Functional role of inducible nitric oxide synthase in the induction of male germ cell apoptosis, regulation of sperm number, and determination of testes size: evidence from null mutant mice.
Inducible nitric oxide synthase (iNOS) through its product, nitric oxide (NO), may contribute to the induction of germ cell apoptosis. Using adult iNOS-deficient mice, we characterized the reproductive hormonal profile and the testicular phenotype. Although there was no difference in body weight, mean testis weights in mutant mice were 30.77% higher, and testicular sperm count was 65.51% higher than control animals. No significant differences were apparent in plasma LH, FSH, and testosterone levels between these mice. Compared with wild-type mice, histomorphometric analysis showed that the mutant mice had a 39.63% increase in the number of pachytene spermatocytes and 33.79% in round spermatids, with no apparent changes in the number of preleptotene spermatocytes and spermatogonia. The incidence of spontaneous germ cell apoptosis detected by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling assay was lower at stages I-IV and XI-XII of iNOS-mutant mice compared with wild-type animals. The rate of germ cell proliferation estimated by quantitative assessment of the bromodeoxyuridine labeled preleptotene spermatocytes showed no significant change between wild-type and iNOS-deficient mice. When applying testicular warming (43 C for 15 min) to mice, the rate of germ cell apoptosis was elevated predominantly at early (I-IV) and late (XI-XII) stages, and less during stages V-VI, VII-VIII, and IX-X at 2 and 6 h after heat exposure in the wild-type mice. In contrast, the rate of apoptosis in mutant mice was markedly decreased at early and late stages 2 and 6 h after heat exposure. Pachytene spermatocytes and early round spermatids were most susceptible to heat-induced apoptosis in both mutant and control animals. Our studies demonstrate that: 1) deficiency of iNOS results in failure to eliminate a small portion of pachytene spermatocytes and round spermatids by apoptosis, resulting in a remarkable increase in testis weight and sperm output; 2) deficiency of iNOS confers partial resistance to heat-induced germ cell apoptosis. These experiments suggest that iNOS plays a physiological role in regulation of germ cell number and in determining testicular size.
Lue
Yanhe
Y
Division of Endocrinology, Department of Medicine, Harbor-University of California-Los Angeles Medical Center, 1000 West Carson Street, Torrance, CA 90509, USA.
Sinha Hikim
Amiya P
AP
Wang
Christina
C
Leung
Andrew
A
Swerdloff
Ronald S
RS
D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D002470:Cell Survival; D006358:Hot Temperature; D008297:Male; D051379:Mice; D008817:Mice, Mutant Strains; D019001:Nitric Oxide Synthase / Q000235:genetics* / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II; D009929:Organ Size; D010641:Phenotype; D013076:Sperm Count; D013090:Spermatocytes / Q000166:cytology*; D013737:Testis / Q000166:cytology*
D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse