Most intriguingly, TPCL was not localized in mitochondria but was in other cellular membranes even though mitochondrial CL was the substrate from which TPCL was synthesized. Midodrine D6 hydrochloride acrosome, a sperm-specific organelle, along with a subset of authentic mitochondrial proteins, including Ant4, Suox, and Spata18. Our data suggest that mitochondria-derived membranes are assembled into the acrosome, challenging the concept that this organelle is strictly derived from the Golgi apparatus and revealing a novel function of mitochondria. Introduction Cardiolipin (CL) has been recognized as mitochondrias own phospholipid. It is intimately associated with proteins of the inner mitochondrial membrane (Beyer and Klingenberg, 1985) and strongly Midodrine D6 hydrochloride supports the clustering of protein complexes to supercomplexes (Zhang et al., 2002; Mileykovskaya and Dowhan, 2014). Lack of CL causes severe mitochondrial dysfunction in yeast (Jiang et al., 2000) and in (Acehan et al., 2011) and leads to early embryonic lethality in mice (Zhang et al., 2011). In humans, CL deficiency is associated with Barth syndrome, a systemic disease involving heart and skeletal muscles (Clarke et al., 2013). CL is rich in unsaturated fatty acids, which are essential for the function of CL in mitochondria (Ren et al., 2014; Lu and Claypool, 2015). This is best exemplified in Barth syndrome where mutations in tafazzin (TAZ) cause a moderate increase in the fatty acid saturation specifically in CL. This leads Midodrine D6 hydrochloride to CL degradation, which in turn has a destabilizing effect on the supercomplexes of oxidative phosphorylation (Xu et al., 2016). Given the importance of unsaturated fatty acids for the function of CL, it was highly unexpected when fully saturated tetrapalmitoyl-CL (TPCL) was first discovered in the testes of rats (Wang et al., 2007). Here, we confirm the presence of TPCL in the testes of humans and mice, suggesting that it is a widespread phenomenon. This raises questions, such as which cell type is TPCL associated with and what is its function? TPCL is strikingly different from unsaturated CL because the latter is one of the most fluid phospholipids whereas the former remains in the gel phase at body temperature (Lewis and McElhaney, Midodrine D6 hydrochloride 2009). Therefore, TPCL is expected to segregate from unsaturated CL, which makes it difficult to conceptualize how the two lipids can coexist in mitochondrial membranes. Results and discussion Testicular germ cells contain a fully saturated CL species Following up on the discovery of TPCL in rat testes (Wang et al., 2007), we analyzed the CL composition of testes from humans and mice, where we confirmed the presence of TPCL (Fig. S1 a). Since fully saturated CL is rather improbable given the established unsaturation of mitochondrial lipids (Horvath and Daum, 2013), we first verified its identity by multistage mass spectrometry (MSn). Our measurements corroborated the structure of a CL molecule with four palmitoyl residues (Fig. S1 b). TPCL was only present in testes and not in other mouse tissues (Fig. 1 a), but in testes it was more abundant than any of the regular CL species (Fig. 1 b). Open in a separate Midodrine D6 hydrochloride window Figure 1. Testicular germ cells contain TPCL. Lipids were extracted from wild-type mouse (C57BL/6) tissues and cells. CL was analyzed by MALDI-TOF MS. (a) Adult mouse tissues. (b) Adult mouse testes. (c) Mouse testes at different ages. (d) Crude testicular cell populations. (e) Different germ cell populations (maturity increases from left to right). (f) Germ cells separated by flow cytometry. Data are mean values SEMs (a: = 3; b: = 8; cCf: = 4). Clec1b Mean values were compared by Students test (a, d, e) and by ANOVA (c). Interestingly, TPCL emerged only after the third week of life (Fig. 1 c), an age in which the first wave of spermatogenesis has advanced to the post-meiotic stage. This implicated germ cell development and raised the question as to which cell type involved in the reproductive process was the source of TPCL. After separating testes into three crude fractions enriched in Leydig, Sertoli, and germ cells (Chang et al., 2011), we found TPCL to be most abundant in the germ cells (Fig. 1 d). Further purification based on tissue adhesion and density revealed that the purest and most mature germ cells contained the highest proportion of TPCL (Fig. 1 e). Finally, we separated germ cells by flow cytometry into spermatogonia, spermatocytes, and spermatids (Fig. S2) and determined that the concentration of TPCL increased along the developmental pathway up to the spermatid stage (Fig. 1 f). Mature sperm, isolated separately from the epididymis, also contained TPCL, albeit in a lower quantity than spermatids, apparently due to its degradation to lyso-TPCL (Fig. 1,.