RECENT PROGRESS IN HORMONE RESEARCH, VOL. 47
Gonadotropin-Releasing Hormone Pulses: Regulators of Gonadotropin Synthesis and Ovulatory Cycles JOHN C . M A R S H A L L , * A L A N C . D A L K I N , ! D A N I E L J.
HAISENLEDER,t
S A N D E R J. P A U L , ! GIROLAMO A . O R T O L A N O , ! A N D ROBERT P.
KELCHI
^Department of Medicine, University of Virginia Health Sciences Center, Virginia 22908 and fDivision of Endocrinology and Metabolism, University Center, Ann Arbor, Michigan 48109
I.
Charlottesville, of Michigan Medical
Introduction
Reproductive function is predominantly controlled by the pituitary gonadotropin hormones, luteinizing hormone (LH), and follicle-stimulating hormone (FSH). Both hormones are composed of two glycoprotein subunits, a common α subunit and distinct β subunits which confer biologic specificity (1). The genes coding for the three gonadotropin subunits are located on different chromosomes (2). The two pituitary gonadotropins act synergistically on the gonads, with FSH predominantly regulating gametogenesis, and LH production of the steroid hormones. Both LH and FSH are secreted by the same pituitary gonadotrope cells and their synthesis and secretion is controlled by the hypothalamic decapeptide, gonadotropin-releasing hormone (GnRH). LH and FSH are known to be secreted differentially in some circumstances, but intensive investigation during the past two decades has failed to provide convincing evidence for other gonadotropinreleasing hormones. Thus, the differential regulation of LH and FSH synthesis and secretion appears to be effected by changes in the pattern of GnRH secretion, together with the direct feedback effects of gonadal steroids and peptides on the gonadotrope cell. Gonadal steroids can alter GnRH secretory patterns and also modify gonadotrope responses to GnRH. Inhibin, a polypeptide also consisting of two subunits and secreted by both the testis and the ovary, circulates in plasma and exerts a selective direct action to predominantly inhibit FSH secretion. Early studies of the patterns of LH secretion in humans revealed that LH is released into the circulation in a series of pulses (3,4), and studies in animals have shown that pulses of LH reflect pulsatile secretion of GnRH by the hypothalamus (5). This pulsatile mode of GnRH secretion is essential for the maintenance of gonadotropin synthesis and release. Administration of GnRH in a continuous manner, or stimulation by long-acting GnRH agonists, results in 155
Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
156
J O H N C. M A R S H A L L ET A L .
desensitization of gonadotropin secretion (6,7). In normal physiologic situations, such as during pubertal maturation and during ovulatory cycles in females, the pattern of pulsatile GnRH secretion changes, with alterations occurring in both the frequency and amplitude of GnRH release (8). Gonadal steroids modify pulsatile GnRH secretion and the pattern of GnRH stimulation appears to be important in the differential synthesis and release of LH and FSH by the pituitary gonadotrope. In this article, we examine the role of the pattern of GnRH stimulation of the gonadotrope in regulating gonadotropin synthesis and secretion. In addition, we review current evidence on the effects of the changes in pulsatile GnRH release during human pubertal maturation and during ovulatory cycles in women, and suggest that the ability to alter the pattern of GnRH stimulation is an essential component of the regulation of reproduction in mammals. In the first section, the regulation of gonadotropin subunit gene expression will be reviewed. In the subsequent section, the proposed role of altered GnRH pulsatile secretion in normal physiology and as a cause of reproductive failure are considered.
II.
Regulation of Gonadotropin Subunit Gene Expression
The cloning of the cDNAs for the α, LH β, and FSH β subunits has allowed direct measurement of steady state mRNA concentrations. The original cDNA clones used in the present studies were generously supplied by Dr. W. W. Chin (rat α and LH β) (9,10) and Dr. R. A. Maurer (FSH β) (11). Steady state subunit mRNA concentrations were measured in cytoplasmic RNA extracts from pituitary tissue. Dot-blot hybridization was performed using saturating amounts of 32 P-labeled cDNA probes, and concentrations of mRNAs are expressed as femtomoles cDNA bound/100 μg pituitary DNA (12). The rates of transcription of the subunit genes were measured by Dr. M. A. Shupnik (University of Virginia, Charlottesville, VA) as previously described (13). A. 1.
Gonadectomy
PHYSIOLOGIC STUDIES
and Gonadal Steroid
Replacement
Initial evidence for differential regulation of the gonadotropin subunit genes was found in studies of the effects of gonadectomy in male and female rats (Fig. 1). Serum LH, FSH, and all three subunit mRNAs increase following gonadectomy in both sexes. However, the timing and magnitude of the changes in serum hormones and steady state mRNAs differ between the sexes (12,14-16). In females, serum LH, and a and LH β subunit mRNAs do not increase until 4 - 7 days after ovariectomy. Thereafter, α mRNA rises to a plateau (5-fold increase)
GONADOTROPIN-RELEASING
HORMONE
PULSES
157
FIG. 1. Pituitary gonadotropin subunit mRNA concentrations following gonadectomy in male and female rats.
after 14 days, but LH β mRNA continues to increase gradually through 30 days (15-fold increase). In contrast, serum FSH and FSH β mRNA increase more rapidly, reaching a plateau (fourfold rise) after 4 days. In male rats, both α and LH β mRNAs increase within 24 hours after orchidectomy. Thereafter, α mRNA plateaus after 14 days while LH β continues to rise through 30 days. Again, FSH β mRNA increases more rapidly than LH β and peak concentrations occur after 7 days, after which FSH β plateaus or even declines (15). Replacement of estradiol (E 2 ) in ovariectomized rats suppresses all three gonadotropin subunit mRNAs (14-16), but if E 2 is replaced in physiologic concentrations, only partial suppression occurs over 7 days (Fig. 2). E 2 replacement at the time of ovariectomy has yielded interesting results and differential effects on subunit gene expression are seen (17). Estradiol prevented the increase in LH β, but α mRNA concentrations continued to increase to a plateau after 10 days. The rise in FSH β mRNA concentrations was only partly suppressed by replacement of E 2 alone, or both E 2 and progesterone (P). Administration of a GnRH antagonist to ovariectomized females, in the presence or absence of E 2
158
J O H N C. MARSHALL ET A L .
INTACT
7d CAST.
7d C • 7dE
FIG. 2. Effect of estradiol ( E 2) replacement by silastic sc implant (to achieve plasma E 2 of 40 pg/ml) in 7-day ovariectomized female rats. 7dC + 7dE, 7 days of E 2 given to rats castrated 7 days earlier. *p < 0.05 vs intact.
and P, abolished the increase in α and LH β mRNAs, but did not prevent the rise in FSH β mRNA. This suggests that the increase in gonadotropin subunit mRNA following ovariectomy, particularly that of α and LH β, is a consequence of increased GnRH secretion. However, the failure of a GnRH antagonist to block the increase in FSH β suggests that additional ovarian hormones are involved in inhibiting FSH β mRNA expression in intact females (17). Definitive evidence in vivo is lacking, but in vitro data suggest that inhibin is required to maintain FSH
GONADOTROPIN-RELEASING
HORMONE
PULSES
159
β mRNA expression at the level found in intact female rats. The addition of inhibin to pituitary cells in culture resulted in a rapid decline in the concentrations of FSH β mRNA (18-20). In male rats, replacement of testosterone (T) at the time of castration prevents the increase in all three subunit mRNAs (21). As shown in Fig. 3 replacement of physiologic concentrations of serum Τ ( ~ 2 . 5 ng/ml) in previously castrated animals suppresses subunit mRNAs to intact values (17). However, when higher
INTACT
7d CAST.
7d C • 7dT
FIG. 3. Effect of testosterone (T) replacement by silastic sc implant (plasma Τ was 2.5 ng/ml) in 7-day orchidectomized male rats. *p < 0.05 vs intact.
160
JOHN C. M A R S H A L L ET A L .
doses of T were given to castrate animals by intermittent injections, FSH β mRNA was not fully suppressed (22,23). These differences were of interest and could reflect the effects of different concentrations of testosterone acting either on FSH β transcription, or on the stability of FSH β mRNA in cytosol. Administration of a GnRH antagonist to castrate male rats reduced FSH β mRNA concentrations with a half-disappearance time of —20 hours. In the presence of testosterone, however, steady state FSH β mRNA concentrations declined more slowly (—50 hours) after a GnRH antagonist, while the rate of decline of α and LH β mRNAs was unchanged (24). These data suggested that Τ may prolong FSH β transcription or stabilize FSH β mRNA, and other studies have suggested that stabilization of mRNA may be the mechanism of action. When a GnRH antagonist was administered for 4 days to castrate male rats, replacement of physiologic concentrations of testosterone increased FSH β mRNA twofold, without altering α or LH β concentrations (25,26). Measurement of FSH β mRNA transcription rates in GnRH antagonist-treated rats showed that transcription was not increased in the presence of testosterone, which suggests that the action of testosterone in increasing FSH β mRNA is exerted at a posttranscriptional level (25). Some studies have also suggested that other compounds may regulate FSH β mRNA stability. In vitro, addition of activin to pituitary cell cultures rapidly increased FSH β mRNA (19), and preliminary data show that this is due to enhanced stability of the FSH β mRNA (27). Thus, while data are presently incomplete, evidence suggests that testosterone and activin may enhance FSH β mRNA stability. It is also possible that compounds such as inhibin decrease FSH β mRNA by actions which include reducing the stability of FSH mRNA in cytosol. Overall, gonadal steroid replacement results in suppression of gonadotropin subunit mRNA concentrations, but the degree and mechanisms of this action may depend on plasma steroid concentrations. Testosterone suppresses pulsatile GnRH secretion and can also modify gonadotrope responses to GnRH. Estradiol has variable effects on pulsatile GnRH secretion in vivo, but estradiol replacement to castrate animals results in reduced subunit mRNA transcription rates (28). In vitro studies showed that estradiol did not reduce α and FSH β transcription rates and actually increased LH beta transcription, suggesting that the predominant actions of estradiol in vivo are exerted by inhibiting hypothalamic GnRH secretion (29,30). The positive actions of estradiol on LH secretion and LH β mRNA expression may be exerted directly at the level of the gonadotrope. While not all evidence is in agreement, the latter view is supported by the effect of estradiol in increasing LH β transcription in vitro, and the presence of an estrogen response element in the 5' region of the rat LH β gene (31). Progesterone, particularly in the presence of estradiol, reduces pulsatile GnRH secretion in vivo (32,33). Thus, progesterone could suppress subunit gene expression via hypothalamic mechanisms in vivo, but may also exert direct nega-
GONADOTROPIN-RELEASING
HORMONE
PULSES
161
tive effects on the gonadotrope. In vitro studies using pituitaries from sheep showed that progesterone directly suppressed FSH β gene transcription (34), although studies using rat pituitary cells suggest that this action may also require the presence of estradiol (35). Thus, at the present time data are incomplete, but it appears likely that gonadal steroids and peptides exert actions both at the hypothalamic level to reduce GnRH secretion and directly on the gonadotrope cell to modify expression of subunit mRNAs, hormone synthesis, and secretion. The relative degree and magnitude of actions at these sites may be modified by steroid concentration and duration of exposure to steroid hormones. 2.
Gonadotropin
Subunit mRNAs during the Rat Estrous
Cycle
Serum LH, FSH, α, and LH and FSH β mRNA subunit concentrations in the rat estrous cycle are shown in Fig. 4. During the 4-day cycle in female rats, serum LH and FSH concentrations remain low, except during the preovulatory surge during the late afternoon and evening of proestrus (36). α, LH β, and FSH β mRNAs show evidence of differential expression during the cycle, with increased expression of both β mRNAs occurring at the time of the gonadotropin surge on proestrus (37,38). On the morning of metestrus, FSH β mRNA was increased twofold, gradually falling to basal levels by the evening, α and LH β mRNAs were stable on metestrus, but both transiently increased twofold during a 12-hour period on diestrus when FSH β was unchanged. These changes in mRNA expression occurred in the absence of increased secretion of LH and FSH. On the afternoon of proestrus, LH β mRNA increased threefold, rising before the preovulatory surge of serum LH. FSH β mRNA increased fourfold, but maximum concentrations occurred 2 hours after the onset of the serum FSH surge. In contrast to these changes in β subunit mRNA concentrations, α mRNA was unchanged during the gonadotropin surge. These data suggest that both coordinate and differential regulation of expression of the three subunit genes occur during the estrous cycle. Coordinate increases in α and LH β mRNA are seen on diestrus, and both β subunit mRNAs increase during the proestrus gonadotropin surges. On metestrus, however, only FSH β mRNA increases when both α and LH β mRNAs are stable. The mechanisms regulating subunit gene expression and hormone secretion during the cycle remain uncertain, but may include the effects of changes in GnRH secretory pattern and the direct actions of ovarian steroids and peptides on the gonadotrope. Secretion of GnRH changes during the cycle, and both the amplitude and frequency of pulsatile GnRH release, are increased during the proestrus LH surge (39,40). This coincides with the increased LH β mRNA concentrations and a twofold increase in the rate of LH β transcription (29), suggesting that the LH β mRNA changes are dependent on GnRH secretion.
162
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SERUM LH (ng/ml)
Gonadotropin-Releasing Hormone Pulses: Regulators of Gonadotropin Synthesis and Ovulatory Cycles JOHN C . M A R S H A L L , * A L A N C . D A L K I N , ! D A N I E L J.
HAISENLEDER,t
S A N D E R J. P A U L , ! GIROLAMO A . O R T O L A N O , ! A N D ROBERT P.
KELCHI
^Department of Medicine, University of Virginia Health Sciences Center, Virginia 22908 and fDivision of Endocrinology and Metabolism, University Center, Ann Arbor, Michigan 48109
I.
Charlottesville, of Michigan Medical
Introduction
Reproductive function is predominantly controlled by the pituitary gonadotropin hormones, luteinizing hormone (LH), and follicle-stimulating hormone (FSH). Both hormones are composed of two glycoprotein subunits, a common α subunit and distinct β subunits which confer biologic specificity (1). The genes coding for the three gonadotropin subunits are located on different chromosomes (2). The two pituitary gonadotropins act synergistically on the gonads, with FSH predominantly regulating gametogenesis, and LH production of the steroid hormones. Both LH and FSH are secreted by the same pituitary gonadotrope cells and their synthesis and secretion is controlled by the hypothalamic decapeptide, gonadotropin-releasing hormone (GnRH). LH and FSH are known to be secreted differentially in some circumstances, but intensive investigation during the past two decades has failed to provide convincing evidence for other gonadotropinreleasing hormones. Thus, the differential regulation of LH and FSH synthesis and secretion appears to be effected by changes in the pattern of GnRH secretion, together with the direct feedback effects of gonadal steroids and peptides on the gonadotrope cell. Gonadal steroids can alter GnRH secretory patterns and also modify gonadotrope responses to GnRH. Inhibin, a polypeptide also consisting of two subunits and secreted by both the testis and the ovary, circulates in plasma and exerts a selective direct action to predominantly inhibit FSH secretion. Early studies of the patterns of LH secretion in humans revealed that LH is released into the circulation in a series of pulses (3,4), and studies in animals have shown that pulses of LH reflect pulsatile secretion of GnRH by the hypothalamus (5). This pulsatile mode of GnRH secretion is essential for the maintenance of gonadotropin synthesis and release. Administration of GnRH in a continuous manner, or stimulation by long-acting GnRH agonists, results in 155
Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
156
J O H N C. M A R S H A L L ET A L .
desensitization of gonadotropin secretion (6,7). In normal physiologic situations, such as during pubertal maturation and during ovulatory cycles in females, the pattern of pulsatile GnRH secretion changes, with alterations occurring in both the frequency and amplitude of GnRH release (8). Gonadal steroids modify pulsatile GnRH secretion and the pattern of GnRH stimulation appears to be important in the differential synthesis and release of LH and FSH by the pituitary gonadotrope. In this article, we examine the role of the pattern of GnRH stimulation of the gonadotrope in regulating gonadotropin synthesis and secretion. In addition, we review current evidence on the effects of the changes in pulsatile GnRH release during human pubertal maturation and during ovulatory cycles in women, and suggest that the ability to alter the pattern of GnRH stimulation is an essential component of the regulation of reproduction in mammals. In the first section, the regulation of gonadotropin subunit gene expression will be reviewed. In the subsequent section, the proposed role of altered GnRH pulsatile secretion in normal physiology and as a cause of reproductive failure are considered.
II.
Regulation of Gonadotropin Subunit Gene Expression
The cloning of the cDNAs for the α, LH β, and FSH β subunits has allowed direct measurement of steady state mRNA concentrations. The original cDNA clones used in the present studies were generously supplied by Dr. W. W. Chin (rat α and LH β) (9,10) and Dr. R. A. Maurer (FSH β) (11). Steady state subunit mRNA concentrations were measured in cytoplasmic RNA extracts from pituitary tissue. Dot-blot hybridization was performed using saturating amounts of 32 P-labeled cDNA probes, and concentrations of mRNAs are expressed as femtomoles cDNA bound/100 μg pituitary DNA (12). The rates of transcription of the subunit genes were measured by Dr. M. A. Shupnik (University of Virginia, Charlottesville, VA) as previously described (13). A. 1.
Gonadectomy
PHYSIOLOGIC STUDIES
and Gonadal Steroid
Replacement
Initial evidence for differential regulation of the gonadotropin subunit genes was found in studies of the effects of gonadectomy in male and female rats (Fig. 1). Serum LH, FSH, and all three subunit mRNAs increase following gonadectomy in both sexes. However, the timing and magnitude of the changes in serum hormones and steady state mRNAs differ between the sexes (12,14-16). In females, serum LH, and a and LH β subunit mRNAs do not increase until 4 - 7 days after ovariectomy. Thereafter, α mRNA rises to a plateau (5-fold increase)
GONADOTROPIN-RELEASING
HORMONE
PULSES
157
FIG. 1. Pituitary gonadotropin subunit mRNA concentrations following gonadectomy in male and female rats.
after 14 days, but LH β mRNA continues to increase gradually through 30 days (15-fold increase). In contrast, serum FSH and FSH β mRNA increase more rapidly, reaching a plateau (fourfold rise) after 4 days. In male rats, both α and LH β mRNAs increase within 24 hours after orchidectomy. Thereafter, α mRNA plateaus after 14 days while LH β continues to rise through 30 days. Again, FSH β mRNA increases more rapidly than LH β and peak concentrations occur after 7 days, after which FSH β plateaus or even declines (15). Replacement of estradiol (E 2 ) in ovariectomized rats suppresses all three gonadotropin subunit mRNAs (14-16), but if E 2 is replaced in physiologic concentrations, only partial suppression occurs over 7 days (Fig. 2). E 2 replacement at the time of ovariectomy has yielded interesting results and differential effects on subunit gene expression are seen (17). Estradiol prevented the increase in LH β, but α mRNA concentrations continued to increase to a plateau after 10 days. The rise in FSH β mRNA concentrations was only partly suppressed by replacement of E 2 alone, or both E 2 and progesterone (P). Administration of a GnRH antagonist to ovariectomized females, in the presence or absence of E 2
158
J O H N C. MARSHALL ET A L .
INTACT
7d CAST.
7d C • 7dE
FIG. 2. Effect of estradiol ( E 2) replacement by silastic sc implant (to achieve plasma E 2 of 40 pg/ml) in 7-day ovariectomized female rats. 7dC + 7dE, 7 days of E 2 given to rats castrated 7 days earlier. *p < 0.05 vs intact.
and P, abolished the increase in α and LH β mRNAs, but did not prevent the rise in FSH β mRNA. This suggests that the increase in gonadotropin subunit mRNA following ovariectomy, particularly that of α and LH β, is a consequence of increased GnRH secretion. However, the failure of a GnRH antagonist to block the increase in FSH β suggests that additional ovarian hormones are involved in inhibiting FSH β mRNA expression in intact females (17). Definitive evidence in vivo is lacking, but in vitro data suggest that inhibin is required to maintain FSH
GONADOTROPIN-RELEASING
HORMONE
PULSES
159
β mRNA expression at the level found in intact female rats. The addition of inhibin to pituitary cells in culture resulted in a rapid decline in the concentrations of FSH β mRNA (18-20). In male rats, replacement of testosterone (T) at the time of castration prevents the increase in all three subunit mRNAs (21). As shown in Fig. 3 replacement of physiologic concentrations of serum Τ ( ~ 2 . 5 ng/ml) in previously castrated animals suppresses subunit mRNAs to intact values (17). However, when higher
INTACT
7d CAST.
7d C • 7dT
FIG. 3. Effect of testosterone (T) replacement by silastic sc implant (plasma Τ was 2.5 ng/ml) in 7-day orchidectomized male rats. *p < 0.05 vs intact.
160
JOHN C. M A R S H A L L ET A L .
doses of T were given to castrate animals by intermittent injections, FSH β mRNA was not fully suppressed (22,23). These differences were of interest and could reflect the effects of different concentrations of testosterone acting either on FSH β transcription, or on the stability of FSH β mRNA in cytosol. Administration of a GnRH antagonist to castrate male rats reduced FSH β mRNA concentrations with a half-disappearance time of —20 hours. In the presence of testosterone, however, steady state FSH β mRNA concentrations declined more slowly (—50 hours) after a GnRH antagonist, while the rate of decline of α and LH β mRNAs was unchanged (24). These data suggested that Τ may prolong FSH β transcription or stabilize FSH β mRNA, and other studies have suggested that stabilization of mRNA may be the mechanism of action. When a GnRH antagonist was administered for 4 days to castrate male rats, replacement of physiologic concentrations of testosterone increased FSH β mRNA twofold, without altering α or LH β concentrations (25,26). Measurement of FSH β mRNA transcription rates in GnRH antagonist-treated rats showed that transcription was not increased in the presence of testosterone, which suggests that the action of testosterone in increasing FSH β mRNA is exerted at a posttranscriptional level (25). Some studies have also suggested that other compounds may regulate FSH β mRNA stability. In vitro, addition of activin to pituitary cell cultures rapidly increased FSH β mRNA (19), and preliminary data show that this is due to enhanced stability of the FSH β mRNA (27). Thus, while data are presently incomplete, evidence suggests that testosterone and activin may enhance FSH β mRNA stability. It is also possible that compounds such as inhibin decrease FSH β mRNA by actions which include reducing the stability of FSH mRNA in cytosol. Overall, gonadal steroid replacement results in suppression of gonadotropin subunit mRNA concentrations, but the degree and mechanisms of this action may depend on plasma steroid concentrations. Testosterone suppresses pulsatile GnRH secretion and can also modify gonadotrope responses to GnRH. Estradiol has variable effects on pulsatile GnRH secretion in vivo, but estradiol replacement to castrate animals results in reduced subunit mRNA transcription rates (28). In vitro studies showed that estradiol did not reduce α and FSH β transcription rates and actually increased LH beta transcription, suggesting that the predominant actions of estradiol in vivo are exerted by inhibiting hypothalamic GnRH secretion (29,30). The positive actions of estradiol on LH secretion and LH β mRNA expression may be exerted directly at the level of the gonadotrope. While not all evidence is in agreement, the latter view is supported by the effect of estradiol in increasing LH β transcription in vitro, and the presence of an estrogen response element in the 5' region of the rat LH β gene (31). Progesterone, particularly in the presence of estradiol, reduces pulsatile GnRH secretion in vivo (32,33). Thus, progesterone could suppress subunit gene expression via hypothalamic mechanisms in vivo, but may also exert direct nega-
GONADOTROPIN-RELEASING
HORMONE
PULSES
161
tive effects on the gonadotrope. In vitro studies using pituitaries from sheep showed that progesterone directly suppressed FSH β gene transcription (34), although studies using rat pituitary cells suggest that this action may also require the presence of estradiol (35). Thus, at the present time data are incomplete, but it appears likely that gonadal steroids and peptides exert actions both at the hypothalamic level to reduce GnRH secretion and directly on the gonadotrope cell to modify expression of subunit mRNAs, hormone synthesis, and secretion. The relative degree and magnitude of actions at these sites may be modified by steroid concentration and duration of exposure to steroid hormones. 2.
Gonadotropin
Subunit mRNAs during the Rat Estrous
Cycle
Serum LH, FSH, α, and LH and FSH β mRNA subunit concentrations in the rat estrous cycle are shown in Fig. 4. During the 4-day cycle in female rats, serum LH and FSH concentrations remain low, except during the preovulatory surge during the late afternoon and evening of proestrus (36). α, LH β, and FSH β mRNAs show evidence of differential expression during the cycle, with increased expression of both β mRNAs occurring at the time of the gonadotropin surge on proestrus (37,38). On the morning of metestrus, FSH β mRNA was increased twofold, gradually falling to basal levels by the evening, α and LH β mRNAs were stable on metestrus, but both transiently increased twofold during a 12-hour period on diestrus when FSH β was unchanged. These changes in mRNA expression occurred in the absence of increased secretion of LH and FSH. On the afternoon of proestrus, LH β mRNA increased threefold, rising before the preovulatory surge of serum LH. FSH β mRNA increased fourfold, but maximum concentrations occurred 2 hours after the onset of the serum FSH surge. In contrast to these changes in β subunit mRNA concentrations, α mRNA was unchanged during the gonadotropin surge. These data suggest that both coordinate and differential regulation of expression of the three subunit genes occur during the estrous cycle. Coordinate increases in α and LH β mRNA are seen on diestrus, and both β subunit mRNAs increase during the proestrus gonadotropin surges. On metestrus, however, only FSH β mRNA increases when both α and LH β mRNAs are stable. The mechanisms regulating subunit gene expression and hormone secretion during the cycle remain uncertain, but may include the effects of changes in GnRH secretory pattern and the direct actions of ovarian steroids and peptides on the gonadotrope. Secretion of GnRH changes during the cycle, and both the amplitude and frequency of pulsatile GnRH release, are increased during the proestrus LH surge (39,40). This coincides with the increased LH β mRNA concentrations and a twofold increase in the rate of LH β transcription (29), suggesting that the LH β mRNA changes are dependent on GnRH secretion.
162
J O H N C. MARSHALL ET A L .
SERUM LH (ng/ml)
