Effect of estradiol cypionate and GnRH treatment on plasma estradiol-17ß concentrations, synchronization of ovulation and on pregnancy rates in suckled beef cows treated with FTAI-based protocols.

Two experiments were conducted to evaluate the effect of different ovulation inducers on E-17ß plasma concentrations, synchronized ovulations and pregnancy rates. In Experiment 1, cows received a progesterone intravaginal device (PID) with 1 g of progesterone (P4) plus 2 mg of estradiol benzoate (EB) (day 0). At PID removal (day 8), cows received 0.150 mg of D-cloprostenol and were randomly assigned to four treatment groups (n = 10/treatment): Group ECP: 1 mg of estradiol cypionate at PID removal, Group EB: 1 mg of EB 24 hr after PID removal, Group GnRH: 10 µg of GnRH 48 hr after PID removal, Group ECP-GnRH: 1 mg of ECP at PID removal plus 10 µg of GnRH 48 hr later. Ultrasonographic examinations were performed to detect the dominant follicle and ovulation. GnRH-treated cows ovulated later (p < .05) compared to ECP- and ECP+GnRH-treated cows. There were effects of treatment, time and their interaction on E-17ß concentrations (p < .05). ECP treatment affected plasma E-17ß concentration, which increased earlier and decreased later compared to treatments without ECP. In Experiment 2, cows received (i) ECP: n = 126; (ii) EB: n = 126; (iii) GnRH: n = 136; (iv) ECP+GnRH: n = 139; FTAI was performed 48-50 hr after PID removal. Pregnancy rates did not differ among ovulation inducers (p > .05; ECP: 54.0%, 68/126; EB: 49.2%, 62/126; GnRH: 40.4%, 55/136; ECP+GnRH: 43.9%, 61/139). In conclusion, ECP administration (ECP and ECP+GnRH treatments) affected E-17ß concentrations, determining its earlier increase and later decrease compared to treatments without ECP (EB and GnRH treatments). ECP+GnRH-treated cows achieved the best distribution of ovulations without affecting pregnancy rates.

Biochemical and steroid concentrations in follicular fluid and blood plasma in different follicular waves of the estrous cycle from normal and superovulated beef cows.

The objectives of the current study were to (i) define the changes in size and number of follicles populations, (ii) determine the follicular fluid (FF) biochemical and steroid concentrations collected from different-sized follicles (5-9 and ≥ 10 mm) and (iii) compare between biochemical and hormonal concentrations of FF with those in blood plasma in relation to the first two follicular waves of the estrous cycle (days 4 and 13) from normal and cows primed for superovulation. After estrus, cows (n=20) were assigned randomly to each of four treatment groups. Group 1: ovariectomy on day 4 (day 0 = ovulation). Group 2: FSH treatment and ovariectomy on day 4. Group 3: dominant follicle ablation (DFA) on day 8 and ovariectomy on day 13. Group 4: DFA on day 8, FSH treatment and ovariectomy on day 13. Blood samples were collected and FF was aspirated and pooled per follicle class within cow to determine glucose, urea, triglycerides, cholesterol, total protein, albumin, lactate dehydrogenase, alkaline phosphatase, gamma-glutamyl transpeptidase, aspartate aminotransferase, creatin phosphokinase, estradiol-17ß and progesterone concentrations. Follicular class×follicular wave interaction was detected for albumin and lactate dehydrogenase. Results showed that FF concentrations of cholesterol increased from medium to large follicles and decreased for urea and aspartate aminotransferase. Tryglycerides and total protein were greater in the second than in the first follicular wave. FSH treatment decreased FF alkaline phosphatase, E2 and P4 concentrations. Quantitative differences between these fluids are discussed with respect to follicular development.

Effect of repeated eCG treatments and ovum pick-up on ovarian response and oocyte recovery during early pregnancy in suckling beef cows.

This study was designed to evaluate in suckling early pregnant beef cows with and without eCG-pre-stimulation: (i) the influence of day gestation (from 40 to 101 days) and the consecutive eCG treatments on the follicular growth induced by means of ultrasound-guided transvaginal follicle ablation (FA; all follicles ≥ 5 mm) and the number and quality oocytes recovered by ovum pick-up (OPU) and (ii) the possible effects of repeated hormonal stimulation and FA/OPU on pregnancy outcome. Twelve suckling early pregnant Angus cows (40 days post fixed-time artificial insemination) were randomly assigned to each of two groups (n=6 group(-1)). Group 1 treatments included: FA (Day 0), eCG (1600 IU; Day 1) and OPU (Day 5). Group 2: as cited Group 1 with no eCG treatment. In both groups, OPU was repeated five times (Days 45, 59, 73, 87 and 101 of gestation). The numbers (mean ± SEM) of class II (5-9 mm; 4.3 ± 0.9) and class III (≥10 mm; 2.5 ± 0.4) follicles visualized per cow per OPU session in eCG-treated cows were greater (P<0.05) than for non-treated cows (0.9 ± 0.1 and 0.9 ± 0.1, respectively). In contrast, the number (mean ± SEM) of class I (<5mm) follicles per cow per OPU session was lower for cows with eCG treatment (2.8 ± 0.4) than for non-treated cows (5.7 ± 0.5). The mean number of aspirated follicles was not significantly different (P<0.05) between eCG-treated cows and non-treated cows at 45 and 59 days of pregnancy. However, the mean number of aspirated follicles was greater (P=0.03) in eCG-treated cows than non-treated cows from 73 day of pregnancy onwards. The numbers (mean ± SEM) of recovered oocytes and viable oocytes/cow/session were greater (P<0.05) for eCG-treated cows (2.2 ± 0.2 and 1.6 ± 0.4, respectively) than for non-treated cows (1.0 ± 0.2 and 0.9 ± 0.2, respectively). No donor pregnancies were lost either during or following OPU procedure. We can conclude that (1) eCG-treated pregnant suckled cows can be a source of oocytes for IVF at least to 100 days of gestation and (2) repeated FA/eCG treatment/OPU procedures did not affect the pregnancy outcome.

Influence of the length of progestagen treatment and the time of oestradiol benzoate application on the ovulatory follicle size and ovulation time in anoestrous and cyclic beef cows.

Previous research from our laboratory in beef cattle suggests that the pre-ovulatory follicle size, maturity and subsequent susceptibility to gonadotropin are influenced by the length of progestagen treatment in artificial insemination programme in beef cows. To test this hypothesis, two experiments were conducted. In experiment 1, 35 anoestrous beef cows received an intravaginal sponge containing 200 mg of medroxyprogesterone acetate. The treatment lasted for 7 (n = 12), 8 (n = 11) or 9 (n = 12) days. Half of the animals in each group were injected with 0.7 mg of oestradiol benzoate (EB) at device removal (0 h) and the other half 24 h later. In experiment 2, 38 cycling beef cows were treated with the same protocols as in experiment 1. Ultrasound examinations were performed to determine the follicular diameter at device removal (dominant follicle), interval to ovulation and ovulatory follicle diameter. The dominant follicle of anoestrous cows with progestagen for 7 days (8.4 ± 1.6 mm) resulted smaller (p < 0.05) than the cows treated for 8 (10.5 ± 1.6 mm) and 9 days (10.6 ± 1.2 mm). However, regardless of the length of the treatments, ovulation time after device removal was longer (p < 0.05) when EB was injected 24 h after withdrawal than at 0 h in anoestrous cows (EB0 = 52.7 ± 4.0 h; EB24 = 70.8 ± 6.2 h) and in cyclic cows (EB0 = 50.0 ± 21.0 h; EB24 = 73.0 ± 20.0 h). In anoestrous cows, the treatment with progestagen for 9 days and EB at 24 h increased the diameter of the ovarian follicle (p = 0.033) but did not affect the diameter of the ovulatory follicle in cyclic cows. In conclusion, increasing the length of progestagen treatment for 8 or 9 days compared to 7 days increased the diameter of the dominant follicle, in anoestrous and cyclic beef cows. Oestradiol benzoate administered at device removal resulted in a shorter interval from device removal to ovulation compared with EB injection 24 h after the end of a progestagen treatment.

Human TRIB2 is a repressor of FOXO that contributes to the malignant phenotype of melanoma cells.

FOXO transcription factors are evolutionarily conserved proteins that orchestrate gene expression programs known to control a variety of cellular processes such as cell cycle, apoptosis, DNA repair and protection from oxidative stress. As the abrogation of FOXO function is a key feature of many tumor cells, regulation of FOXO factors is receiving increasing attention in cancer research. In order to discover genes involved in the regulation of FOXO activity, we performed a large-scale RNA-mediated interference (RNAi) screen using cell-based reporter systems that monitor transcriptional activity and subcellular localization of FOXO. We identified genes previously implicated in phosphoinositide 3-kinase/Akt signaling events, which are known to be important for FOXO function. In addition, we discovered a previously unrecognized FOXO-repressor function of TRIB2, the mammalian homolog of the Drosophila gene tribbles. A cancer-profiling array revealed specific overexpression of TRIB2 in malignant melanoma, but not in other types of skin cancer. We provide experimental evidence that TRIB2 transcript levels correlate with the degree of cytoplasmic localization of FOXO3a. Moreover, we show that TRIB2 is important in the maintenance of the oncogenic properties of melanoma cells, as its silencing reduces cell proliferation, colony formation and wound healing. Tumor growth was also substantially reduced upon RNAi-mediated TRIB2 knockdown in an in vivo melanoma xenograft model. Our studies suggest that TRIB2 provides the melanoma cells with growth and survival advantages through the abrogation of FOXO function. Altogether, our results show the potential of large-scale cell-based RNAi screens to identify promising diagnostic markers and therapeutic targets.

Modifications in host cell cytoskeleton structure and function mediated by intracellular HIV-1 Tat protein are greatly dependent on the second coding exon.

The human immunodeficiency virus type 1 (HIV-1) regulator Tat is essential for viral replication because it achieves complete elongation of viral transcripts. Tat can be released to the extracellular space and taken up by adjacent cells, exerting profound cytoskeleton rearrangements that lead to apoptosis. In contrast, intracellular Tat has been described as protector from apoptosis. Tat gene is composed by two coding exons that yield a protein of 101 amino acids (aa). First exon (1-72aa) is sufficient for viral transcript elongation and second exon (73-101 aa) appears to contribute to non-transcriptional functions. We observed that Jurkat cells stably expressing intracellular Tat101 showed gene expression deregulation 4-fold higher than cells expressing Tat72. Functional experiments were performed to evaluate the effect of this deregulation. First, NF-kappaB-, NF-AT- and Sp1-dependent transcriptional activities were greatly enhanced in Jurkat-Tat101, whereas Tat72 induced milder but efficient activation. Second, cytoskeleton-related functions as cell morphology, proliferation, chemotaxis, polarization and actin polymerization were deeply altered in Jurkat-Tat101, but not in Jurkat-Tat72. Finally, expression of several cell surface receptors was dramatically impaired by intracellular Tat101 but not by Tat72. Consequently, these modifications were greatly dependent on Tat second exon and they could be related to the anergy observed in HIV-1-infected T cells.

Transvaginal follicular aspiration and embryo development in superstimulated early postpartum beef cows and subsequent fertility after artificial insemination.

This study was conducted to investigate in early postpartum suckled beef cows with and without FSH pre-stimulation: (i) the influence of the postpartum period on the number and quality of oocytes recovered by ovum pick-up (OPU), (ii) the overall efficiency of the OPU/IVP embryos from days 30 to 80 postpartum and (iii) if repeated OPU negatively affect fertility following a fixed-time artificial insemination protocol. After parturition suckled Angus cows (n = 30) were divided in three groups (n = 10 group(-1)). All cows were anestrous at the commencement of experimental treatments (30.0 +/- 3.2 days postpartum, mean +/- SD; range 25-34 days). Group 1 treatments included: dominant follicle ablation (DFA), FSH treatment and OPU procedure 5 days after DFA. A total of 9 mg FSH (Ovagen) was administered s.c. once a day over 2 days at equal doses (4.5 + 4.5mg). For fertility test the cows received an intravaginal progesterone treatment from Days 78 to 86 postpartum and were fixed-time artificially inseminated (FTAI) at 56 and 72 h after device removal. Group 2: as cited for Group 1 with no FSH treatment. In both groups, OPU was repeated four times (Days 35, 49, 63 and 77 postpartum) and the collected oocytes classified as viable were in vitro matured, fertilized and presumptive embryos cultured for 8 days. Group 3 (Control FTAI): cows that had not previously aspirations were FTAI as Groups 1 and 2. Pregnancy was diagnosed by means ultrasonography 39 days after FTAI. The numbers (mean +/- SEM) of follicles visible and aspirated at the time of OPU in FSH-treated cows were greater (P < 0.05) than in non-treated cows (10.6 +/- 0.6 and 8.4 +/- 0.4 vs. 8.0 +/- 0.5 and 4.6 +/- 0.3, respectively). Following FSH treatment, the number (mean +/- SEM) of recovered oocytes per cow per OPU session and percentage of viable oocytes were greater in the treated (P < 0.05) than in non-treated animals (3.0 +/- 0.1 and 39.5% vs. 1.5 +/- 0.1 and 30.0%). The cleavage and embryo development rates were similar (P > 0.05) for both groups (14.8 and 6.4% vs. 16.6 and 5.5%). After FTAI the pregnancy rates were not different (P > 0.05) among groups (70, 60 and 90% for Groups 1, 2 and 3, respectively). We can conclude that (1) FSH-treated suckled postpartum cows can be a source of oocytes for in vitro fertilization and (2) repeated DFA/OPU applied during postpartum period did not affect the subsequent fertility following FTAI.

Síndrome del ¿hombre en barril¿. A propósito de un caso / The man-in-the-barrel syndrome. A case report

El síndrome del ¿hombre en barril? (SHB) describe un cuadro clínico poco frecuente de paresia braquial proximal bilateral con movilidad conservada de miembros inferiores y musculatura facial, secundario a una hipotensión arterial sistémica con hipoperfusión encefálica que origina infartos isquémicos bilaterales en territorios limítrofes entre la arteria cerebral media y la anterior, de pronóstico muy grave. Ocurre tras parada cardiopulmonar, shock hipovolémico, sobredosis de narcóticos, hipotensión tras infarto de miocardio u oclusión de la vía aérea, y supone del 5 al 12 % de los accidentes isquémicos. Presentamos el caso de un sujeto de 27 años, diagnosticado de endocarditis por estreptococo con insuficiencia aórtica severa y arteritis de la arteria pulmonar, intervenido mediante sustitución valvular aórtica y limpieza arterial. En el postoperatorio se observa focalidad motora compatible con SHB debido a encefalopatía hipoxoisquémica en territorio frontera bihemisférico. Describimos su evolución y tratamiento, destacando la buena recuperación que ha presentado en contra de lo encontrado en la literatura

The man-in-the-barrel syndrome (MIB) describes an uncommon clinical picture of bilateral proximal brachial paralysis with preserved mobility of the lower limbs and facial muscles, secondary to systemic arterial hypotension with hypoperfusion encephalic, bilateral ischemic strokes originating in territories bordering between the anterior and medial cerebral arteries, with very serious prognosis. It occurs after a cardiopulmonary arrest, hypovolemic shock, overdose of narcotics, hypotension after myocardial infarction or occlusion of the airway and accounts for 5 % to 12 % of the ischemic attacks. We present the case of a 27-year-old man, diagnosed with endocarditis by streptococcus with severe aortic insufficiency and arteritis of the pulmonary artery, with an intervention for aortic valve replacement and arterial cleaning. During post-intervention, motor focality consistent with MIB syndrome due to hypoxoischemic encephalopathy in the bihemispheric border territory was observed. We describe his evolution and treatment, stressing his good recovery on the contrary to that found in the literature (AU)

Effect of progesterone administration on the ovarian response to superovulatory treatments in cattle.

To evaluate ovarian response in Angus cows previously treated with progesterone (P4), animals were randomly assigned to two groups: T600 group (n=14), 600 mg of P4/day. P4 was injected from days 3 to 7 of the estrous cycle. On day 7, superovulatory treatments began. The control group (n=12) was given vehicle only. The superovulatory treatments in the control group began on days 7-9 of the estrous cycle. The superovulatory total treatment dose of 400mg NIH FSH P1 was given twice a day over a 4-day period. Ultrasonography of the ovaries was conducted 3 days preceding the initiation of superovulatory treatment, every 24h. In both groups, an additional ultrasonographic evaluation was made at 24h after the end of superovulatory treatment. Blood samples were collected 4 days preceding the initiation of superovulatory treatment, every 24h. Additional samples were taken from the P600 group for 12 day after of initiation of superovulatory treatment every 24h, except on the fifth day after the initiation of superovulatory treatment. In the P600 group, P4 concentrations were greater than in the control group (P<0.01) and remained over 1 ng/ml up to day 11 after beginning of superovulatory treatment. The diameter of the dominant follicle was larger in the animals of the control group (P<0.01). Cows of the P600 group had a greater number of Class I (3-4mm) follicles (P<0.01). A significant day and treatment effect (P<0.01) were observed in Class II (5-9 mm) follicles. Effects due to treatment on the number of Class III follicles (P<0.05) were observed. In the P600 group, no estrous post-superovulatory was observed and there were no ovulations that occurred. Conversely, 100% of the cows of the control group showed estrous. In the P600 group, there were a greater number of Class III follicles (P<0.01) and a lesser number of Class II follicles (P<0.05) at 24h after the end of superovulatory. In the control group, 66.7% of the cows responded to superovulatory treatments. In conclusion, the daily administration of 600 mg of P4, from days 3 to 7 of the estrous cycle, produces an increase of plasma concentrations of this hormone from day 4, resulting in changes in follicular dynamics (absence of follicles greater than 10mm of diameter and an increase of the population of Class I follicles). As to the ovarian stimulation using Folltropin V in animals receiving a daily injection of 600 mg of P4 from days 3 to 7 of the estrous cycle, a greater population of follicles>or=10mm developed by 24h after superovulatory treatments were completed.

Influence of different doses of progesterone treatments on ovarian follicle status in beef cows.

To determine a dose of progesterone (P4) that allow ovarian follicular wave control, Aberdeen Angus cows were randomly assigned into four groups: T600 (n=5), 600 mg of P4/day; T400 (n=5), 400 mg of P4/day; T200 (n=4), 200mg of P4/day and Control (n=4) (excipient only). Progesterone was injected from day 3 to 9 of estrous cycle. Ultrasonographies and blood sample collections were performed daily from day 2 to 10 and on day 15 of the estrous cycle. Additionally, an ultrasonographic study was conducted on day 13. Progesterone concentrations were different among all groups (P<0.01). The diameter of the dominant follicle was greater for control than for T200, T400 and T600 groups (P<0.01); there was no difference between T200 and T400 (P>0.05), but they had a greater diameter follicle than the T600 group (P<0.01). The growth rate of the dominant follicle between day 3 and 7 of estrous cycle was greater for control group (1.63+/-0.3 mmday(-1)) than for T200 (0.56+/-0.19 mmday(-1), P<0.05), T400 (0.6+/-0.23 mmday(-1), P<0.05) and T600 (0.11+/-0.13 mmday(-1), P<0.01) groups. The mean number of class I follicles (3-4mm) per day for the entire experimental period was less for the control group than for T200 (P<0.05), T400 and T600 (P<0.01) groups (3.7+/-1.3; 5.3+/-1.3; 6.6+/-1.8 and 8.1+/-1.9, respectively). The mean number for the T200 group was less than for T600 (P<0.05) and similar for T400 and T600 groups (P>0.05). The number of class III follicles was greater for control group than for the other groups (P<0.01). T200 and T400 groups had similar numbers of class III follicles (P>0.05) and both had greater numbers of follicles than the T600 group (P<0.05). The diameter of the corpus luteum of the T600 group (15.8+/-1.6 mm) was less than for control (21.0+/-2.5 mm, P<0.01), T200 (19.3+/-2.7 mm, P<0.01) and T400 (20.0+/-2.2 mm) groups (P<0.05). The mean diameter of corpus luteum of T200 was similar to T400 (P>0.05), but different from the control group (P<0.05). In conclusion, the daily intramuscular administration of 200mg or more of progesterone from day 3 to 9 of the estrous cycle indicates that plasma concentrations of progesterone can be used to modify the pattern of follicular development during the follicular wave. From day 5 of the estrous cycle, progesterone concentrations greater than 15 ng/ml (T600 group: 600 mg/day of progesterone from day 3 to 9 of the estrous cycle) inhibit dominant follicle development, increase the class I follicle populations (3-4 mm) and diminish the development of the corpus luteum.

Regulation of mouse embryo development by autocrine throphic factors.

Embryo development depends on maternal and embryonic factors. When occurs in vitro, embryos secrete factors that stimulate their development. The purpose of this study was to investigate the possible effects of embryos at morula stage on mouse embryo development in vitro. To obtain conditioned media (CM), morulas were cultured in groups of 5 (CM5) or 10 (CM10) in microdrops of Ham-F10 culture medium during 24 h and later they were removed. Subsequently, 365 morulas were cultured in CM5 and CM10 or in Ham-F10 media (as control group). No differences in blastocyst formation could be found between embryos cultured for 24h in Ham-F10, CM5 or CM10 (49.66, 53.04, 60.00% respectively). However, CM5 significantly increased differentiation in embryos cultured for 48h as compared to Ham-F10 medium (80.00% and 64.14 respectively). The CM5 caused a significant increase in the hatching rate compared to Ham-F10 evaluated at 78 and 96 h of culture (66.96 vs. 52.41% and 70.43 vs. 55.17%, respectively). After 72, 78 and 96h of culture the hatching rate for embryos cultured in CM10 was significantly higher than that in Ham-F10 (64.76 vs. 47.59%, 67.62 vs. 52.41% and 73.33 vs. 55.17%, respectively). At 48h of culture, differences between CM5, CM10 and Ham-F10 were not observed. These results suggest that preimplantational mouse embryos produce trophic factor/factors that enhance the differentiation and hatching process.

Regulation of mouse embryo development by autocrine throphic factors

Embryo development depends on maternal and embryonic factors. When occurs in vitro, embryos secrete factors that stimulate their development. The purpose of this study was to investigate the possible effects of embryos at morula stage on mouse embryo development in vitro. To obtain conditioned media (CM), morulas were cultured in groups of 5 (CM5) or 10 (CM10) in microdrops of Ham-Fl0 culture medium during 24h and later they were removed. Subsequently, 365 morulas were cultured in CM5 and CM10 or in Ham-F10 media (as control group). No differences in blastocyst formation could be found between embryos cultured for 24h in Ham-F1O, CM5 or CM10 (49.66, 53.04, 60.00% respectively). However, CM5 significantly increased differentiation in embryos cultured for 48h as compared to Ham-FlO medium (80.00% and 64.14 respectively). The CM5 caused a significant increase in the hatching rate compared to Ham-F10 evaluated at 78 and 96h of culture (66.96 vs. 52.41% and 70.43 vs. 55.17%, respectively). After 72, 78 and 96h of culture the hatching rate for embryos cultured in CM10 was significantly higher than that in Ham-F10 (64.76 vs. 47.59%, 67.62 vs. 52.41% and 73.33 vs. 55.17%, respectively). At 48h of culture, differences between CM5, CMl0 and Ham-F10 were not observed. These results suggest that preimplantational mouse embryos produce trophic factor/factors that enhance the differentiation and hatching process

Regulation of mouse embryo development by autocrine throphic factors

Embryo development depends on maternal and embryonic factors. When occurs in vitro, embryos secrete factors that stimulate their development. The purpose of this study was to investigate the possible effects of embryos at morula stage on mouse embryo development in vitro. To obtain conditioned media (CM), morulas were cultured in groups of 5 (CM5) or 10 (CM10) in microdrops of Ham-Fl0 culture medium during 24h and later they were removed. Subsequently, 365 morulas were cultured in CM5 and CM10 or in Ham-F10 media (as control group). No differences in blastocyst formation could be found between embryos cultured for 24h in Ham-F1O, CM5 or CM10 (49.66, 53.04, 60.00% respectively). However, CM5 significantly increased differentiation in embryos cultured for 48h as compared to Ham-FlO medium (80.00% and 64.14 respectively). The CM5 caused a significant increase in the hatching rate compared to Ham-F10 evaluated at 78 and 96h of culture (66.96 vs. 52.41% and 70.43 vs. 55.17%, respectively). After 72, 78 and 96h of culture the hatching rate for embryos cultured in CM10 was significantly higher than that in Ham-F10 (64.76 vs. 47.59%, 67.62 vs. 52.41% and 73.33 vs. 55.17%, respectively). At 48h of culture, differences between CM5, CMl0 and Ham-F10 were not observed. These results suggest that preimplantational mouse embryos produce trophic factor/factors that enhance the differentiation and hatching process

Regulation of mouse embryo development by autocrine throphic factors.

Embryo development depends on maternal and embryonic factors. When occurs in vitro, embryos secrete factors that stimulate their development. The purpose of this study was to investigate the possible effects of embryos at morula stage on mouse embryo development in vitro. To obtain conditioned media (CM), morulas were cultured in groups of 5 (CM5) or 10 (CM10) in microdrops of Ham-F10 culture medium during 24 h and later they were removed. Subsequently, 365 morulas were cultured in CM5 and CM10 or in Ham-F10 media (as control group). No differences in blastocyst formation could be found between embryos cultured for 24h in Ham-F10, CM5 or CM10 (49.66, 53.04, 60.00

respectively). However, CM5 significantly increased differentiation in embryos cultured for 48h as compared to Ham-F10 medium (80.00

and 64.14 respectively). The CM5 caused a significant increase in the hatching rate compared to Ham-F10 evaluated at 78 and 96 h of culture (66.96 vs. 52.41

and 70.43 vs. 55.17

, respectively). After 72, 78 and 96h of culture the hatching rate for embryos cultured in CM10 was significantly higher than that in Ham-F10 (64.76 vs. 47.59

, 67.62 vs. 52.41

and 73.33 vs. 55.17

, respectively). At 48h of culture, differences between CM5, CM10 and Ham-F10 were not observed. These results suggest that preimplantational mouse embryos produce trophic factor/factors that enhance the differentiation and hatching process.

Bioavailability of fluticasone propionate and mometasone furoate aqueous nasal sprays.

OBJECTIVES: To compare the systemic exposure for intranasal mometasone furoate (MF) and fluticasone propionate (FP) aqueous nasal sprays (ANS) in terms of serum and urinary cortisol parameters and plasma pharmacokinetics. METHODS: Twelve healthy subjects completed this three-way, cross-over study. They received FPANS (50 microg/spray), MFANS (50 microg/spray) or placebo ANS, eight sprays per nostril every 8 h for 4 days. Cortisol measurements were made at baseline and day 4. FP and MF plasma concentrations were also measured on day 4. RESULTS: MFANS produced similar mean plasma AUC (123 pmol/l h) to FPANS (112 pmol/l h). Despite the use of high doses, necessary to generate adequate pharmacokinetic data, only minor reductions in cortisol parameters were found, with no difference between FPANS and MFANS. CONCLUSIONS: FP and MF have similar and very low systemic bioavailability when administered intranasally using a high-dose regimen. It is therefore unlikely that therapeutic doses of intranasal FP or MF will produce dissimilar or significant degrees of systemic exposure or systemic effects.

Morphological aspects of in vivo cleavage in Myocastor coypus (coypu).

The objective of the present work was to characterize the in vivo cleavage stage of Myocastor coypus embryos. For this purpose a colpocytological follow-up and controlled mating of 18 females were performed. Specimens from the beginning of the first cleavage to the acquisition of a morula appearance were considered to be in cleavage stage. Embryos in cleavage were collected between days 3 and 6 post-coitus. Of the collected embryos, 80% presented an even number of blastomeres and the remaining 20% an odd number. Embryos from 3 to 7 cells were blastomere associations in a spherical disposition within the zona pellucida. Blastomeres were spherical or ovoid, presenting slight flattening in areas contacting with other blastomeres. Embryos of 8 and 9 cells were as a group of blastomeres slightly elongated, surrounded by a spherical zona pellucida. The percentage of peri-vitelline space occupied by the embryonic mass ranged from 74.1 to 95.8% for all the substages. The cleavage pattern, developed in the oviduct, was of a rotational holoblastic type and asynchronic.

Uso del protocolo Ovsynch en vaquillonas lecheras con o sin cuerpo lúteo funcional al inicio del tratamiento / Use of the Ovsynch protocol in dairy heifers with or without a functional corpus luteum at the initiation of treatment

Con el objetivo de comparar la respuesta ovárica y el porcentaje de preñez de vaquillonas lecheras que recibieron dos tratamientos con doble dosis de prostaglandina, separadas por 11 días, para generar presencia o ausencia de cuerpo lúteo funcional al inicio del protocolo Ovsynch., 22 vaquillonas Holando Argentino fueron distribuidas en dos grupos para recibir dos dosis de 750 µg de tiaprost, los días -22 y -11 (grupo CLF+, n=11) o los días -14 y -3 (grupo CLF-, n=11). La presencia o no de cuerpo lúteo funcional fue confirmada por ecografía y medición de progesterona plasmática, con lo cual dos vaquillonas CLF+ y una CLF- fueron eliminadas. El día 0 ambos grupos recibieron 8 mg de buserelina; el día 7, 750 mg de tiaprost y el día 9, 8 mg de buserelina. El servicio se realizó por inseminación artificial a tiempo fijo a las 15 horas post-tratamiento. El porcentaje de animales con un folículo > = 8 mm al día 0 y su tamaño fueron similares entre grupos (P>0,05). Estos folículos ovularon (94,1 por ciento) o se luteinizaron (5,9 por ciento) luego de la primera inyección de buserelina (P>0,05). El porcentaje de animales con cuerpo lúteo funcional al día 7 fue de 100 por ciento y 90 por ciento, para CLF+ y CLF- (P>0,05), mientras que la concentración de progesterona de los mismos fue superior en CLF+ (3,5 ñ 0,7 vs 2,2 ñ 1,2 ng/ml; P0,05). El diámetro del folículo dominante fue superior en CLF-, tanto al día 7 (P=0,05) como al día 9 (P0,05). Los porcentajes de preñez fueron similares entre grupos (66,7 por ciento y 70 por ciento; P>0,05). En conclusión, la utilización de dos tratamientos con doble dosis de prostaglandina separadas por 11 días, para generar presencia (11 días post-segunda PGF2alfa) o ausencia (3 días post-segunda PGF2alfa) de cuerpo lúteo funcional al inicio del protocolo Ovsynch en vaquillonas lecheras produce respuestas ováricas similares (ovulación, sincronización de la fase luteal y luteólisis), lo que explicaría la similitud entre los porcentajes de preñez de ambos grupos (AU)

Ciliate cryptobiosis: a microbial strategy against environmental starvation.

This review outlines the main features of ciliate resting-cyst formation or encystment. It represents a strategy against several environmental stresses (such as starvation), which involves a highly gene-regulated cell differentiation process and originates a more resistant, differentiated form or resting cyst. This process is mainly characterized by drastic cytoplasmic dehydration that induces a general metabolic rate decrease, intense autophagic activity, the formation of a permeable cyst wall protecting the cell against the adverse environmental conditions, and a gene-silencing mechanism after opening the specific encystment genes.

DNA methylation in ciliates: implications in differentiation processes.

Much experimental evidence on the role of DNA methylation in gene expression has been reported. Here we review reports on DNA methylation in ciliated protozoa, emphasizing its implications in cell differentiation processes. Both types of methylated bases (adenine and cytosine) can be found in macronuclear DNA. The division cycle and conjugation have been studied with regard to adenine methylation, and several different functions have been assigned to the methylation changes detected in these processes. Cytosine methylation changes were analyzed during stomatogenesis of Paramecium and encystment of Colpoda inflata. A comparative analysis with other similar microbial eukaryotic differentiation processes is carried out.

Typological series for ovarian follicles of sexually mature Myocastor coypus (coypu).

We are herein putting forward the results derived from the careful examination of the ovary of a sexually mature Myocastor coypus which was carried out to establish a follicular typological series. Sexually mature virgin females from breeding farms were used and their ovaries were processed by routinely histological techniques. The following analysis criteria were considered for the follicular classification: size of the oocyte in follicles at different stages of development, size of the follicle regarding the number of follicular cells, and follicular morphology. Complementary characteristics were also analyzed: mean follicular diameter, presence and thickness of the pellucid zone, mean size of follicular cells, their shape in all follicular types, presence and extent of the antrum, and presence of thecas. By combining these different criteria, a follicular typological series was obtained according to Pedersen and Peters (1968) nomenclature together with a qualitative and quantitative characterization of the follicular types.