Andres A. Ponce

Activity of Chinchilla laniger spermatozoa collected by electroejaculation and cryopreserved

Because reproductive studies and the application of assisted reproductive techniques are relevant issues for an endangered species such as Chinchila laniger, the availability of a source of viable spermatozoa becomes of utmost importance. In this paper, we evaluate several functional parameters (motility, viability, response to hypoosmotic swelling test and acrosomal integrity) of fresh or frozen-thawed spermatozoa. Electro-ejaculation trials (50-cyc/sec sinusoidal wave was applied for 5 of every 10 sec) were successful in all unanesthetized animals. After volume (108.3 +/- 12.0 microL, n = 15) and concentration (421.8 +/- 34.4 x 10(6) cells/mL, n = 15) measurements, the above mentioned parameters were determined. In frozen-thawed semen samples sperm motility, viability, hypoosmotic swelling test and acrosomal integrity were significantly lower than in fresh semen samples. The results clearly indicated that electro-ejaculation is a useful method for evaluating spermatozoa for genetic analysis or for used in Al in this species. In addition, the cryopreservation procedure in this study preserved adequate levels of functional sperm activity.

Pathogenic Consequences in Semen Quality of an Autoimmune Response against the Prostate Gland: From Animal Models to Human Disease

We have recently proposed an autoimmune etiology in ∼35% of chronic nonbacterial prostatitis patients, the most frequent form of prostatitis observed, because they exhibit IFN-γ-secreting lymphocytes specific to prostate Ags. Interestingly, this particular group of patients, but not the rest of chronic nonbacterial prostatitis patients, also presented striking abnormalities in their semen quality. In this work, we use an experimental animal model of autoimmune prostatitis on Wistar rats developed in our laboratory to investigate when, where, and how sperm cells from autoimmune prostatitis individuals are being damaged. As in patients, a marked reduction in sperm concentration, almost null sperm motility and viability, and an increased percentage of apoptotic spermatozoa were detected in samples from animals with the disease. Prostate-specific autoantibodies as well as elevated levels of NO, TNF-α, and IFN-γ were also detected in their seminal plasma. In contrast, epididymal spermatozoa remain intact, indicating that sperm damage occurs at the moment of joining of prostate secretion to sperm cells during ejaculation. These results were further supported by experiments in which mixture of normal sperm cells with autoimmune seminal plasma were performed. We hypothesize that sperm damage in experimental autoimmune prostatitis can be the consequence of an inflammatory milieu, originally produced by an autoimmune response in the prostate; a diminished prostate functionality, evidenced by reduced levels of citric acid in semen or by both mechanisms simultaneously. Once more, we suggest that autoimmunity to prostate may have consequences on fertility.

Differential effects of pharmacologically generated reactive oxygen species upon functional activity of epididymal mouse spermatozoa.

Several studies indicate that reactive oxygen species (ROS) are involved in defective sperm function pathophysiology. In this study we attempted to determine differentially the effects of xanthine (0.12 mM) plus xanthine oxidase (0.035 U/mL) (X+XO, a ROS promoter system), ROS scavengers (Tiron (TIR, 15 mM); catalase (CAT, 10 micrograms/mL); dimethylsulfoxide (DMSO, 140 mM)), and X+XO plus scavengers on several epididymal mouse spermatozoa functional parameters, incubated in NTPC medium, for 29 min. In the presence of X+XO, progressive gametes significantly diminished. TIR or CAT attenuated this effect, but DMSO did not. Inversely, X+XO increased the bending-forms population; only TIR reversed this phenomenon. The ROS promoter system diminished the viable cell population; all scavengers assayed maintained sperm viability at levels similar to control ones. When exposed to hypoosmotic shock after 29 min incubation with X+XO, the percentage of swollen cells decreased; TIR, CAT, or DMSO did not prevent this effect. Our experiments demonstrate that it is possible to differentiate the deleterious ROS effects upon sperm functional activity. O-2. and H2O2 preferentially seem to modify sperm motility, O-2. exhibiting the greatest ability for generating bending-form gametes, OH-being the most lethal ROS. In addition, sperm membrane clearly appears as the most damaged structure.

INFLUENCE OF PENTOXIFYLLINE ON SPERM MEMBRANE FUNCTIONAL INTEGRITY

In epididymal mouse spermatozoa, the effects of dibutyryl cyclic adenosine monophosphate 1 mmol/L (dbcAMP), pentoxifylline 5 mmol/L (PX), and/or mastoparan 50 mumol/L (MT) were evaluated for the following parameters: percentage of motile cells and response to hypoosmotic shock (HOS). The gametes were incubated during 80 min (A) or 200 min (B) in Tyrodes medium, and the drugs were added during the last 20 min. In A, dbcAMP + PX (61.5 +/- 5.4%; n = 10) enhanced and MT decreased significantly the population of motile cells (13.4 +/- 5.4%; n = 6) (control: 47.6 +/- 3.9%; n = 11). In B, PX significantly increased this parameter and MT plus PX also exerted a significant detrimental effect. Responses to HOS dropped significantly in the presence of PX + MT in A or in B; in this latter condition a similar decrease was evoked by MT alone. A positive correlation between percentages of swollen and motile spermatozoa was detected in A or in B in samples incubated with PX (r = .58, n = 11 and r = .76, n = 10; p < .05, respectively). These results that support that, in mouse sperm tail, PX would preserve functional membrane integrity, a relevant condition for adequate motility.

Chemical compositions and properties of Schinus areira L. essential oil on airway inflammation and cardiovascular system of mice and rabbits

The main purpose was to investigate the effects of essential plant-oil of Schinus areira L. on hemodynamic functions in rabbits, as well as myocardial contractile strength and airways inflammation associated to bacterial endotoxin lipopolysaccharide (LPS) in mice. This study shows the important properties of the essential oil (EO) of S. areira studied and these actions on lung with significant inhibition associated to LPS, all of which was assessed in mice bronchoalveolar lavage fluid and evidenced by stability of the percentage of alveolar macrophages, infiltration of polymorphonuclear leukocytes and tumor necrosis factor-α concentration, and without pathway modifications in conjugated dienes activity. Clinical status (morbidity or mortality), macroscopic morphology and lung/body weight index were unaffected by the administration of the EO S. areira. Furthermore, the ex vivo analysis of isolated hearts demonstrated the negative inotropic action of the EO of S. areira in a mice model, and in rabbits changes in the hemodynamic parameters, such as a reduction of systolic blood pressure. We conclude that EO S. areira could be responsible for modifications on the cardiovascular and/or airway parameters.

In vitro parthenogenesis of mouse oocytes under several experimental conditions

Although the in vitro fertilisation index is a parameter commonly employed to investigate sperm functional activity, little attention has been given to the occurrence of parthenogenesis. The purpose of this study was to study at 6 h or 22 h incubation: (a) the cleavage-related events that occur in in vitro incubated mouse oocytes, in the absence (parthenogenesis) or presence of homologous spermatozoa; (b) the effect of mineral oil, commonly used in in vitro fertilisation assays; (c) the effect of piroxicam, a prostaglandin synthesis inhibitor, on the parthenogenetic rate; and (d) the influence on parthenogenesis of spontaneous loss of the cumulus oophorus coat during incubation. Under the experimental conditions employed, there was parthenogenetic activation and activation due to fertilisation. Both increased in a time-dependent manner. The mineral oil enhanced the parthenogenetic rate at 22 h incubation. However, it did not have any effect when the oocytes were inseminated. Since we can not discriminate how much of this activation was due to fertilisation and how much to parthenogenesis we must be very careful with this comparison. Piroxicam 10(-8) M did not show any effect on the mouse oocyte parthenogenetic rate at neither 6 h or 22 h incubation. Our results suggest that oocyte susceptibility to spontaneous parthenogenetic activation may be modified by the presence of the cumulus and corona radiata cells. In conclusion, we consider that further rigorous studies on these influences are necessary in order to confer more reliability on the results.

Bio-efficacy of the Essential Oil of Oregano (Origanum vulgare Lamiaceae. Ssp. Hirtum)

The aim of this study was to investigate the bioactivity of the essential oil isolated from Origanum vulgare L. (EOv). We analyzed the in vivo anti-inflammatory properties in a mouse-airway inflammation model and the in vitro antimicrobial activity, genotoxicity over the anaphase-telophase with the Allium cepa strain and its cytotoxicity/viability in A549 culture cells. In vivo, EOv modified the levels of tumor necrosis factor -α and viable activated macrophages and was capable to mitigate the effects of degradation of conjugated dienes. In vitro, EOv reduced the viability of cultured A549 cells as well as the mitotic index and a number of chromosomal aberrations; however, it did not change the number of phases. We found that EOv presents antimicrobial activity against different Gram (−) and (+) strains, measured by disc-diffusion test and confirmed with a more accurate method, the AutoCad software. We postulate that EOv presents antibacterial, antioxidant and chemopreventive properties and could be play an important role as bioprotector agent.

Effects of Cecropia pachystachya and Larrea divaricata aqueous extracts in mice

Our studies were performed to investigate the effects of the aqueous extracts of Cecropia pachystachya and Larrea divaricata. These plants are used in folkloric medicine in infusion and were administered orally (0.76 g/kg) to male Albino Swiss mice for 16 days, on drink intake, organ weight/body weight (OW/BW × 100) ratio, histology, broqueoalveolar fluid (BALF) and elevated plus-maze (EPM). Feeding as well as body weight were unaffected by the consumption of these extracts. There were no signs of toxicity in BALF, morbidity or mortality during the study. C. pachystachya caused an increase in relative kidney OW/BW (p ≤ .05 vs control). The macroscopic and microscopic morphologic analyses of the organs were not altered by administration of these plants. A non-anxiolytic-like activity of an aqueous extract prepared from the leaves of C. pachystachya and L. divaricata in EPM was observed. We conclude that the crude aqueous extracts of leaves tested on mice orally did not produce signs of toxicity or behavioral changes in routine histological and clinical evaluation. However, knowledge of the biological activity of many herbal medicine used in Latin American is still deficient and more studies will be needed to elucidate the possible toxic effects.

Biphasic Modulation of Neutrophil Migration by Aqueous Extracts of Uncaria tomentosa in Murine Lung

In the history of medicine, the treatment of disease has been influenced by an intimate contact with nature. A mice model of lipopolysaccharide (LPS)-induced acute lung injury was used to evaluate the protective effects of long-term water-soluble administration ad libitum of Uncaria tomentosa extracts (20 gr/L; UTE) in lung inflammation. Swiss mice had LPS (1,67µg/ml) instilled intranasally 3hs before sacrificed, and were then pre-treated with UTE for 7, 15, 30 or 90 days or with a single dose of dexamethasone (2,5 mg/kg, DX). Inflammatory cell concentration was measured in the bronchoalveolar fluid (BALF) and histology was performed. No acute or chronic toxicity signs were observed in the clinical status. In addition, body weight, food consumption, organ weight, kidney, liver, and lung pathology were not found to be affected by the UTE treatments. UTE or DX significantly reduced the lung edema, exudation and lung injury histology for 7 and 90 day treatments. In addition, pre-treatment with UTE revealed a biphasic attenuated recruitment in BALF from neutrophils at 7 and 90 days induced by endotoxin exposure compared to the control (p<0.05). These data suggest that UTE initially induces a nonspecific response that is transient protection from PMNs migration into the lung mice.

Selection of a high quality subpopulation of frozen thawed bovine spermatozoa by filtration in a Sephadex column. An assessment of functional effects of progesterone

Summary It is accepted that cryopreservation exerts deleterious effects on functional characteristics of mammalian spermatozoa. Conventional procedures for processing frozen-thawed gametes, such as centrifugation, produce additional damage. In the present work, we investigated the efficacy of processing bovine cryopreserved semen by filtration in a Sephadex column (SF group) or by washing by centrifugation (100 g, 10 min, twice) (W group); the results obtained from both procedures were compared to untreated samples (C group). The effects of in vitro addition of progesterone (10 μM, 20 min) upon sperm functional activity were studied also. The evaluated sperm parameters were concentration, motility (progressive or non progressive cells), viability and acrosome reaction. They were measured at time 0 (immediately after processing) or after 4 h incubation in capacitating conditions. Sperm concentration was (× 10−6): 37.5 ± 5.4 in C, 8.3 ± 2.1 in W and 12.5 ± 2.9 in SF. The percentages of motile, progressive, viable or acrosome intact gametes were significantly higher in SF than in W or in C. in SF group, after 4 h incubation in capacitating conditions, progesterone increased significantly the population of acrosome reacted cells whereas this parameter was not modified when the cells were incubated in absence of heparin. Motility and viability were not modified by the hormone. We conclude that Sephadex filtration method is an adequate tool to obtain a subpopulation of spermatozoa with superior quality, as assessed by motility, viability and acrosomal integrity; besides, our results strongly support that, as in other species, progesterone would be a physiological inductor of acrosome reaction in bovine.