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1 Enterprise Interest None

2 Plurihormonal cells of normal anterior pituitary Lubov Mitrofanova¹, Petr Konovalov¹, Julia Krylova², Igor Kvetnoy² ¹Federal Almazov North-West Medical Research Centre, St. Petersburg, Russia ² Ott Research Institute of Obstetrics and Gynecology, St. Petersburg, Russia

3 Hematoxylin and eosin stain shows acidophils, basophils and chromophobic cells in normal adenohypophysis. The introduction of immunohistochemistry in a routine morphological study permitted to divide cells into 6 types releasing different hormones, that each cell type secretes a different hormone or hormones: somatotrophs, prolactin cells, mammosomatotrophs, corticotrophs, thyrotrophs and gonadotrophs [Asa S.L., 2011].

4 Whether plurihormonal cells were more often found in the normal pituitary gland or in adenomas remained to be seen [Kovacs K. et al, 1998]. Under physiological conditions the presence of plurihormonal cells can be related to transdifferentiation which means the conversion of one cell type into another [Felix I. Asa SL., Kovacs K. et al, 1994; Vidal S., Syro L., Horvath E. et al, 1999; Vidal S., Horvath E., Kovacs K. et al, 2000].

5 Literature review has showed that it is not clear if the cells of the normal anterior pituitary of an adult co-express some hormones simultaneously and if it is the case what hormones they are. We hypothesize that the same cells of a normal pituitary gland releases several hormones simultaneously.

6 Objective: to investigate a possible coexpression of hormones by the cells of the normal adenohypophysis in adult human autopsy material

7 Patient Characteristics

8 Methods Histological study: hematoxylin & eosin and Gordon and Sweet's silver staining (for reticular fibers) Immunohistochemistry: - with antibodies against prolactin, thyroid-stimulating hormone (TSH), luteinizing hormone (LH), follicle-stimulating hormone (FSH), adrenocorticotropic hormone (ACTH), growth hormone (GH; in all 10 specimens) - double staining immunohistochemistry with 11 hormone combinations: PRL/TSH, PRL/LH, PRL/FSH, PRL/ACTH, GH/TSH, GH/LH, GH/FSH, GH/ACTH, TSH/LH, TSH/FSH, TSH/ACTH Scanning of immunohistochemical sections with Aperio Digital Pathology Scanner Confocal Laser Scanning Microscopy (CLSM) (Olympus FV1000D, Japan) with a mixture of primary antibodies: ACTH/PRL, FSH/PRL, TSH/PRL, ACTH/GH, and FSH/GH (in 2 cases) Morphometric study with image analyzer Leica Microsystems Qwin V3 Professional. The co-expression coefficient of hormones was defined as the ratio of the double-stained cells to the single-stained ones, expressed in percentage. Statistical analyses (IBM SPSS Statistics 19, version )

9 Results GH LH PRL ACTH Having compared the scanned immunochemical sections of 10 pituitary glands, we found out evident overlaps of expression areas of 4 hormones, namely GH, PRL, LH and ACTH

10 Hormone co-expression in the same anterior pituitary cells, Double staining immunohistochemistry We found that the same cells of the normal adenohypophysis can co-express prolactin with ACTH, TSH, FSH, LH; GH with ACTH, TSH, FSH, LH, and TSH with ACTH, FSH, LH. Hormones PRL GH ACTH LH FSH TSH GH /- --/++ PRL /- +++/- +++/- +++/- +++/- ---/+ ACTH +++/- +++/ /+ LH +++/- --/ /++ FSH +++/- +++/ /+ TSH +++/- --/++ ---/+ ---/+ ---/ * blank cells no information available on double-staining

11 Double staining immunohistochemistry A B A. PRL/TSH, x400. B. PRL/LH, x400. PRL is visualized with red colour, TSH and LH with blue colour, hormone co-expression with marron.

12 The average percentages of anterior pituitary hormone co-expression, Double staining immunohistochemistry

13 Double staining immunohistochemistry The comparison of the average co-expression coefficients of prolactin, GH and TSH with other hormones showed that the TSH co-expression coefficient was significantly the least (9,5±6,9%; 9,6±7,8%; 1,0±1,3% correspondingly)

14 Average percentages of hormone co-expression. Confocal Laser Scanning Microscopy and Double staining immunohistochemistry Hormone mixture ACTH/PRL 34/9 The average percentage of coexpression (%; confocal laser scanning microscopy/doublestaining immunohistochemistry) FSH/PRL 51/8 TSH/PRL 50/13 FSH/GH 40/12 ACTH/GH 55/7

15 Laser Confocal Scanning Microscopy with PRL and ACTH A. Blue fuorescence of cell nuclei (DAPI). B. Green fuorescence of prolactin. C. Red fuorescence of Adrenocorticotropic hormone. D, E. Hormone co-expression is visualized with yellow/orange colour (arrows). A, B, C, D x400. E Zoom.

16 Laser Confocal Scanning Microscopy with PRL and FSH A. Blue fuorescence of cell nuclei (DAPI). B. Green fuorescence of prolactin. C. Red fuorescence of Follicle-stimulating hormone. D, E. Hormone co-expression is visualized with yellow/orange colour (arrows). A, B, C, D x400. E x600.

17 Conclusion Plurihormonality of normal adenohypophysis is an actually existing phenomenon, which refutes the concept one cell type one hormone, as had been accepted before

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