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BOR - Papers in Press, published online ahead of print October 15, 2003.
Biol Reprod 2003, 10.1095/biolreprod.103.020818
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BIOLOGY OF REPRODUCTION 70, 348–355 (2004)
DOI: 10.1095/biolreprod.103.020818
© 2004 by the Society for the Study of Reproduction, Inc.

Female Reproductive Tract

Gonadotropin-Releasing Hormone Receptor Gene Expression During Pubertal Development of Female Rats1

Helena Zapatero-Caballero3, Franco Sanchez-Franco3, Carolina Fernandez-Mendez3, Miriam García-San Frutos3, Luisa M. Botella-Cubells4, and Gumersindo Fernandez-Vazquez2,3

Servicio de Endocrinología,3 Hospital Carlos III, Instituto de Salud Carlos III, 28029 Madrid, Spain Consejo Superior de Investigaciones Cientificas,4 28029 Madrid, Spain


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Appropriate expression of the GnRH receptor (GnRH-R) in gonadotrophs is critical for GnRH signaling and hence for gonadotropin secretion and sexual development. In the present work, we have studied the ontogeny of the steady-state GnRH-R mRNA levels in pituitaries of female rats from Day 5 to Day 55, when sexual maturity is attained. Developmental changes of gonadotropin subunit ({alpha}, FSHß, and LHß) mRNA levels were also assessed. In addition, the role of the endogenous GnRH on the maturational changes of GnRH-R and gonadotropin subunit gene expression was investigated. Messenger RNA levels were determined by Northern blot analysis of total RNA from anterior pituitaries. Amounts of the most abundant (5.0 kilobase [kb]) GnRH-R mRNA increased slowly from Day 5 through the infantile period, to peak at Day 20 ({approx}4-fold increase vs. Day 5). Thereafter the levels of the GnRH-R mRNA decline abruptly by Day 25 (75% decrease vs. Day 20) and then fell slightly until Day 35. Parallel changes were observed on the 4.5-kb transcript of the GnRH-R gene. Alpha subunit mRNA was easily detected at Day 5 and its levels increased quickly through the beginning of the infantile period to peak at Day 10 (3.2-fold increase vs. Day 5); then it decreased by 85% at Day 35. FSHß and LHß mRNA levels rose slowly until Days 15–20, a short time before GnRH-R. Thereafter, the levels of both mRNAs fell until Day 35 (90% decrease vs. Day 15 for FSHß and 50% decrease vs. Day 20 for LHß). To ascertain whether developmental activation of the GnRH-R and gonadotropin subunit gene expression is GnRH dependent, we have studied the effect of blocking the endogenous GnRH action by treating developing female rats with the specific GnRH antagonist cetrorelix (1.5 mg/kg body weight/wk, s.c.) through the infantile (Days 5–20) and the juvenile period (Days 20–35). Cetrorelix completely blocked the rise of levels of the two most abundant species, 5.0 kb and 4.5 kb, of GnRH-R mRNA during the infantile phase and dropped them to almost undetectable levels during the juvenile prepubertal period. Cetrorelix also abolished the developmental rise of gonadotropin ß subunit mRNAs during the two periods of the study. In contrast, {alpha} subunit gene expression tended to decrease, but not significantly, with cetrorelix treatment during the two periods. These data demonstrate that sexual maturation of female rats is advanced by an early and strong induction of GnRH-R and gonadotropin subunit gene expression during the infantile period, followed by weaker persistent activation during puberty. Developmental GnRH-R and gonadotropin ß subunit gene expression is almost entirely GnRH dependent, not only in the juvenile prepubertal stage but also during the infantile period.

follicle-stimulating hormone, gonadotropin-releasing hormone receptor, luteinizing hormone, pituitary, puberty


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Pubertal development in mammals results from a complex cascade of progressive maturational events involving the entire gonadal axis. It is heralded by increasing pulsatile secretion of gonadotropins in response to augmented pulsatile GnRH secretion. The GnRH receptor (GnRH-R) plays an essential role in this process allowing GnRH signaling to the gonadotroph cell.

Based on endocrinological and morphological landmarks, the postnatal development of the female rat has been divided into several phases [1]. 1) The first week after birth is considered as the neonatal period, the time at which the hypophysial portal system becomes established and the ovary acquires steroidal responsiveness to gonadotropins. 2) The infantile period extends from Day 7 to Day 21. By the end of this period, serum FSH levels have already declined and the estrogen negative feedback has become operative. The serum concentration of {alpha}-fetoprotein has decreased considerably, thus allowing an increased uptake of estrogen by tissues; the stimulatory effect of estradiol on gonadotropin release can be unambiguously demonstrated and ovarian follicular development has progressed to follicles with 200–400 cells. 3) The next phase is a juvenile or prepubertal period that extends from Day 21 to the time at which uterine fluid appears for the first time (around Day 32–37), at which time serum GH levels have developed and adult patterns of release and pituitary responsiveness to GnRH have decreased to a minimum. The end of this period is not as well defined as those previously described because it usually continues with the peripubertal period, which begins at variable ages after Day 30. 4) The peripubertal period is characterized by marked increases in uterine weight, accumulation of uterine fluid, and enhanced ovarian responsiveness to gonadotropins; it culminates with the first preovulatory surge of gonadotropins and vaginal opening, events that are followed by the first estrus and initiation of luteal function.

Several aspects of the maturational changes in the hypothalamic-gonadotroph cell unit of female rats have already been described. Hypothalamic proGnRH mRNA levels increase steadily ({approx}3.5-fold) during the first 45 days of life [2] and are accompanied by parallel changes in hypothalamic GnRH content [3]. Moreover, the pituitary content of FSH and LH increase with age, reaching maximum levels of both gonadotropins at Day 20, with a second peak for LH on Day 32 [4]. Also, ligand binding studies have demonstrated that the number of GnRH receptors in female rat pituitaries increases progressively during the neonatal [5] and the infantile stages, peaking at Day 20 ({approx}2-fold), 15 days earlier than in males [6, 7]. Thereafter, it declines abruptly to lower levels at Days 30–50. Moreover, pituitary responsiveness to GnRH follows the same pattern as receptor number [8, 9], indicating its functionality. Very recently, the developmental pattern of the GnRH-R mRNA levels in male rats has been reported, showing a progressive accumulation, to peak at Day 35 with a small decline thereafter [10]. However, little is known regarding the developmental pattern of the GnRH-R gene expression in the female rat at the mRNA level. Studies focused on the infantile period, by using a reverse transcription-polymerase chain reaction (RT-PCR) assay, revealed an increase ({approx}2-fold) of the GnRH-R mRNA levels from Day 8 to Day 12, with a decline thereafter, in a profile similar to FSHß [11]. However, no data are available regarding the juvenile and peripubertal stages.

Since the GnRH-R gene has been cloned, several experiments performed in vitro demonstrated that its expression is directly regulated by two signals, pulsatile GnRH [12, 13] and activin [14, 15]. Because pubertal development is accompanied by increased pulsatile GnRH secretion, this presumably leads to an activation of pituitary GnRH-R gene expression. In fact, in a previous work, we have demonstrated that pubertal activation of the GnRH-R gene expression is entirely GnRH dependent as it is blocked by a GnRH antagonist, both during the infantile and juvenile periods of male rat development [10]. Studies performed in the late juvenile stage of female rats, by using RT-PCR assay, demonstrated that GnRH-R mRNA levels declined in response to a GnRH antagonist [16, 17]. However, other authors reported no changes in the GnRH-R and FSHß gene expression in response to a GnRH antagonist during the infantile period [11]. This questions the role of GnRH in the control of gonadotroph gene expression during the infantile stage of female rat development. Therefore, in order to contribute to a better understanding of the molecular basis of pubertal development, we have investigated the ontogeny of the steady-state GnRH-R mRNA levels in pituitaries of female rats from the infantile period to sexual maturation. Also, changes of the gonadotropin subunit ({alpha}, LHß, and FSHß) mRNA levels were assessed in parallel with those of the GnRH-R. Finally, to clarify the role of the endogenous GnRH on the developmental regulation of the gonadotroph gene expression at different stages of female rat puberty, we have studied the effect of cetrorelix, a specific GnRH antagonist [18–21] on the regulation of the GnRH-R and gonadotropin subunit mRNA levels during the infantile and the juvenile prepubertal periods of the female rat.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Experimental Design

For the ontogenic studies, female rats aged 5 (neonatal-infantile transition), 10, 15, 20, 25, 35, and 55 days (sexually mature rats) were used. In order to obtain sufficient sample for analysis, anterior pituitaries from several rats were pooled as follows: Day 5, six animals/pool; Day 10, four animals/pool; Days 15–25, three animals/pool; Days 35–55, two animals/pool. Sera were pooled as follows: Day 5, four rats/pool; Day 10, three rats/pool; Days 15–25, two animals/pool. For Days 35–55, hormone levels were determined in serum samples from individual rats. Total RNA from four pools of anterior pituitaries per group of age was extracted by the method of Chomczynski and Sacchi [22] and analyzed by Northern blot. Gonadotropin and estradiol levels were measured in pooled sera (six pools/group of age) by specific RIAs.

To investigate the GnRH dependency of gonadotroph gene expression during both the infantile and juvenile stages of sexual development, two sets of experiments were conducted by using the specific GnRH antagonist cetrorelix. In the first set, looking at the infantile period, 5-day-old female rats were treated by weekly s.c. injection (Days 5, 12, and 19) of either vehicle or cetrorelix acetate (1.5 mg/kg body weight [BW]), accordingly to the manufacturer's recommendations, to avoid unnecessary, more frequent injections. The last dose of cetrorelix was administered the day before decapitation to assure appropriate blockade. Both groups of rats, vehicle- and cetrorelix-treated rats, were killed on Day 20. Nontreated 5-day-old female rats were killed to serve as controls of gonadotroph gene expression at this age. In the second set of experiments, 20-day-old female rats were treated as above until Day 35 (injections on Days 20, 27, and 34) (juvenile period). Each experimental group consisted of six pools of 6 to 2 animals, as described for the ontogenic studies. The animals were killed on Day 35. A group of nontreated 20-day-old rats was killed and used as controls of gonadotroph gene expression at this age. Anterior pituitaries were removed to analyze gonadotroph gene expression by Northern blot, and trunk blood was collected for hormone assays.

Animals

Female Wistar rats, 5–55 days old, were obtained from IFFA-CREDO (Charles River Laboratories, Barcelona, Spain). Five-day-old female rats were delivered to our laboratory on Day 4. The other age groups were delivered 4 days before decapitation and maintained in a 12L:12D cycle at 24°C and with food and water ad libitum, either with their respective mothers (10–20 days of age) or housed in groups of three animals/cage (25–55 days of age). For GnRH antagonist experiments, female rats aged 4 (for the infantile period) or 16 days (for the juvenile period) were delivered to our laboratory with their mothers and maintained as above until the day of sacrifice. All animals were killed by decapitation after ether anesthesia between 0900 and 1000 h; trunk blood was collected for hormone assays, and sera were stored at -20°C until assayed. The anterior pituitaries were separated from the intermediate and posterior lobes, frozen on dry ice, and stored at -80°C until assayed. Experimental protocol described within this report was approved by the Animal Research Committee of our institution.

GnRH Antagonist

As a GnRH antagonist, we used cetrorelix acetate (SB-75), generously provided by Asta Medica (Frankfurt/Main, Germany). For injection, cetrorelix was dissolved in distilled water containing 5% mannitol and administered s.c. through a 22-gauge needle.

Northern Blot Analysis

Twenty micrograms of total RNA were electrophoresed on denaturing 1.2% agarose gels containing formaldehyde. RNA was transferred onto a Duralon ultraviolet nylon membrane (Stratagene, La Jolla, CA) by diffusion blotting [23]. Each blot was hybridized with a [32P]dCTP-labeled rat GnRH-R cDNA probe [24] using conditions previously described [25]. Blots were washed, subjected to autoradiography, and subsequently stripped of radioactivity and rehybridized with specific [32P]dCTP-labeled rat cDNA probes for FSHß, LHß and {alpha} glycoprotein subunit. Differences in total RNA loading among samples were corrected by cyclophilin mRNA quantitation in the same samples. The mRNA levels were estimated on the basis of the intensity of the hybridization signal quantitated by a densitometer.

Serum Gonadotropin and Estradiol

Serum LH and FSH were determined by RIA using reagents generously supplied by Dr. A.F. Parlow (National Hormone and Pituitary Program). LH-RP3 and FSH-RP2 were used as reference preparations. LH and FSH were iodinated by the chloramine T method and specific RIAs were performed according to the recommended procedure. The intra-assay variations were 8% and 7% for LH and FSH, respectively. Serum estradiol was measured by RIA using commercial kits obtained from Diagnostic Products Corporation DPC (Los Angeles, CA). The intra-assay variations were 15.3% for concentrations of 10–40 pg/ml. All samples from the same experiment were analyzed in duplicates in one assay.

Statistical Analysis

Data are expressed as the mean ± SD. Statistical analysis of ontogeny experiments was performed by using one-way ANOVA. Those experiments where the effects of two factors (age and cetrorelix treatment) were studied, the two-way ANOVA was performed. In both cases, Bonferroni posttest was used to establish the level of significance between a pair of groups. Statistics were done with the Prism program (GraphPad Software, Inc., San Diego, CA). A P value less than 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
GnRH-R mRNA Levels

The two major species of GnRH-R mRNA, 5.0 and 4.5 kilobase (kb), were present in the pituitaries of 5-day-old female pups. As shown in Figure 1, the levels of both transcripts increased through the infantile period to peak at Day 20 (3.5-fold increase vs. Day 5). Thereafter, the levels of both transcripts declined sharply by Day 25 and then very slowly until Day 35. The other two much less abundant transcripts, 2.5 and 1.3 kb, were almost undetectable and therefore their levels were not quantitated.



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  		FIG. 1. Developmental changes in the GnRH receptor (GnRH-R) mRNA levels in the pituitary of female rats. A) Northern blot of a representative sample for each group of postnatal age. The two major transcripts of the GnRH-R gene are shown. Cyclophilin mRNA levels were used to normalize the results. B) Densitometric quantification of levels of both transcripts. Results, expressed as arbitrary densitometric units (ADU), were calculated as GnRH-R mRNA/cyclophilin mRNA for each sample. Each point represents the mean ± SD of four samples; a: P < 0.01 compared with Day 5; b: not significant (NS) compared with Day 10; c: P < 0.01 compared with Day 20. {alpha}: P < 0.01 compared with Day 5; ß: NS compared with Day 10; {chi}: P < 0.001 compared with Day 20

Gonadotropin Subunit mRNA Levels

As shown in Figure 2, the {alpha} subunit mRNA was easily detected at Day 5 and its levels increased rapidly to peak at Day 10 (3.5-fold increase vs. Day 5). Thereafter, the levels declined slowly until Day 55. FSHß mRNA levels were also easily detected by Day 5 and increased slowly to peak at Day 15 (3-fold increase vs. Day 5), and then declined to very low levels by Days 35 (peripubertal period). LHß mRNA levels followed a similar pattern as FSHß, peaking at Days 15–20 with a smother falling thereafter through the juvenile and peripubertal periods (Fig. 2).



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  		FIG. 2. Developmental changes in gonadotropin subunit (FSHß, LHß, and {alpha}) mRNA levels in the pituitary of female rats. Blot from the experiment depicted in Figure 1 was stripped of radioactivity and rehybridized with specific [32P]dCTP-labeled rat cDNA probes for FSHß, LHß, and {alpha} subunits. A) Northern blot of a representative sample for each group of postnatal age. B) Densitometric quantification of bands was expressed in arbitrary densitometric units (ADU) and calculated as gonadotropin subunit mRNA/cyclophilin mRNA for each sample. Each point represents the mean ± SD of four samples; 1: P < 0.01 compared with Day 5; 2: not significant (NS) compared with Day 10; 3: P < 0.05 compared with Day 20; {alpha}: P < 0.05 compared with Day 5; ß: NS compared with Day 10; {chi}: P < 0.01 compared with Day 20; a: P < 0.01 compared with Day 5; b: NS compared with Day 10; c: P < 0.001 compared with Day 20

Serum Gonadotropin and Estradiol Levels

As shown in Figure 3, serum FSH levels increased progressively to peak at Day 15; then the levels fell to Day 35, when they showed a small peak. In contrast, the circulating LH levels increased from Day 5 to Day 10, fell slowly until Day 25, reaching maximum levels at Day 35, when the estrous cycle has started. Over the course of pubertal development in female rats, serum estradiol levels exhibited a bimodal profile (Fig. 3B). It was detected at Day 5 and increased through the infantile period to reach the first peak at Day 15. Thereafter, it declined to a nadir at Day 25, with a second peak at Day 35.



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  		FIG. 3. Serum hormone levels during sexual development of female rats. A) Gonadotropins (LH and FSH). Values represent the mean ± SD of six samples; a: P < 0.001. compared with Day 5; b: P < 0.001 compared with Day 5; c: P < 0.001 compared with Day 10; {alpha}: P < 0.05 compared with Day 5; ß: P < 0.05 compared with Day 5; {chi}: P < 0.05 compared with Day 10; {delta}: not significant (NS) compared with Day 25. B) Estradiol. Values represent the mean ± SD of six samples; 1: P < 0.001 compared with Day 5; 2: P < 0.05 compared with Day 10; 3: P < 0.001 compared with Day 15; 4: P < 0.001 compared with Day 25

Effect of the GnRH Antagonist Cetrorelix on the GnRH-R Gene Expression

To investigate the role of the endogenous GnRH on the GnRH-R gene expression during different stages of the female rat maturation, developing female rats were treated either with vehicle or the GnRH antagonist cetrorelix during the infantile and juvenile stages of prepubertal development. As shown in Figure 4, cetrorelix treatment completely abolished the rise of the two major species of the GnRH-R mRNA during the infantile period. Cetrorelix also abated the expression of the GnRH-R gene during the juvenile prepubertal period.



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  		FIG. 4. Effect of the GnRH antagonist cetrorelix on developmental activation of the GnRH receptor gene expression in the pituitary of female rats. Animals were treated either with vehicle or the GnRH antagonist (1.5 mg/kg BW/wk, s.c.) during the infantile (Days 5–20) and juvenile (Days 20–35) periods. 5: Nontreated 5-day-old rats; 20: infantile control group; 20A: infantile antagonist-treated group; 35: juvenile control group; 35A: juvenile antagonist-treated group. A) Northern blot of a representative sample for each experimental group. The two major transcripts of the GnRH-R gene (5.0 and 4.5 Kb) are shown. Cyclophilin mRNA levels were used to normalize the results. B) Densitometric quantification of bands was expressed in arbitrary densitometric units (ADU) and calculated as GnRH-R mRNA/cyclophilin mRNA. Each bar represents the mean ± SD of six samples. ** P < 0.01; *** P < 0.001

Effect of the GnRH Antagonist Cetrorelix on the Gonadotropin Subunit Gene Expression (Fig. 5)

Cetrorelix treatment blocked the rise of LHß and FSHß subunit mRNA levels during the infantile period in a similar manner as the GnRH-R mRNA. (LHß mRNA: Control Day 5 = 0.15 ± 0.036 arbitrary densitometric units [ADU], Control Day 20 = 1.08 ± 0.108 ADU, cetrorelix-treated = 0.12 ± 0.02 ADU, P < 0.001 vs. Control Day 2; FSHß mRNA: Control Day 5 = 0.3 ± 0.025 ADU, Control Day 20 = 0.72 ± 0.03 ADU, cetrorelix-treated = 0.096 ± 0.006 ADU, P < 0.001 vs. Control Day 20). Also, cetrorelix treatment abated the expression of gonadotropin ß subunits during the juvenile phase (LHß mRNA: Control Day 20 = 1.08 ± 0.26 ADU, Control Day 35 = 1.32 ± 0.035, cetrorelix-treated = 0.16 ± 0.02 ADU, P < 0.001 vs. Control Day 35; FSHß mRNA: Control Day 20 = 1.85 ± 0.11 ADU, Control Day 35 = 1.08 ± 0.081 ADU, cetrorelix-treated = 0.093 ± 0.006 ADU, P < 0.001 vs. Control Day 35). In contrast, the developmental changes of the {alpha} subunit gene expression was not significantly altered by cetrorelix treatment, during both the infantile period (Control Day 5 = 0.83 ± 0.032 ADU, Control Day 20 = 0.17 ± 0.045 ADU, cetrorelix-treated = 0.65 ± 0.19 ADU, not significant [NS] vs. Control Day 20) and the juvenile period (Control Day 20 = 1.20 ± 0.072 ADU, Control Day 35 = 0.72 ± 0.03 ADU, cetrorelix-treated = 1.04 ± 0.056 ADU, NS vs. Control Day 35).



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  		FIG. 5. Effect of the GnRH antagonist cetrorelix on developmental activation of the gonadotropin subunit (LHß, FSHß, and {alpha}) gene expression in the pituitary of female rats. The animals were treated either with vehicle or the GnRH antagonist (1.5 mg/kg BW/wk, s.c.) during the infantile (Days 5–20) and juvenile (Days 20–35) periods. 5: Nontreated 5-day-old rats; 20: infantile control group; 20A: infantile antagonist-treated group; 35: juvenile control group; 35A: juvenile antagonist-treated group. Blots from the experiment depicted in Figure 4 were stripped of radioactivity and rehybridized with specific [32P]dCTP-labeled rat cDNA probes for FSHß, LHß, and {alpha} subunits. A) Northern blot of a representative sample for each experimental group. Cyclophilin mRNA levels were used to normalize the results. B) Densitometric quantification of bands was expressed in arbitrary densitometric units (ADU) and calculated as GnRH-R mRNA/cyclophilin mRNA. Each bar represents the mean ± SD of six samples. ** P < 0.01; *** P < 0.001

Effect of the GnRH Antagonist on Serum Gonadotropins and Estradiol Levels

Cetrorelix treatment decreased serum LH and FSH concentrations in both the infantile and the juvenile periods of female rat pubertal development (Table 1). Cetrorelix treatment completely abated FSH levels during the two stages of the development. Meanwhile, LH levels are completely abated during the juvenile phase, only a partial blockade (66%) was observed in infancy. Cetrorelix treatment reduced estradiol levels during the juvenile prepubertal period. However, no statistically significant decrease was observed during infancy (Table 2). In cetrorelix-treated animals, the ovaries were atrophied, weighing 20-fold less than nontreated controls (data not shown).


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  		TABLE 1. Effect of the GnRH antagonist cetrorelix on serum gonadotropin levels during sexual maturation of female rats.*


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  		TABLE 2. Effect of the GnRH antagonist cetrorelix on serum estradiol levels during sexual maturation of female rats.*


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
It now seems clear that puberty is a gradual process that is progressively manifested. To our knowledge, the present work shows for the first time the developmental changes in GnRH-R and gonadotropin subunit mRNA levels in female rats from Day 5 to sexual maturity, as determined by Northern blot analysis. The levels of the two major transcripts of the GnRH-R gene, 5.0 and the 4.5 kb [24, 26], rose in parallel to peak at Day 20, at the end of infancy and long before complete sexual maturation was attained. Thereafter, during the juvenile and peripubertal periods, levels of both GnRH-R mRNAs dropped markedly until estrous cycle changes were operative in the mature female rat [27]. Previous work has reported dynamic changes in the GnRH-R mRNA levels during the rat estrous cycle, with a 3-fold increase on the afternoon of proestrus compared with the morning of metaestrus [28]. Therefore, values in 55-day-old rats, in our work, may reflect random estrous cycle values because we have not checked the stage of the estrous cycle in these rats. The ontogenetic patterns of gonadotropin ß subunit gene expression are quite similar to that of the GnRH-R gene. This pattern of gonadotroph gene expression is in agreement with many other reports about other facts of the female rat sexual maturation [for reviews, see 1, 27]. Thus, it parallels the profiles described for GnRH-R number [6, 7], pituitary responsiveness to GnRH [8, 9], pituitary gonadotropin content [4], and serum gonadotropin levels [1, 27], which are exceedingly high during infancy with a marked decline to low levels until the first estrus. Clearly, this female pattern is in contrast with our previously reported ontogenetic profiles of the GnRH-R and gonadotropin subunit gene expression in male rats [10], where relatively lower levels were detected during infancy and maximal levels at the end of the juvenile period (Day 35), close to sexual maturity. This shows a sexual dimorphism in the developmental activation of the gonadotroph gene expression, occurring earlier in female than in male rats.

Inasmuch as pulsatile GnRH is the major positive regulatory signal for GnRH-R gene expression [12, 13], presumably most of the changes in GnRH-R mRNA levels during development might be induced by changes in endogenous GnRH secretion. Our data show that the specific GnRH antagonist cetrorelix completely prevents the rise in levels of both GnRH-R gene transcripts during the infantile period and also abated its expression during the juvenile phase. This is in agreement with previous work indicating that chronic administration of cetrorelix to adult female rats caused a marked decrease in the levels of GnRH-R mRNA by counteracting the stimulatory effect of endogenous GnRH [20]. Also, it has been reported that chronic cetrorelix treatment of female rats from Day 25 to Day 36 down-regulates (by 50%) the expression of the GnRH-R gene, measured by quantitative RT-PCR assay [16, 17]. In the same way as occurred with the GnRH-R gene, cetrorelix prevented the infantile up-regulation of gonadotropin ß subunit mRNAs and also abates their expression during the juvenile prepubertal period. This is congruent with the overwhelming evidence from in vivo and in vitro experiments indicating that LHß and FSHß gene expression is GnRH dependent [29–33]. Also, the dramatic inhibition of FSHß mRNA levels by cetrorelix suggests that pituitary and gonadal activin is not a major contributor to the activation of the FSHß gene expression, both during the infantile and prepubertal periods. However, a caveat to this conclusion must be introduced because great evidence indicates that GnRH can indirectly regulate the FSHß gene through induction of activin and follistatin genes [34]. These data indicate that developmental activation of the GnRH-R and ß-gonadotropin subunit gene expression in female rats is entirely GnRH dependent, even at very early stages of development, such as the infantile period. Thus, gonadotroph gene expression during development is GnRH dependent in both female and male rats. However, our results are in apparent discordance with another report by Wilson et al. using RT-PCR assay [11]. Despite the fact that serum FSH and LH levels decreased in response to the GnRH antagonist, these authors observed that GnRH-R as well as gonadotropin ß subunit mRNAs were not affected by treatment of infantile female rats with a GnRH antagonist along a period of 2 days (Postnatal Days 8–10 and 11–13) [11]. This lack of response to the GnRH antagonist treatment may be attributed to a shorter period of treatment (2 days) in comparison with the present work (15 days). Another possibility is that the gonadotroph has the capacity of responding to GnRH after the 13-day postnatal period. However, this is unlikely because functional GnRH-R [8, 9] and hypothalamic expression of GnRH [2] and its secretion in vitro by hypothalamic fragments [35, 36] have been present since the neonatal period and are maintained during infancy [35]. Therefore, taken together, our results and the great bulk of previous information indicate that the hypothalamic-gonadotroph unit of female rats is very active during the infantile period, with a relative inhibition of neonatal levels of activity during the juvenile prepubertal period, before estrous cyclic activation occurs in sexually mature rats. The most plausible explanation for this infantile activation of the female rat reproductive axis is the presence of high serum levels of {alpha}-fetoprotein, which binds estradiol selectively [37, 38] and renders the estradiol negative feedback inefficient [39]. This allows a progressive activation of the gonadal axis to peak at mid to late infancy. This trend is deflected down during the juvenile period, when the estradiol negative feedback progressively takes place as long as serum {alpha}-fetoprotein levels go down and estradiol production by developing ovaries increases. This negative feedback turns positive when estradiol circulating levels increase to a certain level, leading to the first preovulatory gonadotropin surge at proestrus [40, 41].

The levels of glycoprotein {alpha} subunit mRNA behave slightly differently than those of ß subunits. Alpha subunit mRNA was easily detected during the neonatal period and a more rapid increase is observed at the beginning of the infantile period with a peak at Day 10. It then decreased slowly. These divergent patterns of mRNA levels between {alpha} and ß subunits were also observed in male rats [10]. Alpha subunit gene expression in thyrotropes (not regulated by GnRH) can account for this different pattern of {alpha} subunit gene expression.

The ontogenetic patterns of GnRH-R and gonadotropin ß subunit gene expression, and gonadotropin secretion do not show parallelism with proGnRH mRNA levels [2] and GnRH content in the preoptic area-anterior hypothalamic region of the rat brain [3, 42]. Both hypothalamic proGnRH mRNA and peptide content increased steadily and in parallel from birth to sexual maturity in males and females. In contrast, GnRH secretion, GnRH-R and gonadotropin subunit expression, and gonadotropin secretion are all inhibited during the juvenile period of female rat development. These divergent ontogenetic patterns can be explained by differential actions of estradiol on the proGnRH gene expression and GnRH secretion [see ref. 41 for review]. While the role of estrogen in the regulation of proGnRH gene expression is controversial, low concentrations are clearly inhibitory for GnRH secretion [41]. Thus, rising estradiol production and secretion by the maturing ovaries during the juvenile period does not affect GnRH gene expression and clearly inhibits GnRH secretion, leading to a progressive increase of both proGnRH mRNA and its peptide content in the hypothalamus, while by inhibiting GnRH secretion, gonadotroph gene expression and gonadotropin secretion are decreased, as they are positively regulated by GnRH.

The ontogenetic profiles of serum gonadotropin levels in our experiments, running in parallel with the gonadotroph gene expression, are in agreement with many other reports [1, 6, 7]. An early increase in the infantile period and a later decrease during the juvenile stage until first ovulation, when LH levels became maximal, occurred.

As previously reported [see 1, 27 for reviews], circulating estradiol (E2) levels increase during infancy to reach a first peak on Day 15. However, as mentioned above, this early wave of total circulating E2 does not correlate with free, biologically active E2, which is likely to be low at this time because estrogenization of target organs does not become evident until around 35 days of age, when vaginal opening indicates the rise in circulating free estradiol [1, 4, 8, 27]. E2 levels decrease from Day 15 to Day 25 as long as circulating {alpha}-fetoprotein falls. Thereafter, E2 increased again during pre- and peripuberty to reach a peak during the first estrus, around Days 35–45, reflecting the progressive maturation of ovarian steroidogenesis. As occurred with GnRH-R and ß-gonadotropin subunit gene expression, serum LH and FSH levels dropped in response to the GnRH antagonist during both the infantile and juvenile periods. These results are in accordance with previous reports on infantile [11], juvenile [16, 17], and adult [18] female rats. Although E2 levels tend to decrease in infantile rats treated with the GnRH antagonist, it was not statistically significant. This, together with the fact that serum LH and FSH levels were abated by cetrorelix, suggests that much of the circulating estradiol during the infancy of female rats is from an extraovarian source, mainly from the adrenals, as has been reported previously [43–45]. Conversely, serum estradiol rises during the juvenile prepubertal stage and was suppressed by GnRH antagonist, indicating that the developing ovary at this stage is actively engaged in steroidogenesis, providing much of the circulating estradiol.

On the other hand, {alpha} subunit mRNA levels tend to decrease after cetrorelix treatment during the infancy of female rats. However, this was not statistically significant. This observation was, in some way, unexpected in light of the numerous reports showing a GnRH dependence of {alpha} subunit gene expression in vivo, explored with GnRH antagonist treatment [29, 30, 32, 33]. This different behavior of the {alpha} subunit gene may be explained, at least partially, by the contribution of the {alpha} subunit mRNA expressed in thyrotropes (not regulated by GnRH). Alternatively, other signals that could affect selectively the {alpha} subunit gene expression may overcome the blocking effect of cetrorelix on the GnRH-regulated {alpha} subunit gene expression during sexual maturation.

In summary, this study shows that sexual development in female rats is accompanied by a progressive and concerted expression of GnRH-R and gonadotropin subunit genes in the anterior pituitary to reach maximal rates during infancy and are kept at a minimum during the juvenile prepubertal period. This seems to be the major mechanism for the patterns of synthesis of GnRH-R number, pituitary responsiveness to GnRH, and gonadotropin subunit synthesis and gonadotropin secretion, being also greatest during infancy and lowest during prepuberty. This represents a striking sexual dimorphism in comparison with male rats, where the activation of gonadotroph genes is continuous all along prepubertal development, being greatest in a period closer to sexual maturity. Developmental activation of the expression of the GnRH-R and gonadotropin ß subunit genes and gonadotropin secretion is GnRH dependent, even during the preliminary burst of the infantile activation. Changes in estradiol production and secretion by the maturing ovary, combined with the dramatic variation on serum levels of the estradiol-selective binding protein {alpha}-fetoprotein, provided a plausible explanation for the sexual dimorphic pattern of gonadotroph gene expression in developing female rats. Very high levels of {alpha}-fetoprotein impede estradiol negative feedback on GnRH release, leading to the infantile burst of gonadotroph gene expression that spurs ovarian development. A striking drop in {alpha}-fetoprotein levels, together with increased estradiol synthesis and secretion by the developing ovary, allows a progressive negative feedback on GnRH secretion, responsible for the partial inhibition of the juvenile prepubertal gonadotroph-ovarian axis. The progressive rise of estradiol levels to the threshold necessary for positive feedback on the hypothalamus during the first proestrous surge of gonadotropins, initiates the cyclic nature of mature ovarian function. The apparent GnRH-independent activation of {alpha} subunit gene expression can be due to the contribution of {alpha} subunit mRNA from thyrotropes. Additionally, this raises the question of whether other specific regulatory signals for the pituitary {alpha} subunit gene expression, aside from GnRH and thyrotropin-releasing hormone, could have a role.


   ACKNOWLEDGMENTS
 
We would like to thank Dr. William W. Chin for his generous gift of rat gonadotropin subunit cDNAs and Dr. Ursula B. Kaiser for kindly providing rat GnRH-R cDNA. We are also indebted to Dr. Thomas Reissmann (Asta Medica, Frankfurt/Main, Germany) for his generous gift of cetrorelix. Also, we are grateful to Dr. A. F. Parlow (National Hormone and Pituitary Program) for providing rat FSH and LH reference preparations and the corresponding antibodies.


   FOOTNOTES
 
1 Supported by grants PM95-212 from the Ministerio de Educación y Ciencia and 99/0412 from the Fondo de Investigación Sanitaria (to G.F.V.) and by a predoctoral fellowship from the Instituto de Salud Carlos III (to H.Z.C.) Back

2 Correspondence: Gumersindo Fernandez-Vazquez, Servicio de Endocrinología, Hospital Carlos III, Instituto de Salud Carlos III, Sinesio Delgado 10, Madrid 28029, Spain. FAX: 34-91 733 6614; gfernandez.hciii{at}salud.madrid.org Back

Received: 30 June 2003.

First decision: 25 July 2003.

Accepted: 24 September 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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