The release of late spermatids from the seminiferous epithelium requires the internalization of intercellular junctions by Sertoli cell specific structures called “tubulobulbar complexes” (TBCs). These large, endocytic devices likely evolved from classic clathrin-mediated-endocytosis (CME) machinery, but have several important morphological differences to CME vesicles. Most notable among these differences is that extensive endoplasmic reticulum (ER) membrane contact sites (MCSs) occur with TBCs and not with clathrin-coated pits. One of the well-established functions of ER MCSs is lipid exchange. Previously, we have established that the ORP9 lipid exchange protein is localized to the TBC-ER MCS; however, the function of ORP9 and lipid exchange at the sites is not known. Here we use an in vivo knockdown approach to probe function. The testes of Sprague–Dawley rats were injected with ORP9 targeted siRNA or non-targeted reagents, and the tissues examined by bright field, super-resolution stimulated emission depletion, and electron microscopy. The knockdown of ORP9 was achieved and maintained with daily injections of siRNA for 2-3 day intervals. Compared to controls, sections from ORP9 siRNA-injected testes had longer TBC tubes and fewer fused TBC bulbs. Late spermatids were also abnormally retained in the epithelium of knockdown tissue. These results suggest that ORP9 is necessary for normal TBC bulb vesiculation and fusion, most likely by changing the plasma membrane lipid profile of the TBC. These data also further support the conclusion that TBCs are part of the normal mechanism of sperm release.

中文翻译：

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ORP9 敲低延迟了睾丸中与连接处相关的内吞结构的成熟，并导致精子释放受损†。

从生精上皮释放晚期精子细胞需要通过称为“球管复合物”（TBC）的支持细胞特异性结构将细胞间连接内化。这些大型的内吞装置可能是从经典的网格蛋白介导的内吞 (CME) 机制演变而来的，但与 CME 囊泡有几个重要的形态差异。这些差异中最值得注意的是广泛的内质网 (ER) 膜接触位点 (MCS) 出现在 TBC 中，而不是出现在网格蛋白涂层的凹坑中。ER MCS 的公认功能之一是脂质交换。以前，我们已经确定 ORP9 脂质交换蛋白定位于 TBC-ER MCS；然而，ORP9 和脂质交换位点的功能尚不清楚。在这里，我们使用体内组合式方法来探测功能。向 Sprague-Dawley 大鼠的睾丸注射 ORP9 靶向 siRNA 或非靶向试剂，并通过明场、超分辨率受激发射损耗和电子显微镜检查组织。通过每天注射 siRNA 实现并维持 ORP9 的敲低，间隔为 2-3 天。与对照相比，ORP9 siRNA 注射的睾丸切片具有更长的 TBC 管和更少的融合 TBC 球。晚期精子细胞也异常地保留在击倒组织的上皮中。这些结果表明 ORP9 是正常 TBC 球泡囊泡形成和融合所必需的，最有可能是通过改变 TBC 的质膜脂质谱。这些数据还进一步支持了 TBC 是精子释放正常机制的一部分的结论。