altered by expression of gonadotropin in terms of weight of the anterior pituitary gland. In all cases (Table 2 and 3; Fig. 1) the pituitaries from males contained more gonadotropin than similarly treated females. This is consistent with the observations of Bacon et al. (1964) on the Japanese quail. The pars distalis of the Japanese quail is deeply furrowed and, at least visually, can be divided into cephalic and caudal lobes. Some preliminary experiments were undertaken to determine gonadotropic potency of the separate lobes. Activity was found in both cephalic and caudal portions ; the former is somewhat larger than the latter; e.g., in one experiment mean cephalic lobe weight was 0.744 * 0.033 mg (20)) whereas the weight of the caudal lobe was 0.399 + 0.022 mg (20). The gonadotropic activities for two groups of males were as follows: (1) 20L/4D, cephalic lobe, 5.7 (3.78.7) pg/lobe or 6.2 (4&9,.4)/mg wet wt.; caudal lobe, 3.0 (2.0-4.6) ,pg/lobe or 6.8 (4.6-10.4)/mg wet wt. (2) 20L/4D, cephalic lobe, 5.5 (4.2-7.8) ,pg/lobe or 7.4 (5.7-10.4)/mg wet wt.; caudal lobe, 3.8 (2.7-5.4) pg/lobe or 9.4 (6.6613.4)/mg wet, wt. These results suggest that gonadotropins may occur in about equal concentrations in both lobes; further analysis must await the
FARNER
outcome of current studies combining assays and cytological techniques.
bio-
DISCUSSION
The interpretation of data from the assay of pituitary gonadotropins is difficult, since they represent steady-state levels without yielding direct information on rates of synthesis or release of the hormone. It is possible, however, that the rate of photoperiodically induced gonadal growth in some species of birds may afford an approximation of the rate of hormone release; this is especially so because of the logarithmic relationship between gonadal weight and time (See review, Farner and Follett, 1966). Moreover, in all assay techniques based on increases in gonadal weight in birds there is a logarithmic relation between dose of hormone and the increase in weight. Theoretically, the rate of hormone release in photoperiodically induced growth may represent a constant output during the logarithmic phase of growth. In the present experiment only two photoperiodic treatments were used; no accumulation of gonadotropins occurred under the short daily photoperiod (6L/18D), suggesting that not only release was blocked but also that synthesis must have occurred at a minimal rate. In the quail subjected to 2OL/4D the rate of synthesis apparently exceeded slightly the rate of release during the first 21 days, whereas the reverse was true thereafter. Emphasis has been placed on the separation of the mechanisms of gonadotropin release and synthesis, since there is evidence (Farner and Follett, 1966) in the white-crowned sparrow (Zon#otrichia lseuc,ophrysga&eZii) that the two functions may be controlled partially independently of each other. The data presented in this communication offer a quantitative analysis related to a standard preparation, and thus confidence limits may be calculated for each potency determination and experiments may be compared directly with each other. The results are in general agreement with the semi-quantitative observations of pituitary gonadotropin content during gonadal
PITUITARY
GONADOTROPINS
IN
THE
JAPANESE
QUAIL
731
growth in other birds, including the domestic fowl (Breneman, 1945)) the ringnecked pheasant (Greeley and Meyer, 1953) and, very recently, on the Japanese quail (Tanaka et al., 1965).
neurosecretion in the hypothalamo-hypophysial system of the Japanese quail (Coturniz cotuwk japozica). Gen. Comp. Endocrinol. 6, 111-324. FRAPS, R. M., AKD FEVOLD, H. L. (1955). Delaying action of gonadotropins on ovulation in the hen.
ACKNOWLEDGMENTS We are indebted to Professor James McGinnis and Dr. Jean K. Lauber, Department of Animal Science, Washington State University, who hatched and reared the quail used in these experiments. Standard preparations of gonadotropin were kindly supplied by the Endocrinology Study Section, National Institutes of Health.
FRAPS,
REFERENCES B.~coN, W., CHERMS, F. L., AND MCSKQN, W. H. (1964). Gonadotropin assay of pituitaries from sexually mature male and laying female Cotur?aix quail. Endocrinology 74, 498-500. BENOIT, J. (1935). R81e de l’hypophyse dans l’action stimulante de la lumikre sur le dhveloppement testiculaire chez le Canard. Compt. Rend. Sot.
Biol.
11’8, 672-674.
J.: ASSEXMACBER, I., ASD WALTER, F. X. (1950). Activitk gonadotrope de l’hypophyse du canard domestique, au coure de la rkgreesion testiculaire saisonniere et de la p&pub&&. Compt. Rend. Xoc. Biol. 144, 1403-1407. BLISS, C. I. (1952). “The Statistics of Bioassay with Special Reference to the Vitamins.” Academic Press, New York. BRENEMAN, W. R. (1945). The gonadotropic activity of the anterior pituitary of cockerels. BENOIT,
Endoc%ology
36,
190-199.
W. R., ZELLER, F. J., AND CREEK, R. 0. (1962). Radioactive phosphorus uptake by chick testes as an end-point for gonadotropin assay.
BRENEMAN,
Endocrinology
71,
790-798.
D. S., AND FOLLETT, B. K. (1966). Light and other environmental factors affecting avian reproduction. VII. Biennial Symposium on Animal Reproduction, 1965. J. Animal Sci., (in press). FLOKSHEIM, W. H., VELCOFF, S. M., AKD BODFISH, R. E. (1959). Gonadotrophin assay based on augmentation of radiophosphate uptake by the chick testes. Actn Endockol. 30, 175-182. FOLLEY~, 13. K., AND FARNER, D. S. (1965). The effects of the daily photoperiod of gonadal growth, neurohypophysial hormone content and FARNER,
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R. M., FEVOLD, H. L., ASD N~-ti~a, B. H. (1947) Ovulatory response of the hen to prcsumptive luteinizing and other fractions from fowl anterior pituitary tissue. Anat. Kecor11 ‘99:
571-572. GREELET,
.AND METER, R. Ii. (1953). Seasonal in testis-stimulating activity of male pituitary glands. AU& 70, 350-358. HERRICK, R. B., MCGIBBOS, W. H., AND MCSHAA. W. H. (1962). Gondotropic activit.y of chicken pituitary glands. Endocrinology 71, 487-491. MCFARLAKD, L. Z., MATHER, F. B., ASD W~LSOE: W. 0. (1964). Testicular response to gonadol.rophins and intermittent light in Coturnix reared under limited light. Am. J. Vet. Res. 25, 1531F.,
variation pheasant
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A. V. (1959). Nemoendocrine reflex mechanisms: bird ovulation. I?L “Comparative Endocrinology” (A. Gorbman, ed.), pp. 161-173, Wiley, New York. PARLOW, A. I?. (1964). Differential action of small doses of e&radio1 on gonadotrophins in the rat. NaLBhxDOV,
Endocrinology
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F. B., Wrmos, W. O., ANI: L. Z. (1965). Effect of photoperiods on early growth of gonads and on potency of gonadotropins of the anterior pituitary gland in
TANAIU,
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Coturnix.
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VAN TIENHOVEK, A. (1959). Reproduction in th<, domestic fowl: Physiology of the female. 11~ “Reproduction in Domestic Animals” (H. II. Cole, P. T. Cupps, eds.), Vol. II, pp. 305-342. Academic Press, New York. VAN TIENHCIVEK, A. (1961). Endocrinology of reproduction in birds. 117 “Sex and Int#ernal SoereLions” (W. C. Young, ed.)? 3rd Ed., pp. 198S1169. Williams and Wilkins, Baltimore. TIXIER-VIDAL, A. (1963). Histophgsio;ogie tic l’adknohypophyee des oiseaux. 1,~ “Cytologic de I’AdCnohypophyse” (J. Benoit and C. DaLage, eds.); Colloq. Intern. Cetltw Nntl. Rech. Sci. (Paris) 12’8, 255-273. TISIER-VID.~L, A., HERL.IXT: M., AYSD BENOIT, 3. (1962). La prkhypophyse du Canard P&kin mlilc au tours du cycle annuel. Arch. BioE. (Liege) 73, 318-368.