Marcelo Alfredo Aba

Oestrogen, Progesterone and Oxytocin Receptors and COX-2 Expression in Endometrial Biopsy Samples from the Induction of Ovulation to Luteolysis in Llamas (Lama glama)

Endometrial expression of oestrogen (ERα), progesterone (PR) and oxytocin receptor (OR) and cyclooxygenase-2 (COX-2) was evaluated from the induction of ovulation to luteolysis in llamas. Ovarian activity was daily assessed by ultrasonography in five females. Ovulation was induced immediately after the detection of an ovulatory follicle by a GnRH injection (Day 0). Endometrial samples were obtained by transcervical biopsies from the left and right horns on day 0 and days 4, 8, 10 and 12 post-GnRH. Blood samples were collected daily for progesterone and estradiol-17β determinations by RIA. An immunohistochemical technique was used to study receptors population and COX-2 expression which were then evaluated by two independent observers. The expression of ERα and PR was highest on day 0 in the luminal epithelium and stroma in association with high plasma estradiol-17β concentrations. Thereafter, a decrease in ERα population was registered on day 4 and a new increase of its expression was observed between days 8 and 12 in those cell types. Conversely, PR population was gradually down-regulated until its lowest expression was reached on day 10 post-GnRH in the luminal epithelium. Content of OR was similar throughout the study in all cell types. The expression of COX-2 was highest from day 8 to 12 post-GnRH in the luminal epithelium, in relation to the time of maximal PGF2α release. Both steroid receptors populations and COX-2 expression were similar between horns. Meanwhile, OR expression was higher in the right than in the left uterine horn. In summary, this study showed that the loss of endometrium sensitivity to progesterone by days 8-10 post-induction of ovulation and the concomitant increase of COX-2 expression could play a key role in the mechanism of luteolysis and somehow be related to the short corpus luteum lifespan of llamas.

Development of corpus luteum susceptibility to an analog of prostaglandin F2α, throughout the luteal phase in llamas (Lama glama).

The aim of the present study was to evaluate the susceptibility of the corpus luteum to d-cloprostenol (synthetic analog of PGF(2α)) throughout the luteal phase in llamas. Female llamas (n=43) were induced to ovulate by GnRH injection in the presence of an ovulatory follicle and randomly assigned into one of six groups: control and treated with an injection of d-cloprostenol on Day 3, 4, 5, 6 or 8 post GnRH. Blood samples were collected to determine plasma progesterone concentrations. There was no effect of treatment on animals injected on Day 3 or 4 post-GnRH. In animals treated on Day 5, different responses were observed. No effect of treatment was recorded in 27% of the animals whereas 55% of the llamas showed a transitory decrease followed by a recovery in plasma progesterone concentrations after d-cloprostenol injection, indicative of a resurgence of the corpus luteum, extending the luteal phase a day more than in control animals. In the remaining 18% of the animals injected on Day 5, (corresponding to those exhibiting the greatest plasma progesterone concentrations at the day of injection), complete luteolysis was observed. Plasma progesterone concentrations decreased to below 1 ng ml(-1) 24 h after d-cloprostenol in llamas injected on Day 6 or 8 post-GnRH. In conclusion, the corpus luteum of llamas is completely refractory to PGF(2α) until Day 4 after induction of ovulation, being partially sensitive by Day 5 and fully responsive to PGF(2α), by Day 6 after induction of ovulation.

Endometrial population of oestrogen receptors alpha and beta and progesterone receptors A and B during the different phases of the follicular wave of llamas (Lama glama).

The aim of this study was to characterize the distribution of oestrogen receptor (ER)α and ERβ as well as both progesterone receptors isoforms progesterone receptor (PR) A and PRB in the luminal and glandular epithelia and stroma of the endometrium during the different phases of the follicular wave in llamas. Six llamas were examined by transrectal ultrasonography, and a transcervical biopsy was obtained when a follicle at the growing, plateau and regressing phase was recorded. Blood samples were collected at the time of biopsy for hormone determinations. An immunohistochemical technique was used to study receptor populations. Total positive area was evaluated in the different cell types by Image Analysis. Mean diameter measurements of the largest follicle were 6.9, 8.5 and 5.1 mm (p < 0.001) and mean plasma oestradiol-17β concentrations were 27.9 ± 3.26; 30.0 ± 2.79 and 24.0 ± 1.78 pmol/l (p = 0.32) during the growing, plateau and regressing phases, respectively. Immunostaining of ERα was higher in the luminal epithelium during the plateau and regressing phases (p < 0.05) than during the growing phase. More positive cells to ERβ were observed in the glandular epithelium of the growing and plateau phases (p < 0.05) than during the regressing phase. A higher percentage of cells positive to PRB was recorded in the luminal and glandular epithelia during the plateau phase (p < 0.05), while the PRA immunostaining was similar among phases. In brief, this study showed an increased population of ERα and PRB in the luminal epithelium, and only of PRB in the glandular epithelium at the time when an ovulatory follicle is present. The physiological importance of these changes in llamas remains to be elucidated.

Antiproliferative Effects of Oxytocin and Desmopressin on Canine Mammary Cancer Cells

Neoplasms of the mammary gland represent the most frequent tumor type in the female dog, and according to the histologic criteria, approximately 50% of them are malignant. In the most aggressive cases of mammary cancer, surgery is not enough to warrant a favorable outcome, and adjuvant therapies are needed to improve the patient’s overall survival. The aim of the present study was to evaluate the effects of two peptides on proliferation of a canine mammary cancer cell line derived from a simple carcinoma. The cell line CMT-U27 was grown in 96-well plates, at two cell densities (4 × 103 and 8 × 103 cells/well). Cultures were treated with oxytocin (OT) or desmopressin at five concentrations (10, 50, 100, 500, and 1000 nM). After 72 h of incubation, cell proliferation was determined by the MTT assay. Results showed that with 4 × 103 cells/well, OT at 50, 500, and 1000 nM was growth inhibitory for the cells, being statistically significant at 1000 nM. On the contrary, no antiproliferative effect was observed with 10 or 100 nM. At 8 × 103 cells/well, OT showed a significant antiproliferative effect only with the highest concentration (1000 nM). Desmopressin at 4 × 103 cells/well decreased cell viability at concentrations of 50, 100, 500, and 1000 nM (statistically significant with the highest concentration), while no effect was observed with 10 nM. With 8 × 103 cells/well, this peptide reduced cell growth at 100, 500, and 1000 nM. In conclusion, we suggest that these peptides may be potential and promising compounds for the treatment of dogs with simple carcinomas of the mammary gland. In vivo studies are required to confirm this hypothesis.

Oestrogen and Progesterone Receptors and COX-2 Expression in Endometrial Biopsy Samples During Maternal Recognition of Pregnancy in Llamas (Lama glama).

Endometrial expression of oestrogen receptor-α (ERα), progesterone receptor (PR) and cyclooxigenase-2 (COX-2) was evaluated in non-pregnant and pregnant llamas during the period when luteolysis/maternal recognition of pregnancy is expected to occur. Females (n = 28) were divided into two groups: non-pregnant llamas were induced to ovulate with a Buserelin injection, and endometrial biopsies were obtained on day 8 (n = 5) or 12 (n = 5) post-induction of ovulation. Animals of the pregnant group (n = 18) were mated with a fertile male. Pregnancy was confirmed by the visualization of the embryo collected by transcervical flushing in 5 of 9 animals on day 8 post-mating and by progesterone profile on day 12 post-mating in 4 of 9 animals, when endometrial biopsies were obtained. An immunohistochemical technique was used to evaluate receptors population and COX-2 expression. Pregnant llamas showed a higher percentage of positive cells and stronger intensity for ERα than for non-pregnant llamas in stroma on day 8 and in the luminal epithelium on day 12 post-induction of ovulation, while a deep decrease in endometrial PR population was reported in pregnant llamas on that day in luminal and glandular epithelia and stroma. In the luminal epithelium, COX-2 expression was lower in pregnant than in non-pregnant animals. Briefly, the increase of ERα in pregnant llamas gives further support to the hypothesis that oestrogens are involved in the mechanism of maternal recognition of pregnancy. Endometrial PR decrease in pregnant llamas might be a necessary event to allow the expression of proteins involved in conceptus attachment, a mechanism widely accepted in other species. Moreover, embryo seems to attenuate maternal PGF(2α) secretion during early pregnancy by decreasing the endometrial expression of COX-2 in the luminal epithelium of pregnant llamas.

Oestradiol-17β plasma concentrations after intramuscular injection of oestradiol benzoate or oestradiol cypionate in llamas (Lama glama)

BackgroundLlamas (Lama glama) are induced ovulators and the process of ovulation depends on dominant follicular size. In addition, a close relationship between behavioural estrus and ovulation is not registered in llamas. Therefore, the exogenous control of follicular development with hormones aims to predict the optimal time to mate. Oestradiol-17β (E2) and its esters are currently used in domestic species, including camelids, in synchronization treatments. But, in llamas, there is no reports regarding the appropriate dosages to be used and most protocols have been designed by extrapolation from those recommended for other ruminants. The aim of the present study was to characterize plasma E2 concentrations in intact female llamas following a single intramuscular (i.m.) injection of two oestradiol esters: oestradiol benzoate (EB) and oestradiol cypionate (ECP).MethodsTwelve non pregnant and non lactating sexually mature llamas were i.m. injected on day 0 with 2.5 mg of EB (EB group, n = 6) or ECP (ECP group, n = 6). Blood samples were collected immediately before injection, at 1, 6, 12, 24 h after treatment and then daily until day 14 post injection. Changes in hormone concentrations with time were analyzed in each group by analysis of variance (ANOVA) using a repeated measures (within-SS) design. Plasma E2 concentrations and area under the concentration-time curve (AUC) values were compared between groups by ANOVA. In all cases a Least-Significant Difference test (LSD) was used to determine differences between means. Hormonal and AUC data are expressed as mean ± S.E.M.ResultsPeak plasma E2 concentrations were achieved earlier and were higher in EB group than in ECP group. Thereafter, E2 returned to physiological concentrations earlier in EB group (day 5) than in ECP group (day 9). Although plasma E2 profiles differed over time among groups there were no differences between them on AUC values.ConclusionsThe i.m. injection of a single dose of both oestradiol esters resulted in plasma E2 concentrations exceeding physiological values for a variable period. Moreover, the plasma E2 profiles observed depended on the derivative of oestradiol administered. This basic information becomes relevant at defining treatment protocols including oestrogens in llamas.

Synchronization of time of ovarian follicular development in llamas ( Lama glama ) using a protocol based on GnRH and PGF 2α

The aim of this study was to evaluate the effectiveness of a protocol based on GnRH and PGF2α to synchronize the emergence of a new wave of ovarian follicular development in llamas and, therefore, when a new dominant follicle develops. Llamas (n = 18) were assigned to growing, mature or regressing follicle groups according to the phase of the follicular wave at the beginning of treatment. The protocol was initiated with a GnRH analogue (GnRHa) injection on Day 0 followed 7 days later with a d-cloprostenol injection and a second GnRHa injection on Day 10. Ovulation rate after the first GnRHa treatment, day of new follicle emergence, mean plasma progesterone concentration and percentage of animals with a newly developed dominant follicle ≥ 7 mm on Day 10 were evaluated. Ovulation rate after the first GnRHa was less in the regressing than mature and growing follicle groups and new follicular wave emergence occurred earlier in the regressing follicle group than in the other two groups. Mean plasma progesterone concentration in females that had ovulations after the first GnRHa injection was similar. The percentage of animals that had a new follicle ≥ 7 mm on Day 10 was not different among groups and the overall percentage was 66.6%. The total synchronization rate for development of a new wave of follicular development on Day 10 was greater in females having ovulations after the first GnRHa injection than in those that did not have ovulations. In conclusion, the protocol used in the present study was useful for synchronizing ovarian follicular development in 66% of the llamas regardless of the phase of the follicular wave development at the beginning of treatment.