Re-localization of SNARE proteins in mouse sperm prior to the acrosome reaction

The principal role of SNARE proteins is to arbitrate vesicle fusion to a target membrane. Formation of tripartite SNARE protein complexes between SNARE proteins on opposing membranes is the minimal requirement for membrane fusion. The SNARE protein family is large, consisting of more than 60 members. A member of the SNARE family, syntaxin, is found on the sperm plasma membrane while synaptobrevin, is found on the outer acrosomal membrane. During the sperm acrosome reaction, the outer acrosomal membrane fuses at hundreds of points with the overlying plasma membrane, resulting in release of the acrosomal contents. We hypothesize that syntaxin and synaptobrevin re-localize within the sperm plasma membrane prior to the acrosome reaction to form SNARE complexes and promote membrane fusion at hundreds of specific points. Immunofluorescence was used to localize both syntaxin and synaptobrevin in mouse epididymal sperm before and after capacitation. Sperm were fixed and incubated with antibodies to syntaxin, synaptobrevin and then fluorescent secondary antibodies. Super resolution Structured Illumination Microscopy (SR-SIM) was used to examine samples collected at 0, 10, 30, 60, and 120 min of capacitation time, to obtain 3D images of SNARE localization. Quantification of the images was completed using western blotting and by image analysis, using IMARIS, which interpreted the total syntaxin and synaptobrevin positive volume. Results showed that syntaxin-positive volume and syntaxin content remained the same in capacitated and non-capacitated sperm but the location of syntaxin after capacitation was more restricted to the apical ridge of the plasma membrane overlying the acrosome in more than 90% of the sperm observed. The effect of bicarbonate (HCO3-) and BSA, agents necessary in the medium for capacitation, was also investigated. Bicarbonate (HCO3-), which activates soluble adenylate cyclase, was not necessary for re-localization of syntaxin. On the other hand, BSA, which promotes cholesterol efflux, was required for syntaxin re-localization. In the case of synaptobrevin, the volume of protein remained the same in both capacitated and non-capacitated sperm. Synaptobrevin was found at the apical ridge of the sperm head prior to and following capacitation in approximately 83% of the sperm observed. Our results demonstrate that, unlike syntaxin, synaptobrevin does not shift during the plasma membrane modifications that occur in sperm during capacitation. Syntaxin and synaptobrevin did not co-localize at any time during capacitating and non-capacitating conditions in sperm. Our results will help in identification of pathways that may regulate SNARE localization and function during capacitation, membrane fusion and the acrosome reaction.

DNA Methylation Dynamics in the Female Germline and Maternal-Effect Mutations That Disrupt Genomic Imprinting.

Genomic imprinting is an epigenetic marking process that results in the monoallelic expression of a subset of genes. Many of these 'imprinted' genes in mice and humans are involved in embryonic and extraembryonic growth and development, and some have life-long impacts on metabolism. During mammalian development, the genome undergoes waves of (re)programming of DNA methylation and other epigenetic marks. Disturbances in these events can cause imprinting disorders and compromise development. Multi-locus imprinting disturbance (MLID) is a condition by which imprinting defects touch more than one locus. Although most cases with MLID present with clinical features characteristic of one imprinting disorder. Imprinting defects also occur in 'molar' pregnancies-which are characterized by highly compromised embryonic development-and in other forms of reproductive compromise presenting clinically as infertility or early pregnancy loss. Pathogenic variants in some of the genes encoding proteins of the subcortical maternal complex (SCMC), a multi-protein complex in the mammalian oocyte, are responsible for a rare subgroup of moles, biparental complete hydatidiform mole (BiCHM), and other adverse reproductive outcomes which have been associated with altered imprinting status of the oocyte, embryo and/or placenta. The finding that defects in a cytoplasmic protein complex could have severe impacts on genomic methylation at critical times in gamete or early embryo development has wider implications beyond these relatively rare disorders. It signifies a potential for adverse maternal physiology, nutrition, or assisted reproduction to cause epigenetic defects at imprinted or other genes. Here, we review key milestones in DNA methylation patterning in the female germline and the embryo focusing on humans. We provide an overview of recent findings regarding DNA methylation deficits causing BiCHM, MLID, and early embryonic arrest. We also summarize identified SCMC mutations with regard to early embryonic arrest, BiCHM, and MLID

Sperm release from the oviduct reservoir

Sperm undergo capacitation while traveling through the female reproductive tract, a process including several biochemical and physiological events before fertilization. Also prior to fertilization, sperm bind to the epithelial cells of the oviduct isthmus to form a sperm reservoir. This sperm reservoir regulates sperm capacitation, reduces polyspermy and prolongs fertile lifespan. It has been proposed that oviduct glycans interact with sperm, which leads to the formation of sperm reservoir. Our lab also has previously demonstrated that porcine sperm bind to the oviductal glycans biantennary 6-sialylated Nacetyllactosamine (bi-SiaLN) oligosaccharide and a Lewis X trisaccharide (LeX) to form the sperm reservoir. These glycans are also implicated in the regulation of sperm capacitation by regulating calcium influx and extending sperm life span. My objective was to identify how sperm bound to oviduct glycans are released from the reservoir to fertilize the oocyte. The results of this study indicate that soluble oviduct glycans do not alter sperm capacitation. Progesterone and the related steroid pregnenolone release 40-60% of sperm from oviduct cells and immobilized glycans. Progesterone release requires the ion channel CatSper and protein degradation by proteasomes. Finally, the development of sperm hyperactivation was sufficient to induce sperm release, but complete release also required the function of CatSper and proteasomes. My results are the first to show that hyperactivated motility is sufficient to induce sperm release from the oviduct reservoir and implicate progesterone as an important signal to sperm promoting their release and movement towards the egg

Sperm release from the oviduct reservoir

Sperm undergo capacitation while traveling through the female reproductive tract, a process including several biochemical and physiological events before fertilization. Also prior to fertilization, sperm bind to the epithelial cells of the oviduct isthmus to form a sperm reservoir. This sperm reservoir regulates sperm capacitation, reduces polyspermy and prolongs fertile lifespan. It has been proposed that oviduct glycans interact with sperm, which leads to the formation of sperm reservoir. Our lab also has previously demonstrated that porcine sperm bind to the oviductal glycans biantennary 6-sialylated Nacetyllactosamine (bi-SiaLN) oligosaccharide and a Lewis X trisaccharide (LeX) to form the sperm reservoir. These glycans are also implicated in the regulation of sperm capacitation by regulating calcium influx and extending sperm life span. My objective was to identify how sperm bound to oviduct glycans are released from the reservoir to fertilize the oocyte. The results of this study indicate that soluble oviduct glycans do not alter sperm capacitation. Progesterone and the related steroid pregnenolone release 40-60% of sperm from oviduct cells and immobilized glycans. Progesterone release requires the ion channel CatSper and protein degradation by proteasomes. Finally, the development of sperm hyperactivation was sufficient to induce sperm release, but complete release also required the function of CatSper and proteasomes. My results are the first to show that hyperactivated motility is sufficient to induce sperm release from the oviduct reservoir and implicate progesterone as an important signal to sperm promoting their release and movement towards the egg

Adhesion to oviduct glycans regulates porcine sperm Ca2+ influx and viability.

Before fertilization, sperm bind to epithelial cells of the oviduct isthmus to form a reservoir that regulates sperm viability and capacitation. The sperm reservoir maintains optimum fertility in species, like swine, in which semen deposition and ovulation may not be well synchronized. We demonstrated previously that porcine sperm bind to two oviductal glycan motifs, a biantennary 6-sialylated N-acetyllactosamine (bi-SiaLN) oligosaccharide and 3-O-sulfated Lewis X trisaccharide (suLeX). Here, we assessed the ability of these glycans to regulate sperm Ca2+ influx, capacitation and affect sperm lifespan. After 24 h, the viability of sperm bound to immobilized bi-SiaLN and suLeX was higher (46% and 41% respectively) compared to viability of free-swimming sperm (10-12%). Ca2+ is a central regulator of sperm function so we assessed whether oviduct glycans could affect the Ca2+ influx that occurs during capacitation. Using a fluorescent intracellular Ca2+ probe, we observed that both oviduct glycans suppressed the Ca2+ increase that occurs during capacitation. Thus, specific oviduct glycans can regulate intracellular Ca2+. Because the increase in intracellular Ca2+ was suppressed by oviduct glycans, we examined whether glycans affected capacitation, as determined by protein tyrosine phosphorylation and the ability to undergo a Ca2+ ionophore-induced acrosome reaction. We found no discernable suppression of capacitation in sperm bound to oviduct glycans. We also detected no effect of oviduct glycans on sperm motility during capacitation. In summary, LeX and bi-SiaLN glycan motifs found on oviduct oligosaccharides suppress the Ca2+ influx that occurs during capacitation and extend sperm lifespan but do not affect sperm capacitation or motility