Expression and secretion of plasma membrane Ca2+-ATPase 4a (PMCA4a) during murine estrus: association with oviductal exosomes and uptake in sperm.
Expression and secretion of plasma membrane Ca2+-ATPase 4a (PMCA4a) during murine estrus: association with oviductal exosomes and uptake in sperm.
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DOI：
10.1371/journal.pone.0080181
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Martin-Deleon PA
中科院分区：
文献类型：
--
作者：
Al-Dossary AA;Strehler EE;Martin-Deleon PA
PMCA4是一种膜蛋白，是小鼠精子中主要的Ca2+外排泵，其缺失会导致过度激活的运动性严重丧失和男性不育。我们之前已经证明PMCA4b剪接变体与CASK （Ca2+/ cam依赖性丝氨酸激酶）相互作用，调节精子Ca2+。最近，我们发现PMCA4a异构体除了存在于睾丸中外，还在附睾腔液中分泌并转移到精子中。在这里，我们发现pmca4mrna在阴道、子宫和输卵管的4a和4b变体中都有表达。免疫荧光显示PMCA4a在发情期间表达相似且升高，出现在腺上皮和管腔上皮。Western分析在发情期间收集的阴道（VLF）、子宫（ULF）和输卵管（OLF）的所有组织和腔液（LF）中检测到PMCA4a。黄韧带骨化率是VLF和ULF的9倍和4倍，仅在流星/枯月收集的LF中略有出现。对发情时收集的LF进行超离心分离，发现100%的PMCA4a存在于ULF和OLF的囊泡部分。透射电镜（TEM）显示，黄韧带黄韧带囊泡具有外泌体取向（细胞质侧向内），大小范围为25-100 nm，具有特征性的CD9生物标志物。因此，我们将这些囊泡命名为“输卵管小体”，PMCA4a免疫定位于其上。通过流式细胞术检测，尾端精子在联合LF或外泌体中孵育可导致精子PMCA4a增加3倍，表明体外摄取。我们的结果与增加的需求钙+外排在输卵管一致。他们首次展示了输卵管外泌体的存在，并强调了它们与子宫体和阴道外泌体一起在睾丸后精子获得PMCA4a中的作用，PMCA4a对于过度激活的运动性和生育能力至关重要。
PMCA4, a membrane protein, is the major Ca2+ efflux pump in murine sperm where its deletion leads to a severe loss of hyperactivated motility and to male infertility. We have previously shown that the PMCA4b splice variant interacts with CASK (Ca2+/CaM-dependent serine kinase) in regulating sperm Ca2+. More recently we detected that PMCA4a isoform, in addition to its presence in testis, is secreted in the epididymal luminal fluid and transferred to sperm. Here we show that Pmca4 mRNA is expressed in both the 4a and 4b variants in the vagina, uterus, and oviduct. Immunofluorescence reveals that PMCA4a is similarly expressed and is elevated during estrus, appearing in the glandular and luminal epithelia. Western analysis detected PMCA4a in all tissues and in the luminal fluids (LF) of the vagina (VLF), uterus (ULF), and the oviduct (OLF) collected during estrus. It was ~9- and 4-fold higher in OLF than in VLF and ULF, and only marginally present in LF collected at metestrus/diestrus. Fractionation of the LF collected at estrus, via ultracentrifugation, revealed that 100% of the PMCA4a resides in the vesicular fraction of the ULF and OLF. Transmission electron microscopy (TEM) revealed that OLF vesicles have an exosomal orientation (with the cytoplasmic-side inward), a size range of 25-100 nm, with the characteristic CD9 biomarker. Thus, we dubbed these vesicles “oviductosomes”, to which PMCA4a was immunolocalized. Incubation of caudal sperm in the combined LF or exosomes resulted in up to a ~3-fold increase of sperm PMCA4a, as detected by flow cytometry, indicating in vitro uptake. Our results are consistent with the increased requirement of Ca2+ efflux in the oviduct. They show for the first time the presence of oviductal exosomes and highlight their role, along with uterosomes and vaginal exosomes, in post-testicular sperm acquisition of PMCA4a which is essential for hyperactivated motility and fertility.
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影响因子：
2.5
作者：
Griffiths, Genevieve S.;Galileo, Deni S.;Martin-Deleon, Patricia A.
通讯作者：
Martin-Deleon, Patricia A.
DOI：
10.1073/pnas.0610286104
发表时间：
2007-01-23
影响因子：
11.1
作者：
Qi, Huayu;Moran, Magdalene M.;Clapham, David E.
通讯作者：
Clapham, David E.
影响因子：
3.8
作者：
Griffiths, Genevieve S.;Miller, Kimberly A.;Martin-DeLeon, Patricia A.
通讯作者：
Martin-DeLeon, Patricia A.
DOI：
10.1152/ajpcell.2000.279.5.c1595
发表时间：
2000-11-01
影响因子：
5.5
作者：
Reinhardt, TA;Filoteo, AG;Horst, RL
通讯作者：
Horst, RL
影响因子：
4.8
作者：
Caride, Ariel J.;Filoteo, Adelaida G.;Strehler, Emanuel E.
通讯作者：
Strehler, Emanuel E.