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Pituitary surgery generates pituitary tissue for histology, immunohistochemistry, and molecular biological research. In the last decade, the pathogenesis of pituitary adenomas has been extensively studied in humans, and to a lesser degree in dogs, and tumor oncogenesis has been studied in knock-out mice, often by means of quantitative reversed-transcriptase PCR (RT-qPCR). A precondition of such analyses is that so-called reference genes are stably expressed regardless of changes in disease status or treatment. In this study, the expression of six frequently used reference genes, namely, tata box binding protein (tbp), tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide (ywhaz), hydroxymethylbilane synthase (hmbs), beta-2-microglobulin (b2m), succinate dehydrogenase complex subunit A (sdha), and glyceraldehyde 3 phosphate dehydrogenase 1 (gapdh), was studied in pituitary tissue (normal and adenoma) from three species (humans, mice, and dogs). The stability of expression of these reference genes differed between species and between healthy and diseased tissue within one species. Quantitative analysis based on a single reference gene that is assumed to be stably expressed might lead to wrong conclusions. This cross-species analysis clearly emphasizes the need to evaluate the expression stability of reference genes as a standard and integral aspect of study design and data analysis, in order to improve the validity of the conclusions drawn on the basis of quantitative molecular analyses.

Cushing's disease (CD) or pituitary-dependent hypercortisolism is a common endocrinopathy in dogs, with an estimated prevalence of 1 or 2 in 1000 dogs per year. It is caused by an adrenocorticotropic hormone secreting adenoma in the pars distalis or pars intermedia of the pituitary gland. The pituitary gland is a small endocrine gland located in the pituitary fossa. In the postnatal individual, the hypothalamus-pituitary axis plays a central role in maintaining homeostatic functions, like control of metabolism, reproduction, and growth. Stem cells are suggested to play a role in the homeostatic adaptations of the adult pituitary gland, such as the rapid specific cell-type expansion in response to pregnancy or lactation. Several cell populations have been suggested as pituitary stem cells, such as Side Population cells and cells expressing Sox2 or Nestin. These cell populations are discussed in this review. Also, stem and progenitor cells are thought to play a role in pituitary tumorigenesis, such as the development of pituitary adenomas in dogs. There are limited reports on the role of stem cells in pituitary adenomas, especially in dogs. Further studies are needed to identify and characterize this cell population and to develop specific cell targeting therapeutic strategies as a new way of treating canine CD.

To date, stem/progenitor cells have not been identified in the canine pituitary gland. Cells that efficiently exclude the vital dye Hoechst 33342 can be visualised and identified using fluorescence activated cell sorting (FACS) as a 'side population' (SP), distinct from the main population (MP). Such SPs have been identified in several tissues and display stem/progenitor cell characteristics. In this study, a small SP (1.3%, n=6) was detected in the anterior pituitary glands of healthy dogs. Quantitative PCR indicated significantly higher expression of CD34 and Thy1 in this SP, but no differences in the expression of CD133, Bmi-1, Axin2 or Shh. Pro-opiomelanocortin (POMC) and Lhx3 expression were significantly higher in the MP than in the SP, but no differences in the expression of Tpit, GH or PRL were found. The study demonstrated the existence of an SP of cells in the normal canine pituitary gland, encompassing cells with stem cell characteristics and without POMC expression.

Pituitary-dependent hypercortisolism is a common endocrinopathy in dogs, caused by an adrenocorticotrophic hormone secreting pituitary tumour of the anterior or intermediate lobe. The prognosis of intermediate lobe adenomas is worse than that of anterior lobe adenomas, indicating the possible usefulness of melanotropic markers as prognosticators. Another possible origin of pituitary adenomas is found in cancer stem cells. The aim of the present study was to investigate the expression of melanotroph specific transcription factor paired box protein 7 (Pax7) and stem cell marker and reprogramming factor sex determining region Y-box 2 (Sox2) and to relate their expression to clinical parameters. The mean ± SD of labelling index (LI) for Pax7 was 8.6% ± 21.7% in the adenomas; 1/6 controls had positive staining (LI, 15.2%). For Sox2, the LI in the adenomas was 16.9% ± 15.2% and 19.5% ± 11.6% in the controls. Pax7 expression was significantly higher in enlarged pituitaries, compared to non-enlarged pituitaries (P = 0.05), but Pax7 or Sox2 immunopositivity did not correlate to other clinical parameters such as histological diagnosis, survival time or disease-free interval. Gene expression of Pax7 target genes, such as proconvertase 2 (PC2), pro-opiomelanocortin (POMC), and dopamine D2 receptor (DRD2), was significantly lower in the adenoma samples compared to normal tissue, indicating that Pax7 signalling was not activated in adenomas. It was suggested that Pax7 and Sox2 remain interesting targets for molecular investigations into their role in pituitary tumorigenesis, but were unsuitable as clinical prognosticators in dogs.