Malgorzata Kotwicka

Caspase-3 activation and phosphatidylserine membrane translocation in human spermatozoa: is there a relationship?

The presence of biochemical signs of apoptosis in ejaculated spermatozoa suggests that apoptosis may be one of the pathways for sperm death. The relationship between the phosphatidylserine (PS) externalization and the presence of the active form of caspase-3 (CP-3) was studied in human spermatozoa after exposure to hydrogen peroxide (H(2)O(2)). Semen from 27 normozoospermic men was examined, as the neat semen, after swim-up isolation and after H(2)O(2) incubation, for the translocation of PS, activation of caspase-3 and mitochondrial membrane potential. The percentage of vital spermatozoa expressing PS translocation was lower than the percentage of vital spermatozoa with the active form of the caspase-3, either in neat (4.9 +/- 2.3% versus 19.7 +/- 6.2%, P < 0.001) or in swim-up semen (2.2 +/- 2.3% versus 4.8 +/- 2.9%, P < 0.01). After swim-up isolation, the percentage of vital spermatozoa with active caspase-3 decreased (P < 0.01). After H(2)O(2) stimulation of the swim-up semen fraction, a decrease in the mitochondrial membrane potential was observed (P < 0.001). Only the midpiece revealed PS translocation after H(2)O(2) stimulation, and it was also the only part to reveal the presence of the active form of caspase-3. All spermatozoa expressing the PS translocation revealed the presence of the active form of caspase-3.

XbaI and PvuII Polymorphisms of Estrogen Receptor 1 Gene in Females with Idiopathic Scoliosis: No Association with Occurrence or Clinical Form

Introduction XbaI single nucleotide polymorphism (SNP) (A/G rs934099) in estrogen receptor 1 gene (ESR1) was described to be associated with curve severity in Japanese idiopathic scoliosis (IS) patients and in Chinese patients with both curve severity and predisposition to IS. PvuII SNP (C/T rs2234693) of ESR1 was described to be associated with the occurrence of IS in the Chinese population; however, two replication studies did not confirm the findings. The ESR1 SNPs have never been studied in Caucasian IS patients. Methods Case-control study. 287 females with IS underwent clinical, radiological and genetic examinations. The patients were divided into three groups according to curve progression velocity: non-progressive IS, slowly progressive IS (progression <1° per month), and rapidly progressive IS (progression ≥1° per month). The radiological maximum Cobb angle was measured and surgery rate established. A control group consisted of 182 healthy females. Results All results followed Hardy-Weinberg equilibrium. In the case-control study, genotype frequency in the patients did not differ for the XbaI (AA = 33.5%, AG = 49.1%, GG = 17.4%), nor for the PvuII (TT = 26.8%, TC = 50.2%, CC = 23.0%) comparing to controls (AA = 33.5%, AG = 50.5%, GG = 15.9%) and (TT = 23.1%, TC = 51.1%, CC = 25.8%), respectively, p = 0.3685, p = 0.6046. The haplotype frequency for the patients (AT = 47.1%, GC = 39.2%, AC = 8.9%, GT = 2.8%) did not differ from the controls (AT = 44.8%, GC = 37.4%, AC = 14.0%, GT = 3.8%), p = 0.0645. No difference was found either in XbaI (p = 0.8671) or PvuII (p = 0.3601) allele distribution between the patients and the controls. In the case study, there was no significant difference in genotype frequency for the non-progressive, slowly progressive, and rapidly progressive scoliosis. No difference was found in genotype or haplotype distribution for the mean maximum Cobb angle or the surgery rate. Conclusions No association was found between ESR1 XbaI or ESR1 PvuII SNP and idiopathic scoliosis in Caucasian females. None of the previously reported associations could be confirmed, regarding curve severity, progression or operation rate.

Human Spermatozoa Ultrastructure Assessment in the Infertility Treatment by Assisted Reproduction Technique

The aim of this study was to prospectively investigate the spermatozoa ultrastructure in relation to the results of in vitro fertilization – embryo transer (IVF-ET). Forty-nine consecutive couples admitted for IVF-ET were prospectively evaluated for electron microscopic spermatozoa morphology and the outcome of IVF-ET. Thirty-four couples revealed successful fertilization, defined as presence of two pronuclei 14–16 hours after spermatozoa administration, while the remaining 15 formed the failure group. Spermatozoa fixed with 2.5% glutaraldehyd and embedded in Spurrs resin were analyzed with JAM 100 S transmission electron microscope (TEM) for the following ultrastructure abnormalities: head deformity, cytoplasmic residues, chromatin condensation failures, acrosomal alterations, neck defects, midpiece defects, principal piece and end-piece defects and immature forms. Successful IVF-ET couples revealed a significantly higher percentage of normal spermatozoa utrastructure (32.0±13.1% versus 17.1±13.4%, p < 0.001). Failed IVF-ET couples represented a significantly higher percentage of chromatin condensation failures (9.8±5.1% versus 5.7±5.3%, p < 0.05) and tail defects (16.7±11.5% versus 7.2±7.2%, p < 0.001). A positive correlation between normal ultrastructure spermatozoa percentage and fertilized oocytes percentage was found (r = 0.35, p < 0.05). Our data suggest that spermatozoa TEM findings correlate with IVF-ET results. Ultrastructural estimation of spermatozoa can improve the diagnosis of male fertility and may explain some reasons of failure in assisted reproduction methods. We consider systematic TEM spermatozoa examination in cases with failed IVF-ET prior to intracytoplasmic sperm injection (ICSI).

Expression of ghrelin receptor (GHSR-1a) in rat epididymal spermatozoa and the effects of its activation.

In this study we demonstrated the expression of the ghrelin receptor GHSR-1a in rat spermatids and epididymal spermatozoa, as well as some effects of ghrelin on the spermatozoa in vitro. For the demonstration of GHSR-1a the immunocytochemical, immunofluorescence and Western blotting techniques were applied using three different types of antibodies. The response of spermatozoa to ghrelin was tested in a series of in vitro experiments and their effects were evaluated using confocal microscopy and flow cytometry. GHSR-1a protein was found as expressed in the Golgi and acrosomes of spermatids and acrosome regions or the head cell membrane of epididymal spermatozoa. The GHSR-1a expression in spermatozoa was also confirmed by Western blot. No differences were found in percentage of spermatozoa showing annexin-V binding and expression of active form caspase-3 between control and ghrelin-treated spermatozoa. This result may indicate no pro-apoptotic effects of ghrelin neither at 10(-9) nor 10(-6)mol/L concentration. Ghrelin (10(-6)mol/L) increased free intracellular calcium ion concentration in the rat spermatozoa. Moreover, stimulation with 10(-6)mol/L ghrelin increased, while 10(-4)mol/L ghrelin decreased the number of spermatozoa showing progressive motility. In conclusion, the expression of the GHSR-1a receptor in spermatozoa, as well as ghrelin influences on sperm motility and intracellular calcium ion concentration suggest that such biological effects of ghrelin may be produced under in vivo conditions.

Effects of chlorhexidine, essential oils and herbal medicines (Salvia, Chamomile, Calendula) on human fibroblast in vitro

Antiseptic rinses have been successfully used in inflammatory states of the gums and oral cavity mucosa. Antibacterial effects of chlorhexidine, essential oils and some herbs are well documented. Reaction of host tissue to these substances has much poorer documentation. The aim of the study was to analyse the influence of chlorhexidine (CHX), essential oil (EO: thymol, 0.064%; eucalyptol, 0.092%; methyl salicylate, 0.060%; menthol, 0.042%) mouth rinses and salvia, chamomile and calendula brews on fibroblast biology in vitro. The human fibroblast CCD16 line cells were cultured in incubation media which contained the examined substances. After 24 and 48 hours, the cell morphology, relative growth and apoptosis were evaluated. Exposure of fibroblasts to CHX, EO or salvia caused various changes in cell morphology. Cells cultured for 48 hours with CHX revealed a noticeably elongated shape of while cells cultured in high EO concentration or with salvia were considerably smaller and contracted with fewer projections. Chlorhexidine, EO and salvia reduced the fibroblast proliferation rate and stimulated cell death. Both reactions to EO were dose dependent. Cells exposure to chamomile or calendula brews did not change morphology or proliferation of fibroblasts. The results of this in vitro study showed that in contrast to chamomile and calendula, the brews of EO, CHX or salvia had a negative influence on fibroblast biology.

17β-estradiol and xenoestrogens reveal synergistic effect on mitochondria of human sperm

OBJECTIVES The aim of the study was to investigate the influence of 17β-estradiol (main endogenous estrogen) and selected xenoestrogens (genistein, bisphenol-A), individually and in combination, on the mitochondrial function of human sper-matozoa. In natural environment, human beings are exposed to multiple xenoestrogens, so their impact is combined with endogenous steroids. MATERIAL AND METHODS The effects of ligands on human spermatozoa were assessed regarding the following phenomena: spermatozoa vitality (propidium iodide staining), phosphatidylserine membrane translocation (staining with annexin V marked with fluorescein), mitochondrial membrane potential (using JC-1 fluorochrome), and production of superoxide anion in mitochondria (using MitoSOX RED dye). RESULTS Two-hour incubation of spermatozoa with 17β-estradiol, genistein, and bisphenol-A neither altered cell vitality nor stimulated phosphatidylserine membrane translocation. Incubation of spermatozoa with 17β-estradiol or bisphenol-A sepa-rately, as well as incubation with the three ligands simultaneously, resulted in altered mitochondrial membrane potential. Spermatozoa incubation with the three ligands significantly increased the mitochondrial superoxide anion level. CONCLUSIONS It seems safe to conclude that human spermatozoa mitochondria are target cell structures for both, 17β-estradiol and xenoestrogens. The reaction to the 17β-estradiol and xenoestrogens mixture suggests a synergistic mechanism of action. Xenoestrogens may increase the sensitivity of spermatozoa to 17β-estradiol.

Effects of enamel matrix proteins on adherence, proliferation and migration of epithelial cells: A real‑time in vitro study

Enamel matrix derivative (EMD) can mimic odontogenic effects by inducing the proliferation and differentiation of connective tissue progenitor cells, stimulating bone growth and arresting epithelial cells migration. To the best of our knowledge, there is no data indicating that any active component of EMD reduces epithelial cell viability. The present study examines the impact of commercial lyophilized EMD, porcine recombinant amelogenin (prAMEL; 21.3 kDa) and tyrosine-rich amelogenin peptide (TRAP) on the adherence, proliferation and migration of human epithelial cells in real-time. The tongue carcinoma cell line SCC-25 was stimulated with EMD, porcine recombinant AMEL and TRAP, at concentrations of 12.5, 25 and 50 µg/ml. Cell adherence, migration and proliferation were monitored in real-time using the xCELLigence system. No significant effects of EMD on the morphology, adhesion, proliferation and migration of SCC-25 cells were observed. However, porcine recombinant AMEL had a dose-dependent inhibitory effect on SCC-25 cell proliferation and migration. Predominantly, no notable differences were found between control and TRAP-treated cells in terms of cell adhesion and migration, a decrease in proliferation was observed, but this was not statistically significant. EMD and its active components do not increase the tongue cancer cell viability.

ESR1 and ESR2 genotypes and the age at menarche in idiopathic scoliosis

Background Environmental and genetic factors have influence on the age at menarche (AAM). Disturbance of the AAM in patients with idiopathic scoliosis (IS) were postulated [1]. Estrogen receptor genes 1 and 2 (ESR1, ESR2) single nucleotide polymorphisms (SNP) in IS were suggested to have some association with predisposition to IS [2]. ESR SNPs were reported to have association with AAM in healthily females [3].

Replication study of estrogen receptor 1 XbaI polymorphism in adolescent idiopathic scoliosis (AIS) Caucasian population

Background It has been suggested that XbaI single nucleotide polymorphism (SNP) (A/G rs934099) in estrogen receptor 1 (ESR1) may be associated with curve severity in Japanese AIS patients [1] and with both curve severity and predisposition for AIS in Chinese patients [2]. However, replication studies have not confirmed these findings [3]. The role of the XbaI ESR1 polymorphism rs9340799 in AIS has never been described in Caucasian AIS patients.

Increased expression of PELP1 in human sperm is correlated with decreased semen quality

Proline-, glutamic acid-, and leucine-rich protein 1 (PELP1) is a scaffolding protein involved in both genomic and nongenomic estrogen signal transduction pathways. To date, the role of PELP1 protein has yet to be characterized in human sperm and has not been associated with sperm parameters. To confirm the presence of PELP1 in human sperm, fresh semen samples were obtained from 178 donors. The study was designed to establish both mRNA and protein presence, and protein cellular localization. Additionally, the number of PELP1-positive spermatozoa was analyzed in men with normal and abnormal semen parameters. Sperm parameters were assessed according to the World Health Organization (WHO) 2010 standards. The presence of PELP1 in spermatozoa was investigated using four precise, independent techniques. The qualitative presence of transcripts and protein was assessed using reverse transcription-polymerase chain reaction (RT-PCR) and western blot protocols, respectively. The cellular localization of PELP1 was investigated by immunocytochemistry. Quantitative analysis of PELP1-positive cells was done by flow cytometry. PELP1 mRNA and protein was confirmed in spermatozoa. Immunocytochemical analysis identified the presence of PELP1 in the midpieces of human sperm irrespective of sperm parameters. Becton Dickinson fluorescence-activated cell sorting (FACSCalibur™) analysis revealed a significantly lower number of PELP1-positive cells in males with normal semen parameters versus abnormal samples (42.78% ± 11.77% vs 61.05% ± 21.70%, respectively; P = 0.014). The assessment of PELP1 may be a time-saving method used to obtain information about sperm quality. The results of our study suggest that PEPL1 may be utilized as an indicator of sperm quality; thereby, PELP1 may be an additional biomarker useful in the evaluation of male infertility.