Journal of Histochemistry and Cytochemistry, Vol. 49, 665-666, May 2001, Copyright © 2001, The Histochemical Society, Inc.


BRIEF REPORT

Differential Expression of Estradiol Receptors Alpha and Beta by Gonadotropes During the Estrous Cycle

Gwen V. Childsa,b, Geda Unabiab, and Spogmai Komakb
a Department of Anatomy, University of Arkansas for Medical Science, Little Rock, Arkansas
b Department of Anatomy and Neuroscience, University of Texas Medical Branch, Galveston, Texas

Correspondence to: Gwen V. Childs, Dept. of Anatomy, Univ. of Arkansas for Medical Sciences, 4301 West Markham Street, Slot 510, Little Rock, AR 72205-7199.


  Summary
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This study focused on expression of estradiol receptors (ER) during the estrous cycle. Labeling for ER{alpha} or ß antigens and luteinizing hormone (LH) or follicle-stimulating hormone (FSH) ß-subunits was done on freshly dispersed pituitary cells. The lowest expression of ER{alpha} and ß was seen in estrus (23% and 12%, respectively). Expression increased to 42–54% of pituitary cells by diestrus. In males, cells with ER{alpha} or ß were 37% or 20% of the population, respectively. ER{alpha} or ß and gonadotropin antigens were in 6–9% of pituitary cells from male rats. Early in the cycle (estrus and metestrus), less than 5% of pituitary cells expressed ER{alpha} or ß with gonadotropins. These values doubled to reach a peak of 10% during proestrus (just before ovulation). These data show that a rise in expression of both ER{alpha} and ERß is a part of preovulatory differentiation of pituitary gonadotropes.

(J Histochem Cytochem 49:665–666, 2001)

Key Words: estrous cycle, estrogen receptors, pituitary cells, differentiation


  Introduction
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Summary
Introduction
Literature Cited

Estrogen receptors (ER) were first discovered in the 1960s (Jensen and Jacobson 1960 ; Toft and Gorski 1966 ). Immunocytochemical studies confirmed their predominantly nuclear localization (Greene et al. 1980 ; King and Greene 1984 ). Two types of ER (ERa and b), encoded by separate genes on different chromosomes, have been described (Kuiper et al. 1996 ; Mosselman et al. 1996 ; Tremblay et al. 1997 ; Couse and Korach 1999 ). There are no physiological functions unique to ERb, whereas a number of reproductive functions require ERa (Couse and Korach 1999 ). ER molecules dimerize before they bind to the nuclear sites, suggesting that the two may work cooperatively.Both ER{alpha} and ERß are found in the pituitary of most species (Couse and Korach 1999 ). Most studies agree that gonadotropes are among the ER-expressing cells (Mitchner et al. 1998 ). Because the function of ERß is unknown, this study was initiated to learn more about expression during the rat estrous cycle.

The stage of the cycle was determined by vaginal smears. The ACUC committee, University of Texas Medical Branch, approved the animal care and use protocol. Pituitaries were dispersed, cultured for 1–2 hr, fixed in 2% glutaraldehyde (30 min), and washed four times in phosphate buffer with 4% sucrose (Childs et al. 1994 ). Cells were pretreated with 3% hydrogen peroxide, washed twice in Tris-buffered saline (TBS), and treated with 0.3% Triton X-100 (10 min each), followed by a TBS wash. Nonspecific reactions were blocked with 1% bovine serum albumin and 1% nonfat dry milk in TBS (10 min). Dual immunolabeling involved detection of ER{alpha} or ß with polyclonal rabbit anti-ER{alpha} (1:500) or goat anti-ERß (1:500) (Santa Cruz Biotechnology; Santa Cruz, CA) and streptavidin peroxidase detection kits (DAKO; Carpinteria, CA) that used either a biotinylated anti-rabbit antibody (1:100, 30 min for the anti-ER{alpha}) or anti-goat antibody (1:100, 30 min for the anti-ERß) as second antibody. Peroxidase was detected by nickel-intensified diaminobenzidine (Childs et al. 1994 ). After blocking with 1% normal goat serum, the cells were immunolabeled for luteinizing hormone (LH) or follicle-stimulating hormone (FSH) ß-subunits with 1:30,000 anti-bovine LHß or 1:10,000 anti-human FSHß and DAKO streptavidin–peroxidase kits (amber DAB; Childs et al. 1994 ). ANOVA followed by Fisher's least significant differences test detected significant differences between percentages of each cell type.

Estrous animals had only 12 ± 1% or 23 ± 1% cells with ERß or {alpha}, respectively. A significant rise in expression to 41–42% of the population was seen on the morning of metestrus. Diestrous rats showed a continued rise in ER{alpha}-bearing cells to 54 ± 2%, but no further increase in cells with ERß. Proestrous rats maintained relatively high percentages of ER{alpha} cells (48 ± 1%), whereas those with ERß declined (36 ± 2%). Populations from male rats expressed 37 ± 5% or 20 ± 2% cells with ER{alpha} or ß, respectively.

Pituitary cells with ER{alpha} or ß and LH antigens increased from 4.7 ± 0.6% at estrus to 10 ± 1.6% at proestrus. Percentages of pituitary cells with ER{alpha} or ß and FSH were lower at estrus (2.7 ± 0.2% or 1.3 ± 0.1%, respectively). After remaining below 5% during diestrus, cells with FSH and ER{alpha} or ß increased to reach a peak of 9.5 ± 1% or 8 ± 0.3%, respectively, by proestrus. In male rats, percentages of pituitary cells with ER{alpha} or ß and gonadotropins were similar to peak levels in proestrus. Fig 1 illustrates dual labeling for ERß and FSH antigens in proestrous female rat pituitary cells. In most cells, the labeling is concentrated in the nucleus (see arrows in figure). However some cells exhibit either cytoplasmic or plasma membrane labeling (not shown).



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Figure 1. (A–C) Dual labeling for ERß and FSH in proestrous rat pituitary cells. Labeling is concentrated in the nucleus (arrows). U, unlabeled cells. Bars = 10 µm.

Estrogen positive feedback on pituitary gonadotropes during diestrus promotes differentiation by stimulating the production of gonadotropin-releasing hormone receptors (Childs et al. 1994 ). This is vital for the preovulatory surge of LH and the rise in FSH. The peak expression of ER{alpha} and ß during proestrus may facilitate the positive feedback, and the negative feedback after the surge activity. Our counts agree with previous studies that looked at ER{alpha} only (Kikuta et al. 1993 ). We also show a parallel increase in ERß, suggesting that the two receptors may work cooperatively in the same cells. Studies by Mitchner et al. 1998 suggested that few pituitary cells expressed both types. ERß is sometimes difficult to detect, and the pretreatment steps used in our protocol may better detect dual expression, especially during proestrus.

To summarize, although these studies have not detected a function for ERß, they have shown that this new receptor type increases early in the cycle and that peak expression parallels that seen for ER{alpha}. This points to some cooperative potential for these receptors. The non-parallel expression seen in LH and FSH gonadotropes may reflect expression by monohormonal cells and/or unique functions for each of these receptors in different gonadotrope subtypes.


  Footnotes

Presented in part at the Joint Meeting of the Histochemical Society and the International Society for Analytical and Molecular Morphology, Santa Fe, NM, February 2–7, 2001.

Received for publication December 19, 2000; accepted February 16, 2001.
  Literature Cited
Top
Summary
Introduction
Literature Cited

Childs GV, Unabia G, Miller BT (1994) Cytochemical detection of GnRH binding sites on rat pituitary cells with LH, FSH and GH antigens during diestrous upregulation. Endocrinology 134:1943-1956[Abstract]

Couse JF, Korach KS (1999) Estrogen receptor null mice: what have we learned and where will they lead us? Endocrine Rev 20:358-417[Abstract/Free Full Text]

Greene GL, Fitch FW, Jensen EV (1980) Monoclonal antibodies to estrophilin: probes for the study of estrogen receptors. Proc Natl Acad Sci USA 77:157-161[Abstract]

Kikuta T, Yamamoto K, Namiki H, Hayashi S (1993) Immunocytochemical localization of estrogen receptor in various anterior pituitary hormone cells of adult male and female rats. Acta Histochem Cytochem 26:609-614

King WJ, Greene GL (1984) Monoclonal antibodies localize oestrogen receptor in the nuclei of target cells. Nature 307:745-747[Medline]

Kuiper GG, Enmark E, Pelto–Huikko M, Nilsson S, Gustafsson JÅ (1996) Cloning of a novel receptor expressed in rat prostate and ovary. Proc Natl Acad Sci USA 93:5925-5930[Abstract/Free Full Text]

Jensen EV, Jacobson HI (1960) Fate of steroidal estrogen in target tissues. In Pincus G, Vollmer EP, eds. Biological Activities of Steroids in Relation to Cancer. New York Academic Press, 1, 61-174

Mitchner NA, Garlick C, Ben-Jonathan N (1998) Cellular distribution and gene regulation of estrogen receptors and in the rat pituitary gland. Endocrinology 139:3976-3983[Abstract/Free Full Text]

Mosselman S, Polman J, Dijkema R (1996) ER: identification and characterization of a novel human estrogen receptor. FEBS Lett 392:49-53[Medline]

Toft D, Gorski J (1966) A receptor molecule for estrogens: isolation from the rat uterus and preliminary characterization. Proc Natl Acad Sci USA 55:1574-1581[Medline]

Tremblay GB, Tremblay A, Copeland NG, Gilbert DJ, Jenkins NA, Labrie F, Giguere V (1997) Cloning, chromosomal localization, and functional analysis of the murine estrogen receptor. Mol Endocrinol 11:353-365[Abstract/Free Full Text]