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A small RNA in testis and brain: implications for male germ cell development

¹ Department of Physiology and Pharmacology, State University of New York, Health Science Center at Brooklyn, Brooklyn, New York 11203, USA
² Department of Neurology, State University of New York, Health Science Center at Brooklyn, Brooklyn, New York 11203, USA
³ Institute of Experimental Pathology/Molecular Neurobiology, University of Münster, D-48149 Münster, Germany
⁴ Department of Biology, Queens College and Graduate Center of CUNY, Flushing, New York 11367, USA

View larger version (116K): [in a new window]   Fig. 1. Distribution of BC1 RNA in murine testis. Signal is represented as white autoradiographic silver grains in dark field photomicrographs. Labeling patterns in seminiferous tubules indicate an annular but patchy distribution of BC1 RNA along the tubular periphery (see low-power photomicrograph A with corresponding bright field B). Central adluminal regions are devoid of significant labeling (asterisk in A). Highest relative BC1 labeling appears concentrated in distinct patches overlying several neighboring cells (C-F; arrowheads in D,F) that are located adjacent to the basement membrane. Such cells often show the irregular nuclear outlines typical of enlarged Aal spermatogonia (Huckins, 1971). A sense-strand control is shown in G. Gold-colored silhouettes apparent in these and other dark field photomicrographs are produced by reflections from elongated spermatids in the adluminal regions of seminiferous tubules. (A,C,E,G) Dark field photomicrographs; (B,D,F) bright field photomicrographs. Bar, 100 µm (A,B); 50 µm (G); 30 µm (C-F).

View larger version (29K): [in a new window]   Fig. 2. Northern hybridization with total RNA from developing testes. (A) 10 µg were loaded per lane. No bands other than the ones shown were detected in this and the following northern hybridization experiments using BC1-specific probe HT005. Equal loading was verified as described in Materials and Methods. (B) Labeling intensities were quantified by phosphorimaging (Molecular Dynamics SF).

View larger version (124K): [in a new window]   Fig. 3. BC1 expression in developing seminiferous tubules. (A,B) At postnatal day 7 (p7), BC1 labeling in developing tubules is robust but heterogeneous. Arrows indicate hotspots of BC1 expression in several tubules. Note that more heavily counterstained cells typically exhibit little or no specific BC1 labeling. (C) At p17, only cells in peripheral-most layers show strong labeling; asterisk indicates the position of cells in adluminal layers (presumably spermatocytes) that exhibit little specific labeling. (D) The adult expression pattern is shown for comparison. Exposure time for D was three times longer than for A-C. (A,C,D) Dark field photomicrographs; (B) bright field photomicrograph. Bar: 40 µm (A,B); 50 µm (C,D).

View larger version (101K): [in a new window]   Fig. 4. Cell-type-specific expression of BC1 RNA in seminiferous tubules. In situ hybridization was performed with testes from homozygous (WV/W) germ-line deficient mutant mice (C,D), and with testes from wild-type (+/+) littermates (A,B). Little specific signal is observed in seminiferous tubules of germ-line-deficient animals. Note that most of the brightness associated with cells in C is not caused by silver grains but by reflections from cellular structures. (A,C) Dark field photomicrographs; (B,D) bright field photomicrographs. Bar, 100 µm. (E) Northern hybridization with 10 µg total RNA isolated from germ-line-deficient adult testes (lane 1) or wild-type adult testes (lane 2). BC1 RNA is not detected in germ-line-deficient testes. Equal loading was verified as described in Materials and Methods. (F) Northern hybridization with RNA isolated from enriched populations of spermatogenic cells. Lane 1, total RNA from entire adult testis (total RNA loaded in this and other lanes: 15µg); lane 2, poly(A)+ RNA from entire testis (5 µg); lane 3, poly(A)- RNA from entire testis (25 µg); lane 4, total RNA from cells in the prophase of meiosis (mainly pachytene); lane 5, total RNA from early spermatids; lane 6, total RNA from the fraction containing cytoplasmic fragments of elongating speriment and residual bodies. For the experiment shown in F, a filter used for a previous publication [(Zakeri et al., 1988) see figure 5, third panel)] was reprobed with BC1-specific probe HT005. Arrows in E and F indicate the position of the BC1 RNA band on the two blots.

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