Immunofluorescence Reveals Ubiquitination of Retained Distal Cytoplasmic Droplets on Ejaculated Porcine Spermatozoa

Kuster, Christopher E.; Hess, Rex A.; Althouse, Gary C.

doi:10.1002/j.1939-4640.2004.tb02798.x

Cited by 44 publications

(34 citation statements)

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“…The present data and a recent report by Kuster et al (2004) support a model in which the ubiquitin-dependent proteolytic pathway acts in synergy with the 15-LOX pathway for the degradation of the CD. This synergy could be similar to the respective contributions of the 15-LOX (van Leyen et al 1998) and ubiquitin (Dubiel & Rapoport 1989) pathways to reticulocyte organelle degradation.…”

Section: Discussionsupporting

confidence: 87%

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

Fischer¹,

Leyen²,

Lovercamp³

et al. 2005

Reproduction

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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 a-and b-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. Reproduction (2005) 130 213-222

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Section: Discussionsupporting

confidence: 87%

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

Fischer¹,

Leyen²,

Lovercamp³

et al. 2005

Reproduction

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“…The shape of the CD is related to infertility in some human patients [10] and is thus thought to affect fertilizability in vivo and in vitro. Although the shedding system and function of the CD are still unclear, the protein 15-LOX [19] and molecules recognized by anti-ubiquitin antibody [20] have been reported to be involved in CD function and removal. Further analyses are needed to determine whether the distribution of these molecules is strain-dependent, as is the presence of a CD.…”

Section: Discussionmentioning

confidence: 99%

The Relationship between Sperm Morphology and In Vitro Fertilization Ability in Mice

Kawai

Hata

Сузукі

et al. 2006

Journal of Reproduction and Development

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Abstract.In vitro fertilization (IVF) is widely used in reproduction research, but the sperm of some inbred strains of mice yield low fertilization rates in IVF. To determine the cause of this problem, we examined the effect of epididymal sperm morphology, in particular, tail bending and the presence and type of cytoplasmic droplet (CD), on fertilizability in vitro. Sperm suspensions were obtained from the following five strains: C57BL/6J, BALB/cA, C3H/HeN, DBA/2J, and 129 × 1/SvJ. The sperm were fixed in 10% formalin and three parts of the sperm, namely the head, tail, and CD, were examined. We recorded the proportion of abnormal sperm heads and hairpins at the neck; tails were categorized as straight, proximal bent, or distal bent; and the CDs were categorized as none, lighttype, and heavy-type. Based on these parameters, we determined the correlations between sperm morphology and fertilizability in vitro, as judged by IVF using ICR oocytes. The proportion of sperm with a hairpin neck was higher in strain C57BL/6J, while abnormal head morphology occurred significantly more often in strain BALB/cA. The percentage of sperm with a proximal bent tail was highest in strain DBA/2J and lowest in strain 129 × 1/SvJ. A heavy-type CD was observed more frequently in the 129 × 1/SvJ and C57BL/6J strains than in the other three strains in which a light-type CD predominated. The rank order of the fertilization rates was 129 × 1/SvJ < C57BL/6J < C3H/HeN < BALB/cA < DBA/2J. In addition, fertilization rate was positively correlated with a proximal bent tail, but negatively correlated with a heavy-type CD and distal bent tail. This new classification system establishes that the morphological characteristics of epididymal sperm differ among inbred strains of mice and that tail and CD morphology are closely related to fertilization ability in IVF. Thus, our results provide a novel method for assessing the quality of mouse sperm used for IVF. Key words: Cytoplasmic droplet, Epididymal sperm, In vitro fertilizability, Sperm morphology (J. Reprod. Dev. 52: [561][562][563][564][565][566][567][568] 2006) nbred strains of laboratory mice are the most frequently used animals in basic research, including reproduction and genetic research. Strain C57BL/6 is used in studies on cardiovascular biology, developmental biology, diabetes and obesity, genetics, immunology, neurobiology, and sensorineural research. The BALB/c strain is used to produce monoclonal antibodies. The 129 × 1/Sv strain produces targeted mutations owing to the availability of several embryonic stem cell lines. DBA/2 mice are valuable in cardiovascular biology, neurobiology, and sensorineural research, while strain C3H/HeJ is used as a general-purpose a n i m a l m o d e l f o r c a n c e r , i m m u n o l o g y , inflammation, sensorineural, and cardiovascular research. Recent advances in reproductive techniques have allowed the production of various transgenic and knockout animals that serve as useful models of human diseases and allow

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“…Morphologically, it has also been shown that the cytoplasmic droplet contains numerous vesicular elements and small particles derived from the Golgi apparatus, sperm histones, and ubiquitin (Kuster et al, 2004;Moreno et al, 2000b;Sutovsky, 2003). Live imaging of the Golgi apparatus using Golgi markers, Golgin-97 [ID: T2-182], Golgin-95/GM130 [ID: T2-183], and Golgin-160 [ID: T2-188] revealed that these compo- Fig.…”

Section: Microscopy Research and Techniquementioning

confidence: 98%

Surfing the wave, cycle, life history, and genes/proteins expressed by testicular germ cells. Part 3: Developmental changes in spermatid flagellum and cytoplasmic droplet and interaction of sperm with the zona pellucida and egg plasma membrane

Hermo¹,

Pelletier²,

Cyr³

et al. 2009

Microscopy Res & Technique

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Spermiogenesis constitutes the steps involved in the metamorphosis of spermatids into spermatozoa. It involves modification of several organelles in addition to the formation of several structures including the flagellum and cytoplasmic droplet. The flagellum is composed of a neck region and middle, principal, and end pieces. The axoneme composed of nine outer microtubular doublets circularly arranged to form a cylinder around a central pair of microtubules is present throughout the flagellum. The middle and principal pieces each contain specific components such as the mitochondrial sheath and fibrous sheath, respectively, while outer dense fibers are common to both. A plethora of proteins are constituents of each of these structures, with each playing key roles in functions related to the fertility of spermatozoa. At the end of spermiogenesis, a portion of spermatid cytoplasm remains associated with the released spermatozoa, referred to as the cytoplasmic droplet. The latter has as its main feature Golgi saccules, which appear to modify the plasma membrane of spermatozoa as they move down the epididymal duct and hence may be partly involved in male gamete maturation. The end product of spermatogenesis is highly streamlined and motile spermatozoa having a condensed nucleus equipped with an acrosome. Spermatozoa move through the female reproductive tract and eventually penetrate the zona pellucida and bind to the egg plasma membrane. Many proteins have been implicated in the process of fertilization as well as a plethora of proteins involved in the development of spermatids and sperm, and these are high lighted in this review.

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Immunofluorescence Reveals Ubiquitination of Retained Distal Cytoplasmic Droplets on Ejaculated Porcine Spermatozoa

Cited by 44 publications

References 44 publications

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

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

The Relationship between Sperm Morphology and In Vitro Fertilization Ability in Mice

Surfing the wave, cycle, life history, and genes/proteins expressed by testicular germ cells. Part 3: Developmental changes in spermatid flagellum and cytoplasmic droplet and interaction of sperm with the zona pellucida and egg plasma membrane

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