Original Paper Biol Signals 1992;1:219-227
E. V. YoungLai3 E.C. Todoroff3 W.G. Foster3 G.M. Brown* Departments of Obstetrics and Gynecology, and Biomedical Sciences, McMastcr University, Health Sciences Centre, Hamilton, Ontario, Canada
Light-Related Melatonin Changes in Pituitary Response to Gonadotropin-Releasing Hormone during Sexual Development in the Female Rabbit
Abstract
Keywords
Melatonin GnRH LH FSH Female rabbit Puberty
Introduction
Sexual maturation is characterized by changes in the hypothalamic-pituitary axis re sulting in an increase in gonadotropin-releas
Received: April 7.1992 Accepted: June 3.1992
ing hormone (GnRH) secretion. The pro cesses involved in initiating sexual matura tion are complex and have been most thor oughly investigated in the rat. Hypothalamic content of GnRH [ I ] and pituitary content of
Dr. E.V. YoungLai Department of Obstetrics & Gynecology McMastcr University Faculty of Health Sciences Hamilton. Ontario, L8N3Z5 (Canada)
© 1992 S. Kargcr AG. Basel 1016-0922/92/ 0014-021952.75
Downloaded by: Stockholm University Library 130.237.165.40 - 11/2/2017 2:08:00 AM
................................................
Melatonin levels in the blood of female rabbits were deter mined from the time of weaning to adulthood in a longitudinal study. Blood samples were taken at 3 h after light onset and I h after dark onset and plasma was analysed for melatonin, LH and FSH by radioimmunoassay procedures. The animals were sacrificed on days 25, 32, 39, 51,72, 91 and 120 days of life and pituitaries removed for in vitro incubation of slices to determine their response to GnRH. Circulating melatonin lev els were significantly higher in the dark compared to the light phase and peaked on days 72 and 91 when gonadotropin levels were at a nadir. Melatonin levels on day 120 were lower than those on any other day examined. Basal secretion of LH and FSH by pituitary slices in vitro increased several-fold from day 25 of age to days 51-91 and then decreased by day 120. The pituitary gonadotropin responsiveness to GnRH in vitro was also different: while LH generally increased, FSH accu mulation remained constant after GnRH stimulation. These data suggest that the female rabbit pituitary undergoes changes in sensitivity to GnRH during sexual maturation and that the pineal gland may play a role in this process.
220
Materials and Methods Animals Female New Zealand White rabbits (n = 56) were obtained from local breeders. Animals arrived at 22 days of age and were housed under fixed lighting con ditions (12L: 12D) with food and water available ad libitum. Trunk blood was collected in heparinized tubes at specific times after the onset of light and darkness and stored at - 2 0 ° C until analyzed. Animals were then killed (somnitol 2 ml/kg) on postpartum days 25. 32, 3 9.51.72.91 and 120. Groups of 5-8 female rabbits of specified ages were sacrificed at 10 a.m. (lights on for 3 h) and 8 p.m. (lights off for 1 h). At 8 p.m.. animals were killed in complete darkness (red photosafe light). Culture Technique The anterior pituitarics were sliced at 350 pm us ing a Mcllwain Tissue Chopper. The slices were placed in cold phosphate-buffered saline (PBS) and carefully leased apart under a dissecting microscope using fine tweezers. Individual pituitary slices were then placed into separate wells of tissue culture plates containing 500 pi o f medium. Culture medium consisted of 0.5 ml Medium 199 (Gibco) supplemented with 0.01 .1/ Hepcs buffer (Sigma), glucose (500 mg/100 ml) and sodium pyruvate (0.001 M). In addition, experi mental culture medium contained 2 X I0"'' M GnRH (Factrel). Slices were incubated in a CCL incubator at 37 ° C in a water-saturated atmosphere o f 95 % 0 :/5 % CO;. The amount o f LH and FSH secreted into the medium was determined in 100-pl aliquots removed at 0. 60. 120 and 180 min of incubation. Each aliquot was replaced with 100 pi of fresh medium. Culture medium was diluted with buffer (1% bovine scrum albumin/PBS) and frozen at - 2 0 °C until determina tion of gonadotropins. Gonadotropin content was as sessed by established radioimmunoassay procedures (1 6 .17], The LH standard used was WP360A (Dr. A.F. Parlow). I ng of which is equivalent to 30 pg pure rab bit pituitary LH (EX130GB. Dr. L.E. Reichert). Guinea pig antirabbit LH. 6F GPaLH (Dr. R. Scaramuzi) was used as primary antibody. Purified ovine LH was used for iodination (LER-I056-C2) (Dr. L.E. Reichert). The limit of sensitivity of the LH assay was 42 pg and intra- and interassay coefficients of varia tion were 5.4 and 12.7%, respectively. The FSH was analysed by a homologous radioimmunoassay using reagents provided by Dr. A.F. Parlow. The limit of sen sitivity o f the assay was 300 pg and the intra- and inter-
YoungLai/Todorofl'/Foster/Brown
Melatonin in Female Rabbit Puberty
Downloaded by: Stockholm University Library 130.237.165.40 - 11/2/2017 2:08:00 AM
luteinizing hormone (LH) and follicle-stimu lating hormone (FSH) increase with age [2], Studies in the female rat show that the pattern of LH and FSH response to GnRH also changes during sexual development. Maximal LFI and FSH response to GnRH occurs be tween 10 and 15 days in the female rat fol lowed by a decline prior to first ovulation [1, 3-7], Peak responses to GnRH have been cor related with maximal GnRH receptor bind ing. suggesting a mechanism by which pitu itary responsiveness is altered during sexual maturation [8]. Unlike the rat. the mechanisms leading to sexual maturation in the female rabbit are ill defined. Puberty occurs at about 100 days of age [9. 10] and is associated with reduction of levels of pituitary gonadotropins [11. 12], There is evidence that opioidergic neurons may have a role in controlling puberty since opioids have effects depending on the devel opmental changes in circulating gonadotropin levels [13.14], The in vivo response to GnRH is also age-related [15], While photoperiod can influence seasonal reproduction and pu berty in the wild [9], the influence of light on sexual maturation in the domestic rabbit has not hitherto been investigated. The present study was therefore conducted to determine whether the female rabbit dis plays any photoperiodic differences in the in vitro responsiveness to GnRH and whether the age-related changing patterns of gonado tropin release are related to circulating mela tonin levels. While it could be argued that the rabbit being a reflex ovulator is not a suitable model, the hiatus of gonadotropin secretion between days 72 and 100 corresponds to a similar period during childhood in man and could provide further insight into the univer sality of biological phenomena.
Table 1. Circulating levels o f melatonin. LH and FSH during sexual development in female rabbits from day 25 to day 120 of life (n = 3—7)
Day
25 32 39 51 72 91 120
Melatonin (pg/ml)
LH (ng/ml)
a.m.
p.m.
a.m.
p.m.
a.m.
p.m.
22.98 ± 12.14° 42.68 ±7.76 52.52 + 6.09 39.76 ±13.62 75.00 ± !6 .3 6 b 77.28 ± 2! ,84b 6.67±2.58c
114.80 ± 16.95a 95.27± 26.08 85.78 ±3.23 117.76±32.75 160.54 ±38.17 189.23 ±37.92c 67.22 ±16.75b
0.26 ±0.09 0.50 ±0.14 t .45 ±0.54 2.28 ±1.04 0.24 ±0.03 0.55 ±0.11 1.83 ±0.84
0.37 ±0.30 0.53 ±0.16 l.t0 ± 0 .3 5 1.32 ±0.41 0.48 ±0.17 0.41 ±0.06 1.10 ± 0.44
2.50±0.38a 8.01 ±0.75b 11.00± l.22c 11.89 ± 2.25c 1.18 ± 0.09d 1.16 ±0.23 2.36 ±0.74
1.97 ± 0.29u 7.51 ± l.3 3 b 8.19± l.46b 17.89 ± 6.82b 1.35 ±0.50“ 0.85 ±0.05 3.15 ± 1.73
FSH (ng/ml)
assay coefficients of variation were 6.8 and 16 .1%. respectively. Plasma samples were analysed directly. Incubation media were further diluted with I % BSA/ PBS before analysis. Final results from media were corrected for dilutions at each sampling period. Melatonin was measured by radioimmunoassay (CIDtech Research Inc.) using the method of Grota et al. [ 18] which had less than I % cross-reactivity with a number of melatonin analogues tested. The limit of sensitivity of the assay was 5 pg with intra- and interas say coefficients of variation of 6.7 and 17.4%, respec tively. Statistical analysis was by ANOVA in conjunction with Student's t test using the MINITAB statistical software (Minitab Inc.. Pa.)
Results
Circulating Levels o f Melatonin, LH and FSH Table 1 lists the hormone levels found at different ages in the female rabbit. Melatonin levels were significantly elevated in the dark compared to light (Anova F = 33.57, d.f. = 1, p = 0.000). Analysis of variance for the day light samples gave F = 5.12. d.f. = 6, p = 0.001 and for dark F = 2.37, d.f. = 6. p = 0.055. Melatonin levels at 120 days during the light phase were significantly depressed from those
at 72 (p = 0.015) and 91 (p = 0.049) days and day 72 was higher than day 25 (p = 0.029). In the dark, day 120 levels of melatonin were lower than those on day 25 (p = 0.043) and day 91 (p = 0.042). LH and FSH patterns showed the normal peak values around day 5!. Only FSH levels were significantly ele vated, F = 14.58, d.f. = 6. p = 0.000 (Anova) for daylight and F = 8.09, d.f. = 6, p = 0.000 (Anova) for dark. There was no significant difference between a.m. and p.m. samples for LH (F = 0.46. d.f. = 1. p = 0.501) and FSH (F = 0.0. d.f. = 1, p = 0.96) although LH tended to be lower in the dark at the peak period on days 40 and 51. As shown in figure 1, there appears to be a temporal negative correlation between levels of melatonin and gonadotropins. In the light phase, the mean melatonin levels and mean LH levels had an r value of -0.43 and r for mean melatonin versus FSH was-0.11. In the dark, the corresponding r values were -0.568 for LH and -0.279 for FSH. The most consis tent negative correlation was found between days 51 and 72 and 91 and 120. The mean gonadotropin levels were better correlated: for LH/FSH in the light, r = 0.608 and dark r =
221
Downloaded by: Stockholm University Library 130.237.165.40 - 11/2/2017 2:08:00 AM
Values with different superscripts were significantly different from each other by t test.
Pituitary LH Secretion in Response to GnRH Incubation of female rabbit anterior pitu itary slices with 2 X 10~9 M GnRH caused a significant increase in LH accumulation in
the medium for rabbits aged 25, 39, 72 (light) and days 25, 32 and 39 (dark) (fig. 2). In tissue from animals at 120 days, the overall LH release, both stimulatory and unstimulatory, decreased from those at day 91. There was a significant depression in endogenous secre tion of LH from pituitary slices of rabbits aged 39, 51 and 72 days during the dark phase compared to the light phase (p < 0.05, t test). Analysis of variance indicated that there was
Fig. 1. Peripheral levels o f mel atonin, LH and FSH in female rab bit blood from day 25 to day 120 of life, a During the light phase, b During the dark phase (n = 3-7). Significant differences are noted in table 1.
222
YoungLai/Todoroff/Foster/Brown
Melatonin in Female Rabbit Puberty
Downloaded by: Stockholm University Library 130.237.165.40 - 11/2/2017 2:08:00 AM
0.743. High levels of melatonin in both the light and dark phases were associated with low levels of gonadotropins but were not sta tistically significant.
Pituitary FSH Secretion in Response to GnRH Figure 3 shows the FSH response to GnRH in vitro. Stimulation of FSH accumulation occurred in both light and dark on days 25 and in the light on day 72 (p < 0.008. t test). There was a significant suppression of FSH accumulation in both light and dark on day 91 (p < 0.05, t test) and no significant difference in endogenous secretion between light and dark phases (p = 0.222, d.f. = 1, F = 1.51,
SSS IH-C(Ught)
■ ■ lH-GnRH(Light)
§SS8 LH-C(Dork)
gg
LH-GnRH(0orl
E. V. YoungLai3 E.C. Todoroff3 W.G. Foster3 G.M. Brown* Departments of Obstetrics and Gynecology, and Biomedical Sciences, McMastcr University, Health Sciences Centre, Hamilton, Ontario, Canada
Light-Related Melatonin Changes in Pituitary Response to Gonadotropin-Releasing Hormone during Sexual Development in the Female Rabbit
Abstract
Keywords
Melatonin GnRH LH FSH Female rabbit Puberty
Introduction
Sexual maturation is characterized by changes in the hypothalamic-pituitary axis re sulting in an increase in gonadotropin-releas
Received: April 7.1992 Accepted: June 3.1992
ing hormone (GnRH) secretion. The pro cesses involved in initiating sexual matura tion are complex and have been most thor oughly investigated in the rat. Hypothalamic content of GnRH [ I ] and pituitary content of
Dr. E.V. YoungLai Department of Obstetrics & Gynecology McMastcr University Faculty of Health Sciences Hamilton. Ontario, L8N3Z5 (Canada)
© 1992 S. Kargcr AG. Basel 1016-0922/92/ 0014-021952.75
Downloaded by: Stockholm University Library 130.237.165.40 - 11/2/2017 2:08:00 AM
................................................
Melatonin levels in the blood of female rabbits were deter mined from the time of weaning to adulthood in a longitudinal study. Blood samples were taken at 3 h after light onset and I h after dark onset and plasma was analysed for melatonin, LH and FSH by radioimmunoassay procedures. The animals were sacrificed on days 25, 32, 39, 51,72, 91 and 120 days of life and pituitaries removed for in vitro incubation of slices to determine their response to GnRH. Circulating melatonin lev els were significantly higher in the dark compared to the light phase and peaked on days 72 and 91 when gonadotropin levels were at a nadir. Melatonin levels on day 120 were lower than those on any other day examined. Basal secretion of LH and FSH by pituitary slices in vitro increased several-fold from day 25 of age to days 51-91 and then decreased by day 120. The pituitary gonadotropin responsiveness to GnRH in vitro was also different: while LH generally increased, FSH accu mulation remained constant after GnRH stimulation. These data suggest that the female rabbit pituitary undergoes changes in sensitivity to GnRH during sexual maturation and that the pineal gland may play a role in this process.
220
Materials and Methods Animals Female New Zealand White rabbits (n = 56) were obtained from local breeders. Animals arrived at 22 days of age and were housed under fixed lighting con ditions (12L: 12D) with food and water available ad libitum. Trunk blood was collected in heparinized tubes at specific times after the onset of light and darkness and stored at - 2 0 ° C until analyzed. Animals were then killed (somnitol 2 ml/kg) on postpartum days 25. 32, 3 9.51.72.91 and 120. Groups of 5-8 female rabbits of specified ages were sacrificed at 10 a.m. (lights on for 3 h) and 8 p.m. (lights off for 1 h). At 8 p.m.. animals were killed in complete darkness (red photosafe light). Culture Technique The anterior pituitarics were sliced at 350 pm us ing a Mcllwain Tissue Chopper. The slices were placed in cold phosphate-buffered saline (PBS) and carefully leased apart under a dissecting microscope using fine tweezers. Individual pituitary slices were then placed into separate wells of tissue culture plates containing 500 pi o f medium. Culture medium consisted of 0.5 ml Medium 199 (Gibco) supplemented with 0.01 .1/ Hepcs buffer (Sigma), glucose (500 mg/100 ml) and sodium pyruvate (0.001 M). In addition, experi mental culture medium contained 2 X I0"'' M GnRH (Factrel). Slices were incubated in a CCL incubator at 37 ° C in a water-saturated atmosphere o f 95 % 0 :/5 % CO;. The amount o f LH and FSH secreted into the medium was determined in 100-pl aliquots removed at 0. 60. 120 and 180 min of incubation. Each aliquot was replaced with 100 pi of fresh medium. Culture medium was diluted with buffer (1% bovine scrum albumin/PBS) and frozen at - 2 0 °C until determina tion of gonadotropins. Gonadotropin content was as sessed by established radioimmunoassay procedures (1 6 .17], The LH standard used was WP360A (Dr. A.F. Parlow). I ng of which is equivalent to 30 pg pure rab bit pituitary LH (EX130GB. Dr. L.E. Reichert). Guinea pig antirabbit LH. 6F GPaLH (Dr. R. Scaramuzi) was used as primary antibody. Purified ovine LH was used for iodination (LER-I056-C2) (Dr. L.E. Reichert). The limit of sensitivity of the LH assay was 42 pg and intra- and interassay coefficients of varia tion were 5.4 and 12.7%, respectively. The FSH was analysed by a homologous radioimmunoassay using reagents provided by Dr. A.F. Parlow. The limit of sen sitivity o f the assay was 300 pg and the intra- and inter-
YoungLai/Todorofl'/Foster/Brown
Melatonin in Female Rabbit Puberty
Downloaded by: Stockholm University Library 130.237.165.40 - 11/2/2017 2:08:00 AM
luteinizing hormone (LH) and follicle-stimu lating hormone (FSH) increase with age [2], Studies in the female rat show that the pattern of LH and FSH response to GnRH also changes during sexual development. Maximal LFI and FSH response to GnRH occurs be tween 10 and 15 days in the female rat fol lowed by a decline prior to first ovulation [1, 3-7], Peak responses to GnRH have been cor related with maximal GnRH receptor bind ing. suggesting a mechanism by which pitu itary responsiveness is altered during sexual maturation [8]. Unlike the rat. the mechanisms leading to sexual maturation in the female rabbit are ill defined. Puberty occurs at about 100 days of age [9. 10] and is associated with reduction of levels of pituitary gonadotropins [11. 12], There is evidence that opioidergic neurons may have a role in controlling puberty since opioids have effects depending on the devel opmental changes in circulating gonadotropin levels [13.14], The in vivo response to GnRH is also age-related [15], While photoperiod can influence seasonal reproduction and pu berty in the wild [9], the influence of light on sexual maturation in the domestic rabbit has not hitherto been investigated. The present study was therefore conducted to determine whether the female rabbit dis plays any photoperiodic differences in the in vitro responsiveness to GnRH and whether the age-related changing patterns of gonado tropin release are related to circulating mela tonin levels. While it could be argued that the rabbit being a reflex ovulator is not a suitable model, the hiatus of gonadotropin secretion between days 72 and 100 corresponds to a similar period during childhood in man and could provide further insight into the univer sality of biological phenomena.
Table 1. Circulating levels o f melatonin. LH and FSH during sexual development in female rabbits from day 25 to day 120 of life (n = 3—7)
Day
25 32 39 51 72 91 120
Melatonin (pg/ml)
LH (ng/ml)
a.m.
p.m.
a.m.
p.m.
a.m.
p.m.
22.98 ± 12.14° 42.68 ±7.76 52.52 + 6.09 39.76 ±13.62 75.00 ± !6 .3 6 b 77.28 ± 2! ,84b 6.67±2.58c
114.80 ± 16.95a 95.27± 26.08 85.78 ±3.23 117.76±32.75 160.54 ±38.17 189.23 ±37.92c 67.22 ±16.75b
0.26 ±0.09 0.50 ±0.14 t .45 ±0.54 2.28 ±1.04 0.24 ±0.03 0.55 ±0.11 1.83 ±0.84
0.37 ±0.30 0.53 ±0.16 l.t0 ± 0 .3 5 1.32 ±0.41 0.48 ±0.17 0.41 ±0.06 1.10 ± 0.44
2.50±0.38a 8.01 ±0.75b 11.00± l.22c 11.89 ± 2.25c 1.18 ± 0.09d 1.16 ±0.23 2.36 ±0.74
1.97 ± 0.29u 7.51 ± l.3 3 b 8.19± l.46b 17.89 ± 6.82b 1.35 ±0.50“ 0.85 ±0.05 3.15 ± 1.73
FSH (ng/ml)
assay coefficients of variation were 6.8 and 16 .1%. respectively. Plasma samples were analysed directly. Incubation media were further diluted with I % BSA/ PBS before analysis. Final results from media were corrected for dilutions at each sampling period. Melatonin was measured by radioimmunoassay (CIDtech Research Inc.) using the method of Grota et al. [ 18] which had less than I % cross-reactivity with a number of melatonin analogues tested. The limit of sensitivity of the assay was 5 pg with intra- and interas say coefficients of variation of 6.7 and 17.4%, respec tively. Statistical analysis was by ANOVA in conjunction with Student's t test using the MINITAB statistical software (Minitab Inc.. Pa.)
Results
Circulating Levels o f Melatonin, LH and FSH Table 1 lists the hormone levels found at different ages in the female rabbit. Melatonin levels were significantly elevated in the dark compared to light (Anova F = 33.57, d.f. = 1, p = 0.000). Analysis of variance for the day light samples gave F = 5.12. d.f. = 6, p = 0.001 and for dark F = 2.37, d.f. = 6. p = 0.055. Melatonin levels at 120 days during the light phase were significantly depressed from those
at 72 (p = 0.015) and 91 (p = 0.049) days and day 72 was higher than day 25 (p = 0.029). In the dark, day 120 levels of melatonin were lower than those on day 25 (p = 0.043) and day 91 (p = 0.042). LH and FSH patterns showed the normal peak values around day 5!. Only FSH levels were significantly ele vated, F = 14.58, d.f. = 6. p = 0.000 (Anova) for daylight and F = 8.09, d.f. = 6, p = 0.000 (Anova) for dark. There was no significant difference between a.m. and p.m. samples for LH (F = 0.46. d.f. = 1. p = 0.501) and FSH (F = 0.0. d.f. = 1, p = 0.96) although LH tended to be lower in the dark at the peak period on days 40 and 51. As shown in figure 1, there appears to be a temporal negative correlation between levels of melatonin and gonadotropins. In the light phase, the mean melatonin levels and mean LH levels had an r value of -0.43 and r for mean melatonin versus FSH was-0.11. In the dark, the corresponding r values were -0.568 for LH and -0.279 for FSH. The most consis tent negative correlation was found between days 51 and 72 and 91 and 120. The mean gonadotropin levels were better correlated: for LH/FSH in the light, r = 0.608 and dark r =
221
Downloaded by: Stockholm University Library 130.237.165.40 - 11/2/2017 2:08:00 AM
Values with different superscripts were significantly different from each other by t test.
Pituitary LH Secretion in Response to GnRH Incubation of female rabbit anterior pitu itary slices with 2 X 10~9 M GnRH caused a significant increase in LH accumulation in
the medium for rabbits aged 25, 39, 72 (light) and days 25, 32 and 39 (dark) (fig. 2). In tissue from animals at 120 days, the overall LH release, both stimulatory and unstimulatory, decreased from those at day 91. There was a significant depression in endogenous secre tion of LH from pituitary slices of rabbits aged 39, 51 and 72 days during the dark phase compared to the light phase (p < 0.05, t test). Analysis of variance indicated that there was
Fig. 1. Peripheral levels o f mel atonin, LH and FSH in female rab bit blood from day 25 to day 120 of life, a During the light phase, b During the dark phase (n = 3-7). Significant differences are noted in table 1.
222
YoungLai/Todoroff/Foster/Brown
Melatonin in Female Rabbit Puberty
Downloaded by: Stockholm University Library 130.237.165.40 - 11/2/2017 2:08:00 AM
0.743. High levels of melatonin in both the light and dark phases were associated with low levels of gonadotropins but were not sta tistically significant.
Pituitary FSH Secretion in Response to GnRH Figure 3 shows the FSH response to GnRH in vitro. Stimulation of FSH accumulation occurred in both light and dark on days 25 and in the light on day 72 (p < 0.008. t test). There was a significant suppression of FSH accumulation in both light and dark on day 91 (p < 0.05, t test) and no significant difference in endogenous secretion between light and dark phases (p = 0.222, d.f. = 1, F = 1.51,
SSS IH-C(Ught)
■ ■ lH-GnRH(Light)
§SS8 LH-C(Dork)
gg
LH-GnRH(0orl
