The present study evaluated the major proteome of ram seminal plasma and the main secretions that contribute to its formation, such as the cauda epididymal and accessory sex gland fluids. The study also investigated sperm membrane protein profiles before and after ejaculation. First, semen was collected from six rams (using artificial vagina) to obtain seminal plasma and ejaculated sperm. Then, rams were vasectomized for collection of accessory sex gland fluid (using artificial vagina). Next, rams were slaughtered and cauda epididymal fluid (CEF), seminal vesicle fluid, bulbourethral gland fluid and cauda epididymal sperm were properly collected. Proteins from reproductive fluids and sperm membranes were analyzed by 2-D SDS-PAGE, tandem mass spectrometry and bioinformatics. There we 386 proteins and 256 isoforms identified in all samples. The most abundant seminal plasma proteins were BSP1, BSP5 and spermadhesins (bodhesin-2 and spermadhesin Z13-like). These proteins were present in similar patterns in maps of accessory sexgland fluid, with very low quantities in the CEF and absent in the bulbourethral gland secretion. Thus, practically all BSPs and spermadhesins come from seminal vesicles. Bulbourethral gland fluid brought bactericidal/permeability-increasing protein-containing Family A member 1 isoforms, superoxide dismutase [Cu-Zn] and betamicroseminoprotein to seminal plasma. CEF was the major provider of clusterin, epididymal-specific lipocalin-5-like isoform, epididymal secretory gluthathione peroxidase, epididymal secretory protein E1 and prostaglandin-H2 D-isomerase to seminal plasma. Albumin came from all reproductive fluids. BSPs and spermadhesins were present in 2-D maps of ejaculated sperm but absent in cauda epididymal sperm. These proteins come from the seminal vesicles and bind to sperm at the moment of ejaculation. Other proteins of ejaculated and epididymal sperm membranes were mostly associated to energy production, cell adhesion and proteolytic activity (ATP synthases, disintegrin, metalloproteinase domain-containing protein 32, carboxypeptidase Q and cytosol aminopeptidase). In conclusion, there is a well-orchestrated sequence of events to form the major seminal plasma proteome, with specific contributions from cauda epididymis, seminal vesicles and bulbourethral glands. The present data contribute to a better understanding of male reproductive biology and how sperm functions are affected by the noncellularmicro environment of semen.

中文翻译：

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绘制公羊生殖液和精子膜的主要蛋白质组：从附睾尾到射精

本研究评估了公羊精浆的主要蛋白质组和有助于其形成的主要分泌物，例如附睾尾液和副性腺液。该研究还调查了射精前后的精子膜蛋白谱。首先，从六只公羊（使用人工阴道）收集精液以获得精浆和射出的精子。然后，公羊被切除输精管以收集附属性腺液（使用人工阴道）。接下来，宰杀公羊并适当收集附睾尾液(CEF)、精囊液、尿道球腺液和附睾尾精子。来自生殖液和精子膜的蛋白质通过 2-D SDS-PAGE、串联质谱和生物信息学进行分析。我们在所有样品中鉴定了 386 种蛋白质和 256 种同种型。最丰富的精浆蛋白是 BSP1、BSP5 和精子粘附素（bodhesin-2 和精子粘附素 Z13 样）。这些蛋白质在附属性腺液图中以相似的模式存在，在 CEF 中含量非常低，在尿道球腺分泌物中不存在。因此，几乎所有的 BSP 和精子粘附素都来自精囊。尿道球腺液将杀菌/渗透性增加的含蛋白质的家族 A 成员 1 亚型、超氧化物歧化酶 [Cu-Zn] 和 β-微精蛋白带到精浆中。CEF 是向精浆提供凝聚素、附睾特异性脂质运载蛋白 5 样亚型、附睾分泌型谷胱甘肽过氧化物酶、附睾分泌蛋白 E1 和前列腺素-H2 D-异构酶的主要提供者。白蛋白来自所有生殖液。BSP 和精子粘附素存在于射出精子的二维图中，但在附睾尾精子中不存在。这些蛋白质来自精囊，在射精时与精子结合。射精和附睾精子膜的其他蛋白质主要与能量产生、细胞粘附和蛋白水解活性有关（ATP 合酶、去整合素、含金属蛋白酶结构域的蛋白 32、羧肽酶 Q 和胞质溶胶氨肽酶）。总之，形成主要精浆蛋白质组的事件序列经过精心安排，其中附睾尾、精囊和尿道球腺有特殊贡献。目前的数据有助于更好地了解男性生殖生物学以及精液的非细胞微环境如何影响精子功能。