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The use of electronic devices such as mobile phones has had a long stretch of rapid growth all over the world. Therefore, exposure to radio frequency radiation (RFR) has increased enormously. Here, we aimed to assess the balance between cell death and proliferation and also investigate the involvement of the JNK/p38 MAPK signaling pathway in the testis of rats exposed to 900 MHz RFR in acute and chronic periods (2 h/day, 5 days/week) for 1 or 10 weeks, respectively. The expression of proliferating cell nuclear antigen (PCNA), Bcl-xL, cleaved caspase-3, phosphorylated-JNK (p-JNK), and phosphorylated-p38 (p-p38) was analyzed in line with histopathology and TUNEL analysis in rat testis. There were no histopathological differences between sham and RFR groups in the acute and chronic groups. PCNA expression was not altered between groups in both periods. However, alterations for cleaved caspase-3 and Bcl-xL were observed depending on the exposure period. TUNEL analysis showed a significant increase in the RFR group in the acute period, whereas no difference in the chronic groups for the apoptotic index was reported. In addition, both p-p38 and p-JNK protein expressions increased significantly in RFR groups in both periods. Our study indicated that 900 MHz RFR might result in alterations during acute period exposure for several parameters, but this can be ameliorated in the chronic period in rat testis. Here, we also report the involvement of the p38/JNK-mediated MAPK pathway after exposure to 900 MHz RFR. Hence, this information might shed light in future studies toward detailed molecular mechanisms in male reproduction and infertility.

The study aims to investigate first the presence of Syncytin 2 and its receptor, MFSD2, in human sperm, and second whether the expressions of Syncytin 1, Syncytin 2, and their receptors, SLC1A5 and MFSD2, differ between normozoospermic, asthenozoospermic, oligozoospermic, and oligoasthenozoospermic human sperm samples.The localization patterns and expression levels of syncytins and their receptors were evaluated in normozoospermic (concentration = 88.9 ± 5.5 × 10Syncytins and their receptors visualized by immunofluorescence showed similar staining patterns with slight staining of the tail in all spermatozoa regardless of normozoospermia, asthenozoospermia, oligozoospermia, or oligoasthenozoospermia. The localization patterns were categorized as equatorial segment, midpiece region, acrosome, and post-acrosomal areas. The combined staining patterns were also detected as acrosomal cap plus post acrosomal region, the midpiece plus equatorial segment, and midpiece plus acrosomal region. However, some sperm cells were categorized as non-stained. Both syncytin proteins were most intensely localized in the midpiece region, while their receptors were predominantly present in the midpiece plus acrosomal region. Conspicuously, syncytins and their receptors showed decreased expression in asthenozospermic, oligozoospermic, and oligoasthenozoospermic samples compared to normozoospermic samples.The expression patterns of HERV-derived syncytins and their receptors were identical regardless of the spermatozoa in men with normozoospermia versus impaired semen quality. Further, asthenozoospermia, oligozoospermia, and oligoasthenozoospermia as male fertility issues are associated with decreased expression of both syncytins and their receptors.

CTCFL is expressed in testis, oocytes and embryonic stem cells, and is aberrantly expressed in malignant cells, and is classified as a cancer-testis gene. We have previously shown by using a tetracycline-inducible Ctcfl transgene that inappropriate expression of Ctcfl negatively impacts fetal development and causes early postnatal lethality in the mouse. The affected pups displayed severe vascular abnormalities and localized hemorrhages in the brain evocative of cerebral cavernous malformations (CCM) and arteriovenous malformations (AVM) in humans. Thus, we aim to analyze; a) the presence of CCM-related proteins CCM1/KRIT1, CCM2/malcavernin and CCM3/PDCD10 in Ctcfl transgenic animals and, b) whether there is CTCFL expression in human CCM and AVM tissues. Ctcfl transgenic animals exhibited increased CD31 expression in vascular areas of the dermis and periadnexal regions but no difference was observed for vWF and alpha-SMA expressions. CCM-related proteins CCM1/KRIT1, CCM2/malcavernin and CCM3/PDCD10 were aberrantly expressed in coronal sections of the head in transgenic animals. We also observed CTCFL expression in human CCMs and AVMs. The induced expression of CTCFL resulting in vascular brain malformations in mice combined with the presence of CTCFL in human vascular malformations provide new insights into the role of this gene in vascular development in humans.

Advanced modalities in the treatment of prostate cancer, an important health problem among men, draw attention to the field of cancer immunotherapy. Although the immune system holds promise for prostate cancer treatment, the biological processes associated with prostate cancer cells and immune cells have not yet been fully elucidated. The prostate cancer microenvironment is a complex system containing a large number of immune cells. Among multiple immune cells, natural killer (NK) cells have enormous potential in targeting various cancer cells. NK cells, which are an important part of the innate immune system, play a very important role as a first-line defense against prostate cancer cells. Various strategies have been adopted by researchers to increase the efficiency of NK cells. However, there are many difficulties to increase the therapeutic relevance of NK cells. This review will provide a perspective on the phenotypic and functional properties of NK cells, molecular links between NK cells and prostate cancer cells, and the therapeutic and prognostic significances of NK cells in both advanced hormone naïve prostate cancer and castration-resistant prostate cancer. © 2021 Elsevier Inc. All rights reserved.

The circadian system regulates the daily temporal organization in behavior and physiology, including neuroendocrine rhythms and reproduction. Modern life, however, increasingly impacts this complex biological system. Due to limitations of working with human subjects exposed to shift work schedules, most chronoregulation research has used rodent models. Recent publications in these model systems have emphasized the negative effects of circadian rhythm disruption on both female and male reproductive systems and fertility. Additionally, there is growing concern about the long-term effects of circadian rhythm disruptions during pregnancy on human offspring and their descendants as circadian regulation during pregnancy can also alter epigenetic programing in offspring. However, to truly know if such concerns apply to humans will require retrospective and prospective human studies. Therefore, this review will highlight the latest available evidence regarding potential effects of chronodisruption on both female and male reproductive systems. Additionally, it presents a comprehensive summary of transgenerational and epigenetic effects on adult offspring that result from maternal chronodisruption.

Excessive levels of free radicals diminish the functional integrity of spermatozoa. Levels of reactive oxygen species (ROS) produced by spermatozoa were negatively correlated with the quality of sperm in the original semen. “Intrinsic,” “extrinsic,” and “iatrogenic” sources of ROS production were identified within semen: intrinsic sources of ROS in semen are morphologically abnormal and arrested-maturity spermatozoa and leukocytes. Poor sperm quality showing attributes of arrested sperm maturation is linked to increased ROS generation as a consequence of excess residual cytoplasm related to the arrest of cytoplasmic extrusion in terminal spermiogenesis. In the past 30 years, the Huszar lab has studied several key events of sperm maturation, including cytoplasmic extrusion and expression of the HspA2 chaperone protein. In experiments related to sperm function and fertilizing potential, it has been established that, simultaneously with cytoplasmic extrusion during terminal spermiogenesis, there is a remodeling of the plasma membrane that facilitates the formation of the zona pellucida and hyaluronic acid (HA)-binding sites. The studies with HA immobilized to glass slides or Petri dishes showed that sperm firmly bind to HA. However, not all sperm exhibited HA-binding ability. These data supported the hypothesis that the ability of sperm-HA binding is related to sperm cellular maturity. Although, recent literature on the efficacy with HA selection of human sperm reported a lack of efficacy but with the caution that is either inadequately powered or of low-quality, HA-mediated sperm ICSI selection may increase fertilization rates and paternal contribution of sperm to the embryo even if the man to be treated is affected by seminal ROS production. Indeed, well-designed new studies are needed to evaluate this aspect in terms of potential evaluation and treatment of subfertile men with ROS-affected semen utilizing HA.

Despite numerous advances in the field of cancer cell biology, the origin of cancer cells and the systemic disruptions in the cancer process of normal cells have not yet been fully identified. However, we certainly know that cancer cells carry genetic and molecular similarities of the early embryonic development indicating an interesting relationship between “birth” and “death.” During the embryonic development process, the software of system codes to determine cell fate has been designed. Then, cells containing embryonic residues can be directed as a normal cell, cancer cell, or a stem cell. The transcription factor Yin Yang 1 (YY1) has been reported to play critical roles in many biological processes, such as the embryonic development, cell fate specification, regulation of pluripotency, and oncogenesis. In view of these activities, YY1 has been implicated in both the embryonic development and oncogenesis by acting as a bridge linking these two important biological processes. Therefore, in this review, we primarily focus on YY1 investigations linking the embryonic development, oncogenesis, and stem cell biology. © 2021 Elsevier Inc. All rights reserved.

CTCFL, a paralog of CTCF, also known as BORIS (brother of regulator of imprinted sites), is a testis-expressed gene whose function is largely unknown. Its product is a cancer testis antigen (CTA), and it is often expressed in tumor cells and also seen in two benign human vascular malformations, juvenile angiofibromas and infantile hemangiomas. To understand the function of Ctcfl, we created tetracycline-inducible Ctcfl transgenic mice. We show that Ctcfl expression during embryogenesis results in growth retardation, eye malformations, multiorgan pathologies, vascular defects, and neonatal death. This phenotype resembles prior mouse models that perturb the transforming growth factor β (TGFB) pathway. Embryonic stem (ES) cells with the Ctcfl transgene reproduce the phenotype in ES cell-tetraploid chimeras. Transcriptome sequencing of the Ctcfl ES cells revealed 14 genes deregulated by Ctcfl expression. Bioinformatic analysis revealed the TGFB pathway as most affected by embryonic Ctcfl expression. Understanding the consequence of Ctcfl expression in nontesticular cells and elucidating downstream targets of Ctcfl could explain the role of its product as a CTA and its involvement in two, if not more, human vascular malformations.

The aim of this study was to assess the effect of oral magnesium supplementation (Mg-supp) on blood pressure (BP) and possible mechanism in nitric oxide synthase (NOS) inhibition-induced hypertension model. Hypertension and/or Mg-supp were created by N-nitro-l-arginine methyl ester (25 mg/kg/day by drinking water) and magnesium-oxide (0.8% by diet) for 6 weeks. Systolic BP was measured weekly by tail-cuff method. The effects of hypertension and/or Mg-supp in thoracic aorta and third branch of mesenteric artery constriction and relaxation responses were evaluated. NOS-inhibition produced a gradually developing hypertension and the magnitude of the BP was significantly attenuated after five weeks of Mg-supp. The increased phenylephrine-induced contractile and decreased acetylcholine (ACh)-induced dilation responses were found in both artery segments of hypertensive groups. Mg-supp was restored ACh-relaxation response in both arterial segments and also Phe-constriction response in thoracic aorta but not in mesenteric arteries. The contributions of NO, prostaglandins and K(+) channels to the dilator response of ACh were similar in the aorta of all the groups. The contribution of the NO to the ACh-mediated relaxation response of mesenteric arteries was suppressed in hypertensive rats, whereas this was corrected by Mg-supp. The flow-mediated dilation response of mesenteric arteries in hypertensive rats failed and could not be corrected by Mg-supp. Whereas, vascular eNOS protein and magnesium levels were not changed and plasma nitrite levels were reduced in hypertensive rats. The results of this study showed that Mg-supp lowered the arterial BP in NOS-inhibition induced hypertension model by restoring the agonist-induced relaxation response of the arteries.

PURPOSE: The purpose of this study is to investigate whether erythrocyte-sperm separation medium (ESSM) has effects on human sperm motility, morphology, viability, membrane maturity, acrosome integrity, and nuclear attributes before and after cryopreservation. METHODS: Semen samples from normozoospermic (n = 36) and oligozoospermic (n = 9) patients were analyzed. Samples from the same patient were divided into three aliquots: group 1 and group 2 were resuspended in sperm washing media and ESSM, respectively. Group 3 was resuspended in ESSM with blood sample to mimic the extensive number of erythrocytes in the testicular sperm extraction (TESE) material. All groups were evaluated for sperm concentration, motility, Kruger/Tygerberg strict morphology, viability by eosin-nigrosin staining, membrane maturity by hyaluronic acid-binding assay (HBA), acrosomal integrity by Pisum sativum lectin staining, chromatin maturity by aniline blue staining, and DNA integrity by TUNEL assay before and after cryopreservation. RESULTS: No significant difference was determined between ESSM-treated and ESSM-untreated sperm samples for the sperm parameters tested (p > 0.05). After cryopreservation, total sperm motility and viability decreased regardless of ESSM used. The percentages of sperm with Tygerberg normal morphology, intact acrosome, and HA-bound sperm were found to be lower in oligozoospermic samples before cryopreservation in each group. However, no statistically significant differences were found between oligozoospermic and normozoospermic samples when all groups were compared. Thus, ESSM treatment did not cause a significant change on sperm motility, normal morphology, viability, HA-binding capacity, chromatin maturity, and DNA fragmentation. CONCLUSION: ESSM can enhance the efficiency of sperm retrieval protocol and can also decrease the time required to collect spermatozoa while not affecting sperm morphogenetic properties.

Our aim was the development of a safe sperm cryopreservation New Media (NM), composed of consistent and reproducible components devoid of any animal origin, and evaluation of NM in terms of its effect on sperm structure and function as compared to regularly used yolk media (TYM) (Irvine Scientific).We evaluated patient semen samples and cryopreserved them in duplicates in either NM or TYM. The samples were cryopreserved for either a short term of 1 week or long term of 1 month prior to thawing. The parameters investigated include sperm motility via computer-assisted semen analysis (CASA), sperm concentration, and sperm biomarkers that promote paternal contribution of spermatozoa to fertilization including hyaluronic acid binding, chromatin maturity, apoptotic markers, cytoplasmic retention, and sperm DNA integrity.As compared to TYM, NM was equally capable of sperm cryopreservation with both short-term and long-term storage in media, and after freeze-thaw and gradient processing of sperm. HA binding of sperm was comparable post thaw in both NM and yolk media. There are also no differences observed between the samples cryopreserved in NM or TYM in terms of their aniline blue staining, CK immunocytochemistry, caspase 3 immunostaining, or DNA nick translation.NM has the advantage of being xeno-free, yet in preservation of sperm motility and other sperm attributes, the NM is as effective as the TYM.

Recent developments in the field of insecticide exposure have led to a renewed interest in alternative antioxidant therapy. The present study was to investigate the neuroprotective role of syringic acid (SA, 25 mg/kg/day) on the neurotoxicity and oxidative damage induced by deltamethrin (DTM, 1.28 mg/kg/day during two months) in CA1/3 pyramidal neurons. Animals were divided into 4 groups (n = 16/group) (250–270 g) for control, DTM, SA and DTM + SA. DTM and SA were administered by oral gavage daily. Rats that were given sub-chronic DTM had revealed a significant increase in caspase-3 levels, impaired recognition memory, reduced antioxidant activity and enhanced free radicals in the hippocampus. The results showed that SA ameliorated neurobehavioral alterations, reduced reactive oxygen/nitrogen species, pyknosis in the CA1/3 and increased antioxidant enzyme activity. In conclusion, SA (25 mg/kg/day) had potential neuroprotective and therapeutic impacts against sub-chronic DTM exposure via its antioxidant and antiapoptotic efficacy. Therefore, it can be used as a neuroprotective natural plant-derived agent against DTM-induced neurotoxicity.

Syncytins belong to the Human Endogenous Retrovirus family. The syncytin-1 receptor, SLC1A5, and syncytin-2 receptor, MFSD2, interact with their respective syncytin proteins to induce syncytiotrophoblast formation. However, there is no information about syncytins in gestational diabetic placenta. Therefore, we studied the expression and localization of syncytins and their receptors during normal placental development and in gestational diabetic placenta.Immunohistochemistry and Western-blot methods were performed with antibodies against syncytin-1, syncytin-2, SLC1A5 and MFSD2 in human first trimester placental tissues, normal term and gestational diabetic placentas. Syncytin-1, syncytin-2 and MFSD2 mRNA transcripts were determined by qRT-PCR in normal and diabetic term placentas.Cytoplasmic syncytin-1, syncytin-2, SLC1A5 and MFSD2 immunoreactions were observed in the trophoblastic layers in all placental samples. Some of the stromal cells showed strong cytoplasmic punctate staining. There were significantly weak syncytin-2 and MFSD2 immunoreaction intensities in diabetic placentas by ImageJ analysis, in parallel with decreased syncytin-2 and MFSD2 proteins in diabetic placentas by Western-blot. Protein expression of SLC1A5 increased dramatically in early pregnancy compared to term placenta. Syncytin-1, syncytin-2 and MFSD2 mRNA transcripts showed similar relative expression pattern by qRT-PCR.Syncytins were localized not only in cytotrophoblast cells and the basement membrane of the syncytiotrophoblast but also in the apical microvillous membrane and cytoplasm of syncytiotrophoblast, and some of the stromal cells and endothelium. Decreased syncytin-2 and MFSD2 proteins in gestational diabetic placentas might cause abnormal syncytiotrophoblast formation and possibly be involved in the pathology. Therefore, our study highlights an important potential relationship between syncytins and gestational diabetic placenta.

Environmental exposure to pesticides may cause serious health risks including fertility and reproductive function. The aim of this study was to highlight whether there is a relationship between exposure to abamectin and male fertility parameters of farmworkers. Twenty male farmworkers who were using abamectin and 20 men not exposed to pesticides were recruited as experimental and control groups, respectively. Semen analysis, molecular markers of sperm maturity and serum reproductive hormone levels were evaluated. In experimental group, high plasma abamectin levels were detected. These men have decreased sperm motility. Moreover, diminished molecular markers of sperm maturity, such as decreased hyaluronic acid (HA) binding of sperm, increased numbers of aniline blue positive sperm and increased percentage of creatine kinase (CK) positive sperm, were observed in abamectin-exposed men. Their serum testosterone, LH and FSH levels did not change significantly. We conclude that exposure to abamectin may impair male fertility by effecting semen quality.

Despite the known adverse effects of abamectin pesticide, little is known about its action on male fertility. To explore the effects of exposure to abamectin on male fertility and its mechanism, low (1 mg/kg/day) and high dose (4 mg/kg/day) abamectin were applied to male rats by oral gavage for 1 week and for 6 weeks. Weight of testes, serum reproductive hormone levels, sperm dynamics and histopathology of testes were used to evaluate the reproductive efficiency of abamectin-exposed rats. Abamectin level was determined at high concentrations in plasma and testicular tissues of male rats exposed to this pesticide. The testes weights of animals and serum testosterone concentrations did not show any significant changes after abamectin exposure. Abamectin administration was associated with decreased sperm count and motility and increased seminiferous tubule damage. In addition, significant elevations in the 4-hydroxy-2-nonenal (4-HNE)-modified proteins and poly(ADP-ribose) (PAR) expression, as markers for oxidative stress and poly(ADP-ribose) polymerase (PARP) activation, were observed in testes of rats exposed to abamectin. These results showed that abamectin exposure induces testicular damage and affects sperm dynamics. Oxidative stress-mediated PARP activation might be one of the possible mechanism(s) underlying testicular damage induced by abamectin. (C) 2011 Elsevier Inc. All rights reserved.

The aim of the study was to investigate the arrangements and related localization patterns of different collagen types in the stroma of placental stem villi by immunohistochemistry and electron microscopy. A total of 14 normal human term placental tissue samples were studied. Immunohistochemistry was performed in order to localize collagen types I, III, IV, V and cytokeratin 7 on tissue sections. Parallel tissue samples were examined by transmission electron microscopy. Semi-quantitative analysis of immunolabeling intensities was also performed to determine the distribution of fibers in stem villi stroma. All. collagen types, especially collagen type V, were strongly immunopositive in the triangular areas of the stem villi stroma. However, there was no collagen type I or type III immunolabeling in the subtrophoblastic regions. Membrane collagen type IV immunolabeling was also observed in the stroma of stem villi. Ultrastructurally, collagen fibers showed different configurations in cross, longitudinal, circular, oblique and parallel directions compared to the villous axis. We conclude that the organization of collagen fiber bundles in stem villi shows a very specific arrangement: a compact coat formed by fibrillar bundles between the vascular wait and extravascular stroma of stem villi correlated with the functional activity. (C) 2007 Elsevier GmbH. All rights reserved.

Mammalian target of rapamycin (mTOR) signaling serves as a central regulator of cell growth, proliferation, and survival by interacting with various proteins. To date, few studies implicated mTOR in placenta. Human placenta in gestational diabetes mellitus (GDM) shows several alterations including villous immaturity, impaired placental function, and overgrowth. Hence, we aimed to investigate the expression of mTOR, phospho-mTOR (p-mTOR), and the 2 phosphorylated downstream targets of mTOR, ribosomal protein S6 kinase 1 (p-p70S6K), and eukaryotic initiation factor 4E-binding protein 1 (p-4EBP1) in normal term and gestational diabetic human placentas. Immunohistochemistry and Western blot were performed with antibodies against mTOR, p-mTOR, p-p70S6K, and p-4EBP1 (Thr37/46) in normal and diabetic placentas (n = 6 each) and quantified by ImageJ. All mTOR pathway components that we studied were immunolocalized in both normal and diabetic placenta groups. Syncytiotrophoblast and the vascular wall in villi displayed cytoplasmic mTOR and p-mTOR (S2448) immunoreactivities in all placenta samples. However, increased expression of p70S6K in syncytiotrophoblast and p-4EBP1 (Thr37/46) in villous stromal cells was observed in gestational diabetic placentas. Western blot analysis also confirmed the statistically significant increase in p-p70S6K (T389) expression in diabetic placentas. The altered expression of downstream components of mTOR signaling in gestational diabetic placentas suggests an involvement of mTOR activity in the placental pathology of GDM. However, whether increased nutrient transport via this pathway will stimulate fetal and placental overgrowth is still unknown. Although this is a descriptive study, further studies with a functional analysis to highlight the molecular mechanisms underlying this placental pathology are proposed.

Objective To explore the dimensional attributes of haploid and disomic X-bearing and Y-bearing spermatozoa. Design Morphometric evaluation of more than 2,000 X-bearing and Y-bearing spermatozoa after identification of the genotype with fluorescence in situ hybridization. Setting Academic clinical and research andrology laboratory. Main Outcome Measure(s) Sperm head area, perimeter, long axis, short axis, shape factor, elliptical form factor (long axis/short axis), and tail length. Result(s) We found no differences in dimensions or dimensional distributions between X-bearing and Y-bearing spermatozoa, whether in the native or the decondensed state, or in oligozoospermic or normozoospermic men. There were inconsistent differences and a 70% overlap in the dimensions of haploid and disomic spermatozoa. The other 30% of sperm with disomic nuclei were either smaller or larger compared to haploid spermatozoa. Conclusion(s) There are no differences, or distinguishing characteristics, in dimensions or dimensional distributions between X-bearing and Y-bearing spermatozoa. Dimensional attributes do not discriminate between dysomic and haploid spermatozoa.

To develop a method for delayed assessment of sperm motility, after shipment of semen to a remote laboratory. Sperm in semen were labeled with the MitoTracker(®) Red CM-H(2)XRos reagent, and fixed with 3.7 % formaldehyde by the laboratory technicians at the origin of the semen. This treatment reflected well sperm mitochondrial activity, and the MitoTracker(®) signal was related to sperm motility and velocity for 2-3 days following ejaculation.Sperm motility and velocity were evaluated manually and by computer assisted semen analysis (CASA), respectively. Fluorescence assessment of individual sperm was carried out with the computer assisted Metamorph v4.6.9 program. Emission levels of MitoTracker(®) spermatozoa were studied in room temperature and cooled semen, or in the respective room temperature swim-up sperm fractions following ejaculation, and on the second day (N = 103 samples, 89 men) and third day (N = 10 samples, 8 men).Sperm with optical density (O.D.) ≥0.7 showed close correlations with ejaculatory sperm motility and velocity even after second day (r = 0.92, p 0.001, N = 103 samples). Further, the multiple of sperm motility and velocity was also related to the proportion of high MitoTracker(®) reagent emission sperm (r = 0.83, p 0.001, N = 103 samples). MitoTracker(®) dye fluorescence on the second day accurately reflected the ejaculatory sperm motility (r = 0.90, p 0.001). Thus, a shipping delay would not adversely affect the results.The delayed assessment of sperm motility in samples treated with MitoTracker(®) Red CM-H(2)XRos reagent and shipped to remote laboratory truly reflects the level of sperm motility at the time of the ejaculation.

WOS:000337285300004 PubMed: 24077441 In mammalian species, acquisition of sperm fertilization competence is dependent on the phenomenon of sperm capacitation. One of the key elements of capacitation is protein tyrosine phosphorylation (TP) in various sperm membrane regions. In previous studies performed, the pattern of TP was examined in human sperm bound to zona pellucida of oocytes. In the present comparative study, TP patterns upon sperm binding to the zona pellucida or hyaluronic acid (HA) were investigated in spermatozoa arising from the same semen samples. Tyrosine phosphorylation, visualized by immunofluorescence, was localized within the acrosomal cap, equatorial head region, neck, and the principal piece. Tyrosine phosphorylation has increased in a time-related manner as capacitation progressed, and the phosphorylation pattern was identical within the principal piece and neck, regardless of the sperm bound to the zona pellucida or HA. Thus, the data demonstrated that the patterns of sperm activation-related TP were similar regardless of the spermatozoa bound to zona pellucida or HA. Further, sperm with incomplete development, as detected by excess cytoplasmic retention, failed to exhibit TP.

In this chapter, the laboratory methods for detection of sperm biomarkers that are aimed at identifying arrested sperm development are summarized. These probes include sperm staining with aniline blue for persistent histones, representing a break in the histone-transition protein-protamine sequence, immunocytochemistry with cytoplasmic sperm proteins, highlighting cytoplasmic retention during spermiogenesis, DNA nick translation testing for DNA chain fragmentation due to various reasons, for instance low HspA2 chaperone protein levels, and consequential diminished DNA repair. Finally, we briefly provide references on our work on sperm hyaluronan binding, abnormal Tybergerg sperm morphology, and the increased levels of chromosomal aneuploidies in sperm with developmental arrest. A very interesting aspect of the biomarker field is the discovery (Sati et al, Reprod Biomed Online 16:570-579, 2008) that the various nuclear and cytoplasmic defects detected by the biomarkers are related, and may simultaneously occur within the same spermatozoa as evidenced by a combination of biomarkers, such as aniline blue staining (persistent histones) coupled with cytoplasmic retention, DNA fragmentation, Caspase-3, Tygerberg abnormal morphology, and increased levels of chromosomal aneuploidies. We show examples of this80% overlap in staining patterns within the same spermatozoa.

In this chapter, the laboratory methods for detection of sperm biomarkers that are aimed at identifying arrested sperm development are summarized. These probes include sperm staining with aniline blue for persistent histones, representing a break in the histone-transition protein-protamine sequence, immunocytochemistry with cytoplasmic sperm proteins, highlighting cytoplasmic retention during spermiogenesis, DNA nick translation testing for DNA chain fragmentation due to various reasons, for instance low HspA2 chaperone protein levels, and consequential diminished DNA repair. Finally, we briefly provide references on our work on sperm hyaluronan binding, abnormal Tybergerg sperm morphology, and the increased levels of chromosomal aneuploidies in sperm with developmental arrest. A very interesting aspect of the biomarker field is the discovery (Sati et al, Reprod Biomed Online 16:570-579, 2008) that the various nuclear and cytoplasmic defects detected by the biomarkers are related, and may simultaneously occur within the same spermatozoa as evidenced by a combination of biomarkers, such as aniline blue staining (persistent histones) coupled with cytoplasmic retention, DNA fragmentation, Caspase-3, Tygerberg abnormal morphology, and increased levels of chromosomal aneuploidies. We show examples of this >80% overlap in staining patterns within the same spermatozoa.

In the past two decades, the Huszar laboratory focused upon the objective biochemical markers of sperm cellular maturity and development, and the characteristics of sperm in which cellular development is arrested during spermatogenesis and spermiogenesis. In assays directed to sperm cellular maturity/development, several sperm attributes were studied, including (a) excess cytoplasm (arrested cytoplasmic extrusion), (b) low expression of the HspA2 chaperone protein, a protein of the synaptonemal complex that supports meiosis, as well as the delivery of essential components in developing sperm, such as DNA repair enzymes or chromatin elements, (c) sperm shape (affected by cytoplasmic extrusion and insertion of the tail) according to the criteria detected by the Metamorph computer-assisted program or by the Tygerberg strict morphology, and (d) the relationship between sperm nuclear events (such as presence of chromosomal aneuploidies and excess residual histones). The primary focus of this chapter is related to recent reports that studied human sperm chromatin complexity and sperm HA-binding capacity. The studies suggest that the numerical chromosomal aberrations and persistent histones, i.e., errors in the spermatogenetic and spermiogenetic phases of male germ cell development, are associated. This chromatin deficiency associated with high persistent histones and relative lack of protamines affects DNA folding and renders the sperm DNA more vulnerable. It appears that these two phenomena correspond with each other and, thus, is responsible for defining sperm DNA integrity, fertility, and sterility.

The preventive and therapeutic effects of eicosapentaenoic acid (EPA) on diet-induced hyperlipidemia in rabbits have been investigated. Eighteen New Zealand rabbits were randomly divided into three groups of 6 subjects each; experimental group-I (EG-I) was administered a cholesterol rich diet, experimental group-II (EG-II) was treated with EPA (300 mg/kg/d) following a cholesterol-rich diet and the control group (CG) had a standard diet. Blood samples were collected at day 0 and at the 4th and 12th weeks of EG-II to obtain serum levels of total cholesterol (TC), high density lipid-cholesterol (HDL-C), low density lipid-cholesterol (LDL-C) and triglyceride (TG). From each group tissue samples were collected from the carotid artery for immunohistochemistry and electron microscopy. Our results showed that EPA could significantly lower (p0.001) serum TC, LDL-C, HDL-C and TG levels with a reduction of 35%; 55%; 44% and 51%, respectively. Scanning and transmission electron microscopy results revealed that endothelial damage was more prominent in EG-I when compared to EG-II. The ruptured endothelial lining and damaged cellular surface was increased in EG-I when compared to EG-II. Ultrastructural observations showed that after EPA treatment, the degeneration and cellular surface damage on the endothelium were also decreased. These biochemical and ultrastructural results suggest that EPA is a potential drug which significantly lowers the serum lipid profile and partially repairs endothelial dysfunction due to hyperlipidemia.

Pulmonary surfactant is a complex mixture of phospholipids and four surfactant-associated proteins (SP-A, SP-B, SP-C and SP-D). The biological functions of SP-A and SP-D are primarily twofold, namely surfactant homeostasis and host defense. The hydrophobic surfactant proteins, SP-B and SP-C, are required for achieving the optimal surface tension reducing properties of surfactant by promoting the rapid adsorption of surfactant phospholipids along the alveolar surface. Despite the promising findings, only little is known about the extrapulmonary distribution of these proteins. Therefore, in this study, the presence of SP-A, SP-B, SP-C and SP-D in early human placenta has been investigated. First-trimester placental tissues (22-56 days) were obtained from women undergoing curettage during normal pregnancies. In parallel tissue sections, vimentin, cytokeratin-7 and CD-68 immunostainings were used for the identification of mesenchymal cells, trophoblast cells and Hofbauer cells, respectively. According to immunohistochemistry (IHC) results, SP-A, SP-B, SP-C and SP-D immunoreactivities with different staining intensities were observed in trophoblastic layers of chorionic villous tree, trophoblastic cell columns, stromal cells, Hofbauer cells, angiogenic cell cords and vascular endothelium. Fetal hematopoietic cells showed a variable staining pattern for all four surfactant proteins ranging from none to strong intensity. Western blotting of tissue extracts confirmed our IHC results. The presence of surfactant glycoproteins in early human placenta may yield a very important feature of surfactants during first trimester and enables further studies of the role of surfactants in various pregnancy complications.

Placenta as an ideal model to study angiogenic mechanisms have been established in previous studies. There are two processes, vasculogenesis and angiogenesis, involved in blood vessel formation during placental development. Therefore, blood vessel formation is a crucial issue that might cause vascular malformations. One of the vascular malformations is cerebral cavernous malformation (CCM) in the central nervous system, consisting of endothelium-lined vascular channels without intervening normal brain parenchyma. Three CCM loci have been mapped as Ccm1, Ccm2, Ccm3 genes in CCM. In order to investigate whether CCM proteins participate in blood vessel formation, we report here the expression patterns of CCM2 and CCM3 in developing and term human placenta by means of immunohistochemistry and Western blot analysis. CCM2 and CCM3 were obviously detected in the vascular endothelium during early pregnancy. Moreover, vascular endothelium of stem villi revealed a moderate immunostaining for CCM2 and, to a lesser extent, in the endothelium of mature intermediate villi in term placenta. Interestingly, CCM3 immuno-staining was weakly localized in the endothelium of mature intermediate villi and showed lesser expression going toward stem villi in term placenta. The expression patterns of the proteins were clearly identified in the vascular endothelium of human placenta, suggesting that they might play roles during angiogenesis and vasculogenesis. Furthermore, with this study, CCM2 and CCM3 have been described for the first time in the human placenta.

It has been previously demonstrated that red blood cells (RBC) possess functional nitric oxide (NO) synthesizing mechanisms. RBC are also equipped with variety of intracellular control mechanisms, and respond to mechanical forces and to various biological stimuli by increased release of ATP. Nitric oxide has also been demonstrated to be released from RBC under certain circumstances, and it has been hypothesized that NO synthase (NOS), which is located in both the RBC membrane and cytoplasm, might be activated by mechanical factors. The present study aimed at investigating NOS activation and NO export induced by mechanical stress applied to RBC in suspension. Heparinized venous blood samples were obtained from healthy, adult volunteers and their hematocrit adjusted to 0.4 l/l. The RBC suspensions were equilibrated at room temperature (22+/-2 degrees C) with either room air or made hypoxic (36 mmHg, approximately 70% saturation) using moisturized 100% nitrogen. The samples were then continuously pumped through a glass tube (diameter = 0.06 cm; length = 33 cm) for 30 min using a dual syringe pump to maintain a wall shear stress of 0.5-2 Pa with NO concentrations in the RBC suspensions measured electrochemically. NO concentration significantly increased under the influence of 2 Pa in hypoxic RBC suspensions: 105.0+/-14.2 nM to 127.1+/-12.0 nM as the peak value at 20 min of perfusion. No increase was observed at lower levels of shear stress. Plasma nitrite/nitrate concentrations were measured in samples obtained at five minute intervals. Application of fluid shear stress to hypoxic RBC suspensions resulted in a significant, time-dependent increase of plasma nitrite/nitrate levels, reaching to 14.7+/-1.5 microM from a control value of 11.2+/-1.3 microM. The presence of the non-specific NOS inhibitor L-NAME (10(-3) M) prevented this increment. Additionally, both eNOS and serine 1177 phosphorylated eNOS immuno-fluorescence staining in RBC cytoplasm were shown to increase in response to applied shear stress. Our results support the hypothesis that RBC NO synthase is activated and that export of NO from RBC is enhanced by mechanical stress.

Objective To study the potential relationship between two sperm nuclear attributes: persistence of histones and occurrence of chromosomal aneuploidies. Design The two variables were examined by double probing of the same spermatozoa. Setting Academic Andrology Laboratory. Patient(s) Semen samples subjected for analyses were examined. Intervention(s) We studied >58,000 spermatozoa, in seven men, first with aniline blue histone staining, graded as light (mature sperm), intermediate (moderately immature), and dark (severely arrested maturation). After recording the staining patterns and destaining, the same spermatozoa were subjected to fluorescence in situ hybridization (FISH), using centrometric X, Y, and 17 chromosome probes. Main Outcome Measure(s) Proportions of sperm with light, intermediate, and dark staining were assessed, and ploidy of these sperm was evaluated. Result(s) The aneuploidy frequencies in intermediate versus light (mature) spermatozoa were increased four- to sixfold. In addition, aneuploidy frequencies and proportions of intermediate sperm were related. There was no FISH signal detectable in the darkly stained, severely arrested mature sperm. Conclusion(s) The data suggest that in sperm with arrested maturity and DNA fragmentation, the binding of FISH probes is diminished. DNA damage is further aggravated by the decondensation and denaturation steps of FISH. Thus, there is a strong likelihood that in oligozoospermic men, with a higher proportion of sperm with arrested maturation, the sperm disomy frequencies are historically underestimated.

Objective To assess the distribution of cerebral cavernous malformation-2 (CCM2) and CCM3 proteins in a normal and experimentally induced varicocele model in rat testes. Design Comparative and controlled study. Setting University animal care and operation unit. Animal(s) Wistar male rats for experimental and control groups. Intervention(s) The control group underwent a sham operation. Rats in the experimental groups underwent partial ligation of the renal vein to induce experimental varicocele, and left testicular tissues were analyzed. Main Outcome Measure(s) Tissues were fixed and processed for paraffin-embedded testicular tissues. Levels of CCMs were assessed by immunohistochemistry and Western blot analysis. Result(s) In control and sham-operated testes, CCM2 expression was detected in acrosomes of round spermatids at stages 6–8 and in the heads of elongating spermatids at stages 1–5 and 9–14. CCM3 expression was weakly localized in pachytene spermatocytes at stages 8–12 and strongly immunolocalized in the cytoplasm of round spermatids at stages 1–8 and in elongating spermatid cytoplasm at stages 9–12. Varicocele induction clearly demonstrated histopathological changes due to germ cell degeneration. CCM2 and CCM3 expression increased significantly in varicocele-induced rat testes. Conclusion(s) The expression patterns of two brain proteins were clearly identified in rat testes, suggesting a role during normal and pathological spermatogenesis. This article represents the first demonstration that these proteins have a role outside the central nervous system.

Kinesin superfamily proteins (KIFs) are motor proteins that participate in chromosomal and spindle movements during mitosis and meiosis, and transport membranous organelles and macromolecules fundamental for cellular functions. Although the roles of KIFs in axonal and dendritic transports have been studied extensively, their role in intracellular transport in general is less well known. The diversity of kinesins suggests that each kinesin may have a specific function. Therefore, in this study we aimed to investigate the presence and cellular localization of KIFC1 and KIF17 in normal and pathological human placentas. First-trimester (22–56 days) and normal, preeclamptic (PE), and diabetic-term placental tissues were obtained and further studied by immunohistochemistry (IHC) and Western blot methods. KIFC1 was mainly localized to the syncytiotrophoblast both in early and term placental samples. However, a stronger immunoreactivity was observed both in PE and diabetic placentas compared to normal-term placentas. KIF17 was most intensively localized in developing vascular endothelium in early pregnancy. Even though KIF17 was moderately stained in the endothelium of villi from normal human-term placentas, stronger immunoreactivity was observed in all types of villi of both PE and diabetic placentas. Western blotting of tissue extracts confirmed the IHC results. Here, we demonstrate the presence of KIFC1 and KIF17 in human placenta for the first time. The intense expression of KIFC1 in syncytiotrophoblast and KIF17 in vascular endothelium suggests that both the proteins might be important in a cargo-transport system. An increased expression pattern of both KIFC1 and KIF17 in PE and diabetes might suggest that these proteins may be involved in complex trophoblast functions and placental pathologies. Further studies will clarify the physiological role of KIFs in human placental transport and development.

The aim of this study was to investigate the pleotropic effects of an extract of a traditional herb, Tribulus terrestris (TT), on the lipid profile and vascular endothelium of the abdominal aorta in New Zealand rabbits fed a cholesterol-rich diet. Eighteen rabbits were randomly divided into three groups (n=6 for each). One experimental group (EG-I) was given a cholesterol-rich diet, a second experimental group (EG-II) was treated with TT following a cholesterol-rich diet, and a control group (CG) was fed a standard diet. Blood samples were collected on day 0 and then at weeks 4 and 12 to determine total serum cholesterol (TC), high density lipid-cholesterol (HDL-C), low density lipid-cholesterol (LDL-C) and triglyceride (TG) levels. Tissues were collected from the abdominal aorta for immunohistochemistry and transmission and scanning electron microscopy. In EG-II, the serum lipid profile was significantly lower than that of EG-I at week 12 with a reduction of TC: 65%; LDL-C: 66%; HDL-C: 64%; TG: 55%. Ultrastructural analysis revealed that endothelial damage was more prominent in EG-I compared to EG-II. The ruptured endothelial linings and damaged cellular surfaces increased in EG-I compared to EG-II. Our data indicate that dietary intake of TT can significantly lower serum lipid profiles, decrease endothelial cellular surface damage and rupture and may partially repair the endothelial dysfunction resulting from hyperlipidemia. © 2008 Elsevier GmbH. All rights reserved.

Individual spermatozoa were assessed with pairs of probes for persistent histones and cytoplasmic retention, persistent histones and DNA fragmentation, and persistent histones and apoptotic markers. The individual spermatozoa were treated sequentially with combinations of probes for these cytoplasmic and nuclear biochemical markers. Sperm fields were recorded with computer-assisted imaging, and staining patterns with the two probes in the same spermatozoa were examined and scored as light, intermediate or dark (mature to arrested-maturity spermatozoa). The effects of arrested sperm maturation were similar with respect to the cytoplasmic and nuclear characteristics of spermatozoa in 84% of cells, indicating that cytoplasmic and nuclear attributes of arrested sperm maturation are related. However, there were moderate (intermediate-dark or intermediate-light patterns, 14.5% of cells) or major (light-dark patterns, 1.6% of cells) discrepancies in the intensity of the double staining patterns. Thus, testing with single maturity markers may not be fully reliable. These findings are important with respect to: (i) arrested sperm maturation; (ii) potential efficacy of antioxidant and similar therapeutic strategies in subfertile men, as spermatozoa with infrastructure defects due to mismaturation or maturation arrest are unlikely to respond to interventions; and (iii) detection of adverse male environmental exposures.

Angiopoietins are integral to vasculogenesis and angiogenesis, which play crucial roles in the growth and development of the placenta. The current study assessed expression of angiopoietins (Ang-1 and Ang-2) and their receptors (Tie-1 and Tie-2) during development of the early human placenta. First-trimester placental tissues were obtained from women Undergoing curettage during normal pregnancies. The use of immunochemistry (IHC) showed that Ang-1 was primarily localized to syncytiotrophoblasts where it displayed moderate immunoreactivity, whereas weak immunoreactiviry for Ang-1 was observed in endothelial cells and angiogenic cell cords (ACC). Strong immunoreactivity for Ang-2 Was also found predominantly in syncytiotrophoblasts with lower immunostaining levels evident in cytotrophoblasts. Moderate immunoreactivity for Ang-2 was observed in endothelial cells, ACC and Hofbauer cells. By Contrast, the trophoblastic shell, as well as endothelial cells and ACC exhibited strong staining intensity for Tie-1 with the strongest immunoreactivity for Tie-2 observed in cytotrophoblasts, ACC and endothelial cells. Western blotting of tissue extracts confirmed the IHC results. Previous studies focused on VEGF and its receptors in controlling vasculogenesis and angiogenesis in human placenta. However, the specific localization patterns of angiopoietins and their receptors revealed by the Current study emphasize the importance of these molecules in placental vascular development. Functional studies aimed at identifying the molecular mechanisms of actions of these factors and receptors may prove essential in elucidating the pathophysiology of placental disorders such as intrauterine growth restriction and pre-eclampsia. (C) 2008 Elsevier Ltd. All rights reserved.

Objective To investigate the presence of surfactant protein-A (SP-A); molecular weight 34 kDa and surfactant protein-D (SP-D); and molecular weight 43 kDa in human spermatozoa. Design Prospective, research study. Setting Two universities in Turkey. Patient(s) Semen specimens (n = 10) were obtained from normozoospermic donors. Intervention(s) Human sperm were exposed to an anti–human SP-A polyclonal antibody, and monoclonal antibody, to human SP-D protein. Main Outcome Measure(s) Presence of SP-A and SP-D proteins in human beings. Result(s) Indirect immunofluorescence assays of human sperm indicated the presence of SP-A in the mid piece, the tail, and sometimes at the equatorial region of spermatozoa. A brilliant green light detected SP-D in the tails and acrosome of some sperm. The anti–SP-A antibody detected a single band corresponding to the molecular weight values of 34 kDa in spermatozoa, whereas no band was observed in the negative control. The anti–SP-D antibody showed the expected band at 43 kDa in spermatozoa. Conclusion(s) This is the first report and a novel finding of the presence of surfactant glycoproteins on human spermatozoa.

The aim of the study was to investigate the distribution and differentiation of cell types in the stroma of human placental stem villi (SV). A total of 14 human term placental tissues were studied. Double immunolabeling was performed for desmin-vimentin, desmin-alpha-smooth actin and vimentin-alpha-smooth actin. Cytokeratin 7, proliferating cell nuclear antigen immunolabeling was also performed. Parallel tissue samples were examined by transmission electron microscopy. HSCORE was performed for the semi-quantitative analysis of distribution of cells in the stroma of SV. Vimentin-labeled cells were mostly distributed in the subtrophoblastic area. Desmin-vimentin double immunolabeling was mainly localized in the triangular area and to a lesser degree in the perivascular area and vessel walls (p=or0.001). However, desmin-alpha smooth actin labeling was observed predominantly in the vessel wall and perivascular area. Vimentin-alpha smooth actin immunoreactivity was significantly stronger in the triangular and perivascular areas compared to the vessel walls (p=0.003). Ultrastructurally, cells in the stroma of SV were mesenchyme cells, reticulum cells, fibroblasts, myofibroblasts, smooth muscle cells, and Hofbauer cells, filamented and vacuolated cells. The differentiation of myofibroblasts in the triangular and perivascular areas may play a role in maturation of SV and villous contractility, modulation of the intervillous space and this may have effects on maternofetal placental circulation.

Objective To investigate the localization of tenascin expression in the endometrium of women without endometriosis and in endometriotic implants, and to determine the in vitro regulation of tenascin by E2 in these tissues. Design Experimental laboratory study. Setting University medical center. Patient(s) Reproductive age women with or without endometriosis. Intervention(s) Proliferative (n = 14), and secretory (n = 14) endometrium from women without endometriosis and endometriosis implants (n = 14) were used for immunohistochemical analysis. Endometrial and endometriotic stromal cells were grown in culture and treated with E2, the estrogen receptor antagonist ICI 182 780 (ICI) alone, E2 in combination with ICI, or vehicle (control) for 24 hours, and tenascin expression was analyzed by Western blotting. Main Outcome Measure(s) Expression levels of tenascin in normal endometrium and endometriotic implants and its regulation by E2. Result(s) Tenascin immunostaining revealed an increasing intensity in the stromal cells, starting from normal secretory endometrium, then normal proliferative endometrium, and reaching the highest expression in endometriotic implants. Estradiol induced a significant increase in tenascin protein levels in the endometriotic stromal cells in culture. Conclusion(s) The modulation of tenascin as an extracellular matrix protein by E2 in endometriotic stromal cells may be one of the factors playing a role in the development of endometriosis.

The current concepts of sperm biochemical markers and the central role of the HspA2 chaperone protein, a measure of sperm cellular maturity and fertilizing potential, are reviewed.Because HspA2 is a component of the synaptonemal complex, low HspA2 levels and increased frequency of chromosomal aneuploidies are related in diminished maturity sperm. We also suggest a relationship between HspA2 expression in elongating spermatids and events of late spermiogenesis, such as cytoplasmic extrusion and plasma membrane remodeling that aid the formation of the zona pellucida binding and hyaluronic acid binding sites. The presence of hyaluronic acid receptor on the plasma membrane of mature sperm, coupled with hyaluronic acid coated glass or plastic surfaces, facilitates testing of sperm function and selection of single mature sperm for intracytoplasmic sperm injection. The frequencies of sperm with chromosomal disomy are reduced approximately fourfold to fivefold in hyaluronic acid selected sperm compared with semen sperm, comparable to the increase in such abnormalities in intracytoplasmic sperm injection offspring. Hyaluronic acid binding also excludes immature sperm with cytoplasmic extrusion, persistent histones, and DNA chain breaks.Hyaluronic acid mediated sperm selection is a novel technique that is comparable to sperm zona pellucida binding. Hyaluronic acid selected sperm will also alleviate the risks related to intracytoplasmic sperm injection fertilization with sperm of diminished maturity that currently cause worldwide concern.

We have explored the action of zoledronic acid, which has an apoptotic effect and is used as an agent for treating skeletal metastases and osteoporosis, in the presence of vinblastine, and whether this effect is associated with MRP-1 (multidrug resistance protein-1) expression. HEK (human embryonic kidney) 293 cells were transfected to form the multidrug resistant cell line designated 293MRP (MRP-1 expressing HEK293 cells). Both lines were treated with varying concentrations of vinblastine and zoledronic acid. Apoptosis was determined by the TUNEL (deoxyuridine triphosphate nick end-labeling) method. The type of treatment, MRP-1 expression status, and the type of treatment with respect to MRP-1 expression status significantly affected (P < 0.001) the degree of apoptosis. The largest increase in cytotoxicity was noted in HEK293 cells, when 100 μmol zoledronic acid was added to 4 μg/ml vinblastine (an increment of 80.3%, P < 0.001). This preliminary work shows that zoledronic acid acts synergistically with vinblastine to induce apoptosis in an MRP-1 dependent way. © 2005 Published by Elsevier Ltd on behalf of International Federation for Cell Biology.

In the present study, we present the ultrastructural. and immunohistochemical. properties of the sural nerves of two patients, one of whom was diagnosed as having multiple sclerosis with involvement of the peripheral nervous system (PNS), and the other as having hereditary motor sensory neuropathy type-I with involvement of the central nervous system (CNS). Expression of several extracellutar matrix (ECM) proteins (fibronectin, laminin, and collagen type-IV), intermediate filaments (vimentin) and S-100 protein (marker for the axon-Schwann cell interface) was investigated by means of immunohistochemical. methods. In addition, the tissue samples were evaluated ultrastructurally. Immunohistochemical staining revealed increased expression of the ECM molecules mentioned above in relation with the sural. nerves of the patients. We hypothesize that this enhanced expression is due to Schwann cell-axon interactions. Vimentin expression was different in Schwann cells and S-100 immunostaining was decreased near the Schwann cell-axon interface. Myelin fragmentation, axon vacuolization, onion bulbs, tomoculous formation, axonal. degeneration were found to occur. These results suggest that there is active ECM reorganization in the sural nerve of these patients, and some ultrastructural changes are similar in the damaged axonal organization and in Schwann cells although the changes are not completely the same in the two patients. In conclusion, our study demonstrates that there is an association between the demyelinization process in the CNS and the PNS even though they are affected by different mechanisms. (C) 2004 Elsevier GmbH. All rights reserved.