Cholesterol is an essential component of the mammalian plasma membrane because it promotes membrane stability without comprising membrane fluidity. Given this important cellular role, cholesterol ...levels are tightly controlled at multiple levels. It has been clearly shown that cholesterol redistribution and depletion from the sperm membrane is a key part of the spermatozoon's preparation for fertilization. Some factors that regulate these events are described (e.g., bicarbonate, calcium) but the mechanisms underlying cholesterol export are poorly understood. How does a hydrophobic cholesterol molecule inserted in the sperm plasma membrane enter the energetically unfavorable aqueous surroundings? This review will provide an overview of knowledge in this area and highlight our gaps in understanding. The overall aim is to better understand cholesterol redistribution in the sperm plasma membrane, its relation to the possible activation of a cholesterol transporter and the role of cholesterol acceptors. Armed with such knowledge, sperm handling techniques can be adapted to better prepare spermatozoa for in vitro and in vivo fertilization.
Spermatozoa interact with their immediate environment and this contact remodels the sperm surface in preparation for fertilisation. These fundamental membrane changes will be critically covered in ...this review with special emphasis on the very specific surface destabilisation event, capacitation. This process involves very subtle and intricate modifications of the sperm membrane including removal of suppression (decapacitation) factors and changes in the lateral organisation of the proteins and lipids of the sperm surface. Processing of sperm for assisted reproduction (storage, sex-sorting, etc.) subjects spermatozoa to numerous stressors, and it is possible that this processing overrides such delicate processes resulting in sperm instability and cell damage. To improve sperm quality, novel mechanisms must be used to stabilise the sperm surface during handling. In this review, different types of membrane stress are considered, as well as novel surface manipulation methods to improve sperm stability.
Worldwide over 5 million children have been conceived using assisted reproductive technology, and research has concentrated on increasing the likelihood of ongoing pregnancy. However, studies using ...animal models have indicated undesirable effects of in vitro embryo culture on offspring development and health. In vivo, the oviduct hosts a period in which the early embryo undergoes complete reprogramming of its (epi)genome in preparation for the reacquisition of (epi)genetic marks. We designed an oviduct-on-a-chip platform to better investigate the mechanisms related to (epi)genetic reprogramming and the degree to which they differ between in vitro and in vivo embryos. The device supports more physiological (in vivo-like) zygote genetic reprogramming than conventional IVF. This approach will be instrumental in identifying and investigating factors critical to fertilization and pre-implantation development, which could improve the quality and (epi)genetic integrity of IVF zygotes with likely relevance for early embryonic and later fetal development.
The sperm cell has a unique, polarized, and segregated surface that is modified extensively by the changing environments in both the male and the female reproductive tracts. The sperm cannot refresh ...its surface, as protein translation and membrane recycling by intracellular vesicular transport have ceased upon its maturation. So, how is the sperm surface modified in the reproductive tracts and how do these processes affect fertilization? This review traces these modifications as boar sperm travels from their liberation from the Sertoli cell into the lumen of seminiferous tubules of the testis to the site of fertilization in the ampulla of the oviduct in the sow, via an artificial insemination route. The effect of sperm dilution for artificial insemination, as well as more extensive sperm processing for in vitro fertilization, cryopreservation, or sex sorting, are also discussed with respect to how these procedures affect sperm surface organization and fertilization capacity.
In contrast to various other mammalian species, conventional in vitro fertilization (IVF) with horse gametes is not reliably successful. In particular, stallion spermatozoa fails to penetrate the ...zona pellucida, most likely due to incomplete activation of stallion spermatozoa (capacitation) under in vitro conditions. In other mammalian species, specific capacitation triggers have been described; unfortunately, none of these is able to induce full capacitation in stallion spermatozoa. Nevertheless, knowledge of capacitation pathways and their molecular triggers might improve our understanding of capacitation-related events observed in stallion sperm. When sperm cells are exposed to appropriate capacitation triggers, several molecular and biochemical changes should be induced in the sperm plasma membrane and cytoplasm. At the level of the sperm plasma membrane, (1) an increase in membrane fluidity, (2) cholesterol depletion and (3) lipid raft aggregation should occur consecutively; the cytoplasmic changes consist of protein tyrosine phosphorylation and elevated pH, cAMP and Ca2+ concentrations. These capacitation-related events enable the switch from progressive to hyperactivated motility of the sperm cells, and the induction of the acrosome reaction. These final capacitation triggers are indispensable for sperm cells to migrate through the viscous oviductal environment, penetrate the cumulus cells and zona pellucida and, finally, fuse with the oolemma. This review will focus on molecular aspects of sperm capacitation and known triggers in various mammalian species. Similarities and differences with the horse will be highlighted to improve our understanding of equine sperm capacitation/fertilizing events.
Background: Mammalian sperms are activated in the oviduct. This process, which involves extensive sperm surface remodelling, is required for fertilization and can be mimicked under in vitro ...fertilization conditions (IVF). Methodology/Principal Findings: Here we demonstrate that such treatments caused stable docking and priming of the acrosome membrane to the apical sperm head surface without the emergence of exocytotic membrane fusion. The interacting membranes could be isolated as bilamellar membrane structures after cell disruption. These membrane structures as well as whole capacitated sperm contained stable ternary trans-SNARE complexes that were composed of VAMP 3 and syntaxin 1B from the plasma membrane and SNAP 23 from the acrosomal membrane. This trans-SNARE complex was not observed in control sperm. Conclusions/Significance: We propose that this capacitation driven membrane docking and stability thereof is a preparative step prior to the multipoint membrane fusions characteristic for the acrosome reaction induced by sperm-zona binding. Thus, sperm can be considered a valuable model for studying exocytosis.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
SignificanceHatching from the zona pellucida is a prerequisite for embryo implantation and is less likely to occur in vitro for reasons unknown. Extracellular vesicles (EVs) are secreted by the ...embryo into the culture medium. Yet the role that embryonic EVs and their cargo microRNAs (miRNAs) play in blastocyst hatching has not been elucidated, partially due to the difficulties of isolating them from low amounts of culture medium. Here, we optimized EV-miRNA isolation from medium conditioned by individually cultured bovine embryos and subsequently showed that miR-378a-3p, which was up-regulated in EVs secreted by blastocysts, plays a crucial role in promoting blastocyst hatching. This demonstrates the regulatory effect of miR-378-3p on hatching, which is an established embryo quality parameter linked with implantation.
Motile cilia are molecular machines used by a myriad of eukaryotic cells to swim through fluid environments. However, available molecular structures represent only a handful of cell types, limiting ...our understanding of how cilia are modified to support motility in diverse media. Here, we use cryo‐focused ion beam milling‐enabled cryo‐electron tomography to image sperm flagella from three mammalian species. We resolve in‐cell structures of centrioles, axonemal doublets, central pair apparatus, and endpiece singlets, revealing novel protofilament‐bridging microtubule inner proteins throughout the flagellum. We present native structures of the flagellar base, which is crucial for shaping the flagellar beat. We show that outer dense fibers are directly coupled to microtubule doublets in the principal piece but not in the midpiece. Thus, mammalian sperm flagella are ornamented across scales, from protofilament‐bracing structures reinforcing microtubules at the nano‐scale to accessory structures that impose micron‐scale asymmetries on the entire assembly. Our structures provide vital foundations for linking molecular structure to ciliary motility and evolution.
SYNOPSIS
Diverse organisms and cell types rely on evolutionarily ancient and structurally conserved motile cilia to swim through a broad range of fluid environments. Here, in situ cryo‐electron tomography uncovers structural specialisations of sperm flagella in several mammalian species.
Mammalian sperm flagella are reinforced by micron‐scale accessory structures and nano‐scale microtubule inner proteins.
The connecting piece at the base of the flagellum forms an intricate, asymmetric chamber around the sperm centrioles.
The atypical distal centriole is composed of doublet microtubules splayed out around two singlet microtubules.
Outer dense fibers are directly coupled to axonemal doublet microtubules in the principal piece, but not in the midpiece.
In situ cryo‐electron tomography uncovers structural specialisations of sperm flagella in several mammalian species.
Cysteine-rich secretory proteins (CRISPs) are a subgroup of the CRISP, antigen 5 and PR-1 (CAP) superfamily that is characterized by the presence of a conserved CAP domain. Two conserved histidines ...in the CAP domain are proposed to function as a Zn
-binding site with unknown function. Human CRISP1 is, however, one of the few family members that lack one of these characteristic histidine residues. The Zn
-dependent oligomerization properties of human CRISP1 were investigated using a maltose-binding protein (MBP)-tagging approach in combination with low expression levels in XL-1 Blue bacteria. Moderate yields of soluble recombinant MBP-tagged human CRISP1 (MBP-CRISP1) and the MBP-tagged CAP domain of CRISP1 (MBP-CRISP1
) were obtained. Zn
specifically induced oligomerization of both MBP-CRISP1 and MBP-CRISP1
in vitro. The conserved His142 in the CAP domain was essential for this Zn
dependent oligomerization process, confirming a role of the CAP metal-binding site in the interaction with Zn
. Furthermore, MBP-CRISP1 and MBP-CRISP1
oligomers dissociated into monomers upon Zn
removal by EDTA. Condensation of proteins is characteristic for maturing sperm in the epididymis and this process was previously found to be Zn
-dependent. The Zn
-induced oligomerization of human recombinant CRISP1 may shed novel insights into the formation of functional protein complexes involved in mammalian fertilization.
Metabolic rich and poor conditions are both characterized by elevated free fatty acid levels and have been associated with impaired female fertility. In particular, saturated free fatty acids have a ...dose-dependent negative impact on oocyte developmental competence, while monounsaturated free fatty acids appear less harmful. Cumulus cells seem to protect the oocyte against free fatty acids, and the aim of this study was to determine the mechanism behind this protection In particular, the role of the enzyme stearoyl-CoA desaturase (SCD) that converts saturated into monounsaturated fatty acids was investigated. SCD gene and protein were abundantly expressed in cumulus cells, but expression was low in oocytes. The level of SCD protein expression in cumulus cells did not change when COCs were exposed to saturated stearic acid during maturation. SCD inhibition in the presence of stearic acid significantly reduced the developmental competence of oocytes and increased the incidence of apoptosis in cumulus cells. The esterified oleic/stearic acid ratio of the neutral lipid fraction in cumulus cells decreased in the presence of SCD inhibitors when COCs were exposed to saturated free fatty acids during maturation, indicating the SCD-specific conversion of saturated fatty acids under noninhibiting conditions. The observation that cumulus cells can desaturate the potentially toxic stearic acid into oleic acid via SCD activity provides a mechanistic insight into how the cumulus cells protect the oocyte against toxicity by saturated fatty acid. Summary Sentence Stearoyl-CoA desaturase in bovine cumulus cells converts saturated into monounsaturated fatty acid and protects the oocyte against fatty acid-induced lipotoxicity.
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