Morphological and immunohistochemical characterization of angiogenic and apoptotic factors and the expression of thyroid receptors in the ovary of tilapia Oreochromis niloticus in captivity

Morphological and immunohistochemical characterization of angiogenic and apoptotic factors and the expression of thyroid receptors in the ovary of tilapia Oreochromis niloticus in captivity were studied. The morphological evaluation of the ovaries was performed by histological paraffin embedded and stained with HE. The immunohistochemical expressions of CDC47, VEGF, Flk-1, angiopoietin, Tie-2 and thyroid receptor (TRα) were performed by the technique of streptavidein-biotin-peroxidase. Apoptosis was assessed using the TUNEL kit. The relative expression of thyroid hormone receptors (TRα and TRβ) was assessed by RT-PCR real time. The nuclear expression of CDC47 increased with the stage of maturation of the oocyte and was observed in the follicle cells. Apoptotic bodies were observed in the follicular cells of atretic follicles and postovulatory follicles from the ovaries of 150g and 350g fish. Expression of VEGF and its receptor Flk-1 was also observed in the follicular cells, and the expression of both increased with the maturity of the oocyte, with a higher intensity observed in the full-grown follicle. The expression of angiopoietin and of its receptor (Tie 2) was discrete and moderate respectively. TRα expression was independent of follicular development. However, the 350 g tilapia exhibited higher expression of TRβ compared with the 50 g tilapia. We conclude that the proliferative activity and the expression of VEGF and its receptor increase with follicular maturation and that the TRs expression increases with ovarian maturity in tilapia (Oreochromis niloticus).

Pathological and parasitological aspects of the peacock (Pavo cristatus) infection by Tanaisia(Paratanaisia)bragai

Abstract:Trematodes belonging to the family Eucotylidae, including Tanaisia(Paratanaisia)bragaiSantos, 1934are parasites of the kidney and ureter that affect several species of domestic and wild birds. Tanaisia bragaiis considered a low pathogenic parasite, but high worm burdens may determine clinical complications, including signs of apathy, weight loss, diarrhea and death. This paper describes the first report of infection by T. bragai in peacocks (Pavo cristatus), which constitutes a new host record and offers data on the lesions associated to this parasitism, although the degree of pathogenicity and parasite load may be considered mild. These birds did not exhibit clinical signs of parasitism. The macroscopic exam revealed discreet yellow spots on the liver. In the histological sections of the kidney, specimens of T. bragai were found in the collecting ducts, which were markedly dilated, with a thickened wall. Other findings included a mild inflammatory reaction in the wall of the ducts (but sometimes absent), flattening of lining epithelial cells and small, multifocal points of calcification around the collecting ducts. The microscopic examination of the parasites revealed trematodes with an elongated body, well-developed sub terminal oral sucker, pharynx present, short esophagus, cecum somewhat undulating or not, with blind end, testes symmetrical, equatorial, irregular in shape or slightly lobed, vitelline fields extending in both pre-ovarian and post ovarian fields, uterus very long, intercecal or sometimes overlapping the cecum and containing large quantities of eggs. The present findings suggest the need for further diagnostic studies on the prevalence of this trematode in peacocks as well as pathologic studies for the determination of the potential pathogenicity of this parasite in this species of bird. Moreover, infected peacocks could serve as carriers of T. bragai to be transferred to other bird species, thereby contributing to the dispersion of the parasite.

Vitrification of bovine preantral follicles with dimethylsulfoxide and sucrose plus α-tocopherol

Abstract: The objective of this study was to evaluate the vitrification of bovine preantral follicles with dimethylsulfoxide (D) and sucrose (S) plus α-tocopherol 5mmol/L (T5) or 10mmol/L (T10) and, evaluate the thawed with minimal essential medium (m) with or without sucrose (s). Ovaries of cows were collected from slaughterhouse for the experiment I (n=66) and II (n=51). In the laboratory ovarian fragments were randomly assigned either to fresh control and 8 vitrification treatments (Controle and Dm; Dms, DSm; DSms; DST5m; DST5ms; DST10m; DST10ms). Ovarian fragments were placed in vitrification solution (5 min) and immersed in liquid nitrogen (-196°C), after a week, the fragments were thawed and analyzed. In the experiments I, preantral follicles were morphologically observed for histological evaluation, (normal; degenerated and developing of stage). In the experiment II, preantral follicles were mechanically isolated from ovarian tissue and examined with trypan blue, where dead and live corresponded to stained or non-stained. The treatments DSm, DSms and DST10m were effective in preserving the morphology in situ. However, the viability of isolated preantral follicles after vitrification remained high only in treatment DST10m. Thus, DST10m preserves survival rates and morphological integrity during vitrification of bovine preantral follicles.

Late-acting self-incompatibility in Capparis retusa (Capparaceae), a species of Chaco woodland in NE Argentina

The reproductive biology of Capparis retusa was studied by means of controlled pollination experiments and fluorescence microscopy observations of post-pollination events. Self-pollinated flowers mostly failed to form fruits despite the fact that self-pollen tubes grew to the ovary and penetrated ovules within 24 h. Since embryo development to globular stage was observed in some self-fertilized ovules it was concluded that control of self-fertilization in this species occurs by some kind of post-zygotic mechanism.

Morphology and anatomy of the fruit and seed in development of Chorisia speciosa A. St.-Hil. - Bombacaceae

The structure of the fruit and seed in development of Chorisia speciosa are described with the main purpose of clarifying the origin and nature of the hairs that cover the seeds and aiding future taxonomical and ecological studies of the group. The fruit is an ellipsoid loculicide capsule and presents the exocarp formed by 7-10 cells layers, with very thick walls and evident simple pits. A great number of mucilage secretory cavities and ramified vascular bundles, accompanied by fibers, occur in the parenchymatic mesocarp. The endocarp derives from the ventral epidermis of the ovary wall, whose cells undergo a gradual elongation, become lignified, and constitute the trichomes which cover the mature seeds. The fruit aperture occurs by means of a suture evident in the ovarian wall in the middle region of the carpel leaf. Anatropous and bitegmic ovules, provided by a hypostase, give rise to campilotropous and bitegmic seeds. The testa is uniseriate, the exotegmen is completely formed by macrosclereids, and mucilage secretory cavities occur in the mesotegmen. The endotegmen, which is differentiated in the endothelium, is crushed in the mature seed. The plicate embryo, which occupies practically the entire seminal cavity, is found between endosperm layers, both being rich in lipids.

Meiotic behavior in wild and domesticated species of Passiflora

The meiotic behavior of fourteen Passiflora taxa was analyzed. The species were grouped according to the n value (6, 9 and 12) for statistical studies. Some species presented tetravalent associations or univalent chromosomes in diakinesis, bivalent formation prevailing. The qui-square test revealed significant differences in the chiasma frequency among species for n = 9 and n = 6 groups. There was predominance of interstitial chiasmata in almost all studied species. The n = 12 group was the only one whose meiotic behavior was considered similar due to the quantity of chiasmata per cell, tendency of interstitial chiasma localization. Some species presented meiotic irregularities, such as laggard and precocious chromosomes in meiosis I. In telophase II the percentages of meiotic irregularities was low. Irregularities in the spindle orientation were presented in higher percentages in the end of meiosis II, and were also responsible for post-meiotic abnormal products. The irregularities observed during meiosis can have influence on the percentage of sterile pollen grains and success of interspecific crossings in Passiflora species.

The polytene chromosomes of the mosquito Anopheles bellator compared with those of Anopheles cruzii

A photographic map was made of polytene chromosomes of ovarian nurse cells of Anopheles bellator females. The chromosomes of this species have complete or partial homology with those of A. cruzii, mainly in the telomeric and centromeric regions. Variability at the single band level was observed as asymmetric bands at seven different positions. One inversion (3Ra) was detected in the 3R arm.

Risk factors for decreased bone density in premenopausal women

Osteoporosis is a major health problem. Little is known about the risk factors in premenopause. Sixty 40-50-year old patients with regular menses were studied cross-sectionally. None of the patients were on drugs known to interfere with bone mass. Patients answered a dietary inquiry and had their bone mineral density (BMD) measured. The Z scores were used for the comparisons. A blood sample was taken for the determination of FSH, SHBG, estradiol, testosterone, calcium and alkaline phosphatase. Calcium and creatinine were measured in 24-h urine. A Z score less than -1 was observed for the lumbar spine of 14 patients (23.3%), and for the femur of 24 patients (40%). Patients with a Z score less than -1 for the lumbar spine were older than patients with a Z score ³-1 (45.7 vs 43.8 years) and presented higher values of alkaline phosphatase (71.1 ± 18.2 vs 57.1 ± 14.3 IU/l). Multiple regression analysis showed that a lower lumbar spine BMD was associated with higher values of alkaline phosphatase, lower calcium ingestion, a smaller body mass index (BMI), less frequent exercising, and older age. The patients with a Z score less than -1 for the femur were shorter than patients with a Z score ³-1 (158.2 vs 161.3 cm). Multiple regression analysis showed that a lower femoral BMD was associated with lower BMI, higher alkaline phosphatase and caffeine intake, and less frequent exercising. A lower than expected BMD was observed in a significant proportion of premenopausal women and was associated with lower calcium intake, relatively lower physical activity and lower BMI. We conclude that the classical risk factors for osteoporosis may be present before ovarian failure, and their effect may be partly independent of estrogen levels.

Annual changes in serum calcium and inorganic phosphate levels and correlation with gonadal status of a freshwater murrel, Channa punctatus (Bloch)

Adult Channa punctatus murrels of both sexes (60-80 g) were collected locally from Ramgarh Lake during the second week of every month (10 individuals of each sex/month) throughout the year. Blood samples were collected and analyzed for serum calcium and phosphate levels by the methods of Trinder (1960) and Fiske and Subbarow (1925), respectively. Gonads were fixed to judge the state of maturation of the fish. Males exhibited no change in serum calcium levels throughout the year in correlation with testicular maturation. However, serum phosphate levels exhibited a rise in correlation with the increased gonadosomatic index. Females showed marked seasonal changes in serum calcium and phosphate levels which were associated with ovarian maturation (vitellogenesis).

Synchronization of ovulation in crossbred dairy heifers using gonadotrophin-releasing hormone agonist, prostaglandin F2a and human chorionic gonadotrophin or estradiol benzoate

Girolando (Gir x Holstein) is a very common dairy breed in Brazil because it combines the rusticity of Gir (Bos indicus) with the high milk yield of Holstein (Bos taurus). The ovarian follicular dynamics and hormonal treatments for synchronization of ovulation and timed artificial insemination were studied in Girolando heifers. The injection of a gonadotrophin-releasing hormone (GnRH) agonist was followed 6 or 7 days (d) later by prostaglandin F2a (PGF2a). Twenty-four hours after PGF2a injection either human chorionic gonadotropin (hCG, GPh-d6 and GPh-d7 groups) or estradiol benzoate (EB, GPE-d6 and GPE-d7 groups) was administered to synchronize ovulation and consequently allow timed artificial insemination (AI) 24 and 30 h after hCG and EB injection, respectively. Follicular dynamics in Girolando heifers was characterized by the predominance of three follicular waves (71.4%) with sizes of dominant follicles (10-13 mm) and corpus luteum (approximately 20 mm) similar to those for Bos indicus cattle. In the GnRH-PGF-hCG protocol, hCG administration induced earlier ovulation (67.4 h, P<0.01) compared to the control group (GnRH-PGF) and a better synchronization of ovulation, since most of it occurred within a period of 12 to 17 h. Pregnancy rate after timed AI was 42.8 (3/7, GPh-d6) to 50% (7/14, GPh-d7). In contrast, estradiol benzoate (GnRH-PGF-EB protocol) synchronized ovulation of only 5 of 11 heifers from the GPE-d7 group and of none (0/7) from the GPE-d6 group, which led to low pregnancy rates after timed AI (27.3 and 0%, respectively). However, since a small number of Girolando heifers was used to determine pregnancy rates in the present study, pregnancy rates should be confirmed with a larger number of animals.

Clinical and molecular analysis of human reproductive disorders in Brazilian patients

Several genes that influence the development and function of the hypothalamic-pituitary-gonadal-axis (HPG) have been identified. These genes encode an array of transcription factors, matrix proteins, hormones, receptors, and enzymes that are expressed at multiple levels of the HPG. We report the experience of a single Endocrinology Unit in the identification and characterization of naturally occurring mutations in families affected by HPG disorders, including forms of precocious puberty, hypogonadism and abnormal sexual development due to impaired gonadotropin function. Eight distinct genes implicated in HPG function were studied: KAL, SF1, DAX1, GnRH, GnRHR, FSHß, FSHR, and LHR. Most mutations identified in our cohort are described for the first time in literature. New mutations in SF1, DAX1 and GnRHR genes were identified in three Brazilian patients with hypogonadism. Eight boys with luteinizing hormone- (LH) independent precocious puberty due to testotoxicosis were studied, and all have their LH receptor (LHR) defects elucidated. Among the identified LHR molecular defects, three were new activating mutations. In addition, these mutations were frequently associated with new clinical and hormonal aspects, contributing significantly to the knowledge of the molecular basis of reproductive disorders. In conclusion, the naturally occurring genetic mutations described in the Brazilian families studied provide important insights into the regulation of the HPG.

Symmetric pollen mitosis I and suppression of pollen mitosis II prevent pollen development in Brachiaria jubata (Gramineae)

Microsporogenesis and pollen development were analyzed in a tetraploid (2n = 4x = 36) accession of the forage grass Brachiaria jubata (BRA 007820) from the Embrapa Beef Cattle Brachiaria collection that showed partial male sterility. Microsporocytes and pollen grains were prepared by squashing and staining with 0.5% propionic carmine. The meiotic process was typical of polyploids, with precocious chromosome migration to the poles and laggards in both meiosis I and II, resulting in tetrads with micronuclei in some microspores. After callose dissolution, microspores were released into the anther locule and appeared to be normal. Although each microspore initiated its differentiation into a pollen grain, in 11.1% of them nucleus polarization was not observed, i.e., pollen mitosis I was symmetric and the typical hemispherical cell plate was not detected. After a central cytokinesis, two equal-sized cells showing equal chromatin condensation and the same nuclear shape and size were formed. Generative cells and vegetative cells could not be distinguished. These cells did not undergo the second pollen mitosis and after completion of pollen wall synthesis each gave rise to a sterile and uninucleate pollen grain. The frequency of abnormal pollen mitosis varied among flowers and also among inflorescences. All plants were equally affected. The absence of fertile sperm cells in a considerable amount of pollen grains in this accession of B. jubata may compromise its use in breeding and could explain, at least in part, why seed production is low when compared with the amount of flowers per raceme.

Quantification of fecal estradiol and progesterone metabolites in Syrian hamsters (Mesocricetus auratus)

Alternative methods to the utilization of laboratory animal blood and its by-products are particularly attractive, especially regarding hamsters due to their small size and difficulties in obtaining serial blood samples. Steroid hormone metabolite quantification in feces, widely used in studies of free-ranging or intractable animals, is a non-invasive, non-stressor, economical, and animal saving technique which allows longitudinal studies by permitting frequent sampling of the same individual. The present study was undertaken to determine the suitability of this method for laboratory animals. Estradiol and progesterone metabolites were quantified by radioimmunoassay in feces of intact, sexually mature female Syrian hamsters during the estrous cycle (control) and in feces of superovulated females. Metabolites were extracted by fecal dilution in ethanol and quantified by solid phase radioimmunoassay. Median estrogen and progesterone concentrations were 9.703 and 180.74 ng/g feces in the control group, respectively. Peaks of estrogen (22.44 ± 4.54 ng/g feces) and progesterone (655.95 ± 129.93 ng/g feces) mean fecal concentrations respectively occurred 12 h before and immediately after ovulation, which is easily detected in this species by observation of a characteristic vaginal postovulatory discharge. Median estrogen and progesterone concentrations (28.159 and 586.57 ng/g feces, respectively) were significantly higher in superovulated animal feces (P < 0.0001). The present study demonstrated that it is possible to monitor ovarian activity in Syrian hamsters non-invasively by measuring fecal estradiol and progesterone metabolites. This technique appears to be a quite encouraging method for the development of new endocrinologic studies on laboratory animals.

Dexamethasone-induced reactivation of bovine herpesvirus type 5 latent infection in experimentally infected rabbits results in a broader distribution of latent viral DNA in the brain

Bovine herpesvirus type 5 (BHV-5) is a major agent of meningoencephalitis in cattle and establishes latent infections mainly in sensory nerve ganglia. The distribution of latent BHV-5 DNA in the brain of rabbits prior to and after virus reactivation was studied using a nested PCR. Fifteen rabbits inoculated intranasally with BHV-5 were euthanized 60 days post-inoculation (group A, N = 8) or submitted to dexamethasone treatment (2.6 mg kg-1 day-1, im, for 5 days) and euthanized 60 days later (group B, N = 7) for tissue examination. Two groups of BHV-1-infected rabbits (C, N = 3 and D, N = 3) submitted to each treatment were used as controls. Viral DNA of group A rabbits was consistently detected in trigeminal ganglia (8/8), frequently in cerebellum (5/8), anterior cerebral cortex and pons-medulla (3/8) and occasionally in dorsolateral (2/8), ventrolateral and posterior cerebral cortices, midbrain and thalamus (1/8). Viral DNA of group B rabbits showed a broader distribution, being detected at higher frequency in ventrolateral (6/7) and posterior cerebral cortices (5/7), pons-medulla (6/7), thalamus (4/7), and midbrain (3/7). In contrast, rabbits inoculated with BHV-1 harbored viral DNA almost completely restricted to trigeminal ganglia and the distribution did not change post-reactivation. These results demonstrate that latency by BHV-5 is established in several areas of the rabbit's brain and that virus reactivation leads to a broader distribution of latent viral DNA. Spread of virus from trigeminal ganglia and other areas of the brain likely contributes to this dissemination and may contribute to the recrudescence of neurological disease frequently observed upon BHV-5 reactivation.

Early remodeling of rat cardiac muscle induced by swimming training

The aim of the present investigation was to study the effect of acute swimming training with an anaerobic component on matrix metallopeptidase (MMP) activity and myosin heavy chain gene expression in the rat myocardium. Animals (male Wistar rats, weighing approximately 180 g) were trained for 6 h/day in 3 sessions of 2 h each for 1 to 5 consecutive days (N = 5 rats per group). Rats swam in basins 47 cm in diameter and 60 cm deep filled with water at 33 to 35ºC. After the training period a significant increase (P < 0.05) was observed in the heart weight normalized to body weight by about 22 and 35% in the groups that trained for 96 and 120 h, respectively. Blood lactate levels were significantly increased (P < 0.05) in all groups after all training sessions, confirming an anaerobic component. However, lactate levels decreased (P < 0.05) with days of training, suggesting that the animals became adapted to this protocol. Myosin heavy chain-ß gene expression, analyzed by real time PCR and normalized with GAPDH gene expression, showed a significant two-fold increase (P < 0.01) after 5 days of training. Zymography analysis of myocardium extracts indicated a single ~60-kDa activity band that was significantly increased (P < 0.05) after 72, 96, and 120 h, indicating an increased expression of MMP-2 and suggesting precocious remodeling. Furthermore, the presence of MMP-2 was confirmed by Western blot analysis, but not the presence of MMP-1 and MMP-3. Taken together, our results indicate that in these training conditions, the rat heart undergoes early biochemical and functional changes required for the adaptation to the new physiological condition by tissue remodeling.