Two distinct forms of the 64,000 Mr protein of the cleavage stimulation factor are expressed in mouse male germ cells

Abstract

Polyadenylation in male germ cells differs from that in somatic cells. Many germ cell mRNAs do not contain the canonical AAUAAA in their 3' ends but are efficiently polyadenylated. To determine whether the 64,000 Mr protein of the cleavage stimulation factor (CstF-64) is altered in male germ cells, we examined its expression in mouse testis. In addition to the 64,000 Mr form, we found a related approximately 70,000 Mr protein that is abundant in testis, at low levels in brain, and undetectable in all other tissues examined. Expression of the approximately 70,000 Mr CstF-64 was limited to meiotic spermatocytes and postmeiotic spermatids in testis. In contrast, the 64,000 Mr form was absent from spermatocytes, suggesting that the testis-specific CstF-64 might control expression of meiosis-specific genes. To determine why the 64,000 Mr CstF-64 is not expressed in spermatocytes, we mapped its chromosomal location to the X chromosome in both mouse and human. CstF-64 may, therefore, be absent in spermatocytes because the X chromosome is inactivated during male meiosis. By extension, the testis-specific CstF-64 may be expressed from an autosomal homolog of the X chromosomal gene.

Figure 1

Protein immunoblot detection of CstF-64 in mouse tissues. (A and B) HeLa nuclear extract (lane 1), nuclei from indicated mouse tissues (lanes 2–6 and 8), or whole testicular tubules (lane 7) were examined by protein immunoblotting. Testis tubules were used rather than nuclei because of the difficulty in obtaining germ cell nuclei (66). Immunoblots were incubated with 3A7 (A) or 6A9 (B) monoclonal antibodies. (C) A single immunoblotted membrane was split along one lane (5a/5b), was incubated with 3A7 (Left) or 6A9 (Right), and was reassembled. Lanes contain HeLa nuclear extract (lanes 1 and 6), whole testicular tubules (lanes 2, 5a/5b, and 7), liver (lanes 3 and 8), or thymus (lanes 4 and 9) nuclei. Arrows indicate approximate sizes of CstF-64 proteins.

Figure 2

Immunohistochemical localization of CstF-64 in mouse testis sections. Sections of mouse testis were incubated with 3A7 (A), 6A9 (B), a mixture of 3A7 and 6A9 (C), or an irrelevant control antibody, 2A6 (D). Roman numerals refer to the stage of the seminiferous epithelium. lc, Leydig cell; sc, Sertoli cell; sg, spermatogonia; spc, spermatocyte; rs, round spermatid; es, elongating spermatid. (×100.) (E) Summary of CstF-64 protein expression in mouse germ cells (see also Fig. 5). 3A7 (somatic form) reactivity is in red; 6A9 (testis-specific) is in blue. The diagram is adapted from Russell et al. (38).

Figure 3

FISH analysis of human metaphase chromosomes. Metaphase spreads from peripheral blood were prepared as described in the text. Hybridization was with dJ347M6 containing the human CstF-64 gene. (A) Representative metaphase spread from a female. (B) Metaphase spread from a male. Yellow arrows indicate X chromosomal signals from probe hybridization. The blue arrow indicates signal in an intact interphase cell.

Figure 4

Cstf64 maps in the central region of the mouse X chromosome. (Right) Columns represent the chromosome identified in backcross progeny that inherited from the (C57BL/6J × M. spretus) F1 parent. Shaded boxes represent a C57BL/6J, and white boxes represent a M. spretus allele. The number of offspring inheriting each type of chromosome is listed at the bottom. (Left) Partial X chromosome linkage map showing Cstf64 in relation to linked genes. Recombination distances between loci are shown to the left, and positions of loci in human chromosomes are shown to the right. Human loci cited can be obtained from Genome Data Base, a computerized database maintained by The William H Welch Medical Library of The Johns Hopkins University (Baltimore).

Figure 5

Representation of essential events in mouse spermatogenesis with emphasis on sex chromosome transcription and CstF-64 expression. Stage and times (in days) of spermatogenesis are shown at top. Approximate durations of molecular events (Middle) and CstF-64 protein expression (Bottom) are expressed as bars.