Cytogenetic, Genomic, and Functional Characterization of Pituitary Gonadotrope Cell Lines

Abstract

L β T2 and α T3-1 are important, widely studied cell line models for the pituitary gonadotropes that were generated by targeted tumorigenesis in transgenic mice. L β T2 cells are more mature gonadotrope precursors than α T3-1 cells. Microsatellite authentication patterns, chromosomal characteristics, and their intercellular variation have not been reported. We performed microsatellite and cytogenetic analysis of both cell types at early passage numbers. Short tandem repeat (STR) profiling was consistent with a mixed C57BL/6J × BALB/cJ genetic background, with distinct patterns for each cell type. Spectral karyotyping in α T3-1 cells revealed cell-to-cell variation in chromosome composition and pseudodiploidy. In L β T2 cells, chromosome counting and karyotyping demonstrated pseudotriploidy and high chromosomal variation among cells. Chromosome copy number variation was confirmed by single-cell DNA sequencing. Chromosomal compositions were consistent with a male sex for α T3-1 and a female sex for L β T2 cells. Among L β T2 stocks used in multiple laboratories, we detected two genetically similar but distinguishable lines via STR authentication, L β T2a and L β T2b. The two lines differed in morphological appearance, with L β T2a having significantly smaller cell and nucleus areas. Analysis of immediate early gene and gonadotropin subunit gene expression revealed variations in basal expression and responses to continuous and pulsatile GnRH stimulation. L β T2a showed higher basal levels of Egr1 , Fos , and Lhb but lower Fos induction. Fshb induction reached significance only in L β T2b cells. Our study highlights the heterogeneity in gonadotrope cell line genomes and provides reference STR authentication patterns that can be monitored to improve experimental reproducibility and facilitate comparisons of results within and across laboratories.

DNA-PK Inhibitor Peposertib Amplifies Radiation-Induced Inflammatory Micronucleation and Enhances TGFβ/PD-L1 Targeted Cancer Immunotherapy

“…Cells were fixed with fresh ice-cold methanol:acetic acid (3:1), dropped onto slides, stained with Giemsa, and >60 metaphase spreads/condition were scored for chromosomal aberrations. For spectral karyotyping, fixed cells were processed and analyzed at the Molecular Cytogenetic Core, Albert Einstein College of Medicine, Bronx, NY as described previously ( 17 ). Ten randomly picked metaphase spreads were imaged/analyzed for each treatment condition.…”

GLUT1-mediated glycolysis supports GnRH-induced secretion of luteinizing hormone from female gonadotropes

“…These data demonstrate that in vivo physiological changes in LH secretion are likely tied to gonadotrope cellular metabolism and are responsive to changes in upstream GnRH secretion which regulates the LH surge 20 . mRNA-seq analysis of GnRH-treated LβT2 cells 21 , 22 , a mature C57BL/6 mouse female gonadotrope cell line 23 , indeed demonstrates that GnRH regulates genes associated with gonadotrope cellular metabolism (Supplementary Fig. S1 ).…”

“…The female C57BL/6 mouse-derived LβT2 gonadotrope cell line 23 , 64 was maintained in high-glucose (4.5 g/l) HEPES-buffered DMEM supplemented with penicillin/streptomycin and 10% fetal bovine serum (FBS: FB-11, Omega Scientific, CA) at 37 °C in a humidified atmosphere of 5% CO 2 . Lentivirus transduced LβT2 cells were maintained under the same conditions with puromycin (0.25 µg/ml)-supplemented DMEM.…”

Lipopolysaccharide-induced chronic inflammation increases female serum gonadotropins and shifts the pituitary transcriptomic landscape

“…The female C57BL/6 mouse-derived LβT2 gonadotrope cell line ( 24 , 25 ) was maintained in high-glucose (4.5 g/l) HEPES-buffered DMEM supplemented with penicillin/streptomycin and 10% fetal bovine serum (FBS: FB-11, Omega Scientific, CA) at 37°C in a humidified atmosphere of 5% CO2. To test the effects of LPS, LβT2 cells were seeded at 2 × 10 5 cells per cm 2 , cultured for 24 h, and pretreated with serum-free DMEM for 12–16 h prior to LPS treatment.…”