Regulation of hamster sperm hyperactivation by extracellular Na+

Abstract Mammalian sperm motility has to be hyperactivated to be fertilization-competent. Hyperactivation is regulated by extracellular environment. Osmolality of mammalian semen is higher than that in female reproductive tract; however, the effect of them on hyperactivation has not been investigated. So we investigated the effect of osmotic environment on hyperactivation using hamster spermatozoa at first. Increase in the osmolality of the media (∼370 mOsm) by increasing the concentration of NaCl (∼150 mmol/L) caused the delay of the expression of hyperactivation. When NaCl concentration varied in the same range (75–150 mmol/L) whereas the osmolality was fixed at 370 mOsm by adding mannitol, the delay of hyperactivation occurred dependent on NaCl concentration. Increase in NaCl concentration also caused suppression of curvilinear velocity, bend angle, and sliding velocity of the flagellum at the onset of incubation, suggesting that NaCl concentration affect both activation and hyperactivation in hamster spermatozoa. Hamster sperm intracellular Ca2+ concentration decreased as extracellular NaCl concentration increased, whereas membrane potential and intracellular pH were unaffected by extracellular NaCl concentration. SN-6 and SEA0400, inhibitors of Na+-Ca2+ exchanger (NCX), increased intracellular Ca2+ and accelerated hyperactivation in the presence of 150 mmol/L NaCl. Tyrosine phosphorylation on fibrous sheath proteins was unaffected by extracellular NaCl concentration. These results suggest that extracellular Na+ suppresses hamster sperm hyperactivation by reducing intracellular Ca2+ via an action of NCX in a tyrosine phosphorylation-independent manner. It seems that the removal of suppression by extracellular Na+ leads to the expression of hyperactivated motility.

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Integration of Sperm Motility and Chemotaxis Screening with a Microchannel-Based Device

Clinical Chemistry ◽

10.1373/clinchem.2010.146902 ◽

2010 ◽

Vol 56 (8) ◽

pp. 1270-1278 ◽

Cited By ~ 62

Author(s):

Lan Xie ◽

Rui Ma ◽

Chao Han ◽

Kai Su ◽

Qiufang Zhang ◽

...

Keyword(s):

Sperm Motility ◽

Reproductive Tract ◽

Cumulus Cells ◽

Female Reproductive Tract ◽

Straight Channel ◽

Vitro Fertilization ◽

Mammalian Sperm ◽

Reaction Capability ◽

Mammalian Female

BACKGROUND Sperm screening is an essential step in in vitro fertilization (IVF) procedures. The swim-up method, an assay for sperm motility, is used clinically to select the ideal sperm for subsequent manipulation. However, additional parameters, including acrosome reaction capability, chemotaxis, and thermotaxis, are also important indicators of mammalian sperm health. To monitor both sperm motility and chemotaxis simultaneously during sperm screening, we designed and constructed a microdevice comprising a straight channel connected with a bibranch channel that mimics the mammalian female reproductive tract. METHODS The width and length of the straight channel were optimized to select the motile sperms. We selectively cultured cumulus cells in the bibranch channel to generate a chemoattractant-forming chemical gradient. Sperm chemotaxis was represented by the ratio of the sperm swimming toward different branches. RESULTS The percentage of motile sperms improved from 58.5% (3.8%) to 82.6% (2.9%) by a straight channel 7 mm in length and 1 mm in width. About 10% of sperms were found to be chemotactically responsive in our experiment, which is consistent with previous studies. CONCLUSIONS For the first time, we achieved the combined evaluation of both sperm motility and chemotaxis. The motile and chemotactically responsive sperms can easily be enriched on a lab-on-a-chip device to improve IVF outcome.

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Inhibition of mouse fertilization in vivo by intra-oviductal injection of an anti-equatorin monoclonal antibody

Reproduction ◽

10.1530/rep.0.1220649 ◽

2001 ◽

pp. 649-655 ◽

Cited By ~ 19

Author(s):

K Yoshinaga ◽

DK Saxena ◽

T Oh-oka ◽

I Tanii ◽

K Toshimori

Keyword(s):

Monoclonal Antibody ◽

Reproductive Tract ◽

Female Reproductive Tract ◽

Pathological Changes ◽

Histological Studies ◽

Control Side ◽

Mammalian Sperm ◽

The Right ◽

And Control

The monoclonal antibody mMN9 recognizes an antigenic molecule, equatorin, which is localized at the equatorial segment of the mammalian sperm acrosome. Our previous results using an IVF system indicated that mMN9 blocked sperm-oocyte fusion. Antibody-containing and control solutions were injected directly into the right and left oviductal ampullae, respectively, of anaesthetized female mice to assess the effect of mMN9 on fertilization in vivo. After hCG treatment, the females were mated, and their oviductal eggs and implanted embryos were examined. mMN9 was retained in the oviductal lumen at 20 h after injection. The rates of fertilization and concomitant pregnancy were significantly lower than in the control side (P < 0.05). In addition, histological studies showed no evidence of pathological changes in the female reproductive tract after the injections. These results indicate that mMN9 inhibits mouse fertilization significantly under in vivo conditions and that this injection method should be useful for studying the effects of antibodies and agents on fertilization in vivo.

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Role of spermine in mammalian sperm capacitation and acrosome reaction

Biochemical Journal ◽

10.1042/bj2780025 ◽

1991 ◽

Vol 278 (1) ◽

pp. 25-28 ◽

Cited By ~ 21

Author(s):

S Rubinstein ◽

H Breitbart

Keyword(s):

Binding Sites ◽

Acrosome Reaction ◽

Reproductive Tract ◽

Female Reproductive Tract ◽

Sperm Capacitation ◽

Amino Groups ◽

Binding Properties ◽

Mammalian Sperm ◽

Energy Dependent

The binding properties of seminal polyamines to ram spermatozoa and their possible role in sperm capacitation and the acrosome reaction were studied. Binding and release of [14C]spermine from ram spermatozoa occurred at a rate faster than in somatic cells and were not energy-dependent. Release of bound spermine was further facilitated by heparin, a constituent of the female reproductive tract which was reported to induce capacitation and the acrosome reaction. High- and low-affinity polyamine-binding sites were identified, of which the high-affinity site was specific to polyamines with three or more amino groups. We also found that spermine inhibited the acrosome reaction and propose that it is the major seminal decapacitating factor. Since precise timing of capacitation and the acrosome reaction are critical for successful fertilization, it is suggested that the role of seminal spermine is to prevent premature capacitation and the acrosome reaction.

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Effect of flagellar beating pattern on sperm rheotaxis and boundary-dependent navigation

10.1101/2020.01.20.913145 ◽

2020 ◽

Author(s):

Meisam Zaferani ◽

Farhad Javi ◽

Amir Mokhtare ◽

Alireza Abbaspourrad

Keyword(s):

Reproductive Tract ◽

Complex Structure ◽

Clinical Importance ◽

Microfluidic System ◽

Female Reproductive Tract ◽

Circular Motion ◽

Sperm Cells ◽

Progressive Motility ◽

Mammalian Sperm ◽

Correct Path

AbstractThe study of navigational mechanisms used by mammalian sperm inside a microenvironment yields better understanding of sperm locomotion during the insemination process, which aids in the design of tools for overcoming infertility. Near- and far-field hydrodynamic interactions with nearby boundaries and rheotaxis are known to be some of the steering strategies that keep sperm on the correct path toward the egg. However, it is not known how the beating patterns of sperm may influence these navigational strategies. In this study, we investigate the effect of flagellar beating pattern on navigation of sperm cells both theoretically and experimentally using a two-step approach. We first isolate bovine sperm based on their rheotactic behavior in a zone with quiescent medium using a microfluidic system. This step ensures that the swimmers are able to navigate upstream and have motilities higher than a selected value, even though they feature various flagellar beating patterns. We then explore the flagellar beating pattern of these isolated sperm and their subsequent influence on boundary-dependent navigation. Our findings indicate that rheotaxis enables sperm to navigate upstream even in the presence of circular motion in their motility, whereas boundary-dependent navigation is more sensitive to the circular motion and selects for progressive motility. This finding may explain the clinical importance of progressive motility in semen samples for fertility, as the flow of mucus may not be sufficiently strong to orient the sperm cells throughout the process of insemination.SignificanceFinding the egg and moving toward it while traversing the complex structure of the female reproductive tract is necessary for mammalian sperm. Previous studies have shown how sperm use navigational steering mechanisms that are based on swimming upstream (i.e. rheotaxis) and along the boundaries of the female reproductive tract. We demonstrate that the performance of theses navigational mechanisms is associated with the primary characteristics of sperm motility. In fact, sperm rheotaxis is more sensitive to the motility and thus average velocity of sperm while navigation via rigid boundaries is more sensitive to the flagellar beating pattern and selects for symmetric beating. Our results can be expanded to other autonomous microswimmers and their subsequent navigation mechanisms.

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PKA and CaMKII mediate PI3K activation in bovine sperm by inhibition of the PKC/PP1 cascade

Reproduction ◽

10.1530/rep-13-0560 ◽

2014 ◽

Vol 147 (3) ◽

pp. 347-356 ◽

Cited By ~ 28

Author(s):

H Rotfeld ◽

P Hillman ◽

D Ickowicz ◽

H Breitbart

Keyword(s):

Tyrosine Phosphorylation ◽

Reproductive Tract ◽

Significant Inhibition ◽

Female Reproductive Tract ◽

Phosphatidylinositol 3 Kinase ◽

Biochemical Processes ◽

Bovine Sperm ◽

Pi3k Activation ◽

Dependent Protein ◽

Camkii Activation

To enable fertilization, spermatozoa must undergo several biochemical processes in the female reproductive tract, collectively called capacitation. These processes involve protein kinase A (PKA)-dependent protein tyrosine phosphorylation including phosphatidylinositol-3-kinase (PI3K). It is not known how PKA, a serine/threonine (S/T) kinase, mediates tyrosine phosphorylation of proteins. We recently showed that inhibition of S/T phosphatase 1 (PP1) causes a significant increase in phospho-PI3K. In this study, we propose a mechanism by which PKA and PP1 mediate an increase in PI3K tyrosine phosphorylation and implicate calmodulin-dependent kinase II (CaMKII) in this process. Inhibition of sperm PP1 or PKC, stimulated CaMKII phosphorylation/activation, and inhibition of PKC enhanced PP1 phosphorylation/inactivation. Inhibition of CaMKII, using KN-93, caused significant reduction in phospho-PP1, indicating its activation. Moreover, KN-93 prevented the dephosphorylation/inactivation of PKC. We therefore suggest that CaMKII inhibits PKC, leading to PP1 inhibition and the reciprocal auto-activation of CaMKII. Thus, CaMKII can regulate its own activation by inhibiting the PKC/PP1 cascade. Inhibition of Src family kinases (SFK) caused significant inhibition of CaMKII and PP1 phosphorylation, suggesting that SFK activity results in PP1 inhibition and CaMKII activation. Activation of sperm PKA by 8Br-cAMP revealed an increase in phospho-CaMKII, which was inhibited by PKA inhibitor. Tyrosine phosphorylation of PI3K was stimulated by 8Br-cAMP and by PKC or PP1 inhibition and was abrogated by CaMKII inhibition. Furthermore, phosphorylation/activation of the tyrosine kinase Pyk2 was enhanced by PP1 inhibition, and this activation is blocked by CaMKII inhibition. Thus, PKA activates Src, which inhibits PP1, leading to CaMKII and Pyk2 activation, resulting in PI3K tyrosine phosphorylation/activation.

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Biochemical and Structural Characterization of Apolipoprotein A-I Binding Protein, a Novel Phosphoprotein with a Potential Role in Sperm Capacitation

Endocrinology ◽

10.1210/en.2007-0582 ◽

2008 ◽

Vol 149 (5) ◽

pp. 2108-2120 ◽

Cited By ~ 30

Author(s):

Kula N. Jha ◽

Igor A. Shumilin ◽

Laura C. Digilio ◽

Olga Chertihin ◽

Heping Zheng ◽

...

Keyword(s):

Cholesterol Efflux ◽

Reproductive Tract ◽

Binding Protein ◽

Protein A ◽

Density Lipoprotein ◽

Female Reproductive Tract ◽

Apolipoprotein A ◽

The Media

The physiological changes that sperm undergo in the female reproductive tract rendering them fertilization-competent constitute the phenomenon of capacitation. Cholesterol efflux from the sperm surface and protein kinase A (PKA)-dependent phosphorylation play major regulatory roles in capacitation, but the link between these two phenomena is unknown. We report that apolipoprotein A-I binding protein (AI-BP) is phosphorylated downstream to PKA activation, localizes to both sperm head and tail domains, and is released from the sperm into the media during in vitro capacitation. AI-BP interacts with apolipoprotein A-I, the component of high-density lipoprotein involved in cholesterol transport. The crystal structure demonstrates that the subunit of the AI-BP homodimer has a Rossmann-like fold. The protein surface has a large two compartment cavity lined with conserved residues. This cavity is likely to constitute an active site, suggesting that AI-BP functions as an enzyme. The presence of AI-BP in sperm, its phosphorylation by PKA, and its release during capacitation suggest that AI-BP plays an important role in capacitation possibly providing a link between protein phosphorylation and cholesterol efflux.

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Membrane Remodeling and Matrix Dispersal Intermediates During Mammalian Acrosomal Exocytosis

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.765673 ◽

2021 ◽

Vol 9 ◽

Author(s):

Miguel Ricardo Leung ◽

Ravi Teja Ravi ◽

Bart M. Gadella ◽

Tzviya Zeev-Ben-Mordehai

Keyword(s):

Plasma Membrane ◽

Reproductive Tract ◽

Morphological Changes ◽

Female Reproductive Tract ◽

Cell Integrity ◽

Membrane Fission ◽

Mammalian Sperm ◽

Acrosomal Exocytosis ◽

Plausible Mechanism ◽

Structural Mechanisms

To become fertilization-competent, mammalian sperm must undergo a complex series of biochemical and morphological changes in the female reproductive tract. These changes, collectively called capacitation, culminate in the exocytosis of the acrosome, a large vesicle overlying the nucleus. Acrosomal exocytosis is not an all-or-nothing event but rather a regulated process in which vesicle cargo disperses gradually. However, the structural mechanisms underlying this controlled release remain undefined. In addition, unlike other exocytotic events, fusing membranes are shed as vesicles; the cell thus loses the entire anterior two-thirds of its plasma membrane and yet remains intact, while the remaining nonvesiculated plasma membrane becomes fusogenic. Precisely how cell integrity is maintained throughout this drastic vesiculation process is unclear, as is how it ultimately leads to the acquisition of fusion competence. Here, we use cryoelectron tomography to visualize these processes in unfixed, unstained, fully hydrated sperm. We show that paracrystalline structures within the acrosome disassemble during capacitation and acrosomal exocytosis, representing a plausible mechanism for gradual dispersal of the acrosomal matrix. We find that the architecture of the sperm head supports an atypical membrane fission–fusion pathway that maintains cell integrity. Finally, we detail how the acrosome reaction transforms both the micron-scale topography and the nanoscale protein landscape of the sperm surface, thus priming the sperm for fertilization.

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Of mice and women: advances in mammalian sperm competition with a focus on the female perspective

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.2020.0082 ◽

2020 ◽

Vol 375 (1813) ◽

pp. 20200082

Author(s):

Renée C. Firman

Keyword(s):

Sperm Competition ◽

Reproductive Tract ◽

Female Reproductive Tract ◽

Selective Force ◽

Mammal Species ◽

Female Reproductive Success ◽

Positive Effects ◽

Mammalian Sperm ◽

Made In ◽

Female Perspective

Although initially lagging behind discoveries being made in other taxa, mammalian sperm competition is now a productive and advancing field of research. Sperm competition in mammals is not merely a ‘sprint-race’ between the gametes of rival males, but rather a race over hurdles; those hurdles being the anatomical and physiological barriers provided by the female reproductive tract, as well as the egg and its vestments. With this in mind, in this review, I discuss progress in the field while focusing on the female perspective. I highlight ways by which sperm competition can have positive effects on female reproductive success and discuss how competitive outcomes are not only owing to dynamics between the ejaculates of rival males, but also attributable to mechanisms by which female mammals bias paternity toward favourable sires. Drawing on examples across different species—from mice to humans—I provide an overview of the accumulated evidence which firmly establishes that sperm competition is a key selective force in the evolution of male traits and detail how females can respond to increased sperm competitiveness with increased egg resistance to fertilization. I also discuss evidence for facultative responses to the sperm competition environment observed within mammal species. Overall, this review identifies shortcomings in our understanding of the specific mechanisms by which female mammals ‘select’ sperm. More generally, this review demonstrates how, moving forward, mammals will continue to be effective animal models for studying both evolutionary and facultative responses to sperm competition. This article is part of the theme issue ‘Fifty years of sperm competition’.

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Release of Porcine Sperm from Oviduct Cells is Stimulated by Progesterone and Requires CatSper

Scientific Reports ◽

10.1038/s41598-019-55834-z ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 13

Author(s):

Sergio A. Machado ◽

Momal Sharif ◽

Huijing Wang ◽

Nicolai Bovin ◽

David J. Miller

Keyword(s):

Reproductive Tract ◽

Sperm Storage ◽

Female Reproductive Tract ◽

Cell Aggregates ◽

Sperm Release ◽

Intracellular Ca ◽

Sperm Reservoir ◽

Hormonal Milieu ◽

Oviduct Cell ◽

Sperm Functions

AbstractSperm storage in the female reproductive tract after mating and before ovulation is a reproductive strategy used by many species. When insemination and ovulation are poorly synchronized, the formation and maintenance of a functional sperm reservoir improves the possibility of fertilization. In mammals, the oviduct regulates sperm functions, such as Ca2+ influx and processes associated with sperm maturation, collectively known as capacitation. A fraction of the stored sperm is released by unknown mechanisms and moves to the site of fertilization. There is an empirical association between the hormonal milieu in the oviduct and sperm detachment; therefore, we tested directly the ability of progesterone to induce sperm release from oviduct cell aggregates. Sperm were allowed to bind to oviduct cells or an immobilized oviduct glycan and then challenged with progesterone, which stimulated the release of 48% of sperm from oviduct cells or 68% of sperm from an immobilized oviduct glycan. The effect of progesterone on sperm release was specific; pregnenolone and 17α-OH-progesterone did not affect sperm release. Ca2+ influx into sperm is associated with capacitation and development of hyperactivated motility. Progesterone increased sperm intracellular Ca2+, which was abrogated by blocking the sperm–specific Ca2+ channel CatSper with NNC 055-0396. NNC 055-0396 also blocked the progesterone-induced sperm release from oviduct cells or immobilized glycan. An inhibitor of the non-genomic progesterone receptor that activates CatSper similarly blocked sperm release. This is the first report indicating that release of sperm from the sperm reservoir is induced by progesterone action through CatSper channels.

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Simultaneous knockout of Slo3 and CatSper1 abolishes all alkalization- and voltage-activated current in mouse spermatozoa

The Journal of General Physiology ◽

10.1085/jgp.201311011 ◽

2013 ◽

Vol 142 (3) ◽

pp. 305-313 ◽

Cited By ~ 35

Author(s):

Xu-Hui Zeng ◽

Betsy Navarro ◽

Xiao-Ming Xia ◽

David E. Clapham ◽

Christopher J. Lingle

Keyword(s):

Membrane Potential ◽

Patch Clamp ◽

Reproductive Tract ◽

Outward Current ◽

Female Reproductive Tract ◽

Resting Membrane Potential ◽

Patch Clamp Technique ◽

Mouse Sperm ◽

Mammalian Sperm ◽

K Current

During passage through the female reproductive tract, mammalian sperm undergo a maturation process termed capacitation that renders sperm competent to produce fertilization. Capacitation involves a sequence of changes in biochemical and electrical properties, the onset of a hyperactivated swimming behavior, and development of the ability to undergo successful fusion and penetration with an egg. In mouse sperm, the development of hyperactivated motility is dependent on cytosolic alkalization that then results in an increase in cytosolic Ca2+. The elevation of Ca2+ is thought to be primarily driven by the concerted interplay of two alkalization-activated currents, a K+ current (KSPER) composed of pore-forming subunits encoded by the Kcnu1 gene (also termed Slo3) and a Ca2+ current arising from a family of CATSPER subunits. After deletion of any of four CATSPER subunit genes (CATSPER1–4), the major remaining current in mouse sperm is alkalization-activated KSPER current. After genetic deletion of the Slo3 gene, KSPER current is abolished, but there remains a small voltage-activated K+ current hypothesized to reflect monovalent flux through CATSPER. Here, we address two questions. First, does the residual outward K+ current present in the Slo3 −/− sperm arise from CATSPER? Second, can any additional membrane K+ currents be detected in mouse sperm by patch-clamp methods other than CATSPER and KSPER? Here, using mice bred to lack both SLO3 and CATSPER1 subunits, we show conclusively that the voltage-activated outward current present in Slo3 −/− sperm is abolished when CATSPER is also deleted. Any leak currents that may play a role in setting the resting membrane potential in noncapacitated sperm are likely smaller than the pipette leak current and thus cannot be resolved within the limitation of the patch-clamp technique. Together, KSPER and CATSPER appear to be the sole ion channels present in mouse sperm that regulate membrane potential and Ca2+ influx in response to alkalization.

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