scholarly journals 15-Lipoxygenase is a component of the mammalian sperm cytoplasmic droplet

Reproduction ◽  
2005 ◽  
Vol 130 (2) ◽  
pp. 213-222 ◽  
Author(s):  
K A Fischer ◽  
K Van Leyen ◽  
K W Lovercamp ◽  
G Manandhar ◽  
M Sutovsky ◽  
...  
Keyword(s):  
Semen Quality ◽  
Mammalian Species ◽  
Immune Serum ◽  
Residual Body ◽  
Cytoplasmic Droplet ◽  
Mammalian Sperm ◽  
Proteolytic Pathway ◽  
Cellular Debris ◽  
Ubiquitin Conjugating Enzyme ◽  
Boar Spermatozoa

Lipoxygenases (LOXs) are a family of enzymes capable of peroxidizing phospholipids. A member of the LOX family of enzymes, 15-LOX, participates in the degradation of mitochondria and other organelles within differentiating red blood cells, the reticulocytes. The present study provides biochemical and immunocytochemical evidence for the presence of 15-LOX in the sperm cytoplasmic droplet (CD). Testicular, epididymal and ejaculated spermatozoa were evaluated for the presence of 15-LOX using an affinity-purified immune serum raised against a synthetic peptide corresponding to the C-terminal sequence of rabbit reticulocyte 15-LOX. Western blotting revealed an appropriate single band of ~81 kDa in boar spermatozoa but not in boar seminal plasma. When ejaculated boar spermatozoa were subjected to separation on a 45/90% Percoll gradient, 15-LOX co-migrated with the immotile sperm and cellular debris/CD fractions, but not with the motile sperm fraction containing morphologically normal spermatozoa without CDs. Varied levels of 15-LOX were expressed in ejaculated sperm samples from boars with varied semen quality. By immunofluorescence, prominent 15-LOX immunoreactivity was found within the residual body in the testis and within the CDs from caput, corpus and cauda epididymal and ejaculated spermatozoa. Components of the ubiquitin-dependent proteolytic pathway, which is thought to facilitate both spermiogenesis and reticulocyte organelle degradation, were also detected in the sperm CD. These included ubiquitin, the ubiquitin-conjugating enzyme E2, the ubiquitin C-terminal hydrolase PGP 9.5, and various 20S proteasomal core subunits of the α- and β-type. The 15-LOX and various components of the ubiquitin–proteasome pathway were also detected in sperm CDs of other mammalian species, including the human, mouse, stallion and wild babirusa boar. We conclude that 15-LOX is prominently present in the mammalian sperm CD and thus may contribute to spermiogenesis, CD function or CD removal.

Localization of actin in boar spermatozoa using myosin S1-colloidal gold

1989 ◽  
Vol 47 ◽  
pp. 978-979
Author(s):  
J. J. Bozzola ◽  
R. N. Peterson ◽  
W. P. Hunt
Keyword(s):  
Colloidal Gold ◽  
Actin Polymerization ◽  
Mammalian Species ◽  
Specific Inhibitor ◽  
Sperm Cells ◽  
Exact Form ◽  
Mammalian Sperm ◽  
Predominant Type ◽  
Myosin S1 ◽  
Boar Spermatozoa

Actin has been reported to be present in the sperm cells of a number of mammalian species. The location and form of actin (monomeric or polymeric) is variable from species to species with monomeric or G-actin being the predominant type. The cytoskeleton is thought to be involved in maintaining the structural/chemical specialization of the plasma and acrosomal membranes of mammalian sperm cells, since actin has been localized in various regions. Cytochalasin D, a specific inhibitor of actin polymerization, inhibits fertilization of hamster eggs, indicating that F-actin may play an important role in the fertilization process. Since the exact form and location of actin has not been resolved, we used the myosin SI subfragment coupled to colloidal gold to localize F-actin in boar caput and cauda epididymal as well as ejaculated sperm cells.The sperm cells were washed in an F-actin stabilizing buffer (pH 7.0) consisting of 5 mM Na2HP04, 5 mM NaH2P04, 135 mM NaCl, 5 mM MgCl2, 2 mM EGTA, 0.005% PMSF, 0.005% PTAME, and 1 mg/ml soybean trypsin inhibitor.

scholarly journals Compartmentalization of membrane trafficking, glucose transport, glycolysis, actin, tubulin and the proteasome in the cytoplasmic droplet/Hermes body of epididymal sperm

Open Biology ◽  
2015 ◽  
Vol 5 (8) ◽  
pp. 150080 ◽  
Author(s):  
Catherine E. Au ◽  
Louis Hermo ◽  
Elliot Byrne ◽  
Jeffrey Smirle ◽  
Ali Fazel ◽  
...  
Keyword(s):  
Membrane Trafficking ◽  
Glucose Transporter ◽  
Molecular Level ◽  
Mammalian Species ◽  
Cytoskeletal Proteins ◽  
Epididymal Sperm ◽  
Quantitative Electron Microscopy ◽  
Cytoplasmic Droplet ◽  
Germ Cell Differentiation

Discovered in 1909 by Retzius and described mainly by morphology, the cytoplasmic droplet of sperm (renamed here the Hermes body) is conserved among all mammalian species but largely undefined at the molecular level. Tandem mass spectrometry of the isolated Hermes body from rat epididymal sperm characterized 1511 proteins, 43 of which were localized to the structure in situ by light microscopy and two by quantitative electron microscopy localization. Glucose transporter 3 (GLUT-3) glycolytic enzymes, selected membrane traffic and cytoskeletal proteins were highly abundant and concentrated in the Hermes body. By electron microscope gold antibody labelling, the Golgi trafficking protein TMED7/p27 localized to unstacked flattened cisternae of the Hermes body, as did GLUT-3, the most abundant protein. Its biogenesis was deduced through the mapping of protein expression for all 43 proteins during male germ cell differentiation in the testis. It is at the terminal step 19 of spermiogenesis that the 43 characteristic proteins accumulated in the nascent Hermes body.

scholarly journals The sperm protein SPACA4 is required for efficient fertilization in mice

2021 ◽  
Author(s):  
Sarah Herberg ◽  
Yoshitaka Fujihara ◽  
Andreas Blaha ◽  
Karin Panser ◽  
Kiyonari Kobayashi ◽  
...  
Keyword(s):  
Zona Pellucida ◽  
Knockout Mice ◽  
Mammalian Species ◽  
Wild Type ◽  
Sperm Protein ◽  
Mammalian Sperm ◽  
Fundamental Process ◽  
Mammalian Fertilization

Fertilization is the fundamental process that initiates the development of a new individual in all sexually reproducing species. Despite its importance, our understanding of the molecular players that govern mammalian sperm-egg interaction is incomplete, partly because many of the essential factors found in non-mammalian species do not have obvious mammalian homologs. We have recently identified the Ly6/uPAR protein Bouncer as a new, essential fertilization factor in zebrafish (Herberg et al., 2018). Here, we show that Bouncer's homolog in mammals, SPACA4, is also required for efficient fertilization in mice. In contrast to fish, where Bouncer is expressed specifically in the egg, SPACA4 is expressed exclusively in the testis. Male knockout mice are severely sub-fertile, and sperm lacking SPACA4 fail to fertilize wild-type eggs in vitro. Interestingly, removal of the zona pellucida rescues the fertilization defect of Spaca4-deficient sperm in vitro, indicating that SPACA4 is not required for the interaction of sperm and the oolemma but rather of sperm and zona pellucida. Our work identifies SPACA4 as an important sperm protein necessary for zona pellucida penetration during mammalian fertilization.

scholarly journals Binding of boar spermatozoa to oviductal epithelium in vitro in relation to sperm morphology and storage time

Reproduction ◽  
2006 ◽  
Vol 131 (2) ◽  
pp. 311-318 ◽  
Author(s):  
D Waberski ◽  
F Magnus ◽  
F Ardón ◽  
A M Petrunkina ◽  
K F Weitze ◽  
...  
Keyword(s):  
Semen Quality ◽  
Binding Capacity ◽  
Storage Period ◽  
Sperm Function ◽  
Sperm Binding ◽  
Oviductal Epithelium ◽  
Semen Storage ◽  
Boar Semen ◽  
Boar Spermatozoa

In vitro short-term storage of boar semen for up to 72 h before insemination negatively affects fertility, but this often remains undetected during semen quality assessment. One important sperm function is the ability to form the functional sperm reservoir in the oviduct. In the present study, we used the modified oviductal explant assay to study sperm binding to oviductal epithelium in vitro in diluted boar semen stored for 24 or 72 h. First, we determined the kinetics of in vitro sperm binding to oviductal epithelium in relation to co-incubation time of sperm and oviductal tissue pieces. Then, we studied how the binding of sperm to oviductal epithelium was affected by in vitro semen storage and by differences among individual boars. Sperm binding after different incubation times was significantly higher when semen was stored 24 h than after 72-h storage (P < 0.05), and peaked at 30–90 min of incubation. Sperm binding differed between boars (n = 44), and was negatively correlated to the percentage of sperm with cytoplasmic droplets (R = −0.51, P < 0.001). There were no significant changes in motility, acrosome integrity and propidium iodide stainability during the 72-h storage period. However, sperm-binding indices were significantly lower after 72 h in vitro storage than after 24-h storage in sperm from boars with normal semen quality (P < 0.05); in contrast, the binding capacity of sperm from boars with higher percentages of morphologically altered sperm remained at a low level. The sperm-binding capacity of sperm from four of the five boars with known subfertility was lower than the mean binding index minus one standard deviation of the boar population studied here. It is concluded that changes in the plasma membrane associated with in vitro ageing reduce the ability of stored boar sperm to bind to the oviductal epithelium. This study shows the potential of sperm–oviduct binding as a tool to assess both male fertility and changes in sperm function associated with in vitro ageing.

scholarly journals Calpain modulates capacitation and acrosome reaction through cleavage of the spectrin cytoskeleton

Reproduction ◽  
2010 ◽  
Vol 140 (5) ◽  
pp. 673-684 ◽  
Author(s):  
Yadira Bastián ◽  
Ana L Roa-Espitia ◽  
Adela Mújica ◽  
Enrique O Hernández-González
Keyword(s):  
Plasma Membrane ◽  
Guinea Pig ◽  
Acrosome Reaction ◽  
Mammalian Species ◽  
Physiological Changes ◽  
Signal Transducer ◽  
Mammalian Sperm ◽  
Important Player ◽  
Calpain 2 ◽  
Spectrin Cytoskeleton

Research on fertilization in mammalian species has revealed that Ca2+is an important player in biochemical and physiological events enabling the sperm to penetrate the oocyte. Ca2+is a signal transducer that particularly mediates capacitation and acrosome reaction (AR). Before becoming fertilization competent, sperm must experience several molecular, biochemical, and physiological changes where Ca2+plays a pivotal role. Calpain-1 and calpain-2 are Ca2+-dependent proteases widely studied in mammalian sperm; they have been involved in capacitation and AR but little is known about their mechanism. In this work, we establish the association of calpastatin with calpain-1 and the changes undergone by this complex during capacitation in guinea pig sperm. We found that calpain-1 is relocated and translocated from cytoplasm to plasma membrane (PM) during capacitation, where it could cleave spectrin, one of the proteins of the PM-associated cytoskeleton, and facilitates AR. The aforementioned results were dependent on the calpastatin phosphorylation and the presence of extracellular Ca2+. Our findings underline the contribution of the sperm cytoskeleton in the regulation of both capacitation and AR. In addition, our findings also reveal one of the mechanisms by which calpain and calcium exert its function in sperm.

scholarly journals Cryopreservation of boar semen

2020 ◽  
Vol 65 (No. 4) ◽  
pp. 115-123
Author(s):  
Marija Jovičić ◽  
Eva Chmelíková ◽  
Markéta Sedmíková
Keyword(s):  
Animal Species ◽  
Semen Quality ◽  
Egg Yolk ◽  
High Rate ◽  
Freezing And Thawing ◽  
Long Term Storage ◽  
Boar Semen ◽  
Boar Spermatozoa ◽  
Term Storage

Sperm cryopreservation is the best technology for long-term storage of the semen. However, the damage of boar spermatozoa by cryopreservation is more severe than in other animal species and a standardized freezing protocol for efficient cryopreservation has not been established yet. Semen quality and freezability vary greatly between breeds as well as between individual boars and even the season. Boar spermatozoa are sensitive to low temperatures; they sustain damage and a high rate of mortality and freezing/thawing the boar semen may strongly impair the sperm function and decrease the semen quality. The freezability of boar semen can be influenced by a cryopreservation procedure, and also by using various additives to freezing and thawing extenders such as antioxidants. In order to obtain acceptable results after thawing the boar semen, it is necessary to combine an optimal amount of additives (glycerol, egg yolk, sugars, antioxidants), cooling and warming velocities.

Cell degeneration and gill remodelling during basidiocarp development in the fungus Coprinus cinereus

1991 ◽  
Vol 69 (6) ◽  
pp. 1161-1169 ◽  
Author(s):  
Benjamin C. Lu
Keyword(s):  
Hydrolytic Enzymes ◽  
Multivesicular Body ◽  
Coprinus Cinereus ◽  
High Dose ◽  
Residual Body ◽  
Multivesicular Bodies ◽  
Cell Degeneration ◽  
Cellular Debris ◽  
Extensive Cell ◽  
Residual Bodies

The early mushroom gill development in a primordium of Coprinus cinereus was studied by electron microscopy. Extensive cell degeneration and cell death were found in gill cavities but not within gill domains. These degenerative cells were found to contain multivesicular and membranous residual bodies, suggesting that the multivesicular bodies are part of the cell degeneration. Cellular debris was observed in intercellular spaces probably as a consequence of cell lysis. The presence of multivesicular bodies was also observed in cells shortly before Coprinus basidiocarps underwent autolysis: a high dose of hydrolytic enzymes can be extracted from such basidiocarps. The high numbers of multivesicular bodies, the membranous residual bodies, and the cellular debris in the primordial tissues are manifestations of cell degeneration that may be a prerequisite to gill remodelling during early primordial development. Key words: cell degeneration, gill remodelling, multivesicular body, residual body, mushroom development.

scholarly journals Elimination of apoptotic boar spermatozoa using magnetic activated cell sorting

2014 ◽  
Vol 83 (1) ◽  
pp. 13-18 ◽  
Author(s):  
Janko Mrkun ◽  
Tamara Dolenšek ◽  
Tanja Knific ◽  
Anja Pišlar ◽  
Marjan Kosec ◽  
...  
Keyword(s):  
Cell Sorting ◽  
Semen Quality ◽  
Annexin V ◽  
Alexa Fluor ◽  
Liquid Storage ◽  
Boar Semen ◽  
And Control ◽  
Boar Spermatozoa ◽  
First Time ◽  
Magnetic Activated Cell Sorting

One of the features of apoptosis is the externalization of phosphatidylserine which could be used to remove apoptotic cells from semen preparations. Magnetic-activated cell sorting using annexin V-conjugated microbeads which bind to phosphatidylserine could be used to enhance semen quality. Twelve boar semen samples after 3 days of liquid storage at 16­­–17 °C were subjected to magnetic-activated cell sorting. Bound and unbound fractions and control samples were subjected to flow cytometry following the staining of spermatozoa with Annexin V conjugated with Alexa Fluor 488 and propidium iodide. Four subpopulations were obtained: live, early apoptotic live, late apoptotic, early necrotic dead and late necrotic dead. The frequency of early apoptotic and late necrotic spermatozoa was significantly higher (P< 0.05) in bound (14.1 ± 10.6% and 24.1 ± 10.2%, respectively) than in unbound fractions (3.4 ± 2.1% and 12.7 ± 3.1%) and control (3.5 ± 1.6% and 12.0 ± 5.0%). The lowest concentration of live spermatozoa was found in the bound fraction (10.6 ± 8.0 %), which differed significantly (P< 0.05) from the control. In unbound fractions there was a significantly higher concentration (P< 0.05) of morphologically normal spermatozoa (31.8 ± 12.6%) compared to bound ones (5.9 ± 7.3%). A significantly (P< 0.05) lower proportion of morphologically normal spermatozoa was observed in both fractions compared to control (67.2 ± 17.0%). Boar spermatozoa were separated by the above method for the first time, however, the results showed this method to be inappropriate for boar semen separation under the tested conditions.

scholarly journals COMPARATIVE ANALYSIS OF MAMMALIAN SPERM MOTILITY

1972 ◽  
Vol 53 (2) ◽  
pp. 561-573 ◽  
Author(s):  
David M. Phillips
Keyword(s):  
High Speed ◽  
Mammalian Species ◽  
Chinese Hamster ◽  
Small Radius ◽  
Radius Of Curvature ◽  
Large Radius ◽  
Cross Sectional ◽  
Mammalian Sperm ◽  
High Speed Cinematography ◽  
Pass Through

Spermatozoa of several mammalian species were studied by means of high-speed cinematography and electron microscopy. Three types of motile patterns were observed in mouse spermatozoa. The first type involved an asymmetrical beat which seemed to propel the sperm in circular paths. The second type involved rotation of the sperm and appeared to allow them to maintain straight paths. In the third type of pattern, the sperm appeared to move by crawling on surfaces in a snakelike manner. Spermatozoa of rabbit and Chinese hamster also had an asymmetrical beat which sometimes caused them to swim in circles. In spite of the asymmetry of the beat, these spermatozoa were also able to swim in straight paths by rotating around a central axis as they swam. Spermatozoa of some species appeared very flexible; their flagella formed arcs with a very small radius of curvature as they beat. Spermatozoa of other species appeared very stiff, and their flagella formed arcs with a very large radius of curvature. The stiffness of the spermatozoan appeared to correlate positively with the cross-sectional area of the dense fibers. This suggests that the dense fibers may be stiff elastic elements. Opossum sperm become paired as they pass through the epididymis. Pairs of opossum spermatozoa beat in a coordinated, alternating manner.

scholarly journals Novel Control of S Phase of the Cell Cycle by Ubiquitin-conjugating Enzyme H7

2009 ◽  
Vol 20 (1) ◽  
pp. 1-9 ◽  
Author(s):  
Elizabeth A. Whitcomb ◽  
Edward J. Dudek ◽  
Qing Liu ◽  
Allen Taylor
Keyword(s):  
Cell Cycle ◽  
S Phase ◽  
Regulatory Proteins ◽  
Protein Levels ◽  
Proteolytic Pathway ◽  
Dynamic Cell ◽  
Ubiquitin Conjugating Enzyme ◽  
Phase Progression ◽  
Cycle Dependent ◽  
Cycle Regulation

Timely degradation of regulatory proteins by the ubiquitin proteolytic pathway (UPP) is an established paradigm of cell cycle regulation during the G2/M and G1/S transitions. Less is known about roles for the UPP during S phase. Here we present evidence that dynamic cell cycle–dependent changes in levels of UbcH7 regulate entrance into and progression through S phase. In diverse cell lines, UbcH7 protein levels are dramatically reduced in S phase but are fully restored by G2. Knockdown of UbcH7 increases the proportion of cells in S phase and doubles the time to traverse S phase, whereas UbcH7 overexpression reduces the proportion of cells in S phase. These data suggest a role for UbcH7 targets in the completion of S phase and entry into G2. Notably, UbcH7 knockdown was coincident with elevated levels of the checkpoint kinase Chk1 but not Chk2. These results argue that UbcH7 promotes S phase progression to G2 by modulating the intra-S phase checkpoint mediated by Chk1. Furthermore, UbcH7 levels appear to be regulated by a UPP. Together the data identify novel roles for the UPP, specifically UbcH7 in the regulation of S phase transit time as well as in cell proliferation.

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