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At least 19 records

Novel localization of Aurora A kinase in mouse testis suggests multiple roles in spermatogenesis

Highlights: • Aurora A kinase is a previously identified mitotic kinase. • Developing male germ cells express Aurora A including spermatogonia, spermatocytes, and spermatids. • Aurora A kinase and its activated form are localized to the flagella of sperm. • Activated Aurora A kinase is enriched in motile sperm of the epididymis suggesting a role in sperm motility. Male germ cells are transformed from undifferentiated stem cells into spermatozoa through a series of highly regulated steps together termed spermatogenesis. Spermatogonial stem cells undergo mitosis and differentiation followed by two rounds of meiotic division and then proceed through a series of dramatic cell shape changes to form highly differentiated spermatozoa. Using indirect immunofluorescence, we investigated a role for the mitotic kinase, Aurora A (AURKA), in these events through localization of this protein in mouse testis and spermatozoa. AURKA is expressed in several cell types in the testis. Spermatogonia and spermatocytes express AURKA as expected based on the known role of this kinase in cell division. Surprisingly, we also found AURKA localized to spermatids and the flagellum of spermatozoa. Total AURKA and activated AURKA are expressed in different compartments of the sperm flagellum with total AURKA found in the principal piece and its phosphorylated and activated form found in the sperm midpiece. In addition, active AURKA is enriched in the flagellum of motile sperm isolated from cauda epididymis. These results provide evidence for a unique role for AURKA in spermatogenesis and sperm motility. Defining the signaling mechanisms that govern spermatogenesis and sperm cell function is crucial to understanding and treating male infertility as well as for development of new contraceptive strategies.

60 APPLIED LIFE SCIENCES↗

Exposure to non‐ionizing electromagnetic radiation of public risk prevention instruments threatens the quality of spermatozoids

Contents The use of artificial insemination in cattle breeding has evolved to global extent, and insemination doses are often shipped via air transport which requires strict radiation‐based examinations. For the determination of effect of non‐ionizing radiation (NIR), to which are beings frequently exposed due to protection of airport or cultural event security, freshly ejaculated and cryopreserved bovine spermatozoa were used as experimental model. Following radiation with hand‐held metal detector in various exposition times (0, 10 s, 15, 30 and 60 min—groups FR, FR10, FR15, FR30 and FR60) the spermatozoa underwent motility and DNA fragmentation analyses. Study on cryoconserved semen treated with NIR was performed in time intervals 0, 10 s, 1 and 5 min (insemination doses radiated before cryoconservation—CB, CB10, CB1, CB5; samples radiated after freezing—CA, CA10, CA1 and CA5). Fresh semen and insemination doses radiated after cryoconservation showed significantly lower total and progressive motility. No effect on motility parameters was detected in semen extended with cryopreservative medium and radiated prior to freezing. Surprisingly, NIR showed a potential to stimulate spermatozoa velocity; however, the effect was modulated throughout the post‐thawing incubation. Based on the DNA fragmentation assay, sperm DNA stayed intact. Present study underlines the potential harm of NIR, which is frequently used in everyday life, with overall adverse impact on human and animal reproduction. Current study also points out on interesting short‐term spermatozoa stimulation induced by NIR.

Tirpak, Filip↗

pH-dependent Ca+2 oscillations prevent untimely acrosome reaction in human sperm

Highlights: • The external pH maintained during capacitation influences Acrosome Reaction. • Ca{sup 2+} oscillations prevent AR and are more frequent at external pH 6.5. • Pharmacological inhibition of Ca{sup 2+} oscillations restored Acrosome Reaction. During transit through the female reproductive tract, sperm encounter metabolites and environmental conditions that modulate various processes leading to fertilization. Intracellular Ca{sup 2+} dynamics regulate the acrosome reaction (AR), which involves exocytosis of the acrosomal granule, a prerequisite for successful fertilization. We explored the ability of progesterone, prostanglandin-E1, and GABA to induce Ca{sup 2+} mobilization and AR in single human spermatozoa capacitated under external pH (pH{sub e}) conditions found in different regions of the female reproductive tract (pH{sub e} 6.5, 7.4 and 8.0). The highest percentage of AR induction, regardless of the inducer, occurred when sperm were capacitated at pH{sub e} 7.4. Interestingly, at pH{sub e} 6.5 a high percentage of cells exhibit Ca{sup 2+} oscillations, which prevent AR. These oscillations involve extracellular and intracellular Ca{sup 2+} channels. Pharmacological inhibition of Ca{sup 2+} oscillations restores the ability of spermatozoa to undergo the AR when exposed to progesterone, even if capacitated at pH{sub e} 6.5.

60 APPLIED LIFE SCIENCES↗

Maturation and fertilization of echinoderm eggs: Role of actin cytoskeleton dynamics

Highlights: • Meiotic maturation of starfish oocytes involves the rearrangement of actin filaments and intracellular Ca{sup 2+} increases. • The actin cytoskeleton plays a role in optimizing the Ca{sup 2+} release mechanisms during oocyte maturation. • F-actin dynamics regulates Ca{sup 2+} signaling and cortical granules exocytosis at fertilization in starfish and sea urchin eggs. • Monospermic fertilization is heavily dependent upon the integrity of the actin cytoskeleton of the egg cortex. Starfish and sea urchin are excellent models to study the mechanisms that regulate oocyte maturation and egg activation. Hormonal stimulation of starfish oocytes and their following interaction with spermatozoa induce rapid changes of F-actin and Ca{sup 2+} increases which are prerequisites for normal fertilization and development. Fully grown oocytes isolated from the gonads of starfish contain a large nucleus (∼60–70 μm) (termed germinal vesicle, GV), which is arrested at the first prophase of meiosis. If inseminated, these immature oocytes are penetrated by additional spermatozoa. However, starfish oocytes naturally shed into the sea have already initiated the (meiotic) maturation and are normally fertilized between GV breakdown and the extrusion of the first polar body. This is considered the optimum period to ensure monospermic instead of polyspermic fertilization. By contrast, sea urchin eggs are fertilized only after being fully matured, i.e., at the end of the two meiotic divisions. Here, we provide a comparative review of the role of the actin cytoskeleton in oocyte maturation and fertilization in starfish and sea urchin. It has become increasingly evident that the exquisite regulation of the cortical F-actin is involved in nearly all aspects of the molecular events taking place during the progression of meiotic maturation and fertilization.

60 APPLIED LIFE SCIENCES↗

A gravitational test of wave reinforcement versus fluid density models

Spermatozoa, protozoa, and algae form macroscopic patterns somewhat analogous to thermally driven convection cells. These bioconvective patterns have attracted interest in the fluid dynamics community, but whether in all cases these waves were gravity driven was unknown. There are two conflicting theories, one gravity dependent (fluid density model), the other gravity independent (wave reinforcement theory). The primary objectives of the summer faculty fellows were to: (1) assist in sample collection (spermatozoa) and preparation for the KC-135 research airplane experiment; and (2) to collaborate on ground testing of bioconvective variables such as motility, concentration, morphology, etc., in relation to their macroscopic patterns. Results are very briefly given.

Johnson, Jacqueline Umstead↗

Genetic effects on heavy ions in drosophila

Drosophila sex-linked recessive lethal mutation test was used to study the dose response relation and relative biological effectiveness of heavy ions. The experiments were performed using the heavy ion beams at BEVALAC of Lawrence Berkeley Laboratory. These experiments were undertaken according to the proposed milestones and included Ne-20, A-40 and Fe-65 ions with respective energies of 600 MeV, 840 MeV and 850 MeV. At these energies several doses of these radiations ranging from 20 to 1280 R were used. Space radiation exposure to astronauts is supposed to be quite low and therefore very low dose experiments i.e., 20 R, were also performed for the three ions. The mutation response was measured in all germ cell types i.e., spermatozoa, spermatids, spermatocytes and spermatogonia of treated Drosophila males. A linear dose frequency relation was observed for most of the range except at high doses where the saturation effect was observed. Also, a very significant difference was observed among the sensitivity of the four germ cell stages where spermatozoa and spermatids were more sensitive. At the higher doses of this range, most of the spermatogonia and spermatocytes were killed. Although comparative and identical experiments with X-rays or neutrons have not been performed, the compassion of our data with the ones available in literature suggest that the heavy ions have a high rbe and that they are several times more effective than low LET X-rays. The rbe compared to neutrons however appears to be only slightly higher.

Kale, P. G.↗

Adaptation of Motility Analysis Apparatus for Space Science and Microgravity Ground-Based Experiments

Previous space flight studies have described unfavorable effects of microgravity on testicular morphology and spermatogenesis (Cosmos 1887 Biosputnik flight, 9/29/87 - 10/12/87). The flight animals demonstrated small reductions in testicular and epididymal size, the phenomenon explained as resulting water loss. Yet, light microscopic histological preparations revealed few spermatozoa in the rete testis of the flight males compared to control animals. The cause for this finding was subjectively assessed to be due to "the anatomical dislocation of the organs... and a disturbance in testicular blood supply". Unfortunately, the reported effects of microgravity on the reproductive processes (particularly within males) are few and divergent. If habitation in space is a futuristic goal, more objective testing (of male and female gametes) in a microgravity environment will provide insight to the developmental potential of these reproductive cells. As part of the Marshall Space Flight Centers' Summer Faculty Fellowship Program within the Biophysics Branch, a key component of the research investigation was to develop a test to evaluate individual cell motility and orientation in varying gravitational environments, using computerized assessment of sperm cell concentration, morphology and motility to provide objective, quantitative experimental control. In previous work performed jointly by the author and a NASA colleague, it has been shown that macroscopic motile aggregates of spermatozoa were not altered by the absence of microgravity. Variations in the number of normal versus abnormal sperm due to microgravity influences have yet to be established. It is therefore of interest to monitor the cytoskeletal matrix (microtubulin) of these organisms as a possible indicator of cell viability and/or function.

Johnson, Jacqueline U.↗

Pigment epithelium derived factor (PEDF) expression in the male tract of Wistar rats

Highlights: • PEDF expression is detected in epididymal tract of male rats. • PEDF expression was not detected in rat testes. • PEDF expression was decreased by using flutamide. • PEDF expression was recovered after flutamide cessation. Pigment epithelium derived factor (PEDF) expression has been described in many organs as showing neurotrophic, anti-angiogenic, anti-apoptotic, anti-inflammatory, anti-oxidant and pro-cell survival properties. However, references to its activity in the male reproductive system are scarce. We aimed to characterize the expression of PEDF in the male reproductive tract of Wistar rats by using RT-PCR, western blot and immunostaining and also evaluate the effect of flutamide in PEDF expression. We found that PEDF is expressed in the epididymis, prostate and seminal vesicles in Wistar rats, but notably not in the testes. Under the effect of flutamide PEDF expression decreased, recovering by suppressing the antiandrogen. The epididymis is an essential organ in sperm maturation-storages. The role of PEDF in this physiological process has not been fully elucidated yet, but considering that in other systems PEDF has anti-apoptotic, anti-oxidants and pro-cell survival properties, its expression along the epididymis could play a role in the protection of spermatozoa while they are stored.

60 APPLIED LIFE SCIENCES↗

The genomic effects of cell phone exposure on the reproductive system

Highlights: • Electromagnetic fields (EMF) causes an imbalance between pro-oxidant and antioxidant mechanismsEMF increases reactive oxygen species production. • Exposure to EMF leads to disruptions in spermatogenic cells. • Exposure to GSM-modulated EMF causes an increase in superoxide formation levels in human spermatozoa. Humans are exposed to increasing levels of electromagnetic fields (EMF) at various frequencies as technology advances. In this context, improving understanding of the biological effects of EMF remains an important, high priority issue. Although a number of studies in this issue and elsewhere have focused on the mechanisms of the oxidative stress caused by EMF, the precise understanding of the processes involved remains to be elucidated. Due to unclear results among the studies, the issue of EMF exposure in the literature should be evaluated at the genomic level on the reproductive system. Based on this requirement, a detail review of recently published studies is necessary. The main objectives of this study are to show differences between negative and positive effect of EMF on the reproductive system of animal and human. Extensive review of literature has been made based on well known data bases like Web of Science, PubMed, MEDLINE, Google Scholar, Science Direct, Scopus. This paper reviews the current literature and is intended to contribute to a better understanding of the genotoxic effects of EMF emitted from mobile phones and wireless systems on the human reproductive system, especially on fertility. The current literature reveals that mobile phones can affect cellular functions via non-thermal effects. Although the cellular targets of global system for mobile communications (GSM)-modulated EMF are associated with the cell membrane, the subject is still controversial. Studies regarding the genotoxic effects of EMF have generally focused on DNA damage. Possible mechanisms are related to ROS formation due to oxidative stress. EMF increases ROS production by enhancing the activity of nicotinamide adenine dinucleotide (NADH) oxidase in the cell membrane. Further detailed studies are needed to elucidate DNA damage mechanisms and apoptotic pathways during oogenesis and spermatogenesis in germ cells exposed to EMF.

63 RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTAN↗

Biological patterns: Novel indicators for pharmacological assays

Variable gravity testing using the KC-135 demonstrated clearly that biological pattern formation was definitely shown to result from gravity alone, and not from oxygen gradients in solution. Motile pattern formation of spermatozoa are driven by alternate mechanisms, and apparently not affected by short-term changes in gravity. The chemical effects found appear to be secondary to the primary effect of gravity. Cryopreservation may be the remedy to the problem of 'spare' or 'standing order' biological samples for testing of space lab investigations, but further studies are necessary.

Johnson, Jacqueline U.↗

Ground testing of bioconvective variables such as morphological characterizations and mechanisms which regulate macroscopic patterns

Conditions simulating low- and high-gravity, reveal changes in macroscopic pattern formation in selected microorganisms, but whether these structures are gravity dependent is not clear. Two theories have been identified in the fluid dynamics community which support macroscopic pattern formation. The first one is gravity dependent (fluid density models) where small concentrated regions of organisms sink unstably, and the second is gravity independent (wave reinforcement theory) where organisms align their movements in concert, such that either their swimming strokes beat in phase or their vortices entrain neighbors to follow parallel paths. Studies have shown that macroscopic pattern formation is consistent with the fluid density models for protozoa and algae and wave reinforcement hypothesis for caprine spermatozoa.

Johnson, Adriel D.↗

Physiological Anatomical Rodent Experiment (PARE): 04 Flight Support

Rats were shipped to Kennedy Space Center (KSC), Florida, on day 2 of gestation [(G2) (day 1 = morning on which spermatozoa are present in the vagina)] and laparotomized on G7 to determine the number of implantation sites in each uterine horn. On G8, ten pregnant rats meeting flight criteria (at least five implantation sites each horn) were placed into NASA flight cages (animal enclosure modules, AEMS, five rats per cage), loaded onto the mid-deck of the space shuttle Atlantis, and on G9 (November 3, 1994) they were launched into orbit. On gestation day 20, (November 14, 1994) the shuttle returned to Edwards Air Force Base, California and the flight animals were recovered from the shuttle and subjected to unilateral hysterectomy to provide fetal material from one horn to investigators identified by NASA to study selected fetal parameters. After unilateral hysterectomy, the animals were allowed to recover and deliver vaginally. There were three groups (n = 10 each group) of control animals housed at KSC in the study. A synchronous control group, delayed 24 hours with reference to the flight group, received the same surgeries and was housed five rats per AEM and exposed to all flight conditions (identical temperatures, lighting and humidity) except microgravity. Vivarium control group I did not receive any surgery and was housed in the vivarium in clear polycarbonate cages. Vivarium control group 2 received only a unilateral hysterectomy on day 20 of gestation but otherwise was housed like the vivarium control group 1. Control groups were allowed to complete their pregnancy and deliver fetuses from the remaining horn (Synchronous control and vivarium control group 2) or horns (Vivarium control group 1). After surgery, the dams were euthanized and tissues recovered, and the neonates were assigned to foster dams.

Burden, Hubert W.↗

Evaluation of Uterine Contractility by Magnetic Resonance Imaging in Women Undergoing Embolization of Uterine Fibroids

Purpose: To assess uterine contractility using ultrafast magnetic resonance imaging (cine MRI) before and after uterine fibroid embolization (UFE). Materials and Methods: This is a prospective study of uterine contractility in 26 patients (age 30–41 years) undergoing UFE for symptomatic uterine fibroids. Cine MRI was performed before and 6 months after UFE. Two radiologists evaluated uterine contractility and classified it as absent, ordered, or disordered. Patients were then grouped into three distinct patterns of progression: unchanged contractility (group A), modified contractility (B), and loss of contractility (C). These findings were then confronted with factors that might have interfered with uterine contractility pattern (uterine volume, location of dominant fibroid, fibroid/myometrium index, and fibroid necrosis pattern). Results: Of the 26 patients, 8 (30.7%) had no contractility before the procedure, while 18 (69.2%) exhibited some form of contractility (11 [61%] ordered, 7 [39%] disordered). All 8 patients who had no contractility at baseline exhibited contractility after UFE (5 ordered, 3 disordered). Of the 11 who had ordered contractility at baseline, 9 remained ordered and 2 lost contractility after UFE. Of the 7 with disordered contractility at baseline, 1 remained disordered, 5 progressed to ordered contractility, and 1 lost contractility. Overall, 10 patients (38%) had no change in contractility after UFE (group A), 13 (50%) had a positive change (group B), and 3 (11%) lost contractility (group C). The potential interference factors assessed had no statistically significant effect in any group. Conclusion: In women of reproductive age with symptomatic fibroids, uterine contractility improved significantly after UFE. Level of Evidence: Level 3—non-randomized controlled cohort/follow-up study.

62 RADIOLOGY AND NUCLEAR MEDICINE↗

Effects of Varicocele Treatment on Sperm Conventional Parameters: Surgical Varicocelectomy Versus Sclerotherapy

Introduction: Varicocele is often associated with impaired sperm parameters. Different procedures have been developed for varicocele treatment. The aim of this study was to evaluate the effects of varicocele treatment on conventional sperm parameters. Materials and Methods: We compared two different techniques of intervention: surgical varicocelectomy and sclerotherapy. We also evaluated the number of varicocele recurrences and the pregnancy rate. We included 102 patients (mean age 29.8 ± 0.8 years) with ultrasound diagnosis of varicocele. We excluded patients whose ultrasound evaluation and/or sperm parameters were not known before and after varicocele correction. We divided the patients (excluding 8 with azoospermia) into two subgroups: surgical varicocelectomy (n = 44) and sclerotherapy (n = 50). For each patient, we compared conventional sperm parameters before and after varicocele correction. Results: After varicocele correction, we found a significant improvement in sperm concentration, total count and total motility. Considering the two subgroups, baseline sperm parameters did not differ significantly. Sperm concentration and total count increased significantly after varicocele correction by varicocelectomy. Varicocele correction by sclerotherapy resulted in a significant increase in sperm concentration, progressive and total motility. We found varicocele recurrence in 32% of patients who underwent varicocelectomy and in 19.7% of patients undergoing sclerotherapy. The pregnancy rate was higher after sclerotherapy (28%) than after surgical varicocelectomy (13%). Conclusion: Varicocele treatment must be recommended when other causes of infertility have been treated. Our results suggest the use of sclerotherapy for varicocele repair. Level of Evidence: 2 b.

62 RADIOLOGY AND NUCLEAR MEDICINE↗

Structural and Functional Analysis of the Spermatogenic Epithelium in Rats Exposed to Titanium Dioxide Nanoparticles

We studied immunohistochemical and morphometric characteristics of the spermatogenic epithelium in rats against the background of peroral administration of nanoparticles containing titanium dioxide. Substantial degenerative changes in the spermatogenic epithelium were revealed: thinning, disorganization of layers, and detachment of sperm cells from the basement membrane. Immunohistochemical analysis revealed reduced proliferative activity and differentiation potential of epithelial cells, which was confirmed by changes in the expression of Ki-67 and c-kit markers. Our data attest to unfavorable effects of titanium dioxide nanoparticles on the structural and functional characteristics of the reproductive system in male rats leading to spermatogenesis disturbances.

60 APPLIED LIFE SCIENCES↗

Induction of immune-related gene expression by seminal exosomes in the porcine endometrium

Highlights: • Exosomes are present in sperm-rich fraction of porcine seminal plasma (SP). • Exosomes present in SP regulate immune-related genes in the porcine uterus. • Seminal exosomes may pave the way for sperm and fertilized egg transport. Seminal plasma (SP) is considered as a vehicle to carry sperm into female reproductive tract, of which functions have not been completely understood. This study aimed to identify the function of seminal exosomes on porcine endometrium. Exosomes were isolated from the sperm-rich fraction of boar semen and were confirmed by the expression of exosome marker HSP70 and size distribution using nano-sight tracking analysis. Porcine endometrial epithelial cells (EECs) were then treated with seminal exosomes, and RNA extracted were subjected to global expression analysis. Transcripts related to “immune response”, “inflammatory response” and their associated signaling pathways were up-regulated in EECs treated with seminal exosome, whereas those associated with “steroid biosynthesis”, “metabolic pathways” and “T cell differentiation” were down-regulated. The decrease in PMVK, SC5D, INSIG1, HSD17B7, NSDHL, HMGCR, SQLE and FDFT1, and increase in CCL20, TNFSF15, AMCFII, CXCL2 and CXCL8 were also found in the endometrium from the naturally mated pigs. Moreover, changes in exosome-induced CYP24A1, EBP, CCL20, AMCFII and IL1A expression were not regulated by the exosome removed SP. These observations indicated that exosomes present in SP are involved in the immune-related gene regulation in the uterus, which could pave the passage for sperm and possibly fertilized eggs.

60 APPLIED LIFE SCIENCES↗

Decrease in the cytosolic NADP+-dependent isocitrate dehydrogenase activity through porcine sperm capacitation

Highlights: • IDPc is phosphorylated at tyrosine residues during porcine sperm capacitation. • The tyrosine phosphorylated IDPc showed a significantly lowered enzymatic activity. • IDPc is mainly localized to the principal piece of the porcine sperm flagellum. • The decrease in IDPc activity is involved in the increased levels of ROS. • The decrease in IDPc activity induced the induction of the hyperactivated motility. In order to understand the molecular mechanisms involved in the sperm capacitation, we have identified the proteins tyrosine-phosphorylated during the capacitation especially in conjunction with the regulation of the levels of reactive oxygen species (ROS) in sperm. In the present study, the effects of the tyrosine phosphorylation of cytosolic NADP{sup +}-dependent isocitrate dehydrogenase (IDPc) on its catalytic activity and on the levels of ROS in sperm have been studied. The tyrosine phosphorylated IDPc showed a significantly lowered enzymatic activity. The immunocytochemical analyses using the highly specific antisera against IDPc revealed that IDPc was mainly localized to the principal piece of the porcine sperm flagellum. As IDPc is one of the major NADPH regenerating enzymes in porcine sperm, it is strongly suggested that the decrease in IDPc activity is involved in the increased levels of ROS, which results in the induction of hyperactivated flagellar movement and capacitation.

60 APPLIED LIFE SCIENCES↗