Investigation of epididymal proteins and general sperm membrane characteristics of Formosan pangolin (Manis pentadactyla pentadactyla)

Abstract Background Formosan Pangolin (Manis pentadactyla pentadactyla) is one of the three subspecies of Chinese pangolins, it is also an isolated sub-species naturally habitat in Taiwan. Despite earlier report on successful breeding of Sunda (Manis javanica) pangolin, breeding of Formosan pangolins in zoo captive populations is still challenging due to unknown reproductive characterizations of this species in both male and female populations. Results We characterized for the first time, reproductive tract of male Formosan pangolin. We showed pangolin epididymis was a collagen-enriched organ with apparent segmented sub-regions similar to other mammals. However, unlike most mammals exhibited two V-ATPase subunits, Formosan pangolin exhibited only V-ATPase subunit 2. This specific V-ATPase subunit extended its cellular localization throughout the cytoplasm of epididymal clear cells, suggesting pH regulation of luminal microenvironment might be different from other mammals. Electron micrographs showed rod-shaped pangolin sperm cells with multi-lamellar membrane structure at the sperm head. Similar to well-defined capacitation and acrosome reaction membrane changes in other mammals, we reported three distinct patterns (homogenous, punctuated and faded) of pangolin sperm head membrane changes. The concurrent increase in phosphotyrosine protein expression detected at the sperm mid-piece/tail and the emergence of punctuated membrane aggregates likely representing three sperm activation stages, namely inactivated, capacitated and acrosome reacted status of pangolin sperm. Conclusion By revealing unique epididymal V-ATPase distribution and sperm membrane dynamics in Formosan pangolin, we would understand better the fundamental aspects of reproduction parameters of Formosan pangolin.

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Sub-cellular patterns of SPE-6 localization reveal unexpected complexities in C. elegans sperm activation and sperm function

G3 Genes|Genome|Genetics ◽

10.1093/g3journal/jkab288 ◽

2021 ◽

Author(s):

Jackson J Peterson ◽

Claire E Tocheny ◽

Gaurav Prajapati ◽

Craig W LaMunyon ◽

Diane C Shakes

Keyword(s):

Plasma Membrane ◽

Cellular Localization ◽

Reproductive Tract ◽

Female Reproductive Tract ◽

Sperm Function ◽

Temperature Sensitive ◽

Casein Kinase 1 ◽

Sperm Activation ◽

C Elegans ◽

Extracellular Signals

Abstract To acquire and maintain directed cell motility, Caenorhabditis elegans sperm must undergo extensive, regulated cellular remodeling, in the absence of new transcription or translation. To regulate sperm function, nematode sperm employ large numbers of protein kinases and phosphatases, including SPE-6, a member of C. elegans’ highly expanded casein kinase 1 superfamily. SPE-6 functions during multiple steps of spermatogenesis, including functioning as a “brake” to prevent premature sperm activation in the absence of normal extracellular signals. Here we describe the sub-cellular localization patterns of SPE-6 during wildtype C. elegans sperm development and in various sperm activation mutants. While other members of the sperm activation pathway associate with the plasma membrane or localize to the sperm’s membranous organelles, SPE-6 surrounds the chromatin mass of unactivated sperm. During sperm activation by either of two semiautonomous signaling pathways, SPE-6 redistributes to the front, central region of the sperm’s pseudopod. When disrupted by reduction-of-function alleles, SPE-6 protein is either diminished in a temperature-sensitive manner (hc187) or is mis-localized in a stage-specific manner (hc163). During the multistep process of sperm activation, SPE-6 is released from its perinuclear location after the spike stage in a process that does not require fusion of membranous organelles with the plasma membrane. After activation, spermatozoa exhibit variable proportions of perinuclear and pseudopod-localized SPE-6, depending on their location within the female reproductive tract. These findings provide new insights regarding SPE-6’s role in sperm activation and suggest that extracellular signals during sperm migration may further modulate SPE-6 localization and function.

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ACTN4 Mediates SEPT14 Mutation-Induced Sperm Head Defects

Biomedicines ◽

10.3390/biomedicines8110518 ◽

2020 ◽

Vol 8 (11) ◽

pp. 518

Author(s):

Yu-Hua Lin ◽

Chia-Yen Huang ◽

Chih-Chun Ke ◽

Ya-Yun Wang ◽

Tsung-Hsuan Lai ◽

...

Keyword(s):

Mass Spectrometry ◽

Cell Model ◽

Membrane Dynamics ◽

Sperm Head ◽

Cellular Processes ◽

Germ Cell Line ◽

Head Formation ◽

Fourth Component ◽

Related Proteins ◽

Cellular Components

Septins (SEPTs) are highly conserved GTP-binding proteins and the fourth component of the cytoskeleton. Polymerized SEPTs participate in the modulation of various cellular processes, such as cytokinesis, cell polarity, and membrane dynamics, through their interactions with microtubules, actin, and other cellular components. The main objective of this study was to dissect the molecular pathological mechanism of SEPT14 mutation-induced sperm head defects. To identify SEPT14 interactors, co-immunoprecipitation (co-IP) and nano-liquid chromatography-mass spectrometry/mass spectrometry were applied. Immunostaining showed that SEPT14 was significantly localized to the manchette structure. The SEPT14 interactors were identified and classified as (1) SEPT-, (2) microtubule-, (3) actin-, and (4) sperm structure-related proteins. One interactor, ACTN4, an actin-holding protein, was selected for further study. Co-IP experiments showed that SEPT14 interacts with ACTN4 in a male germ cell line. SEPT14 also co-localized with ACTN4 in the perinuclear and manchette regions of the sperm head in early elongating spermatids. In the cell model, mutated SEPT14 disturbed the localization pattern of ACTN4. In a clinical aspect, sperm with mutant SEPT14, SEPT14A123T (p.Ala123Thr), and SEPT14I333T (p.Ile333Thr), have mislocalized and fragmented ACTN4 signals. Sperm head defects in donors with SEPT14 mutations are caused by disruption of the functions of ACTN4 and actin during sperm head formation.

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Seminal vesicle proteins SVS3 and SVS4 facilitate SVS2 effect on sperm capacitation

Reproduction ◽

10.1530/rep-15-0551 ◽

2016 ◽

Vol 152 (4) ◽

pp. 313-321 ◽

Cited By ~ 8

Author(s):

Naoya Araki ◽

Natsuko Kawano ◽

Woojin Kang ◽

Kenji Miyado ◽

Kaoru Yoshida ◽

...

Keyword(s):

Seminal Vesicle ◽

Reproductive Tract ◽

Female Reproductive Tract ◽

The Other ◽

Sperm Capacitation ◽

Other Hand ◽

Sperm Membrane ◽

Vesicle Secretion ◽

Mammalian Spermatozoa

Mammalian spermatozoa acquire their fertilizing ability in the female reproductive tract (sperm capacitation). On the other hand, seminal vesicle secretion, which is a major component of seminal plasma, inhibits the initiation of sperm capacitation (capacitation inhibition) and reduces the fertility of the capacitated spermatozoa (decapacitation). There are seven major proteins involved in murine seminal vesicle secretion (SVS1-7), and we have previously shown that SVS2 acts as both a capacitation inhibitor and a decapacitation factor, and is indispensable forin vivofertilization. However, the effects of SVSs other than SVS2 on the sperm have not been elucidated. Since mouseSvs2–Svs6genes evolved by gene duplication belong to the same gene family, it is possible that SVSs other than SVS2 also have some effects on sperm capacitation. In this study, we examined the effects of SVS3 and SVS4 on sperm capacitation. Our results showed that both SVS3 and SVS4 are able to bind to spermatozoa, but SVS3 alone showed no effects on sperm capacitation. On the other hand, SVS4 acted as a capacitation inhibitor, although it did not show decapacitation abilities. Interestingly, SVS3 showed an affinity for SVS2 and it facilitated the effects of SVS2. Interaction of SVS2 and spermatozoa is mediated by the ganglioside GM1 in the sperm membrane; however, both SVS3 and SVS4 had weaker affinities for GM1 than SVS2. Therefore, we suggest that separate processes may cause capacitation inhibition and decapacitation, and SVS3 and SVS4 act on sperm capacitation cooperatively with SVS2.

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Altered expression of the 100 kDa subunit of the Dictyosteliumvacuolar proton pump impairs enzyme assembly, endocytic function and cytosolic pH regulation

Journal of Cell Science ◽

10.1242/jcs.115.9.1907 ◽

2002 ◽

Vol 115 (9) ◽

pp. 1907-1918 ◽

Cited By ~ 3

Author(s):

Tongyao Liu ◽

Christian Mirschberger ◽

Lilian Chooback ◽

Quyen Arana ◽

Zeno Dal Sacco ◽

...

Keyword(s):

Proton Pump ◽

Catalytic Domain ◽

Ph Regulation ◽

Proton Pumps ◽

Cytosolic Ph ◽

Acid Media ◽

Dynamic Regulation ◽

Atpase Subunit ◽

Altered Expression ◽

Mutant Cells

The vacuolar proton pump (V-ATPase) appears to be essential for viability of Dictyostelium cells. To investigate the function of VatM, the 100 kDa transmembrane V-ATPase subunit, we altered its level. By means of homologous recombination, the promoter for the chromosomal vatM gene was replaced with the promoter for the act6 gene, yielding the mutant strain VatMpr. The act6 promoter is much more active in cells growing axenically than on bacteria. Thus, transformants were selected under axenic growth conditions, then shifted to bacteria to determine the consequences of reduced vatM expression. When VatMpr cells were grown on bacteria,the level of the 100 kDa V-ATPase subunit dropped, cell growth slowed, and the A subunit, a component of the peripheral catalytic domain of the V-ATPase,became mislocalized. These defects were complemented by transformation of the mutant cells with a plasmid expressing vatM under the control of its own promoter. Although the principal locus of vacuolar proton pumps in Dictyostelium is membranes of the contractile vacuole system, mutant cells did not manifest osmoregulatory defects. However, bacterially grown VatMpr cells did exhibit substantially reduced rates of phagocytosis and a prolonged endosomal transit time. In addition, mutant cells manifested alterations in the dynamic regulation of cytosolic pH that are characteristic of normal cells grown in acid media, which suggested that the V-ATPase also plays a role in cytosolic pH regulation.

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Time-resolved Fluorescence and Generalized Polarization: Innovative tools to assess bull sperm membrane dynamics during slow freezing

Cryobiology ◽

10.1016/j.cryobiol.2019.10.196 ◽

2019 ◽

Vol 91 ◽

pp. 69-76

Author(s):

Shaliha Bechoua ◽

Pascale Winckler ◽

Audrey Jossier ◽

Caroline Peltier ◽

Frédéric Delize ◽

...

Keyword(s):

Membrane Dynamics ◽

Slow Freezing ◽

Time Resolved Fluorescence ◽

Generalized Polarization ◽

Time Resolved ◽

Sperm Membrane ◽

Bull Sperm

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KSper, a pH-sensitive K+ current that controls sperm membrane potential

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.0702018104 ◽

2007 ◽

Vol 104 (18) ◽

pp. 7688-7692 ◽

Cited By ~ 137

Author(s):

Betsy Navarro ◽

Yuriy Kirichok ◽

David E. Clapham

Keyword(s):

Membrane Potential ◽

Acrosome Reaction ◽

Reproductive Tract ◽

Female Reproductive Tract ◽

Male Reproductive Tract ◽

Sperm Flagellum ◽

K Current ◽

Sperm Membrane ◽

Principal Piece ◽

Mammalian Spermatozoa

Mature mammalian spermatozoa are quiescent in the male reproductive tract. Upon ejaculation and during their transit through the female reproductive tract, they undergo changes that enable them to fertilize the egg. During this process of capacitation, they acquire progressive motility, develop hyperactivated motility, and are readied for the acrosome reaction. All of these processes are regulated by intracellular pH. In the female reproductive tract, the spermatozoan cytoplasm alkalinizes, which in turn activates a Ca2+-selective current (ICatSper) required for hyperactivated motility. Here, we show that alkalinization also has a dramatic effect on membrane potential, producing a rapid hyperpolarization. This hyperpolarization is primarily mediated by a weakly outwardly rectifying K+ current (IKSper) originating from the principal piece of the sperm flagellum. Alkalinization activates the pHi-sensitive IKSper, setting the membrane potential to negative potentials where Ca2+ entry via ICatSper is maximized. IKSper is one of two dominant ion currents of capacitated sperm cells.

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Cloning of the V-ATPase subunit G in plant: functional expression and sub-cellular localization 1

FEBS Letters ◽

10.1016/s0014-5793(98)01252-6 ◽

1998 ◽

Vol 437 (3) ◽

pp. 287-292 ◽

Cited By ~ 31

Author(s):

David Rouquié ◽

Colette Tournaire-Roux ◽

Wojciech Szponarski ◽

Michel Rossignol ◽

Patrick Doumas

Keyword(s):

Cellular Localization ◽

Functional Expression ◽

Atpase Subunit

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Changes in distribution of labile zinc in mouse spermatozoa during maturation in the epididymis assessed by the fluorophore Zinquin

Reproduction Fertility and Development ◽

10.1071/rd9961097 ◽

1996 ◽

Vol 8 (7) ◽

pp. 1097 ◽

Cited By ~ 41

Author(s):

PD Zalewski ◽

X Jian ◽

LL Soon ◽

WG Breed ◽

RF Seamark ◽

...

Keyword(s):

Sodium Dodecyl Sulfate ◽

Reproductive Tract ◽

Regional Distribution ◽

Sodium Dodecyl ◽

Sperm Head ◽

Male Reproductive Tract ◽

Intracellular Location ◽

And Function ◽

Triton X

The Zn(II)-specific fluorophore Zinquin was used to determine the regional distribution of free or loosely-bound Zn(II) in mouse spermatozoa. Spermatozoa from the testes exhibited bright fluorescence over the entire head; those from the caput epididymides generally fluoresced more brightly in the post-acrosomal region; and spermatozoa from the caudae epididymides fluoresced less brightly, with foci of fluorescence over the sperm head which were lost after extraction with Triton X-100 and hence appeared to be membrane-associated. Treatment of cauda sperm with sodium dodecyl sulfate resulted in a bright uniform Zinquin fluorescence in the heads, similar to that observed in caput sperm, indicating that the two types of sperm have similar amounts of head Zn(II) but that the availability of Zn(II) for binding Zinquin is different. By contrast, the intensity of tail fluorescence was similar in spermatozoa from different regions of the male reproductive tract and was largely unaffected by Triton X-100 extraction, consistent with an intracellular location. Similar differences were observed between caput sperm and cauda sperm in the rat. It is concluded that visualization and measurement of free or loosely-bound Zn(II) in subcellular compartments of spermatozoa should facilitate investigation of the role of this metal in the development and function of spermatozoa and abnormalities that might accompany infertility and Zn(II) deficiency.

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Sperm Selection for ICSI: Do We Have a Winner?

Cells ◽

10.3390/cells10123566 ◽

2021 ◽

Vol 10 (12) ◽

pp. 3566

Author(s):

Domenico Baldini ◽

Daniele Ferri ◽

Giorgio Maria Baldini ◽

Dario Lot ◽

Assunta Catino ◽

...

Keyword(s):

Reproductive Tract ◽

Female Reproductive Tract ◽

Sperm Cells ◽

Cell Selection ◽

Sperm Selection ◽

Gold Standard Method ◽

Advantages And Disadvantages ◽

Selection Processes ◽

Sperm Membrane ◽

Selection For

In assisted reproductive technology (ART), the aim of sperm cells’ preparation is to select competent spermatozoa with the highest fertilization potential and in this context, the intracytoplasmic sperm injection (ICSI) represents the most applied technique for fertilization. This makes the process of identifying the perfect spermatozoa extremely important. A number of methods have now been developed to mimic some of the natural selection processes that exist in the female reproductive tract. Although many studies have been conducted to identify the election technique, many doubts and disagreements still remain. In this review, we will discuss all the sperm cell selection techniques currently available for ICSI, starting from the most basic methodologies and continuing with those techniques suitable for sperm cells with reduced motility. Furthermore, different techniques that exploit some sperm membrane characteristics and the most advanced strategy for sperm selection based on microfluidics, will be examined. Finally, a new sperm selection method based on a micro swim-up directly on the ICSI dish will be analyzed. Eventually, advantages and disadvantages of each technique will be debated, trying to draw reasonable conclusions on their efficacy in order to establish the gold standard method.

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2. Immunolocalization of Cystatin‐Related Epididymal Spermatogenic (CRES) Protein in Mouse Spermatozoa

Inquiry@Queen's Undergraduate Research Conference Proceedings ◽

10.24908/iqurcp.7533 ◽

2017 ◽

Author(s):

Marvin Ferrer

Keyword(s):

Serine Proteases ◽

Anterior Pituitary ◽

Reproductive Tract ◽

Sperm Head ◽

Immunogold Electron Microscopy ◽

Male Reproductive Tract ◽

Sperm Tail ◽

Enzymatic Regulation ◽

Sperm Development ◽

Developmental Analysis

CRES is an inhibitor of a specific member of an enzyme family called serine proteases. It is thus thought to play a role in enzymatic regulation. It has been shown to be expressed only in the testes, epididymis, sperm, and gonadotropic cells of the anterior pituitary gland. Localization of CRES in sperm is an important step in determining its role within sperm. Previous studies have shown CRES to be localized in the mouse acrosomal cap: a vesicle on the tip of the sperm head which releases its contents upon initial sperm‐egg contact. The present study shows that developmental analysis of spermatogenesis via immunohistochemistry is incompatible with the conclusion that CRES is localized in the acrosomal cap. CRES expression begins in the elongating spermatid stage of sperm development; by this point, the acrosomal cap has been completed and it has never been shown that further additions to it can occur. Western blot and immunogold electron microscopy shows that CRES is localized in the sperm tail. It is also possible that further CRES is placed on the outer sperm membranes on the head as it travels through the epididymis, which is a part of the male reproductive tract.

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