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• W2160996976 abstract "Gonadotropin-releasing hormone (GnRH) is encoded by the proGnRH gene which contains four exons and three introns. In this study, two immortalized GnRH-expressing cell lines (Gn11 and NLT) were characterized. The NLT and Gn11 cells, derived from a same brain tumor in a transgenic mouse, display neuronal morphology and neuron-specific markers. However, NLT cells secrete much higher levels of GnRH than Gn11 cells. To delineate the mechanism underlying this difference, reverse transcriptase-polymerase chain reaction and RNase protection assays were performed to examine proGnRH gene expression. While the mature proGnRH mRNA was predominately expressed in NLT cells, Gn11 cells express an abundant short transcript. Sequence analysis revealed that this short transcript contains exons 1, 3, and 4, but not exon 2, which encodes the GnRH decapeptide. RNase protection assays demonstrated that NLT cells express much higher levels of mature proGnRH mRNA than Gn11 cells. The lower level of GnRH secreting capacity in Gn11 cells is due, in part, to decreased expression of mature proGnRH mRNA. When proGnRH gene expression in the mouse brain was examined, the same short splicing variant was observed in the olfactory area and preoptic area-anterior hypothalamus. But the prevalent transcript in these regions was the mature proGnRH mRNA. In contrast, only the mature proGnRH mRNA was found in the caudal hypothalamus. These results suggest that alternative splicing may be one of the mechanisms regulating proGnRH gene expression in the animal brain. Gonadotropin-releasing hormone (GnRH) is encoded by the proGnRH gene which contains four exons and three introns. In this study, two immortalized GnRH-expressing cell lines (Gn11 and NLT) were characterized. The NLT and Gn11 cells, derived from a same brain tumor in a transgenic mouse, display neuronal morphology and neuron-specific markers. However, NLT cells secrete much higher levels of GnRH than Gn11 cells. To delineate the mechanism underlying this difference, reverse transcriptase-polymerase chain reaction and RNase protection assays were performed to examine proGnRH gene expression. While the mature proGnRH mRNA was predominately expressed in NLT cells, Gn11 cells express an abundant short transcript. Sequence analysis revealed that this short transcript contains exons 1, 3, and 4, but not exon 2, which encodes the GnRH decapeptide. RNase protection assays demonstrated that NLT cells express much higher levels of mature proGnRH mRNA than Gn11 cells. The lower level of GnRH secreting capacity in Gn11 cells is due, in part, to decreased expression of mature proGnRH mRNA. When proGnRH gene expression in the mouse brain was examined, the same short splicing variant was observed in the olfactory area and preoptic area-anterior hypothalamus. But the prevalent transcript in these regions was the mature proGnRH mRNA. In contrast, only the mature proGnRH mRNA was found in the caudal hypothalamus. These results suggest that alternative splicing may be one of the mechanisms regulating proGnRH gene expression in the animal brain. GnRH 1The abbreviations used are: GnRH, gonadotropin-releasing hormone; GAP, GnRH-associated peptide; RT-PCR, reverse transcriptase-polymerase chain reaction; SV40-Tag, simian virus 40 T antigen; MAP-2, microtubule-associated protein-2; RIA, radioimmunoassay; bp, base pair(s). 1The abbreviations used are: GnRH, gonadotropin-releasing hormone; GAP, GnRH-associated peptide; RT-PCR, reverse transcriptase-polymerase chain reaction; SV40-Tag, simian virus 40 T antigen; MAP-2, microtubule-associated protein-2; RIA, radioimmunoassay; bp, base pair(s). neurons in the hypothalamus play an essential role in the regulation of mammalian reproduction. These neurons originate from precursor cells in the olfactory placode and migrate to their target sites during embryonic development (1Schwanzel-Fukuda M. Pfaff D.W. Nature. 1989; 338: 161-164Crossref PubMed Scopus (935) Google Scholar, 2Wray S. Gant P. Gainer H. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 8132-8136Crossref PubMed Scopus (619) Google Scholar). The most prominent axonal projection of GnRH neurons is to the median eminence, where GnRH is released and transported via the hypothalamic hypophyseal portal vessels to the anterior pituitary to stimulate the synthesis and release of luteinizing hormone and follicle-stimulating hormone (3Silverman A.J. Knobil E. Neill J.D. The Physiology of Reproduction. Raven Press, Ltd., New York1988: 1283-1304Google Scholar). The regulation of GnRH neuronal activities has been difficult to study, however, due to their scattered distribution and paucity in cell number (3Silverman A.J. Knobil E. Neill J.D. The Physiology of Reproduction. Raven Press, Ltd., New York1988: 1283-1304Google Scholar, 4Wray S. Hoffman G.A. J. Comp. Neurol. 1986; 252: 522-531Crossref PubMed Scopus (129) Google Scholar, 5Merchanthaler I. Gorcs T. Setalo G. Petrusz P. Flerko B. Cell Tissue Res. 1984; 237: 15-29PubMed Google Scholar). Recently, this laboratory and Mellonet al. (6Mellon P.L. Windle J.J. Goldsmith P.C. Padula C.A. Roberts J.L. Weiner R.I. Neuron. 1990; 5: 1-10Abstract Full Text PDF PubMed Scopus (891) Google Scholar, 7Radovick S. Wray S. Lee E. Nichols D.K. Nakayama Y. Weintraub B.D. Westphal H. Cutler G.B. Wondisford F.E. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3402-3406Crossref PubMed Scopus (193) Google Scholar) have generated immortalized GnRH-expressing neuronal cell lines by targeted tumorigenesis. In our laboratory, targeting the expression of the simian virus 40 T antigen (SV40-Tag) to the GnRH neurons with the human GnRH gene 5′-upstream regulatory sequence resulted in the development of an olfactory tumor in one of the transgenic mice (7Radovick S. Wray S. Lee E. Nichols D.K. Nakayama Y. Weintraub B.D. Westphal H. Cutler G.B. Wondisford F.E. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3402-3406Crossref PubMed Scopus (193) Google Scholar). Two GnRH immunoreactive cell lines (Gn11 and NLT) were subsequently derived from this tumor. Characterization of the NLT and Gn11 cells demonstrated that these cells display neuronal morphology and neuron-specific markers, such as microtubule-associated peptide 2 (MAP-2) and Tau protein (8Fischer I. Richter-Landsberg C. Safaei R. Exp. Cell Res. 1991; 194: 195-201Crossref PubMed Scopus (39) Google Scholar, 9Mavilia C. Couchie D. Mattei M.G. Nivez M.P. Nunez J. J. Neurochem. 1993; 61: 1073-1081Crossref PubMed Scopus (27) Google Scholar). Solution hybridization-RNase protection assays and RT-PCR analysis indicated that these cells express proGnRH mRNA and are able to synthesize and secrete GnRH as demonstrated by RIA. Therefore, these cell lines provide a suitablein vitro model for the study of GnRH neuronal activity and its regulation.Although the NLT and Gn11 cells were derived from the same tumor, RIA measurement of GnRH concentrations demonstrated that the NLT cells secrete about 10 times higher levels of GnRH than the Gn11 cells. In an attempt to understand the molecular basis responsible for generating this difference, we characterized the expression of the proGnRH gene in these cell lines by RT-PCR and solution hybridization-RNase protection assays. In addition, the expression of proGnRH gene expression in the mouse olfactory and preoptic area-hypothalamus was also examined. Our results demonstrate that a splicing variant lacking exon 2 of the proGnRH gene was present in Gn11 cells but not in NLT cells. This same splicing variant was also found in the olfactory and preoptic area-anterior hypothalamus, but not in the caudal hypothalamus of the mouse brain. This is the first demonstration that alternative splicing of the primary proGnRH gene transcript occurs in the immortalized GnRH-expressing neuronal cell lines and in the animal forebrain.DISCUSSIONOur data indicate that the NLT and Gn11 cells are neuronal in phenotype, express proGnRH mRNA, and secrete GnRH into the medium. Since one major difficulty in the study of GnRH neuronal activities is their low abundance and scattered distribution, these cell lines provide a convenient in vitro model for the study of GnRH secretion and regulation of gene expression. Using the Gn11 and NLT cell lines as a model, we found that although both cell lines were derived from a same tumor in a transgenic mice, they display heterogeneity in the secretion of GnRH and expression of proGnRH gene. Our data provide evidence that the lower GnRH secreting capacity found in the Gn11 cells is due, in part, to the prevalence of a splicing variant lacking exon 2 which encodes the GnRH decapeptide. Moreover, we extended this finding to the animal forebrain by demonstrating that this splicing variant is also expressed in the olfactory and preoptic area/anterior hypothalamus, although the prevalent form of transcripts in these areas is the mature proGnRH mRNA. Therefore, these results suggest that alternative splicing of the primary transcript may contribute to the regulation of proGnRH gene expression in the animal forebrain.Various lines of evidence indicate that proGnRH gene expression is regulated in a tissue-specific pattern. In various peripheral tissues, including the placenta, mammary gland, testes, and ovary (12Radovick S. Wondisford F.E. Nakayama Y. Yamada M. Cutler G.B. Weintraub B.D. Mol. Endocrinol. 1990; 4: 476-480Crossref PubMed Scopus (107) Google Scholar, 13Seeburg P.H. Adelman J.P. Nature. 1984; 311: 666-668Crossref PubMed Scopus (377) Google Scholar, 14Goubau S. Bond C.T. Adelman J.P. Misra V. Hynes M.F. Schultz G.A. Murphy B.D. Endocrinology. 1992; 130: 3098-3100Crossref PubMed Scopus (64) Google Scholar, 17Dong K.W. Yu K.L. Roberts J.L. Mol. Endocrinol. 1993; 7: 1654-1666Crossref PubMed Scopus (98) Google Scholar), the expression and processing of the primary transcript is significantly different from that in the hypothalamus in that a greater proportion of the transcripts in these tissues contains intron A. Moreover, a major transcriptional start site upstream of that used in the hypothalamus is utilized for transcription of the proGnRH gene in these tissues (14Goubau S. Bond C.T. Adelman J.P. Misra V. Hynes M.F. Schultz G.A. Murphy B.D. Endocrinology. 1992; 130: 3098-3100Crossref PubMed Scopus (64) Google Scholar, 17Dong K.W. Yu K.L. Roberts J.L. Mol. Endocrinol. 1993; 7: 1654-1666Crossref PubMed Scopus (98) Google Scholar). In the immature T lymphocyte cell line Nb2, in addition to the mature GnRH mRNA, the alternatively spliced form of transcript is also found, which lacks exon 2 that encodes the GnRH decapeptide (15Wilson T.M. Yu-Lee L. Kelley M.R. Mol. Endocrinol. 1995; 9: 44-53Crossref PubMed Google Scholar). Because the splicing process affects the coding capacity of transcripts directly, its efficiency and accuracy are obviously critical for normal functions of GnRH neurons. Our present data demonstrate that even among GnRH neurons located in different regions of the central nervous system, the primary transcript is differentially processed. For GnRH neurons located in the olfactory area where Gn11 and NLT cells were derived, and those in the preoptic area-anterior hypothalamus, in addition to the prevalent mature mRNA, a short splicing variant lacking exon 2 of proGnRH gene is also produced, whereas only the mature proGnRH mRNA is generated by GnRH neurons located in the caudal hypothalamus. This finding is in agreement with the emerging evidence that the expression of proGnRH gene is differentially regulated in different regions of the central nervous system. For example, in the human brain, in situhybridization studies suggest the presence of three distinct subtypes of GnRH neurons with pronounced differences in morphology, labeling density, and location (18Rance N.E. Young III, W.S. McMullen N.T. J. Comp. Neurol. 1994; 339: 573-586Crossref PubMed Scopus (86) Google Scholar). The number of GnRH neurons detected byin situ hybridization and immunocytochemistry has also been shown to vary in response to ovariectomy (19King J.C. Rubin B.S. Conn P.M. Crowley Jr., W.F. Symposium on Modes of Action of GnRH and GnRH Analogs February 26 to March 2, 1991, Scottsdale, AZ. Springer-Verlag, New York1992: 161-178Google Scholar, 20King J.C. Kugel G. Zahniser D. Wooledge K. Damassa D.A. Alexsavich B. Peptides. 1987; 8: 721-735Crossref PubMed Scopus (60) Google Scholar), steroid treatment (21Shivers B.D. Harlan R.E. Morrell J.I. Pfaff D.W. Neuroendocrinology. 1983; 36: 1-12Crossref PubMed Scopus (173) Google Scholar), and during the estrous cycle (22Lee W.-S. Abbud R. Smith M.S. Hoffman G.E. Endocrinology. 1992; 130: 3101-3103Crossref PubMed Scopus (61) Google Scholar, 23Hiatt E.S. Brunetta P.G. Seiler G.R. Barney S.A. Selles W.D. Wooledge K.H. King J.C. Endocrinology. 1992; 130: 1030-1043PubMed Google Scholar). Recent studies of the rat preovulatory luteinizing hormone surge demonstrate that the number of GnRH-expressing cells fluctuates during the periovulatory period, and peak numbers of GnRH-expressing cells are attained at different time points in the preoptic area versus GnRH neurons in the more rostral regions (24Porkka-Heiskanen T. Urban J.H. Turek F.W. Levine J.E. J. Neurosci. 1994; 14: 5548-5558Crossref PubMed Google Scholar). Taken together, these studies support the presence of heterogeneous populations of GnRH neurons in the mammalian central nervous system. Our data suggest that alternative splicing may be one of the mechanisms by which heterogeneous populations of GnRH neurons can be generated in the animal brain.Studies of RNA splicing mechanisms suggest that differences in the activities/amounts of general splicing factors or the presence of specialized proteins may participate in the regulation of alternative splicing (25Maniatis T. Science. 1991; 251: 33-34Crossref PubMed Scopus (163) Google Scholar). In addition, a number of cis-acting sequences that influence splice site recognition have been identified, which include intron size, exon sequence, alternative branch points, pyrimidine content of 3′ acceptor sites, and secondary structure of the pre-mRNA (26McKeown M. Annu. Rev. Cell Biol. 1992; 8: 139-181Crossref Scopus (180) Google Scholar). The difference in processing the primary proGnRH gene transcript between the Gn11 versus NLT cells and between the GnRH neurons in the caudal hypothalamus versusthose in the olfactory and preoptic areas may reflect the differential capacity of different GnRH neurons to recognize exon 2 in the proGnRH pre-mRNA. In the Gn11 cells and GnRH neurons located in the olfactory area and preoptic area-anterior hypothalamus, the splicing machinery either recognizes exon 2 in the primary transcript to generate the mature mRNA or ignores it, resulting in the formation of a shorter species of transcript lacking exon 2. Whether the difference in splicing of the primary transcript is due to the presence or absence of specific splicing factors in the Gn11 cellsversus NLT cells or due to the difference in the activities in general splicing factors assembled in the spliceosomes inside the nucleus remains to be determined.Although the shorter transcript found in the Gn11 cells and in GnRH neurons in the forebrain lacks the normal ATG translation initiation codon located in exon 2, examination of exon 3 sequence indicates that another ATG translation initiation codon is present in exon 3. The methionine at position +25 of the proGnRH peptide may act as a translation initiation signal for the remaining 45 amino acids of the GAP. However, the Kozak sequence (16Kozak M. Nucleic Acids Res. 1987; 15: 8125-8148Crossref PubMed Scopus (4151) Google Scholar), which is preserved as a purine (G) located at 3 nucleotides upstream of the ATG in the larger transcript, is not present in the smaller transcript. Whether this splicing variant is translated into a protein in Gn11 cells and the translational efficiency has yet to be determined.Since the NLT cells express higher levels of mature proGnRH mRNA and secrete larger quantities of GnRH, these cells have the advantage over the Gn11 cells for studying the regulation of proGnRH gene expression and GnRH secretion by various factors. Recent studies from this laboratory using the NLT cell line have demonstrated that these cells express type-I receptor for insulin-like growth factor (27Zhen, S., and Radovick, S. (1996) 26th Annual Meeting Neuroscience, November 19, 1996, Washington, D. C., Slide 379.12.Google Scholar) and the receptor for epidermal growth factor (28Zhen S. Su E. Zakaria M. Radovick S. 10th International Congress on Endocrinology, June 12–15, 1996, San Francisco, CA. 1996; (,): 1-346Google Scholar). These findings are interesting since both insulin-like growth factor-1 and epidermal growth factor have been shown to regulate GnRH neuronal activitiesin vivo and have been suggested to play an important role in the control of pubertal development (29Hiney J.K. Ojeda S.R. Dees W.L. Neuroendocrinology. 1991; 54: 420-423Crossref PubMed Scopus (188) Google Scholar, 30Ma Y.J. Junier M.P. Costa M.E. Ojeda S.R. Neuron. 1992; 9: 657-670Abstract Full Text PDF PubMed Scopus (185) Google Scholar). However, to date, the molecular mechanisms by which these growth factors regulate GnRH neuronal activities remain largely unknown, due to the fact that less than 1500 GnRH neurons are present in the animal brain (4Wray S. Hoffman G.A. J. Comp. Neurol. 1986; 252: 522-531Crossref PubMed Scopus (129) Google Scholar, 5Merchanthaler I. Gorcs T. Setalo G. Petrusz P. Flerko B. Cell Tissue Res. 1984; 237: 15-29PubMed Google Scholar). Therefore, the availability of NLT cells should greatly enhance our ability to explore the molecular mechanisms that mediate the regulation of proGnRH gene expression and GnRH secretion. GnRH 1The abbreviations used are: GnRH, gonadotropin-releasing hormone; GAP, GnRH-associated peptide; RT-PCR, reverse transcriptase-polymerase chain reaction; SV40-Tag, simian virus 40 T antigen; MAP-2, microtubule-associated protein-2; RIA, radioimmunoassay; bp, base pair(s). 1The abbreviations used are: GnRH, gonadotropin-releasing hormone; GAP, GnRH-associated peptide; RT-PCR, reverse transcriptase-polymerase chain reaction; SV40-Tag, simian virus 40 T antigen; MAP-2, microtubule-associated protein-2; RIA, radioimmunoassay; bp, base pair(s). neurons in the hypothalamus play an essential role in the regulation of mammalian reproduction. These neurons originate from precursor cells in the olfactory placode and migrate to their target sites during embryonic development (1Schwanzel-Fukuda M. Pfaff D.W. Nature. 1989; 338: 161-164Crossref PubMed Scopus (935) Google Scholar, 2Wray S. Gant P. Gainer H. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 8132-8136Crossref PubMed Scopus (619) Google Scholar). The most prominent axonal projection of GnRH neurons is to the median eminence, where GnRH is released and transported via the hypothalamic hypophyseal portal vessels to the anterior pituitary to stimulate the synthesis and release of luteinizing hormone and follicle-stimulating hormone (3Silverman A.J. Knobil E. Neill J.D. The Physiology of Reproduction. Raven Press, Ltd., New York1988: 1283-1304Google Scholar). The regulation of GnRH neuronal activities has been difficult to study, however, due to their scattered distribution and paucity in cell number (3Silverman A.J. Knobil E. Neill J.D. The Physiology of Reproduction. Raven Press, Ltd., New York1988: 1283-1304Google Scholar, 4Wray S. Hoffman G.A. J. Comp. Neurol. 1986; 252: 522-531Crossref PubMed Scopus (129) Google Scholar, 5Merchanthaler I. Gorcs T. Setalo G. Petrusz P. Flerko B. Cell Tissue Res. 1984; 237: 15-29PubMed Google Scholar). Recently, this laboratory and Mellonet al. (6Mellon P.L. Windle J.J. Goldsmith P.C. Padula C.A. Roberts J.L. Weiner R.I. Neuron. 1990; 5: 1-10Abstract Full Text PDF PubMed Scopus (891) Google Scholar, 7Radovick S. Wray S. Lee E. Nichols D.K. Nakayama Y. Weintraub B.D. Westphal H. Cutler G.B. Wondisford F.E. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3402-3406Crossref PubMed Scopus (193) Google Scholar) have generated immortalized GnRH-expressing neuronal cell lines by targeted tumorigenesis. In our laboratory, targeting the expression of the simian virus 40 T antigen (SV40-Tag) to the GnRH neurons with the human GnRH gene 5′-upstream regulatory sequence resulted in the development of an olfactory tumor in one of the transgenic mice (7Radovick S. Wray S. Lee E. Nichols D.K. Nakayama Y. Weintraub B.D. Westphal H. Cutler G.B. Wondisford F.E. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3402-3406Crossref PubMed Scopus (193) Google Scholar). Two GnRH immunoreactive cell lines (Gn11 and NLT) were subsequently derived from this tumor. Characterization of the NLT and Gn11 cells demonstrated that these cells display neuronal morphology and neuron-specific markers, such as microtubule-associated peptide 2 (MAP-2) and Tau protein (8Fischer I. Richter-Landsberg C. Safaei R. Exp. Cell Res. 1991; 194: 195-201Crossref PubMed Scopus (39) Google Scholar, 9Mavilia C. Couchie D. Mattei M.G. Nivez M.P. Nunez J. J. Neurochem. 1993; 61: 1073-1081Crossref PubMed Scopus (27) Google Scholar). Solution hybridization-RNase protection assays and RT-PCR analysis indicated that these cells express proGnRH mRNA and are able to synthesize and secrete GnRH as demonstrated by RIA. Therefore, these cell lines provide a suitablein vitro model for the study of GnRH neuronal activity and its regulation. Although the NLT and Gn11 cells were derived from the same tumor, RIA measurement of GnRH concentrations demonstrated that the NLT cells secrete about 10 times higher levels of GnRH than the Gn11 cells. In an attempt to understand the molecular basis responsible for generating this difference, we characterized the expression of the proGnRH gene in these cell lines by RT-PCR and solution hybridization-RNase protection assays. In addition, the expression of proGnRH gene expression in the mouse olfactory and preoptic area-hypothalamus was also examined. Our results demonstrate that a splicing variant lacking exon 2 of the proGnRH gene was present in Gn11 cells but not in NLT cells. This same splicing variant was also found in the olfactory and preoptic area-anterior hypothalamus, but not in the caudal hypothalamus of the mouse brain. This is the first demonstration that alternative splicing of the primary proGnRH gene transcript occurs in the immortalized GnRH-expressing neuronal cell lines and in the animal forebrain. DISCUSSIONOur data indicate that the NLT and Gn11 cells are neuronal in phenotype, express proGnRH mRNA, and secrete GnRH into the medium. Since one major difficulty in the study of GnRH neuronal activities is their low abundance and scattered distribution, these cell lines provide a convenient in vitro model for the study of GnRH secretion and regulation of gene expression. Using the Gn11 and NLT cell lines as a model, we found that although both cell lines were derived from a same tumor in a transgenic mice, they display heterogeneity in the secretion of GnRH and expression of proGnRH gene. Our data provide evidence that the lower GnRH secreting capacity found in the Gn11 cells is due, in part, to the prevalence of a splicing variant lacking exon 2 which encodes the GnRH decapeptide. Moreover, we extended this finding to the animal forebrain by demonstrating that this splicing variant is also expressed in the olfactory and preoptic area/anterior hypothalamus, although the prevalent form of transcripts in these areas is the mature proGnRH mRNA. Therefore, these results suggest that alternative splicing of the primary transcript may contribute to the regulation of proGnRH gene expression in the animal forebrain.Various lines of evidence indicate that proGnRH gene expression is regulated in a tissue-specific pattern. In various peripheral tissues, including the placenta, mammary gland, testes, and ovary (12Radovick S. Wondisford F.E. Nakayama Y. Yamada M. Cutler G.B. Weintraub B.D. Mol. Endocrinol. 1990; 4: 476-480Crossref PubMed Scopus (107) Google Scholar, 13Seeburg P.H. Adelman J.P. Nature. 1984; 311: 666-668Crossref PubMed Scopus (377) Google Scholar, 14Goubau S. Bond C.T. Adelman J.P. Misra V. Hynes M.F. Schultz G.A. Murphy B.D. Endocrinology. 1992; 130: 3098-3100Crossref PubMed Scopus (64) Google Scholar, 17Dong K.W. Yu K.L. Roberts J.L. Mol. Endocrinol. 1993; 7: 1654-1666Crossref PubMed Scopus (98) Google Scholar), the expression and processing of the primary transcript is significantly different from that in the hypothalamus in that a greater proportion of the transcripts in these tissues contains intron A. Moreover, a major transcriptional start site upstream of that used in the hypothalamus is utilized for transcription of the proGnRH gene in these tissues (14Goubau S. Bond C.T. Adelman J.P. Misra V. Hynes M.F. Schultz G.A. Murphy B.D. Endocrinology. 1992; 130: 3098-3100Crossref PubMed Scopus (64) Google Scholar, 17Dong K.W. Yu K.L. Roberts J.L. Mol. Endocrinol. 1993; 7: 1654-1666Crossref PubMed Scopus (98) Google Scholar). In the immature T lymphocyte cell line Nb2, in addition to the mature GnRH mRNA, the alternatively spliced form of transcript is also found, which lacks exon 2 that encodes the GnRH decapeptide (15Wilson T.M. Yu-Lee L. Kelley M.R. Mol. Endocrinol. 1995; 9: 44-53Crossref PubMed Google Scholar). Because the splicing process affects the coding capacity of transcripts directly, its efficiency and accuracy are obviously critical for normal functions of GnRH neurons. Our present data demonstrate that even among GnRH neurons located in different regions of the central nervous system, the primary transcript is differentially processed. For GnRH neurons located in the olfactory area where Gn11 and NLT cells were derived, and those in the preoptic area-anterior hypothalamus, in addition to the prevalent mature mRNA, a short splicing variant lacking exon 2 of proGnRH gene is also produced, whereas only the mature proGnRH mRNA is generated by GnRH neurons located in the caudal hypothalamus. This finding is in agreement with the emerging evidence that the expression of proGnRH gene is differentially regulated in different regions of the central nervous system. For example, in the human brain, in situhybridization studies suggest the presence of three distinct subtypes of GnRH neurons with pronounced differences in morphology, labeling density, and location (18Rance N.E. Young III, W.S. McMullen N.T. J. Comp. Neurol. 1994; 339: 573-586Crossref PubMed Scopus (86) Google Scholar). The number of GnRH neurons detected byin situ hybridization and immunocytochemistry has also been shown to vary in response to ovariectomy (19King J.C. Rubin B.S. Conn P.M. Crowley Jr., W.F. Symposium on Modes of Action of GnRH and GnRH Analogs February 26 to March 2, 1991, Scottsdale, AZ. Springer-Verlag, New York1992: 161-178Google Scholar, 20King J.C. Kugel G. Zahniser D. Wooledge K. Damassa D.A. Alexsavich B. Peptides. 1987; 8: 721-735Crossref PubMed Scopus (60) Google Scholar), steroid treatment (21Shivers B.D. Harlan R.E. Morrell J.I. Pfaff D.W. Neuroendocrinology. 1983; 36: 1-12Crossref PubMed Scopus (173) Google Scholar), and during the estrous cycle (22Lee W.-S. Abbud R. Smith M.S. Hoffman G.E. Endocrinology. 1992; 130: 3101-3103Crossref PubMed Scopus (61) Google Scholar, 23Hiatt E.S. Brunetta P.G. Seiler G.R. Barney S.A. Selles W.D. Wooledge K.H. King J.C. Endocrinology. 1992; 130: 1030-1043PubMed Google Scholar). Recent studies of the rat preovulatory luteinizing hormone surge demonstrate that the number of GnRH-expressing cells fluctuates during the periovulatory period, and peak numbers of GnRH-expressing cells are attained at different time points in the preoptic area versus GnRH neurons in the more rostral regions (24Porkka-Heiskanen T. Urban J.H. Turek F.W. Levine J.E. J. Neurosci. 1994; 14: 5548-5558Crossref PubMed Google Scholar). Taken together, these studies support the presence of heterogeneous populations of GnRH neurons in the mammalian central nervous system. Our data suggest that alternative splicing may be one of the mechanisms by which heterogeneous populations of GnRH neurons can be generated in the animal brain.Studies of RNA splicing mechanisms suggest that differences in the activities/amounts of general splicing factors or the presence of specialized proteins may participate in the regulation of alternative splicing (25Maniatis T. Science. 1991; 251: 33-34Crossref PubMed Scopus (163) Google Scholar). In addition, a number of cis-acting sequences that influence splice site recognition have been identified, which include intron size, exon sequence, alternative branch points, pyrimidine content of 3′ acceptor sites, and secondary structure of the pre-mRNA (26McKeown M. Annu. Rev. Cell Biol. 1992; 8: 139-181Crossref Scopus (180) Google Scholar). The difference in processing the primary proGnRH gene transcript between the Gn11 versus NLT cells and between the GnRH neurons in the caudal hypothalamus versusthose in the olfactory and preoptic areas may reflect the differential capacity of different GnRH neurons to recognize exon 2 in the proGnRH pre-mRNA. In the Gn11 cells and GnRH neurons located in the olfactory area and preoptic area-anterior hypothalamus, the splicing machinery either recognizes exon 2 in the primary transcript to generate the mature mRNA or ignores it, resulting in the formation of a shorter species of transcript lacking exon 2. Whether the difference in splicing of the primary transcript is due to the presence or absence of specific splicing factors in the Gn11 cellsversus NLT cells or due to the difference in the activities in general splicing factors assembled in the spliceosomes inside the nucleus remains to be determined.Although the shorter transcript found in the Gn11 cells and in GnRH neurons in the forebrain lacks the normal ATG translation initiation codon located in exon 2, examination of exon 3 sequence indicates that another ATG translation initiation codon is present in exon 3. The methionine at position +25 of the proGnRH peptide may act as a translation initiation signal for the remaining 45 amino acids of the GAP. However, the Kozak sequence (16Kozak M. Nucleic Acids Res. 1987; 15: 8125-8148Crossref PubMed Scopus (4151) Google Scholar), which is preserved as a purine (G) located at 3 nucleotides upstream of the ATG in the larger transcript, is not present in the smaller transcript. Whether this splicing variant is translated into a protein in Gn11 cells and the translational efficiency has yet to be determined.Since the NLT cells express higher levels of mature proGnRH mRNA and secrete larger quantities of GnRH, these cells have the advantage over the Gn11 cells for studying the regulation of proGnRH gene expression and GnRH secretion by various factors. Recent studies from this laboratory using the NLT cell line have demonstrated that these cells express type-I receptor for insulin-like growth factor (27Zhen, S., and Radovick, S. (1996) 26th Annual Meeting Neuroscience, November 19, 1996, Washington, D. C., Slide 379.12.Google Scholar) and the receptor for epidermal growth factor (28Zhen S. Su E. Zakaria M. Radovick S. 10th International Congress on Endocrinology, June 12–15, 1996, San Francisco, CA. 1996; (,): 1-346Google Scholar). These findings are interesting since both insulin-like growth factor-1 and epidermal growth factor have been shown to regulate GnRH neuronal activitiesin vivo and have been suggested to play an important role in the control of pubertal development (29Hiney J.K. Ojeda S.R. Dees W.L. Neuroendocrinology. 1991; 54: 420-423Crossref PubMed Scopus (188) Google Scholar, 30Ma Y.J. Junier M.P. Costa M.E. Ojeda S.R. Neuron. 1992; 9: 657-670Abstract Full Text PDF PubMed Scopus (185) Google Scholar). However, to date, the molecular mechanisms by which these growth factors regulate GnRH neuronal activities remain largely unknown, due to the fact that less than 1500 GnRH neurons are present in the animal brain (4Wray S. Hoffman G.A. J. Comp. Neurol. 1986; 252: 522-531Crossref PubMed Scopus (129) Google Scholar, 5Merchanthaler I. Gorcs T. Setalo G. Petrusz P. Flerko B. Cell Tissue Res. 1984; 237: 15-29PubMed Google Scholar). Therefore, the availability of NLT cells should greatly enhance our ability to explore the molecular mechanisms that mediate the regulation of proGnRH gene expression and GnRH secretion. Our data indicate that the NLT and Gn11 cells are neuronal in phenotype, express proGnRH mRNA, and secrete GnRH into the medium. Since one major difficulty in the study of GnRH neuronal activities is their low abundance and scattered distribution, these cell lines provide a convenient in vitro model for the study of GnRH secretion and regulation of gene expression. Using the Gn11 and NLT cell lines as a model, we found that although both cell lines were derived from a same tumor in a transgenic mice, they display heterogeneity in the secretion of GnRH and expression of proGnRH gene. Our data provide evidence that the lower GnRH secreting capacity found in the Gn11 cells is due, in part, to the prevalence of a splicing variant lacking exon 2 which encodes the GnRH decapeptide. Moreover, we extended this finding to the animal forebrain by demonstrating that this splicing variant is also expressed in the olfactory and preoptic area/anterior hypothalamus, although the prevalent form of transcripts in these areas is the mature proGnRH mRNA. Therefore, these results suggest that alternative splicing of the primary transcript may contribute to the regulation of proGnRH gene expression in the animal forebrain. Various lines of evidence indicate that proGnRH gene expression is regulated in a tissue-specific pattern. In various peripheral tissues, including the placenta, mammary gland, testes, and ovary (12Radovick S. Wondisford F.E. Nakayama Y. Yamada M. Cutler G.B. Weintraub B.D. Mol. Endocrinol. 1990; 4: 476-480Crossref PubMed Scopus (107) Google Scholar, 13Seeburg P.H. Adelman J.P. Nature. 1984; 311: 666-668Crossref PubMed Scopus (377) Google Scholar, 14Goubau S. Bond C.T. Adelman J.P. Misra V. Hynes M.F. Schultz G.A. Murphy B.D. Endocrinology. 1992; 130: 3098-3100Crossref PubMed Scopus (64) Google Scholar, 17Dong K.W. Yu K.L. Roberts J.L. Mol. Endocrinol. 1993; 7: 1654-1666Crossref PubMed Scopus (98) Google Scholar), the expression and processing of the primary transcript is significantly different from that in the hypothalamus in that a greater proportion of the transcripts in these tissues contains intron A. Moreover, a major transcriptional start site upstream of that used in the hypothalamus is utilized for transcription of the proGnRH gene in these tissues (14Goubau S. Bond C.T. Adelman J.P. Misra V. Hynes M.F. Schultz G.A. Murphy B.D. Endocrinology. 1992; 130: 3098-3100Crossref PubMed Scopus (64) Google Scholar, 17Dong K.W. Yu K.L. Roberts J.L. Mol. Endocrinol. 1993; 7: 1654-1666Crossref PubMed Scopus (98) Google Scholar). In the immature T lymphocyte cell line Nb2, in addition to the mature GnRH mRNA, the alternatively spliced form of transcript is also found, which lacks exon 2 that encodes the GnRH decapeptide (15Wilson T.M. Yu-Lee L. Kelley M.R. Mol. Endocrinol. 1995; 9: 44-53Crossref PubMed Google Scholar). Because the splicing process affects the coding capacity of transcripts directly, its efficiency and accuracy are obviously critical for normal functions of GnRH neurons. Our present data demonstrate that even among GnRH neurons located in different regions of the central nervous system, the primary transcript is differentially processed. For GnRH neurons located in the olfactory area where Gn11 and NLT cells were derived, and those in the preoptic area-anterior hypothalamus, in addition to the prevalent mature mRNA, a short splicing variant lacking exon 2 of proGnRH gene is also produced, whereas only the mature proGnRH mRNA is generated by GnRH neurons located in the caudal hypothalamus. This finding is in agreement with the emerging evidence that the expression of proGnRH gene is differentially regulated in different regions of the central nervous system. For example, in the human brain, in situhybridization studies suggest the presence of three distinct subtypes of GnRH neurons with pronounced differences in morphology, labeling density, and location (18Rance N.E. Young III, W.S. McMullen N.T. J. Comp. Neurol. 1994; 339: 573-586Crossref PubMed Scopus (86) Google Scholar). The number of GnRH neurons detected byin situ hybridization and immunocytochemistry has also been shown to vary in response to ovariectomy (19King J.C. Rubin B.S. Conn P.M. Crowley Jr., W.F. Symposium on Modes of Action of GnRH and GnRH Analogs February 26 to March 2, 1991, Scottsdale, AZ. Springer-Verlag, New York1992: 161-178Google Scholar, 20King J.C. Kugel G. Zahniser D. Wooledge K. Damassa D.A. Alexsavich B. Peptides. 1987; 8: 721-735Crossref PubMed Scopus (60) Google Scholar), steroid treatment (21Shivers B.D. Harlan R.E. Morrell J.I. Pfaff D.W. Neuroendocrinology. 1983; 36: 1-12Crossref PubMed Scopus (173) Google Scholar), and during the estrous cycle (22Lee W.-S. Abbud R. Smith M.S. Hoffman G.E. Endocrinology. 1992; 130: 3101-3103Crossref PubMed Scopus (61) Google Scholar, 23Hiatt E.S. Brunetta P.G. Seiler G.R. Barney S.A. Selles W.D. Wooledge K.H. King J.C. Endocrinology. 1992; 130: 1030-1043PubMed Google Scholar). Recent studies of the rat preovulatory luteinizing hormone surge demonstrate that the number of GnRH-expressing cells fluctuates during the periovulatory period, and peak numbers of GnRH-expressing cells are attained at different time points in the preoptic area versus GnRH neurons in the more rostral regions (24Porkka-Heiskanen T. Urban J.H. Turek F.W. Levine J.E. J. Neurosci. 1994; 14: 5548-5558Crossref PubMed Google Scholar). Taken together, these studies support the presence of heterogeneous populations of GnRH neurons in the mammalian central nervous system. Our data suggest that alternative splicing may be one of the mechanisms by which heterogeneous populations of GnRH neurons can be generated in the animal brain. Studies of RNA splicing mechanisms suggest that differences in the activities/amounts of general splicing factors or the presence of specialized proteins may participate in the regulation of alternative splicing (25Maniatis T. Science. 1991; 251: 33-34Crossref PubMed Scopus (163) Google Scholar). In addition, a number of cis-acting sequences that influence splice site recognition have been identified, which include intron size, exon sequence, alternative branch points, pyrimidine content of 3′ acceptor sites, and secondary structure of the pre-mRNA (26McKeown M. Annu. Rev. Cell Biol. 1992; 8: 139-181Crossref Scopus (180) Google Scholar). The difference in processing the primary proGnRH gene transcript between the Gn11 versus NLT cells and between the GnRH neurons in the caudal hypothalamus versusthose in the olfactory and preoptic areas may reflect the differential capacity of different GnRH neurons to recognize exon 2 in the proGnRH pre-mRNA. In the Gn11 cells and GnRH neurons located in the olfactory area and preoptic area-anterior hypothalamus, the splicing machinery either recognizes exon 2 in the primary transcript to generate the mature mRNA or ignores it, resulting in the formation of a shorter species of transcript lacking exon 2. Whether the difference in splicing of the primary transcript is due to the presence or absence of specific splicing factors in the Gn11 cellsversus NLT cells or due to the difference in the activities in general splicing factors assembled in the spliceosomes inside the nucleus remains to be determined. Although the shorter transcript found in the Gn11 cells and in GnRH neurons in the forebrain lacks the normal ATG translation initiation codon located in exon 2, examination of exon 3 sequence indicates that another ATG translation initiation codon is present in exon 3. The methionine at position +25 of the proGnRH peptide may act as a translation initiation signal for the remaining 45 amino acids of the GAP. However, the Kozak sequence (16Kozak M. Nucleic Acids Res. 1987; 15: 8125-8148Crossref PubMed Scopus (4151) Google Scholar), which is preserved as a purine (G) located at 3 nucleotides upstream of the ATG in the larger transcript, is not present in the smaller transcript. Whether this splicing variant is translated into a protein in Gn11 cells and the translational efficiency has yet to be determined. Since the NLT cells express higher levels of mature proGnRH mRNA and secrete larger quantities of GnRH, these cells have the advantage over the Gn11 cells for studying the regulation of proGnRH gene expression and GnRH secretion by various factors. Recent studies from this laboratory using the NLT cell line have demonstrated that these cells express type-I receptor for insulin-like growth factor (27Zhen, S., and Radovick, S. (1996) 26th Annual Meeting Neuroscience, November 19, 1996, Washington, D. C., Slide 379.12.Google Scholar) and the receptor for epidermal growth factor (28Zhen S. Su E. Zakaria M. Radovick S. 10th International Congress on Endocrinology, June 12–15, 1996, San Francisco, CA. 1996; (,): 1-346Google Scholar). These findings are interesting since both insulin-like growth factor-1 and epidermal growth factor have been shown to regulate GnRH neuronal activitiesin vivo and have been suggested to play an important role in the control of pubertal development (29Hiney J.K. Ojeda S.R. Dees W.L. Neuroendocrinology. 1991; 54: 420-423Crossref PubMed Scopus (188) Google Scholar, 30Ma Y.J. Junier M.P. Costa M.E. Ojeda S.R. Neuron. 1992; 9: 657-670Abstract Full Text PDF PubMed Scopus (185) Google Scholar). However, to date, the molecular mechanisms by which these growth factors regulate GnRH neuronal activities remain largely unknown, due to the fact that less than 1500 GnRH neurons are present in the animal brain (4Wray S. Hoffman G.A. J. Comp. Neurol. 1986; 252: 522-531Crossref PubMed Scopus (129) Google Scholar, 5Merchanthaler I. Gorcs T. Setalo G. Petrusz P. Flerko B. Cell Tissue Res. 1984; 237: 15-29PubMed Google Scholar). Therefore, the availability of NLT cells should greatly enhance our ability to explore the molecular mechanisms that mediate the regulation of proGnRH gene expression and GnRH secretion. We thank Drs. Andrew Wolfe, Laurie Cohen, and Marjorie Zakaria for comments on the manuscript, Eric Su for technical assistance, and Drs. William Chin and Rupert Yip in the Division of Genetics/Brigham & Women's Hospital for support of the immunocytochemistry." @default.
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