探討精子特異性蛋白質SMZ-1及SMZ-2調節線蟲成精作用

Abstract

在有性生殖的動物中，精子產生於高度特異性的雄性生殖系統，因此在雄性生殖系統中，必定有特殊的調控者參與精子製造的過程。利用線蟲進行蛋白質體分析，SMZ-1及SMZ-2兩個帶有PDZ domain且極為相像的蛋白質，被發現大量表現於精子的染色質。儘管在過去帶有PDZ domain的蛋白質已經被知道參與在細胞訊息傳遞的過程中，然而PDZ domain在成精作用中所扮演的角色尚未被發掘。 為了探討SMZ-1及SMZ-2兩者在線蟲成精作用中扮演的角色，利用CRISPR/Cas9技術，獲得了兩隻基因型不一樣且不會表現SMZ-2的線蟲株，接著藉由交配產生smz-1、smz-2雙重基因剔除線蟲株。首先透過計算雌雄同體線蟲後代產生率和孵化率來分析SMZ-1、SMZ-2是否參與在線蟲生殖發育的過程之中，發現僅剔除smz-1或smz-2其中一基因的線蟲株可以正常產生後代，然而在smz-1; smz-2基因雙剔除的線蟲株無法產生可計數的後代。表示SMZ-1和SMZ-2參與線蟲後代生殖發育並且帶有相同的功能。 接著觀察smz-1;smz-2線蟲株的子宮，發現充滿了未受精的卵，代表著在沒有SMZ-1和SMZ-2的情況下線蟲無法正常受精，而無法受精的原因可能源於精子或卵子的缺陷。為了區分無法受精的原因，我們先將不孕的smz-1;smz-2雌雄同體線蟲株與帶有正常smz-1、smz-2基因的雄性線蟲進行交配，此時smz-1;smz-2線蟲株可以正常受孕，暗示著SMZ-1和SMZ-2僅在精子扮演功能。以性腺只製造精子或卵子的線蟲株進行西方墨點法分析SMZ-1、SMZ-2表現，發現SMZ-1、SMZ-2只表現在製造精子的性腺。由以上實驗結果可以知道SMZ-1、SMZ-2的功能在於製造具有功能的正常精子。 為了瞭解SMZ-1、SMZ-2缺失的情況下對於精子形成過程的影響，我們將smz-1;smz-2雄蟲性腺固定染色，發現與野生株相比smz-1;smz-2在性腺中沒有精子形成。接著透過曠時攝影的方式觀察smz-1;smz-2的精原細胞，發現細胞不僅無法進到第一次中期，同時smz-1;smz-2也製造出體積較小的精原細胞。由於細胞進入中期需要紡錘絲形成，再利用固定染色及曠時攝影的方式，發現紡錘絲在smz-1;smz-2精原細胞中無法正常維持結構。透過以上實驗結果，同時缺乏SMZ-1和SMZ-2使得細胞無法開始減數分裂，進而影響精子形成。

In sexually reproducing animals, sperm are generated in highly-specialized male germline. Therefore, male-specific regulators should be participated in functional sperm production. SMZ-1 and SMZ-2, two highly identical PDZ domain proteins, were found abundant in spermatogenic chromatin through proteomic study in C. elegans. While PDZ domain-containing proteins are known for acting as scaffolds for cell signal transduction, little is known about the roles of PDZ domain-containing proteins in sperm generation. Since smz-2 deletion strain was not available, we first used CRISPR/Cas9 techniques to generate two smz-2 deletion strains. We then crossed smz-2 deletion worms with smz-1 deletion worms to generate double mutants. To test the roles of SMZ-1 and SMZ-2 in fertility, we compared total brood size and hatch rate of single and double mutant hermaphrodites. We found that worms bearing either smz-1 or smz-2 single deletion can normally generate offspring. However smz-1; smz-2 hermaphrodites do not have any countable progeny. The results show that SMZ-1 and SMZ-2 are functionally redundant in fertility. Through examination of smz-1; smz-2 hermaphrodite uterus revealed the uterus filled with unfertilized oocytes. This suggests fertilization defects happened to smz-1; smz-2 hermaphrodites, which can result from defects in sperm or oocyte function. To distinguish where the defects take place, we examined SMZ-1 and SMZ-2 protein expression in worms generating either sperm or oocytes. Immunoblotting analyses showed that SMZ-1 and SMZ-2 are highly expressed in sperm but not in oocytes or somatic cells. In agreement to this, the infertility in smz-1; smz-2 double mutant hermaphrodites can be rescued by mating with wild type males. These results indicate SMZ-1 and SMZ-2 only function in sperm. In order to define the origin defects of smz-1; smz-2 in sperm generation, we examined fixed smz-1; smz-2 male gonad. We found smz-1; smz-2 male germline failed to generate mature sperm comparing with wild type, suggesting there are meiotic division defects. Time-lapse recording of smz-1; smz-2 spermatocytes showed that chromosomes do not align and the cells are failed to progress into metaphase to enter meiotic division. Additionally, smz-1; smz-2 male tends to generate smaller spermatocytes than wild type male does. Since spindle is a key component for cell to proceed into metaphase, we examined the formation of spindles through staining and time-lapse recording. The results show that smz-1; smz-2 male failed to maintain spindle morphology. In conclusion, we proposed SMZ-1/2 are important to spermatogenesis, absence of the two proteins can result in the failure of generating sperm.