Luteinizing hormone deficiency and female infertility in mice lacking the transcription factor NGFI-A (Egr-1)

Science

Volumn 273, Issue 5279, 1996, Pages 1219-1221

  Lee, Stephen L ^a   Sadovsky, Yoel ^a   Swirnoff, Alexander H ^a   Polish, Jeffrey A ^a   Goda, Pam ^a   Gavrilina, Galina ^a   Milbrandt, Jeffrey ^a  

^a WASHINGTON UNIVERSITY SCHOOL OF MEDICINE   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

ACTIVIN; FOLLITROPIN; INHIBIN; LUTEINIZING HORMONE; MESSENGER RNA; TRANSCRIPTION FACTOR; ZINC FINGER PROTEIN;

ANIMAL CELL; ANIMAL EXPERIMENT; ARTICLE; CONTROLLED STUDY; FEMALE; FEMALE INFERTILITY; FOLLITROPIN RELEASE; GENE EXPRESSION; GONADOTROPIN SECRETING CELL; HORMONE DEFICIENCY; HYPOPHYSIS DISEASE; IMMEDIATE EARLY GENE; MOUSE; NONHUMAN; OVARIECTOMY; PRIORITY JOURNAL; PROMOTER REGION; SIGNAL TRANSDUCTION; TRANSCRIPTION REGULATION;

ANIMALIA;

EID: 0029812609     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.273.5279.1219     Document Type: Article

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32. Mice were housed and bred in a specific pathogen-free facility with a 12-hour light-dark cycle. Procedures were conducted in compliance with the guidelines of the Animal Studies Committee of Washington University and the National Institutes of Health. Adult mice (older than 6 weeks) were given an intraperitoneal injection of PMS gonadotropin (5 IU) (Sigma). Where indicated, hCG (5 IU) was given 48 hours later. Progesterone, estrogen, and testosterone concentrations were determined by radioimmunoassay.
33. Preparation of mouse tissue for histology was done as described (8). For immunofluorescence, frozen pituitary sections were incubated in buffer [0.5 M tris (pH 7.5) and 0.3% Triton X-100] containing rabbit antibody to rat FSH (lot AFP-C0972881; 1:300) and guinea pig antibody to rat LH-β (lot AFP-22238790; 1:300) for 2 hours at 25°C, and then in buffer containing indocarbocyanine-conjugated donkey antibody to rabbit immunoglobulin G (IgG) (1:100) and fluorescein isothiocyanate-conjugated donkey antibody to guinea pig IgG (1:50) (Jackson ImmunoResearch, West Grove, PA).
34. 32P]deoxycytidine 5′-triphosphate, with primers for the following genes (GenBank accession numbers are in parentheses); rat LH-β (D00576), 5′-GCCGGCCTGTCAACGCAACC-3′, 5′-GAGGGCCACAGGGAAGGAGA-3′; rat FSH-β (D00577), 5′-TGAACTGACCAACATCACC-3′, 5′-ACTATCACACTTGCCACAGT-3′; rat β-actin (J00691), 5′-TATGGAGAAGATTTGGCACC-3′, 5′-GTCCAGACGCAGGATGGCAT-3′; and mouse NGFI-A (M20157), 5′-ACCCGGCCCAGCAAGACACC-3′, 5′-GGGCACAGGGGATGGGAATG-3′. Portions of the reaction were subjected to electrophoresis in a 5% nondenaturing polyacrylamide gel and were quantified by Phosphorimager (Molecular Dynamics) or x-ray film (Kodak). Control experiments verified a linear relation between the input RNA and the radioactive signal. For protein blots, pituitary lysates were resolved on an SDS-polyacrylamide gel (12%) and analyzed essentially as described (16). Rabbit antiserum to rat LH (NIDDK-rLH-I-9) was used at a dilution of 1:1000. Horseradish peroxidase-conjugated goat antibody to rabbit IgG (Cappel, Durham, NC) was used at a dilution of 1:10,000 and detected by enhanced chemiluminescence (Amersham).
35. Reporter constructs of the LH-β promoter containing a wild-type or mutated NGFI-A site were constructed in pL(KS)b-LUC (17) with standard methods. CMV-NGFI-A (17), CMV-SF1 (14), and an LH-β reporter construct were transfected along with a constitutive β-galactosidase reporter (RSV-βGal) by means of the calcium phosphate method (17) in six-well plates containing αT3 or CV-1 cells. Luciferase activity was determined 48 hours later, as described (17), and normalized to β-Gal activity (Galacto-Light; Tropix, Bedford, MA) according to the manufacturer's protocol.
36. We thank J. Baenziger for the αT3 cells; K. Tung for performing the testosterone measurements; F. Naftolin, R. Maurer, and members of the Milbrandt laboratory (W. Tourtellotte, J. Svaren, P. Crawford, B. Svetson, and K. Woodson) for helpful discussion and comments; and E. Sadovsky and S. Audrain for technical assistance. Antibodies to FSH and LH were obtained through the National Hormone and Pituitary Program, the National Institute of Diabetes and Digestive and Kidney Diseases, the National Institute of Child Health and Human Development, and the U.S. Department of Agriculture. Supported by NIH grant PO1 CA53524 to J.M. J.M. is an established investigator of the American Heart Association.