Cyp11a1基因剔除小鼠睪丸發育與精子生成作用異常

Abstract

Cyp11a1基因主要表現在腎上腺皮質及性腺，參與類固醇荷爾蒙生成的第一關鍵步驟。睪丸中生成雄性荷爾蒙即需要CYP11A1的作用，對於雄性特徵的發育與維持相當重要。由於Cyp11a1基因剔除 (Cyp11a1-/-) 會使小鼠在出生後不久死於腎上腺功能不足。我們利用皮質類固醇的補充及腎上腺移植的方法，使Cyp11a1-/- 小鼠存活至八週，藉以探討Cyp11a1在睪丸發育與精子生成作用的重要性。我們發現Cyp11a1-/- 小鼠雙側睪丸較小且皆無法沉降至陰囊區域，位置呈現右低左高。右側與左側睪丸的平均重量分別比野生型Cyp11a1+/+ 減少了72% 及90%。以精母細胞 (spermatocyte) 的標誌蛋白synaptonemal complex protein 3 (SCP3) 進行免疫螢光染色，我們發現Cyp11a1-/- 小鼠睪丸的細精管中有粗絲期 (pachytene) 或偶絲期 (zygotene) 精母細胞堆積的現象，其出現比例分別為50%、15%。以lectin染劑觀察精細胞 (spermatid)，結果顯示Cyp11a1-/- 小鼠右側睪丸中圓型及長型精細胞的數目大量減少，而左側睪丸中則沒有看到精細胞。Cyp11a1-/- 小鼠細精管中沒有看到發育至後期S14-16的精細胞，說明其精子生成作用可能停滯在S13時期的精細胞。此外，我們也發現這些生殖細胞發育異常的程度與睪丸的大小有關。而Cyp11a1-/- 睪丸中與Sertoli cell相關的WT1及SOX9表現與野生型小鼠相比並沒有差異。TUNEL結果顯示，Cyp11a1-/- 小鼠睪丸中出現凋亡訊號的細精管比例顯著增加，細精管內的凋亡細胞數也變多。其睪丸中cleaved caspase 3、cleaved caspase 7及p53的蛋白質量皆有增加。在Cyp11a1-/- 小鼠的血清中沒有測到睪固酮，然而在其睪丸中卻有少量的睪固酮存在。由上述結果說明Cyp11a1基因對於雄性素的生成相當重要，在睪丸的發育以及精子生成過程中亦扮演著重要的角色。

The Cyp11a1 gene, which is mainly expressed in adrenal cortex and gonads, is involved in the first and rate-limiting step of steroids biosynthesis. In testes, Cyp11a1 is needed for the production of androgens which control the development and maintenance of male characteristics. Cyp11a1 knockout mice (Cyp11a1-/-) die early after birth because of adrenal failure. To investigate the role of Cyp11a1 in testicular development and spermatogenesis, Cyp11a1-/- mice were rescued with steroid injection and subcutaneous adrenal transplantation to survive to 8 weeks of age. We found that the testes of Cyp11a1-/- mice were not descended into the scrotal region, with the left testis located at a higher intra-abdominal position. In the Cyp11a1-/- mice, the weight of testis were reduced by 73% in the right-side and 90% in the left-side relative to wild-type. Immunostaining with synaptonemal complex protein 3 (SCP3) exhibited spermatocytes accumulation arrested at pachytene (50%) or zygotene (15%) stage in a number of tubules of knockout mice. Spermatids were stained with lectin and the results showed that the number of spermatids, including round and elongated spermatids, were markedly reduced in the right testis and no signal was detected in the left testis in the Cyp11a1-/- mice. In addition, the steps 14-16 spermatid in late stage were not found in the Cyp11a1-/- testis, suggesting that a arrest of spermatogenesis at step 13. We found that the extent of defective germ cell development was correlated to the size of testis. The levels of WT1 and Sox9, the Sertoli cell markers, showed no significant difference between Cyp11a1-/- and control testis. TUNEL assay revealed that the percentage of tubules with TUNEL-positive cells was greatly increased in Cyp11a1-/- testis compared to control mice (83% vs 11%). In addition, the number of apoptotic cells in tubules was also increased in Cyp11a1-/- mice. Increased expressions of cleaved caspase 3, cleaved caspase 7 and P53 were found in Cyp11a1-/- testis. In Cyp11a1-/- mice, the level of testosterone was not be detected in the serum; however, low level of testosterone still was detected in the testis. In conclusion, Cyp11a1 gene is essential for androgen production which plays a critical role in testicular development and spermatogenesis.