- Email: [email protected]
Inhibin in the Hen1
Inhibin in the Hen1 P. A. JOHNSON
ABSTRACT The role of the gonadal hormone inhibin has not been extensively examined in the hen. A bovine radioimmunoassay kit has been used to assess plasma levels of immunoreactive inhibin throughout the ovulatory cycle, in response to follicle removal, and after stimulation of follicle growth. Homologous chicken radioimmunoassays for luteinizing hormone (LH) and follicle-stimulating hormone (FSH) were used to assay plasma gonadotropins. No significant peak of immunoreactive inhibin was detected throughout the ovulatory cycle in spite of a preovulatory surge of LH. Removal of preovulatory follicles caused a decrease of plasma immunoreactive inhibin whereas stimulation of follicle growth with equine chorionic gonadotropin increased plasma immunoreactive inhibin levels. Associated with the fall in inhibin level upon follicle removal is an acute rise in FSH with no concomitant change in LH. (Key words: reproduction, inhibin, ovulation, follicle, follicle-stimulating hormone) 1993 Poultry Science 72:955-958
INTRODUCTION
Mammalian inhibin is a dimeric protein hormone produced by the ovary and testis and composed of an a- (molecular weight 18,000) and a /3-subunit (molecular weight 14,000). The /3-subunit exists in two forms (/3A and /3g), which are distinct but quite similar. Therefore, depending upon the 0subunit involved, inhibin exists as inhibinA or inhibin-B. Both subunits, when joined by disulfide bonds, are required for biological activity in suppressing folliclestimulating hormone (FSH) secretion from the pituitary (Ling et al., 1988). Interestingly, combination of two /3-subunits gives rise to the hormone activin, a substance found to release FSH from the pituitary (Ling et al, 1988). In addition to its role in suppressing FSH, inhibin has been shown to exert paracrine effects within the ovary (Woodruff et al, 1990).
Received for publication August 3, 1992. Accepted for publication December 31, 1992. iThis work was supported in part by a grant from the Cornell Biotechnology Program and by USDA grant Number 90-37240-5716.
There are few reports characterizing inhibin in birds. Initial studies indicated that chicken testes preparations were effective in selectively reducing the postcastration rise in FSH in adult male rats (Bandivdekar et al, 1982, 1984). More recently, Lewis et al. (1989) reported that inhibin activity (as measured by heterologous RIA) decreased in roosters after castration and that the mRNA for the asubunit of inhibin hybridized with a human a-inhibin cDNA. Johnson and Wang (1993) have found hybridization between mRNA from granulosa cells and the porcine a-inhibin cDNA. Two studies have investigated the bioactivity of chicken ovarian inhibin. Both of these studies demonstrated a selective suppression of FSH when granulosa cell conditioned medium (charcoal-stripped) was incubated with rat (Akashiba et al, 1988) or sheep (Tsonis et al, 1988) anterior pituitary cells. Akashiba et al. (1988) demonstrated a somewhat greater amount of biologically active inhibin in the granulosa of the Fi follicle as compared with that of the F3 follicle. In the study by Tsonis et al. (1988), both granulosa and theca were tested and although theca cell
955
Downloaded from http://ps.oxfordjournals.org/ at Ernst Mayr Library of the Museum Comp Zoology, Harvard University on March 12, 2015
Department of Animal Science, 202 Morrison Hall, Cornell University, Ithaca, New York 14853
956
JOHNSON
conditioned medium suppressed FSH, it was less potent than granulosa cell conditioned medium.
Figure 1A - Time 0
Figure 1B - Time +6h
Inhibin
WMwh FSH
a
»
•H b
i
• ^1
Sham
^H *
F,
u i—^^^^^^^^|
In these studies, Single Comb White Leghorn hens in their 1st yr of lay were used. A bovine RIA kit for inhibin containing reagents developed by Robertson et al (1988) was obtained as a gift from the National Institute of Child Health and Human Development of the National Institutes of Health and was validated for use with chicken plasma (Johnson et al, 1993). Plasma luteinizing hormone (LH) was assayed using a RIA for chicken LH (Sharp et al, 1987) and FSH was also assayed with a homologous chicken FSH RIA (Krishnan et al, in press). For surgical removal of follicles, a laparotomy was performed on the left side below the ribs. Sham-operated animals served as controls. Plasma levels of inhibin were assessed throughout the ovulatory cycle of five hens. Blood samples were collected from five hens at 2-h intervals beginning at 1600 h on the day of lay of the last egg of the sequence through the following (pause) day. No significant peak of immunoreactive inhibin was detected throughout the cycle in spite of a distinct preovulatory peak of LH at 2400 h (Johnson et al, 1993). In sheep, the association between increased inhibin level and preovulatory follicular development has been somewhat controversial. Some workers have shown increased inhibin secretion during the follicular phase (Findlay et al, 1990) and others have demonstrated little change (Mann et al, 1989; Campbell et al, 1990). This observation of minimal change has suggested that inhibin may function as a more long-term regulator of FSH levels as compared to estradiol (Mann et al, 1989). This may be the case in the hen. Removal of three or four of the largest preovulatory follicles was shown to significantly decrease plasma inhibin level by 6 h after removal and this depression was maintained through 24 h. In addition, enhancement of follicular growth with
F,- F2
F,- F,
Follicle Removal
FIGURE 1. A. Plasma levels (X ± SE) of immunoreactive inhibin and follicle-stimulating hormone (FSH, nanograms per milliliter) at Time 0 relative to surgery in hens with various numbers of preovulatory follicles removed. No differences in levels of inhibin or FSH were detected among the groups at this time. B. Plasma levels (x ± SE) of immunoreactive inhibin and FSH (nanograms per milliliter) at +6 h relative to surgery in hens with various numbers of preovulatory follicles removed. Means with no common letters are different (P < .05). Plasma levels of immunoreactive inhibin were progressively lowered with increasing numbers of follicles removed. Plasma levels of FSH were significantly increased at +6 h in all groups with foUicles removed as compared to the sham-operated group (indicated by asterisk).
equine chorionic gonadotropin (75 IU/ day) significantly increased the plasma concentration of inhibin concomitant with increased follicle growth 0ohnson et al, 1993). In order to determine more precisely the contributions of individual folli-
Downloaded from http://ps.oxfordjournals.org/ at Ernst Mayr Library of the Museum Comp Zoology, Harvard University on March 12, 2015
INTERRELATIONSHIP AMONG INHIBIN, FOLLICLE-STIMULATING HORMONE, AND LUTEINIZING HORMONE IN THE HEN
SYMPOSIUM: CURRENT ADVANCES IN REPRODUCTION
ACKNOWLEDGMENTS
The author is grateful to Gabe Bialy of the National Institute of Child Health and Human Development of the National Institutes of Health for the inhibin RIA kit and to Peter Sharp (Edinburgh, Scotland) and John Proudman (USDA Animal Hormone Program) for the reagents for the LH and FSH RIA, respectively.
REFERENCES Akashiba, H., K. Taya, S. Sasamoto, H. Goto, M. Kamiyoshi, and K. Tanaka, 1988. Secretion of inhibin by chicken granulosa cells in vitro. Poultry Sci. 67:1625-1631. Bandivdekar, A. H., S. Vijayalakshmi, S. B. Moodbidri, and A. R. Sheth, 1982. Low-molecularweight inhibin from sheep, human, rat and chicken testes. J. Androl. 3:140-143. Bandivdekar, A. H., S. B. Moodbidri, and A. R. Sheth, 1984. FSH receptor binding inhibiting activity associated with low molecular weight inhibin from sheep, human, rat and chicken. Experientia 40:994-995. Campbell, B. K., G. E. Mann, A. S. McNeilly, and D. T. Baird, 1990. The pattern of ovarian inhibin, estradiol and androstenedione secretion during the estrous cycle of the ewe. Endocrinology 127: 227-235. Findlay, J. K., I. J. Clarke, and D. M. Robertson, 1990. Inhibin concentrations in ovarian and jugular venous plasma and the relationship of inhibin with follicle-stimulating hormone and luteinizing hormone during the ovine estrous cycle. Endocrinology 126:528-535. Johnson, P. A., and S.-Y. Wang, 1993. Characterization and quantitation of mRNA for the inhibin a-subunit in the granulosa layer of the domestic hen. Gen. Comp. Endocrinol, (in press). Johnson, P. A., S.-Y. Wang, and C. Brooks, 1993. Characterization of a source and levels of plasma immunoreactive inhibin during the ovulatory cycle of the domestic hen. Biol. Reprod. 48:262-267. Krishnan, K. A., J. A. Proudman, D. J. Bolt, and J. M. Bahr, 1993. Development of an homologous radioimmunoassay for chicken folliclestimulating hormone and measurement of plasma FSH during the ovulatory cycle. Comp. Biochem. Physiol, (in press). Lewis, W. M., K. M. Muster, T. L. Martin, J.L.H. Ireland, S. Bursian, R. Balander, A. J. Mason, and J. J. Ireland, 1989. Evidence for inhibin in roosters. Biol. Reprod. 40(Suppl. l):109.(Abstr.) Ling, N., N. Ueno, S.-Y. Ying, F. Esch, S. Shimasaki, M. Hotta, P. Cuevas, and R. Guillemin, 1988. Inhibins and activins. Vitam. Horm. 44:1-46. Mann, G. E., A. S. McNeilly, and D. T. Baird, 1989. Source of ovarian inhibin secretion during the estrous cycle of the sheep. J. Endocrinol. 123: 181-188. Robertson, D. M., M. Giacometti, L. M. Foulds, J. Lahnstein, N. K. Goss, M.T.W. Hearn, and D. M. de Kretser, 1989. Isolation of inhibin a-subunit precursor proteins from bovine follicular fluid. Endocrinology 125:2141-2149. Robertson, D. M., C. G. Tsonis, R. I. McLachlan, D. J. Handelsman, R. Leask, D. T. Baird, A. S. McNeilly, S. Hayward, D. L. Healy, J. K. Findlay, H. G. Burger, and D. M. de Kretser, 1988. Comparison of inhibin immunological and in vitro biological activities in human serum. J.
Downloaded from http://ps.oxfordjournals.org/ at Ernst Mayr Library of the Museum Comp Zoology, Harvard University on March 12, 2015
cles to plasma inhibin, the largest preovulatory follicles were selectively removed. Animals were sham-operated or the Fi, Fx + F2, Fx + F2 + F3, or Fx + F 2 + F 3 + F4 follicles were moved (n = four or five per group). Figure 1A depicts the plasma inhibin and FSH levels in all groups of hens at Time 0, immediately prior to surgery. In Figure IB, these same parameters are depicted at +6 h after surgery. No difference in plasma inhibin or FSH was observed among the groups at Time 0. At +6 h, however, it is seen that increasing the number of follicles removed caused a progressive decline in plasma inhibin. Associated with follicle removal, regardless of the number of follicles removed, was a rise in plasma FSH. The LH values (data not shown) were not different following follicle removal. The antibody in the RIA used to measure immunoreactive inhibin in the hen was made against the intact inhibin molecule. It has been found to crossreact with pro-a- and free a-subunit (Robertson et al., 1990). The results of studies in the hen showed that plasma inhibin level progressively declined with increasing numbers of follicles removed but plasma FSH did not change in a related fashion. It is possible that the Fj follicle is releasing biologically active inhibin and the less mature follicles are secreting free asubunit. Further experiments will be necessary to test this question. The present studies demonstrate that the large preovulatory follicles are a source of immunoreactive inhibin in the hen. Furthermore, associated with a fall in inhibin level is an acute rise in FSH with no concomitant change in LH.
957
958
JOHNSON
Clin. Endocrinol. Metab. 67:438-443. Sharp, P. J., I. C. Dunn, and R. T. Talbot, 1987. Sex differences in the LH responses to chicken LHRH-I and -II in the domestic fowl. J. Endocrinol. 115:323-331. Tsonis, C. G., P. J. Sharp, and A. S. McNeilly, 1988. Inhibin bioactivity and pituitary cell mitogenic
activity from cultured chicken ovarian granulosa and thecal/stromal cells. J. Endocrinol. 116:293-299. Woodruff, T. K., R. J. Lyon, S. E. Hansen, G. C. Rice, and J. P. Mather, 1990. Inhibin and activin locally regulate rat ovarian folliculogenesis. Endocrinplogy 127:3196-3205. Downloaded from http://ps.oxfordjournals.org/ at Ernst Mayr Library of the Museum Comp Zoology, Harvard University on March 12, 2015
ABSTRACT The role of the gonadal hormone inhibin has not been extensively examined in the hen. A bovine radioimmunoassay kit has been used to assess plasma levels of immunoreactive inhibin throughout the ovulatory cycle, in response to follicle removal, and after stimulation of follicle growth. Homologous chicken radioimmunoassays for luteinizing hormone (LH) and follicle-stimulating hormone (FSH) were used to assay plasma gonadotropins. No significant peak of immunoreactive inhibin was detected throughout the ovulatory cycle in spite of a preovulatory surge of LH. Removal of preovulatory follicles caused a decrease of plasma immunoreactive inhibin whereas stimulation of follicle growth with equine chorionic gonadotropin increased plasma immunoreactive inhibin levels. Associated with the fall in inhibin level upon follicle removal is an acute rise in FSH with no concomitant change in LH. (Key words: reproduction, inhibin, ovulation, follicle, follicle-stimulating hormone) 1993 Poultry Science 72:955-958
INTRODUCTION
Mammalian inhibin is a dimeric protein hormone produced by the ovary and testis and composed of an a- (molecular weight 18,000) and a /3-subunit (molecular weight 14,000). The /3-subunit exists in two forms (/3A and /3g), which are distinct but quite similar. Therefore, depending upon the 0subunit involved, inhibin exists as inhibinA or inhibin-B. Both subunits, when joined by disulfide bonds, are required for biological activity in suppressing folliclestimulating hormone (FSH) secretion from the pituitary (Ling et al., 1988). Interestingly, combination of two /3-subunits gives rise to the hormone activin, a substance found to release FSH from the pituitary (Ling et al, 1988). In addition to its role in suppressing FSH, inhibin has been shown to exert paracrine effects within the ovary (Woodruff et al, 1990).
Received for publication August 3, 1992. Accepted for publication December 31, 1992. iThis work was supported in part by a grant from the Cornell Biotechnology Program and by USDA grant Number 90-37240-5716.
There are few reports characterizing inhibin in birds. Initial studies indicated that chicken testes preparations were effective in selectively reducing the postcastration rise in FSH in adult male rats (Bandivdekar et al, 1982, 1984). More recently, Lewis et al. (1989) reported that inhibin activity (as measured by heterologous RIA) decreased in roosters after castration and that the mRNA for the asubunit of inhibin hybridized with a human a-inhibin cDNA. Johnson and Wang (1993) have found hybridization between mRNA from granulosa cells and the porcine a-inhibin cDNA. Two studies have investigated the bioactivity of chicken ovarian inhibin. Both of these studies demonstrated a selective suppression of FSH when granulosa cell conditioned medium (charcoal-stripped) was incubated with rat (Akashiba et al, 1988) or sheep (Tsonis et al, 1988) anterior pituitary cells. Akashiba et al. (1988) demonstrated a somewhat greater amount of biologically active inhibin in the granulosa of the Fi follicle as compared with that of the F3 follicle. In the study by Tsonis et al. (1988), both granulosa and theca were tested and although theca cell
955
Downloaded from http://ps.oxfordjournals.org/ at Ernst Mayr Library of the Museum Comp Zoology, Harvard University on March 12, 2015
Department of Animal Science, 202 Morrison Hall, Cornell University, Ithaca, New York 14853
956
JOHNSON
conditioned medium suppressed FSH, it was less potent than granulosa cell conditioned medium.
Figure 1A - Time 0
Figure 1B - Time +6h
Inhibin
WMwh FSH
a
»
•H b
i
• ^1
Sham
^H *
F,
u i—^^^^^^^^|
In these studies, Single Comb White Leghorn hens in their 1st yr of lay were used. A bovine RIA kit for inhibin containing reagents developed by Robertson et al (1988) was obtained as a gift from the National Institute of Child Health and Human Development of the National Institutes of Health and was validated for use with chicken plasma (Johnson et al, 1993). Plasma luteinizing hormone (LH) was assayed using a RIA for chicken LH (Sharp et al, 1987) and FSH was also assayed with a homologous chicken FSH RIA (Krishnan et al, in press). For surgical removal of follicles, a laparotomy was performed on the left side below the ribs. Sham-operated animals served as controls. Plasma levels of inhibin were assessed throughout the ovulatory cycle of five hens. Blood samples were collected from five hens at 2-h intervals beginning at 1600 h on the day of lay of the last egg of the sequence through the following (pause) day. No significant peak of immunoreactive inhibin was detected throughout the cycle in spite of a distinct preovulatory peak of LH at 2400 h (Johnson et al, 1993). In sheep, the association between increased inhibin level and preovulatory follicular development has been somewhat controversial. Some workers have shown increased inhibin secretion during the follicular phase (Findlay et al, 1990) and others have demonstrated little change (Mann et al, 1989; Campbell et al, 1990). This observation of minimal change has suggested that inhibin may function as a more long-term regulator of FSH levels as compared to estradiol (Mann et al, 1989). This may be the case in the hen. Removal of three or four of the largest preovulatory follicles was shown to significantly decrease plasma inhibin level by 6 h after removal and this depression was maintained through 24 h. In addition, enhancement of follicular growth with
F,- F2
F,- F,
Follicle Removal
FIGURE 1. A. Plasma levels (X ± SE) of immunoreactive inhibin and follicle-stimulating hormone (FSH, nanograms per milliliter) at Time 0 relative to surgery in hens with various numbers of preovulatory follicles removed. No differences in levels of inhibin or FSH were detected among the groups at this time. B. Plasma levels (x ± SE) of immunoreactive inhibin and FSH (nanograms per milliliter) at +6 h relative to surgery in hens with various numbers of preovulatory follicles removed. Means with no common letters are different (P < .05). Plasma levels of immunoreactive inhibin were progressively lowered with increasing numbers of follicles removed. Plasma levels of FSH were significantly increased at +6 h in all groups with foUicles removed as compared to the sham-operated group (indicated by asterisk).
equine chorionic gonadotropin (75 IU/ day) significantly increased the plasma concentration of inhibin concomitant with increased follicle growth 0ohnson et al, 1993). In order to determine more precisely the contributions of individual folli-
Downloaded from http://ps.oxfordjournals.org/ at Ernst Mayr Library of the Museum Comp Zoology, Harvard University on March 12, 2015
INTERRELATIONSHIP AMONG INHIBIN, FOLLICLE-STIMULATING HORMONE, AND LUTEINIZING HORMONE IN THE HEN
SYMPOSIUM: CURRENT ADVANCES IN REPRODUCTION
ACKNOWLEDGMENTS
The author is grateful to Gabe Bialy of the National Institute of Child Health and Human Development of the National Institutes of Health for the inhibin RIA kit and to Peter Sharp (Edinburgh, Scotland) and John Proudman (USDA Animal Hormone Program) for the reagents for the LH and FSH RIA, respectively.
REFERENCES Akashiba, H., K. Taya, S. Sasamoto, H. Goto, M. Kamiyoshi, and K. Tanaka, 1988. Secretion of inhibin by chicken granulosa cells in vitro. Poultry Sci. 67:1625-1631. Bandivdekar, A. H., S. Vijayalakshmi, S. B. Moodbidri, and A. R. Sheth, 1982. Low-molecularweight inhibin from sheep, human, rat and chicken testes. J. Androl. 3:140-143. Bandivdekar, A. H., S. B. Moodbidri, and A. R. Sheth, 1984. FSH receptor binding inhibiting activity associated with low molecular weight inhibin from sheep, human, rat and chicken. Experientia 40:994-995. Campbell, B. K., G. E. Mann, A. S. McNeilly, and D. T. Baird, 1990. The pattern of ovarian inhibin, estradiol and androstenedione secretion during the estrous cycle of the ewe. Endocrinology 127: 227-235. Findlay, J. K., I. J. Clarke, and D. M. Robertson, 1990. Inhibin concentrations in ovarian and jugular venous plasma and the relationship of inhibin with follicle-stimulating hormone and luteinizing hormone during the ovine estrous cycle. Endocrinology 126:528-535. Johnson, P. A., and S.-Y. Wang, 1993. Characterization and quantitation of mRNA for the inhibin a-subunit in the granulosa layer of the domestic hen. Gen. Comp. Endocrinol, (in press). Johnson, P. A., S.-Y. Wang, and C. Brooks, 1993. Characterization of a source and levels of plasma immunoreactive inhibin during the ovulatory cycle of the domestic hen. Biol. Reprod. 48:262-267. Krishnan, K. A., J. A. Proudman, D. J. Bolt, and J. M. Bahr, 1993. Development of an homologous radioimmunoassay for chicken folliclestimulating hormone and measurement of plasma FSH during the ovulatory cycle. Comp. Biochem. Physiol, (in press). Lewis, W. M., K. M. Muster, T. L. Martin, J.L.H. Ireland, S. Bursian, R. Balander, A. J. Mason, and J. J. Ireland, 1989. Evidence for inhibin in roosters. Biol. Reprod. 40(Suppl. l):109.(Abstr.) Ling, N., N. Ueno, S.-Y. Ying, F. Esch, S. Shimasaki, M. Hotta, P. Cuevas, and R. Guillemin, 1988. Inhibins and activins. Vitam. Horm. 44:1-46. Mann, G. E., A. S. McNeilly, and D. T. Baird, 1989. Source of ovarian inhibin secretion during the estrous cycle of the sheep. J. Endocrinol. 123: 181-188. Robertson, D. M., M. Giacometti, L. M. Foulds, J. Lahnstein, N. K. Goss, M.T.W. Hearn, and D. M. de Kretser, 1989. Isolation of inhibin a-subunit precursor proteins from bovine follicular fluid. Endocrinology 125:2141-2149. Robertson, D. M., C. G. Tsonis, R. I. McLachlan, D. J. Handelsman, R. Leask, D. T. Baird, A. S. McNeilly, S. Hayward, D. L. Healy, J. K. Findlay, H. G. Burger, and D. M. de Kretser, 1988. Comparison of inhibin immunological and in vitro biological activities in human serum. J.
Downloaded from http://ps.oxfordjournals.org/ at Ernst Mayr Library of the Museum Comp Zoology, Harvard University on March 12, 2015
cles to plasma inhibin, the largest preovulatory follicles were selectively removed. Animals were sham-operated or the Fi, Fx + F2, Fx + F2 + F3, or Fx + F 2 + F 3 + F4 follicles were moved (n = four or five per group). Figure 1A depicts the plasma inhibin and FSH levels in all groups of hens at Time 0, immediately prior to surgery. In Figure IB, these same parameters are depicted at +6 h after surgery. No difference in plasma inhibin or FSH was observed among the groups at Time 0. At +6 h, however, it is seen that increasing the number of follicles removed caused a progressive decline in plasma inhibin. Associated with follicle removal, regardless of the number of follicles removed, was a rise in plasma FSH. The LH values (data not shown) were not different following follicle removal. The antibody in the RIA used to measure immunoreactive inhibin in the hen was made against the intact inhibin molecule. It has been found to crossreact with pro-a- and free a-subunit (Robertson et al., 1990). The results of studies in the hen showed that plasma inhibin level progressively declined with increasing numbers of follicles removed but plasma FSH did not change in a related fashion. It is possible that the Fj follicle is releasing biologically active inhibin and the less mature follicles are secreting free asubunit. Further experiments will be necessary to test this question. The present studies demonstrate that the large preovulatory follicles are a source of immunoreactive inhibin in the hen. Furthermore, associated with a fall in inhibin level is an acute rise in FSH with no concomitant change in LH.
957
958
JOHNSON
Clin. Endocrinol. Metab. 67:438-443. Sharp, P. J., I. C. Dunn, and R. T. Talbot, 1987. Sex differences in the LH responses to chicken LHRH-I and -II in the domestic fowl. J. Endocrinol. 115:323-331. Tsonis, C. G., P. J. Sharp, and A. S. McNeilly, 1988. Inhibin bioactivity and pituitary cell mitogenic
activity from cultured chicken ovarian granulosa and thecal/stromal cells. J. Endocrinol. 116:293-299. Woodruff, T. K., R. J. Lyon, S. E. Hansen, G. C. Rice, and J. P. Mather, 1990. Inhibin and activin locally regulate rat ovarian folliculogenesis. Endocrinplogy 127:3196-3205. Downloaded from http://ps.oxfordjournals.org/ at Ernst Mayr Library of the Museum Comp Zoology, Harvard University on March 12, 2015