Fig 2: Immunohistochemistry of GM-CSF in pig accessory sex glands. Immunolabelling in (a) prostate, present in supranuclear cytoplasm and small vesicles (arrow) in the lumen; (b) corpora amylacea (arrow); (c) vesicles in lumen (asterisk); (d–f) Seminal vesicle with staining in protruding apical membranes (arrows) and (g) the bulbourethral gland where immunostaining was observed in vascular smooth muscle, but not in secretory epithelium (asterisk). Scale bars: (a) 20 µm; and (b–g) 50 µm.

Fig 3: Immunohistochemistry of GM-CSF in pig testes. Positive immunolabelling is seen in (a) the interstitium (Leydig cells) and the residual bodies of elongated spermatids. (b) Detail of positivity in residual cytoplasm derived from spermatids (arrows). (c) Expression in cytoplasmic droplets attached to neck region of immature spermatozoa (arrow). (d) Strong cytoplasmic immunostaining in Leydig cells (arrows) and weak in Sertoli cells (arrowhead). Scale bars: (a) 100 µm; (b and c) 20 µm; and (d) 50 µm.

Fig 4: Immunohistochemistry of GM-CSF in pig epididymis. Immunoreactivity in (a) supranuclear region of the principal cells (arrow) of the epithelium from the caput segment. Arrowheads identifies apically-stained cells, and intralumenal vesicles positive (b) to GM-CSF (the relative size of the arrows used illustrate differences in vesicle size and arrowheads mark apical protuberances of principal cells). Insert: gross arrow: positive vesicle; narrow arrows: smaller positive vesicles inside the vesicles. Scale bars: (a) 50 µm; and (b detail) 20 µm.

Fig 5: Proportion of spermatozoa hierarchically clustered in two populations according to the expression of GM-CSF in the viable and nonviable sperm population. a,b Indicate significant differences (p < 0.01) between cluster 1 and 2. The cluster 1 gathered the spermatozoa showing low/absent fluorescence intensity (4.45 ± 0.17 RU; ranging from 0.0 to 15.33 RU) corresponding with a low/absent GM-CSF expression. The cluster 2 gathered those sperm displaying high fluorescence intensities (29.81 ± 1.82 RU; ranging from 15.67 to 84.0 RU).