Modulation of Growth Hormone Pulsatility by Sex Steroids in Female Goats
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内分泌学杂志 2005年第6期
Department of Veterinary Physiology and Animal Resource Science Center, Veterinary Medical Science, The University of Tokyo, Tokyo 113-8657, Japan
Address all correspondence and requests for reprints to: Masugi Nishihara, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan. E-mail: amnishi@mail.ecc.u-tokyo.ac.jp.
Abstract
GH is secreted in a pulsatile manner, the pattern of which plays an important role in the regulation of growth and metabolism. Sex steroids are also known to participate in metabolic regulation. The present study was undertaken to elucidate the relationship between changes in GH pulsatility and metabolic transition during the estrous cycle in goats. From ovariectomized (OVX) and intact females in the early luteal, late luteal, and follicular phases, blood samples were taken every 15 min for 24 h, and plasma GH was measured by RIA. In the early luteal phase, GH was secreted in a distinct pulsatile manner, the pattern of which was similar to that in OVX goats, whereas the GH pulse frequency, amplitude, and area under the curve (AUC) were decreased in the late luteal phase. In the follicular phase, the GH pulse frequency, amplitude, and AUC were significantly larger than those in the late luteal phase. The regularity of GH pulsatility was highest and lowest in the early and late luteal phases, respectively. Both IGF-I and free fatty acid levels in the plasma were higher in the follicular than the luteal phase. Subcutaneous injection of estradiol to OVX goats increased the GH pulse amplitude and AUC, whereas the implantation of progesterone for 5 d decreased those parameters. These results suggest that the pulsatile pattern of GH secretion in goats varies with sex steroid levels and thereby affects IGF-I secretion and lipolysis during the estrous cycle.
Introduction
GH IS SECRETED IN A pulsatile manner, the pattern of which is sexually dimorphic in most mammalian species (1, 2). For example, male rats exhibit regular high amplitude GH pulses with relatively low interpulse GH levels (3), whereas females have lower amplitude pulses with higher interpulse levels (4, 5). These sex-specific patterns of GH secretion may be related to the expression of GH bioactivity. It has been shown that intermittent GH administration is more effective than continuous GH infusion in stimulating somatic growth (6, 7). In addition, we have shown that transgenic rats lacking distinct GH pulses exhibited fat accumulation and insulin resistance (8, 9, 10). Hence, the GH secretory patterns appear to play an important role in the regulation of metabolism as well as growth between the sexes.
The relationship between endogenous GH pulsatile patterns and sex steroids remains controversial. Previous studies demonstrated that estradiol stimulated GH release in sheep (11) and humans (12), whereas it decreased the pulse amplitude and increased basal GH levels in rats (13). The effect of progesterone on GH pulsatility has not been well understood. On the other hand, androgen administration increased the mean levels and pulse amplitude of GH in humans (14) and rats (15), respectively. We have previously shown, however, that castration of male goats increased the pulse amplitude without affecting interpulse interval and area under the curve of GH secretion (16). These observations suggest that sex steroids indeed affect the pulsatile pattern of GH secretion, but the effects may vary among species.
IGF-I is synthesized in not only the liver but also many other tissues and has potential paracrine and autocrine actions in response to GH. GH administration induces the increase of skeletal tissue IGF-I mRNA (17) and serum IGF-I levels (18). In particular, frequent treatments with GH are more effective in increasing serum IGF-I than bolus administration, even with the same amount of GH (19). Estradiol is also a potent stimulator of IGF-I gene expression in the uterus (20) and serum IGF-I levels (21). Moreover, IGF-I mRNA expression peaked at estrus in the muscle layers of the ovine uterus and oviducts (22). These findings suggest that locally produced IGF-I accounts for the changes in plasma IGF-I concentrations during the estrous cycle. However, the association between serum IGF-I levels and GH pulsatility during the estrous cycle has not been fully elucidated.
Although GH pulses are known to be susceptible to stress in general (23), we have previously shown that male Shiba goats exhibit strictly regular GH pulses of 5-h intervals over 24 h (16). They were Japanese native miniature goats of nonseasonal breed that had been bred as a closed colony for experimental uses (24) and were regarded as a suitable experimental model for the analysis of GH pulsatility (16). In the present study, to clarify the changes in GH pulsatility during the inherent estrous cycle, we characterized the episodic GH release and IGF-I concentrations in several phases of the estrous cycle of Shiba goats. To assess the role of the gonadal steroids in modulating GH pulsatility, we also examined the effects of estradiol and progesterone exposure on GH release in ovariectomized (OVX) goats.
Materials and Methods
Animals
Adult female Shiba goats (2–4 yr of age) with regular estrous cycles or OVX goats for at least 6 months were used in this study. During the experimental periods, they were loosely restrained by being tied to stanchions indoors and kept at a temperature of 23 ± 2 C with the lights on from 0600 to 1800 h (12-h light, 12-h dark cycle). They were allowed free access to food and water. All experiments were conducted according to the guidelines for the care and use of laboratory animals, the Graduate School of Agriculture and Life Sciences, University of Tokyo.
Experimental procedures
First, plasma estradiol and progesterone levels were determined during the estrous cycle using seven intact goats. Blood samples (5 ml) were drawn every other day from the jugular vein, and plasma was separated and stored at –20 C until assayed for estradiol and progesterone. Plasma IGF-I and free fatty acid (FFA) levels were also measured as described below. Based on the plasma levels of sex steroids, the estrous cycle was divided into three phases, i.e. the early luteal, late luteal, and follicular phases (1–8, 9–18, and 19–21 d after the estrous behavior, respectively; see Results).
To determine the characteristics of the GH pulses in each phase of the estrous cycle in intact goats (n = 5) and those in OVX goats (n = 5), blood samples (1 ml) were collected at 15-min intervals for 24 h (0600–0600 h) through indwelling jugular catheters (Argyle Medical Catheter, 18G and 70 cm in length, Nihon Sherwood, Tokyo, Japan). The light was kept on during blood sampling to calm the animals. After centrifugation, plasma was separated and stored at –20 C until it was assayed for GH. The next 24-h blood sampling was done after an interval of at least 6 d.
Next, the OVX goats (n = 5) bearing the chronic iv catheter were sc administered either 1 mg of estradiol (Sigma, St. Louis, MO) dissolved in sesame oil or sesame oil alone (1 ml) at 1200 h. Blood samples (1 ml) were collected at 15-min intervals from 6 h before to 18 h after the injection (0600–0600 h). A separate group of OVX goats (n = 5) were sc implanted with a SILASTIC-brand (Dow Corning, Midland, MI) packet (50 x 75 mm, wall thickness 3 mm) containing progesterone (Sigma) together with a short SILASTIC-brand tubing (3.35 mm inner diameter, 4.65 outer diameter, 20 mm length including 5 mm SILASTIC-brand glue on both sides) containing estradiol to mimic the steroidal milieu during the luteal phase (25). Control animals (n = 3) received identical capsules that contained cholesterol. All the implantation procedures were carried out under anesthesia with a bolus iv injection of a mixture of xylazine HCl and ketamine HCl. Five days after the implantation, blood samples were withdrawn every 15 min for 24 h (0600–0600 h). Plasma was separated by centrifugation and stored at –20 C until further use.
Hormone assays
Plasma GH concentrations were measured by double-antibody RIA using antibovine GH (bGH) antiserum. The parallelism between goat plasma and bGH has been reported previously (26). The bGH standard preparation and hormone for iodination were supplied by the National Institute of Diabetes and Digestive and Kidney Diseases (Bethesda, MD). Antiserum to bGH generated in monkeys was supplied by Dr. K. Hodate (Kitasato University, Towada, Japan). The minimum concentration of GH that could be detected was 1 ng/ml, and the maximum assay range was 512 ng/ml. Intra- and interassay coefficients of variance (CVs) for GH assay were 11.6 and 12.3%, respectively.
Plasma estradiol concentrations were measured with a commercial enzyme immunoassay (EIA) kit (Cayman Chemical, Ann Arbor, MI) according to the assay protocol supplied by the manufacturer. In this EIA, 1 ml plasma was extracted with 2 ml diethylether, and the extracts were redissolved in 200 μl of the buffer and then subjected to the EIA. The range of linearity was 1.6–200 pg. Intra- and interassay CVs were 9.8 and 8.5%, respectively. Plasma progesterone concentrations were measured by RIA using specific antibodies generated in our laboratory as described previously (27). Plasma (50 μl) was extracted with 2 ml diethylether and the extracts were assayed for progesterone. The range of linearity was 50–1600 pg, and the intra- and interassay CVs were 8.6 and 10.5%, respectively.
Plasma IGF-I concentrations were measured by the double-antibody RIA (17). The IGF-I standard preparation was purchased from Biomedical Technologies Inc. (Stoughton, MA). Radioiodinated IGF-I was purchased from Amersham Biosciences (Buckinghamshire, UK). Antiserum against IGF-I generated in rabbits was supplied by the NIDDK. The plasma samples were first extracted with an acid-ethanol solution according to the method described by Daughaday et al. (28). Samples were mixed with an acid-ethanol solution for 30 sec. After standing for 30 min at room temperature, the samples were centrifuged at 1860 x g for 30 min at 4 C. An aliquot of the supernatant from each sample was neutralized with Tris base. Intra- and interassay CVs for IGF-I assay were both 7.4%.
Plasma FFA concentrations were measured with a commercial assay kit using the acyl-CoA synthetase and acyl-CoA oxidase method (Wako Pure Chemical, Osaka, Japan). The assay was performed according to the assay protocol supplied by the manufacturer. The range of linearity was 0.1–2.0 mEq/liter, and the intraassay CV was 2.7% (this assay was performed only once).
Pulse analysis and statistics
GH pulses were identified by means of the PULSAR computer program (29). The cut-off criteria for pulse determination, G1, G2, G3, G4, and G5 were 4.40, 2.20, 1.92, 1.46, and 1.13, respectively. The parameters of the pulsatile GH calculated for each sampling session were as follows: 1) pulse frequency, the mean number of pulses per 24 or 6 h; 2) pulse amplitude, the mean height of all amplitudes derived from the difference between peak and baseline; 3) pulse width, the mean length of pulse peak criteria; 4) area under the curve (AUC), the mean of all GH values during the individual pulse peak criteria. Group comparisons were performed by Tukey-Kramer’s test or the unpaired t test.
In addition, the regularity of GH secretory pattern was determined by approximate entropy (ApEn) test. ApEn comprises a class of model- and scale-independent irregularity statistics designed to monitor the relative orderliness of patterns in individual time series (30, 31, 32). In the present study, we calculated ApEn based on r = 0.2 and m = 1. These parameter sets have been validated for a number of hormonal time series (30, 31, 32). Group comparisons were performed by Fisher’s protected least significant difference test (P < 0.05). Statistical significance was considered to be at P < 0.05.
Results
Plasma sex steroid levels during the estrous cycle in goats
The intact female goats used in this study had a regular estrous cycle of 21 d. Plasma estradiol and progesterone levels were measured throughout the estrous cycle, and according to the steroid levels, the estrous cycle was divided into three phases: the early luteal, late luteal, and follicular phase (1–8, 9–18, and 19–21 d after the estrous behavior, respectively). Plasma concentrations of estradiol and progesterone during the estrous cycle are presented in Fig. 1, and those in each phase are summarized in Table 1. Plasma estradiol concentrations in the follicular phase were markedly higher than those in the early and late luteal phases. Plasma progesterone levels in the late luteal phase were significantly higher than those in the early luteal and follicular phases.
FIG. 1. Changes in plasma progesterone and estradiol levels during the estrous cycle. Each square and vertical bar represents the mean and SEM (n = 7), respectively.
TABLE 1. Plasma concentration of sex steroids during the goat estrous cycle
Characteristics of GH pulses during the estrous cycle in goats
Representative 24-h plasma GH profiles in OVX and intact goats in each phase of the estrous cycle are shown in Fig. 2. Figure 3 illustrates the results of the characteristics of the GH pulses according to the PULSAR algorithm. In OVX goats, GH was secreted in a pulsatile manner, which involves the recurrence of obvious peaks followed by troughs of virtually no GH secretion. Plasma GH profiles in the early luteal phase did not significantly differ from those of OVX goats. In the late luteal phase, pulse amplitude and AUC were significantly decreased as compared with the early luteal phase, whereas the pulse frequency and width remained unchanged. On the other hand, in the follicular phase, the pulse frequency and AUC were significantly increased as compared with all the other groups. The pulse amplitude in the follicular phase was also significantly larger than that in the late luteal phase. The pulse width was not different among all the experimental groups. These data were also analyzed by ApEn test that quantifies the subpattern reproducibility of successive measurements. Higher ApEn denotes greater disorderliness or irregularity of patterns. As shown in Fig. 4, the regularity of GH pulsatility was highest in the early luteal phase and lowest in the late luteal phase during the estrous cycle.
FIG. 2. Individual representative 24 h plasma GH profiles in OVX and intact female goats of the early luteal (EL), late luteal (LL), and follicular (FO) phases of the estrous cycle. Open triangles indicate GH pulses identified by the PULSAR algorithm.
FIG. 3. Characteristics of GH pulses in OVX and intact female goats of the early luteal (EL), late luteal (LL), and follicular (FO) phases of the estrous cycle. Each column and vertical bar represents the mean and SEM (n = 5), respectively. Values with different superscripts are significantly different (P < 0.05).
FIG. 4. Regularity of GH pulsatility assessed by the ApEn test in the early luteal (EL), late luteal (LL), and follicular (FO) phases of the estrous cycle. Each circle and horizontal bar represents the individual data and the mean (n = 5), respectively. Values of the mean and SEM are also presented. Values with different superscripts are significantly different (P < 0.05).
Effects of sex steroids on GH pulses in OVX goats
The representative plasma GH profiles after estradiol injection to OVX goats are shown in Fig. 5A. Figure 5B presents the results of the GH pulse analysis using the PULSAR algorithm for every 6-h period. Plasma estradiol levels increased to 13.3 ± 1.9 pg/ml (mean ± SEM, n = 5) 1 h after the injection and remained high until 18 h after the injection (17.5 ± 2.2 pg/ml). Although the acute exposure to estradiol did not affect GH pulsatility during the first 6 h-period after treatment, it significantly increased the pulse amplitude and AUC in the second and third 6-h periods after treatment. The frequency and width of GH pulses, however, were not affected by estradiol injection. The injection of sesame oil alone did not affect any of the parameters of GH pulsatility (n = 3, data not shown).
FIG. 5. Changes in GH pulses after sc injection of estradiol (1 mg) in OVX goats. A, Representative plasma GH profiles after the injection of estradiol. Arrows indicate the time of injection. Open triangles indicate GH pulses identified by PULSAR algorithm. B, Characteristics of GH pulses during the 6 h before injection (Pre) and 0–6, 6–12, and 12–18 h after injection. Each column and vertical bar represents the mean and SEM (n = 5), respectively. *, P < 0.05 vs. Pre.
Figure 6A shows the representative plasma GH profiles for 24 h 5 d after the implantation of the progesterone-containing SILASTIC-brand capsule in OVX goats, and the results of the GH pulse analysis are presented in Fig. 6B. The plasma progesterone levels in goats implanted with progesterone on the day of the experiment were 8.6 ± 0.8 ng/ml (mean ± SEM, n = 5). The implantation of the progesterone-containing capsule plus a small estradiol implant significantly decreased the pulse amplitude and AUC as compared with implantation of the cholesterol-containing capsule, whereas it did not affect the frequency or the width of the GH pulses.
FIG. 6. Changes in GH pulses 5 d after the implantation of a SILASTIC-brand capsule containing cholesterol (Chol) or progesterone (P) plus a small estradiol implant in OVX goats. A, Representative plasma GH profiles after the implantation of Chol or P. Open triangles indicate GH pulses identified by PULSAR algorithm. B, Characteristics of GH pulses. Each column and vertical bar represents the mean and SEM (n = 3 for Chol, and 5 for P), respectively. *, P < 0.05 vs. Chol.
Plasma IGF-I and FFA levels during the estrous cycle in goats
The changes in the plasma IGF-I levels during the estrous cycle are shown in Fig. 7A. Although plasma IGF-I levels in the luteal phase remained low, they increased in the follicular phase. All possible comparisons were made through Tukey-Kramer test, and it was found that the peak level of plasma IGF-I on d 22 (the day of estrus) was significantly higher than those on d 4–18. The changes in the plasma FFA levels are shown in Fig. 7B, which were similar to those of the plasma IGF-I levels. The plasma FFA levels also peaked on d 22, which was significantly higher than those on d 8–12.
FIG. 7. Changes in plasma IGF-I (A) and FFA (B) levels during the estrous cycle. Each square and vertical bar represents the mean and SEM (n = 5), respectively. Values with different superscripts are significantly different (P < 0.05).
Discussion
The present study demonstrated that plasma GH profiles in female Shiba goats, either intact or OVX, exhibit distinct pulsatility consisting of conspicuous pulses separated by trough periods of virtually no GH secretion. In the previous reports of female rats and sheep, they secreted GH in an irregular pulsatile manner with high nadir or rather erratic interpulse intervals ranging from 3 to 6 h (1). There may be a species difference in the basal pattern of GH secretion in female animals. Alternatively, the possibility that the GH pulsatility in female goats is more resistant than those in females of other species to environmental factors such as stress cannot be ruled out. The present study also demonstrated for the first time that the pulsatility of GH secretion in female goats varies during the estrous cycle.
GH secretory profiles in the early luteal phase, when both estrogen and progesterone levels were low, were similar to those in OVX goats. All the parameters analyzed, i.e. pulse frequency, width, amplitude, and AUC, did not differ between the early luteal phase and the OVX goats. Then pulse frequency, amplitude, and AUC were markedly decreased in the late luteal phase when the progesterone levels were high. The regularity of GH pulsatility was also decreased in the late luteal phase. In contrast, in the follicular phase when plasma estradiol concentrations were elevated, the GH pulses were abruptly enhanced as for pulse frequency, amplitude, and AUC, and the regularity of pulsatility became higher. These changes in the characteristics of GH pulses were almost exactly repeated in two consecutive cycles for all three of the goats that were studied for more than one cycle (data not shown). Our current data confirmed in part the previous studies in humans (33) and sheep (34), in which the integrated serum GH concentrations in the late follicular phase were higher than those in the luteal phase. Although it has been shown that there was no significant change in GH pulse profiles during the estrous cycle in rats (5) and goats (35), female Shiba goats appear to exhibit considerable changes in GH pulsatility during the estrous cycle.
The sc injection of estradiol to OVX goats did not affect the GH pulses during the 6 h after the treatment, but thereafter it significantly increased the pulse amplitude and AUC, whereas the pulse frequency and width remained unchanged. This is consistent at least partially with the previous reports showing increased plasma GH levels after estradiol administration in several species including primates (21) and ruminants (11, 36). In contrast, the implantation of progesterone together with a small estradiol implant for 5 d decreased the same parameters of the GH pulses. The profiles of GH secretion after treatment with sex steroids are in accordance with the characteristics of GH pulses in each phase of the goat estrous cycle. It is therefore probable that the pulsatility of GH secretion during the inherent estrous cycle changes, depending on the suppressive effect of endogenous progesterone as well as the stimulatory effect of endogenous estradiol.
How sex steroids could modulate GH output remains poorly understood. Regulation of GH secretion is mainly under the control of two hypothalamic peptides, GHRH and somatostatin (SRIF), which interact reciprocally at the level of the pituitary to generate GH pulses (37, 38). Although estradiol decreased GHRH mRNA expression without affecting SRIF mRNA in rats (2), c-fos expression in both GHRH and SRIF neurons was increased in the follicular phase in sheep (39). Thus, it is currently difficult to correlate estrogen’s effects on the hypothalamic peptides and GH pulses. The possibility that estrogen affects GH pulsatility by directly acting on the pituitary also cannot be ruled out because transcription factor pituitary transcription factor-1, which is involved in the expression of GH mRNA, is up-regulated by estrogen (40). However, because the majority of GHRH and SRIF neurons, and also the pituitary somatotropes, do not express estrogen receptors (39, 41), the effect of estrogen on these cells, if any, may be indirect.
The amplitude and frequency of GH pulses are known to be important determinants of GH bioactivity (7). In the present study, both plasma IGF-I and FFA levels changed in a similar pattern during the estrous cycle and peaked on the day of estrus. Further studies in the OVX steroid-treated goats would be necessary to establish the ability of steroid treatments to acutely alter GH secretion and, subsequently, metabolism. However, considering the lipolytic action of GH (42), as well as the stimulatory action on IGF-I release, it is reasonable to speculate that the observed changes in plasma IGF-I and FFA levels well reflected the changes in GH pulsatility during the estrous cycle. It is therefore suggested that lipolysis is stimulated during the follicular phase when the GH pulses are robust, and the accumulation of fat occurs during the late luteal phase when the GH pulses are suppressed. Interestingly, it has also been shown in humans that free fatty acid concentrations are higher in the follicular than in the luteal phase (43). In addition to lipid metabolism, GH plays an important role in regulating reproductive function. It was recently reported that GH release preceded the LH surge (36) and acted as a cogonadotropin to induce gametogenesis and ovulation (44). Hence, the stimulation of GH pulses by estrogen may be involved in follicular development and the ovulatory processes. IGF-I also participates in reproductive cell replication and differentiation (45) as well as protein/carbohydrate metabolism, and IGF-I receptors are localized in the granulosa cells and epithelium of the endometrial glands of the uterus (20, 22). Because both GH and estrogen are potent stimulators of IGF-I gene expression and secretion (21), they may synergistically increase IGF-I levels in the follicular phase, which in turn contributes to follicular and uterus wall development.
In conclusion, the GH secretory pattern of female goats varies during the estrous cycle according to the changes in plasma sex steroid levels. It is suggested that estrogen enhanced the amplitude and AUC of the GH pulses, whereas progesterone reduced them and thereby affected the lipid metabolism and circulating IGF-I levels to attain the appropriate metabolic milieu for each reproductive stage. Further studies are needed, however, to clarify the mechanisms underlying the actions of sex steroids on GH pulsatility.
Acknowledgments
We thank Mr. Kazumi Shinozaki and Ms. Miyuki Koori (Animal Resource Science Center, University of Tokyo) for their help with animal care. We are grateful to Dr. Yutaka Kubo (Internal Medicine, Tokyo Women’s Medical University Daini Hospital) for his help with ApEn analysis. We are also grateful to the NIDDK for the supply of bGH and IGF-I RIA materials.
References
Jansson JO, Eden S, Isaksson O 1985 Sexual dimorphism in the control of growth hormone secretion. Endocr Rev 6:128–150
Robinson IC, Gevers EF, Bennett PA 1998 Sex differences in growth hormone secretion and action in the rat. Growth Horm IGF Res 8(Suppl B):39–47
Tannenbaum GS, Martin JB 1976 Evidence for an endogenous ultradian rhythm governing growth hormone secretion in the rat. Endocrinology 98:562–570
Saunders A, Terry LC, Audet J, Brazeau P, Martin JB 1976 Dynamic studies of growth hormone and prolactin secretion in the female rat. Neuroendocrinology 21:193–203
Clark RG, Carlsson LM, Robinson IC 1987 Growth hormone secretory profiles in conscious female rats. J Endocrinol 114:399–407
Clark RG, Jansson JO, Isaksson O, Robinson ICAF 1985 Intravenous growth hormone: growth response to patterned infusions in hypophysectomized rats. J Endocrinol 104:53–61
McMahon CD, Radcliff RP, Lookingland KJ, Tucker HA 2001 Neuroregulation of growth hormone secretion in domestic animals. Domest Anim Endocrinol 20:65–87
Ikeda A, Matsuyama S, Nishihara M, Tojo H, Takahashi M 1994 Changes in endogenous growth hormone secretion and onset of puberty in transgenic rats expressing human growth hormone gene. Endocr J 41:523–529
Ikeda A, Chang KT, Matsumoto Y, Furuhata Y, Nishihara M, Takahashi M 1998 Obesity and insulin resistance in hGH transgenic rats. Endocrinology 139:3057–3063
Furuhata Y, Yonezawa T, Takahashi M, Nishihara M 2002 Impaired insulin signaling in the liver of transgenic rats with low circulating growth hormone levels. J Endocrinol 172:127–136
Malven PV, Haglof SA, Jiang H 1995 Serum concentrations of luteinizing hormone, growth hormone, and prolactin in untreated and estradiol-treated ovariectomized ewes after immunoneutralization of hypothalamic neuropeptide Y. J Anim Sci 73:2105–2112
Ho KY, Evans WS, Blizzard RM, Veldhuis JD, Merriam GR, Samojlik E, Furlanetto R, Rogol AD, Kaiser DL, Thorner MO 1987 Effects of sex and age on the 24-hour profile of growth hormone secretion in man: importance of endogenous estradiol concentrations. J Clin Endocrinol Metab 64:51–58
Painson JC, Thorner MO, Krieg RJ, Tannenbaum GS 1992 Short-term adult exposure to estradiol feminizes the male pattern of spontaneous and growth hormone-releasing factor-stimulated growth hormone secretion in the rat. Endocrinology 130:511–519
Veldhuis JD, Metzger DL, Martha Jr PM, Mauras N, Kerrigan JR, Keenan B, Rogol AD, Pincus SM 1997 Estrogen and testosterone, but not a nonaromatizable androgen, direct network integration of the hypothalamo-somatotrope (growth hormone)-insulin-like growth factor I axis in the human: evidence from pubertal pathophysiology and sex-steroid hormone replacement. J Clin Endocrinol Metab 82:3414–3420
Painson JC, Veldhuis JD, Tannenbaum GS 2000 Single exposure to testosterone in adulthood rapidly induces regularity in the growth hormone release process. Am J Physiol Endocrinol Metab 278:E933–E940
Mogi K, Li JY, Suzuki M, Sawasaki T, Takahashi M, Nishihara M 2002 Characterization of GH pulsatility in male Shiba goats: effects of postpubertal castration and KP102. Endocr J 49:145–151
Isgaard J, Carlsson L, Isaksson OG, Jansson JO 1988 Pulsatile intravenous growth hormone (GH) infusion to hypophysectomized rats increases insulin-like growth factor I messenger ribonucleic acid in skeletal tissues more effectively than continuous GH infusion. Endocrinology 123:2605–2610
Maiter D, Underwood LE, Maes M, Davenport ML, Ketelslegers JM 1988 Different effects of intermittent and continuous growth hormone (GH) administration on serum somatomedin-C/insulin-like growth factor I and liver GH receptors in hypophysectomized rats. Endocrinology 123:1053–1059
Jorgensen JO, Moller N, Lauritzen T, Christiansen JS 1990 Pulsatile versus continuous intravenous administration of growth hormone (GH) in GH-deficient patients: effects on circulating insulin-like growth factor-I and metabolic indices. J Clin Endocrinol Metab 70:1616–1623
Murphy LJ, Friesen HG 1988 Differential effects of estrogen and growth hormone on uterine and hepatic insulin-like growth factor I gene expression in the ovariectomized hypophysectomized rat. Endocrinology 122:325–332
Copeland KC, Johnson DM, Kuehl TJ, Castracane VD 1984 Estrogen stimulates growth hormone and somatomedin-C in castrate and intact female baboons. J Clin Endocrinol Metab 58:698–703
Wathes DC, Reynolds TS, Robinson RS, Stevenson KR 1998 Role of the insulin-like growth factor system in uterine function and placental development in ruminants. J Dairy Sci 81:1778–1789
Delitala G, Tomasi P, Virdis R 1987 Prolactin, growth hormone and thyrotropin-thyroid hormone secretion during stress states in man. Baillieres Clin Endocrinol Metab 1:391–414
Mori Y, Kano Y 1984 Changes in plasma concentrations of LH, progesterone and oestradiol in relation to the occurrence of luteolysis, oestrus and time of ovulation in the Shiba goat (Capra hircus). J Reprod Fertil 72:223–230
Okada M, Takeuchi Y, Mori Y 1998 Estradiol-dependency of sexual behavior manifestation at the post-LH surge period in ovariectomized goat. J Reprod Dev 44:53–58
Hashizume T, Kanematsu S 1991 Effects of cholecystokinin octapepitide on the release of growth hormone in perifused pituitary and hypothalamus of goats. Anim Sci Technol 62:343–350
Matsuyama S, Shiota K, Takahashi M 1990 Possible role of transforming growth factor-? as a mediator of luteotropic action of prolactin in rat luteal cell cultures. Endocrinol 127:1561–1567
Daughaday WH, Parker KA, Borowsky S, Trivedi B, Kapadia M 1982 Measurement of somatomedin-related peptides in fetal, neonatal, and maternal rat serum by insulin-like growth factor (IGF) I radioimmunoassay, IGF-II radioreceptor assay (RRA), and multiplication-stimulating activity RRA after acid-ethanol extraction. Endocrinology 110:575–581
Merriam GR, Wachter KW 1982 Algorithms for the study of episodic hormone secretion. Am J Physiol 243:E310–E318
Veldhuis JD, Fletcher TP, Gatford KL, Egan AR, Clarke IJ 2002 Hypophyseal-portal somatostatin (SRIH) and jugular venous growth hormone secretion in the conscious unrestrained ewe. Neuroendocrinology 75:83–91
Pincus SM, Gevers EF, Robinson IC, van den Berg G, Roelfsema F, Hartman ML, Veldhuis JD 1996 Females secrete growth hormone with more process irregularity than males in both humans and rats. Am J Physiol 270:E107–E115
Pincus SM 1991 Approximate entropy as a measure of system complexity. Proc Natl Acad Sci USA 88:2297–2301
Faria AC, Bekenstein LW, Booth Jr RA, Vaccaro VA, Asplin CM, Veldhuis JD, Thorner MO, Evans WS 1992 Pulsatile growth hormone release in normal women during the menstrual cycle. Clin Endocrinol (Oxf) 36:591–596
Landefeld TD, Suttie JM 1989 Changes in messenger ribonucleic acid concentrations and plasma levels of growth hormone during the ovine estrous cycle and in response to exogenous estradiol. Endocrinology 125:1474–1478
Hashizume T, Ohtsuki K, Matsumoto N 2000 Plasma insulin-like growth factor-I concentrations increase during the estrous phase in goats. Domest Anim Endocrinol 18:253–263
Scanlan N, Skinner DC 2002 Estradiol modulation of growth hormone secretion in the ewe: no growth hormone-releasing hormone neurons and few somatotropes express estradiol receptor . Biol Reprod 66:1267–1273
Robinson IC, Gevers EF, Bennett PA 1998 Sex differences in growth hormone secretion and action in the rat. Growth Horm IGF Res 8(Suppl B):39–47
Murray HE, Simonian SX, Herbison AE, Gillies GE 1999 Correlation of hypothalamic somatostatin mRNA expression and peptide content with secretion: sexual dimorphism and differential regulation by gonadal factors. J Neuroendocrinol 11:27–33
Scanlan N, Dufourny L, Skinner DC 2003 Somatostatin-14 neurons in the ovine hypothalamus: colocalization with estrogen receptor and somatostatin-28 (1–12) immunoreactivity, and activation in response to estradiol. Biol Reprod 69:1318–1324
Gonzalez-Parra S, Chowen JA, Garcia-Segura LM, Argente J 1996 In vivo and in vitro regulation of pituitary transcription factor-1 (Pit-1) by changes in the hormone environment. Neuroendocrinology 63:3–15
Scott CJ, Tilbrook AJ, Simmons DM, Rawson JA, Chu S, Fuller PJ, Ing NH, Clarke IJ 2000 The distribution of cells containing estrogen receptor- (ER-) and ER-? messenger ribonucleic acid in the preoptic area and hypothalamus of the sheep: comparison of males and females. Endocrinology 141:2951–2962
Takahashi S, Satozawa N 2002 The 20-kD human growth hormone reduces body fat by increasing lipolysis and decreasing lipoprotein lipase activity. Horm Res 58:157–164
Buffenstein R, Poppitt SD, McDevitt RM, Prentice AM 1995 Food intake and the menstrual cycle: a retrospective analysis, with implications for appetite research. Physiol Behav 58:1067–1077
Eckery DC, Moeller CL, Nett TM, Sawyer HR 1997 Localization and quantification of binding sites for follicle-stimulating hormone, luteinizing hormone, growth hormone, and insulin-like growth factor I in sheep ovarian follicles. Biol Reprod 57:507–513
Jones JI, Clemmons DR 1995 Insulin-like growth factors and their binding proteins: biological actions. Endocr Rev 16:3–34(Tomohiro Yonezawa, Kazuta)
Address all correspondence and requests for reprints to: Masugi Nishihara, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan. E-mail: amnishi@mail.ecc.u-tokyo.ac.jp.
Abstract
GH is secreted in a pulsatile manner, the pattern of which plays an important role in the regulation of growth and metabolism. Sex steroids are also known to participate in metabolic regulation. The present study was undertaken to elucidate the relationship between changes in GH pulsatility and metabolic transition during the estrous cycle in goats. From ovariectomized (OVX) and intact females in the early luteal, late luteal, and follicular phases, blood samples were taken every 15 min for 24 h, and plasma GH was measured by RIA. In the early luteal phase, GH was secreted in a distinct pulsatile manner, the pattern of which was similar to that in OVX goats, whereas the GH pulse frequency, amplitude, and area under the curve (AUC) were decreased in the late luteal phase. In the follicular phase, the GH pulse frequency, amplitude, and AUC were significantly larger than those in the late luteal phase. The regularity of GH pulsatility was highest and lowest in the early and late luteal phases, respectively. Both IGF-I and free fatty acid levels in the plasma were higher in the follicular than the luteal phase. Subcutaneous injection of estradiol to OVX goats increased the GH pulse amplitude and AUC, whereas the implantation of progesterone for 5 d decreased those parameters. These results suggest that the pulsatile pattern of GH secretion in goats varies with sex steroid levels and thereby affects IGF-I secretion and lipolysis during the estrous cycle.
Introduction
GH IS SECRETED IN A pulsatile manner, the pattern of which is sexually dimorphic in most mammalian species (1, 2). For example, male rats exhibit regular high amplitude GH pulses with relatively low interpulse GH levels (3), whereas females have lower amplitude pulses with higher interpulse levels (4, 5). These sex-specific patterns of GH secretion may be related to the expression of GH bioactivity. It has been shown that intermittent GH administration is more effective than continuous GH infusion in stimulating somatic growth (6, 7). In addition, we have shown that transgenic rats lacking distinct GH pulses exhibited fat accumulation and insulin resistance (8, 9, 10). Hence, the GH secretory patterns appear to play an important role in the regulation of metabolism as well as growth between the sexes.
The relationship between endogenous GH pulsatile patterns and sex steroids remains controversial. Previous studies demonstrated that estradiol stimulated GH release in sheep (11) and humans (12), whereas it decreased the pulse amplitude and increased basal GH levels in rats (13). The effect of progesterone on GH pulsatility has not been well understood. On the other hand, androgen administration increased the mean levels and pulse amplitude of GH in humans (14) and rats (15), respectively. We have previously shown, however, that castration of male goats increased the pulse amplitude without affecting interpulse interval and area under the curve of GH secretion (16). These observations suggest that sex steroids indeed affect the pulsatile pattern of GH secretion, but the effects may vary among species.
IGF-I is synthesized in not only the liver but also many other tissues and has potential paracrine and autocrine actions in response to GH. GH administration induces the increase of skeletal tissue IGF-I mRNA (17) and serum IGF-I levels (18). In particular, frequent treatments with GH are more effective in increasing serum IGF-I than bolus administration, even with the same amount of GH (19). Estradiol is also a potent stimulator of IGF-I gene expression in the uterus (20) and serum IGF-I levels (21). Moreover, IGF-I mRNA expression peaked at estrus in the muscle layers of the ovine uterus and oviducts (22). These findings suggest that locally produced IGF-I accounts for the changes in plasma IGF-I concentrations during the estrous cycle. However, the association between serum IGF-I levels and GH pulsatility during the estrous cycle has not been fully elucidated.
Although GH pulses are known to be susceptible to stress in general (23), we have previously shown that male Shiba goats exhibit strictly regular GH pulses of 5-h intervals over 24 h (16). They were Japanese native miniature goats of nonseasonal breed that had been bred as a closed colony for experimental uses (24) and were regarded as a suitable experimental model for the analysis of GH pulsatility (16). In the present study, to clarify the changes in GH pulsatility during the inherent estrous cycle, we characterized the episodic GH release and IGF-I concentrations in several phases of the estrous cycle of Shiba goats. To assess the role of the gonadal steroids in modulating GH pulsatility, we also examined the effects of estradiol and progesterone exposure on GH release in ovariectomized (OVX) goats.
Materials and Methods
Animals
Adult female Shiba goats (2–4 yr of age) with regular estrous cycles or OVX goats for at least 6 months were used in this study. During the experimental periods, they were loosely restrained by being tied to stanchions indoors and kept at a temperature of 23 ± 2 C with the lights on from 0600 to 1800 h (12-h light, 12-h dark cycle). They were allowed free access to food and water. All experiments were conducted according to the guidelines for the care and use of laboratory animals, the Graduate School of Agriculture and Life Sciences, University of Tokyo.
Experimental procedures
First, plasma estradiol and progesterone levels were determined during the estrous cycle using seven intact goats. Blood samples (5 ml) were drawn every other day from the jugular vein, and plasma was separated and stored at –20 C until assayed for estradiol and progesterone. Plasma IGF-I and free fatty acid (FFA) levels were also measured as described below. Based on the plasma levels of sex steroids, the estrous cycle was divided into three phases, i.e. the early luteal, late luteal, and follicular phases (1–8, 9–18, and 19–21 d after the estrous behavior, respectively; see Results).
To determine the characteristics of the GH pulses in each phase of the estrous cycle in intact goats (n = 5) and those in OVX goats (n = 5), blood samples (1 ml) were collected at 15-min intervals for 24 h (0600–0600 h) through indwelling jugular catheters (Argyle Medical Catheter, 18G and 70 cm in length, Nihon Sherwood, Tokyo, Japan). The light was kept on during blood sampling to calm the animals. After centrifugation, plasma was separated and stored at –20 C until it was assayed for GH. The next 24-h blood sampling was done after an interval of at least 6 d.
Next, the OVX goats (n = 5) bearing the chronic iv catheter were sc administered either 1 mg of estradiol (Sigma, St. Louis, MO) dissolved in sesame oil or sesame oil alone (1 ml) at 1200 h. Blood samples (1 ml) were collected at 15-min intervals from 6 h before to 18 h after the injection (0600–0600 h). A separate group of OVX goats (n = 5) were sc implanted with a SILASTIC-brand (Dow Corning, Midland, MI) packet (50 x 75 mm, wall thickness 3 mm) containing progesterone (Sigma) together with a short SILASTIC-brand tubing (3.35 mm inner diameter, 4.65 outer diameter, 20 mm length including 5 mm SILASTIC-brand glue on both sides) containing estradiol to mimic the steroidal milieu during the luteal phase (25). Control animals (n = 3) received identical capsules that contained cholesterol. All the implantation procedures were carried out under anesthesia with a bolus iv injection of a mixture of xylazine HCl and ketamine HCl. Five days after the implantation, blood samples were withdrawn every 15 min for 24 h (0600–0600 h). Plasma was separated by centrifugation and stored at –20 C until further use.
Hormone assays
Plasma GH concentrations were measured by double-antibody RIA using antibovine GH (bGH) antiserum. The parallelism between goat plasma and bGH has been reported previously (26). The bGH standard preparation and hormone for iodination were supplied by the National Institute of Diabetes and Digestive and Kidney Diseases (Bethesda, MD). Antiserum to bGH generated in monkeys was supplied by Dr. K. Hodate (Kitasato University, Towada, Japan). The minimum concentration of GH that could be detected was 1 ng/ml, and the maximum assay range was 512 ng/ml. Intra- and interassay coefficients of variance (CVs) for GH assay were 11.6 and 12.3%, respectively.
Plasma estradiol concentrations were measured with a commercial enzyme immunoassay (EIA) kit (Cayman Chemical, Ann Arbor, MI) according to the assay protocol supplied by the manufacturer. In this EIA, 1 ml plasma was extracted with 2 ml diethylether, and the extracts were redissolved in 200 μl of the buffer and then subjected to the EIA. The range of linearity was 1.6–200 pg. Intra- and interassay CVs were 9.8 and 8.5%, respectively. Plasma progesterone concentrations were measured by RIA using specific antibodies generated in our laboratory as described previously (27). Plasma (50 μl) was extracted with 2 ml diethylether and the extracts were assayed for progesterone. The range of linearity was 50–1600 pg, and the intra- and interassay CVs were 8.6 and 10.5%, respectively.
Plasma IGF-I concentrations were measured by the double-antibody RIA (17). The IGF-I standard preparation was purchased from Biomedical Technologies Inc. (Stoughton, MA). Radioiodinated IGF-I was purchased from Amersham Biosciences (Buckinghamshire, UK). Antiserum against IGF-I generated in rabbits was supplied by the NIDDK. The plasma samples were first extracted with an acid-ethanol solution according to the method described by Daughaday et al. (28). Samples were mixed with an acid-ethanol solution for 30 sec. After standing for 30 min at room temperature, the samples were centrifuged at 1860 x g for 30 min at 4 C. An aliquot of the supernatant from each sample was neutralized with Tris base. Intra- and interassay CVs for IGF-I assay were both 7.4%.
Plasma FFA concentrations were measured with a commercial assay kit using the acyl-CoA synthetase and acyl-CoA oxidase method (Wako Pure Chemical, Osaka, Japan). The assay was performed according to the assay protocol supplied by the manufacturer. The range of linearity was 0.1–2.0 mEq/liter, and the intraassay CV was 2.7% (this assay was performed only once).
Pulse analysis and statistics
GH pulses were identified by means of the PULSAR computer program (29). The cut-off criteria for pulse determination, G1, G2, G3, G4, and G5 were 4.40, 2.20, 1.92, 1.46, and 1.13, respectively. The parameters of the pulsatile GH calculated for each sampling session were as follows: 1) pulse frequency, the mean number of pulses per 24 or 6 h; 2) pulse amplitude, the mean height of all amplitudes derived from the difference between peak and baseline; 3) pulse width, the mean length of pulse peak criteria; 4) area under the curve (AUC), the mean of all GH values during the individual pulse peak criteria. Group comparisons were performed by Tukey-Kramer’s test or the unpaired t test.
In addition, the regularity of GH secretory pattern was determined by approximate entropy (ApEn) test. ApEn comprises a class of model- and scale-independent irregularity statistics designed to monitor the relative orderliness of patterns in individual time series (30, 31, 32). In the present study, we calculated ApEn based on r = 0.2 and m = 1. These parameter sets have been validated for a number of hormonal time series (30, 31, 32). Group comparisons were performed by Fisher’s protected least significant difference test (P < 0.05). Statistical significance was considered to be at P < 0.05.
Results
Plasma sex steroid levels during the estrous cycle in goats
The intact female goats used in this study had a regular estrous cycle of 21 d. Plasma estradiol and progesterone levels were measured throughout the estrous cycle, and according to the steroid levels, the estrous cycle was divided into three phases: the early luteal, late luteal, and follicular phase (1–8, 9–18, and 19–21 d after the estrous behavior, respectively). Plasma concentrations of estradiol and progesterone during the estrous cycle are presented in Fig. 1, and those in each phase are summarized in Table 1. Plasma estradiol concentrations in the follicular phase were markedly higher than those in the early and late luteal phases. Plasma progesterone levels in the late luteal phase were significantly higher than those in the early luteal and follicular phases.
FIG. 1. Changes in plasma progesterone and estradiol levels during the estrous cycle. Each square and vertical bar represents the mean and SEM (n = 7), respectively.
TABLE 1. Plasma concentration of sex steroids during the goat estrous cycle
Characteristics of GH pulses during the estrous cycle in goats
Representative 24-h plasma GH profiles in OVX and intact goats in each phase of the estrous cycle are shown in Fig. 2. Figure 3 illustrates the results of the characteristics of the GH pulses according to the PULSAR algorithm. In OVX goats, GH was secreted in a pulsatile manner, which involves the recurrence of obvious peaks followed by troughs of virtually no GH secretion. Plasma GH profiles in the early luteal phase did not significantly differ from those of OVX goats. In the late luteal phase, pulse amplitude and AUC were significantly decreased as compared with the early luteal phase, whereas the pulse frequency and width remained unchanged. On the other hand, in the follicular phase, the pulse frequency and AUC were significantly increased as compared with all the other groups. The pulse amplitude in the follicular phase was also significantly larger than that in the late luteal phase. The pulse width was not different among all the experimental groups. These data were also analyzed by ApEn test that quantifies the subpattern reproducibility of successive measurements. Higher ApEn denotes greater disorderliness or irregularity of patterns. As shown in Fig. 4, the regularity of GH pulsatility was highest in the early luteal phase and lowest in the late luteal phase during the estrous cycle.
FIG. 2. Individual representative 24 h plasma GH profiles in OVX and intact female goats of the early luteal (EL), late luteal (LL), and follicular (FO) phases of the estrous cycle. Open triangles indicate GH pulses identified by the PULSAR algorithm.
FIG. 3. Characteristics of GH pulses in OVX and intact female goats of the early luteal (EL), late luteal (LL), and follicular (FO) phases of the estrous cycle. Each column and vertical bar represents the mean and SEM (n = 5), respectively. Values with different superscripts are significantly different (P < 0.05).
FIG. 4. Regularity of GH pulsatility assessed by the ApEn test in the early luteal (EL), late luteal (LL), and follicular (FO) phases of the estrous cycle. Each circle and horizontal bar represents the individual data and the mean (n = 5), respectively. Values of the mean and SEM are also presented. Values with different superscripts are significantly different (P < 0.05).
Effects of sex steroids on GH pulses in OVX goats
The representative plasma GH profiles after estradiol injection to OVX goats are shown in Fig. 5A. Figure 5B presents the results of the GH pulse analysis using the PULSAR algorithm for every 6-h period. Plasma estradiol levels increased to 13.3 ± 1.9 pg/ml (mean ± SEM, n = 5) 1 h after the injection and remained high until 18 h after the injection (17.5 ± 2.2 pg/ml). Although the acute exposure to estradiol did not affect GH pulsatility during the first 6 h-period after treatment, it significantly increased the pulse amplitude and AUC in the second and third 6-h periods after treatment. The frequency and width of GH pulses, however, were not affected by estradiol injection. The injection of sesame oil alone did not affect any of the parameters of GH pulsatility (n = 3, data not shown).
FIG. 5. Changes in GH pulses after sc injection of estradiol (1 mg) in OVX goats. A, Representative plasma GH profiles after the injection of estradiol. Arrows indicate the time of injection. Open triangles indicate GH pulses identified by PULSAR algorithm. B, Characteristics of GH pulses during the 6 h before injection (Pre) and 0–6, 6–12, and 12–18 h after injection. Each column and vertical bar represents the mean and SEM (n = 5), respectively. *, P < 0.05 vs. Pre.
Figure 6A shows the representative plasma GH profiles for 24 h 5 d after the implantation of the progesterone-containing SILASTIC-brand capsule in OVX goats, and the results of the GH pulse analysis are presented in Fig. 6B. The plasma progesterone levels in goats implanted with progesterone on the day of the experiment were 8.6 ± 0.8 ng/ml (mean ± SEM, n = 5). The implantation of the progesterone-containing capsule plus a small estradiol implant significantly decreased the pulse amplitude and AUC as compared with implantation of the cholesterol-containing capsule, whereas it did not affect the frequency or the width of the GH pulses.
FIG. 6. Changes in GH pulses 5 d after the implantation of a SILASTIC-brand capsule containing cholesterol (Chol) or progesterone (P) plus a small estradiol implant in OVX goats. A, Representative plasma GH profiles after the implantation of Chol or P. Open triangles indicate GH pulses identified by PULSAR algorithm. B, Characteristics of GH pulses. Each column and vertical bar represents the mean and SEM (n = 3 for Chol, and 5 for P), respectively. *, P < 0.05 vs. Chol.
Plasma IGF-I and FFA levels during the estrous cycle in goats
The changes in the plasma IGF-I levels during the estrous cycle are shown in Fig. 7A. Although plasma IGF-I levels in the luteal phase remained low, they increased in the follicular phase. All possible comparisons were made through Tukey-Kramer test, and it was found that the peak level of plasma IGF-I on d 22 (the day of estrus) was significantly higher than those on d 4–18. The changes in the plasma FFA levels are shown in Fig. 7B, which were similar to those of the plasma IGF-I levels. The plasma FFA levels also peaked on d 22, which was significantly higher than those on d 8–12.
FIG. 7. Changes in plasma IGF-I (A) and FFA (B) levels during the estrous cycle. Each square and vertical bar represents the mean and SEM (n = 5), respectively. Values with different superscripts are significantly different (P < 0.05).
Discussion
The present study demonstrated that plasma GH profiles in female Shiba goats, either intact or OVX, exhibit distinct pulsatility consisting of conspicuous pulses separated by trough periods of virtually no GH secretion. In the previous reports of female rats and sheep, they secreted GH in an irregular pulsatile manner with high nadir or rather erratic interpulse intervals ranging from 3 to 6 h (1). There may be a species difference in the basal pattern of GH secretion in female animals. Alternatively, the possibility that the GH pulsatility in female goats is more resistant than those in females of other species to environmental factors such as stress cannot be ruled out. The present study also demonstrated for the first time that the pulsatility of GH secretion in female goats varies during the estrous cycle.
GH secretory profiles in the early luteal phase, when both estrogen and progesterone levels were low, were similar to those in OVX goats. All the parameters analyzed, i.e. pulse frequency, width, amplitude, and AUC, did not differ between the early luteal phase and the OVX goats. Then pulse frequency, amplitude, and AUC were markedly decreased in the late luteal phase when the progesterone levels were high. The regularity of GH pulsatility was also decreased in the late luteal phase. In contrast, in the follicular phase when plasma estradiol concentrations were elevated, the GH pulses were abruptly enhanced as for pulse frequency, amplitude, and AUC, and the regularity of pulsatility became higher. These changes in the characteristics of GH pulses were almost exactly repeated in two consecutive cycles for all three of the goats that were studied for more than one cycle (data not shown). Our current data confirmed in part the previous studies in humans (33) and sheep (34), in which the integrated serum GH concentrations in the late follicular phase were higher than those in the luteal phase. Although it has been shown that there was no significant change in GH pulse profiles during the estrous cycle in rats (5) and goats (35), female Shiba goats appear to exhibit considerable changes in GH pulsatility during the estrous cycle.
The sc injection of estradiol to OVX goats did not affect the GH pulses during the 6 h after the treatment, but thereafter it significantly increased the pulse amplitude and AUC, whereas the pulse frequency and width remained unchanged. This is consistent at least partially with the previous reports showing increased plasma GH levels after estradiol administration in several species including primates (21) and ruminants (11, 36). In contrast, the implantation of progesterone together with a small estradiol implant for 5 d decreased the same parameters of the GH pulses. The profiles of GH secretion after treatment with sex steroids are in accordance with the characteristics of GH pulses in each phase of the goat estrous cycle. It is therefore probable that the pulsatility of GH secretion during the inherent estrous cycle changes, depending on the suppressive effect of endogenous progesterone as well as the stimulatory effect of endogenous estradiol.
How sex steroids could modulate GH output remains poorly understood. Regulation of GH secretion is mainly under the control of two hypothalamic peptides, GHRH and somatostatin (SRIF), which interact reciprocally at the level of the pituitary to generate GH pulses (37, 38). Although estradiol decreased GHRH mRNA expression without affecting SRIF mRNA in rats (2), c-fos expression in both GHRH and SRIF neurons was increased in the follicular phase in sheep (39). Thus, it is currently difficult to correlate estrogen’s effects on the hypothalamic peptides and GH pulses. The possibility that estrogen affects GH pulsatility by directly acting on the pituitary also cannot be ruled out because transcription factor pituitary transcription factor-1, which is involved in the expression of GH mRNA, is up-regulated by estrogen (40). However, because the majority of GHRH and SRIF neurons, and also the pituitary somatotropes, do not express estrogen receptors (39, 41), the effect of estrogen on these cells, if any, may be indirect.
The amplitude and frequency of GH pulses are known to be important determinants of GH bioactivity (7). In the present study, both plasma IGF-I and FFA levels changed in a similar pattern during the estrous cycle and peaked on the day of estrus. Further studies in the OVX steroid-treated goats would be necessary to establish the ability of steroid treatments to acutely alter GH secretion and, subsequently, metabolism. However, considering the lipolytic action of GH (42), as well as the stimulatory action on IGF-I release, it is reasonable to speculate that the observed changes in plasma IGF-I and FFA levels well reflected the changes in GH pulsatility during the estrous cycle. It is therefore suggested that lipolysis is stimulated during the follicular phase when the GH pulses are robust, and the accumulation of fat occurs during the late luteal phase when the GH pulses are suppressed. Interestingly, it has also been shown in humans that free fatty acid concentrations are higher in the follicular than in the luteal phase (43). In addition to lipid metabolism, GH plays an important role in regulating reproductive function. It was recently reported that GH release preceded the LH surge (36) and acted as a cogonadotropin to induce gametogenesis and ovulation (44). Hence, the stimulation of GH pulses by estrogen may be involved in follicular development and the ovulatory processes. IGF-I also participates in reproductive cell replication and differentiation (45) as well as protein/carbohydrate metabolism, and IGF-I receptors are localized in the granulosa cells and epithelium of the endometrial glands of the uterus (20, 22). Because both GH and estrogen are potent stimulators of IGF-I gene expression and secretion (21), they may synergistically increase IGF-I levels in the follicular phase, which in turn contributes to follicular and uterus wall development.
In conclusion, the GH secretory pattern of female goats varies during the estrous cycle according to the changes in plasma sex steroid levels. It is suggested that estrogen enhanced the amplitude and AUC of the GH pulses, whereas progesterone reduced them and thereby affected the lipid metabolism and circulating IGF-I levels to attain the appropriate metabolic milieu for each reproductive stage. Further studies are needed, however, to clarify the mechanisms underlying the actions of sex steroids on GH pulsatility.
Acknowledgments
We thank Mr. Kazumi Shinozaki and Ms. Miyuki Koori (Animal Resource Science Center, University of Tokyo) for their help with animal care. We are grateful to Dr. Yutaka Kubo (Internal Medicine, Tokyo Women’s Medical University Daini Hospital) for his help with ApEn analysis. We are also grateful to the NIDDK for the supply of bGH and IGF-I RIA materials.
References
Jansson JO, Eden S, Isaksson O 1985 Sexual dimorphism in the control of growth hormone secretion. Endocr Rev 6:128–150
Robinson IC, Gevers EF, Bennett PA 1998 Sex differences in growth hormone secretion and action in the rat. Growth Horm IGF Res 8(Suppl B):39–47
Tannenbaum GS, Martin JB 1976 Evidence for an endogenous ultradian rhythm governing growth hormone secretion in the rat. Endocrinology 98:562–570
Saunders A, Terry LC, Audet J, Brazeau P, Martin JB 1976 Dynamic studies of growth hormone and prolactin secretion in the female rat. Neuroendocrinology 21:193–203
Clark RG, Carlsson LM, Robinson IC 1987 Growth hormone secretory profiles in conscious female rats. J Endocrinol 114:399–407
Clark RG, Jansson JO, Isaksson O, Robinson ICAF 1985 Intravenous growth hormone: growth response to patterned infusions in hypophysectomized rats. J Endocrinol 104:53–61
McMahon CD, Radcliff RP, Lookingland KJ, Tucker HA 2001 Neuroregulation of growth hormone secretion in domestic animals. Domest Anim Endocrinol 20:65–87
Ikeda A, Matsuyama S, Nishihara M, Tojo H, Takahashi M 1994 Changes in endogenous growth hormone secretion and onset of puberty in transgenic rats expressing human growth hormone gene. Endocr J 41:523–529
Ikeda A, Chang KT, Matsumoto Y, Furuhata Y, Nishihara M, Takahashi M 1998 Obesity and insulin resistance in hGH transgenic rats. Endocrinology 139:3057–3063
Furuhata Y, Yonezawa T, Takahashi M, Nishihara M 2002 Impaired insulin signaling in the liver of transgenic rats with low circulating growth hormone levels. J Endocrinol 172:127–136
Malven PV, Haglof SA, Jiang H 1995 Serum concentrations of luteinizing hormone, growth hormone, and prolactin in untreated and estradiol-treated ovariectomized ewes after immunoneutralization of hypothalamic neuropeptide Y. J Anim Sci 73:2105–2112
Ho KY, Evans WS, Blizzard RM, Veldhuis JD, Merriam GR, Samojlik E, Furlanetto R, Rogol AD, Kaiser DL, Thorner MO 1987 Effects of sex and age on the 24-hour profile of growth hormone secretion in man: importance of endogenous estradiol concentrations. J Clin Endocrinol Metab 64:51–58
Painson JC, Thorner MO, Krieg RJ, Tannenbaum GS 1992 Short-term adult exposure to estradiol feminizes the male pattern of spontaneous and growth hormone-releasing factor-stimulated growth hormone secretion in the rat. Endocrinology 130:511–519
Veldhuis JD, Metzger DL, Martha Jr PM, Mauras N, Kerrigan JR, Keenan B, Rogol AD, Pincus SM 1997 Estrogen and testosterone, but not a nonaromatizable androgen, direct network integration of the hypothalamo-somatotrope (growth hormone)-insulin-like growth factor I axis in the human: evidence from pubertal pathophysiology and sex-steroid hormone replacement. J Clin Endocrinol Metab 82:3414–3420
Painson JC, Veldhuis JD, Tannenbaum GS 2000 Single exposure to testosterone in adulthood rapidly induces regularity in the growth hormone release process. Am J Physiol Endocrinol Metab 278:E933–E940
Mogi K, Li JY, Suzuki M, Sawasaki T, Takahashi M, Nishihara M 2002 Characterization of GH pulsatility in male Shiba goats: effects of postpubertal castration and KP102. Endocr J 49:145–151
Isgaard J, Carlsson L, Isaksson OG, Jansson JO 1988 Pulsatile intravenous growth hormone (GH) infusion to hypophysectomized rats increases insulin-like growth factor I messenger ribonucleic acid in skeletal tissues more effectively than continuous GH infusion. Endocrinology 123:2605–2610
Maiter D, Underwood LE, Maes M, Davenport ML, Ketelslegers JM 1988 Different effects of intermittent and continuous growth hormone (GH) administration on serum somatomedin-C/insulin-like growth factor I and liver GH receptors in hypophysectomized rats. Endocrinology 123:1053–1059
Jorgensen JO, Moller N, Lauritzen T, Christiansen JS 1990 Pulsatile versus continuous intravenous administration of growth hormone (GH) in GH-deficient patients: effects on circulating insulin-like growth factor-I and metabolic indices. J Clin Endocrinol Metab 70:1616–1623
Murphy LJ, Friesen HG 1988 Differential effects of estrogen and growth hormone on uterine and hepatic insulin-like growth factor I gene expression in the ovariectomized hypophysectomized rat. Endocrinology 122:325–332
Copeland KC, Johnson DM, Kuehl TJ, Castracane VD 1984 Estrogen stimulates growth hormone and somatomedin-C in castrate and intact female baboons. J Clin Endocrinol Metab 58:698–703
Wathes DC, Reynolds TS, Robinson RS, Stevenson KR 1998 Role of the insulin-like growth factor system in uterine function and placental development in ruminants. J Dairy Sci 81:1778–1789
Delitala G, Tomasi P, Virdis R 1987 Prolactin, growth hormone and thyrotropin-thyroid hormone secretion during stress states in man. Baillieres Clin Endocrinol Metab 1:391–414
Mori Y, Kano Y 1984 Changes in plasma concentrations of LH, progesterone and oestradiol in relation to the occurrence of luteolysis, oestrus and time of ovulation in the Shiba goat (Capra hircus). J Reprod Fertil 72:223–230
Okada M, Takeuchi Y, Mori Y 1998 Estradiol-dependency of sexual behavior manifestation at the post-LH surge period in ovariectomized goat. J Reprod Dev 44:53–58
Hashizume T, Kanematsu S 1991 Effects of cholecystokinin octapepitide on the release of growth hormone in perifused pituitary and hypothalamus of goats. Anim Sci Technol 62:343–350
Matsuyama S, Shiota K, Takahashi M 1990 Possible role of transforming growth factor-? as a mediator of luteotropic action of prolactin in rat luteal cell cultures. Endocrinol 127:1561–1567
Daughaday WH, Parker KA, Borowsky S, Trivedi B, Kapadia M 1982 Measurement of somatomedin-related peptides in fetal, neonatal, and maternal rat serum by insulin-like growth factor (IGF) I radioimmunoassay, IGF-II radioreceptor assay (RRA), and multiplication-stimulating activity RRA after acid-ethanol extraction. Endocrinology 110:575–581
Merriam GR, Wachter KW 1982 Algorithms for the study of episodic hormone secretion. Am J Physiol 243:E310–E318
Veldhuis JD, Fletcher TP, Gatford KL, Egan AR, Clarke IJ 2002 Hypophyseal-portal somatostatin (SRIH) and jugular venous growth hormone secretion in the conscious unrestrained ewe. Neuroendocrinology 75:83–91
Pincus SM, Gevers EF, Robinson IC, van den Berg G, Roelfsema F, Hartman ML, Veldhuis JD 1996 Females secrete growth hormone with more process irregularity than males in both humans and rats. Am J Physiol 270:E107–E115
Pincus SM 1991 Approximate entropy as a measure of system complexity. Proc Natl Acad Sci USA 88:2297–2301
Faria AC, Bekenstein LW, Booth Jr RA, Vaccaro VA, Asplin CM, Veldhuis JD, Thorner MO, Evans WS 1992 Pulsatile growth hormone release in normal women during the menstrual cycle. Clin Endocrinol (Oxf) 36:591–596
Landefeld TD, Suttie JM 1989 Changes in messenger ribonucleic acid concentrations and plasma levels of growth hormone during the ovine estrous cycle and in response to exogenous estradiol. Endocrinology 125:1474–1478
Hashizume T, Ohtsuki K, Matsumoto N 2000 Plasma insulin-like growth factor-I concentrations increase during the estrous phase in goats. Domest Anim Endocrinol 18:253–263
Scanlan N, Skinner DC 2002 Estradiol modulation of growth hormone secretion in the ewe: no growth hormone-releasing hormone neurons and few somatotropes express estradiol receptor . Biol Reprod 66:1267–1273
Robinson IC, Gevers EF, Bennett PA 1998 Sex differences in growth hormone secretion and action in the rat. Growth Horm IGF Res 8(Suppl B):39–47
Murray HE, Simonian SX, Herbison AE, Gillies GE 1999 Correlation of hypothalamic somatostatin mRNA expression and peptide content with secretion: sexual dimorphism and differential regulation by gonadal factors. J Neuroendocrinol 11:27–33
Scanlan N, Dufourny L, Skinner DC 2003 Somatostatin-14 neurons in the ovine hypothalamus: colocalization with estrogen receptor and somatostatin-28 (1–12) immunoreactivity, and activation in response to estradiol. Biol Reprod 69:1318–1324
Gonzalez-Parra S, Chowen JA, Garcia-Segura LM, Argente J 1996 In vivo and in vitro regulation of pituitary transcription factor-1 (Pit-1) by changes in the hormone environment. Neuroendocrinology 63:3–15
Scott CJ, Tilbrook AJ, Simmons DM, Rawson JA, Chu S, Fuller PJ, Ing NH, Clarke IJ 2000 The distribution of cells containing estrogen receptor- (ER-) and ER-? messenger ribonucleic acid in the preoptic area and hypothalamus of the sheep: comparison of males and females. Endocrinology 141:2951–2962
Takahashi S, Satozawa N 2002 The 20-kD human growth hormone reduces body fat by increasing lipolysis and decreasing lipoprotein lipase activity. Horm Res 58:157–164
Buffenstein R, Poppitt SD, McDevitt RM, Prentice AM 1995 Food intake and the menstrual cycle: a retrospective analysis, with implications for appetite research. Physiol Behav 58:1067–1077
Eckery DC, Moeller CL, Nett TM, Sawyer HR 1997 Localization and quantification of binding sites for follicle-stimulating hormone, luteinizing hormone, growth hormone, and insulin-like growth factor I in sheep ovarian follicles. Biol Reprod 57:507–513
Jones JI, Clemmons DR 1995 Insulin-like growth factors and their binding proteins: biological actions. Endocr Rev 16:3–34(Tomohiro Yonezawa, Kazuta)