Cyclooxygenase 2 messenger RNA levels in canine follicular cells: interrelationship with GDF-9, BMP-15, and progesterone.

Cyclooxygenase 2 (COX-2) encoded by the Cox-2 gene within the periovulatory follicles is a critical mediator of oocyte development. Growth differentiation factor 9 (GDF-9) and bone morphogenetic protein 15 (BMP-15) participate in the modulation of certain target genes in the ovary, possibly influencing the Cox-2 gene expression. However, this relationship has not been characterized in canines. This study aimed to examine the possible relationships among BMP-15, GDF-9, progesterone, and Cox-2 gene expression in granulosa-cumulus cells in dogs. Granulosa cells from antral follicles and their corresponding cumulus-oocyte complexes and follicular fluid (FF) were separately obtained from 56 ovaries collected from adult bitches at estrus (n = 15) and proestrus (n = 13) after ovariohysterectomy. Total RNA extraction was performed in follicular cells, and Cox-2 gene expression was assessed by quantitative PCR analysis. Progesterone, BMP-15, and GDF-9 were determined in the FF samples using ELISA assays. Cumulus-oocyte complexes were subjected to in vitro maturation (IVM) with or without (control) recombinant GDF-9 and BMP-15. After 72 h of culture, Cox-2 transcript analyses were performed in cumulus cells via quantitative PCR. Data were evaluated by ANOVA. An increase (P < 0.05) in Cox-2 messenger RNA levels was observed in follicular cells from follicles at estrus with respect to those at proestrus. However, the levels of BMP-15 and GDF-9 in FF decreased (P < 0.05), whereas progesterone increased (P < 0.05) from the proestrus phase to the estrus phase. The expression of Cox-2 gene in cumulus cells was 4-fold greater (P < 0.01) than that in the control when both growth factors were added to the IVM culture. In conclusion, although BMP-15 together with GDF-9 appears to upregulate the levels of Cox-2 transcripts during IVM, the inverse relationship of these paracrine factors with Cox-2 gene expression and the positive correlation of progesterone with Cox-2 transcripts suggest that the high progesterone levels could be more relevant in the local mechanisms regulating the Cox-2 gene expression.

Ram semen deterioration by short-term exposure to high altitude is prevented by improvement of antioxidant status.

Ovine reproduction efficiency in herds at high altitude (ha) is lower than that at low altitude (la). In ewes, ha effects are due to hypoxia and oxidative stress. Our aim was to establish the effect of antioxidant vitamin supplementation on semen traits and antioxidant status of rams exposed to short or long time ha. A total of 32 rams native to la (~500 m) were used, 16 were kept at la and the other 16 were brought to ha (~3600 m), where they were placed in the same flock as the ha native rams (n=16). Half of the animals in each group were supplemented daily with vitamins C 600 mg and E 450 IU per os, during the entire experimental period, starting the 4th day after animal's arrival at ha (day 0). At days 0, 30 and 60 of treatment, blood and semen samples were collected for evaluation of antioxidant status and semen standard characteristics. Data were compared within each experimental time by analysis of variance using a general linear model. Elevated concentrations of oxidative stress biomarkers were present in blood from animals maintained at ha. Ejaculates from ha exposed rams showed decreased sperm concentration, progressive motility and viability, in addition to decreased antioxidant status in seminal fluid. A total of 30 days of oral supplementation with vitamins C and E prevented some ha negative effects on semen characteristics, mainly in recently ha exposed rams. It is concluded that exposure of rams to ha negatively affects semen quality, where oxidative stress plays a predominant role. These effects are mainly prevented by oral supplementation of vitamins C and E, which constitutes a simple and cheap alternative to improve semen quality of rams when they are moved to ha.

Fertility of a high-altitude sheep model is compromised by deficiencies in both preovulatory follicle development and plasma LH availability.

At high altitude, hypoxia and/or oxidative stress may compromise fertility. This study tested the relative effect of short- or long-term exposure to high-altitude hypobaric hypoxia and oxidative stress in sheep on preovulatory follicle dynamics and gonadotrophin secretion. Thus, growth dynamics, stereidogenic function and competence to ovulate of preovulatory follicles, as well as FSH and LH availability throughout the entire oestrous cycle, were compared among sheep native from low and high altitude, and sheep newcomers to high altitude. The results indicates that short-term exposure in sheep newcomers to high altitude has a deleterious effect on both the ovarian function (affecting preovulatory follicular development) and the pituitary function (diminishing plasma LH availability). On the other hand, there were no detected differences in the preovulatory follicular development in sheep adapted to high altitude for generations and, conversely, LH secretion was increased, which suggests an adaptive mechanism. The treatment with antioxidant agents during a relative short period for the time of folliculogenesis (approximately 1 month and a half) changed substantially the development of preovulatory follicles in short-term exposed sheep to similar patterns than in sheep native and living to both high and low altitude. These results highlight the role of oxidative stress in the detriment of the reproductive function in individuals recently exposed to high-altitude hypoxic environment.

Cytologic, hormonal, and ultrasonographic correlates of the menstrual cycle of the New World monkey Cebus apella.

Few reports on the reproductive physiology of Cebus apella have been published. In this study we characterized menstrual cycle events by means of vaginal cytology, ultrasonography (US), and hormonal measurements in serum during three consecutive cycles in 10 females, and assessed the probability that ovulation would occur in the same ovary in consecutive cycles in 18 females. The lengths and phases of the cycles were determined according to vaginal cytology. Taking the first day of endometrial bleeding as the first day of the cycle, the mean cycle length +/- SEM was 19.5+/-0.4 days, with follicular and luteal phases lasting 8.2+/-0.2 and 11.3+/-0.4 days, respectively. The follicular phase included menstruation and the periovulatory period, which was characterized by the presence of a large number of superficial eosinophilic cells in the vaginal smear. The myometrium, endometrium, and ovaries were clearly distinguished on US examination. During each menstrual cycle a single follicle was recruited at random from either ovary. The follicle grew from 3 mm to a maximum diameter of 8-9 mm over the course of 8 days, in association with increasing estradiol (E(2)) serum levels (from 489+/-41 to 1600+/-92 pmol/L). At ovulation, the mean diameter of the dominant follicle usually decreased by >20%, 1 day after the maximum E(2) level was reached. Ovulation was associated with an abrupt fall in E(2), a decreased number of eosinophilic cells, the presence of leukocytes and intermediate cells in the vaginal smear, and a progressive increase in progesterone (P) levels that reached a maximum of 892+/-65 nmol/L on days 3-6 of the luteal phase. The menstrual cycle of Cebus apella differs in several temporal and quantitative aspects from that in humans and Old World primates, but it exhibits the same correlations between ovarian endocrine and morphologic parameters.

Plasma progesterone in alpaca (Lama pacos) during pregnancy, parturition and early postpartum.

Plasma progesterone concentration during pregnancy and the early postpartum period was measured by radioimmunoassay in five alpacas (Lama pacos), of the Huacaya breed, whose pregnancy length had a mean of 344.8 +/- 4.4 days. Concentration of progesterone increased (P < 0.001) from low premating values (0.11 +/- 0.06 nmol/l) to greater values at 30 days of pregnancy (8.05 +/- 1.13 nmol/l) and remained high up to 2 months of pregnancy. A slight transitory decline was observed between 3 to 7 months. Concentration of plasma progesterone dropped markedly during the 72 h before parturition especially, at the day of parturition.

Day-night changes in c-fos expression in the fetal sheep suprachiasmatic nucleus at late gestation.

The suprachiasmatic nucleus (SCN) is a circadian oscillator in mammals and shows day-night changes in metabolic activity. To investigate whether the fetal sheep SCN behaves as a circadian oscillator, day-night changes in c-fos expression, a marker of neuronal activity, were measured. Eight fetal sheep were sacrificed at 135 days gestation--four at day-time (1200 hours) and four at night-time (2400 hours). Fetal brains were fixed, removed and cut in 40-microns serial coronal sections. Alternate sections were incubated with anti-Fos antibody (1:500) and Fos expression was revealed with extra-avidin-peroxidase and 3,3'-diaminobenzidine or stained with cresyl violet. The number of Fos-immunoreactive (Fos-ir) neurons per mm2 in the rostral, intermediate and caudal regions of the fetal sheep SCN was counted. Fetuses sacrificed in the day-time showed a higher number of Fos-ir neurons per mm2 (mean +/- s.e.; 516.7 +/- 60.1) in the three regions of the SCN than fetuses sacrificed at night-time (140.5 +/- 21.8). In addition, at night-time Fos-ir neurons were mainly localized to the ventrolateral area of the SCN. These findings demonstrate day-night changes in molecular activity consistent with the presence of a circadian oscillator in the fetal sheep SCN.

The retino-hypothalamic tract is involved in prolactin regulation in fetal sheep.

To investigate the role of the retino-hypothalamic tract on fetal prolactin regulation, we examined the effect of ocular enucleation on fetal plasma prolactin. Eleven fetuses of Suffolk ewes were chronically catheterized during fall, and six of them were subjected to bilateral ocular enucleation. All ewes were kept at 12h:12h light:dark cycle (lights on at 0800 and off at 2000). The experiments were performed 5-9 days after surgery (GA control fetuses 125 +/- 1.5, optical enucleation 121.3 +/- 1.5 days). Blood samples were taken from fetuses hourly around the clock, and plasma prolactin and cortisol were measured by radioimmunoassay (RIA). Luteinizing hormone (LH) and Growth hormone (GH) were measured in pooled plasma samples from control and enucleated fetuses by RIA. Average plasma prolactin was 5-fold lower in enucleated than in control fetuses (9.6 +/- 0.5 and 54.2 +/- 3.3 ng/ml, SEM; P < 0.005). Both control and enucleated fetuses presented circadian rhythm of prolactin with acrophase between 1400 and 1830 h. An enucleated fetus was tested for response of prolactin to TRH. Prolactin increased as described in the literature. There was no change in plasma concentration of cortisol, LH or GH after ocular enucleation. Our data indicate that the optical pathway participates in prolactin regulation in the fetal sheep.

Ontogeny of the circadian variation of plasma prolactin in sheep.

The ontogeny of circadian rhythms is unknown. The newborn sheep has a circadian rhythm of temperature; to study the ontogeny of other rhythms, we examined the 24-h variation of plasma prolactin concentration in fetal and newborn sheep. To this effect, we measured plasma prolactin concentration in chronically catheterized fetuses (n = 7) and in newborn lambs raised under short day nycthemeral (12 light:12 dark, n = 13) or constant light conditions (n = 5). Indwelling catheters were implanted into the jugular vein and carotid artery of late gestation fetuses (0.9 gestation) and newborns (5-29 days old). Experiments were performed 4 or more days after surgery. Lambs were kept in a canvas sling and were fed cow's milk either by mouth or through a nasogastric catheter at established time intervals. Haematocrit, pH, and blood gases were measured before and after the experiments in all cases and remained within normal values. Lights were on and room temperature was maintained constant during the whole experiment. Samples were obtained every 1-2 h for 24 h in fetuses and newborn lambs under nycthemeral conditions and every hour for 48 h in newborn lambs kept under constant light. Plasma prolactin was measured by radioimmunoassay. The presence of a 24 h rhythm was determined by Cosinor analysis. Fetuses, aged 129 +/- 6 days (SD) n = 7, showed a variation in plasma prolactin concentration with a period of 24 h that fits the equation: plasma prolactin (ng ml-1) = 97.0 + 15.4 cos 15 (t-23.0), P = 0.035.(ABSTRACT TRUNCATED AT 250 WORDS)