Avila Frank W, Wolfner Mariana F
Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY, USA.
J Insect Physiol. 2017 Aug;101:66-72. doi: 10.1016/j.jinsphys.2017.06.015. Epub 2017 Jul 1.
Sperm storage in the mated female reproductive tract (RT) is required for optimal fertility in numerous species with internal fertilization. In Drosophila melanogaster, sperm storage is dependent on female receipt of seminal fluid proteins (SFPs) during mating. The seminal fluid protein Acp36DE is necessary for the accumulation of sperm into storage. In the female RT, Acp36DE localizes to the anterior mating plug and also to a site in the common oviduct, potentially "corralling" sperm near the entry sites into the storage organs. Genetic studies showed that Acp36DE is also required for a series of conformational changes of the uterus that begin at the onset of mating and are hypothesized to move sperm towards the entry sites of the sperm storage organs. After Acp36DE is transferred to the female RT, the protein is cleaved by the astacin-metalloprotease Semp1. However, the effect of this cleavage on Acp36DE's function in sperm accumulation into storage is unknown. We used mass spectrometry to identify the single cleavage site in Acp36DE. We then mutated this site and tested the effects on sperm storage. Mutations of Acp36DE's cleavage site that slowed or prevented cleavage of the protein slowed the accumulation of sperm into storage, although they did not affect uterine conformational changes in mated females. Moreover, the N-terminal cleavage product of Acp36DE was sufficient to mediate sperm accumulation in storage, and it did so faster than versions of Acp36DE that could not be cleaved or were only cleaved slowly. These results suggest that cleavage of Acp36E may increase the number of bioactive molecules within the female RT, a mechanism similar to that hypothesized for Semp1's other substrate, the seminal fluid protein ovulin.
在许多体内受精的物种中，精子储存在已交配雌性的生殖道（RT）中是实现最佳生育能力所必需的。在黑腹果蝇中，精子储存取决于雌性在交配过程中接受精液蛋白（SFP）。精液蛋白Acp36DE是精子积累到储存部位所必需的。在雌性生殖道中，Acp36DE定位于前交配栓以及共同输卵管中的一个位点，可能在精子进入储存器官的入口部位附近“聚集”精子。遗传学研究表明，Acp36DE对于交配开始时子宫发生的一系列构象变化也是必需的，据推测这些变化会使精子移向精子储存器官的入口部位。Acp36DE转移到雌性生殖道后，该蛋白会被阿斯他汀金属蛋白酶Semp1切割。然而，这种切割对Acp36DE在精子积累到储存部位过程中的功能的影响尚不清楚。我们使用质谱法鉴定了Acp36DE中的单个切割位点。然后我们对该位点进行突变并测试其对精子储存的影响。Acp36DE切割位点的突变减缓或阻止了该蛋白的切割，从而减缓了精子积累到储存部位的速度，尽管它们并不影响已交配雌性的子宫构象变化。此外，Acp36DE的N端切割产物足以介导精子在储存部位的积累，而且其作用速度比无法切割或切割缓慢的Acp36DE版本更快。这些结果表明，Acp36E的切割可能会增加雌性生殖道内生物活性分子的数量，这一机制类似于针对Semp1的另一种底物——精液蛋白卵黄蛋白原所假设的机制。
