Spam1-associated transmission ratio distortion in mice: Elucidating the mechanism

BACKGROUND: While transmission ratio distortion, TRD, (a deviation from Mendelian ratio) is extensive in humans and well-documented in mice, the underlying mechanisms are unknown. Our earlier studies on carriers of spontaneous mutations of mouse Sperm Adhesion Molecule 1 (Spam1) suggested that TRD results from biochemically different sperm, due to a lack of transcript sharing through the intercellular cytoplasmic bridges of spermatids. These bridges usually allow transcript sharing among genetically different spermatids which develop into biochemically and functionally equivalent sperm. OBJECTIVES: The goals of the study were to provide support for the lack of sharing (LOS) hypothesis, using transgene and null carriers of Spam1, and to determine the mechanism of Spam1-associated TRD. METHODS: Carriers of Spam1-Hyal5 BAC transgenes were mated with wild-type female mice and the progeny analyzed for TRD by PCR genotyping. Sperm from transgene and Spam1 null carriers were analyzed using flow cytometry and immunocytochemistry to detect quantities of Spam1 and/or Hyal5. Transgene-bearing sperm with Spam1 overexpression were detected by fluorescence in situ hybridization. In wild-type animals, EM studies of in situ transcript hybridization of testis sections and Northern analysis of biochemically fractionated testicular RNA were performed to localize Spam1 transcript. Finally, AU-rich motifs identified in the 3' UTR of Spam1 RNA were assayed by UV cross-linking to determine their ability to interact with testicular RNA binding proteins. RESULTS: The Tg8 line of transgene carriers had a significant (P < 0.001) TRD, due to reduced fertilizing ability of transgene-bearing sperm. These sperm retained large cytoplasmic droplets engorged with overexpressed Spam1 or Hyal5 protein. Caudal sperm from transgene carriers and caput sperm of null carriers showed a bimodal distribution of Spam1, indicating that the sperm in a male were biochemically different with respect to Spam1 quantities. Spam1 RNA was absent from the bridges, associated exclusively with the ER, and was shown to be anchored to the cytoskeleton. This compartmentalization of the transcript, mediated by cytoskeletal binding, occurs via protein interactions with 3' UTR AU-rich sequences that are likely involved in its stabilization. CONCLUSION: We provide strong support for the LOS hypothesis, and have elucidated the mechanism of Spam1-associated TRD

Epididymosomes: transfer of fertility‑modulating proteins to the sperm surface

Publisher's PDF.A variety of glycosylphosphatidylinositol (GPI)‑linked proteins are acquired on spermatozoa from epididymal luminal fluids (ELF) during sperm maturation. These proteins serve roles in immunoprotection and in key steps of fertilization such as capacitation, acrosomal exocytosis and sperm‑egg interactions. Their acquisition on sperm cells is mediated both by membrane vesicles (epididymosomes, EP) which were first reported to dock on the sperm surface, and by lipid carriers which facilitate the transfer of proteins associated with the membrane‑free fraction of ELF. While the nonvesicular fraction is more efficient, both pathways are dependent on hydrophobic interactions between the GPI‑anchor and the external lipid layer of the sperm surface. More recently proteomic and hypothesis‑driven studies have shown that EP from several mammals carry transmembrane (TM) proteins, including plasma membrane Ca2+‑ATPase 4 (PMCA4). Synthesized in the testis, PMCA4 is an essential protein and the major Ca2+ efflux pump in murine spermatozoa. Delivery of PMCA4 to spermatozoa from bovine and mouse EP during epididymal maturation and in vitro suggests that the docking of EP on the sperm surface precedes fusion, and experimental evidence supports a fusogenic mechanism for TM proteins. Fusion is facilitated by CD9, which generates fusion–competent sites on membranes. On the basis of knowledge of PMCA4’s interacting partners a number of TM and membrane‑associated proteins have been identified or are predicted to be present, in the epididymosomal cargo deliverable to spermatozoa. These Ca2+‑dependent proteins, undetected in proteomic studies, play essential roles in sperm motility and fertility, and their detection highlights the usefulness of the hypothesis‑driven approach.University of Delaware. Department of Biological Sciences

Effectiveness of a walnut-enriched diet on murine sperm: involvement of reduced peroxidative damage

A walnut supplement for a Western-style diet in men was shown to improve sperm motility, vitality, and morphology. To gain further insights into factors underlying this improvement, we administered a parallel walnut-enriched diet to mice [including those with a defect in sperm motility due to deletion of Plasma Membrane Ca2+-ATPase 4 (Pmca4−/−)] to determine if there is a similar improvement that is accompanied by reduced sperm membrane peroxidative damage. Although sperm vitality and acrosome reaction rate were unaffected, the diet led to a significant improvement in motility (P < 0.05) and morphology (P < 0.04) in wild-type sperm and in morphology (P < 0.01) in Pmca4−/−, confirming the diet’s efficacy, which appeared to be more modest in mice than in humans. In both strains of mice, the diet resulted in a significant decrease in sperm lipid peroxidation (oxidative stress) levels, but did not rescue the significantly increased apoptotic levels seen in the testis and epididymis of Pmca4 nulls. Our findings support the effectiveness of walnuts on sperm quality, associated with reduced peroxidative damage; and suggest that oxidative stress is involved in the mechanism(s) underlying male reproductive defects in Pmca4−/−

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 Ca(2+) 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 Ca(2+). 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 Ca(2+) 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

Clusterin Facilitates Exchange of Glycosyl Phosphatidylinositol-Linked SPAM1 Between Reproductive Luminal Fluids and Mouse and Human Sperm Membranes1

Glycosyl phosphatidylinositol (GPI)-linked proteins, which are involved in post-testicular maturation of sperm and have a role in fertilization, are acquired on the sperm surface from both vesicular and membrane-free soluble fractions of epididymal luminal fluid (LF) and uterine LF. Herein, we investigate the mechanism of uptake of these proteins from the soluble fraction of LFs using sperm adhesion molecule 1 (SPAM1) as a model. Ultracentrifugation and native Western blot analysis of the soluble fraction revealed that SPAM1 is present in low-molecular-weight (monomeric) and high-molecular-weight (oligomeric) complexes. The latter are incapable of transferring SPAM1 and may serve to produce monomers. Monomers are stabilized by hydrophobic interactions with clusterin (CLU), a lipid carrier that is abundantly expressed in LFs. We show that CLU is involved in the transfer of SPAM1 monomers, whose delivery was decreased by anti-CLU antibody under normal and apolipoprotein-enhanced conditions. Coimmunoprecipitation revealed an intimate association of CLU with SPAM1. Both plasma and recombinant CLU had a dose-related effect on transfer efficiency: high concentrations reduced and low concentrations enhanced delivery of SPAM1 to human and mouse sperm membranes, reflecting physiological states in the epididymal tract. We propose a lipid exchange model (akin to the lipid-poor model for cholesterol efflux) for the delivery of GPI-linked proteins to sperm membranes via CLU. Our investigation defines specific conditions for membrane-free GPI-linked protein transfer in vitro and could lead to technology for improving fertility or treating sperm pathology by the addition of relevant GPI-linked proteins critical for successful fertilization in humans and domestic animals

Detection of PMCA4a in reproductive luminal fluids and its acquisition on caudal sperm

<p><b>A</b>) Representative Western blot of FLFs collected during pro-estrus and estrus and metestrus and diestrus (40 µg proteins loaded). The ~128 kDa PMCA4a is seen in pro-estrus and estrus and is marginally present at metestrus and diestrus. Caudal epididymal luminal fluid was used as a positive control. The membrane was stripped and re-probed for HSC70 as a loading control. <b>B</b>) Western blots of VLF, ULF, and OLF recovered after superovulation demonstrate the presence of the ~128 kDa PMCA4a. Sperm protein was used as a positive control. The membrane was stripped and re-probed for β-actin as a loading control. <b>C</b>) Quantitation of Western blot data shown in B; the relative expression was determined using VLF as 1. The data represent the mean (±SEM) of a minimum of three independent experiments, and the intensity was quantified by Image J software. ANOVA and <i>t</i>-tests were performed on the mean and <i>P</i> values were calculated. *<i>P</i> = 0.03 indicates a significantly increase amount of PMCA4a in OLF compared to that in VLF. <b>D</b>) A peak shift of fluorescence intensity to the right, indicates increase amounts of PMCA4a in sperm incubated in FLF compared to PBS for 2 h and treated as described in Materials and Methods. </p

Indirect Immunofluorescence of PMCA4a in the murine myometrium of the uterus during the estrous cycle

<p>In addition to the endometrium, the muscles and mesothelium of the myometrium (M*, M, respectively) were positively stained for PMCA4a. Elevated levels of PMCA4a immunoreactivity were detected at the boundaries of the epithelial cells lining the pro-estrus uterine glands (g). The nuclei were visualized by staining with Draq-5 (blue). Negative controls (NC) of diestrus, pro-estrus, estrus, and metestrus phases are respectively shown (d, h, l, and p). The images were captured using confocal microscopy and a 20x (a plan-Apochromatic) objective lens. GE = glandular epithelium; L = lumen; ST= stroma. Bar = 100 µm (same scale for all micrographs).</p