Ribonucleases endowed with specific toxicity for spermatogenic layers

Abstract

Bovine seminal ribonuclease (BS-RNase) is a dimer in which the subunits are cross-linked by disulfide bonds between Cys31 of one subunit and Cys32 of the other. Dimeric BS-RNase is resistant to ribonuclease inhibitor (RI), a protein endogenous to mammalian cells, and is toxic to a variety of cell types. Monomeric BS-RNase (like its homolog, RNase A) is bound tightly by RI and is not cytotoxic. The three-dimensional structure of the RI·RNase A complex suggests that carboxymethylation of C32S BS-RNase (to give MCM31) or C31S BS-RNase (MCM32) could diminish affinity for RI. We find that MCM31 and MCM32 are not only resistant to RI, but are also aspermatogenic to mice. In contrast to the aspermatogenic activity of dimeric BS-RNase, that of MCM31 and MCM32 is directed only at spermatogenic layers. Intratesticular injection of MCM31 or MCM32 affects neither the diameter of seminiferous tubules nor the weight of testes. Also in contrast to wild-type BS-RNase, MCM31 and MCM32 are not toxic to other cell types. Direct immunofluorescence reveals that MCM31 and MCM32 bind only to spermatogonia and primary spermatocytes. This cell specificity makes MCM31 and MCM32 of potential use in seminoma therapy and contraception.

FIG. 1

Interactions between ribonuclease inhibitor and ribonucleases. (A) Interactions between residues 6, 7 and 31 of porcine RI, and residues 31 and 32 of bovine RNase A in the RI·RNase A complex (26). (B) Residues 31 and 32 of MCAM, MCM31 and MCM32. In human angiogenin, which like RNase A binds tightly to RI (29), the residues analogous to 31 and 32 are both arginine.

FIG. 2

Inhibition of ribonucleolytic activity by RI. Inhibition was assessed by observing the ability of a ribonuclease to degrade rRNA in the absence (A) or presence (B) of RI. Lane 1, no ribonuclease; lane 2, BS-RNase from seminal plasma; lane 3, dimeric BS-RNase from E. coli; lane 4, monomeric C32S BS-RNase; lane 5, monomeric C31S BS-RNase; lane 6, MCM31; and lane 7, MCM32.

FIG. 3

Effect of various forms of BS-RNase on the proliferation in culture of MLC-stimulated human lymphocytes. Proliferation was evaluated by the incorporation of [6-³H]thymidine into cellular DNA. Values are the mean from three cultures and are reported as a percent of the control, which was the mean value from cultures containing no exogenous ribonuclease. Data were recorded 6 days after addition of ribonuclease to the culture.

FIG. 4

Effect of various forms of BS-RNase on the survival of 6-day bovine embryos in culture. Each assay began with 15 embryos except for that of BS-RNase (E. coli), which began with 7 embryos.

FIG. 5

Effect of various forms of BS-RNase on mouse spermatogenesis. Values are an average from 13 (MCM31), 12 (MCM32), 6 (BS-RNase, seminal plasma; PBS) or 4 (BS-RNase, E. coli) injected testes and are reported as a percent of the control, which is from the non-injected testes of the same mice. Data were recorded 10 days after injection. Data for MCAM are from (25).

FIG. 6

Effect of MCM31 and MCM32 on mice testes. Images were obtained 10 days after intratesticular injection of PBS (A). Spermatogenic epithelium, including all spermatogenic cells and spermatozoa, are normal. (B) MCM31 (50 μg); (C) MCM32 (50 μg). Spermatogonia are intact but primary spermatocytes are degenerated. Bar, 20 μm.

FIG. 7

Localization of MCM31 in mouse testis. Images were obtained by direct immunofluorescence 2 hr after injection of MCM31 (80 μg). (A) Visualization with FITC-conjugated normal IgG. (B) Visualization with FITC-conjugated IgG against BS-RNase. Bar, 20 μm.