Argentinian plant extracts with relaxant effect on the smooth muscle of the corpus cavernosum of Guinea pig

Extracts of different polarity from Baccharis trimera, Haplopappus rigidus Huperzia saururus, Maytenus ilicifolia, Satureja parvifolia and Senecio eriophyton were tested for their relaxant activity on smooth muscle using L-phenylephrine precontracted strips of corpus cavernosum obtained from Guinea pigs. Highly significant and dose dependent results were obtained with the dichloromethane extracts of H. saururus (87% of relaxation at the dose of 10 mg/ml), S. parvifolia (95% of relaxation at 2.5 mg/ml) and S. eriophyton (94% of relaxation at 5 mg/ml). Similar effects were observed with the methanol extracts of H. saururus (88% of relaxation at 10 mg/ml) and S. parvifolia (84% of relaxation at 10 mg/ml). These results were comparable to those obtained with the dichloromethane and methanol extracts of the well known Mexican species Turnera diffusa. Moreover, the aqueous extract of H. rigidus and the aqueous and methanol extracts of S. eriophyton were highly effective in a dose dependent manner (more than 90% of relaxation at the dose of 10 mg/ml). Significant results, but with a lower overall relaxant activity (about 70% of relaxation at 10 mg/ml), could also be obtained with the aqueous extract of S. parvifolia and with the dichlormethane and methanol extracts of B. trimera and M. ilicifolia. The positive controls with Sildenafil citrate at doses ranging from 0.35 to 35 microg/ml yielded moderate effects (up to 46% of relaxation at 35 microg/ml). The effects observed in the present study seem to validate the folk medicinal use of the tested plants and open new ways in the search for natural products with vasodilatory effects.     

Effectiveness of near-infrared spectroscopy as a non-invasive tool to discriminate spectral profiles of in vitro cultured oocytes from goats

Here, we aimed to discriminate between the spectral profiles of spent culture media after oocyte in vitro maturation (IVM) and culture (IVC) from goats of different ages subjected to repeated hormonal treatments. The profiles were discriminated using near infrared (NIR) spectroscopy combined with multivariate methods. A total of 19 goats (young = 10; old = 9) were subjected to serial hormonal stimulation (HS) with gonadotropins. Cumulus oophorus complexes (COCs) were collected using laparoscopic ovum pick-up (LOPU) and subjected to IVM and parthenogenetic activation. The initial embryos were subjected to IVC. Spent culture media were collected after oocyte IVM and on day 2 of IVC and analyzed using NIR spectroscopy. NIR spectral data were interpreted through chemometric methods, such as principle component analysis (PCA) and partial least square discriminant analysis (PLS-DA). The results of PCA analysis clearly showed a separation in the spectral profiles between the experimental groups (HS sessions; young and old animals) both after IVM and IVC. Overall, the main absorption bands were attributed to the C-H group second overtone, first overtone of O-H and N-H, and C-H combinations and may serve as molecular markers. On the other hand, the spectral data obtained using PLS-DA models provided a better classification of the groups. The results showed the possibility of discriminating young and old groups as well as the three HS sessions with high specificity, sensitivity, and accuracy using NIR spectra. Thus, the culture medium analysis using NIR spectroscopy combined with multivariate methods indicated the dissimilarities between the groups and provided an insight into the in vitro development of goat oocytes. This technique serves as an efficient, objective, rapid, and non-invasive method to discriminate spectral profiles.     

Glycerin supplementation strategies for three or seven days affects oxidative stress,follicle dynamics and ovulatory response in Morada Nova sheep

This study examined the effect of glycerin supply strategies in different short-term protocols on follicular dynamics and ovulatory rate in Morada Nova sheep. Eighteen Morada Nova ewes with body condition > 2.9 had their estrus and follicular waves synchronized using three injections of prostaglandin analogue at seven-day intervals. All animals received the same diet during 21 days, which consisted of a total mixed ration (TMR) based on chopped elephant grass and concentrate twice daily. In the control group (n=9), ewes were fed the TMR diet. In the other four groups, ewes received 150 mL of glycerol daily, supplied as an oral drench or mixed in the TMR during three or seven days prior to the application of the third PGF2 alfa analogue. These groups were named as follows: Drench3d (n=10), Drench7d (n=8), TMR3d (n=9) and TMR7d (n=9). Follicle dynamics were monitored by ultrasonography, and plasma glucose and glutathione peroxidase levels were measured at the third prostaglandin administration. Six days after the final PGF2 alfa analogue dose, ovulatory rate was measured by laparoscopy. Glucose was higher (P< 0.001) in the glycerin-treated groups than in control group (83.7 ± 1.7 vs. 68.4 ± 4.5 mg. dL^-1; P < 0.001). Ewes in the TMR3d, Drench7d and TMR7d groups had a greater (P < 0.001) number of large follicles (≥ 3 < 5 mm), and the presence of follicles larger than 5 mm was observed. In the same groups, at the third PGF2 alfa analogue dose, a greater (P < 0.001) number of growing follicles (> 3 mm) and a larger size of the largest follicle (P < 0.001) were also recorded. Ovulation rate was 30% higher in the groups that received glycerin for seven days (1.6 ± 0.1 53 vs. 1.1 ± 0.1; P < 0.05), and they also exhibited a 38% reduction in glutathione peroxidase. Thus, the use of glycerin in Morada Nova sheep as a source of energy in short-term supplementation for increase ovulation rate is an efficient strategy when provided for seven days, either orally or in the feed.     

PSMA-Specific CAR-Engineered T Cells Eradicate Disseminated Prostate Cancer in Preclinical Models

Immunology-based interventions have been proposed as a promising curative chance to effectively attack postoperative minimal residual disease and distant metastatic localizations of prostate tumors. We developed a chimeric antigen receptor (CAR) construct targeting the human prostate-specific membrane antigen (hPSMA), based on a novel and high affinity specific mAb. As a transfer method, we employed last-generation lentiviral vectors (LV) carrying a synthetic bidirectional promoter capable of robust and coordinated expression of the CAR molecule, and a bioluminescent reporter gene to allow the tracking of transgenic T cells after in vivo adoptive transfer. Overall, we demonstrated that CAR-expressing LV efficiently transduced short-term activated PBMC, which in turn were readily stimulated to produce cytokines and to exert a relevant cytotoxic activity by engagement with PSMA⁺ prostate tumor cells. Upon in vivo transfer in tumor-bearing mice, CAR-transduced T cells were capable to completely eradicate a disseminated neoplasia in the majority of treated animals, thus supporting the translation of such approach in the clinical setting.     

Flavonoids from Achyrocline satureioides with relaxant effects on the smooth muscle of Guinea pig corpus cavernosum.

Ethanol extract of the aerial parts of Achyrocline satureioides (Lam.) DC. (Asteraceae) showed a significant, dose dependent, relaxant effect on the smooth muscle of corpus cavernosum strips, obtained from Guinea pig (65.5 +/- 4.1% of relaxation at the dose of 25.0 mg/ml). Bioassay guided fractionation of this extract furnished two flavonoids, quercetin and quercetin 3-methyl ether, with important vasorelaxing effects on the corpus cavernosum strips (79.8 +/- 8.4 and 66.0 +/- 4.8% of relaxation respectively at the dose of 0.075 mg/ml). Two methyl derivatives of quercetin obtained by synthesis, quercetin 3,7,3',4'-tetramethylether and quercetin 3,5,7,3',4'-pentamethylether, showed similar relaxant effects at the dose of 0.075 mg/ml (86.4 +/- 8.5 and 67.31 +/- 1.4% of relaxation respectively). The results show that the ethanol extract of A. satureioides and the assayed compounds exhibit significant vasorelaxing properties. Additionally, it is shown that the number of methyl groups in the quercetin nucleus has no significant influence on the effectiveness of these compounds.     

Isolated ovine primordial follicles cryopreserved in different concentrations of ethylene glycol

Cryopreservation of primordial follicles represents an opportunity to preserve female gametes, and consequently to protect the reproductive capacity of humans and animals, as well as to safeguard genetic material from endangered animal species or rare breeds. The aim of this work was to assess the toxicity of different concentrations of ethylene glycol (EG) to primordial follicles, and verify the viability of these follicles after the freezing-thawing procedure. Primordial follicles were isolated from ovine ovaries and exposed to different EG concentrations to evaluate the cryoprotectant (CPA) toxicity before and after cryopreservation. After isolation of primordial follicle (control), the number (mean+/-S.E.M.) of viable primordial follicles/ml was 3764+/-795.21. The number of viable follicles in the toxicity test using EG at 0, 0.5, 1.0, 1.5, 2.0, and 2.5 M was 1096+/-447.9, 960+/-446.67, 948+/-366.14, 832+/-313.59, 856+/-280.67, and 700+/-255.02, respectively. The number of viable follicles at concentrations of 2.5 M was less than for controls. After cryopreservation, the numbers decreased to 0+/-0, 148+/-85.46, 764+/-246.69, 824+/-291.9, 844+/-296.27, and 588+/-200.65, respectively for 0, 0.5, 1.0, 1.5, 2.0, and 2.5 M EG. The number of viable follicles at 0, 0.5, and 2.5 M was less than for controls. In conclusion, after the freezing and thawing procedure, concentrations of 1.0, 1.5, and 2.0 M EG can be successfully used for the cryopreservation of isolated follicles in sheep.     

Birth of normal kids after microinjection of pronuclear embryos in a transgenic goat (Capra hircus) production program in Brazil

This pilot project was designed to determine if normal kids could be produced after microinjection in pronuclear embryos and subsequent transfer to recipients in a transgenic goat program in Brazil. Twelve donors of the Saanen breed and 17 recipients of an undefined breed were used. The estrus of both donors and recipients was synchronized by a standard progestagen treatment and superovulation obtained by six pFSH injections. Donors in estrus were mated with fertile Saanen bucks. Zygotes were recovered surgically by flushing oviducts. The recovered zygotes with visible pronuclei were microinjected with 500 to 1000 copies of the human G-CSF gene. Two or four embryos were surgically transferred into the oviducts of recipients. One recipient became pregnant and two kids were born. No transgenic goat was identified after PCR analysis. Even though transgenic goats were not obtained, this experiment establishes the basis of a synchronization and superovulation regimen for use in goats raised in Brazil, for the purpose of collecting and manipulating the pronuclear embryos. This project also showed that microinjected one-cell goat embryos can survive to produce live young following surgical transfer.     

Hypothalamic excitatory amino acid system during sexual maturation in female rats

The present results indicate that during sexual maturation the APOA-MBH from rats of 30 days of age released significantly higher quantities of GnRH than the tissue from 16-day-old rats (P < 0.01). The addition of NMDA, an agonist of the excitatory amino acids system (EAAs), to the medium after 30 min of incubation significantly increased (P < 0.01) the GnRH release in normal rats of both ages and this increase was significantly (P < 0.01) higher in 30-day-old rats (to 661%) than in rats of 16 days of age (to 273%). The administration of estrogen-progesterone (EP) to rats of 16 days of age did not modify the GnRH release response to NMDA. On the contrary, at 30 days of age EP administration significantly potentiated the GnRH release response to NMDA since while in the control group NMDA increased the GnRH release to 630%, in the EP-pretreated group this was to around 4700% (P < 0.01). EP pretreatment of prepubertal rats decreases the hypothalamic release of aspartate and glutamate, the excitatory amino acids involved in NMDA neurotransmission and glycine but increases EAAs release in peripubertal rats. On the basis of these results it is proposed that the increase in EAAs release by the hypothalamus is directly connected with the onset of puberty and that the maturation of the positive feedback effect of ovarian hormones on gonadotropin secretion is related to the maturation of the capacity of EP to increase hypothalamic EAAs. Before this maturational event EP inhibits EAAs release as well as gonadotropin release (prepubertal rats). NMDA receptor stimulation leads to a positive mechanism which increases the release of Asp and Glu from APOA-MBH both in prepubertal and peripubertal rats, but EP potentiates this mechanism only in peripubertal rats. This could be an additional neuroendocrine mechanism involved in the increase of gonadotropin during sexual maturation which induces the onset of puberty and the preovulatory discharge of these pituitary hormones.     

Quantitative and qualitative analysis of the effectiveness of a mechanical method for the isolation of preantral follicles from ovine ovaries

The preantral follicles are the major source of oocytes and its utilization has been investigated as an important tool to store large numbers of female gametes for further utilization in reproductive programs. The aim of the present study was to perform quantitative and qualitative analyses of the efficacy of a mechanical method for isolating of preantral follicles from the ovaries of fetuses and from nonpregnant and pregnant ewes, using as reference the population of preantral follicles in situ. In the isolation method the ovaries were cut into fragments in the tissue chopper. Then, the suspension was filtered through nylon mesh filters. The number of isolated follicles per ovary was 1655, 4735 and 4770, respectively, for the fetus, nonpregnant ewe and pregnant ewe. The number of in situ preantral follicles per ovary was 32961, 16627 and 17794, respectively, for the fetus, nonpregnant ewe and pregnant ewe. The follicle recovery rate (number of isolated preantral follicles/number of in situ preantral follicles x 100) was higher in adult ewes (26 and 28%, respectively, for nonpregnant and pregnant ewes) than in fetuses (5%). Histological analysis showed that very few preantral follicles (less than 0.26% in situ and 0.46% after the isolation procedure) were degenerated. In conclusion, this study showed that a mechanical method could be used effectively to isolate a large number of intact ovine preantral follicles. In the future, with improvements in culture systems, the isolation of a great number of oocytes enclosed in preantral follicles will make a valuable contribution to the rare breeds and endangered species, agricultural efficiency and basic research in folliculogenesis.