Impairment of spermatogenesis in mice lacking a functional aromatase (cyp 19) gene

Abstract

It is well established that spermatogenesis is controlled by gonadotrophins and testosterone. However, a role for estrogens in male reproduction recently was suggested in adult mice deficient in estrogen receptor alpha. These mice became infertile primarily because of an interruption of fluid reabsorption by the efferent ductules of the epididymis, thus leading to a disruption of the seminiferous epithelium [Hess, R. A., Bunick, D., Lee, K. H., Bahr, J., Taylor, J. A., Korach, K. S., and Lubahn, D. B. (1997) Nature (London) 390, 509-512]. Despite the demonstration of the aromatase enzyme, which converts androgens to estrogens, and estrogen receptors within the rodent seminiferous epithelium, the role of aromatase and estrogen in germ cell development is unknown. We have investigated spermatogenesis in mice that lack aromatase because of the targeted disruption of the cyp19 gene (ArKO). Male mice deficient in aromatase were initially fertile but developed progressive infertility, until their ability to sire pups was severely impaired. The mice deficient in aromatase developed disruptions to spermatogenesis between 4.5 months and 1 year, despite no decreases in gonadotrophins or androgens. Spermatogenesis primarily was arrested at early spermiogenic stages, as characterized by an increase in apoptosis and the appearance of multinucleated cells, and there was a significant reduction in round and elongated spermatids, but no changes in Sertoli cells and earlier germ cells. In addition, Leydig cell hyperplasia/hypertrophy was evident, presumably as a consequence of increased circulating luteinizing hormone. Our findings indicate that local expression of aromatase is essential for spermatogenesis and provide evidence for a direct action of estrogen on male germ cell development and thus fertility.

Figure 1

Testicular morphology. (a) All four 1-year-old w/t animals had testes with normal morphology. (b) Major disruptions to spermatogenesis were evident in the testes of 1-year-old ArKO mice, the site of disruption appearing to be early spermiogenesis with symplasts (arrowhead) and degenerating early spermatids visible. (c) Most animals also had some tubules with normal morphology (n), adjacent to tubules with spermiogenic arrest (s). (d) In tubules in which spermiogenic disruption was evident, there also appeared to be impaired acrosomal development. An example is shown in which spermatids with abnormal acrosome development are seen in a stage IV–V tubule. Multiple acrosomal vesicles were noted (arrowheads), and in some cases acrosomes failed to uniformly spread over the spermatid nuclei. (e) All animals showed evidence of what appeared to be Leydig cell hyperplasia/hypertrophy (L). (Scale bars: a–c and e, 20 μm; d, 10 μm.)

Figure 2

Morphology of cauda epididymis in 1-year-old animals. (a) One-year-old w/t cauda epididymis with an abundance of sperm. (b) The lumen of the 1-year-old ArKO mice showing absence of sperm; three of the seven have no elongated spermatids visible in the cauda. (c) The ArKO animals that have undergone spermiogenic arrest show evidence of degenerating germ cells, presumably round spermatids (arrowheads). (Scale bar: 20 μm.)

Figure 3

Germ cells numbers (millions per testis) in ArKO and w/t mice at the two age groups. (a) The numbers of spermatogonia, spermatocytes, round spermatids, and elongated spermatids did not differ in the 4.5-month-old ArKO mice as compared with the w/t mice. (b) No significant decreases were seen in spermatogonia (P = 0.06) and spermatocyte populations (P = 0.08) in 1-year-old ArKO mice; however, there were significantly less round and elongated spermatids present in the epithelium of the ArKO mice (P < 0.05). Data expressed as mean ± SD.

Figure 4

TUNEL assay for apoptosis in the seminiferous tubules of a w/t 1-year-old stage XII–I tubule with no evidence of apoptotic cells. (b) One-year-old ArKO stage XII–I tubule with multiple stained germ cells in the adluminal compartment (arrowheads). (Scale bar: 20 μm.)