Epididymal SPAM1 is a marker for sperm maturation in the mouse

Sperm adhesion molecule 1 (SPAM1), is a glycosyl phoshatidylinositol-linked sperm membrane protein that is dually expressed in testis and epididymis. Epididymal SPAM1 is secreted in all three regions of the epididymis in all mammalian species studied, including humans. It shares the same molecular mass and neutral hyaluronidase activity as the testicular and sperm isoforms that are responsible for the penetration of the cumulus during fertilization. Using wild-type (W/T) sperm and those from mice homozygous for either a null (Spam1-/-) or mutant Spam1 allele, which results in decreased mRNA and protein, we demonstrate that sperm binding of epididymal SPAM1 occurs in vitro after exposure to W/T sperm-free epididymal luminal fluid (ELF). Binding or adsorption that occurred after incubation at room temperature or 32 degrees C was detected immunocytochemically and confirmed quantitatively using flow cytometry. The localization of SPAM1 on the plasma membrane of Spam1-null sperm mimicked that seen in the W/T. The remarkable increase in binding on W/T caudal sperm indicates that they are not fully saturated with SPAM1 during storage, and suggests that uptake of epididymal SPAM1 in vivo augments testicular SPAM1. Spam1-null sperm exposed to W/T ELF for 45-60 min during in vitro capacitation to allow epididymal SPAM1 binding showed a highly significant (P < 0.001) increase in cumulus penetration after 6-7 h compared to those incubated in ELF from null males. Similarly, the number of cumulus-free oocytes was also highly significantly greater (P < 0.001) than that for sperm capacitated in W/T SPAM1-antibody-inhibited ELF. Because epididymal SPAM1 uptake significantly increases cumulus penetration, we conclude that it is a marker of sperm maturation.     

Detection of extracellular vesicles in the mouse vaginal fluid: Their delivery of sperm proteins that stimulate capacitation and modulate fertility

Extracellular vesicles (EVs) were isolated by ultracentrifugation of vaginal luminal fluid (VLF) from superovulated mice and identified for the first time using transmission electron microscopy. Characterized by size and biochemical markers (CD9 and HSC70), EVs were shown to be both microvesicular and exosomal and were dubbed as “Vaginosomes” (VGS). Vaginal cross-sections were analyzed to visualize EVs in situ: EVs were present in the lumen and also embedded between squamous epithelial and keratinized cells, consistent with their endogenous origin. Western blots detected Plasma membrane Ca²⁺-ATPase 1 (PMCA1) and tyrosine-phosphorylated proteins in the VGS cargo and also in uterosomes. Flow cytometry revealed that following coincubation of caudal sperm and VLF for 30 min, the frequencies of cells with the highest Sperm adhesion molecule 1 (SPAM1), PMCA1/4, and PMCA1 levels increased 16.4-, 8.2-, and 27-fold, respectively; compared with control coincubated in phosphate buffered saline (PBS). Under identical conditions, sperm tyrosine-phosphorylated proteins were elevated ~3.3-fold, after VLF coincubation. Progesterone-induced acrosome reaction (AR) rates were significantly (p < 0.001) elevated in sperm coincubated with VGS for 10–30 min, compared with PBS. Sperm artificially deposited in the vaginas of superovulated females for these periods also showed significant (p < 0.01) increases in AR rates, compared with PBS. Thus in vitro and in vivo, sperm acquire from the vaginal environment factors that induce capacitation, explaining recent findings for their acrosomal status in the isthmus. Overall, VGS appear to deliver higher levels of proteins involved in preventing premature capacitation and AR than those promoting them. Our findings which have implications for humans open the possibility of new approaches to infertility treatment with exosome therapeutics.     

Compartmentalization of prion isoforms within the reproductive tract of the ram

Cellular prion protein (Prp(C)) is a glycoprotein usually associated with membranes via its glycosylphosphatidylinositol (GPI) anchor. The trans-conformational form of this protein (Prp(SC)) is the suggested agent responsible for transmissible neurodegenerative spongiform encephalopathies. This protein has been shown on sperm and in the reproductive fluids of males. Antibodies directed against the C-terminal sequence near the GPI-anchor site, an N-terminal sequence, and against the whole protein showed that the Prp isoforms were compartmentalized within the reproductive tract of the ram. Immunoblotting with the three antibodies showed that the complete protein and both N- and C-terminally truncated and glycosylated isoforms are present within cauda epididymal fluid and seminal plasma. Moreover, we demonstrate that in these fluids, the Prp(C) isoforms are both in a soluble state as well as associated with small membranous vesicles (epididymosomes). We also report that only one major glycosylated 25 kDa C-terminally truncated Prp(C) isoform is associated with sperm from the testis, cauda epididymis, and semen, and this form is also present in the sperm cytoplasmic droplets that are released during maturation. In sperm, this C-terminal truncated form was found to be associated with membrane lipid rafts present in the mature sperm, suggesting a role for it in the terminal stages of sperm maturation.     

CD9-Positive Microvesicles Mediate the Transfer of Molecules to Bovine Spermatozoa during Epididymal Maturation

Acquisition of fertilization ability by spermatozoa during epididymal transit occurs in part by the transfer of molecules from membranous vesicles called epididymosomes. Epididymosomes are heterogeneous in terms of both size and molecular composition. Exosomes and other related small membranous vesicles (30–120 nm) containing tetraspanin proteins on their surface are found in many biological fluids. In this study, we demonstrate that these vesicles are present in bovine cauda epididymal fluid as a subpopulation of epididymosomes. They contain tetraspanin CD9 in addition to other proteins involved in sperm maturation such as P25b, GliPr1L1, and MIF. In order to study the mechanism of protein transfer to sperm, DilC12-labeled unfractionated epididymosomes or CD9-positive microvesicles were coincubated with epididymal spermatozoa, and their transfer was evaluated by flow cytometry. CD9-positive microvesicles from epididymal fluid specifically transferred molecules to spermatozoa, whereas those prepared from blood were unable to do so. The CD9-positive microvesicles transferred molecules to the same sperm regions (acrosome and midpiece) as epididymosomes, with the same kinetics; however, the molecules were preferentially transferred to live sperm and, in contrast to epididymosomes, Zn²⁺ did not demonstrate potentiated transfer. Tetraspanin CD9 was associated with other proteins on the membrane surface of CD9-positive microvesicles according to coimmunoprecipitation experiments. CD26 cooperated with CD9 in the molecular transfer to sperm since the amount of molecules transferred was significantly reduced in the presence of specific antibodies. In conclusion, CD9-positive microvesicles are present in bovine cauda epididymal fluid and transfer molecules to live maturing sperm in a tissue-specific manner that involves CD9 and CD26.     

Expression and Secretion of Plasma Membrane Ca2+-ATPase 4a (PMCA4a) during Murine Estrus: Association with Oviductal Exosomes and Uptake in Sperm

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 (Ca^2+/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.     

The roles of the epididymis and prostasomes in the attainment of fertilizing capacity by stallion sperm

The epididymis is a long, tightly coiled tube within the lumen of which sperm matures. Sperm maturation involves morphological and biochemical changes in the sperm plasma membrane in response to epididymal secretions and their various proteins. Some of these proteins become outer membrane components while others become integral membrane proteins; transfer of some proteins to the sperm plasma membrane may be mediated by epididymosomes. Nevertheless, the molecular pathways by which spermatozoa acquire fertilizing capacity during their transit through the epididymis remain ambiguous. In a recent study of stallion epididymal sperm, we found that sperm harvested from different parts of the epididymis (caput, corpus and cauda) had a varying, but generally poor, ability to undergo the acrosome reaction in vitro. At ejaculation, however, sperm mix with seminal plasma which contains various components, including the small membranous vesicles known as prostasomes, that may enable the sperm to undergo physiological activation. Seminal plasma components may have a 'washing' effect and help to remove 'de-capacitation' factors that coat the sperm during storage in the cauda epididymis; alternatively seminal plasma and prostasomes may contain factors that more directly promote sperm activation. This article reviews current information on the roles of epididymal and accessory gland fluids on the acquisition of fertilizing capacity by stallion sperm.     

Vesicular transfer of membrane components to bovine epididymal spermatozoa

Epididymosomes (apocrine secreted epididymal vesicles) are assumed to play a crucial role in sperm maturation. Our aim has been to analyze the fusogenic properties of bovine epididymosomes and their involvement in the transfer of membrane components (lipids, proteins, plasma membrane Ca²⁺-ATPase 4 [PMCA4]) into bovine sperm. The fusogenic properties of epididymosomes with spermatozoa were investigated in vitro by using octadecyl rhodamine-B (R18)-labeled epididymosomes. Spermatozoa isolated from the epididymal caput showed a higher fusion rate than those taken from the cauda. The fusion rate was dependent on pH and time. Furthermore, the lipid and protein content in spermatozoa changed during epididymal transit and after in vitro fusion with epididymosomes. Following the in vitro fusion of caput spermatozoa with epididymosomes, the cholesterol/total phospholipid ratio of the sperm plasma membrane decreased. The effect was comparable with the cholesterol/total phospholipid ratio of native cauda spermatozoa. Co-incubation experiments of spermatozoa with biotinylated epididymosomes additionally revealed that proteins were transferred from epididymosomes to sperm. To examine the potential transfer of epididymis-derived PMCA4 to spermatozoa, immunofluorescence analysis and Ca²⁺-ATPase activity assays were performed. In caput spermatozoa, the PMCA4 fluorescence signal was slightly raised and Ca²⁺-ATPase activity increased after in vitro fusion. Thus, our experiments indicate significant changes in the lipid and protein composition of epididymal sperm following interaction with epididymosomes. Moreover, our results substantiate the presumption that PMCA4 is transferred to spermatozoa via epididymosomes.     

附睾小体功能蛋白及sRNA研究进展

精液质量是反映男性生殖健康最基本和最重要的指标。哺乳动物的精子需在附睾中经历一系列复杂的结构与功能的变化才能成熟并具有潜在受精能力。精子成熟是由大量转录因子、激素等信号分子协同调控的复杂生理过程。近年来越来越多的证据表明附睾小体(epididymosomes)中的功能蛋白和sRNAs(small RNAs)可参与调节精子成熟及受精等过程。本文主要综述了附睾小体中功能蛋白及两类主要的sRNAs(tRNAs和miRNAs)的生物学作用,以期为男性不育等疾病的治疗提供一定的理论指导和新的治疗思路。     

SPAM1 (PH-20) protein and mRNA expression in the epididymides of humans and macaques: utilizing laser microdissection/RT-PCR

Background  

The Sperm Adhesion Molecule 1 (SPAM1) is an important sperm surface hyaluronidase with at least three functions in mammalian fertilization. Previously our laboratory reported that in the mouse, in addition to its expression in the testis, Spam1 is synthesized in the epididymis where it is found in membranous vesicles in the principal cells of the epithelium in all three regions. Since SPAM1 is widely conserved among mammals the aim of the study was to determine if its expression pattern in the epididymis is conserved in rodents and primates.     

Evidence that anandamide-signaling regulates human sperm functions required for fertilization

Ejaculated mammalian sperm require several hours exposure to secretions in female reproductive tracts, or incubation in appropriate culture medium in vitro, before acquiring the capacity to fertilize eggs. Arachidonylethanolamide (AEA), also known as anandamide, is a novel lipid-signal molecule that is an endogenous agonist (endocannabinoid) for cannabinoid receptors. We now report that AEA is present in human seminal plasma, mid-cycle oviductal fluid, and follicular fluid analyzed by high-performance liquid chromatography/mass spectrometry. Sperm are sequentially exposed to these reproductive fluids as they move from the vagina to the site of fertilization in the oviduct. Specific binding of the potent cannabinoid agonist [(3)H]CP-55,940 to human sperm was saturable (K(D) 9.71 +/- 1.04 nM), suggesting that they express cannabinoid receptors. R-methanandamide [AM-356], a potent and metabolically stable AEA analog, and (-)delta(9) tetrahydrocannabinol (THC), the major psychoactive constituent of Cannabis, modulated capacitation and fertilizing potential of human sperm in vitro. AM-356 elicited biphasic effects on the incidence of hyperactivated sperm motility (HA) between 1 and 6 hr of incubation: at (2.5 nM) it inhibited HA, while at (0.25 nM) it stimulated HA. Both AM-356 and THC inhibited morphological alterations over acrosomal caps between 2 and 6 hr (IC(50) 5.9 +/- 0.6 pM and 3.5 +/- 1.5 nM, respectively). Sperm fertilizing capacity, measured in the Hemizona Assay, was reduced 50% by (1 nM) AM-356. These findings suggest that AEA-signaling may regulate sperm functions required for fertilization in human reproductive tracts, and imply that smoking of marijuana could impact these processes. This study has potential medical and public policy ramifications because of the incidence of marijuana abuse by adults in our society, previously documented reproductive effects of marijuana, and the ongoing debate about medicinal use of marijuana and cannabinoids.     

ADAM7 is associated with epididymosomes and integrated into sperm plasma membrane

During epididymal transit, mammalian sperm acquire selected proteins secreted by the epididymis. We previously showed that a disintegrin and metalloprotease (ADAM) 7 is expressed specifically in the epididymis and transferred to the sperm surface during epididymal transit. Here, we show that mouse ADAM7 secreted to the epididymal lumen is associated with membranous vesicles known as epididymosomes. Furthermore, we found that ADAM7 can be transferred directly from epididymal vesicles to sperm and that it is an integral plasma membrane protein in sperm. Thus, our study provides new information regarding the unique mode of secretion and interaction of ADAM7 during the epididymis-to-sperm transfer process.     

Sperm storage and viability in Photinus fireflies

In many species females mate with and store sperm from multiple males, and some female insects have evolved multiple compartments for sperm storage. Sperm storage and sperm viability were investigated in two firefly species, Photinus greeni and P. ignitus, which differ in the morphology of the female reproductive tract. Although the primary spermatheca is similar in both species, P. greeni females have an additional, conspicuous outpocketing within the bursa copulatrix whose potential role in sperm storage was investigated in this study. An assay that distinguishes between live and dead sperm was used to examine sperm viability in male seminal vesicles and sperm storage sites within the female reproductive tract. For both Photinus species, sperm from male seminal vesicles showed significantly higher viability compared to sperm from the primary spermatheca of single mated females. In single mated P. greeni females, sperm taken from the channel outpocketing (secondary spermatheca) showed significantly higher viability compared to sperm from the primary spermatheca. This sperm viability difference was not evident in double mated females. There were no significant differences between P. greeni and P. ignitus females in the viability of sperm from the primary spermatheca. These studies contribute to our understanding of post-mating processes that may influence paternity success, and suggest that sexual conflict over control of fertilizations may occur in multiply mated firefly females.     

Sperm stock and mating of males in a parasitoid wasp

Hymenoptera are haplodiploid insects, consequently sex ratio depends on female's sperm management which itself arises from the reproductive capacity of neighbouring males. To study the influence of ageing on male reproductive potential, laboratory experiments were conducted on Dinarmus basalis (Hymenoptera, Pteromalidae) males, a tropical wasp in which sperm counts are known to constrain sex ratio. Two groups of virgin males were compared: 1-day and 30-days old. Parameters recorded were sperm quantity and viability in seminal vesicles, shape of testis, mating ability in both individual and competitive situations and sperm stored by females after male multiple mating. Older males had twice as much sperm as young males, but their reproductive capacities did not differ. They were able to copulate with 20 successive virgin females in a short period. Sperm stored in spermathecae decreased with female mating order. In competition, old and young males had the same access to females. The difference between old and young males was visible at the level of reproductive tract: young males have functional testis and old males have empty non-functional testis. Spermatozoa are kept viable in male seminal vesicles for long periods. In this species, the reproductive potential of males is not altered by ageing. At the population level this may represent an adaptation for maintaining continuous reserves of sperm at the disposal of females.     

Semen quantity and quality correlate with bank vole males’ social status

Laboratory studies reveal that in several rodent species the females prefer dominant males as mating partners. Here we investigate the correlation between bank vole males’ social rank and their sperm quality and quantity. We used agonistic encounters to determine males’ social status. Sperm quality was assessed by its motility, viability, maturity, morphology and sperm tail membrane integrity. Relatively more dominant males were heavier than males of lower social status. The males’ social position affected the testes, seminal vesicles and coagulation gland development. The weights of these reproductive organs were significantly higher in more dominant males than in more subordinate males. Sperm counts and the values of the other parameters describing sperm quality were higher in high-ranking males than in subordinates. Our results suggest that bank vole females benefit from choosing and mating with high-ranking males by obtaining more and better-quality sperm.     

哺乳动物精浆胞外囊泡及各组分的功能

精浆胞外囊泡是一种存在于精浆的膜性囊泡,按分泌器官分为附睾小体和前列腺小体。囊泡可与精子细胞膜发生融合,通过传递内容物或介导信号通路进而调节精子功能。它含有多种活性物质,其中蛋白质组分可影响精子活力以及顶体反应,并有清除损伤精子和促进细胞粘附的作用;脂质组分具有调节靶细胞质膜稳定性的作用;核酸组分主要参与免疫反应、跨代遗传及男性不育;离子则是多种酶的辅助因子,在调节酶活性和精浆微环境中发挥重要作用。不同组分对精子功能的影响不尽相同,本文将对此方面的研究进展进行详尽的综述,以期为该领域相关研究人员提供一定的参考。     

Examining the potential for resource-dependent female reproductive fluid-sperm interactive effects in a livebearing fish

Sexually selected traits can be costly to produce and maintain. The amount of resources available to an individual is therefore expected to influence investment in costly sexual traits. While resource-dependent expression of sexually selected traits has traditionally been examined in males, resource limitation can also influence how sexual selection operates in females. Female reproductive fluids are thought to be costly to produce and may play an important role in shaping the outcome of postcopulatory sexual selection by influencing sperm performance. However, we know surprisingly little about whether and how female reproductive fluids are influenced by resource limitation. Here, we examine if resource restriction influences female reproductive fluid-sperm interactive effects in the pygmy halfbeak (Dermogenys collettei), a small internally fertilizing freshwater fish where females store sperm. After experimentally altering female diets (high vs. restricted diets), we compared how female reproductive fluids influence two key metrics of sperm quality: sperm viability and velocity. While female reproductive fluids enhanced sperm viability and velocity, we found no evidence that female diet influenced the interactive effect between female reproductive fluids and sperm viability or velocity. Our findings build on the growing evidence that female reproductive fluids influence sperm performance and call for further attention to be devoted to understanding how resource quantity and quality influence how female reproductive fluids affect sperm performance.     

Lateral diffusion of membrane-spanning and glycosylphosphatidylinositol-linked proteins: toward establishing rules governing the lateral mobility of membrane proteins.

In the plasma membrane of animal cells, many membrane-spanning proteins exhibit lower lateral mobilities than glycosylphosphatidylinositol (GPI)-linked proteins. To determine if the GPI linkage was a major determinant of the high lateral mobility of these proteins, we measured the lateral diffusion of chimeric membrane proteins composed of normally transmembrane proteins that were converted to GPI-linked proteins, or GPI-linked proteins that were converted to membrane-spanning proteins. These studies indicate that GPI linkage contributes only marginally (approximately twofold) to the higher mobility of several GPI-linked proteins. The major determinant of the high mobility of these proteins resides instead in the extracellular domain. We propose that lack of interaction of the extracellular domain of this protein class with other cell surface components allows diffusion that is constrained only by the diffusion of the membrane anchor. In contrast, cell surface interactions of the ectodomain of membrane-spanning proteins exemplified by the vesicular stomatitis virus G glycoprotein reduces their lateral diffusion coefficients by nearly 10-fold with respect to many GPI-linked proteins.