Research Article: Steroid hormone receptors ERα and PR characterised by immunohistochemistry in the mare adrenal gland

Date Published: July 22, 2009

Publisher: BioMed Central

Author(s): Ylva Hedberg Alm, Sayamon Sukjumlong, Hans Kindahl, Anne-Marie Dalin.


Sex steroid hormone receptors have been identified in the adrenal gland of rat, sheep and rhesus monkey, indicating a direct effect of sex steroids on adrenal gland function.

In the present study, immunohistochemistry using two different mouse monoclonal antibodies was employed to determine the presence of oestrogen receptor alpha (ERalpha) and progesterone receptor (PR) in the mare adrenal gland. Adrenal glands from intact (n = 5) and ovariectomised (OVX) (n = 5) mares, as well as uterine tissue (n = 9), were collected after euthanasia. Three of the OVX mares were treated with a single intramuscular injection of oestradiol benzoate (2.5 mg) 18 – 22 hours prior to euthanasia and tissue collection (OVX+Oe). Uterine tissue was used as a positive control and showed positive staining for both ERalpha and PR.

ERalpha staining was detected in the adrenal zona glomerulosa, fasciculata and reticularis of all mare groups. Ovariectomy increased cortical ERalpha staining intensity. In OVX mares and one intact mare, positive ERalpha staining was also detected in adrenal medullary cells. PR staining of weak intensity was present in a low proportion of cells in the zona fasciculata and reticularis of all mare groups. Weak PR staining was also found in a high proportion of adrenal medullary cells. In contrast to staining in the adrenal cortex, which was always located within the cell nuclei, medullary staining for both ERalpha and PR was observed only in the cell cytoplasm.

The present results show the presence of ERalpha in the adrenal cortex, indicating oestradiol may have a direct effect on mare adrenal function. However, further studies are needed to confirm the presence of PR as staining in the present study was only weak and/or minor. Also, any possible effect of oestradiol treatment on the levels of steroid receptors cannot be determined by the present study, as treatment time was of a too short duration.

Partial Text

Activation of the hypothalamic-pituitary-adrenal (HPA) axis, with the release of ACTH and cortisol, as occurs during stress, often has an inhibitory effect on the reproductive system [1-3]. The interaction between the HPA axis and the hypothalamic-pituitary-gonadal (HPG) axis may act in both ways, with reproductive hormones also influencing adrenal function. The presence of adrenal sex steroid hormone receptors in the adrenal gland may give an answer to whether sex steroid hormones can act directly on the adrenal gland.

This preliminary study was part of a much larger study investigating adrenal steroid hormone production in mares [11,12]. All of the procedures of this larger study were approved by the Ethical Committee for Experimental Studies with Animals. The animals euthanized in the present study had either been used in the larger study or were mares used in the teaching of veterinary students and were destined for euthanasia regardless. Permission was granted for the collection of organs from all mares used in the present study.

The positive ERα and PR staining observed in uterine tissue in the present study supports that the monoclonal antibodies that were used correctly identified the receptor proteins. Although the present study did not attempt to investigate the effect of oestrous cycle stage on receptor staining, it was noted that the mare in oestrus showed the strongest staining intensity and highest proportion of stained cells for ERα in all of the uterine compartments studied. This is in accordance with other studies in the mare [16-18], ewe [14], mouse [19] and sow [15] that have showed that ERs are, in general, up-regulated by oestrogens. In studies performed in mares, strong staining for ER was found in cell nuclei of the endometrial connective tissue stroma prior to ovulation [16,18], with either weak [20] or strong [16] nuclear staining for ER in luminal and glandular epithelia during that same period.

The present study demonstrated the presence of both ERα and PR immunostaining in the cortex of the mare adrenal gland, although for PR, only weak staining were observed in a minor proportion of cells. To our knowledge, this is the first time ERα and PR in equine adrenal tissue have been investigated. Ovariectomy resulted in stronger cortical ERα immunostaining. The presence of PR and ERα staining in the cytoplasm of adrenal medullary cells was unexpected. Again, ovariectomy influenced the amount of ERα observed, with only one intact mare demonstrating ER staining in the medulla. It is unclear if the steroid receptors found in the mare adrenal gland have any biological effect, and, in particular for PR, further studies are clearly need to verify the presence of this receptor in equine adrenal tissue.

ACTH: adrenocorticotrophic hormone; ERα: oestrogen receptor alpha; ERβ: oestrogen receptor beta; HPA: hypothalamo-pituitary adrenal axis; HPG: hypothalamo-pituitary gonadal axis; IHC: immunohistochemistry; i.m.: intramuscularly; LBA: ligand binding assay; mRNA: messenger ribonucleic acid; OVX: ovariectomised; OVX + Oe: oestradiol treated and ovariectomised; PR: progesterone receptor.

The authors declare that they have no competing interests.

AMD; HK and YHA conceived of the study, participated in its design and collected the adrenal gland and uterine tissues (including weighing and judging reproductive status). SS and YHA performed the immunohistochemistry procedures. SS judged the staining intensities for PR and ER in adrenal and uterine tissue. YHA carried out the hormone analyses and drafted the manuscript. All authors read and approved the final manuscript.