Influence of Gonadotropic Releasing Hormone, Luteinizing Hormone and Pineal Extracts on Testes of Immature Cockerels M. J. DARRE and P. C. HARRISON Department of Animal Science, University of Illinois at Urbana-Champaign, 124 Animal Sciences Laboratory, Urbana, Illinois 68101 (Received for publication April 6, 1977)
Poultry Science 56:2016-2021, 1977 INTRODUCTION
Progonadotropic or antigonadotropic functions have been attributed to the vertebrate pineal. Reiss et al. (1963) demonstrated that in immature or hypophysectomized adult rats, crude aqueous bovine pineal extracts stimulated genital growth, whereas in mature-intact rats an inhibitory effect was found. Balemans (1972) has also reported age-dependent effects of pineal compounds. Injections of pineal indolic compounds (5-methoxytryptophole and melatonin) into juvenile birds stimulated testis growth and comb growth, but in maturing and adult cocks both indolic compounds had inhibitory effects. Injection of melatonin-free extract of bovine pineals into adult rats on the day of unilateral ovariectomy resulted in inhibition of compensatory ovarian hypertrophy that was comparable to the antigonadotropic effects of 30 fig. of crystalline melatonin injected in the same manner(Benson et al., 1971). Sephadex separation of components of crude ovine pineal extracts by Ebels (1975) resulted in stimulatory and inhibitory fractions in the same pineal samples. The pineal has been demonstrated to modulate the action of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in animals with hypophyseal/gonadotropic hypofunction (Moszkowska and Ebels, 1971). White et al. (1974) found that crude extracts of ovine, bovine and porcine pineal glands contained 4 to 10 times as much gonadotropin-releasing activ-
ity as that reported in hypothalami of corresponding species. This led to the hypothesis that the pineal gland may also serve as a storage site for hypothalamic releasing hormones. The effects of gonadotropic-releasing hormone (GnRH) and crude extracts of avian pineals, alone or in combination, indicated that chicken pineal extracts inhibited the potency of synthetic GnRH for inducing premature ovulation in the fowl (Harrison et al., 1975). Our study was designed to determine the effects of an extract of pineals from mature hens, exogenous GnRH and LH, alone or in combination, on testicular uptake of P-32 in immature S.C.W.L. cockerels of different ages. MATERIALS AND METHODS Pineal glands from mature laying hens were collected and placed directly on dry ice at time of slaughter. Pineal homogenates were prepared in 0.1 N HC1 and adjusted to pH 7.0 with 0.1 N NaOH. The homogenate was centrifuged at 5800 X for 30 minutes and the supernatant was decanted and diluted to a dosage of 14 pineal equivalents by weight. Synthetic porcine GnRH was o b t a i n e d from Merck and Co. (MSD-L-361-050-OOB05) and synthetic porcine LH from the National Institutes of Health (NIH-LH-S-318) for use in our experiments. The GnRH and LH were administered subcutaneously, either alone or in combination with pineal extract (PE), into two-, seven-, and ten-day-old S.C.W.L. cockerels. Sixty minutes
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ABSTRACT Two, seven, and ten-day-old S.C.W.L. cockerels were injected with pineal extract (PE), gonadotropic releasing hormone (GnRH) and luteinizing hormone (LH), alone or in combination, to determine the effect of PE on the action of exogenous GnRH and LH on the testis in immature cockerels. Radioactive phosphorus (P-32) uptake by the testis was used as an assay for gonadotropic activity. GnRH had no effects on cockerels aged two days. However, LH caused increased P-32 uptake compared to the saline control at this age. In seven- and ten-day-old cockerels, PE in combination with GnRH resulted in lower P-32 c.p.m./g. of testis compared to the non-PE treated birds at the same doses of GnRH. On the other hand, PE did not cause any decrease in testis response to LH. These results indicate that PE is acting to inhibit release of gonadotropins by GnRH.
PINEAL EXTRACTS AND GONADOTROPINS
doses in combination with PE at the same dose as above. Another 60 ten-day-old cockerels were divided into six groups of 10 birds for the LH treatment. Three groups were given LH at 0 jUg., 10 ixg. and 20 jug., and the remaining three groups were injected with these doses plus PE at the same dose as above.
RESULTS
The gonad response (defined as P-32 uptake by the testis expressed as c.p.m./g. testes) of two-day-old cockerels to GnRH was not different from that of the saline control at any dose (Table 1). Although an apparently increased testis response to PE administered alone was found, the testes weights and total c.p.m. between the saline and saline plus PE treated groups were not different. GnRH appeared to Birds were fasted for 12 hours prior to first have no discernable effect on testis concentrainjections. All birds were kept in an environ- tion of P-32 in the two-day-old cockerels. LH mental chamber at 35 C. under a 12L:12D administered to the two-day-old cockerels inlighting regime. Individuals were injected in creased testis response (P<.05) compared to the random order and killed 18 hours after the first saline control when administered either alone or in combination with PE (Table 1). No injection. The first experiment consisted of 80 two- differences were found between the PE- and day-old cockerels divided into groups of 10 non-PE-treated birds at the same dose of LH injected with GnRH or GnRH plus PE. Four when the c.p.m./g. data were analyzed. Comgroups received GnRH at a dose of 0 ng., 25 pared to the saline controls, testis weight ng., 50 ng. and 75 ng., respectively. Another increased (P<.05), but no between-group differfour groups were given these same doses of ences at the same dose of LH were found. GnRH plus PE at a dose of 14 pineal equiva- Similar results were obtained when average lents. Another 80 two-day-old cockerels were c.p.m. data were analyzed. Pineal extract apdivided into groups of 10 for treatment with peared to have no direct influence on testis LH and LH plus PE. LH at 0 jug., 10 Mg-, 20 \xg. response to exogenous LH in two-day-old cockand 30 \xg. was administered to the other four erels. groups. In seven-day-old cockerels, GnRH plus PEIn a second experiment, 150 seven-day-old treated groups at 75 ng. and 100 ng. of GnRH cockerels were divided into 10 groups of 15 resulted in a lower testis response (P<.05) birds each. GnRH was administered to five compared to GnRH alone at the same doses groups at 0 ng., 25 ng., 50 ng., 75 ng. and 100 (Table 2). Exogenous LH injected into sevenng. The remaining five groups received these day-old cockerels resulted in an increased testis doses of GnRH plus PE at the same dose as response (P<.05) compared to the saline control and pineal extract did not have any effect above. A third experiment was conducted using 60 on this response. In ten-day-old cockerels, the seven-day-old cockerels to test the effects of maximum testis response to exogenous GnRH LH and LH plus PE. LH at 0 ;Ug., 10 /ig. and 20 was found at the 75 ng. dose (Table 3). GnRH l±%. was tested alone and in combination with in combination with PE resulted in lower PE at the same dose as above in groups of 10 (P<.05) testis responses at the 50 ng. and 75 ng. doses of GnRH compared to the GnRH birds. In the final experiment, 30 ten-day-old alone at these doses. Exogenous LH injected cockerels were divided into groups of five birds into ten-day-old cockerels resulted in a higher each. Three groups received GnRH at 0 ng., 50 (P<.05) testis response at 10 jig. and 20 ng. ng. and 75 ng., and three groups received these compared to the saline control.
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following these injections five juCi. of P-32, as orthophosphoric acid, was injected subcutaneously into each bird. All injections were given in a 1.0 ml. volume with 0.80% NaCl. solution as the injection vehicle. Phosphorus-32 uptake by the testis, as described by Breneman et al. (1962), was used to measure testis response to test solutions. Eighteen hours following these injections the birds were killed, the testes removed and paired weights determined. Testes were then solubilized in 1.0 ml. of Soluene 100 (Packard) for five hours, after which 10.0 ml. of Aquasol Scintillation Coctail (New England Nuclear) were added to each sample. The P-32 activity was counted with a Packard Tri-Carb liquid scintillation counter for five minutes. The results were expressed as P-32 counts per minute per gram of testis. Significance of differences was tested using Student's t test.
2017
LH
12.0 17.5 19.9 19.5
LH dose
OMg10 M g. 20 Mg. 30 Mg.
16.5 21.0 19.5 21.8
± ± ± ±
2.0 a t> 1.5 a 1.2 a 1.3ac
Values represent mean of ten birds and the s t a n d a r d error of t h e m e a n .
All PE t r e a t m e n t s received 14 pineal equivalents b y weight.
±0.72 ± 1.3a ± 1.6 a + 1.4 a
Indicates difference ( P < . 0 5 ) from non-PE-treated within dose.
i n d i c a t e s difference ( P < . 0 5 ) from saline plus PE c o n t r o l .
4.7 8.2 9.1 10.0
LH
LH + PE ±0.5 ± 0.7a ± 0.9a ± 1.0 a
pineal
±0.5 ± 1.2ac ± 0.4ac ±0.5C
4.0 6.5 7.4 7.5
±0.5 ±0.5ac ± 0.7ac ± 0.6ac
L H + PE
4.0 8.7 7.7 6.0
G n R H + PE
to GnRH, LH, and
Average testis weights (mg.)
cockerels
+ 0.5 ±0.3a ± 0.7a ± 0.4
4.7 7.6 7.0 5.0
16.5 ± 2.0*b 8.4 + 1 . 0 a c 11.0±0.5C 10.0 ± 0 . 6 a b e
of two-day-old
GnRH
responses
G n R H + PE
i n d i c a t e s difference ( P < . 0 5 ) from saline c o n t r o l .
1
1
12.0 10.6 11.0 12.9
Ong. 25 ng. 50 ng. 75 ng.
±0.7' ± 0.4 ±0.4 ± 1.0
GnRH
G n R H dose
Testis P-3 2 (c.p.m. X 10" 3 /g.)
T A B L E 1.—Gonadal
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3
2
1
Indicates difference (P-C05) from saline plus PE control.
Indicates difference (P<.05) from non-PE-treated within dose.
Indicates difference (P<.05) from saline control.
Values represent mean of ten birds ± the standard error of the mean.
Values represent mean of fifteen birds ± the standard error of the mean.
All PE treatments received 14 pineal equivalents by weight.
2 3 . 0 + 1.4 41.0 ± 3 . 1 a c 64.3 + 4 . 0 a c
25.1 + l . l 3 34.1 ± 0.4a 61.0 + 4 . 3 a
OMg10 Mg. 2 0 Mg.
0.1ab 0.2C 0.2C 0.1abc 0.1abc
LH + PE
± ± ± + ±
LH
1.9 2.9 2.6 2.2 2.3
LH dose
0.12 0.3a 0.2 0.2 0.1
G n R H + PE
2.8 3.4 3.0 2.8 3.0
± ± ± ± ±
GnRH
Ong. 25 ng. SOng. 75 ng. 1 0 0 ng.
G n R H dose
Testis P - 32 (c:.p.m X 1C»"3/g.)
±0.7 + 0.7 ± 1.4 + 1.1 ± 0.9
1 7 . 2 + 1.0 18.8 ± 6.0 1 9 . 6 + 1.2
LH
15.3 14.1 14.4 14.5 14.7
GnRH
19.0 ± 1.1 2 0 . 8 ± 1.4 a 1 8 . 8 + 1.2
LH + PE
17.8 + 0 . 8 a b 13.9±0.9C 14.6 ± l.OC 18.3 ± l . l a b 16.8 + 0.9
G n R H + PE
Average testis i weights (mg.)
TABLE 2.—Gonadal responses of seven-day-old cockerels to GnRH, LH, and pineal
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LH
22.5 ± 3.0 26.4 ± 2.6b 55.6 ± 5 . 6 a c
LH
2 1 . 9 ± 1.9 3 41.2 ± 3.9a 57.6 + 4 . 5 a
LH
3
2
1
Indicates difference (P<.05) from saline plus PE control.
Indicates difference (P<.05) from non-PE-treated within dose.
Indicates difference (P<.05) from saline control.
Values represent mean of ten birds ± the standard error of the mean.
Values represent mean of fifteen birds ± the standard error of the mean.
All PE treatments received 14 pineal equivalents by weight.
OMg10 ng. 2 0 Mg.
+ PE
3.6 ± 0.2 2.5 ± 0 . 2 a b c 3.7 ± 0.2b
3.6 + 0.2 2 3.8 + 0.3 4.6 ± 0.2^
Ong. 50 ng. 75 ng.
dose
G n R H + PE
GnRH
G n R H dose
Testis P— 32 (c .p.m. X 1Cr3/g.)
5.1 + 0.4 6.2 ± 0.4 5.2 + 0.6
LH
LH 4.5 ± 0.5 11.8 ± 0 . 6 a b c 4.6 + 0.7
+ PE
2 2 . 4 ± 3.6 27.1 ± 0 . 6 b 19.5 ± 1.3
GnRH + PE
Average testis weights (mg.)
cockerels to GnRH, LH, and pineal e
22.1 ± 2.1 18.0 ± 2.3 18.5 + 2.4
GnRH
TABLE 3.—Gonadal responses often-day-old
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PINEAL EXTRACTS AND GONADOTROPINS DISCUSSION
Lowered ( P < . 0 5 ) testis response of t h e G n R H + PE-treated seven- and ten-day-old cockerels suggests t h a t PE m a y interfere with t h e ability of G n R H t o cause release of LH from t h e pituitary, since t h e r e was n o effect on t h e response t o LH injected in these birds. Orts et al. ( 1 9 7 4 ) f o u n d t h a t crude extracts of rat pineals could inhibit t h e 24- t o 48-hour postcastration rise in serum LH in rats. T h e y f o u n d t h a t this LH inhibitory activity occurred in a melatonin-free extract. Blask and Reiter ( 1 9 7 5 ) reported t h a t pituitary LH levels were depressed in blind-anosmic rats. Pinealectomy, however, was effective in reversing these effects. Avian pineal extracts were f o u n d t o inhibit t h e p o t e n c y of synthetic G n R H for inducing p r e m a t u r e ovulation in t h e fowl (Harrison et al., 1 9 7 5 ) . Data presented here further substantiate t h e h y p o t h e s i s t h a t PE influences t h e p o t e n c y of G n R H induced release of LH from t h e pituitary. T h e mechanism of such a response has n o t been d e t e r m i n e d .
REFERENCES Balemans, M. G. M., 1972. Age dependent effects of 5-methoxytryptophol and melatonin on testis and comb growth of the White Leghorn. J. Neural Transm. 33:179-194. Benson, B., M. J. Mathews and A. E. Rodin, 1971. A melantonin-free extract of bovine pineal with antigonadotropic activity. Life Sci. 10(1):607—612. Blask, D. E., and R. J. Reiter, 1975. Pituitary and plasma LH and prolactin levels in female rats rendered blind and anosmic: influence of the pineal gland. Biol. Reprod. 12:329-334. Breneman, W. R., F. J. Zeller and R. O. Creek, 1962. Radioactive phosphorus uptake by chick testis as an endpoint for gonadotropin assay. Endocrinol. 71:790-798. Ebels, L, 1975. Pineal factors other than melatonin. Gen. Comp. Endocr. 25:189-198. Harrison, P. C , L. A. Cogburn and D. E. Brown, 1975. Influence of pineal extract on ovulation induction by luteinizing hormone/follicle stimulating hormone releasing hormone (LHRH/FSHRH. Poultry Sci. 54:1773. Moszkowska, A., and I. Ebels, 1971. The influence of the pineal body on the gonadotropic function of the hypophysis. J. Neural Visceral Rel. Supplement X:160-176. Orts, R. J., B. Benson and B. F. Cook, 1974. LH inhibitory properties of aqueous extracts of rat pineal glands. Life Sci. 14:1501-1510. Reiss, M., R. H. Davis, M. B. Sideman, I. Mauer and E. S. Plichta, 1963. Action of pineal extracts on the gonads and their function. J. Endocrinol. 27:107-118. Sharp, P. J., 1975. A comparison of variations in plasma luteinizing hormone concentrations in male and female domestic chickens (Gallus domesticus) from hatch to sexual maturity. J. Endocrinol. 67:211-223. White, W. F., M. T. Hedlund, G. F. Weber, R. H. Rippel, E. S. Johnson and J. F. Wilber, 1974. The pineal gland: a supplemental source of hypothalamic releasing hormones. Endocrinology 94:1422— 1426.
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Absence of a response t o exogenous G n R H in two-day-old cockerels and variability of response t o G n R H in seven-day-old cockerels m a y b e a t t r i b u t e d t o l o w levels of pituitary g o n a d o t r o p i n s available for release in birds of this age. This h y p o t h e s i s is s u p p o r t e d b y findings of Sharp ( 1 9 7 5 ) , in which t h e first peak of LH in t h e cockerel is at a b o u t seven t o ten days of age, before which very l o w levels of LH are found. These findings were tested in our seven- and ten-day-old cockerels. Increased testis response ( P < . 0 5 ) was f o u n d in G n R H treated groups a t b o t h ages. However, there was a response t o e x o g e n o u s LH a t all ages.
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