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Luteinizing Hormone Release and Androgen Production of Avian Hybrids in Response to Luteinizing Hormone Releasing Hormone Injection1
Luteinizing Hormone Release and Androgen Production of Avian Hybrids in Response to Luteinizing Hormone Releasing Hormone Injection1 G. F. MATHIS, W. H. BURKE, and L. R. McDOUGALD Department of Poultry Science, University of Georgia, Athens, Georgia 30602 (Received for publication April 27, 1982)
1983 Poultry Science 62:715-717
INTRODUCTION
MATERIALS AND METHODS
Male avian hybrids are not only sterile but often lack secondary sexual characteristics (Shaklee and Knox, 1954; Basrur, 1972; Admundson and Lorenz, 1957). Basrur (1969) hypothesized that this sterility in male avian hybrids was due to a genetic imbalance in spermatids, causing a failure of spermatogenesis. Pheasant-chicken hybrids conditioned to a long photoperiod had inactive testes and low testosterone levels compared to chickens and pheasants on the same treatment (Purohit et al, 1978). They concluded that sterility in this avian hybrid was due to incomplete meiosis and a deficiency of trophic hormones, which control the sexual processes such as production of testosterone. In this study we further examined sterility of male avian hybrids by determining: a) circulating levels of the pituitary trophic hormone, luteinizing hormone (LH); b) the pituitary gland's ability to release LH when stimulated by hypothalamic luteinizing hormone releasing hormone (LHRH); c) circulating androgen levels; and d) the testes ability to produce androgens when stimulated by LH.
Pooled semen from guinea fowl (Numida meleagris) was inseminated into White Leghorn hens (Gallus gallus domesticus) to produce five hybrids. These hybrids were battery brooded and later maintained in cages with continuous light until used for this study at 10 months of age. Peafowl-guinea fowl hybrids were produced by natural mating of a peacock and guinea hen. They were grown in an outside pen for 18 months with a natural photoperiod of 14 hr of daylight when the experiment was conducted. Five guinea fowl-chicken hybrids and four peafowl-guinea fowl were bled via the brachial vein and immediately given an intravenous injection of 10 /Ug of LHRH (Beckman) per bird. The experiment with the guinea fowlchicken hybrids was repeated after a 2-week interval using 4 of the 5 birds. Heparinized blood samples were then taken 10, 25, and 60 min after LHRH administration. Blood samples were centrifuged at 1,760 x g for 10 min, and plasma was removed and frozen until hormone assays were done. Luteinizing hormone was measured in samples taken from the first experiment with the guinea fowl-chicken hybrids and the peafowl-guinea fowl hybrids. Androgens were measured in the samples from the guinea fowl-chicken hybrids at both bleeding periods. The LH was determined by using a turkey LH assay (Burke et al, 1979; El Hala-
1 Supported by state and Hatch funds allocated to the Georgia Agricultural Experiment Stations of the University of Georgia.
715
Downloaded from http://ps.oxfordjournals.org/ at National Institute of Education Library, Serials Unit on June 18, 2015
ABSTRACT The levels of luteinizing hormone (LH) and androgens were measured in sterile avian hybrids. Guinea fowl-chicken and peafowl-guinea fowl hybrids were bled before and after injection with LH- releasing hormone (LHRH). The preinjection LH levels for the guinea fowlchicken hybrids were below or at the very lower limit of the assay sensitivity and the peafowlguinea fowl hybrids averaged 1.3 ng/ml. Within 10 min after LHRH injection, LH had increased dramatically in both hybrids and then began to slowly decline. Angrogen levels in the guinea fowl-chicken hybirds increased from 16.2 pg/ml to 95.2 pg/ml and continued to increase, reaching 287 pg/ml at the last bleeding 60 min after injection. (Key words: avian hybrids, androgens, luteinizing hormone, luteinizing hormone releasing hormone)
716
MATHIS ET AL.
RESULTS
Levels of LH in three of the five guinea fowl chicken hybrids were below the detection limits
of the RIA prior to LHRH injection and were at the very lower limit of the assays sensitivity in the other 2 birds (defined as 2 standard deviations below the mean of the 100% bound tubes, equivalent to .14 ng/ml based on 50 (A aliquots that were assayed). The mean is given in Table 1 as nondetectable (ND). Within 10 min of LHRH injection, LH levels had increased significantly to '7.48 ng/ml. They then fell during the following 50 min but still remained above basal values. Preinjection LH levels of the peafowl-guinea fowl hybrid averaged 1.3 ng/ml, and they increased to 7.46 ng/ml following LHRH injection. Levels fell at each subsequent bleeding time, reaching 2.64 ng/ml 1 hr after LHRH injection. The antiserum used for the "testosterone" assay shows substantial crossreaction with dihydrotestosterone (78%), androstenedione (30%), and androstendiol (13%) (N. G. Zimmerman, personal communication). Thus, the values we present should best be considered an estimate of "androgen" rather than "testosterone". Because we were interested in the responsiveness of the testes, these nonspecific values were satisfactory. The standard curve for the assay was run from 7.8 to 500 pg with a midpoint (50% bound) at 60 pg. The least detectable dose of hormone, taken as a sample with counts 2 standard deviations below the 100% bound tubes, was 5 pg. Recovery of testosterone in the ether extract averaged 67.7 ± 3.2% (X ± SD). Androgen levels were low in the guinea fowl-chicken cross before LHRH treatment, averaging 16.2 pg/ml. Within 10 min of LHRH treatment, androgen levels had increased to 95.2 pg/ml. They continued to increase, reach-
TABLE 1. Luteinizing hormone (LH) and androgens in blood of avian hybrids after LH-releasing hormone (LHRH) treatment Time after LHRH injection (min) Hybrid
Hormone
Guinea fowl-chicken Guinea fowl-chicken
LH (ng/ml) Androgen (pg/ml) LH (ng/ml)
Peafowl-guinea fowl
5 5
NDC> 16.2 ± 4.0a>'
4
1.3 ± .4 C
10
25
60
7.5+ .7a 95.2+17.5b
1.7+ .lb 213.0 ± 37.8 C
1.2+ .lb 287.0 ± 64.0 C
7.5 +
1.8a
4.2 ±
' ' Groups with same superscript letter are not significantly different (P<.05). 1
LH was undetectable in 3 of the 5 birds and barely detectable in 2 birds.
2
Values are means ± SE.
l.lc
2.6 ±
1.3 b c
Downloaded from http://ps.oxfordjournals.org/ at National Institute of Education Library, Serials Unit on June 18, 2015
wani et al, 1980a). Turkey LH lot B25B (Burke et al., 1979) was used as the standard and for iodination. Androgens were measured by radioimmunoassay (RIA) using antisera kindly provided by G. P. Birrenkott, Clemson University, and characterized by N. G. Zimmerman, University of Wisconsin,. Prior to assay, samples were extracted with diethyl ether. The ether extract was dried and then dissolved in phosphate buffered saline containing . 1 % gelatin. Extraction efficiency was determined on each sample by the addition of 3 H-testosterone several hours prior to extraction. Samples were incubated with antisera and testosterone tracer, then free testosterone was removed by use of dextran coated charcoal. After removal of charcoal by centrifugation, supernatants were poured into liquid scintillation vials, mixed with 10 ml of scintillation cocktail, and counted in a Packard liquid scintillation counter. Potency estimates were calculated using log-logit analysis and then corrections for recovery were made. The data of these studies were subjected to analysis of variance using the Statistical Analysis System (Barr et al., 1976); treatment means were compared by Duncan's multiple range tests (Duncan, 1955). Androgen values obtained from four of the guinea fowl-chicken hybrids from two separate trials were averaged for each individual at each time, and the average values were used in the statistical analysis. Values from the first trial were used for the fifth individual.
RESEARCH NOTE
ing levels of 213 pg/ml at 25 min and 287 pg/ml at 60 min. DISCUSSION
The peafowl-guinea fowl hybrids appear to have higher basal LH levels than the guinea fowl-chicken cross, however, caution must be exercised in this regard, because the samples were assayed at different times. It is clear that these hybrids also respond to LHRH injection by rapid LH release. These findings support those of Purohit et al. (1978) showing low testosterone levels in
another avian hybrid, the pheasant-chicken, and support their suggestion of deficiency of at least one of the trophic hormones, LH. One could speculate that central nervous system mechanisms for the regulation of trophic hormone release may be undeveloped. Because a functional hypothalamic noradrenergic system seems essential for gonadotrophin release in several avian species (El Halawani et al, 1980a,b), failure of maturation of this central nervous system component may be involved in the immature state of the hybrids. These findings do not refute the suggestion of Basrur (1969) that sterility in the avian hybrid is due to a genetic imbalance in spermatids, but they provide evidence that very low release of trophic hormones also contributes to gonadal underdevelopment. REFERENCES Asmundson, V. S., and F. W. Lorenz, 1957. Hybrids of ring-necked pheasant, turkeys, and domestic fowl. Poultry Sci. 36:1323-1334. Barr, A. J., J. H. Goodnight, J. P. Sail, and T. T. Helwig, 1976. A users guide to SAS 76. SAS Inst., Inc., Raleigh, NC. Basrur, P. K., 1969. Hybrid sterility. Pages. 107-131 in Comparative Mammalian Cytogenetics. K. Benirschke, ed. Springer-Verlag, New York, NY. Basrur, P. K., 1972. Differentiation of seminiferous epithelium in chicken-pheasant hybrid. Can. J. Genet. Cytol. 14:720. (Abstr.) Burke, W. H., P. Licht, H. Papkoff, and A. Bona Gallo, 1979. Isolation and characterization of luteinizing hormone and follicle-stimulating hormone from pituitary glands of the turkey (Meleagris gallopavo). Gen. Comp. Endocrinol. 37:508— 520. Duncan, D. B., 1955. Multiple range and multiple F tests. Biometrics 11:1-42. El Halawani, M. E., W. H. Burke, and L. A. Ogren, 1980a. Age-dependent changes in hypothalamic catecholamine turnover rate following castration in turkeys. Gen. Comp. Endocrinol. 42:290— 296. El Halawani, M. E., W. H. Burke, and L. A. Ogren, 1980b. Involvement of catecholaminergic mechanisms in the photoperiodically induced rise in serum luteinizing hormone of Japanese quail (Coturnix coturnix japonica). Gen. Comp. Endocrinol. 4 1 : 1 4 - 2 1 . Purohit, V. D., P. K. Basrur, and V. G. Smith, 1978. Testosterone levels in blood plasma of male chicken-pheasant hybrids. Poultry Sci. 57: 513-517. Shaklee, W. E., and C. W. Knox, 1954. Hybridization of the pheasant and fowl. J. Hered. 45:183-190.
Downloaded from http://ps.oxfordjournals.org/ at National Institute of Education Library, Serials Unit on June 18, 2015
The testes perform two main functions, production of spermatozoa and secretion of steroid hormones (predominantly testosterone). The production of spermatozoa is dependent on two trophic hormones from the pituitary, LH and follicle-stimulating hormone (FSH). Luteinizing hormone influences spermatogenesis indirectly by acting on the Leydig cells and stimulating androgen production and acts in concert with FSH to carry spermatogenesis to completion. The immature stage of the gonads in avian hybrids suggests a lack of stimulation by the pituitary hormones, a lack of sensitivity of the testes to the pituitary hormones, or both. The present studies were directed toward describing the endocrine status of the hybrids available to us. The very low levels of LH in the blood of guinea fowl-chicken hybrids taken together with the responsiveness of their pituitaries to stimulation suggest that their immature state may be due to failure of the hypothalamic-pituitary axis to mature. Although in this study we have used LHRH as the test for the pituitary's ability to release LH, we did not control for the possible nonspecific release of LH due to handling and injection. The time course and magnitude of the LH release in response to LHRH injection suggests that it, in fact, was a specific response. In either case, the pituitary of these hybrids clearly has the ability to release LH. The responsiveness of the Leydig cells to the LH released upon stimulation indicates that these cells will secrete androgens under appropriate trophic stimulation, again suggesting a failure of the reproductive system at some higher level of control.
717
1983 Poultry Science 62:715-717
INTRODUCTION
MATERIALS AND METHODS
Male avian hybrids are not only sterile but often lack secondary sexual characteristics (Shaklee and Knox, 1954; Basrur, 1972; Admundson and Lorenz, 1957). Basrur (1969) hypothesized that this sterility in male avian hybrids was due to a genetic imbalance in spermatids, causing a failure of spermatogenesis. Pheasant-chicken hybrids conditioned to a long photoperiod had inactive testes and low testosterone levels compared to chickens and pheasants on the same treatment (Purohit et al, 1978). They concluded that sterility in this avian hybrid was due to incomplete meiosis and a deficiency of trophic hormones, which control the sexual processes such as production of testosterone. In this study we further examined sterility of male avian hybrids by determining: a) circulating levels of the pituitary trophic hormone, luteinizing hormone (LH); b) the pituitary gland's ability to release LH when stimulated by hypothalamic luteinizing hormone releasing hormone (LHRH); c) circulating androgen levels; and d) the testes ability to produce androgens when stimulated by LH.
Pooled semen from guinea fowl (Numida meleagris) was inseminated into White Leghorn hens (Gallus gallus domesticus) to produce five hybrids. These hybrids were battery brooded and later maintained in cages with continuous light until used for this study at 10 months of age. Peafowl-guinea fowl hybrids were produced by natural mating of a peacock and guinea hen. They were grown in an outside pen for 18 months with a natural photoperiod of 14 hr of daylight when the experiment was conducted. Five guinea fowl-chicken hybrids and four peafowl-guinea fowl were bled via the brachial vein and immediately given an intravenous injection of 10 /Ug of LHRH (Beckman) per bird. The experiment with the guinea fowlchicken hybrids was repeated after a 2-week interval using 4 of the 5 birds. Heparinized blood samples were then taken 10, 25, and 60 min after LHRH administration. Blood samples were centrifuged at 1,760 x g for 10 min, and plasma was removed and frozen until hormone assays were done. Luteinizing hormone was measured in samples taken from the first experiment with the guinea fowl-chicken hybrids and the peafowl-guinea fowl hybrids. Androgens were measured in the samples from the guinea fowl-chicken hybrids at both bleeding periods. The LH was determined by using a turkey LH assay (Burke et al, 1979; El Hala-
1 Supported by state and Hatch funds allocated to the Georgia Agricultural Experiment Stations of the University of Georgia.
715
Downloaded from http://ps.oxfordjournals.org/ at National Institute of Education Library, Serials Unit on June 18, 2015
ABSTRACT The levels of luteinizing hormone (LH) and androgens were measured in sterile avian hybrids. Guinea fowl-chicken and peafowl-guinea fowl hybrids were bled before and after injection with LH- releasing hormone (LHRH). The preinjection LH levels for the guinea fowlchicken hybrids were below or at the very lower limit of the assay sensitivity and the peafowlguinea fowl hybrids averaged 1.3 ng/ml. Within 10 min after LHRH injection, LH had increased dramatically in both hybrids and then began to slowly decline. Angrogen levels in the guinea fowl-chicken hybirds increased from 16.2 pg/ml to 95.2 pg/ml and continued to increase, reaching 287 pg/ml at the last bleeding 60 min after injection. (Key words: avian hybrids, androgens, luteinizing hormone, luteinizing hormone releasing hormone)
716
MATHIS ET AL.
RESULTS
Levels of LH in three of the five guinea fowl chicken hybrids were below the detection limits
of the RIA prior to LHRH injection and were at the very lower limit of the assays sensitivity in the other 2 birds (defined as 2 standard deviations below the mean of the 100% bound tubes, equivalent to .14 ng/ml based on 50 (A aliquots that were assayed). The mean is given in Table 1 as nondetectable (ND). Within 10 min of LHRH injection, LH levels had increased significantly to '7.48 ng/ml. They then fell during the following 50 min but still remained above basal values. Preinjection LH levels of the peafowl-guinea fowl hybrid averaged 1.3 ng/ml, and they increased to 7.46 ng/ml following LHRH injection. Levels fell at each subsequent bleeding time, reaching 2.64 ng/ml 1 hr after LHRH injection. The antiserum used for the "testosterone" assay shows substantial crossreaction with dihydrotestosterone (78%), androstenedione (30%), and androstendiol (13%) (N. G. Zimmerman, personal communication). Thus, the values we present should best be considered an estimate of "androgen" rather than "testosterone". Because we were interested in the responsiveness of the testes, these nonspecific values were satisfactory. The standard curve for the assay was run from 7.8 to 500 pg with a midpoint (50% bound) at 60 pg. The least detectable dose of hormone, taken as a sample with counts 2 standard deviations below the 100% bound tubes, was 5 pg. Recovery of testosterone in the ether extract averaged 67.7 ± 3.2% (X ± SD). Androgen levels were low in the guinea fowl-chicken cross before LHRH treatment, averaging 16.2 pg/ml. Within 10 min of LHRH treatment, androgen levels had increased to 95.2 pg/ml. They continued to increase, reach-
TABLE 1. Luteinizing hormone (LH) and androgens in blood of avian hybrids after LH-releasing hormone (LHRH) treatment Time after LHRH injection (min) Hybrid
Hormone
Guinea fowl-chicken Guinea fowl-chicken
LH (ng/ml) Androgen (pg/ml) LH (ng/ml)
Peafowl-guinea fowl
5 5
NDC> 16.2 ± 4.0a>'
4
1.3 ± .4 C
10
25
60
7.5+ .7a 95.2+17.5b
1.7+ .lb 213.0 ± 37.8 C
1.2+ .lb 287.0 ± 64.0 C
7.5 +
1.8a
4.2 ±
' ' Groups with same superscript letter are not significantly different (P<.05). 1
LH was undetectable in 3 of the 5 birds and barely detectable in 2 birds.
2
Values are means ± SE.
l.lc
2.6 ±
1.3 b c
Downloaded from http://ps.oxfordjournals.org/ at National Institute of Education Library, Serials Unit on June 18, 2015
wani et al, 1980a). Turkey LH lot B25B (Burke et al., 1979) was used as the standard and for iodination. Androgens were measured by radioimmunoassay (RIA) using antisera kindly provided by G. P. Birrenkott, Clemson University, and characterized by N. G. Zimmerman, University of Wisconsin,. Prior to assay, samples were extracted with diethyl ether. The ether extract was dried and then dissolved in phosphate buffered saline containing . 1 % gelatin. Extraction efficiency was determined on each sample by the addition of 3 H-testosterone several hours prior to extraction. Samples were incubated with antisera and testosterone tracer, then free testosterone was removed by use of dextran coated charcoal. After removal of charcoal by centrifugation, supernatants were poured into liquid scintillation vials, mixed with 10 ml of scintillation cocktail, and counted in a Packard liquid scintillation counter. Potency estimates were calculated using log-logit analysis and then corrections for recovery were made. The data of these studies were subjected to analysis of variance using the Statistical Analysis System (Barr et al., 1976); treatment means were compared by Duncan's multiple range tests (Duncan, 1955). Androgen values obtained from four of the guinea fowl-chicken hybrids from two separate trials were averaged for each individual at each time, and the average values were used in the statistical analysis. Values from the first trial were used for the fifth individual.
RESEARCH NOTE
ing levels of 213 pg/ml at 25 min and 287 pg/ml at 60 min. DISCUSSION
The peafowl-guinea fowl hybrids appear to have higher basal LH levels than the guinea fowl-chicken cross, however, caution must be exercised in this regard, because the samples were assayed at different times. It is clear that these hybrids also respond to LHRH injection by rapid LH release. These findings support those of Purohit et al. (1978) showing low testosterone levels in
another avian hybrid, the pheasant-chicken, and support their suggestion of deficiency of at least one of the trophic hormones, LH. One could speculate that central nervous system mechanisms for the regulation of trophic hormone release may be undeveloped. Because a functional hypothalamic noradrenergic system seems essential for gonadotrophin release in several avian species (El Halawani et al, 1980a,b), failure of maturation of this central nervous system component may be involved in the immature state of the hybrids. These findings do not refute the suggestion of Basrur (1969) that sterility in the avian hybrid is due to a genetic imbalance in spermatids, but they provide evidence that very low release of trophic hormones also contributes to gonadal underdevelopment. REFERENCES Asmundson, V. S., and F. W. Lorenz, 1957. Hybrids of ring-necked pheasant, turkeys, and domestic fowl. Poultry Sci. 36:1323-1334. Barr, A. J., J. H. Goodnight, J. P. Sail, and T. T. Helwig, 1976. A users guide to SAS 76. SAS Inst., Inc., Raleigh, NC. Basrur, P. K., 1969. Hybrid sterility. Pages. 107-131 in Comparative Mammalian Cytogenetics. K. Benirschke, ed. Springer-Verlag, New York, NY. Basrur, P. K., 1972. Differentiation of seminiferous epithelium in chicken-pheasant hybrid. Can. J. Genet. Cytol. 14:720. (Abstr.) Burke, W. H., P. Licht, H. Papkoff, and A. Bona Gallo, 1979. Isolation and characterization of luteinizing hormone and follicle-stimulating hormone from pituitary glands of the turkey (Meleagris gallopavo). Gen. Comp. Endocrinol. 37:508— 520. Duncan, D. B., 1955. Multiple range and multiple F tests. Biometrics 11:1-42. El Halawani, M. E., W. H. Burke, and L. A. Ogren, 1980a. Age-dependent changes in hypothalamic catecholamine turnover rate following castration in turkeys. Gen. Comp. Endocrinol. 42:290— 296. El Halawani, M. E., W. H. Burke, and L. A. Ogren, 1980b. Involvement of catecholaminergic mechanisms in the photoperiodically induced rise in serum luteinizing hormone of Japanese quail (Coturnix coturnix japonica). Gen. Comp. Endocrinol. 4 1 : 1 4 - 2 1 . Purohit, V. D., P. K. Basrur, and V. G. Smith, 1978. Testosterone levels in blood plasma of male chicken-pheasant hybrids. Poultry Sci. 57: 513-517. Shaklee, W. E., and C. W. Knox, 1954. Hybridization of the pheasant and fowl. J. Hered. 45:183-190.
Downloaded from http://ps.oxfordjournals.org/ at National Institute of Education Library, Serials Unit on June 18, 2015
The testes perform two main functions, production of spermatozoa and secretion of steroid hormones (predominantly testosterone). The production of spermatozoa is dependent on two trophic hormones from the pituitary, LH and follicle-stimulating hormone (FSH). Luteinizing hormone influences spermatogenesis indirectly by acting on the Leydig cells and stimulating androgen production and acts in concert with FSH to carry spermatogenesis to completion. The immature stage of the gonads in avian hybrids suggests a lack of stimulation by the pituitary hormones, a lack of sensitivity of the testes to the pituitary hormones, or both. The present studies were directed toward describing the endocrine status of the hybrids available to us. The very low levels of LH in the blood of guinea fowl-chicken hybrids taken together with the responsiveness of their pituitaries to stimulation suggest that their immature state may be due to failure of the hypothalamic-pituitary axis to mature. Although in this study we have used LHRH as the test for the pituitary's ability to release LH, we did not control for the possible nonspecific release of LH due to handling and injection. The time course and magnitude of the LH release in response to LHRH injection suggests that it, in fact, was a specific response. In either case, the pituitary of these hybrids clearly has the ability to release LH. The responsiveness of the Leydig cells to the LH released upon stimulation indicates that these cells will secrete androgens under appropriate trophic stimulation, again suggesting a failure of the reproductive system at some higher level of control.
717