Quantification of PERF 15 mRNA in tissue sections from rat testes

Abstract

We previously conducted basic research to quantify in situ hybridization (ISH) signals in rat testes. In this experimental model, we selected ribosomal RNA (rRNA) as the hybridizable RNA in paraffin sections, since it allowed us to easily analyze ISH signals expressed with digoxygenin (DIG)-labeled probes quantitatively through "posterization" of the images. We applied this method to analyze the quantification of transcript, PERF 15 mRNA. PERF 15 is expressed specifically in the testes and localized in the rigid cytoskeletal structure of the sperm head, and has been considered to be involved in the apoptotic process of spermatogenic cells. Quantification of the signals may help to clarify the detailed function of PERF 15. We further analyzed the signals concomitant with a confocal laser scanning microscope. The peak of PERF 15 mRNA expression was found in diplotene spermatocytes, and the amount of PERF 15 mRNA was greatest in late pachytene and diplotene spermatocytes and early spermatids, followed by early pachytene spermatocytes, and then late spermatids. PERF 15 may be involved in the events leading to meiotic division, in which apoptosis is also involved. The present study may help to determine the concentration of mRNA in tissue sections.

Fig. 1

Northern blot analysis to confirm the probe specificity. When the 10 µg of poly (A)⁺ RNA from the adult rat testes was hybridized with the PERF 15 cRNA probe, a band of 0.8 kb was detected (lane 2). No other band was detected in the rat testes. DIG-prelabeled markers as a size control (lane 1).

Fig. 2

Detection of PERF 15 mRNA with sense (A) and antisense (B) DIG-labeled probes in rat seminiferous tubules. Roman numerals represent the stages of each seminiferous tubule. Bars=200 µm (A), 100 µm (B).

Fig. 3

High magnification of seminiferous tubules showing early stages (A–D, stages XIV, II–III, IV, V) and mid-late stages (E–H, stages VIII, X, XII–XIII, XIV). PERF 15 mRNA in tubules was detected by the anti-DIG-AP system (A and E) and by the anti-DIG-HRP system (C and G). Images detected in A and E were separated into five tones from dark to light in B and F. D and H show PAS-hematoxylin stain. Roman numerals represent the stages of each seminiferous tubule. Bar=100 µm.

Fig. 3

High magnification of seminiferous tubules showing early stages (A–D, stages XIV, II–III, IV, V) and mid-late stages (E–H, stages VIII, X, XII–XIII, XIV). PERF 15 mRNA in tubules was detected by the anti-DIG-AP system (A and E) and by the anti-DIG-HRP system (C and G). Images detected in A and E were separated into five tones from dark to light in B and F. D and H show PAS-hematoxylin stain. Roman numerals represent the stages of each seminiferous tubule. Bar=100 µm.

Fig. 4

Quantitative analysis of PERF 15 mRNA detected in the rat seminiferous tubules. The vertical columns numbered with Roman numerals show cell association in cross-sectioned tubules (stages). The developmental progression of a cell is followed horizontally from left to right, and continues from the left of the cycle map one row up. Pl (preleptotene), L (leptotene), Z (zygotene), P (pachytene) and D (diplotene) show the subdivision of the prophase of the first meiotic division. Arabic numerals represent steps of spermatid differentiation. Spermatogonia are not indicated in the cycle map. m2°m, secondary spermatocytes. Original cyclic map from Russell et al. (1990), with permission.

Fig. 5

Quantification of signals. The signal intensity in the cytoplasm of each spermatogenic cell (primary and secondary spermatocytes and spermatids) was measured by LSM 510 software. A histogram of the intensity was obtained from the regions of interest in the cytoplasm, defined by the closed drawing elements and extracted, then converted to tables and transferred to an Excel spreadsheet. The signal intensity was calculated and expressed as the mean pixel intensity (0–255). a: significantly different compared with b, c, d and e (p<0.05). b: significantly different compared with a, d and e (p<0.05). c: significantly different compared with a and e (p<0.05).