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Detection of Luteinizing Hormone β Messenger Ribonucleic Acid (RNA) in Individual Gonadotropes after Castration: Use of a New in Situ Hybridization Method with a Photobiotinylated Complementary RNA Probe

Department of Anatomy and Neurosciences, The University of Texas Medical Branch Galveston, Texas 77550
Department of Medicine, Brigham and Women's Hospital and Howard Hughes Medical Institute and Department of Medicine, Massachusetts General Hospital, Harvard Medical School Boston, Massachusetts, 02115

Address requests for reprints to: Gwen V. Childs, Ph.D., Department of Anatomy and Neurosciences, The University of Texas Medical Branch, 200 University Boulevard, Galveston, Texas 77550.

Abstract

Patterns of gonadotropin storage in individual gonadotropes change with alterations in the physiological state. After castration in the male rat, there is a 2.5-fold increase in the percentage of gonadotropes and an increase in the proportion of gonadotropes storing both LH and FSH. In addition, there are 6- to 8-fold increases in the pituitary concentrations of LH β subunit mRNAs. In order to determine whether these changes are due to increases in the number of gonadotropes containing subunit mRNA, or the amount of mRNA per cell or both, an in situ hybridization technique using a photobiotinylated rat LHβ cRNA probe (bio-LHβ-cRNA) was applied to detect LHβ mRNA in fixed whole rat pituitary cells from intact or castrated rats. After hybridization, the bio-LHβ-cRNA was localized with either avidin-biotin peroxidase complex or the fluorescent streptavidin phycoprobe methods. The cells containing LHβ mRNA were then counted and the amount of mRNA per cell was measured by video microdensitometry. Ten percent of the anterior pituitary cells from intact animals contained LHβ mRNA. After castration (2–4 weeks) this percentage rose to 19–24.5%. Image and microdensitometric analyses showed that castration produced a 1.9-fold increase in the amount of LHβ mRNA per cell, and a 2.2-fold increase in the area of cells containing LHβ mRNA. Hence, castration resulted in an increase in the level of LHβ mRNA per cell as well as the number of LHβ mRNA-containing cells. When in situ hybridization was followed by immunocytochemistry in cells from intact rats, 83% of gonadotropes that stained for LHβ and 80% of gonadotropes that stained for FSHβ contained LHβ mRNA whereas after castration 99% of LHstoring and 93% of FSH-storing cells contained LHβ mRNA. This new in situ hybridization protocol is rapid and allows quantification of mRNA within individual gonadotropes. In addition, since the hybridization protocol does not apparently alter the gonadotropin antigens, the hormone content of the same gonadotrope may be defined by immunocytochemistry.

FOOTNOTES

The study was supported by NIH Grant R01-HD-15472 (to G.V.C.) and HD-19938 (to W.W.C.). This study was presented in platform session at an INSERM CONFERENCE on The Anterior Pituitary Gland—Fundamental and Pathological Aspects, Seillac, France, 1987 and in a Poster at the Cell Biology Meetings, St. Louis, MO, 1987.

Received for publication August 28, 1987. Accepted for publication October 9, 1987.

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