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Oviposition pattern of Japanese quail following hypothalamic lesioning with super-absorbent polymer
GENERAL
AND
COMPARATIVE
72, 424-430 (1988)
ENDOCRINOLOGY
Oviposition Pattern of Japanese Quail following Hypothalamic Lesioning with Super-Absorbent Polymer M. OHTAAND K. HOMMA Department
of Veterinary
Physiology,
College
of Agriculture, Osaka 591, Japan
University
of Osaka
Prefecture,
Sakai
City,
Accepted July 13, 1988 The functions of two hypothalamic areas in controlling the female reproductive cycle were investigated by the intracerebral injection of a new type of water-absorbent polymer of high capacity (super-absorbent polymer). After injection of a minute amount of the polymer into the brain tissue, bulging of the polymer produces a discrete lesion at the site of injection. Two lines (T- and J-lines) of Japanese quail were used; T-line, having a characteristic free-running oviposition pattern irrespective of the environmental 14LlOD, and J-line, having a regular oviposition pattern which synchronized with 14LlOD. Lesions at the preoptic area were without effect in birds of J-line, but the oviposition of T-line was changed from free-running to regular. Lesions at the posterodorsal part of the infundibular complex were without effect in T-line, but the regular oviposition pattern of J-line became free-running. These results suggest that relative dominancy between the two hypothalamic areas may determine basic pattern of oviposition through modification of the ovulation cycle. o 1988 Academic
Press, Inc.
Ovipositional patterns of female quail under 16 hr light and 8 hr darkness (16L8D) have been classified into the following three types (van Tienhoven and Planck, 1973; Konishi, 1980). Type A: ovipositional time is synchronized to the photoperiod and the interval between any successive ovipositions is close to 24 hr. Type B: the pattern of oviposition is analogous to that of the chicken. There is a intersequence pause. Type C: the oviposition takes place without intersequence pause at intervals longer than 24 hr irrespective of the environmental light and dark, like the birds exposed to continuous light. Recently we reported the presence of two lines (T- and J-lines) in Japanese quail (Maruyama et al., 1984). Intact birds of Jand T-lines showed ovipositional patterns of type A and C, respectively. When these birds were subjected to partial or complete hypothalamic deafferentation, their patterns of oviposition were modified depend-
ing upon the type of the cuts (Maruyama et al., 1984). The release of LH from the pituitary gland is under the control of gonadotropinreleasing hormone (GnRH; for review see Sharp, 1982). In male quail, at least two discrete hypothalamic regions control seasonal growth of the testes (Davies and Follett, 1975a, b; Oliver and Bayle, 1976; Follett, 1978; Ohta and Homma, 1987). One is located in the preoptic area (POA), adjacent to the ventral projection of the septomesencephalic tract, while the other is located in the posterodorsal part of the infundibular nuclear complex (INC), some 3.5 mm caudal to the POA. In female quail, GnRH-containing systems have been found in both the POA and the INC (Stetson, 1972; Davies, 1980), but physiological roles of these areas have not been clarified. In the present study, functions of these two hypothalamic areas in controlling the female reproduction were investigated by 424
0016~6480/88 $1.50 Copyright 0 1988 by Academic Press, Inc. AU rights of reproduction in any form reserved.
OVIPOSITION
the intracerebral water-absorbent (super-absorbent MATERIALS
CHANGES
FOLLOWING
injection of a new type of polymer of high capacity polymer, SAP).
HYPOTHALAMIC
LESION
-
425
AC
AND METHODS
Animals. Two lines of female Japanese quail (Coturnti coturnix japonica) were used. One (T-line) was originated in the Toyohashi area and was bred as a closed colony, and the other (J-line) was a commercial line of a local hatchery near Tokyo (Johnan Quail Farm). Birds for experiments were reared in individual cages, 18 x 10 x 18 cm, under 14 hr of light and 10 hr of dark (14LlOD, lights on from 0700 to 2100 hr). The oviposition in birds of T-line took place without intersequence pause at intervals a few hours longer than 24 hr irrespective of the environmental 14L10D cycle. This pattern of oviposition was tentatively named as a free-running pattern. The time of oviposition in birds of J-line was clustered within a narrow range under the same environment (named as a regular pattern). The birds of T-line were darker in the plumage color than those of J-line, but in the other respects these two lines were virtually indistinguishable. Water-absorbent polymer of high capacity (superabsorbent polymer). SAP (polyvinyl alcoholpolyacrylic acid copolymer, Sumikagel S-50 type, Sumitomo Chemical Co., Ltd., Osaka) is the newly synthesized material which absorbs water to form a hydrogel and swells several hundred times of the original weight. The SAP swells 40-60 times that of the original weight with 0.9% NaCl. The swelled gel was stable for at least 6 months as a tight mass. About 1 )~l of the SAP (original weight of the SAP is about 0.025 mg), preswelled to 40 times with saline, was injected at a speed of about 1 (~1 per min. The SAP is nearly maximally swollen when implanted. Stereotaxic injection of the polymer.Four experimental groups were made after checking their natural patterns of oviposition. The bird was anesthetized by an intravenous injection of 25 mglkg body weight of urethane solution and the head was fixed in a stereotaxic instrument modified for quail. A small hole was drilled in the tops of the skull, and a stainless tube (21 gauge) was lowered down into the brain referring to the coordinates in the brain atlas (Bay16 et al., 1974). The sites of polymer injection (see Fig. 1) were: (1) the area just rostral to the POA (group A), (2) the POA (group B), (3) the posterodorsal part of the INC (group C), and (4) both the POA and the posterodorsal part of the INC (group D). Recording of oviposition. Laying time of each bird was recorded continuously with a microcomputer (PC8001 and its peripherals, Nippon Electrics Co., Ltd., Tokyo). During the experimental periods, food and water were given ad libitum. The room temperature was set at 23 t 1” and the light intensity at the floor of
FIG. 1. Schematic representation of the hypothalamus in Japanese quail. @$Preoptic area (POA); .: j posterodorsal part of the infundibular nuclear complex (INC); AC, anterior commissure; PVN, paraventricular nucleus; AH, anterior hypothalamus; INC, infundibular nuclear complex; OC, optic chiasma; PN, pars nervosa; and PD, pars distalis of the pituitary. the cages was adjusted to 300 lux. White fluorescent tubes were the light source. On-off time of the lights was regulated by a 24-hr type electric timer. Histological verification of the brain. At the end of the experiments, birds were killed, and brain tissues were fixed by transcardial perfusion with 10% formolsaline. Serial frozen section, 50 p,rn thick, were stained with 1% cresyl violet (Nissl’s stain). Tissue preparations were microscopically observed to examine the site of polymer injection and the histological changes in the hypothalamus. Statistics. The statistical significance was assessed by Duncan’s multiple range test (Duncan, 1955; Kramer, 1956). A P value of CO.01 defined statistical significance.
RESULTS
After the injection of about 1 p,f of the polymer into the brain tissue, bulging of the polymer produced a discrete lesion at the site of injection which was readily detected histologically (Figs. 2 and 3). All figures illustrate the response of one bird which was representative of its experimental group. The lesions by the pressure of the gel were localized to the sites of polymer injection, and no derivative effect was found in the surroundings anatomically. On the average, the time lag from the injection of the polymer to the resumption of egg production was about 5 days longer in group C than that in groups A and B, when
426
OHTA
AND
FIG. 2. Sagittal section of hypothalamus of representative quail showing the site of stereotaxic polymer injection. The POA is lesioned with the polymer. About 1 ~1 of polymer swelled to 40 times with saline was injected at a speed of 1 p,l per min. OC, optic chiasma. Black bar = 1.00 mm.
the delay of nonlayers (group D) was not counted (Table 1). Lesions produced at the area just rostra1 to the POA were without effect on the ovi-
HOMMA
position cycles of the two lines. The patterns of oviposition of T- and J-line remained free-running and regular, respectively. Although the latent time from the injection of the polymer to the resumption of lay was greater than 5 days (Table I>, perhaps this would be nonspecific effects of the surgical procedure. Two typical patterns of oviposition are illustrated in Figs. 4 and 5. The time of oviposition in birds of J-line (Fig. 5) was clustered early in the evening. Lesions at the POA were without effect in birds of J-line (Fig. 6), but the pattern of oviposition of T-line was changed from free-running to regular (Fig. 7). Lesions at the posterodorsal part of the INC were without effect in T-line (Fig. 8), but the pattern of oviposition of J-line was changed from regular to free-running although the regular pattern of oviposition was observed for 7-11 days (mean !z SE = 8.2 + 0.4 days) after the resumption of egg production (Fig. 9). Double lesions at the POA and the posterodorsal part of the INC resulted in cessation of egg production (not illustrated). DISCUSSION
FIG. 3. Sagittal section of hypothalamus of representative quail showing the lesion of the posterodorsal part of the INC with the polymer. About 1 p.1of polymer swelled to 40 times with saline was injected at a speed of 1 ul per min. OC, optic chiasma. Black bar = 1.OOmm.
Double lesions with SAP at the POA and the posterodorsal part of the JNC always resulted in the cessation of egg production, while after single lesions either at the POA or the posterodorsal part of the INC, egg production ensued. These results suggest that the active systems of GnRH neurons are different between the two lines and the relative dominancy between the two sets of GnRH neurons may determine the basic pattern of oviposition. Electric lesions of either the POA or the INC are known to induce prolonged cessation of ovulation in the laying chicken and quail (Ralph, 1959; Ralph and Fraps, 1959; Egge and Chiasson, 1963; Stetson, 1972; Davies, 1980). However, electric lesions reported by several laboratories (Ralph, 1959;
OVIPOSITION
CHANGES
FOLLOWING
HYPOTHALAMIC
1
TABLE
LATENT
TIME
IN DAYS
FROM THE INJECTION
OF SAP
TO THE RESUMPTION
Groups (sites of lesioning with SAP) (A) Area just rostra1 to POA T-line J-line (B) POA T-line J-line (C) Posterodorsal part of INC T-line J-line (D) POA and posterodorsal part of INC T-line J-line
OF EGG PRODUCTION
Number of birds
Latent time” (day)
7 7
5.4 2 0.4* 5.3 2 0.4*
9 8
5.6 t 0.2* 5.4 2 0.2*
8 10
10 1 + 0.4** 10:9 2 as**
8 7
e-b -
a Mean lr SE. b Cessation of egg production. *,** Means within the same column with different superscripts are significantly
Ralph and Fraps, 1959; Egge and Chiasson, 1963) were quite extensive and resistant to delineate. By these reasons, it was also reported that even when less extensive lesions were placed in either the POA or the INC of laying quail, ovulation ceased (Stetson, 1972; Davies, 1980). Partial or complete hypothalamic deafferentation in male quail (Ohta and Homma, 1987) and female quail (Davies, 1980) reveals that the anterior hypothalamus has neural connection to the INC.
427
LESION
different (P < 0.01).
Therefore, in electric lesioning, effects of electric current on the POA and the INC cannot be ignored. We had reported previously that when female quail received continuous illumination to the hypothalamus with radioluminous beads under short-day environment, the initiation of egg production was markedly advanced while the rhythm of oviposition was synchronized to the environmental photoperiods like the intact controls (Homma and Sakakibara, 1971). At that time we thought that this indicated dominant roles of the POA, or most plausible
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4. Oviposition cycles in T-line: Lesions at the area just rostra1 to the POA. The oviposition remained free-running. Successive days are plotted from top to bottom and the records have been double plotted over 48-hr interval. The black bars at the top of oviposition graph indicate the hours of darkness. 4P, polymer injection.
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FIG. 5. Oviposition cycles in J-line: Lesions at the area just rostra1 to the POA. The oviposition remained regular and the time of oviposition was clustered early in the evening. Others are the same as Fig. 4.
428
OHTA
AND
HOMMA 0
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FIG. 6. Oviposition cycles in J-line: Lesions at the POA. The oviposition remained regular. Others are the same as Fig. 4.
FIG. 8. Oviposition cycles in T-line: Lesions at the posterodorsal part of the INC. The oviposition remained free-running. Others are the same as Fig. 4.
retinal photoreception, for the determination of ovulation time. Whereas under the same photoperiod, blinded quail also exhibited regular oviposition, suggesting the possibility that the deep photoreceptor can regulate timing of oviposition. In the present study, however, the activity of the POA was resistant to be influenced by the environmental light and dark. Thus, the INC which is known at the site of deep photoreceptors (Oliver et al., 1978; Ohta et al., 1984) may function as the major site for regulating the time of ovulation and the POA may be an endogenous circadian oscillator. After single lesions at the posterodorsal part of the INC, the resumption of egg production of the two lines was delayed significantly, in comparison with single lesions at the POA: the natural pattern of oviposition
of J-line was observed for several days before the regular pattern of oviposition became free-running. These results support that for egg production in the Japanese quail, the INC may play the pivotal role and GnRH neurons in the INC may be distributed extensively. An important question to be resolved concerns the control of the POA. In male quail, the POA is not photosensitive in itself, thus the retinal photoreceptor is thought to be involved in the control systems of the POA (Oliver and Bayle, 1977; Oliver et al., 1978; Homma et al., 1979; Ohta et al., 1984). In the present experiments, however, the retinal signals failed to entrain the activity of the POA. Experiments of hypothalamic deafferentation 0
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““5 FIG. 7. Oviposition cycles in T-line: Lesions at the POA. The oviposition was changed from free-running to regular. Others are the same as Fig. 4.
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-
-P
30
FIG. 9. Oviposition cycles in J-line: Lesions at the posterodorsal part of the INC. The oviposition was changed from regular to free-running. Others are the same as Fig. 4.
OVIPOSITION
CHANGES
FOLLOWING
clearly showed that the axons communicating between the POA and the INC were important for determining the basic pattern of oviposition (Maruyama et aZ., 1984) and regulatory neurons in the retina and anterior hypothalamus had neural connection to the posterior side of the INC (Ohta and Homma, 1987). Therefore, the afferent inputs from the INC to the POA may control the output of the POA: the afferent inputs from retina to the POA and/or the INC may be the subcontrol system for the adjustments of the POA. The possibility of communication between the POA and the INC via cerebrospinal fluid may be precluded because the natural patterns of oviposition were unchanged when the polymer was infused into the third ventricle. Additional research is needed to determine the precise roles of the POA and the INC in the control of reproductive activity in birds and to establish how these parts of hypothalamus are functionally linked. The SAP would be useful for these neuroendocrine studies. ACKNOWLEDGMENTS We would like to thank Dr. Hitoshi Aikawa, University of Tokyo, for the technical assistance and histological advice. A part of this research was financially supported by the grant from Scientific Research from the Ministry of Education, Science, and Culture of Japan No. 61760251 to M.O.
REFERENCES BaylC, J. D., Ramade, F., and Oliver, J. (1974). Stereotaxic topography of the brain of the quail (Coturnix coturnix japonica). J. Physiol. 68, 219241. Davies, D. T. (1980). The neuroendocrine control of gonadotrophin release in the Japanese quail. III. The role of the tuberal and anterior hypothalamus in the control of ovarian development and ovulation. Proc. R. Sot. London B 206,421-437. Davies, D. T., and Follett, B. K. (1975a). The neuroendocrine control of gonadotrophin release in the Japanese quail. I. The role of the tuberal hypothalamus. Proc. R. Sot. London B 191, 285-301. Davies, D. T., and Follett, B. K. (1975b). The neuro-
HYPOTHALAMIC
LESION
429
endocrine control of the gonadotrophin release in the Japanese quail. II. The role of the anterior hypothalamus. Proc. R. Sot. London 3 191, 303315. Duncan, D. B. (1955). Multiple range and multiple F tests. Biometrics 11, l-42. Egge, A. S., and Chiasson, R. B. (1963). Endocrine effects of diencephalic lesions in the white leghorn hen. Gen. Camp. Endocrinol. 3, 346-361. Follett, B. K. (1978). Photoperiodism and seasonal breeding in birds and mammals. In “Control of Ovulation” (D. B. Crighton, N. B. Haynes, G. R. Foxcroft, and G. E. Lamming, Eds.), pp. 267294, Butterworths, London. Homma, K., and Sakakibara, Y. (1971). Encephalic photoreceptors and their significance in photoperiodic control of sexual acitivity in Japanese quail. In “Biochronometry” (M. Menaker, Ed.), pp. 333-341. Natl. Acad. Sci., Washington, D.C. Homma, K., Ohta, M., and Sakakibara, Y. (1979). Photoinducible phase of the Japanese quail detected by direct stimulation of the brain. In “Biological Rhythms and their Central Mechanism” (M. Suds, 0. Hayashi, and H. Nakagawa, Eds.), pp. 85-94. Elsevier-North Holland, Amsterdam. Konishi, T. (1980). Circadian rhythm of ovipositional time in Japanese quail. In “Biological Rhythms in Birds” (Y. Tanabe, K. Tanaka, and T. Ookawa, Eds.), pp. 79-90. Japan Sci. Sot. Press, Tokyo/ Springer-Verlag, Berlin. Kramer, C. Y. (1956). Extension of multiple range tests to group means with unequal numbers of replications. Biometrics 12, 307-310. Maruyama, S., Ohta, M., and Homma, K. (1984). Spreading and clustering of oviposition pattern in Japanese quail after partial or complete hypothalamic deafferentation of the infundibular complex. Japan. Poultry Sci. 21, 301-312. Ohta, M., and Homma, K. (1987). Detection of neural connection to the infundibular complex by partial or complete hypothalamic deafferentation in male quail. ken. &np. Endocrinol. 68, 286-292. Ohta, M., Wada, M., and Homma, K. (1984). Induction of rapid testicular growth in Japanese quail by phasic electrical stimulation of the hypothalamic photosensitive area. J. Comp. Physiol. A 154, 583-589. Oliver, J., and Bay& J. D. (1976). The involvement of the preoptic-suprachiasmatic region in the photosexual reflex in quail. Effects of selective lesions or photic stimulations. .I. Physiol. 72, 627-637. Oliver, J., and Bayle, J. D. (1977). Preoptic multiunit activity correlates of the photosexual reflex in quail. Neurosci. Lett. 6, 317-322. Oliver, J., Bouillt, C., Herbutt, S., and Bay& J. D. (1978). Retrograde transport horseradish peroxidase from the preoptic anterior hypothalamic
430
OHTA
AND
region to retinal ganglion cells in quail. Neurosci. Lett. 9, 291-295. Ralph, C. L. (1959). Some effects of hypothalamic lesions on gonadotropin release in the hen. Anat. Rec. 134, 411-431. Ralph, C. L., and Fraps, R. M. (1959). Long-term effects of diencephalic lesions on the ovary of the hen. Amer. J. Physiol. 197, 1279-1283. Sharp, P. J. (1983). Hypothalamic control of gonadotrophin secretion in birds. In “Progress in Nonmammalian Brain Research” (G. Nistico and L.
HOMMA
Bolis, Eds.), Voi 3, pp. 124-176. CRC Press, Boca Raton, FL. Stetson, M. H. (1972). Hypothalamic regulation of gonadotropin release in female Japanese quail. Z. Zellforsch. Mikrosk. Anat. 130, 41 l-428. van Tienhoven, A., and Planck, R. J. (1973). The effect of light on avian reproductive activity. In “Handbook of Physiology, Section 7, Endocrinology, II, Part I” (R. 0. Creep and E. B. Astwood, Eds.), pp. 79-107. Amer. Physiol. Sot., Washington D.C.
AND
COMPARATIVE
72, 424-430 (1988)
ENDOCRINOLOGY
Oviposition Pattern of Japanese Quail following Hypothalamic Lesioning with Super-Absorbent Polymer M. OHTAAND K. HOMMA Department
of Veterinary
Physiology,
College
of Agriculture, Osaka 591, Japan
University
of Osaka
Prefecture,
Sakai
City,
Accepted July 13, 1988 The functions of two hypothalamic areas in controlling the female reproductive cycle were investigated by the intracerebral injection of a new type of water-absorbent polymer of high capacity (super-absorbent polymer). After injection of a minute amount of the polymer into the brain tissue, bulging of the polymer produces a discrete lesion at the site of injection. Two lines (T- and J-lines) of Japanese quail were used; T-line, having a characteristic free-running oviposition pattern irrespective of the environmental 14LlOD, and J-line, having a regular oviposition pattern which synchronized with 14LlOD. Lesions at the preoptic area were without effect in birds of J-line, but the oviposition of T-line was changed from free-running to regular. Lesions at the posterodorsal part of the infundibular complex were without effect in T-line, but the regular oviposition pattern of J-line became free-running. These results suggest that relative dominancy between the two hypothalamic areas may determine basic pattern of oviposition through modification of the ovulation cycle. o 1988 Academic
Press, Inc.
Ovipositional patterns of female quail under 16 hr light and 8 hr darkness (16L8D) have been classified into the following three types (van Tienhoven and Planck, 1973; Konishi, 1980). Type A: ovipositional time is synchronized to the photoperiod and the interval between any successive ovipositions is close to 24 hr. Type B: the pattern of oviposition is analogous to that of the chicken. There is a intersequence pause. Type C: the oviposition takes place without intersequence pause at intervals longer than 24 hr irrespective of the environmental light and dark, like the birds exposed to continuous light. Recently we reported the presence of two lines (T- and J-lines) in Japanese quail (Maruyama et al., 1984). Intact birds of Jand T-lines showed ovipositional patterns of type A and C, respectively. When these birds were subjected to partial or complete hypothalamic deafferentation, their patterns of oviposition were modified depend-
ing upon the type of the cuts (Maruyama et al., 1984). The release of LH from the pituitary gland is under the control of gonadotropinreleasing hormone (GnRH; for review see Sharp, 1982). In male quail, at least two discrete hypothalamic regions control seasonal growth of the testes (Davies and Follett, 1975a, b; Oliver and Bayle, 1976; Follett, 1978; Ohta and Homma, 1987). One is located in the preoptic area (POA), adjacent to the ventral projection of the septomesencephalic tract, while the other is located in the posterodorsal part of the infundibular nuclear complex (INC), some 3.5 mm caudal to the POA. In female quail, GnRH-containing systems have been found in both the POA and the INC (Stetson, 1972; Davies, 1980), but physiological roles of these areas have not been clarified. In the present study, functions of these two hypothalamic areas in controlling the female reproduction were investigated by 424
0016~6480/88 $1.50 Copyright 0 1988 by Academic Press, Inc. AU rights of reproduction in any form reserved.
OVIPOSITION
the intracerebral water-absorbent (super-absorbent MATERIALS
CHANGES
FOLLOWING
injection of a new type of polymer of high capacity polymer, SAP).
HYPOTHALAMIC
LESION
-
425
AC
AND METHODS
Animals. Two lines of female Japanese quail (Coturnti coturnix japonica) were used. One (T-line) was originated in the Toyohashi area and was bred as a closed colony, and the other (J-line) was a commercial line of a local hatchery near Tokyo (Johnan Quail Farm). Birds for experiments were reared in individual cages, 18 x 10 x 18 cm, under 14 hr of light and 10 hr of dark (14LlOD, lights on from 0700 to 2100 hr). The oviposition in birds of T-line took place without intersequence pause at intervals a few hours longer than 24 hr irrespective of the environmental 14L10D cycle. This pattern of oviposition was tentatively named as a free-running pattern. The time of oviposition in birds of J-line was clustered within a narrow range under the same environment (named as a regular pattern). The birds of T-line were darker in the plumage color than those of J-line, but in the other respects these two lines were virtually indistinguishable. Water-absorbent polymer of high capacity (superabsorbent polymer). SAP (polyvinyl alcoholpolyacrylic acid copolymer, Sumikagel S-50 type, Sumitomo Chemical Co., Ltd., Osaka) is the newly synthesized material which absorbs water to form a hydrogel and swells several hundred times of the original weight. The SAP swells 40-60 times that of the original weight with 0.9% NaCl. The swelled gel was stable for at least 6 months as a tight mass. About 1 )~l of the SAP (original weight of the SAP is about 0.025 mg), preswelled to 40 times with saline, was injected at a speed of about 1 (~1 per min. The SAP is nearly maximally swollen when implanted. Stereotaxic injection of the polymer.Four experimental groups were made after checking their natural patterns of oviposition. The bird was anesthetized by an intravenous injection of 25 mglkg body weight of urethane solution and the head was fixed in a stereotaxic instrument modified for quail. A small hole was drilled in the tops of the skull, and a stainless tube (21 gauge) was lowered down into the brain referring to the coordinates in the brain atlas (Bay16 et al., 1974). The sites of polymer injection (see Fig. 1) were: (1) the area just rostral to the POA (group A), (2) the POA (group B), (3) the posterodorsal part of the INC (group C), and (4) both the POA and the posterodorsal part of the INC (group D). Recording of oviposition. Laying time of each bird was recorded continuously with a microcomputer (PC8001 and its peripherals, Nippon Electrics Co., Ltd., Tokyo). During the experimental periods, food and water were given ad libitum. The room temperature was set at 23 t 1” and the light intensity at the floor of
FIG. 1. Schematic representation of the hypothalamus in Japanese quail. @$Preoptic area (POA); .: j posterodorsal part of the infundibular nuclear complex (INC); AC, anterior commissure; PVN, paraventricular nucleus; AH, anterior hypothalamus; INC, infundibular nuclear complex; OC, optic chiasma; PN, pars nervosa; and PD, pars distalis of the pituitary. the cages was adjusted to 300 lux. White fluorescent tubes were the light source. On-off time of the lights was regulated by a 24-hr type electric timer. Histological verification of the brain. At the end of the experiments, birds were killed, and brain tissues were fixed by transcardial perfusion with 10% formolsaline. Serial frozen section, 50 p,rn thick, were stained with 1% cresyl violet (Nissl’s stain). Tissue preparations were microscopically observed to examine the site of polymer injection and the histological changes in the hypothalamus. Statistics. The statistical significance was assessed by Duncan’s multiple range test (Duncan, 1955; Kramer, 1956). A P value of CO.01 defined statistical significance.
RESULTS
After the injection of about 1 p,f of the polymer into the brain tissue, bulging of the polymer produced a discrete lesion at the site of injection which was readily detected histologically (Figs. 2 and 3). All figures illustrate the response of one bird which was representative of its experimental group. The lesions by the pressure of the gel were localized to the sites of polymer injection, and no derivative effect was found in the surroundings anatomically. On the average, the time lag from the injection of the polymer to the resumption of egg production was about 5 days longer in group C than that in groups A and B, when
426
OHTA
AND
FIG. 2. Sagittal section of hypothalamus of representative quail showing the site of stereotaxic polymer injection. The POA is lesioned with the polymer. About 1 ~1 of polymer swelled to 40 times with saline was injected at a speed of 1 p,l per min. OC, optic chiasma. Black bar = 1.00 mm.
the delay of nonlayers (group D) was not counted (Table 1). Lesions produced at the area just rostra1 to the POA were without effect on the ovi-
HOMMA
position cycles of the two lines. The patterns of oviposition of T- and J-line remained free-running and regular, respectively. Although the latent time from the injection of the polymer to the resumption of lay was greater than 5 days (Table I>, perhaps this would be nonspecific effects of the surgical procedure. Two typical patterns of oviposition are illustrated in Figs. 4 and 5. The time of oviposition in birds of J-line (Fig. 5) was clustered early in the evening. Lesions at the POA were without effect in birds of J-line (Fig. 6), but the pattern of oviposition of T-line was changed from free-running to regular (Fig. 7). Lesions at the posterodorsal part of the INC were without effect in T-line (Fig. 8), but the pattern of oviposition of J-line was changed from regular to free-running although the regular pattern of oviposition was observed for 7-11 days (mean !z SE = 8.2 + 0.4 days) after the resumption of egg production (Fig. 9). Double lesions at the POA and the posterodorsal part of the INC resulted in cessation of egg production (not illustrated). DISCUSSION
FIG. 3. Sagittal section of hypothalamus of representative quail showing the lesion of the posterodorsal part of the INC with the polymer. About 1 p.1of polymer swelled to 40 times with saline was injected at a speed of 1 ul per min. OC, optic chiasma. Black bar = 1.OOmm.
Double lesions with SAP at the POA and the posterodorsal part of the JNC always resulted in the cessation of egg production, while after single lesions either at the POA or the posterodorsal part of the INC, egg production ensued. These results suggest that the active systems of GnRH neurons are different between the two lines and the relative dominancy between the two sets of GnRH neurons may determine the basic pattern of oviposition. Electric lesions of either the POA or the INC are known to induce prolonged cessation of ovulation in the laying chicken and quail (Ralph, 1959; Ralph and Fraps, 1959; Egge and Chiasson, 1963; Stetson, 1972; Davies, 1980). However, electric lesions reported by several laboratories (Ralph, 1959;
OVIPOSITION
CHANGES
FOLLOWING
HYPOTHALAMIC
1
TABLE
LATENT
TIME
IN DAYS
FROM THE INJECTION
OF SAP
TO THE RESUMPTION
Groups (sites of lesioning with SAP) (A) Area just rostra1 to POA T-line J-line (B) POA T-line J-line (C) Posterodorsal part of INC T-line J-line (D) POA and posterodorsal part of INC T-line J-line
OF EGG PRODUCTION
Number of birds
Latent time” (day)
7 7
5.4 2 0.4* 5.3 2 0.4*
9 8
5.6 t 0.2* 5.4 2 0.2*
8 10
10 1 + 0.4** 10:9 2 as**
8 7
e-b -
a Mean lr SE. b Cessation of egg production. *,** Means within the same column with different superscripts are significantly
Ralph and Fraps, 1959; Egge and Chiasson, 1963) were quite extensive and resistant to delineate. By these reasons, it was also reported that even when less extensive lesions were placed in either the POA or the INC of laying quail, ovulation ceased (Stetson, 1972; Davies, 1980). Partial or complete hypothalamic deafferentation in male quail (Ohta and Homma, 1987) and female quail (Davies, 1980) reveals that the anterior hypothalamus has neural connection to the INC.
427
LESION
different (P < 0.01).
Therefore, in electric lesioning, effects of electric current on the POA and the INC cannot be ignored. We had reported previously that when female quail received continuous illumination to the hypothalamus with radioluminous beads under short-day environment, the initiation of egg production was markedly advanced while the rhythm of oviposition was synchronized to the environmental photoperiods like the intact controls (Homma and Sakakibara, 1971). At that time we thought that this indicated dominant roles of the POA, or most plausible
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4. Oviposition cycles in T-line: Lesions at the area just rostra1 to the POA. The oviposition remained free-running. Successive days are plotted from top to bottom and the records have been double plotted over 48-hr interval. The black bars at the top of oviposition graph indicate the hours of darkness. 4P, polymer injection.
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FIG. 5. Oviposition cycles in J-line: Lesions at the area just rostra1 to the POA. The oviposition remained regular and the time of oviposition was clustered early in the evening. Others are the same as Fig. 4.
428
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FIG. 6. Oviposition cycles in J-line: Lesions at the POA. The oviposition remained regular. Others are the same as Fig. 4.
FIG. 8. Oviposition cycles in T-line: Lesions at the posterodorsal part of the INC. The oviposition remained free-running. Others are the same as Fig. 4.
retinal photoreception, for the determination of ovulation time. Whereas under the same photoperiod, blinded quail also exhibited regular oviposition, suggesting the possibility that the deep photoreceptor can regulate timing of oviposition. In the present study, however, the activity of the POA was resistant to be influenced by the environmental light and dark. Thus, the INC which is known at the site of deep photoreceptors (Oliver et al., 1978; Ohta et al., 1984) may function as the major site for regulating the time of ovulation and the POA may be an endogenous circadian oscillator. After single lesions at the posterodorsal part of the INC, the resumption of egg production of the two lines was delayed significantly, in comparison with single lesions at the POA: the natural pattern of oviposition
of J-line was observed for several days before the regular pattern of oviposition became free-running. These results support that for egg production in the Japanese quail, the INC may play the pivotal role and GnRH neurons in the INC may be distributed extensively. An important question to be resolved concerns the control of the POA. In male quail, the POA is not photosensitive in itself, thus the retinal photoreceptor is thought to be involved in the control systems of the POA (Oliver and Bayle, 1977; Oliver et al., 1978; Homma et al., 1979; Ohta et al., 1984). In the present experiments, however, the retinal signals failed to entrain the activity of the POA. Experiments of hypothalamic deafferentation 0
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FIG. 9. Oviposition cycles in J-line: Lesions at the posterodorsal part of the INC. The oviposition was changed from regular to free-running. Others are the same as Fig. 4.
OVIPOSITION
CHANGES
FOLLOWING
clearly showed that the axons communicating between the POA and the INC were important for determining the basic pattern of oviposition (Maruyama et aZ., 1984) and regulatory neurons in the retina and anterior hypothalamus had neural connection to the posterior side of the INC (Ohta and Homma, 1987). Therefore, the afferent inputs from the INC to the POA may control the output of the POA: the afferent inputs from retina to the POA and/or the INC may be the subcontrol system for the adjustments of the POA. The possibility of communication between the POA and the INC via cerebrospinal fluid may be precluded because the natural patterns of oviposition were unchanged when the polymer was infused into the third ventricle. Additional research is needed to determine the precise roles of the POA and the INC in the control of reproductive activity in birds and to establish how these parts of hypothalamus are functionally linked. The SAP would be useful for these neuroendocrine studies. ACKNOWLEDGMENTS We would like to thank Dr. Hitoshi Aikawa, University of Tokyo, for the technical assistance and histological advice. A part of this research was financially supported by the grant from Scientific Research from the Ministry of Education, Science, and Culture of Japan No. 61760251 to M.O.
REFERENCES BaylC, J. D., Ramade, F., and Oliver, J. (1974). Stereotaxic topography of the brain of the quail (Coturnix coturnix japonica). J. Physiol. 68, 219241. Davies, D. T. (1980). The neuroendocrine control of gonadotrophin release in the Japanese quail. III. The role of the tuberal and anterior hypothalamus in the control of ovarian development and ovulation. Proc. R. Sot. London B 206,421-437. Davies, D. T., and Follett, B. K. (1975a). The neuroendocrine control of gonadotrophin release in the Japanese quail. I. The role of the tuberal hypothalamus. Proc. R. Sot. London B 191, 285-301. Davies, D. T., and Follett, B. K. (1975b). The neuro-
HYPOTHALAMIC
LESION
429
endocrine control of the gonadotrophin release in the Japanese quail. II. The role of the anterior hypothalamus. Proc. R. Sot. London 3 191, 303315. Duncan, D. B. (1955). Multiple range and multiple F tests. Biometrics 11, l-42. Egge, A. S., and Chiasson, R. B. (1963). Endocrine effects of diencephalic lesions in the white leghorn hen. Gen. Camp. Endocrinol. 3, 346-361. Follett, B. K. (1978). Photoperiodism and seasonal breeding in birds and mammals. In “Control of Ovulation” (D. B. Crighton, N. B. Haynes, G. R. Foxcroft, and G. E. Lamming, Eds.), pp. 267294, Butterworths, London. Homma, K., and Sakakibara, Y. (1971). Encephalic photoreceptors and their significance in photoperiodic control of sexual acitivity in Japanese quail. In “Biochronometry” (M. Menaker, Ed.), pp. 333-341. Natl. Acad. Sci., Washington, D.C. Homma, K., Ohta, M., and Sakakibara, Y. (1979). Photoinducible phase of the Japanese quail detected by direct stimulation of the brain. In “Biological Rhythms and their Central Mechanism” (M. Suds, 0. Hayashi, and H. Nakagawa, Eds.), pp. 85-94. Elsevier-North Holland, Amsterdam. Konishi, T. (1980). Circadian rhythm of ovipositional time in Japanese quail. In “Biological Rhythms in Birds” (Y. Tanabe, K. Tanaka, and T. Ookawa, Eds.), pp. 79-90. Japan Sci. Sot. Press, Tokyo/ Springer-Verlag, Berlin. Kramer, C. Y. (1956). Extension of multiple range tests to group means with unequal numbers of replications. Biometrics 12, 307-310. Maruyama, S., Ohta, M., and Homma, K. (1984). Spreading and clustering of oviposition pattern in Japanese quail after partial or complete hypothalamic deafferentation of the infundibular complex. Japan. Poultry Sci. 21, 301-312. Ohta, M., and Homma, K. (1987). Detection of neural connection to the infundibular complex by partial or complete hypothalamic deafferentation in male quail. ken. &np. Endocrinol. 68, 286-292. Ohta, M., Wada, M., and Homma, K. (1984). Induction of rapid testicular growth in Japanese quail by phasic electrical stimulation of the hypothalamic photosensitive area. J. Comp. Physiol. A 154, 583-589. Oliver, J., and Bay& J. D. (1976). The involvement of the preoptic-suprachiasmatic region in the photosexual reflex in quail. Effects of selective lesions or photic stimulations. .I. Physiol. 72, 627-637. Oliver, J., and Bayle, J. D. (1977). Preoptic multiunit activity correlates of the photosexual reflex in quail. Neurosci. Lett. 6, 317-322. Oliver, J., Bouillt, C., Herbutt, S., and Bay& J. D. (1978). Retrograde transport horseradish peroxidase from the preoptic anterior hypothalamic
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AND
region to retinal ganglion cells in quail. Neurosci. Lett. 9, 291-295. Ralph, C. L. (1959). Some effects of hypothalamic lesions on gonadotropin release in the hen. Anat. Rec. 134, 411-431. Ralph, C. L., and Fraps, R. M. (1959). Long-term effects of diencephalic lesions on the ovary of the hen. Amer. J. Physiol. 197, 1279-1283. Sharp, P. J. (1983). Hypothalamic control of gonadotrophin secretion in birds. In “Progress in Nonmammalian Brain Research” (G. Nistico and L.
HOMMA
Bolis, Eds.), Voi 3, pp. 124-176. CRC Press, Boca Raton, FL. Stetson, M. H. (1972). Hypothalamic regulation of gonadotropin release in female Japanese quail. Z. Zellforsch. Mikrosk. Anat. 130, 41 l-428. van Tienhoven, A., and Planck, R. J. (1973). The effect of light on avian reproductive activity. In “Handbook of Physiology, Section 7, Endocrinology, II, Part I” (R. 0. Creep and E. B. Astwood, Eds.), pp. 79-107. Amer. Physiol. Sot., Washington D.C.