Expression, immunolocalization and processing of fertilins ADAM-1 and ADAM-2 in the boar (sus domesticus) spermatozoa during epididymal maturation

Fertilin alpha (ADAM-1) and beta (ADAM-2) are integral membrane proteins of the ADAM family that form a fertilin complex involved in key steps of the sperm-oocyte membrane interaction. In the present work, we analyzed the presence of ADAM-1 and ADAM-2 mRNAs, the spermatozoa proteins' processing and their sub-cellular localization in epididymal samples from adult boars. ADAM-1 and ADAM-2 mRNAs were highly produced in the testis, but also in the vas efferens and the epididymis. On immunoblots of sperm extracts, ADAM-1 subunit appeared as a main reactive band of ~50-55 kDa corresponding to occurrence of different isoforms throughout the epididymal duct, especially in the corpus region where isoforms ranged from acidic to basic pI. In contrast, ADAM-2 was detected as several bands of ~90 kDa, ~75 kDa, ~50-55 kDa and ~40 kDa. The intensity of high molecular mass bands decreased progressively in the distal corpus where lower bands were also transiently observed, and only the ~40 kDa was observed in the cauda. The presence of bands of different molecular weights likely results from a proteolytic processing occurring mainly in the testis for ADAM-1, and also throughout the caput epididymis for ADAM-2. Immunolocalization showed that fertilin migrates from the acrosomal region to the acrosomal ridge during the sperm transit from the distal corpus to the proximal cauda. This migration is accompanied by an important change in the extractability of a part of ADAM-1 from the sperm membrane. This suggests that the fertilin surface migration may be triggered by the biochemical changes induced by the epididymal post-translational processing of both ADAM1 and ADAM-2. Different patterns of fertilin immunolocalization then define several populations of spermatozoa in the cauda epididymis. Characterization of such fertilin complex maturation patterns is an important step to develop fertility markers based on epididymal maturation of surface membrane proteins in domestic mammals

Biotecnologia reproductiva en porcí: estat actual i reptes de futur

La biotecnologia reproductiva en porcí inclou les diverses tècniques d'anàlisi de la qualitat seminal i les tècniques de reproducció assistida. Els objectius fonamentals són garantir la seguretat biològica, permetre'n la traçabilitat i incrementar (o estabilitzar) el rendiment reproductiu. Entre les tècniques d'anàlisi de la qualitat seminal destaquem les de determinació de qualitat espermàtica (concentració, motilitat, viabilitat, integritat de membranes i del DNA), les de control de l'estat sanitari (PCR-RT per a detecció de virus i bacteris) i les de determinació del poder fecundant i de la resistència osmòtica. Entre les tècniques de reproducció assistida es practiquen la inseminació artificial (cervical, postcervical i intrauterina), la fecundació in vitro, la injecció intracitoplasmàtica d'espermatozoides, la vitrificació embrionària, la transferència embrionària no quirúrgica, la criopreservació espermàtica, el sexatge d'espermatozoides i d'embrions, el clonatge reproductiu i terapèutic i la transgènesi.Reproductive biotechnology in porcine includes several techniques of analysis of the seminal quality and techniques of assisted reproduction. The main goals are guaranteeing the biological security, allowing the traceability and increasing (or stabilizing) the reproductive yield. Among the techniques of analysis of the seminal quality we highlight those of sperm quality (concentration, motility, viability, integrity of membranes and DNA), those of sanitary control (PCR-RT for the detection of virus and bacteria) and those of determination of fertilizing ability and osmotic resistance. Among the assisted reproduction techniques, there is artificial insemination (cervical, postcervical and intrauterine), in vitro fertilization, intracytoplasmic injection of spermatozoa, embryonic vitrification, non surgical embryonic transfer, sperm cryopreservation, spermatozoa and embryos sexing, reproductive and therapeutic cloning, and transgenity

A Review on the Role of Bicarbonate and Proton Transporters during Sperm Capacitation in Mammals

Alkalinization of sperm cytosol is essential for plasma membrane hyperpolarization, hyperactivation of motility, and acrosomal exocytosis during sperm capacitation in mammals. The plasma membrane of sperm cells contains different ion channels implicated in the increase of internal pH (pHi) by favoring either bicarbonate entrance or proton efflux. Bicarbonate transporters belong to the solute carrier families 4 (SLC4) and 26 (SLC26) and are currently grouped into Na+/HCO3− transporters and Cl−/HCO3− exchangers. Na+/HCO3− transporters are reported to be essential for the initial and fast entrance of HCO3− that triggers sperm capacitation, whereas Cl−/HCO3− exchangers are responsible for the sustained HCO3− entrance which orchestrates the sequence of changes associated with sperm capacitation. Proton efflux is required for the fast alkalinization of capacitated sperm cells and the activation of pH-dependent proteins; according to the species, this transport can be mediated by Na+/H+ exchangers (NHE) belonging to the SLC9 family and/or voltage-gated proton channels (HVCN1). Herein, we discuss the involvement of each of these channels in sperm capacitation and the acrosome reaction

Glycocalyx characterisation and glycoprotein expression of Sus domesticus epididymal sperm surface samples

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Blocking NHE Channels Reduces the Ability of In Vitro Capacitated Mammalian Sperm to Respond to Progesterone Stimulus

Few data exist about the presence and physiological role of Na+/H+ exchangers (NHEs) in the plasma membrane of mammalian sperm. In addition, the involvement of these channels in the ability of sperm to undergo capacitation and acrosomal reaction has not been investigated in any mammalian species. In the present study, we addressed whether these channels are implicated in these two sperm events using the pig as a model. We also confirmed the presence of NHE1 channels in the plasma membrane of ejaculated sperm by immunofluorescence and immunoblotting. The function of NHE channels during in vitro capacitation was analyzed by incubating sperm samples in capacitating medium for 300 min in the absence or presence of a specific blocker (DMA; 5-(N,N-dimethyl)-amiloride) at different concentrations (1, 5, and 10 µM); acrosome exocytosis was triggered by adding progesterone after 240 min of incubation. Sperm motility and kinematics, integrity of plasma and acrosome membranes, membrane lipid disorder, intracellular calcium and reactive oxygen species (ROS) levels, and mitochondrial membrane potential (MMP) were evaluated after 0, 60, 120, 180, 240, 250, 270, and 300 min of incubation. NHE1 localized in the connecting and terminal pieces of the flagellum and in the equatorial region of the sperm head and was found to have a molecular weight of 75 kDa. During the first 240 min of incubation, i.e., before the addition of progesterone, blocked and control samples did not differ significantly in any of the parameters analyzed. However, from 250 min of incubation, samples treated with DMA showed significant alterations in total motility and the amplitude of lateral head displacement (ALH), acrosomal integrity, membrane lipid disorder, and MMP. In conclusion, while NHE channels are not involved in the sperm ability to undergo capacitation, they could be essential for triggering acrosome exocytosis and hypermotility after progesterone stimulus

HVCN1 but Not Potassium Channels Are Related to Mammalian Sperm Cryotolerance

Little data exist about the physiological role of ion channels during the freeze–thaw process in mammalian sperm. Herein, we determined the relevance of potassium channels, including SLO1, and of voltage-gated proton channels (HVCN1) during mammalian sperm cryopreservation, using the pig as a model and through the addition of specific blockers (TEA: tetraethyl ammonium chloride, PAX: paxilline or 2-GBI: 2-guanidino benzimidazole) to the cryoprotective media at either 15 °C or 5 °C. Sperm quality of the control and blocked samples was performed at 30- and 240-min post-thaw, by assessing sperm motility and kinematics, plasma and acrosome membrane integrity, membrane lipid disorder, intracellular calcium levels, mitochondrial membrane potential, and intracellular O2−⁻ and H2O2 levels. General blockade of K+ channels by TEA and specific blockade of SLO1 channels by PAX did not result in alterations in sperm quality after thawing as compared to control samples. In contrast, HVCN1-blocking with 2-GBI led to a significant decrease in post-thaw sperm quality as compared to the control, despite intracellular O2−⁻ and H2O2 levels in 2-GBI blocked samples being lower than in the control and in TEA- and PAX-blocked samples. We can thus conclude that HVCN1 channels are related to mammalian sperm cryotolerance and have an essential role during cryopreservation. In contrast, potassium channels do not seem to play such an instrumental role