Spexin expression in normal rat tissues

Abstract

Spexin is a highly conserved peptide which was recently identified through the bioinformatics approach. Immunohistochemical analysis of its expression has not yet been performed. Thus, in this study, we examined spexin location in a wide range of rat organs by both RT-PCR and IHC. RT-PCR identified spexin mRNA in all tissues examined. Spexin immunoreaction was mainly cytoplasmic. Spexin was immunohistochemically detected, although with different staining intensities, in epithelia and glands of skin and respiratory, digestive, urinary, and reproductive systems. Smooth muscle cells showed weak immunostaining, and connective tissue was negative. In the central nervous system, neuronal groups showed different intensities for reaction product. Immunoreaction was also found in ganglionic cells of both trigeminal and superior cervical ganglia and in photoreceptor, inner nuclear, and ganglionic layers of the retina. In the endocrine system, spexin immunoreaction was detected in the hypothalamic paraventricular and supraoptic nuclei; adenohypophysis, thyroid, and parathyroid glands; adrenal cortex and medulla (mainly ganglionic cells); Leydig cells; and thecal, luteal, and interstitial cells of the ovary. Because of its widespread expression, spexin is probably involved in many different physiological functions; in particular, location of spexin in neurons and endocrine cells suggests its roles as neurotransmitter/neuromodulator and endocrine factor.

Figure 1

Amino acid sequences of human, mouse, and rat spexin preprohormones. The signal and mature peptides are marked. Black color indicates 100% homology. Gray color indicates homology more than 50%, but less than 100%.

Figure 2

Qualitative RT-PCR analysis of spexin and housekeeping gene (porphobilinogen deaminase; PBGD) expression in different tissues. 1, DNA molecular marker size; 2, esophagus; 3, stomach; 4, small intestine; 5, liver; 6, pancreas; 7, lung; 8, skeletal muscle; 9, heart; 10, uterus; 11, thymus; 12, spleen; 13, kidney; 14, urinary bladder; 15, brain; 16, hypothalamus; 17, hypophysis; 18, thyroid; 19, adrenal gland; 20, testis; and 21, ovary.

Figure 3

Quantitative PCR results of relative mRNA expression level in different tissues. Obtained results were normalized to PBGD reference gene.

Figure 4

Anti-spexin immunohistochemical staining of rat normal tissues. (A) Skin (sg, sebaceous glands), (B) lung, (C) skeletal muscle, (D) heart, (E) bone and cartilage region of the growth plate, (F) bone marrow, (G) thymus (m, medulla; c, cortex), (H) spleen (wp, white pulp; rp, red pulp), and (I–J) lymph node (gc, germinal centers; pf, parafollicular zone). Bars: A,C–E,G,H,J = 75 μm; B,F = 37.5 μm; I = 300 μm.

Figure 5

Anti-spexin immunohistochemical staining of rat digestive system. (A) Salivary gland, (B) esophagus, (C) stomach, (D) small intestine, (E) pancreas, and (F) liver. Bars: A–C,E,F = 75 μm; D = 150 μm.

Figure 6

Anti-spexin immunohistochemical staining of rat genitourinary system. (A) Kidney, (B) urinary bladder, (C) uterus, (D) vagina, (E) epididymis, and (F) prostate. Bars: A = 23.8 μm; B = 37.5 μm; C–F = 75 μm.

Figure 7

Anti-spexin immunohistochemical staining of rat nervous system and eye. (A) Cerebral cortex and hippocampal field, (B) cerebellar cortex, (C) brainstem nucleus, (D) choroidal plexus, (E) trigeminal ganglion, (F) superior cervical ganglion, (G) retina (gcl, ganglion cell layer; ipl, inner plexiform layer; inl, inner nuclear layer; opl, outer plexiform layer; onl, outer nuclear layer; is, inner segments in the photoreceptor layer), and (H) cornea (ce, corneal epithelium; cs, corneal stroma). Bars: A = 150 μm; B,F,G = 75 μm; C–E,H = 37.5 μm.

Figure 8

Anti-spexin immunohistochemical staining of rat endocrine tissues. (A) Paraventricular nucleus, (B) supraoptic nucleus (oc, optic chiasma), (C) hypophysis (al, anterior lobe; il, intermediate lobe), (D) thyroid gland, (E) adrenal gland (m, medulla; c, cortex), (F) pancreas (iL, islets of Langerhans), (G) testis (L, Leydig cells), and (H) ovary (gc, granulosa cells; tf, theca folliculi; ig, interstitial gland; cl, corpus luteum). Bars: A,B,F = 75 μm; C,D,G,H = 37.5 μm; E = 600 μm.