RESUMO

The beta-nerve growth factor (ß-NGF) from llama seminal plasma exerts ovulatory and luteotrophic effects following intramuscular or intrauterine infusion in llamas and alpacas. In this study, we investigate the in vitro effect of llama ß-NGF on the expression of genes involved in angiogenesis and progesterone synthesis as well as progesterone release in preovulatory llama granulosa cells; we also determine whether these changes are mediated via the ERK1/2 signaling pathway. From adult female llamas, we collected granulosa cells from preovulatory follicles by transvaginal ultrasound-guided follicle aspiration; these cells were pooled and incubated. After 80% confluence, the cultured granulosa cells were treated with ß-NGF, ß-NGF plus the MAPK inhibitor U0126, or luteinizing hormone, and the abundance of angiogenic and steroidogenic enzyme mRNA transcripts were quantified after 10 and 20 h by RT-qPCR. We also quantified the progesterone concentration in the media after 48 h by radioimmunoassay. We found that application of ß-NGF increases the abundance of mRNA transcripts of the vascular endothelial growth factor (VEGFA) and the steroidogenic enzymes cytochrome P450 side-chain cleavage (P450scc/CYP11A1), steroidogenic acute regulatory protein (STAR), and 3ß-hydroxysteroid dehydrogenase (HSD3B1) at 10 and 20 h of treatment. Application of the MAPK inhibitor U0126 resulted in downregulation of the genes encoding these enzymes. ß-NGF also enhanced progesterone synthesis, which was prevented by the prior application of the MAPK inhibitor U0126. Finally, western blot analysis confirmed that ß-NGF activates the ERK1/2 signaling pathway. In conclusion, our results indicate that ß-NGF exerts direct luteotropic effects on llama ovarian tissue via the ERK 1/2 pathway.

RESUMO

Copulation produces different stimuli in the female reproductive tract in camelids, which lead to ovulation. Expression of ß-nerve growth factor (ß-NGF) and its specific receptor, tropomyosin receptor kinase A (TrKA), was studied comparing the oviductal microenvironment of mated and nonmated llamas. ß-NGF and TrKA were expressed in the llama ampulla, isthmus, and utero-tubal-junction (UTJ), and they were mainly colocalized in the apical region of the oviductal mucosa. A TrKA immunosignal was also found in muscle cells and blood vessels, with the highest mark in UTJ muscle cells of copulated females. Both ß-NGF and TrKA transcripts were expressed in the three oviductal segments. Relative TrKA abundance did not differ between mated and nonmated females, but relative ß-NGF abundance was higher in the UTJ of copulated females (p < .05). ß-NGF might not be secreted into the oviductal fluid (OF) since the protein was not found in the OF of mated or nonmated females. Therefore, it can be concluded that the llama oviduct expresses the ß-NGF/TrKA system and that an increase in ß-NGF gene expression in the UTJ 24 h after copulation along with an increase in TrKA protein expression may indicate an important role in the gamete transport and fertilization process in llamas.

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RESUMO

To provide new insights into the mechanisms through which seminal plasma proteins can protect sperm from damage caused during refrigeration, we evaluate the possibility that ß-NGF can contribute to the improvement of sperm quality after cooling. First, ß-NGF was detected in refrigerated sperm and compared with unrefrigerated sperm by western blotting of the proteins adsorbed by sperm, showing that native ß-NGF is still present even 24 h after cooling only as an active form. Then, the effect of exogenous ß-NGF on sperm quality after cooling was evaluated. A total of 12 ejaculates from male llamas (three ejaculates per male), were obtained by electro-ejaculation, diluted 4:1 with buffer Hepes-balanced salt solution and centrifuged at 800 × g for 8 min to remove the seminal plasma. Sperm were suspended in Tris-citrate-fructose-egg yolk diluent for a final concentration of 30 ×106/ml and cooled at 5°C for 24 h. After refrigeration, the extended sperm were equilibrated for 5 min at 37°C and divided into the following subgroups: sperm samples without treatment (control) and sperm samples supplemented with exogenous human ß-NGF (10, 100, and 500 ng/ml). At 5, 30, and 60 min of incubation sperm were evaluated for sperm viability (using eosin/nigrosin stain), sperm motility and vigor (observed under light microscopy), and mitochondrial activity (using the JC-1 fluorescent marker). Vigor data were analyzed with the nonparametric Kruskal-Wallis test. The rest of the variables were analyzed with a mixed models approach. Mean comparisons were performed using Fisher's LSD test with a confidence level of 95%. A principal components analysis was performed to analyze the relationships between variables. Treatment of 24 h cooled sperm with 10 or 100 ng/ml of human ß-NGF increased the percentage of total motility and vigor (p < 0.05). Besides, an incubation time of 60 min would be adequate to improve sperm quality, since all variables are positively related. The significant improvement observed in the motility and vigor of post-refrigerated sperm suggests that supplementation with exogenous ß-NGF may be profitable for the improvement of cooled llama sperm.

RESUMO

ß-Nerve growth factor (ß-NGF) is a seminal plasma element, responsible for inducing ovulation in camelids. The main organ of ß-NGF production remains nondescript. The aims of this study were to (a) characterize gene expression and protein localization of ß-NGF and its main receptor tyrosine kinase receptor A (TrKA) in the llama male reproductive tract, and (b) determine whether the seminal ß-NGF interacts with ejaculated sperm by localizing ß-NGF and TrKA in epididymal, ejaculated, and acrosome-reacted (AR) sperms and, additionally, by identifying ß-NGF presence in sperm-adsorbed proteins (SAP). Both ß-NGF and TrkA transcripts are widely expressed along the male reproductive tract, with a higher expression level of ß-NGF at prostate (p < 0.05). ß-NGF immunolabeling was only positive for prostate, whereas TrKA label was present in epithelial and muscular cells of testis, prostate, bulbourethral glands, and epididymis. Using an immunofluorescent technique, ß-NGF was colocalized with TrKA in the middle piece of ejaculated and AR sperm. However, only TrKA was observed in epididymal sperm indicating that ß-NGF could have a seminal origin. This was also confirmed by the identification of four ß-NGF isoforms in SAP. This study extends the knowledge about the participation of ß-NGF/TrkA in llama reproduction, providing evidence that may have roles in the regulation of sperm physiology.

Assuntos

RESUMO

Oviductal molecules have the potential to improve the reproductive biotechnologies. In camelids, knowledge and assessment of the oviductal environment are necessary to successfully develop species-specific reproductive technologies, especially because of the camelids reproductive particularities. Among the oviductal factors, the matrix metalloproteinases/tissue inhibitor of matrix metalloproteinases system (MMPs/TIMPs) should be investigated more thoroughly due to their participation in reproductive processes. Consequently, the current study assayed gene and protein expression of MMPs throughout the llama oviduct. MMPs zymogen and active forms in the oviductal fluid were also characterized. MMP2 and MMP9 transcripts were detected in ampulla, isthmus, utero-tubal junction and papilla, being MMP2 and MMP9 2.15 and 1.10 folds higher in papilla than in ampulla, respectively. In addition, differences in immunolocalization of MMP2 and MMP9 between the epithelial mucosa layers of the oviductal segments were observed. The presence of MMPs in the epithelium suggests their secretion into the oviductal lumen. Coincidently, bands of 62 and 94 kDa, corresponding to MMP2 and MMP9 were detected by zymography in the oviductal fluid. Treatment with an exogenous activator (APMA) suggests that they are present as proMMPs. TIMP2 and TIMP1, the specific inhibitors of MMP2 and MMP9, respectively, were expressed in each oviductal segment, indicating a well-regulated control of MMP proteolytic activity in the oviduct. These findings prove that the llama oviduct produces and secretes MMPs into the oviductal lumen, suggesting that these enzymes may have an unknown role in the preparation of the oviductal environment for gametes, fertilization and early embryo development in camelids.

Las moléculas oviductales tienen el potencial para mejorar las biotecnologías reproductivas. En los camélidos, debido a sus peculiares características reproductivas, el conocimiento del ambiente oviductal constituye una herramienta útil para el desarrollo de tecnologías reproductivas específicas para estas especies. Entre los factores oviductales de interés se encuentran las metaloproteasas de matriz (MMPs) y sus inhibidores específicos (TIMPs), los cuales han sido involucrados en diferentes procesos reproductivos. Por estas razones, en este trabajo se caracterizó la expresión génica y proteica de MMP2 y MMP9 en el oviducto de llama. Además, se analizó la presencia de las formas activas e inactivas (zimógenos) de estas enzimas en el fluido oviductal. Se observó que todos los segmentos oviductales, ámpula, istmo, unión útero-tubal y papila, expresan MMP2 y MMP9, siendo los niveles de expresión de MMP2 y MMP9 más elevados en papila respecto a ámpula; 2,15 y 1,10 veces respectivamente. Asimismo, se observaron diferencias en la distribución de las MMPs a nivel de la mucosa entre los segmentos oviductales. Consecuentemente, bandas con actividad gelatinolítica de 62 y 94 kDa, se detectaron en el fluido oviductal, las cuales corresponderían a las formas inactivas de la MMP2 y la MMP9, respectivamente. Los inhibidores específicos de MMP2 y MMP9; TIMP2 y TIMP1, también se detectaron en los segmentos oviductales, indicando su probable participación en la regulación de la actividad proteolítica de las MMPs en el oviducto de llama. En conjunto, los datos de este trabajo demuestran que el oviducto de la llama produce y secreta MMPs al lumen oviductal; sugiriendo que estas enzimas pueden participar en la preparación del ambiente oviductal para la recepción de los gametos, la fecundación y el desarrollo embrionario temprano en camélidos.

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