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Biomedical subjects

M C Gil

Publications and source records attributed to M C Gil.

At least 19 recordsLinked to original sources
PubMed2007

Phosphatidylinositol 3-kinase pathway regulates sperm viability but not capacitation on boar spermatozoa.

Phosphatidylinositol 3-kinase (PI3-K) plays an important role in cell survival in somatic cells and recent data pointed out a role for this kinase in sperm capacitation and acrosome reaction (AR). This study was undertaken to evaluate the role of PI3-K pathway on porcine spermatozoa capacitation, AR, and viability using two unrelated PI3-K inhibitors, LY294002 and wortmannin. In boar spermatozoa, we have identified the presence of PDK1, PKB/Akt, and PTEN, three of the main key components of the PI3-K pathway. Incubation of boar sperm in a capacitating medium (TCM) caused a significant increase in the percentage of capacitated (25 +/- 2 to 34 +/- 1% P < 0.05, n = 6) and acrosome reacted (1 +/- 1 to 11 +/- 1% P < 0.01, n = 6) spermatozoa compared with sperm in basal medium (TBM). Inhibition of PI3-K did affect neither the capacitation status nor AR nor protein p32 tyrosine phosphorylation of boar spermatozoa incubated in TBM or TCM. Boar sperm viability in TBM was significantly decreased by 40 and 20% after pretreatment with LY294002 or wortmannin, respectively. Similar results were observed after incubation of boar spermatozoa in TCM. Treatment of boar spermatozoa with the analog of cAMP, 8Br-cAMP significantly prevented the reduction on sperm viability. Our results provide evidence for an important role of the PI3-K pathway in the regulation of boar sperm viability and suggests that other signaling pathways different from PI3-K must be activated downstream of cAMP to contribute to regulation of sperm viability. Finally, in our conditions the PI3-K pathway seems not related with boar sperm capacitation or AR.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed2006

Standardization of sample preparation, staining and sampling methods for automated sperm head morphometry analysis of boar spermatozoa.

The accuracy of computer-assisted sperm morphometry analysis (CASMA) depends on the careful preparation, fixation and staining of spermatozoa. The efficiency of CASMA may be enhanced by developing optimized protocols. The aim of the present study was to evaluate the influence of sperm washing and the use of three staining techniques [rapid Panoptic, Hemacolor and Harris's Haematoxylin (HH)] on image-processing accuracy and boar sperm head morphometry. Sperm washing had a significant effect on samples stained with rapid Panoptic, increasing the percentage of correctly binarized sperm heads and the contrast between cells and background. However, rapid Panoptic yielded the lowest percentage of properly digitized sperm heads. HH provided the highest cell/background contrast, and also greater sperm head staining intensity, but discrimination of sperm midpieces was considered insufficient. Hemacolor occupied an intermediate position, providing acceptable colour intensity and satisfactory cell/background contrast. Use of different staining procedures prompted dimensional differences in sperm head morphometry. Significant differences between animals were observed for all morphometric parameters. Low within-animal variation coefficients reflected a homogeneous sperm head population. Between-animal variation coefficients were relatively high for Hemacolor and HH, and significantly high for the rapid Panoptic stain. Using Panoptic and HH, stable morphometric measurements required at least 100 properly digitized sperm heads rather than 200, while Hemacolor required only 50 spermatozoa. These results indicate that both washing of semen and staining procedures significantly affect the accuracy of image processing and sperm head dimensions. Hemacolor and HH proved to be the best staining techniques for evaluating sperm head dimensions in boar.

Animals↗
PubMed2005

Changes in tyrosine phosphorylation associated with true capacitation and capacitation-like state in boar spermatozoa.

Capacitation is defined as a series of events that render boar sperm competent to fertilize, either in vivo or in vitro. Moreover, preliminary stages of cryopreservation of spermatozoa involving cooling to 5 degrees C have been shown to induce capacitation-like changes in boar spermatozoa. Capacitation of boar spermatozoa is accompanied by protein phosphorylation, however the relationship between both processes is poorly understood. Capacitation status was assessed by chlortetracycline (CTC) staining. Changes in protein tyrosine phosphorylation were examined in pre-cleared whole cell lysates using a specific anti-phosphotyrosine monoclonal antibody. Our results in boar spermatozoa show a significant positive correlation between p32 tyrosine phosphorylation levels and percentage of capacitated (CTC pattern B) spermatozoa. Moreover, incubation of boar spermatozoa with two unrelated tyrosine kinase inhibitors induces a significant reduction in the percentages of capacitated and acrosome-reacted (AR) boar spermatozoa and a reduction in the p32 tyrosine phosphorylation. In our conditions, cooling boar spermatozoa to 5 degrees C and rewarming to 39 degrees C in a noncapacitating medium results in similar CTC staining patterns to those obtained after incubation of boar sperm for 1 or 4 hr at 39 degrees C in a capacitating medium. However, cooled-rewarmed fails to induce an increase in p32 tyrosine phosphorylation in boar spermatozoa. Moreover, CTC staining patterns of cooled-rewarmed spermatozoa do not change after incubation with a tyrosine kinase inhibitor. In conclusion, our results show a direct relationship between capacitation and tyrosine phosphorylation and suggest that p32 tyrosine phosphorylation levels could be used as a marker of the true capacitation changes observed in boar spermatozoa. Moreover, our results show that true capacitation and capacitation-like changes induced after cooling involve alternative intracellular tyrosine phosphorylation pathways in boar spermatozoa.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed2005

Boar sperm velocity and motility patterns under capacitating and non-capacitating incubation conditions.

This study compares the velocity and motility of boar sperm under capacitating and non-capacitating incubation conditions. Aliquots of pooled, washed boar sperm were incubated in either Tyrode's complete medium (TCM; a capacitating medium), Ca2+-free TCM (TCM-Ca2+), or Ca2+ and NaHCO3-free TCM (Tyrode's basal medium [TBM]; a non-capacitating medium). Motility patterns were determined every hour over a 3h period of incubation at 38 degrees C. Capacitation status was assessed by the chlortetracycline assay after 1 and 3h of incubation. Experiments were repeated five times. Compared to the TBM control, a significant increase was seen in the percentage of capacitated sperm after 1h of incubation in TCM: the kinematics of these sperm cells were favorably modified. However, the motility patterns of sperm cells incubated in TCM and TCM-Ca2+ were very similar. Under capacitating conditions (TCM), the coefficients of linearity (LIN) and straightness (STR) significantly increased over time (LIN values were significantly different after 3h of incubation, while STR values were significantly different after only 2 h). Significant correlations were seen between LIN and the percentage of cells showing the B pattern (r = 0.334, P < 0.05) and the number of acrosome reacted spermatozoa (r = 0.301, P < 0.05). This suggests that capacitated boar spermatozoa may have a species-specific motility pattern.

Animals↗
PubMed2005

Inhibition of phosphatidylinositol 3-kinase modifies boar sperm motion parameters.

Motility is the most widely used indicator of sperm quality. Besides modulation by the cAMP pathway little is known regarding the intracellular pathways that regulate boar sperm motility. Recently the role of phosphatidylinositol 3-kinase (PI3-K) in the regulation of human sperm motility has been described. Therefore, the aim of this study was to investigate the role of PI3-K in boar sperm kinematics by using the specific PI3-K inhibitor, LY294002. Boar sperm was incubated up to 1 h in non-capacitating medium in the presence or absence of the cAMP analog, 8Br-cAMP or the PI3-K inhibitor, LY294002 or both. Boar sperm incubated in capacitating medium was treated in the presence or absence of LY294002. First, we have clearly identified that PI3-K is present in whole lysates of boar spermatozoa. Inhibition of PI3-K significantly increased boar sperm straight-line velocity, circular velocity and average velocity without an effect on the percentage of progressively motile spermatozoa in both media. Inhibition of PI3-K induced the same effects on boar sperm velocities as activation of the cAMP/protein kinase A (PKA) pathway and treatment with the PI3-K inhibitor, LY294002 had neither summatory nor synergic effects on boar sperm motion parameters when treated simultaneously with the cAMP analog 8Br-cAMP. Our data suggest that PI3-K plays a negative role, regulating boar sperm motion parameters through a possible inhibition of the cAMP/PKA activating pathway, and since some Computer Aided Sperm Analysis (CASA)-derived parameters have been related to field fertility our results point to the possibility of modulating sperm motile quality by modifying the PI3-K cellular pathway.

Animals↗
PubMed2005

Structure-based assessment of BRCA1 and BRCA2 mutations in a small Spanish population.

Breast cancer is the most frequent cancer type among women in the world. There is abundant evidence, that alterations in the breast cancer susceptibility genes, BRCA1 and BRCA2, are present in a large proportion of families with multiple cases of breast and ovarian cancer. Our aim was to better understand the molecular and structural insights associated with the most frequent alterations, found in the BRCA1 and BRCA2 genes, within our patients. We analyzed 50 breast cancer cases. Of those, 24% were found to carry deleterious mutations, and up to 10 rare variants of unknown significance were detected. The frequency of polymorphic changes was considerably different between genes, BRCA1 (55%) and BRCA2 (19.4%). By looking at the protein level, the changes observed within the BRCA1 and BRCA2 genes could affect not only the nucleic location of the protein but also disrupt the folding and, therefore, the tumor suppressor function.

BRCA1 Protein↗
PubMed2003

Genital lesions in an outbreak of caprine contagious agalactia caused by Mycoplasma agalactiae and Mycoplasma putrefaciens.

This paper reports on the genital lesions observed in adult male and female goats from a commercial flock in the Extremadura region of southwestern Spain, following an outbreak of contagious agalactia syndrome caused by Mycoplasma agalactiae and M. putrefaciens. Although both species were isolated from several organs, M. putrefaciens was the only agent isolated from the genital lesions reported here, characterized by desquamative salpingitis and cystic catarrhal metritis in females and by testicular degeneration in males. Mycoplasma putrefaciens was isolated from the testes of only one of the males examined.

Animals↗
PubMed2001

Effect of vulvomucosal injection of D-cloprostenol at weaning and at insemination on reproductive performance of sows during the low fertility summer season under field conditions.

The aim of this study was to evaluate the hypothesis that the intravulvar injection of a PGF2alpha analogue at weaning just prior to insemination may minimise the effects of summer infertility in pigs. From July to September 1999, two groups of 30 sows were randomly formed each month. The experimental group comprised sows receiving 37.5 microg of a PGF2alpha analogue, D-cloprostenol, in 0.5 ml injected into the vulvar lips at weaning and at insemination. Group 2 sows received 0.5 ml of saline solution injected into the vulvar lips and served as controls. The percentage of sows in oestrus within 7 days after weaning in treated sows was 27.93% higher than in controls (P<0.001). Fertility for treated sows was also significantly increased by the treatment (P<0.01). However, neither the percentage of inseminated sows that farrowed nor litter size was affected by treatment.

Animals↗
PubMed2001

CD99 expression is positively regulated by Sp1 and is negatively regulated by Epstein-Barr virus latent membrane protein 1 through nuclear factor-kappaB.

Epstein-Barr virus (EBV)-encoded latent membrane protein-1 (LMP1) is highly expressed in Hodgkin and Reed-Sternberg (H-RS) cells from patients with EBV-associated Hodgkin disease. It was previously demonstrated that CD99 can be negatively regulated by LMP1 at the transcriptional level, and the decreased expression of CD99 in a B lymphocyte cell line generates H-RS-like cells. In this study, detailed dissection of the CD99 promoter region was performed to search regulatory factor(s) involved in the expression of the gene. Using various mutant constructs containing deletions in the promoter region, it was revealed that the maximal promoter activity was retained on 5'-deletion to the position -137 from the transcriptional initiation site. Despite the presence of multiple putative Sp1-binding sites in the promoter region, the site located at -95 contributes heavily as a positive cis-acting element to its basal promoter activity. However, on examination of the involvement of the positive-acting Sp1-binding site of the promoter for the repressive activity of LMP1, it appeared to be dispensable. Instead, the repressive effect was mapped to the nuclear factor (NF)-kappaB activation domains in the cytoplasmic carboxyl terminus of LMP1 despite the absence of the NF-kappaB consensus sequences in the CD99 promoter region. Furthermore, the decreased CD99 promoter activity by LMP1 was markedly restored when NF-kappaB activity was inhibited. Taken together, these data suggest that Sp1 activates, whereas LMP1 represses, transcription from the CD99 promoter through the NF-kappaB signaling pathway, and they might aid in the understanding of the molecular mechanisms of viral pathogenesis in EBV-positive Hodgkin disease. (Blood. 2001;97:3596-3604)

12E7 Antigen↗
PubMed2001

Effects of simultaneous kidney-pancreaticoduodenal transplantation on diabetes-induced renal insufficiency in rats.

An investigation of the functional and histological changes was done after en-bloc kidney-pancreaticoduodenal transplantation (kpdt) in the diabetes-induced, renal insufficient Lewis rats. For donor preparation, an end-to-side portocaval shunt was performed, and the aortic, vena caval segments, and ureter-bladder patch were obtained. They were anastomosed microsurgically to recipient's aorta, vena cava, and bladder in end-to-side fashion. Of 15 diabetes-induced kpdt rats, 14 survived. Two of the 14 surviving rats showed ischemic necrosis. The remaining 12 transplants showed well-preserved glomeruli and Langerhans islets for 5 months postoperatively. Biochemical data comparing diabetic and sham-operated rats (six rats each), six diabetic controls, and 12 kpdt rats showed no significant statistical difference at said observation period. The diabetes-induced kpdt rats showed improvement of following biochemical data: within 1 week postoperatively, the glucose level fell from 300 to 115 mg/dL; BUN level from >20 to <20 mg/dL; the creatinine level from 1.5 to <1.2 mg/dL. The insulin level returned to normal, 1.1 ng/mL, in 2 weeks. The results demonstrate that the kpdt model is an effective and successful operative technique in diabetic rats and may provide effective therapeutic methods for diabetes-induced renal insufficiency.

Animals↗
PubMed2001

Social-physiological compliance as a determinant of team performance.

A cybernetic model of behavior predicts that team performance may depend on physiological compliance among participants. This laboratory study tested if compliance in electrodermal activity (EDA), heart rate or breathing in two-person teams (N=16) was predictive of team performance or coordination in a continuous tracking task simulating teleoperation. Visual contact among participants was manipulated. Physiological compliance was scored with weighted coherence and cross correlation. Separate multiple regression analyses revealed that the task completion time was predicted by coherence measures for EDA and heart, but only at a trend level for breathing. Task completion time was also predicted by heart cross correlation. Team tracking error was predicted by coherence measures for EDA, heart and breathing, and also heart cross correlation. While social-visual contact did not have an impact, physiological compliance was predictive of improved performance, with coherence robust over all three physiological measures. Heart cross correlation showed the strongest predictive relationships. These results provide evidence that physiological compliance among team members may benefit team performance. While further study is needed, physiological compliance may someday provide a needed tool for the study of team work, and an objective means to guide the ergonomic design of complex sociotechnical systems requiring a high degree of team proficiency.

Adolescent↗
PubMed1999

Influence of age on stress responses to metabolic cage housing in rats.

1. We studied the effect of isolation stress in 3- and 12-month-old rats individually housed in metabolic cages for 7 days. Urine (24 hr) was collected daily from one group of animals of each age. The other group was tested in an open field and on a hot plate on days 1 and 7. 2. Total deambulation in the open-field test was lower in young than in older rats both on day 1 (54.7 +/- 9.9 vs 80 +/- 8.9 crossings/session; P < 0.04) and on day 7 (21 +/- 9 vs 48 +/- 7 crossings per session; P < 0.04) and decreased significantly in the two groups when tested on day 7 (P < 0.03). Latency to paw-licking in the hot-plate test was longer in young than in older animals on day 1 (14 +/- 2 vs 8 +/- 4 sec; P < 0.05) but was similar in the two groups on day 7. 3. Urinary excretions of norepinephrine (NE) and epinephrine (E) were determined by HPLC with electrochemical detection. Urinary NE in day 1 was similar in young and older animals (2627 +/- 828 vs 3069 +/- 598 ng/24 hr). In young animals NE excretion decreased along the study and was significantly (P < 0.02) lower than on day 1 during the last 3 days of the study. Conversely, in older animals urinary excretion of NE remained similar throughout the study. On day 7 urinary excretion of NE in older animals was about two fold that in young rats. Urinary E was similar in young and older rats (341 +/- 127 vs 532 +/- 256 ng/24 hr) on day 1 and showed a tendency to increase throughout the study. 4. Urinary monoamine oxidase inhibitory (IMAO) activity was determined by testing the ability of urine extracts to inhibit rat liver MAO activity in vitro and was higher in young than in older animals throughout the study (day 1, 54.8 +/- 4.2 vs 25.1 +/- 5.1%; P < 0.02). In young rats excretion of IMAO was significantly higher during the last 3 days of the study than on day 1 (P < 0.05). In older animals urinary IMAO showed a tendency to increase at the end of the study. 5. Isolation stress caused by housing rats in metabolic cages results in different behavioral and metabolic responses in young and older animals. Young animals exhibit a lower locomotor and analgesic response and excrete lower amounts of NE and higher IMAO activity in the urine than older rats. The metabolic and behavioral responses to isolation stress are highly dependent on the age of the animals tested. These results should be taken into consideration when designing experiments requiring the use of metabolic cages.

Age Factors↗
PubMed1999

Mastitis caused by Mycoplasma mycoides subspecies mycoides (large colony type) in goat flocks in Spain.

We describe three different outbreaks of mastitis caused by M. mycoides subspecies mycoides LC type (Mmm LC) in three goat flocks from the Extremadura Region of south-west Spain. Thirty-two fast-growing isolates were obtained on Hayflick's and Friis's media with inhibitors from different specimens. All were identified as Mmm LC in spite of their cultural, biochemical and serological features.

Animals↗
PubMed1998

Expression of C5 protein, the protein component of Escherichia coli RNase P, from the tac promoter.

The accurate function of C5 protein, the protein component of Escherichia coli RNase P, is uncertain in vivo. A controllable expression system for C5 protein was constructed which can be used to investigate effects of C5 protein on various cellular functions including biosynthesis of RNase P in vivo. The semisynthetic rnpA gene encoding C5 protein was fused to the tac promoter of the pKK223-3 expression vector. This tac promoter expression system produced a high level of C5 protein upon induction with isopropyl-beta-D-thiogalacto-pyranoside. When the overexpressed C5 protein was purified and used for reconstitution of RNase P, the reconstituted enzyme was active. The N-terminal amino acid of the overexpressed C5 protein was leucine specified by the second codon of the rnpA gene. The more controllable expression system was constructed by introducing the lacIq gene into the vector sequence itself.

Bacterial Proteins↗