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Neuroendocrinology |
Servicio de Endocrinología, Hospital Carlos III, Instituto de Salud Carlos III, 28029 Madrid, Spain
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 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 kb) GnRH-R mRNA increased slowly from Day 5 through the infantile and the juvenile periods, to peak at Day 35 (12-fold increase vs. Day 5). Thereafter, the levels of the GnRH-R mRNA decline slightly until Day 55 (33% decrease vs. 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 progressively through the infantile period (2.5-fold increase) and peaked at Day 25 (3.3-fold increase vs. Day 5) with a smooth nonstatistically significant increment until Day 35; then it decreased by 41.5% at Day 55. FSHß and LHß mRNA levels rose slowly until Day 25. A sharp rise occurred thereafter to reach maximum levels at Day 35 (5.8-fold for FSHß and 3.8-fold for LHß vs. Day 25). Thereafter, the levels of both mRNAs fell until Day 55 (44.1% decrease for FSHß and 37.1% decrease for LHß vs. Day 35). 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 male rats with the specific GnRH antagonist cetrorelix (1.5 mg/kg body weight/week, s.c.) through the infantile (Days 5–20) and the juvenile periods (Days 20–35). Cetrorelix completely blocked the rise of levels of the two most abundant species, 5.0 kb and 4.5 kb, of the GnRH-R mRNA, during both the infantile and the juvenile periods. Cetrorelix also abolished the developmental rise of the gonadotropin ß subunit mRNAs during the two periods of the study. In contrast, the subunit gene expression was not altered by cetrorelix treatment during any of the two periods. These data demonstrate that sexual maturation of male rats is accompanied by a progressive and concerted induction of GnRH-R and gonadotropin subunit gene expression. Developmental activation of GnRH-R and gonadotropin ß subunit genes is GnRH dependent. The apparent GnRH-independent regulation of the -glycoprotein subunit mRNA levels may be due to the contribution of thyrotropes and perhaps to the presence of exclusive regulatory signals for this gene.
gene expression, GnRH receptor, pubertal development
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Based on endocrinological and morphological landmarks, the postnatal development of the male rat has been divided into several phases [1]. The first week after birth is considered as the neonatal period, a period during which the hypophysial portal system becomes established and the gonocytes in the seminiferous tubules terminate their period of mitotic quiescence. The infantile period extends from Day 7 to Day 21, when Sertoli cells stop dividing, the blood-testis barrier becomes established, and serum FSH levels begin to increase. The juvenile period begins at Day 21 and ends at Day 35, when serum FSH levels become maximally elevated and serum testosterone levels begin to rise. The pubertal period is initiated at Day 35 and ends around Day 55–60, when mature sperm are observed in the vas deferens.
Several aspects of the maturational changes in the hypothalamic-gonadotrope cell unit of male rats have already been described. Hypothalamic proGnRH mRNA levels increase steadily (
3-fold) during the first 45 days of life [2] and are accompanied by parallel changes in hypothalamic GnRH content [3, 4]. Moreover, the pituitary content of FSH and LH increases with age, reaching maximum levels at Days 40–50 [5]. Also, ligand binding studies have demonstrated that the number of GnRH receptors in male rat pituitaries increases progressively during neonatal [6], infantile, and juvenile prepubertal stages, peaking at Day 30–35 (2-fold) [7, 8]. Although activation of the GnRH-R gene expression could be the most likely responsible for this increase, other mechanisms either at the translational (altered mRNA translational efficiency) or at posttranslational levels (i.e., receptor recycling or degradation) can be involved. In this regard, it has been reported that GnRH is able to increase the GnRH-R number on the surface of the murine cell line T3 [9] without changes on the GnRH-R mRNA levels. Moreover, in the same cell line, other authors showed that GnRH increases protein synthesis through a cap-dependent activation of mRNA translation [10]. Since the GnRH-R gene has been cloned, several works performed in vitro have demonstrated that its expression is directly regulated by two signals, pulsatile GnRH in primary pituitary cultures [11, 12] and activin in the murine gonadotrope cell line T3 [13, 14], pulsatile GnRH being the most relevant one. Because pubertal development is accompanied by increased pulsatile GnRH secretion, this presumably leads to an activation of pituitary GnRH-R gene expression. 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 male rats from the infantile period to sexual maturation. Also, changes of the gonadotropin subunit (, LHß, and FSHß) mRNA levels were assessed in parallel with those of the GnRH-R. Finally, to confirm that developmental changes of the GnRH-R and gonadotropin subunit gene expression are GnRH dependent, we have studied the effect of cetrorelix, a specific GnRH antagonist [15–17] on the pubertal regulation of the GnRH-R and gonadotropin subunit mRNA levels.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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For the ontogenic studies, male rats aged 5 (neonatal-infantile transition), 10, 15, 20, 25, 30, 35, 40, 45, 50, 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 30–55, two animals/pool. Sera were pooled as follows: Day 5, four rats/pool; Day 10, three rats/pool; Days 15–25, two rats/pool. From Days 30–55, hormone levels were determined in serum samples from individual rats. Total RNA from four pools of anterior pituitaries per age group was extracted by the method of Chomczynski and Sacchi [18] and analyzed by Northern blot. Gonadotropin and total and free testosterone levels were measured in pooled sera (six pools/age group) by specific RIAs.
To investigate the GnRH dependency of gonadotrope 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 male 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]) according to the manufacturer's recommendations to avoid unnecessarily 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, were killed on Day 20. Nontreated 5-day-old male rats were killed to serve as controls of gonadotrope gene expression at this age. In the second set of experiments, 20-day-old male 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 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 gonadotrope gene expression at this age. Anterior pituitaries were removed to analyze gonadotrope gene expression by Northern blot and trunk blood was collected for hormone assays.
Animals
Male Wistar rats, 5–55 days old, were obtained from IFFA-CREDO (Charles River laboratories, Barcelona, Spain). Five-day-old male rats were delivered to our laboratory on Day 4. The other age groups were delivered 4 days before decapitation and were maintained in a 12L:12D cycle at 24°C with food and water ad libitum, either with their respective mothers (groups of 10–20 days) or housed in groups of 3 animals/cage (25–55 days). For GnRH antagonist experiments, male 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 they were killed. All animals were killed by decapitation after ether anesthesia between 9 and 10 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. The experimental protocol described within this article 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 UV nylon membrane (Stratagene, La Jolla, CA) by diffusion blotting [19]. Each blot was hybridized with a [³²P]dCTP-labeled rat GnRH-R cDNA probe [20] using conditions previously described [21]. Blots were washed, subjected to autoradiography, and subsequently stripped off radioactivity and rehybridized with specific [³²P]dCTP-labeled rat cDNA probes for FSHß, LHß, and 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 Testosterone
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 procedure recommended by the National Institutes of Health. The intraassay variations were 8% and 7% for LH and FSH, respectively. Serum total and free testosterone were measured by RIA using commercial kits obtained from Diagnostic Products Corporation DPC (Los Angeles, CA). The intraassay variations for total testosterone assay were 5% for total testosterone concentrations of 100–400 ng/dl, 10% for concentrations of 50–100 ng/dl, and 15% for concentrations of 10–20 ng/dl. The intraassay variation for free testosterone assay was 4%. All samples from the same experiment were analyzed in duplicate 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, the 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.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The two major species of GnRH-R mRNA, 5.0 and 4.5 kb, were present in the pituitaries of 5-day-old male pups. As shown in Figure 1, the levels of both transcripts increased very slowly through the infantile period. A more rapid rise occurred through the juvenile period to peak at Day 35. Thereafter, the levels of both transcripts declined slightly until Day 55. 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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Gonadotropin Subunit mRNA Levels
As shown in Figure 2, the subunit mRNA was easily detected at Day 5 and its level increased slowly until Day 15, when it incremented faster up to Day 25; then a mild nonstatistically significant increase occurred to Day 35. Thereafter, the levels fell to Day 55. FSHß and LHß mRNA levels increased slowly until Day 25, when the levels of both mRNAs rose sharply to peak at Day 35 (Fig. 2). Then the levels of both mRNAs declined to Day 55.
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Serum Gonadotropin and Testosterone Levels
As shown in Figure 3, serum FSH levels showed a tendency to decrease from Day 5 to Day 15, with a progressive rise until Day 45, 10 days after the peak of the FSHß mRNA. In contrast, the circulating LH levels were quite variable and did not show significant changes with increasing age. Serum total testosterone was barely detectable during neonatal and infantile periods and was undetected from Day 25 to Day 35, when it began to rise until Day 50 (Fig. 3B). Serum-free testosterone levels paralleled those of total testosterone until Day 45 and remained unchanged thereafter (Fig. 3B).
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Effect of the GnRH Antagonist Cetrorelix on the GnRH-R Gene Expression
To investigate whether maturational activation of the GnRH-R gene expression is GnRH dependent, developing male 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 two periods of the study.
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Effect of the GnRH Antagonist Cetrorelix on the Gonadotropin Subunit Gene Expression
In a similar way as occurred with the GnRH-R mRNA levels, cetrorelix treatment also prevented the rise of levels of FSHß and LHß subunit mRNAs (Fig. 5) during both the infantile and juvenile periods (FSHß mRNA, infantile period: control Day 5 = 4.30 ± 0.11 arbitrary densitometric units [ADU], control Day 20 = 9.70 ± 0.30 ADU, cetrorelix-treated = 0.5 ± 0.01 ADU, P < 0.001 vs. control Day 20; juvenile period: control Day 20 = 4.50 ± 0.34 ADU, control Day 35 = 12.5 ± 0.8 ADU, cetrorelix treated = 1.10 ± 0.10 ADU, P < 0.001 vs. control Day 35; and LHß mRNA, infantile period: control Day 5 = 4.1 ± 0.30 ADU, control Day 20 = 8.2 ± 0.28 ADU, cetrorelix treated = 0.5 ± 0.14 ADU, P < 0.001 vs. control Day 20; juvenile period: control Day 20 = 6.2 ± 0.10 ADU, control Day 35 = 13.50 ± 0.80, cetrorelix treated = 3.70 ± 0.10 ADU, P < 0.001 vs. control Day 35). In contrast, the developmental induction of subunit gene expression was not altered by cetrorelix treatment during either the infantile period (control Day 5 = 5.6 ± 0.6 ADU, control Day 20 = 10.35 ± 0.21 ADU, cetrorelix treated = 11.5 ± 0.32 ADU, not significant vs. control Day 20) or the juvenile period (control Day 20 = 7.8 ± 0.42 ADU, control Day 35 = 12.09 ± 0.16 ADU, cetrorelix treated = 11.90 ± 0.13 ADU, not significant vs. control Day 35).
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Effect of the GnRH Antagonist on the Serum Gonadotropin and Testosterone Levels
Cetrorelix treatment abated serum LH and FSH concentrations to very low or undetectable levels in both the infantile and the juvenile periods of pubertal development (Table 1). Testosterone and free testosterone levels remained low to undetectable until Day 35. Cetrorelix treatment aborted the initial rise of total and free testosterone levels observed in control animals at Day 35 (Table 2). In cetrorelix-treated animals, the testicles remained in the abdomen and weighed 20-fold less than in nontreated controls (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The ontogenetic patterns of gonadotropin ß subunit gene expression are quite similar to those of the GnRH-R gene. However, the juvenile acceleration started several days later than the GnRH-R mRNA. This fact is not surprising taking into account that appropriate expression of GnRH receptors in gonadotropes may allow the subsequent expression of gonadotropin subunits in response to GnRH. Again, the progressive increase of serum testosterone, acting as negative feedback on GnRH secretion, may explain the decline of gonadotropin ß subunit mRNAs that occurred after Day 35. The levels of glycoprotein subunit mRNA behave slightly different than those of ß subunits. Alpha subunit mRNA was easily detected during the neonatal period, and a more rapid increase is observed around Day 15 (the infantile-juvenile transition) and peaks at Day 25. These divergent patterns of mRNA levels between and ß subunits may be due to different pulse frequency of GnRH at different developmental stages, favoring subunit mRNA accumulation. In this regard, it has been reported that 8-min GnRH pulse intervals increased and had little or no effect on LHß and FSHß mRNA in vitro [26]. In addition, subunit gene expression in thyrotropes (not regulated by GnRH) can account for this different pattern of the subunit gene expression. The temporal sequence of gonadotropin subunit gene expression, being subunit first, followed by LHß and FSHß, seems to fit with the embryonic ontogenetic pattern of expression of the three subunits [27, 28]. Pituitary weight increases 3- to 4-fold from infancy to puberty [7]. Literature about the relative contribution of different cell populations to total pituitary cellular mass during ontogeny is scarce [29], but some studies pointed out that GnRH is mitogenic for gonadotrophs in vitro [30]. Therefore, it is possible that populations of gonadotropes are relatively more represented in the anterior pituitary during puberty, contributing, at least in part, to the dramatic elevation of the gonadotrope mRNAs observed in the present work. In fact, anterior pituitaries of male rats treated with cetrorelix weigh 15% less than controls (data not shown). On the other hand, anesthetics, including ether, can affect the expression of several genes. However, with the exception of early immediate genes, it is unlike that measurements of other mRNAs, such as GnRH-R and gonadotropin subunit, could be affected because tissues were obtained and frozen less than 3 min after ether exposure.
The developmental pattern of circulating FSH levels was similar to that previously reported, with relatively low levels during infancy, to increase markedly during the juvenile and pubertal stages (for a review, see [1]). In contrast, as previously reported [1], LH levels are more variable and do not show a clear pattern. Luteinizing hormone pulsatility, more evident than that of FSH, may contribute to this variability. Even though it has been published that long-term (hours) exposure to ether anesthesia can alter gonadotropin levels in rats, other studies show no differences after brief (up to 30 min) exposures [31, 32]. Thus, it is unlikely that anesthesia affected LH and FSH levels in the present work.
Inasmuch as pulsatile GnRH is the major positive regulatory signal for GnRH-R gene expression, presumably most of the increment of GnRH-R mRNA levels throughout development is due to the progressive increase of endogenous GnRH secretion as young male rats approach sexual maturity. Our data show that the specific GnRH antagonist cetrorelix completely prevents the rise of levels of both GnRH-R gene transcripts during the infantile and juvenile periods. This unequivocally proves that progressive developmental activation of the GnRH-R gene expression in male rats is entirely GnRH dependent, even at very early stages of development such as during the infantile period. This is in agreement with previous works indicating that chronic administration of cetrorelix to adult male rats caused a marked decrease in the GnRH-R number [33] as well as in the levels of GnRH-R mRNA [15] by counteracting the stimulatory effect of endogenous GnRH [16, 17]. 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 reverse transcription-polymerase chain reaction (RT-PCR) assay [34]. However, another report, also using RT-PCR, showed that GnRH-R as well as gonadotropin ß subunit mRNAs were not affected by treatment of infantile female rats with a GnRH antagonist [35]. These different responses can be attributed to sexual dimorphism and to different experimental protocols, such us different antagonists and different developmental periods of treatment.
Although not evaluated in the present work, evidence from in vitro studies showing that GnRH up-regulates the GnRH-R mRNA through transcriptional activation rather than modulation of the mRNA stability [36] suggests that developmental up-regulation of GnRH-R mRNA levels by endogenous GnRH takes place mainly at the transcriptional level.
In a similar manner as occurred with the GnRH-R gene, cetrorelix completely prevented the developmental up-regulation of gonadotropin ß subunit mRNAs during both the infantile and juvenile prepubertal periods. This is congruent with the overwhelming evidence from in vivo and in vitro experiments indicating that LHß and FSHß gene expression is GnRH dependent [37–41]. 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 in the prepubertal period. However, a caveat to this conclusion must be introduced because significant evidence indicates that GnRH can indirectly regulate the FSHß gene through induction of activin and follistatin genes [42]. In agreement with the blocking effect on gonadotropin ß subunit gene expression, cetrorelix also abated serum LH and FSH concentrations and aborted the initial rise of serum total and free testosterone levels. In contrast with the gonadotropin ß subunits, the GnRH antagonist did not affect subunit mRNA levels during either the infantile or the juvenile periods. This observation was, in some ways, unexpected in light of the numerous reports showing a GnRH dependence of subunit gene expression in vivo, explored with GnRH antagonist treatment [37, 39, 41]. This different behavior of the subunit gene may be explained, at least partially, by the contribution of the subunit mRNA expressed in thyrotropes (not regulated by GnRH). Alternatively, other signals that could affect selectively the subunit gene expression may overcome the blocking effect of cetrorelix on the GnRH-regulated subunit gene expression during sexual maturation.
In summary, this study shows that sexual development in male rats is accompanied by a progressive and concerted activation of GnRH-R and gonadotropin subunit genes in the anterior pituitary through the infantile and juvenile periods to puberty. This seems to be the major mechanism for the augmented synthesis of GnRH-R number and gonadotropin subunits during sexual maturation of male rats. Developmental activation of the expression of the GnRH-R and gonadotropin ß subunit genes is GnRH dependent. The apparent GnRH-independent activation of subunit gene expression may be due to the contribution of subunit mRNA from thyrotropes. Additionally, this gives rise to the question of whether other specific regulatory signals for the pituitary subunit gene expression, aside from GnRH and TRH, could have a role.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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
Received: 26 June 2002.
First decision: 18 July 2002.
Accepted: 5 December 2002.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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T3–1 cells is associated with unchanged receptor messenger RNA (mRNA) levels and altered mRNA activity. Mol Endocrinol 1993 7:1625-1633[Abstract]This article has been cited by other articles:
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  J. P. Moore ,Jr. and S. J. Winters Weaning and the Developmental Changes in Follicle-Stimulating Hormone, Pituitary Adenylate Cyclase-Activating Polypeptide, and Inhibin B in the Male Rat Biol Reprod, April 1, 2008; 78(4): 752 - 760. [Abstract] [Full Text] [PDF]
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 S. J Winters and J. P Moore Intra-pituitary regulation of gonadotrophs in male rodents and primates Reproduction, July 1, 2004; 128(1): 13 - 23. [Abstract] [Full Text] [PDF]
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H. Zapatero-Caballero, F. Sanchez-Franco, C. Fernandez-Mendez, M. Garcia-San Frutos, L. M. Botella-Cubells, and G. Fernandez-Vazquez Gonadotropin-Releasing Hormone Receptor Gene Expression During Pubertal Development of Female Rats Biol Reprod, February 1, 2004; 70(2): 348 - 355. [Abstract] [Full Text] [PDF]
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, P < 0.05 compared with Day 35
, P < 0.001 compared with Day 35; a, P < 0.05 compared with Day 5; b, not significant compared with Day 20; c, P < 0.001 compared with Day 25; d, P < 0.001 compared with Day 35
