Clinical adaptation of the sperm ubuquitin tag immunoassay (SUTI): relationship of sperm ubiquitylation with sperm quality in gradient-purified semen samples from 93 men from a general infertility clinic population

Ozanon, Christophe; Chouteau, J.; Šutovský, Peter

doi:10.1093/humrep/dei013

Cited by 31 publications

(23 citation statements)

References 29 publications

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“…After a 1-h migration, the overlying swim-up sperm were collected, washed twice in Dulbecco's phosphate-buffered saline, and stored at Ϫ20°C. Sperm from a fertile sperm bank donor (Fairfax Cryobank, Fairfax, VA) were washed and used for lectin and antibody binding studies as previously described (18).…”

Section: Methodsmentioning

confidence: 99%

“…Fixed sperm were incubated overnight at 4°C with anti-Lewis y monoclonal antibody diluted 1:50, washed, and incubated for 40 min at room temperature with a mixture containing the TRITC-conjugated goat anti-mouse IgM (Zymed Laboratories Inc., San Francisco, CA) diluted 1/100. Acrosomal integrity was determined by the addition of fluorescein isothiocyanate-conjugated Arachis hypogaea (peanut) lectin (Sigma) diluted 1/100 into TRITCconjugated goat anti-mouse IgM/4Ј,6-diamidino-2-phenylindole mixture as described previously (18). Coverslips with the processed sperm were mounted on microscopy slides in VectaShield medium (Vector Laboratories, Burlingame, CA) and examined under a Nikon Eclipse 800 microscope (Nikon Inc., Melville, NY) with epifluorescence and differential interference contrast optics and a CoolSnap HQ CCD camera (Roper Scientific, Tucson, AZ) operated by MetaMorph software (Universal Imaging Corp., Downington, PA).…”

Section: Ms and Ms/ms Analyses Of Permethylated Humanmentioning

confidence: 99%

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Expression of Bisecting Type and Lewisx/Lewisy Terminated N-Glycans on Human Sperm

Pang

Tissot

Drobnis

et al. 2007

Journal of Biological Chemistry

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Human sperm lack major histocompatibility class I molecules, making them susceptible to lysis by natural killer (NK) cells. Major histocompatibility class I negative tumor cells block NK cell lysis by expressing sufficient amounts of bisecting type N-glycans on their surfaces. Therefore, sperm could employ the same strategy to evade NK cell lysis. The total N-glycans derived from sperm were sequenced using ultrasensitive mass spectrometric and conventional approaches. Three major classes of N-glycans were detected, (i) high mannose, (ii) biantennary bisecting type, and (iii) biantennary, triantennary, and tetraantennary oligosaccharides terminated with Lewis x and Lewis y sequences. Immunostaining of normal sperm showed that glycoproteins bearing Lewis y sequences are localized to the acrosome and not the plasma membrane. In contrast, defective sperm showed distinct surface labeling with antiLewis y antibody. The substantial expression of high mannose and complex type N-glycans terminated with Lewis x and Lewis y sequences suggests that sperm glycoproteins are highly decorated with ligands for DC-SIGN. Based on previous studies, the addition of such carbohydrate signals should inhibit antigenspecific responses directed against sperm glycoproteins in both the male and female reproductive systems. Thus, the major N-glycans of human sperm are associated with the inhibition of both innate and adaptive immune responses. These results provide more support for the eutherian fetoembryonic defense system hypothesis that links the expression of carbohydrate functional groups to the protection of gametes and the developing human in utero. This study also highlights the usefulness of glycomic profiling for revealing potential physiological functions of glycans expressed in specific cell types.Studies performed over the past four decades indicate that conditions are not optimal for human sperm in the female reproductive system (1). Women release neutrophils, monocytes, and lymphocytes from the specialized mucosal surface of their cervix during the leukocyte reaction after coitus (2, 3). This sequestration occurs specifically in response to contact with sperm but not seminal plasma (2, 3). Increased immunological awareness related to histocompatibility and its implications for reproduction in eutherians initially raised concerns about how foreign human sperm are tolerated in the female reproductive system (4). Early studies suggested that human sperm express MHC 3 class I molecules that define self, suggesting the possibility that histocompatibility based responses must be suppressed in the female reproductive tract (5). However, more recent studies confirm that sperm precursors down-modulate their MHC class I molecules, yielding mature sperm that are MHC class I negative cells (5). This absence enables these gametes to evade histocompatibility based responses mediated by MHC class I-restricted cytotoxic T lymphocytes (6).This lack of MHC class I molecules does not make sperm completely invulnerable to cell mediated responses...

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Section: Methodsmentioning

confidence: 99%

Section: Ms and Ms/ms Analyses Of Permethylated Humanmentioning

confidence: 99%

Expression of Bisecting Type and Lewisx/Lewisy Terminated N-Glycans on Human Sperm

Pang

Tissot

Drobnis

et al. 2007

Journal of Biological Chemistry

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“…Removal of the beads (together with bound cells) would thus yield a theoretically higher quality sample. This would be especially relevant if the defective sperm cells in question could not be identified by simple visualization (1). Indeed, although spermatozoa from patients possess multiple structural and functional defects, usually evaluated by light microscopy (2), this approach can be limited, because, for example, spermatozoa with normal morphology can, nevertheless, have DNA defects or be positive for apoptotic markers (3).…”

mentioning

confidence: 99%

“…This study also demonstrated that ubiquitinated spermatozoa found in the ejaculate, and which may have escaped phagocytosis, are characterized by major defects in the head or axoneme. Furthermore, more recent studies proposed that this biomarker could be used [a] to assess sperm quality by flow cytometry, with poor quality samples yielding higher ubiquitination, which would translate to higher fluorescence intensities (11), and [b] that the assay could also help select spermatozoa for ART, as a complement for classical separations (1). Surprisingly, results obtained by other authors suggest that sperm ubiquitination positively correlates with normal semen parameters in human sperm (especially morphology), thus implying that there may be a functional role for ubiquitination in normal sperm (14).…”

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confidence: 99%

Characterization of human sperm populations using conventional parameters, surface ubiquitination, and apoptotic markers

Varum

Bento

Sousa

et al. 2007

Fertility and Sterility

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Objective: To directly compare distinct assays proposed to monitor human sperm quality and possibly preselect sperm populations for assisted reproductive technology (ART). Design: Analysis of human sperm sample quality using several methodologies. Setting: Academic and clinical institutions. Patient(s): Samples from consenting patients undergoing routine semen analysis or ART. Interventions: Human sperm samples were analyzed in terms of World Health Organization parameters and processed for annexin V, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling of DNA (TUNEL), and the sperm-ubiquitin tag immunoassay (SUTI). Samples were analyzed both by flow cytometry and fluorescence microscopy. Main Outcome Measure(s): Correlations among apoptotic markers (outer leaflet phosphatidylserine exposure, membrane integrity, and DNA fragmentation), external ubiquitination, and semen parameters in human spermatozoa. Result(s):Nonviable sperm, TUNEL-positive cells, and ubiquitin fluorescence intensity means inversely correlate with semen parameters. Apoptotic markers do not correlate with sperm surface ubiquitination. Normozoospermic samples have a higher number of viable cells and lower DNA fragmentation compared with samples with abnormal parameters. Nonviable sperm are more prevalent in samples with low counts and poor morphology but not low motility. Not all sperm with morphologic abnormalities present surface ubiquitination. Conclusion(s):Sperm quality is inversely correlated with lack of viability, DNA fragmentation, and ubiquitin fluorescence intensity means. However, none of the apoptotic markers correlate with ubiquitin labeling. Elimination of defective sperm cells prior to ART using surface markers (annexin V, ubiquitin) seems unwarranted at this stage. (Fertil Steril 2007;87:572-83.

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“…FC technique would be easily coupled with the analysis of spermatozoa extracellular ubiquitination (eUb) level since this indicator was found to be negatively correlated with their fertilization ability (Sutovsky et al 2002;Hodjat et al 2008). The eUb level determination would then improve the quality control in livestock reproduction and clinical medicine (Ozanon et al 2005).…”

Section: Introductionmentioning

confidence: 99%

Cryopreservation of fluorescence activated cell sorted boar spermatozoa based on extracellular ubiquitination

Petelák¹,

Krylov²

2016

CZECH J ANIM SCI

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ABSTRACT:The present study is focused on the methodology of fluorescence activated cell sorting (FACS) of spermatozoa stained by the antibody against extracellular surface marker ubiquitin (eUb) and subsequent protocol for their long term storage in liquid nitrogen (LN). High level of spermatozoa surface ubiquitination has been previously discussed as a negative quality marker. From a general point of view, any other outer membrane antigen would be compatible with our approach. Regarding our experimental design we found that only those insemination doses with at least 40% of motile spermatozoa after freezing and thawing (F/T) in the egg-yolk medium with lactose are suitable for the subsequent antibody staining and FACS. The sorting rate was sufficient for the preparation of up to 20 spermatozoa aliquots for intracytoplasmic sperm injections (ICSI). Two significantly different groups with good freezability were prepared and stored in LN (0.73% contamination of spermatozoa with high eUb level in non-ubiquitinated group and reversely 6.65% spermatozoa without eUb in highly ubiquitinated group). Sperm viability after FACS varied from 11 to 28% regardless of the used media (P = 0.15). Required viability of F/T sorted spermatozoa was obtained by using Solusem ® extender as a load and collection medium. In this case 12% of viable spermatozoa with progressive motility in low eUb level group and 7% in high eUb level group (P < 0.05) were detected. Our approach allows obtaining sufficient number of viable spermatozoa for subsequent artificial fertilization by ICSI. This procedure could be used for a wide variety of spermatozoa sorting based on different surface markers.

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Clinical adaptation of the sperm ubuquitin tag immunoassay (SUTI): relationship of sperm ubiquitylation with sperm quality in gradient-purified semen samples from 93 men from a general infertility clinic population

Cited by 31 publications

References 29 publications

Expression of Bisecting Type and Lewisx/Lewisy Terminated N-Glycans on Human Sperm

Expression of Bisecting Type and Lewisx/Lewisy Terminated N-Glycans on Human Sperm

Characterization of human sperm populations using conventional parameters, surface ubiquitination, and apoptotic markers

Cryopreservation of fluorescence activated cell sorted boar spermatozoa based on extracellular ubiquitination

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