Tao YX, Lei ZM, Woodworth SH, Rao ChV. Novel expression of luteinizing ... Sciences. New York: John Wiley and Sons; 1978: 203-253. 28. Rao ChV, Mitra S.
BIOLOGY OF REPRODUCTION 56, 501-507 (1997)
Novel Expression of Functional Luteinizing Hormone/Chorionic Gonadotropin Receptors in Cultured Glial Cells from Neonatal Rat Brains' A.A. AL-Hader, 3 4 Z.M. Lei,3 and Ch.V. Rao 2 3,
Laboratory of Molecular Reproductive Biology and Medicine,3 Department of Obstetrics and Gynecology, University of Louisville Health Sciences Center, Louisville, Kentucky 40292 Department of Physiology and Biochemistry, 4 Jordan University of Science and Technology, Irbid, Jordan ABSTRACT Adult rat brains contain LH/hCG receptors, and these receptors are functional in neuroendocrine regulation and behaviors. Since glial cells are important for development, maturation, and functioning of the brain, we tested the hypothesis that these cells from neonatal rat brains may also contain functional LH/hCG receptors. Reverse-transcriptase polymerase chain reaction amplified an expected 256 base-pair LH/hCG receptor fragment from glial cells. This fragment can bind to LH/hCG receptor cDNA in Southern blotting. Northern blotting demonstrated that glial cells contain a major 2.6-kilobase (kb) and a minor 4.3-kb transcript of LH/hCG receptors. Western immunoblotting demonstrated that glial cells also contain an 80-kDa receptor protein and that its levels are significantly higher insecondary and tertiary glial cells than in primary glial cells. Immunocytochemistry confirmed that LH/hCG receptor immunostaining is present in glial cells. Since glial cells are quite active in synthesis of prostaglandins (PGs), we investigated the effect of highly purified hCG on PGD2 and PGE2 levels. The results showed that culturing secondary glial cells for three days with highly purified hCG resulted ina dose-dependent and hormonespecific increase in PGD 2 and a decrease in PGE 2 levels in the medium as compared to control levels. In summary, we conclude that cultured glial cells from neonatal rat brains contain functional LH/hCG receptors. Through regulation of PG synthesis, LH and hCG may influence glial cell functions that are important for neonatal brain development and function. INTRODUCTION Mammalian brain contains neurons and glia, which consists of astrocytes, oligodendrocytes (macroglia), and microglial cells [1]. For many years, glia has simply been considered to represent a mechanical support for neurons. But it is now known that glial cells play important structural and functional roles in the developing as well as in the mature brain. For example, they outnumber neurons by 9 to 1 and make up more than half of the brain volume [24]. In developing brain, glial cells direct the migration and differentiation of neurons [5-7]. In the developing and/or mature brain, glial cells produce myelin, provide nutrients for neurons, are capable of bidirectional communication with other glial cells and neurons, aid in transmission of neuronal signals, and produce proteins and neurotrophic factors-such as nerve growth factor, brain-derived growth factor, and glial-derived growth factor-that are necessary for the health and survival of surrounding neurons [2-4, Accepted September 30, 1996. Received July 25, 1996. 'This work was partially supported by a Fulbright award to Dr. ALHader for sabbatical leave. 2 Correspondence: Ch.V. Rao, Department of Ob/Gyn, 438 MDR Building, University of Louisville Health Sciences Center, Louisville, KY 40292. FAX: (502) 852-0881.
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8-12]. Recent data from our laboratory demonstrated that LH/hCG receptors are present in the epithelial cell layer lining the choroid plexus and brain ventricles (ependyma) (both of which are considered a part of glia), as well as in neurons in hippocampus, dentate gyrus, hypothalamus, and cerebellum in adult rat brain [13]. The neuronal LH/hCG receptors are functional in neuroendocrine regulation and behaviors [14-19]. The receptors in choroid plexus and ependymal tanycytes are functional in transport of peripheral hCG into cerebrospinal fluid and then to various brain areas, respectively [16]. Because glial cells are important for the development, maturation, and functioning of the developing brain, we tested the hypothesis that glial cultures established from neonatal rat brains may also contain functional LH/hCG receptors. MATERIALS AND METHODS Materials The following items were obtained as gifts: highly purified hCG (CR-127; 14 900 IU/mg), human FSH (AFP87929B, 1683 IU/mg), and x (CR-125) and 3 (CR-125; 29 IU/mg) subunits of hCG from the National Hormone and Pituitary Program supported by NIDDK, NICHHD, and USDA (Rockville, MD); polyclonal LH/hCG receptor antibody raised against a synthetic N-terminus amino acid sequence of 15-38 and the corresponding synthetic receptor peptide from Dr. Patrick Roche from the Mayo Clinic (Rochester, MN); and a full-length porcine LH/hCG receptor cDNA from Dr. Hugues Loosfelt at the Hopital de Bicetre (Bicetre, France). The following items were purchased: trypsin, DNase, and soybean trypsin inhibitor from Sigma Chemical Company (St. Louis, MO); all the cell culture supplies from GIBCO-BRL (Grand Island, NY); polyclonal anti-glial fibrillary acidic protein (GFAP) and anti-neuron specific enolase antibodies from DAKO Corp. (Carpenteria, CA); AMV reverse transcriptase, Taq DNA polymerase, and in vitro transcription kits from Promega Corp. (Madison, WI); nusieve agarose from FMC Bioproducts (Rockland, ME); micro-fast track mRNA isolation kits from Invitrogen Corp. (San Diego, CA); random priming cDNA labeling kits from U.S. Biochemicals (Cleveland, OH); Immobilon-P membranes and miniblot system from Millipore Corp. (Bedford, MA); rainbow protein molecular weight standards and enhanced chemiluminescence detection system kits from Amersham Corp. (Arlington Heights, IL); the kits for protein determination by Bradford's method from Bio-Rad Laboratories (Melville, NY); and enzyme immunoassay kits for prostaglandin (PG) D 2 and PGE2 from Cayman Chemical Co. (Ann Arbor, MI). The sources of other items are the same as previously described [13, 14].
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Glial Cell Cultures Newborn or one-day-old pups of Sprague-Dawley rats (Charles River Laboratories, Portage, MI) were killed under diethyl ether anesthesia, and the craniums were opened under sterile conditions. The whole brains were removed and placed in cold isotonic buffer solution, which consisted of 137 mM NaCl, 5.4 mM KC1, 0.2 mM Na2 HPO 4, 0.2 mM KH2PO 4, 5.5 mM glucose, 59 mM sucrose, 0.02 mM phenol red dye, 250 ptg/L fungizone, 100 mg/L streptomycin, and 100 000 U/L penicillin G, pH 7.2 [20]. Under a dissection microscope, the cerebral hemispheres and subcortical structures (hippocampus and basal ganglia) with overlying cortex were removed. The remaining cerebral cortex was carefully cleaned of meninges and blood vessels and placed in fresh cold isotonic buffer solution. Tissue pooled from 12-14 pups was minced with iris scissors and suspended in 5.0 ml of isotonic buffer solution containing 0.005% Trypsin and 0.004% DNase in an Erlenmeyer flask. The contents of the flask were gently mixed and incubated at 37°C in a shaking water bath. After 7 min, the contents were again gently mixed and additional DNase was added if needed. Undissociated tissue was allowed to settle, and the supernatants were carefully removed and combined with 10 ml of defined medium (DM) prepared according to Ahmed et al. [20]. It consisted of Dulbecco's minimal essential medium, trace minerals, biotin, nucleosides, amino acids, DL-lipoic acid, vitamin B12, putrescine-transferrin, selenium, insulin, and fatty acids [20]. The DM also contained soybean trypsin inhibitor to block trypsin activity. The supernatants were centrifuged for 5-10 min at 300 x g, the cell pellets were resuspended in 10 ml of DM and filtered through sterile cheesecloth, and cell numbers were counted in a hemocytometer and plated at a density of 1200/mm 2 on poly-D-lysine-coated 100-mm culture dishes. Cells were allowed to attach for 20 min at 37 ° C in 5% CO 2 and saturated humidity. Then the cells were cultured with DM containing 10% fetal bovine serum (FBS), and the media were changed every 3 days until the cells reached confluence in about 10-14 days. Then the cells were detached from the culture dishes by treating with 0.1% trypsin at room temperature and were replated on poly-D-lysinecoated tissue culture chambers for immunocytochemistry, 12-well culture dishes for the measurements of PGs, or 100-mm culture dishes for studies on LH/hCG receptors. Replated cells were grown in basic DM containing 10% FBS until they reached confluence and were then washed and maintained in serum-free DM for 3 days. Then the cells were cultured for 3 days in the presence or absence of highly purified hCG or other hormones as indicated in figure legends. In the case of immunocytochemistry, the replated cells were grown for 3 days and then used. When tertiary glial cells were needed, the secondary glial cells were allowed to regrow to confluence and were then detached, replated, and allowed to again regrow to confluence before use. The purity of secondary glial cultures, as determined by immunostaining for GFAP, was about 94%. The remaining cells were mostly neurons as determined by immunostaining for neuron-specific enolase. Unless otherwise indicated, all the experiments were performed on cells from secondary glial cultures.
Reverse Transcription-Polymerase Chain Reaction (RT-PCR)/Southern Blotting RT-PCR was performed as previously described [21]. Briefly, total RNA was isolated by a single-step acid gua-
nidinium thiocyanate-chloroform extraction method [22], and 5 Ixg of RNA was reverse-transcribed into cDNA using primer 2 (5'-TCTGGTTCTGGAGCACA-3'). One-seventh of the cDNA was amplified in 35 cycles using primers 1 (5'-AATTCACGAGCCTCCTGGTC-3') and 2. The primers were designed from the rat LH/hCG receptor cDNA sequence at the 5'-end from 846 to 866 base pairs (bp; primer 1) and at the 3'-end from 1101 to 1081 bp (primer 2). Both template-omission and RT-omission controls were used. The size of the amplified product was determined by running a 123-bp DNA ladder in an adjacent lane. Instead of sequencing the PCR product, we performed Southern blotting to confirm its identity. Southern blotting was performed using a full-length LH/hCG receptor cDNA labeled with 32p by a random-priming method using a kit. Northern Blotting Northern blotting was performed using 10-,Lg aliquots of mRNA isolated from the glial cells [13, 23]. Briefly, mRNA was electrophoresed in agarose gels, transferred to nitrocellulose membranes, and hybridized for 18 h at 65°C with 32 P-labeled riboprobe transcribed from LH/hCG receptor cDNA. The membranes were then washed twice at 65°C with double-strength SSC (single-strength SSC = 150 mM sodium chloride and 15 mM sodium citrate, pH 7.0) containing 1% SDS and twice more with double-strength SSC containing 0.1% SDS. The washed membranes were exposed to x-ray film for 10 days at -80°C with intensifying screens. Western Immunoblotting Western immunoblotting was performed as previously described [13, 24, 25]. Briefly, the cells were homogenized for 1 min in 10 mM PBS, pH 7.4, containing 5 mM Nethylmaleimide and 0.2 mM phenylmethanesulfonyl fluoride. The homogenates were centrifuged at 4°C for 15 min g. Fifty-microgram aliquots of protein in superat 120 natants were separated by discontinuous 7.5% SDS-polyacrylamide gel electrophoresis under reducing conditions and then electroblotted to Immobilon P membranes. Receptors were detected by using a 1:1000 dilution of a polyclonal LH/hCG receptor antibody and an enhanced chemiluminescence detection system. The relative optical densities of the bands were determined in a linear range by a Bio-Rad densitometer. The receptor antibody preabsorbed with excess receptor peptide was used for a procedural control. Immunocytochemistry This procedure was performed by an avidin-biotin immunoperoxidase method [13, 26]. Briefly, the glial cells were plated on two-well chamber slides, fixed in Bouin's solution for 5 min, and immunostained with a 1:350 dilution of LH/hCG receptor antibody. The receptor antibody preabsorbed with the corresponding excess receptor peptide was used for the procedural control. Measurement of PGD 2 and PGE2 The PGD 2 and PGE 2 levels in 50-p1 aliquots of media in duplicate were measured by corresponding enzyme immunoassays using kits. The instructions provided by the kit manufacturer were followed. The samples for PGD2 measurement were immediately treated with methoxime (MOX) HC1 to convert unstable PGD 2 into stable PGD 2-
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FIG. 2. The levels of LH/hCG receptors in primary, secondary, and tertiary glial cells. The inset shows a representative Western blot with the sequence of lanes corresponding to the bars. Each bar represents the mean and its standard error. Asterisks indicate significant differences as compared to the control (*p < 0.05; **p < 0.01).
FIG. 1. RT-PCR/Southern blotting (A), Northern blotting (B), and Western immunoblotting (C)for LH/hCG receptors in glial cells. Lanes 1, 2, 4, and 5 are glial cells, lane 2 is RNA omission control, lane 3 is rat testis used as a positive control tissue, and lane 5 is an antibody preabsorption control.
MOX. Then the samples were stored at -80°C until assayed within 2 wk. The samples for PGE 2 measurement were stored at -80°C until assayed within 4 wk. The detection limit was 3.24 pg/ml for the PGD 2 assay and 29 pg/ml for the PGE 2 assay. The intra- and interassay coefficients of variation for both assays were less than 10%. The specificity of the PGD 2 antibody was 100% for PGD 2MOX, 0.2% for PGD 2, and < 0.01% for PGE 2-MOX and PGFIa-MOX. The specificity of the PGE 2 antibody was 100% for PGE 2, 43% for PGE 3, 18.7% for PGE 1, 1% for PGF 1., and < 0.01% for PGA. Even though the PGE 2 antibody has a fairly high cross-reactivity with PGE 3 and PGE 1, these PGs are not present in biological fluids in sufficient amounts to interfere with the assay results. Replication of the Experiments and Statistical Analysis All the experiments were performed in duplicate and repeated three times on different glial cultures. The data were analyzed by one-way analysis of variance and Tukey-Kramer multiple comparison tests [27]. RESULTS We first used RT-PCR to determine the possible presence of LH/hCG receptor transcripts in glial cells. As shown in Figure 1A, this procedure amplified only an expected 256-bp LH/hCG receptor fragment from the glial cells (lane 1) and the adult rat testes (lane 3), used as a positive control tissue. The omissions of either template (lane 2) or reverse transcriptase (data not shown) resulted in the absence of amplification from glial cells. The bottom of lanes 1 and 3
shows that an amplified fragment can bind to a full-length LH/hCG receptor cDNA in Southern blotting, indicating that it came from receptor mRNA in the cells. We next used Northern blotting to determine the size of LH/hCG receptor transcripts in secondary glial cells. As shown in Figure B, glial cells contained a major 2.6-kilobase (kb) and a minor 4.3-kb receptor transcript. The above data led us to use Western immunoblotting to determine the possible presence of LH/hCG receptor protein in glial cells. As shown in Figure 1C, glial cells also contained an 80-kDa receptor protein (lane 3) that was not detected when the receptor antibody preabsorbed with the corresponding excess receptor peptide was used (lane 4). Figure 2 shows that cells from secondary and tertiary glial cultures contained significantly higher LH/hCG receptor protein levels than did the cells from primary glial cultures. Since about 6% of the cells in secondary glial cultures are neurons, we performed immunocytochemistry to determine cellular localization of LH/hCG receptor immunostaining. As shown in Figure 3A, astrocytes contained receptor immunostaining, and a considerable amount of it was present in and around the nucleus of the cells. Preabsorption of the receptor antibody with the corresponding receptor peptide resulted in the absence of immunostaining (Fig. 3B). Neurons also contained receptor immunostaining, but the relative differences between them and glial cells were not determined. We determined the functional relevance of LH/hCG receptors by investigating the effect of highly purified hCG on the synthesis of PGD 2 and PGE 2 by glial cells. First, untreated glial cells synthesized both PGs, producing about twice as much PGD 2 (230 ± 18 pg/ml) as PGE 2 (110 ± 12 pg/ml). Culturing these cells with hCG resulted in a significant increase in PGD2 levels at 100 and 250 ng/ml (Fig. 4). Figure 5 shows that the effect of hCG on PGD 2 levels was not mimicked by FSH or by isolated ot and subunits of hCG. Figure 6 shows that PGE 2 levels decreased after treatment with 50-250 ng/ml hCG. This decreasing effect was also not mimicked by FSH or by isolated ot and 3 subunits of hCG (Fig. 7).
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FIG. 3. Immunocytochemistry for LH/hCG receptors in glial cultures. Unabsorbed receptor antibody was used in A, and the receptor antibody preabsorbed with the excess corresponding receptor peptide was used in B. x600.
DISCUSSION
The RT-PCR amplified a receptor fragment of expected size capable of binding to rat LH/hCG receptor cDNA, suggesting that the glial cells indeed contain receptor transcripts. Northern blotting used to size the receptor tran-
scripts revealed that glial cells contained a major 2.6-kb and a minor 4.3-kb receptor transcript. The relative abundance of receptor transcripts as determined by RT-PCR/ Southern blotting was much higher in the testes than in the glial cells. Previous studies have shown that tissues such as liver, kidney, spleen, lung, heart, and skeletal smooth
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FIG. 5. Hormone specificity of hCG effect on media PGD 2 levels in glial cells. The cells were cultured for 3 days in the presence or absence of 250 ng/ml of various hormones, and then PGD 2 levels were measured. Each bar represents the mean and its standard error. *Significant difference as compared to the control (p < 0.05).
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FIG. 6. Dose dependency of hCG effect on media PGE2 levels in glial cells. The cells were cultured for 3 days in the presence or absence of increasing concentrations of hCG, and then PGE, levels were measured. Each bar represents the mean and its standard error. Asterisks indicate significant differences as compared to the control (*p < 0.01; **p < 0.001). Control levels were 119 ± 12 pg/ml.
muscle do not contain any receptor transcripts ([13, 18, 21] Thompson et al., submitted for publication). Glial cells showed no detectable 1.8-kb receptor transcript, which was previously found in whole adult rat brains and in mouse immortalized hypothalamic GnRH-containing GT1-7 neurons [13, 14]. The major receptor transcript found in glial cells was the same as that found in whole adult brain (2.6 kb) and different from the 1.8-kb major transcript in GT1-7 neurons [13, 14]. These findings suggest that the factors that determine the presence of multiple receptor transcripts, including which transcript is major or which are minor, are different in glial cells as compared to the adult brain and GT1-7 neurons. Despite the presence of two transcripts, glial cells, like whole adult rat brain and GT1-7 neurons, contained an 80-kDa receptor protein [13, 14]. Although primary, secondary, and tertiary glial cells contained the receptor protein, the levels were higher in secondary and tertiary glial cells than in primary glial cells. This increase could have been due to the increasing uniformity of glial cells with increasing passage in culture and/or due to recovery of the cells from the dispersion procedure during reculturing. Our preliminary study has shown that cerebral cortex and cerebellum of intact fetal rat brain, which contain glial cells as a majority, immunostain for LH/hCG receptors. Thus, these data provide indirect evidence that glial cells in intact fetal rat brain contain LH/hCG receptors. This finding, plus the fact that the epithelial cells lining the choroid plexus and ependymal tanycytes, which are considered part of the glia in adult rat brain [13], also contain LH/hCG receptors, minimizes the possibility that glial cell LH/hCG receptors are artifacts of cell culture. The nuclei of astrocytes contain considerable amounts of LH/hCG receptor immunostaining. Whether this apparent perinuclear immunostaining is due to a natural thickness of the cells in the nuclear region and/or an intrinsic receptor distribution is not known. Previous studies have demonstrated, however, that functional LH/hCG receptors are also present in part in the nucleus of the target cells [28-32]. Concerning the functional relevance, we investigated the effect of highly purified hCG on PGD 2 and PGE 2 levels for several reasons. First, neonatal brain contains high levels
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FIG. 7. Hormone specificity of hCG effect on media PGE2 levels in glial cells. The cells were cultured for 3 days in the presence or absence of 250 ng/ml of various hormones, and then PGE2 levels were measured. Each bar represents the mean and its standard error. *Significant difference as compared to the control (p < 0.05).
of cyclooxygenase-2 (COX-2) enzyme and produces high levels of PGs [33-35]. Second, glial cells synthesize higher amounts of PGs than do neurons in neonatal brain [36, 37]. Finally, hCG is known to regulate PGD 2 and PGE 2 synthesis in the adult rat brain [15]. The present results demonstrate that secondary glial cells can synthesize both PGs, producing twice as much PGD 2 as PGE 2. Culturing secondary glial cells for 3 days in the presence of increasing concentrations of highly purified hCG resulted in an increase in PGD 2 and a decrease in PGE 2 levels in the culture medium as compared to the controls. The hCG effect was hormone-specific: FSH had no effect. The hCG effect requires the conformation of hCG dimer, shown by the lack of effect of ao and 3 subunits of hCG on either PGD 2 or PGE 2 levels. The functional significance of hCG's inhibition of PGE2 synthesis may lie in the fact that PGE 2 inhibits the proliferation of glial cells from neonatal rat brains [38]. Thus, by decreasing PGE 2, hCG may promote the controlled proliferation of glial cells that may be required for neonatal brain development, maturation, and function. The role of PGD 2 in glial cells from neonatal brain is not known. However, the effects of PGD 2 are usually the opposite of those of PGE 2 in the brain. For example, whereas PGD 2 promotes sleep, lowers body temperature, and inhibits GnRH secretion, PGE 2 has the opposite effects [39-41]. In view of these examples, it is possible that PGD 2 may stimulate proliferation of glial cells. Thus, it should not be surprising that hCG treatment increases PGD 2 while it decreases PGE 2 levels. The ability of hCG to increase PGD 2 and decrease PGE 2 levels suggests that the primary targets of hCG regulation are corresponding synthases rather than COX-2. Although it was known that glial cells produce numerous regulatory molecules such as PGs and others, it was not known until the present study what hormones regulated the glial cell production of these molecules. The current data suggest that LH and hCG could be involved in this regulation. Concerning the source of LH/hCG for glial receptors, it is not known whether neonatal rat brain can make hCG or LH or similar peptides. However, a number of studies demonstrate that several areas of the adult brain can make LH [42-47]. The function of this nonanterior pituitary source
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of brain LH is not known. The structural and functional maturation of the developing brain is obscure, and it is only recently that investigators have begun to unravel the potential factors involved and their molecular mechanisms of action. Our contribution to this area is to show that LH or hCG or similar peptides that neonatal rat brain may produce could also play a role in brain development. In summary, cultured glial cells from neonatal rat brains contain functional LH/hCG receptors. These findings may suggest new insights into the potential roles of LH and hCG in structural and functional maturation of neonatal brain. REFERENCES 1. Angevine J. The nervous tissue. In: Bloom W, Fawacett DW (eds.), A Textbook of Histology. Philadelphia, PA: W.B. Saunders Co.; 1975: 333-385. 2. Somjen G. Nervenkitt: notes on the history of the concept of neuroglia. Glia 1988; 1:2-9. 3. Smith S. Neuromodulatory astrocytes. Curr Biol 1994; 4:807-810. 4. Travis J. Glia: the brain's other cells. Science 1994; 266:970-972. 5. Rakic P Neuron-glia relationship during granule cell migration in developing cerebellar cortex: a Golgi and electron microscopic study in Macacus rhesus. J Comp Neurol 1971; 141:282-312. 6. Hatten ME, Liem RKH. Astroglial cells provide a template for the positioning of developing cerebellar neurons in vitro. J Cell Biol 1981; 90:622-630. 7. Gasser UE, Hatten ME. Neuron-glia interactions of rat hippocampal cells in vito: glial guided neuronal migration and neuronal regulation of glial differentiation. J Neurosci 1990; 10:1276-1285. 8. Banker GA. Trophic interactions between astroglial cells and hippocampal neurons in culture. Science 1980; 209:809-810. 9. Lindsay RM. Adult rat brain astrocytes support survival of both NGFdependent and NGF-insensitive neurons. Nature 1987; 282:80-82. 10. Hatten ME, Lynch M, Rydel RE, Sanchez J, Joseph-Silverstein J, Moscatelli D, Rifkin DB. In vitro neurite extension by granule neurons is dependent upon astroglial-derived fibroblast growth factor. Dev Biol 1988; 125:280-289. 11. van der Pal RHH, Koper JW, van Golde LMG, Lopes Cardozo M. Effects of insulin and insulin-like growth factor (IGFI) on oligodendrocyte-enriched glial cultures, J Neurosci Res 1988; 19:483-490. 12. Kadle R, Suksang C, Roberson ED, Fellows RE. Identification of an insulin-like factor in astrocyte conditioned medium. Brain Res 1988; 460:60-67. 13. Lei ZM, Rao ChV, Kornyei JL, Licht P, Hiatt ES. Novel expression of human chorionic gonadotropin/luteinizing hormone receptor gene in brain. Endocrinology 1993; 132:2262-2270. 14. Lei ZM, Rao ChV. Novel presence of luteinizing hormone/human chorionic gonadotropin (hCG) receptors and the down regulating action of hCG on gonadotropin releasing hormone gene expression in immortalized hypothalamic GT1-7 neurons. Mol Endocrinol 1994; 8: 1111-1121. 15. Toth P, Lukacs H, Hiatt ES, Reid RH, Iyer V, Rao ChV. Administration of human chorionic gonadotropin affects sleep wake phases and other associated behaviors in cycling female rats. Brain Res 1994; 654:181-190. 16. Lukacs H, Hiatt ES, Lei ZM, Rao ChV. Peripheral and intracerebroventricular administration of human chorionic gonadotropin alters several hippocampus associated behaviors in cycling female rats. Horm Behav 1995; 29:42-58. 17. Lei ZM, Rao ChV. Signaling and transacting factors in the transcriptional inhibition of gonadotropin releasing hormone gene by human chorionic gonadotropin in immortalized hypothalamic GT1-7 neurons. Mol Cell Endocrinol 1995; 109:151-157. 18. Huang ZH, Lei ZM, Rao ChV. Immortalized anterior pituitary caT3 gonadotropes contain functional luteinizing hormone/human chorionic gonadotropin receptors. Mol Cell Endocrinol 1995; 114:217-222. 19. Li X, Lei ZM, Rao ChV. Human chorionic gonadotropin down-regulates the expression of gonadotropin releasing hormone receptor gene in GTI-7 neurons. Endocrinology 1996; 137:899-904. 20. Ahmed Z, Walker PS, Fellows RE. Properties of neurons from dissociated fetal rat brain in serum-free culture. J Neurosci 1983; 3:24482462. 21. Tao YX, Lei ZM, Woodworth SH, Rao ChV. Novel expression of
22. 23. 24. 25.
26.
27. 28.
29.
30. 31.
32. 33.
34. 35.
36. 37.
38.
39.
40.
41. 42. 43.
44. 45.
luteinizing hormone/chorionic gonadotropin receptor gene in rat prostates. Mol Cell Endocrinol 1995; 11:R9-R12. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium-thiocyanate-phenol-chloroform extraction. Anal Biochem 1987; 162:156-159. Sambrook J, Fritsch EE Maniatis T. Molecular Cloning-A Laboratory Manual, ed 2. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory; 1989: 7.12-7.52, 10.27-10.36. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4 . Nature 1970; 227:680-685. Dunn SD. Effects of the modification of transfer buffer composition and the renaturation of proteins in gels on the recognition of proteins on western blots by monoclonal antibodies. Anal Biochem 1986; 157: 144-153. Reshef E, Lei ZM, Rao ChV, Pridham DD, Chegini N, Luborsky JL. The presence of gonadotropin receptors in nonpregnant human uterus, human placenta, fetal membranes and decidua. J Clin Endocrinol & Metab 1990; 70:421-430. Daniel WW. Biostatistics: A Foundation for Analysis in the Health Sciences. New York: John Wiley and Sons; 1978: 203-253. Rao ChV, Mitra S. Gonadotropin and prostaglandins binding sites in nuclei of bovine corpora lutea. Biochim Biophys Acta 1979; 584:454466. Rao ChV, Mitra S, Carman FR Jr. Characterization of gonadotropin binding sites in the intracellular organelles of bovine corpora lutea and comparison with those in plasma membranes. J Biol Chem 1981; 256:2628-2634. Ramani N, Rao ChV. Direct stimulation of nucleoside triphosphatase activity in bovine luteal nuclear membranes by human chorionic gonadotropin. Endocrinology 1987; 2468-2473. Toledo A, Ramani N, Rao ChV. Direct stimulation of nucleoside triphosphatase activity in human ovarian nuclear membranes by human chorionic gonadotropin. J Clin Endocrinol & Metab 1987; 65:305309. Oechsli M, Rao ChV, Chegini N. Human chorionic gonadotropin increases chromatin solubility in isolated bovine and human luteal nuclei. Biol Reprod 1989; 41:753-760. Tsubokura S, Watanabe Y, Ehara H, Imamura K, Sugimoto O, Ragamiyama H, Yamamoto S, Hayaishi O. Localization of prostaglandin endoperoxide synthase in neurons and glia in monkey brain. Brain Res 1991; 543:15-24. Peri KG, Hardy P, Li DY, Varma DR, Chemtob S. Prostaglandin G/H synthase-2 is a major contributor of brain prostaglandins in the newborn. J Biol Chem 1995; 41:24615-24620. Jones SA, Adamson SL, Bishai I, Lees J, Engelberts D, Coceani E Eicosanoids in the third ventricular cerebrospinal fluid of fetal and newborn sheep. Am J Physiol 1993; 264:R135-R142. Seregi A, Keller M, Jackisch R, Hertting G. Comparison of prostanoid synthesizing capacity in homogenates from primary neuronal and astroglial cell cultures. Biochem Pharmacol 1984; 33:3315-3318. Keller M, Jackisch R, Seregi A, Hertting G. Comparison of prostanoid forming capacity of neuronal and astroglial cells in primary cultures. Neurochem Int 1985; 7:655-665. DuBois JH, Boloton C, Cuzner L. The production of prostaglandin and the regulation of cell division in neonate rat primary mixed glial cultures. J Neuroimmunol 1986; 11:277-285. Ueno R, Narumiya S, Ogorochi T, Nakayama T, Ishikawa Y, Hayaishi O. Role of prostaglandin D2 in the hypothermia of rats caused by bacterial lipopolysaccharide. Proc Natl Acad Sci USA 1982; 79:60936097. Kinoshita F, Nakai Y, Katakami H, Imura H, Shimizu T, Hayaishi O. Suppressive effect of prostaglandin (PG) D2 on pulsatile luteinizing hormone release in conscious castrated rats. Endocrinology 1982; 110: 2207-2209. Hayaishi O. Sleep-wake regulation by prostaglandins D2 and E2. J Biol Chem 1988; 263:14593-14596. Croxatto H, Arrau J, Croxatto H. Luteinizing hormone-like activity in human median eminence extracts. Nature 1964; 204:584-585. Antunes JL, Carmel PW, Zimmerman EA, Ferin M. The pars tuberalis of the rhesus monkey secretes luteinizing hormone. Brain Res 1979; 166:49-55. Gross DS, Page RB. Luteinizing hormone and follicle-stimulating hormone production in the pars tuberalis of hypophysectomized rats. Am J Anat 1979; 156:285-291. Hostetter G, Gallo RV, Brownfield MS. Presence of immunoreactive luteinizing hormone in the rat forebrain. Neuroendocrinology 1981; 33:241-245.
GLIAL CELL LH/hCG RECEPTORS 46. Emanuele N, Connick E, Howell T, Anderson J, Hojvat S, Baker G, Souchek J, Kirsteins L, Lawrence AM. Hypothalamic luteinizing hormone (LH): characteristics and response to hypophysectomy. Biol Reprod 1981; 25:321-326.
507
47. Emanuele N, Anderson J, Andersen E, Connick E, Baker G, Kirsteins L, Lawrence AM. Extrahypothalamic brain luteinizing hormone: characterization by radioimmunoassay, chromatography, radioligand assay and bioassay. Neuroendocrinology 1983; 36:254-260.
