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Endocrinology Vol. 140, No. 5 2280-2287
Copyright © 1999 by The Endocrine Society

ARTICLES

Mechanisms for the Regulation of Gonadotropin-Releasing Hormone Gene Expression in the Developing Mouse1

Andrea C. Gore, James L. Roberts and Marie J. Gibson

Neurobiology of Aging Laboratories (A.C.G.), Arthur M. Fishberg Research Center for Neurobiology (A.C.G., J.L.R., M.J.G.), Henry L. Schwartz Department of Geriatrics and Adult Development (A.C.G.), and Department of Medicine (M.J.G.), Mount Sinai School of Medicine, New York, New York 10029

Address all correspondence and requests for reprints to: Andrea C. Gore, Ph.D., Neurobiology of Aging Laboratories, Box 1639, Mount Sinai School of Medicine, New York, New York 10029. E-mail: gore{at}msvax.mssm.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The release of GnRH peptide from neuroterminals in the median eminence increases during postnatal development. We were interested in determining the biosynthetic component contributing to the regulation of GnRH decapeptide levels, and ascertaining the molecular mechanism for these changes. Male and female C57bl/6 mice, from embryonic day (E)16 through postnatal day (P)60, were killed, and the preoptic area-anterior hypothalamus was dissected out. Cytoplasmic and nuclear RNA were extracted separately. Levels of GnRH messenger RNA (mRNA) and primary transcript were quantitated in individual preoptic area-anterior hypothalamus cytoplasmic and nuclear fractions, respectively, by ribonuclease protection assays. Serum LH levels were assayed by RIA. GnRH mRNA levels in the cytoplasm increased gradually and significantly during postnatal development in both males and females, reaching a peak at P55 in females and P40 in males. GnRH primary transcript levels in the nucleus, an index of GnRH gene transcription, changed in a completely different manner developmentally, and they differed between male and female mice. GnRH primary transcript levels in males were quite low until P5, when they underwent an increase of approximately 4-fold, between P5 and P7. They continued to increase through P15, at which time they reached adult levels. In females, GnRH primary transcript levels were high at E16, decreased to a nadir at P5, and then underwent an increase of approximately 5-fold to P7, which were comparable with adult levels. The large and sexually dimorphic changes in GnRH primary transcript between E16 and P7, in the absence of similar changes in GnRH mRNA, suggest that differential mechanisms, such as gene transcription and mRNA stability, play a role in determining levels of GnRH mRNA at different stages of development.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
INCREASES in pulsatile GnRH release during development play a critical role in the timing of the onset of puberty. Evidence for such a role is provided by reports that administration of GnRH to immature animals can result in precocious puberty (1, 2). Hypothalamic GnRH content increases steadily during postnatal development (3, 4, 5, 6), and it has been reported that changes in GnRH biosynthesis, as determined by messenger RNA (mRNA) and proGnRH peptide levels, also increase during the postnatal period, through puberty (5, 7, 8). Similarly, GnRH release, as measured by push-pull perfusion, increases during pubertal development in female rhesus monkeys (9, 10, 11) and rats (12). Thus, developmental changes in GnRH mRNA and peptide precursor levels seem to roughly parallel those of GnRH peptide levels and release.

The biosynthetic mechanism(s) responsible for developmental changes in GnRH mRNA levels is (are) currently unknown. Our laboratory and others have previously reported that levels of GnRH mRNA, both in the animal and in hypothalamic cell lines, can be regulated by transcriptional, as well as posttranscriptional, mechanisms (8, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22). With respect to ontogeny, we have reported that GnRH mRNA levels increase during pubertal development in female rats, whereas GnRH primary transcript (an indicator of GnRH gene transcription) (23) does not change at this time, suggesting that the pubertal increase in GnRH mRNA levels is caused by a posttranscriptional mechanism, such as mRNA stability (8, 13). However, it is not known whether such a mechanism is of equal importance during other periods of development, particularly the perinatal period, during which changes in neuronal and glial circuitry occur and which is the critical period for sexual differentiation in rodents (24, 25, 26, 27). Therefore, in the present study, we first performed a highly detailed quantitative analysis of GnRH mRNA levels in developing male and female mice, from embryonic day (E)16 through postnatal day (P)60. We then measured GnRH primary transcript levels in these mice as a means of determining whether alterations in GnRH gene transcription are a mechanism responsible for those observed changes in GnRH mRNA levels.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Male and female C57bl/6 mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and housed at the Center for Laboratory Animal Science at Mount Sinai School of Medicine. To provide embryos or pups through P28, breeding pairs were established. Mice raised in our colony were weaned at P21. For mice to be used at P30 through P60, animals were purchased directly from the vendor at P23 through P53 and were allowed 1 week to recover from the shipping process. In some cases, mice from our in-house colony were allowed to mature and were compared with age-matched mice shipped from the vendor, and no differences in GnRH gene expression were observed (unpublished observation). Mice were housed in same-sex groups of 2–3/cage in a room with a controlled temperature and light cycle (12 h light, 12 h dark, lights on at 0700 h) and were provided with food and water ad libitum. Female mice raised in our colony were monitored daily for the timing of vaginal opening, which was determined to occur between P32 and P40. Five to 6 animals were used for each group.

Animals were killed rapidly by decapitation, the brain removed, chilled on ice, and the preoptic area-anterior hypothalamus (POA-AH) dissected, as follows (7): The caudal border of the dissection was made by a coronal cut just posterior to the entry point of the optic chiasm. The rostral border was made by a coronal cut at the posterior third of the olfactory tubercle. This coronal section (~3-mm thick in adults, and adjusted accordingly for younger animals) was laid rostral side up, on a chilled glass plate. Then, an isosceles triangle-shaped cut was made with the apex of the triangle just under the midline of the corpus callosum, and the two legs of the triangle passing through the anterior commissure (7). This dissection should include virtually all GnRH perikarya (28, 29). A stainless steel brain slicer (model RBM-4000, Activational Systems, Warren, MI) was used for brains of mice P20 and older. Brains were snap-frozen in liquid Freon on dry ice and were stored at -70 C until use. Trunk bloods were collected, allowed to clot, and centrifuged; and serum was stored at -70 C.

RNA extraction and ribonuclease (RNase) protection assay
RNA from frozen POA-AH dissections was extracted, as described previously (16, 30). Cytoplasmic RNA and nuclear RNA from individual POA-AH dissections were suspended in 20 µl of hybridization solution [0.1 M EDTA (pH 8) and 4 M guanidine thiocyanate; final pH, 7.5] for RNase protection assay. To measure GnRH mRNA in the cytoplasm, a murine GnRH cDNA clone, 443 bp in length, spanning the EcoO109I and XbaI restriction sites and subcloned into a Bluescript KS(+) vector (Stratagene, La Jolla, CA), was used (23, 31). Cyclophilin mRNA levels in the same cytoplasmic fraction were measured using a 111-bp clone, spanning from the PstI and XmnI restriction sites, and subcloned into a Bluescript KS(+) vector (7). GnRH primary transcript levels in the nucleus were measured using a proGnRH genomic fragment covering 383 bp of the intron A-exon 2-intron B junction (A2B), and subcloned into the SpeI and HindIII restriction sites of a Bluescript SK(+) vector (14, 23).

Solution hybridization/RNase protection assay was performed as described previously (16, 30). Briefly, GnRH probes were labeled with [{alpha}-32P]uridine 5'-triphosphate to high specific activity (~1,300,000 cpm/ng) and cyclophilin probe to low specific activity (~60,000 cpm/ng) in a final vol of 25 µl (20 µl of RNA and 5 µl of probe). Cytoplasmic samples were incubated with the GnRH cDNA clone and cyclophilin probes in the same tubes. For standard curves, probes were mixed with increasing known amounts of reference RNAs (GnRH cDNA, 0–2 pg; cyclophilin, 0–250 pg; A2B, 0–1 pg). Samples and standards were allowed to hybridize for 16–18 h at 30 C; the remainder of the assay was conducted exactly as described previously (16, 30). Gels were exposed to x-ray film for 18–48 h to produce an autoradiogram, and to a phosphor imaging screen (Molecular Dynamics, Inc., Sunnyvale, CA) for 18 h for quantitation. The amount of radioactivity in each sample was determined by comparison with the amount of reference RNA calculated by regression analysis.

RIA of LH
LH in serum samples was determined in single samples (because of low blood volumes from immature mice) in the laboratory of Dr. Marie J. Gibson, by double-antibody RIA using the rat LH RP-3 standard from the National Hormone and Pituitary Program of the NIDDK. The antibody used was NIDDK-anti-rLH-S-10, at a dilution of 1:250,000. The sensitivity of the assay was 0.32 ng/ml, at a serum vol of 25 µl. All samples were analyzed in a single assay, and the intraassay coefficient of variation was 5.6%.

Statistical analyses
Changes in mRNA or LH levels were analyzed by 2-way ANOVA, with age and sex as variables. Post hoc comparisons were performed using Fisher’s protected least-significant-difference analysis. Significance was set at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Changes in GnRH mRNA levels during development
GnRH mRNA levels in the POA-AH cytoplasmic fraction increased significantly during development in male and female mice. Levels of cytoplasmic GnRH mRNA were calculated by three different methods: 1) normalized to cyclophilin mRNA levels in the same dissection, to account for loss of RNA during the assay; 2) normalized per microgram RNA in the dissection, as determined by spectrophotometric analysis, to account for differences in the size and/or extent of the dissection; and 3) expressed per animal in individual POA-AH dissections, to enable comparisons with GnRH primary transcript levels in the corresponding nuclear fractions. This latter method of expression is less influenced by minor fluctuations in the size of the dissection, because it reflects absolute levels of GnRH mRNA in a single animal. Expression by all three methods yielded similar ontogenic patterns of GnRH mRNA levels, which varied in magnitude but not in developmental timing.

When normalized to cyclophilin mRNA levels in the same sample, GnRH mRNA levels underwent approximately 4- to 5-fold increases during postnatal development. An autoradiogram showing cytoplasmic GnRH and cyclophilin mRNAs in representative individual POA-AH dissections is shown in Fig. 1aGo. Two-way ANOVA indicated a significant effect of age (P < 0.0001) but no significant effect of sex (P = 0.29), and a significant interaction of sex and age on GnRH mRNA levels (P < 0.0001; Fig. 2Go).



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  		Figure 1. Autoradiogram showing GnRH and cyclophilin mRNAs in the cytosolic fractions (A) and GnRH primary transcript RNA in the nuclear fractions (B) of individual POA-AH dissections from female and male mice at representative ages. The age of the animals, in P, is shown at the bottom.

 


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  		Figure 2. Changes in GnRH mRNA and cyclophilin mRNA (inset) in developing female (A) and male (B) mice. a, P < 0.01 vs. E16, P0, P5, and P7; P < 0.02 vs. P10. b, P < 0.01 vs. P15. c, P < 0.05 vs. P20, P25, and P28. d, P < 0.01 vs. P30 and P32; P < 0.05 vs. P35 and P40. e, P < 0.05 vs. P5 and P7. f, P < 0.005 vs. E16, P5, and P7; P < 0.05 vs. P0 and P10. g, P < 0.0001 vs. all preceding ages. h, P < 0.005 vs. P28, P30, and P32. i, P < 0.03 vs. P40. j, P < 0.01 vs. P28, P30, and P35–P55; P < 0.05 vs. P32. *, P < 0.05 vs. corresponding male; **, P < 0.0005 vs. corresponding male. Inset, changes in cyclophilin mRNA during development in female (A) and male (B) mice. 1, P < 0.05 vs. P25–P60. 2, P < 0.05 vs. P7–P35. 3, P < 0.01 vs. P45. *, P < 0.05 vs. corresponding male.

 
For female mice, post hoc analysis indicated that GnRH mRNA levels normalized to cyclophilin mRNA did not undergo any significant changes from E16 through P15 (Fig. 2aGo). GnRH mRNA levels in P20 mice were significantly higher than in E16, P0, P5, P7, and P10 mice (a, P < 0.005, 0.01, 0.005, 0.005 and 0.02, respectively). While levels of GnRH mRNA in P15 females did not differ significantly from those in P20 mice (P = 0.06), they were significantly higher in P25 than P15 mice (b, P < 0.05). Female mice aged P20 through P40 had GnRH mRNA levels that did not differ from each other. P45 mice had significantly higher GnRH mRNA levels than P20, P25, and P28 mice (c, P < 0.05), but did not differ from P30 through P35 females. P30 mice had similar GnRH mRNA levels to mice aged P32 through P50 but were significantly lower than P55 (d, P < 0.01) and P60 (P < 0.05) female mice. P35 and P40 female mice also had GnRH mRNA levels lower than those in P55 mice (P < 0.05) but while P35 mice did not differ from P60 mice, GnRH mRNA levels in P40 mice were significantly lower than those in P60 mice (P < 0.05). Overall, these data indicate gradual, significant increases in GnRH mRNA levels, with the highest levels observed at P55–60.

For male mice, GnRH mRNA levels normalized to cyclophilin mRNA levels also underwent significant, gradual increases during development (Fig. 2bGo). They did not differ from E16 through P10, and the only difference from E16 through P20 occurred in P15 male mice compared with P5 and P7 males (e, P < 0.05). GnRH mRNA levels were significantly elevated at P25 compared with E16 through P10 (f, P < 0.005, 0.03, 0.0001, 0.001 and 0,05 for E16, P0, P5, P7, and P10, respectively). A more abrupt elevation occurred at P28, at which age GnRH mRNA levels were significantly elevated compared with all previous ages (g, P < 0.001). They did not undergo any further increases until P40, at which age GnRH mRNA levels were highest, and were significantly elevated compared with P28, P30 and P32 (h, P < 0.005). Then, GnRH mRNA levels decreased, with a significant difference between P40 and P55 (i, P < 0.03), and continued to decrease with significantly lower levels at P60 than all preceding ages from P28 through P55 [j, P < 0.01 (P28, P30), 0.03 (P32), 0.0001 (P35 through P50), 0.005 (P55)].

While there was no significant effect of sex on GnRH mRNA levels, a significant interaction of these variables was determined by two-way ANOVA (P < 0.0001). Given this significant interaction, we investigated differences in GnRH mRNA levels between males and females at different ages by post hoc analysis. The results revealed that GnRH mRNA levels were significantly higher in male than female mice at P28 (P < 0.02), P35 (P < 0.05), P40 (P < 0.0005), and P45 (P < 0.03), and higher in females than in males at P60 (P < 0.0001).

Cyclophilin mRNA levels were measured in the same cytoplasmic mRNA fractions as GnRH mRNA. While cyclophilin mRNA levels did not vary as dramatically as GnRH mRNA levels, with changes of only about 2-fold occurring developmentally, a significant effect of age (P < 0.03), sex (P < 0.02) and an interaction of age and sex (P < 0.0001) was observed. For female mice, cyclophilin mRNA levels were significantly lower at E16 and P0 than at 25 through P60 (1, P < 0.05). Levels at P45, 50, and 55 were significantly higher than those at P7 through P35 (2, P < 0.05). Cyclophilin mRNA levels in females were also lower at P60 than at P45 (3, P < 0.01). For male mice, cyclophilin mRNA levels were significantly higher at P5 than at P20, 35, and 50 (4, P < 0.05). Sex differences in cyclophilin mRNA levels were also observed at P35, 45, 50, and 55 (P < 0.05).

Because cyclophilin mRNA levels in the present study were observed to undergo significant developmental changes in POA-AH dissections, GnRH mRNA levels were also determined in femtograms GnRH mRNA per microgram total cytoplasmic RNA. As analyzed by this method GnRH mRNA levels underwent an increase of approximately 15-fold from E16–P60 in females (Fig. 3aGo), and a similar increase from E16–P40 in males (Fig. 3bGo). ANOVA indicated that GnRH mRNA/µg total cytoplasmic RNA varied significantly with age (P < 0.0001) but not sex (P = 0.661), and that there was a significant interaction of age with sex (P < 0.005). Post hoc analysis indicated that GnRH mRNA levels were significantly elevated in female mice at P25, compared with E16, P0, and P10 (a, P < 0.05); at P30, compared with E16 through P20, and P28 (b, P < 0.05); at P32, compared with P15 and younger (c, P < 0.05); at P35, compared with P28 and younger (d, P < 0.05); at P40, P55, and P60, compared with P32 and younger (e, P < 0.05); and at P50, compared with P40 and younger (f, P < 0.05). For male mice, GnRH mRNA levels were elevated at P28, compared with E16 through P10 and P20 (g, P < 0.05); and at P40, compared with all ages from E16 through P35, and P55 and 60 (h, P < 0.05). Significant differences between male and female mice were observed at P28 and P55 (1, P < 0.05) and at P40 and P60 (**, P < 0.01).



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  		Figure 3. Changes in GnRH mRNA, normalized to total micrograms RNA in the POA-AH, in developing female (A) and male (B) mice, and GnRH mRNA levels in femtograms in female (C) and male (D) mice. a, P < 0.05 vs. E16–P20 and P28. b, P < 0.05 vs. P15 and younger. c, P < 0.05, compared with P28 and younger. d, P < 0.05, compared with P32 and younger. e, P < 0.05 vs. P40 and younger. f, P < 0.05 vs. P40 and younger. g, P < 0.05 vs. E16–P10 and P20. h, P < 0.05, compared with all ages from E16–P35, and P55 and P60. i, P < 0.05 vs. E16–P15. j, P < 0.05 vs. all earlier ages. k, P < 0.005 vs. all other ages. l, P < 0.05 vs. E16–P10. m, P < 0.05 vs. P20 and younger. n, P < 0.05 vs. all ages except P45 and P50. GnRH mRNA levels (femtograms) then decreased from this peak at P40–P60. *, P < 0.05 vs. corresponding male; **, P < 0.01 vs. corresponding male.

 
Because the size of the dissection was difficult to control in extremely young animals, data were further analyzed as femtograms GnRH mRNA per animal (the cytoplasmic fraction from a POA-AH dissected from a single animal). Because the dissection should include virtually all of the POA-AH population of GnRH perikarya (28, 29), this number should be relatively unaffected by dissection size. As shown in Fig. 3Go, c and d, ontogenic patterns of GnRH mRNA per animal were similar to those expressed per microgram total RNA (Fig. 3Go, a and b), with highest levels at P60 in females and P40 in males. GnRH mRNA levels, in femtograms, underwent increases of approximately 14-fold in females from E16 through P60 and approximately 15-fold in males from E16 through the peak at P40. ANOVA indicated significant effects of age (P < 0.0001 but not sex (P = 0.152), and a significant interaction of age with sex (P < 0.0005) on femtograms GnRH mRNA. Post hoc analysis demonstrated that GnRH mRNA levels (femtograms) were significantly higher at P20 in females, compared with E16 through P15 (i, P < 0.05). Further elevations in GnRH mRNA (femtograms) occurred at P45, at which age significantly higher levels than those at all earlier ages were observed (j, P < 0.05). GnRH mRNA levels (femtograms) were significantly higher at P60 than at all other ages (k, P < 0.005). For males, GnRH mRNA levels (femtograms) were first elevated at P25, compared with animals at E16 through P10 (l, P < 0.05); at P35, compared with P20 and younger (m, P < 0.05); and at P40, compared with all ages except P45 and P50 (n, P < 0.05). GnRH mRNA levels (femtograms) then decreased from this peak at P40 through P60. Significant differences between males and females occurred at P28 and P40, when levels were higher in males than in females (P < 0.005), and on P60, when levels were higher in females than in males (P < 0.0001).

Changes in GnRH primary transcript levels during development
An autoradiogram showing GnRH primary transcript in nuclear fractions of representative individual POA-AHs is shown in Fig. 1bGo. GnRH primary transcript levels in the POA-AH of individual animals changed significantly during development in male and female mice (Fig. 4Go). Two-way ANOVA indicated a significant effect of age (P < 0.0001) and sex (P < 0.005), as well as a significant interaction of sex and age on GnRH primary transcript levels (P < 0.0001).



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  		Figure 4. Changes in GnRH primary transcript in developing female (A) and male (B) mice. a, P < 0.05 vs. E16, P7, P10, and all subsequent ages. b, P < 0.02 vs. E16, P7, and P10; P < 0.01 vs. all subsequent ages. c, P < 0.05 vs. E16; P < 0.01 vs. P7, P10, P25, and all subsequent ages. d, P < 0.05 vs. P25 and all subsequent ages. e, P < 0.05 vs. P7 and all subsequent ages. f, P < 0.05 vs. P15 and P20; P < 0.001 vs. all subsequent ages except P25. g, P < 0.05 vs. P28, P32, and all subsequent ages. *, P < 0.05 vs. corresponding male; **, P < 0.005 vs. corresponding male.

 
In female mice, GnRH primary transcript levels changed most dramatically from E16 through P7 (Fig. 4aGo). Post hoc analysis revealed that GnRH primary transcript levels were significantly lower on P0, compared with E16 (a, P < 0.03) and with all other ages beginning on P7 through P60 (a, P < 0.03). Levels of GnRH primary transcript reached a nadir on P5, at which age they were significantly lower than at all other ages except P0 (b, P < 0.05). They then underwent a significant increase between P5 and P7, at which time adult levels were reached. Transient increased levels of GnRH primary transcript were observed at P15 (c, P < 0.05 vs. E16; P < 0.005 vs. all other ages except P20) and P20 (d, P < 0.05 vs. P28 through P60).

A different pattern of GnRH primary transcript levels was observed in male mice, in which significant fluctuations also occurred (Fig. 4bGo). Unlike females, in which GnRH primary transcript levels reached a nadir at P5, in males, GnRH primary transcript levels were at their lowest at E16, and they increased during the first two postnatal weeks of development. E16, P0, and P5 mice had GnRH primary transcript levels that did not differ from one another but were significantly different from all other ages (e, P < 0.05, compared with P7; P < 0.005 vs. all other ages). GnRH primary transcript levels increased from P5–P7, continued to increase, and at P15 were significantly higher than at P7 (f, P < 0.03). Except for a transient dip at P25 (g, P < 0.05 vs. P28, P32 through P60), GnRH primary transcript levels remained at this P15 level through P60.

A significant interaction of sex and age on GnRH primary transcript was determined by 2-way ANOVA (P < 0.0001). Post hoc analysis indicated that GnRH primary transcript levels were significantly higher in females than in males on E16 (P < 0.005) and P15 (P < 0.05) and higher in males than in females on P28 (P < 0.01), P32 (P < 0.005), P40 (P < 0.005), P50 (P < 0.005), P55 (P < 0.001), and P60 (P < 0.03). A comparison of levels of GnRH primary transcript with GnRH mRNA is presented in Table 1Go.


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  		Table 1. Mole ratio of GnRH primary transcript:mRNA in developing mice

 
Changes in serum LH levels during development
An overall effect of age (P < 0.02) but not sex (P = 0.065), and a significant interaction of age with sex (P < 0.0005), were observed for serum LH levels in developing mice.

LH levels underwent significant changes with development in female mice. Levels of LH were significantly higher at P5 than at P30 through P60 (P < 0.02; Fig. 5aGo). LH levels at P10 were significantly higher than at all other ages (P < 0.0002). A significant difference between females and males at P10 was also observed (P < 0.0001).



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  		Figure 5. Changes in serum LH in developing female (A) and male (B) mice. a, P < 0.02 vs. P30–P60. b, P < 0.0002 vs. P0, P5, P20, P30, P40, P50, P60, and male P10 mice.

 
LH levels in male mice were consistently low and often close to the limit of assay sensitivity (Fig. 5bGo). No significant effects of age on LH levels in male mice were observed.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The results of the present study indicate that GnRH gene expression changes significantly during development from the late embryonic period through adulthood. Several observations were made that will be discussed in detail below: 1) GnRH mRNA levels increase gradually and significantly during postnatal development and are similar between males and females, except that levels of GnRH mRNA peak earlier in male (P40) than in female (P55–60) mice; 2) GnRH primary transcript levels, an index of GnRH gene transcription (23), change differentially from GnRH mRNA levels and differ dramatically between male and female mice during the perinatal period; and 3) serum LH levels differ between male and female mice, and change developmentally.

GnRH mRNA levels were analyzed by three methods. When levels of GnRH mRNA in the POA-AH of developing male and female mice are normalized to cyclophilin mRNA levels, they increase gradually, with increases of approximately 4-fold occurring in males and females. The first significant elevation in GnRH mRNA levels was observed to occur at P20 in females and at P15 in males, before the onset of puberty in mice (32, 33). In female mice, GnRH mRNA levels peak at P55–60, after the day of vaginal opening in our colony, which occurs between P32 and P40; and it is possible that levels continue to increase after P60, although this was not determined in the present study. The observed timing of vaginal opening in our mice is similar to that observed in this strain in another study (33). That study (33) also reported that reproductive cyclicity did not occur until approximately P58, which is similar to the peak of GnRH mRNA observed in the present study. In males, GnRH mRNA levels reach a peak at P40 and decline thereafter until P60; again it is unknown whether they continue to decrease beyond P60. The results of this study are similar to those in rats studied by Jakubowski et al. (7), who reported significant changes in GnRH mRNA levels from P15–P30 in females and from P15–P45 in males. Another group found that GnRH mRNA levels increased from P21–P23 in male rats (5), and our laboratory previously observed increases in GnRH mRNA levels in female rats during the peripubertal period from P25–P45 (8, 13). Thus, the observation that GnRH mRNA levels increase steadily during postnatal development in mice, with the first significant increases occurring after P15 (34), are consistent with these rat studies, suggesting related changes of this transcript in both rodent species.

Cyclophilin mRNA levels in the POA-AH were found to undergo small, but significant, changes during development. Another study reported that cyclophilin mRNA levels did not change significantly during postnatal development in rats (7), and our laboratory has observed that, in general, cyclophilin mRNA levels are proportional to the total micrograms of RNA in a POA-AH dissection (8, 13). Postnatal differences in cyclophilin mRNA levels were also not detected in our laboratory, in another study using neonatal rats (34). We attribute at least some of the differences in cyclophilin mRNA levels in the present study to difficulties in making completely accurate POA-AH dissections in very young (E16 and P0) mice, in which the brain matrix could not be used. This could affect the ratio of GnRH mRNA to cyclophilin mRNA, as well as the ratio of cyclophilin to total RNA.

Because of the developmental differences in cyclophilin mRNA levels, we also expressed GnRH mRNA per microgram of total RNA in the POA-AH dissections, as well as in femtograms of GnRH mRNA per animal. Overall, the developmental pattern of GnRH mRNA, as determined by these methods, is similar to that determined by expressing GnRH mRNA per cyclophilin mRNA, and the peaks occur at similar ages (P55–P60 in females and P40 in males). However, the magnitude of the increase in GnRH mRNA levels is greater when expressed per microgram of total RNA in the POA-AH, as well as per animal, with increases of approximately 15-fold occurring in both sexes, from the nadir at E16 to the respective peaks in females and males.

To determine the mechanism for the ontogenic increase in GnRH mRNA, GnRH primary transcript levels were measured in the nuclear fraction of the same POA-AH dissections in which GnRH mRNA levels had been measured in the cytoplasm. For both male and female mice, GnRH primary transcript attains essentially adult levels by P7–P15, long before the onset of puberty in mice (32, 33). The molecular mechanism by which the GnRH transcriptional apparatus matures during early development is a subject under intense investigation. Several transcription factors of the POU homeodomain family, such as Brn-2 and SCIP, have been shown to mediate inhibition of GnRH gene transcriptional activity (35). Though the specific developmental expression of the POU domain factors in GnRH neurons is yet to be determined, these factors undergo differential developmental expression, with higher levels observed earlier in development (36). In the present study, adult levels of transcriptional activity of the proGnRH gene are reached during a developmental period when GnRH mRNA is still at immature levels. Indeed, the bulk of the changes in GnRH mRNA occur after P7–P15. Thus, the transcriptional apparatus is in place for transcribing the proGnRH RNA, and subsequent developmental changes in GnRH mRNA levels probably occur by a posttranscriptional mechanism. This observation is consistent with other reports from our laboratory indicating that most of the regulation of GnRH mRNA in rats after P10 occurs independently of changes in GnRH gene transcription (8, 16).

GnRH primary transcript levels differed considerably between neonatal male and female mice. In males, GnRH primary transcript levels are lowest at E16 and remain at this low level until P7. In contrast, female mice at E16 have adult GnRH primary transcript levels; they then decrease to a nadir at P5, and then they undergo a large increase back to adult levels at P7. Interestingly, the large increase in GnRH gene transcription that occurs from P5–P7 in both sexes is quite similar to that reported by another laboratory using a transgenic mouse containing a gene construct comprising the 5'-flanking region of the human GnRH gene fused to the luciferase reporter gene (37). That group observed that GnRH gene transcription, as measured by luciferase activity, increased dramatically from P3–P10. This report by Wolfe et al. (37) also lends support to the likelihood that GnRH primary transcript levels in the animal are, in fact, reflective of GnRH gene transcription, as has been reported in the GT1 cell lines (23).

Differences in GnRH primary transcript levels in the present study, at E16, may be attributable to the fact that the late embryonic/early postnatal period is the critical period for sexual differentiation in mice (25, 27, 38). During this period, testosterone levels are elevated in male mice (39) and rats (40), with no such similar elevation of steroid hormones in female mice (39). Thus, the male reproductive axis could be subjected to steroid hormone negative feedback that is not experienced in females, resulting in a suppression of hypothalamic GnRH neurons or their inputs prenatally. The higher levels of GnRH gene transcription in perinatal females is also consistent with reports of transiently elevated gonadotropin levels in neonatal female, but not male, rodents. Indeed, it has been reported that LH and FSH levels are low in newborn and perinatal male rats (3, 41, 42) and mice (32, 43) but high in newborn female mice (39) and rats (3, 41, 42, 44). The results of the present study are consistent with these findings, in that we observed higher serum LH levels in neonatal females, compared with male mice. Thus, there may be a possible relationship between activated GnRH gene transcription in perinatal female mice, and subsequent GnRH and LH release. However, the nature of this relationship remains to be determined.

In conclusion, we have observed changes in GnRH gene expression during development in mice. The greatest alterations in GnRH mRNA levels occurred after P15, with gradual increases in both females and males. For GnRH primary transcript, large alterations occurred prior to P10, with an initial decrease to a nadir at P5, and then an abrupt increase to P7 in females, and with initially low levels and a large increase at P7–P10 in males. Thus, the mechanisms for the regulation of GnRH mRNA levels are sexually dimorphic; furthermore, the regulation of GnRH mRNA levels after P7 seems to be primarily independent of GnRH gene transcription. We hypothesize that ontogenic differences in GnRH mRNA stability are responsible for the regulation of GnRH mRNA levels in rodents, because such mechanisms play a role in the regulation of GnRH mRNA levels in the hypothalamic GT1–7 cell line (14, 21). Studies are currently underway to address whether GnRH mRNA stability is altered developmentally in the mouse.


   Acknowledgments
 
We would like to acknowledge the help of Areta Dobrjansky for assistance with the LH RIA, and Dr. Mariann Blum for helpful discussions.


   Footnotes
 
1 All experiments were conducted in accord with Guidelines for the Care and Use of Experimental Animals, using protocols approved by the Institutional Animal Care and Use Committee at Mount Sinai School of Medicine (Grant 97–014NA). This report was made possible by funds granted by the National Science Foundation (IBN-9723398, to A.C.G.). A preliminary version of this work was reported at the 25th Annual Meeting of The Society for Neuroscience, San Diego, California, 1995 (Abstract 112.8). Back

Received September 17, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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