Tissue-specific functions of individual glutathione peroxidases.

https://onlinelibrary.wiley.com/doi/pdf/10.1002/j.1939-4640.2002.tb02599.x

Sperm chromatin structure assay: its clinical use for detecting sperm DNA fragmentation in male infertility and comparisons with other techniques.

The selenoprotein phospholipid hydroperoxide glutathione peroxidase (PHGPx) changes its physical characteristics and biological functions during sperm maturation PHGPx exists as a soluble peroxidase in spermatids but persists in mature spermatozoa as an enzymatically inactive, oxidatively cross-linked, insoluble protein In the midpiece of mature spermatozoa, PHGPx protein represents at least 50 percent of the capsule material that embeds the helix of mitochondria The role of PHGPx as a structural protein may explain the mechanical instability of the mitochondrial midpiece that is observed in selenium deficiency

https://science.sciencemag.org/content/sci/285/5432/1393.full.pdf

Dual Function of the Selenoprotein PHGPx During Sperm Maturation

Reactive oxygen metabolites are known to disrupt sperm-oocyte fusion, sperm movement, and DNA integrity; however, the relative sensitivities of these elements to oxidative stress are unknown. In this study these factors were assessed in human spermatozoa exposed to increasing levels of oxidative stress achieved through the stimulation of endogenous oxidant generation with NADPH or direct exposure to hydrogen peroxide. At low levels of oxidative stress, DNA fragmentation was significantly reduced while the rates of sperm-oocyte fusion were significantly enhanced. As the level of oxidative stress increased, the spermatozoa exhibited significantly elevated levels of DNA damage (p < 0.001) and yet continued to express an enhanced capacity for sperm-oocyte fusion. At the highest levels of oxidative stress, extremely high rates of DNA fragmentation were observed but the spermatozoa exhibited a parallel loss in their capacities for movement and oocyte fusion. These studies emphasize how redox mechanisms can either enhance or disrupt the functional and genomic integrity of human spermatozoa depending on the intensity of the oxidative stimulus. Because these qualities are affected at different rates, spermatozoa exhibiting significant DNA damage are still capable of fertilizing the oocyte. These results may have long-term implications for the safety of assisted conception procedures in cases associated with oxidative stress.

/pdf/relative-impact-of-oxidative-stress-on-the-functional-57r02fvx60.pdf

Relative Impact of Oxidative Stress on the Functional Competence and Genomic Integrity of Human Spermatozoa

Protamines are the major nuclear sperm proteins. The human sperm nucleus contains two types of protamine: protamine 1 (P1) encoded by a single-copy gene and the family of protamine 2 (P2) proteins (P2, P3 and P4), all also encoded by a single gene that is transcribed and translated into a precursor protein. The protamines were discovered more than a century ago, but their function is not yet fully understood. In fact, different hypotheses have been proposed: condensation of the sperm nucleus into a compact hydrodynamic shape, protection of the genetic message delivered by the spermatozoa, involvement in the processes maintaining the integrity and repair of DNA during or after the nucleohistone-nucleoprotamine transition and involvement in the epigenetic imprinting of the spermatozoa. Protamines are also one of the most variable proteins found in nature, with data supporting a positive Darwinian selection. Changes in the expression of P1 and P2 protamines have been found to be associated with infertility in man. Mutations in the protamine genes have also been found in some infertile patients. Transgenic mice defective in the expression of protamines also present several structural defects in the sperm nucleus and have variable degrees of infertility. There is also evidence that altered levels of protamines may result in an increased susceptibility to injury in the spermatozoan DNA causing infertility or poor outcomes in assisted reproduction. The present work reviews the articles published to date on the relationship between protamines and infertility.

/pdf/protamines-and-male-infertility-2zxvclca2k.pdf

Protamines and male infertility.

Mammalian spermatozoa undergo maturation as they pass through the epididymis. Maturation is accompanied by the oxidation of thiols to disulfides. Disulfides are probably involved in sperm chromatin condensation and tail structure stabilization. In this work, we used the fluorescent thiol-labeling agent monobromobimane to determine the changes occurring in thiols and disulfides in rat sperm heads and tails during maturation. Spermatozoa were obtained from testis, epididymis (caput, corpus, cauda, and vas deferens), and ejaculate. Intact spermatozoa were labeled with monobromobimane, with or without pretreatment with dithiothreitol. Labeling was evaluated microscopically, and quantitative analysis was carried out spectrofluorimetrically with labeled globin used as a standard. Samples were also analyzed by gel electrophoresis. The total amount of thiols and disulfides remained the same during the entire period of sperm maturation (26 +/- 0.5 nmoles thiols + disulfides/10(6) spermatozoa). However, the reactive thiols decreased markedly between the corpus and the cauda (from greater than 90% of total in testis and 75% in corpus to about 25% in cauda), with little or no further change in vas deferens and ejaculated sperm. Trypsin treatment followed by sucrose gradient was used to separate the heads from the tails. Thiols comprised 84% of the total SH + SS in the heads and 74% in the tails of caput spermatozoa, decreasing to 14% and 45%, respectively, in cauda sperm. Thus, the decrease in reactive thiols involved both heads and tails-oxidation to disulfides being very marked in the head. Electrophoresis revealed that oxidation of thiols to disulfides occurred in many protein fractions during maturation in the epididymis.

Dynamics of the thiol status of rat spermatozoa during maturation: analysis with the fluorescent labeling agent monobromobimane.

The thiol-disulfide status in proteins of human spermatozoa categorized as normozoospermic, teratozoospermic and asthenozoospermic was examined. Washed spermatozoa were incubated with or without dithiothreitol (DTT) to reduce disulfides (SS) to thiols (SH), and then labelled with the specific fluorescence thiol labelling agent monobromobimane (mBBr). The SH and SS in intact labelled spermatozoa were evaluated by fluorescence microscopy and by flow cytometry analysis; mBBr-labelled spermatozoa were solubilized and sperm proteins analysed by gel electrophoresis (SDS-PAGE for non-basic, whole sperm proteins and acid urea-PAGE for sperm nuclear basic proteins). Microscopy and flow cytometry showed that normozoospermic samples (having normal sperm count, morphology and motility) contained both SH and SS, with more SS than SH. Heterogeneity in the proportion of SH/(SH plus SS) was observed among spermatozoa within the ejaculates. The total SH plus SS was similar among the ejaculates, with some variability in SH/(SH plus SS) noted among them. SDS-PAGE of solubilized normozoospermic cells showed differences in the SH and SS content of the protein bands. Acid urea-PAGE of basic proteins isolated from normozoospermic samples showed protamines P1 and P2 and traces of non-protamine basic proteins. P1 and P2 contained SH and SS, with variability in SH/(SH plus SS) observed among the samples. Teratozoospermic samples (in which > 90% of the spermatozoa exhibited abnormal morphology) were similar in thiol-disulfide status to normozoospermic samples, but contained non-protamine basic proteins in addition to protamines.(ABSTRACT TRUNCATED AT 250 WORDS)

/pdf/thiol-disulfide-status-of-human-sperm-proteins-19mzbg26v3.pdf

Thiol-disulfide status of human sperm proteins.

The fluorescent labeling agent monobromobimane (mBBr) was used to label thiols and disulfides (after reduction of sperm disulfides by dithiothreitol) in intact spermatozoa. Bimane-labeled sperm of several mammalian species were analyzed by flow cytometry (FCM) and examined by fluorescent microscopy. FCM analysis showed sperm thiol oxidation to disulfides during epididymal maturation. FCM of labeled mature spermatozoa showed differences among species in the sperm thiol content. Heterogeneity in thiol content of sperm within individual samples was also observed. In addition, FCM patterns showed heterogeneity among and within samples in the content of disulfides and their resistance to reduction. FCM analysis reflected the microscopic appearance of the labeled spermatozoa. FCM analysis of bimane-labeled spermatozoa offers a convenient method for the study of sperm thiol-disulfide status and permits detection of sperm subpopulations within an individual sample. FCM analysis of mBBr-labeled spermatozoa may serve as a test to evaluate sperm quality.

Sperm analysis by flow cytometry using the fluorescent thiol labeling agent monobromobimane

Mammalian spermatozoa mature while passing through the epididymis. Maturation is accompanied by thiol oxidation to disulfides. In rats, sperm become motile and fertile in the cauda. We have previously demonstrated that rat caput sperm contain mostly thiols and that upon passage from the corpus to the cauda epididymidis, sperm protein thiols are oxidized. The present work was undertaken to study the role of the regions of the epididymis in sperm maturation as reflected in the thiol status, fertility, and motility of the spermatozoa. The distal caput epididymidis of mature albino rats was ligated on one side. After 5 days, sperm were isolated from the ligated caput and from caput and cauda of the control side. Thiol groups in sperm, epididymal luminal fluid (EF), and epididymal tissue were labeled using the fluorescent thiol-labeling agent monobromobimane. After ligation, changes were observed in a) sperm proteins, sperm nuclear proteins, and epididymal fluid by electrophoresis; b) epididymal tissues by histochemistry; c) progressive motility by phase microscopy; and d) fertilizing ability after insemination into uteri of immature females. We found that after ligation, caput sperm thiols, especially protamine thiols, are oxidized, rendering them similar to mature sperm isolated from the cauda epididymidis. Spermatozoa from ligated caput epididymidis gain progressive motility and partial fertilizing ability. Morphology of epithelial cells of ligated caput is similar to that of cauda cells. However, other changes in caput EF and epithelium induced by ligation render the ligated caput epididymidis different from either control caput or cauda. Hence, sperm thiol oxidation, along with the development of fertilizing ability, can occur in sperm without necessity for sperm transit through the corpus and cauda epididymidis.

/pdf/effects-of-caput-ligation-on-rat-sperm-and-epididymis-2b00schwpu.pdf

Effects of caput ligation on rat sperm and epididymis: protein thiols and fertilizing ability.

The passage of spermatozoa along the epididymis is characterized by a gradual stabilization of intracellular organelles mainly through the oxidation of thiol groups. In this study, we examined the relationship between the thiol-disulfide status of human spermatozoa (using a specific fluorescent probe, monobromobimane) and routine semen analysis parameters. Fluorescence intensity was measured by spectrofluorimeter and its frequency distribution within samples, using a fluorescence-activated cell sorter. The mean proportion of reactive thiols SH/(SS + SH) in 29 semen samples was 29.8% +/- 2.5%. When comparing thiol labeling patterns, oligozoospermic samples differed from normozoospermic ones (P less than 0.05). However, within the normozoospermic group, no correlation was found between thiol-labeling patterns and routine sperm parameters or fertilizing capacity in vitro. No difference in thiol labeling patterns was found between "swim-up" and "whole semen" preparations.

Thiol status in human sperm.

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