Leyla Sati

Selectivity of hyaluronic acid binding for spermatozoa with normal Tygerberg strict morphology

During spermiogenesis, a plasma membrane remodelling step facilitates formation of sperm zona pellucida and hyaluronic acid (HA) binding sites. Enrichment of Tygerberg normal spermatozoa in HA-bound versus semen sperm fractions was postulated. Semen was placed on the uncoated A side and HA-coated B side of a semen chamber. After 15 min, the HA binding score (proportion of HA-bound motile spermatozoa) was assessed on the B side, and unbound spermatozoa were removed by gentle rinsing. Following Diff-Quick staining, sperm morphology of A and B sides was evaluated by three blinded investigators at Yale and Tygerberg. The proportion of Tygerberg normal spermatozoa was higher in HA-bound versus semen spermatozoa (n = 63 subjects) with a 3.04-fold improvement (95% confidence limits: 1.9-4.7) in 37 teratozoospermic men, comparable with a 4.2-fold enrichment in zona pellucida-bound spermatozoa reported earlier by the Tygerberg group. The morphology scores of three investigators were different but related, indicating that the variations reflect individual-to-individual differences in the perception of shape normality. The selection power of HA and zona pellucida for normal spermatozoa are similar. The sperm biomarkers of creatine phosphokinase (reflecting retained cytoplasm in arrested maturity spermatozoa) and chaperone protein HspA2 (heat shock protein) were proportional with sperm HA binding. As HA binding reflects sperm maturity and function, the combination of Tygerberg morphology and HA binding is likely to improve male infertility management.

The Distribution of Angiopoietin-1, Angiopoietin-2 and Their Receptors Tie-1 and Tie-2 in the Very Early Human Placenta

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 immunohistochemistry (IHC) showed that Ang-1 was primarily localized to syncytiotrophoblasts where it displayed moderate immunoreactivity, whereas weak immunoreactivity 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.

Double probing of human spermatozoa for persistent histones, surplus cytoplasm, apoptosis and DNA fragmentation.

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.

Effects of abamectin exposure on male fertility in rats: potential role of oxidative stress-mediated poly(ADP-ribose) polymerase (PARP) activation.

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 (1mg/kg/day) and high dose (4 mg/kg/day) abamectin were applied to male rats by oral gavage for 1week and for 6weeks. 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.

Double probing individual human spermatozoa: aniline blue staining for persistent histones and fluorescence in situ hybridization for aneuploidies

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.

Influence of Tribulus terrestris extract on lipid profile and endothelial structure in developing atherosclerotic lesions in the aorta of rabbits on a high-cholesterol diet☆

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.

Effect of abamectin exposure on semen parameters indicative of reduced sperm maturity: a study on farmworkers in Antalya (Turkey)

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.

The Presence of Kinesin Superfamily Motor Proteins KIFC1 and KIF17 in Normal and Pathological Human Placenta

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.

Methodology of aniline blue staining of chromatin and the assessment of the associated nuclear and cytoplasmic attributes in human sperm.

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.

The Pattern of Tyrosine Phosphorylation in Human Sperm in Response to Binding to Zona Pellucida or Hyaluronic Acid

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.