Gene expression of growth factor BMP15, GDF9, FGF2 and their receptors in bovine follicular cells / Expresión génica del factor de crecimiento BMP15, GDF9, FGF2 y sus receptores en células foliculares bovinas

ABSTRACT Objective. Evaluate mRNA expression of GDF9, BMP15, FGF2 and their main receptors, transforming growth factor beta receptor 1 (TGFβ-R1), bone morphogenetic protein receptor, type IB (BMPR-IB) and fibroblast growth factor receptor 2 (FGFR2) in bovine follicular cells. Materials and methods. Total RNA was isolated from pooled samples of oocytes (OOs), cumulus cells (CCs) of cumulus oocyte complexes (COCs) and follicular cell pellets (PCs) of 70 ovaries obtained from 96 beef heifers, collected at a local abattoir. The expression pattern of growth factors and their receptors in follicular bovine cells was evaluated by reverse transcriptase polymerase chain reaction (RT-PCR). Results. The mRNA transcripts encoding GDF9, BMP15, FGF2, TGFβ-R1, BMPR-IB and FGFR2 genes were detected, by RT-PCR, in all studied cells. This is the first time that the expression of TGFβ-R1 and BMPR-IB receptors is reported in bovine oocytes. Conclusions. The presence of growth factors and receptor transcripts in the studied cells indicate that these factors could act as paracrine and autocrine regulators of folliculogenesis.

RESUMEN Objetivo. Evaluar la expresión de los factores de crecimiento GDF9, BMP15, FGF2 y sus principales receptores: el receptor 1 del factor de crecimiento transformante beta (TGFβ-R1), el receptor de la proteína morfogénica del hueso tipo IB (BMPR-IB) y el receptor del factor de crecimiento fibroblástico 2 (FGFR2) en células foliculares bovinas. Materiales y métodos. Se realizó la extracción de ARN total de pooles de ovocitos (OOs) y células del cumulus (CCs) de complejos cumulus-ovocito (COCs) y del pellet de células foliculares (PCs) provenientes de 70 ovarios obtenidos de 96 vaquillonas para carne, colectados en un frigorífico local. Los patrones de expresión de los factores de crecimiento y sus receptores en las células foliculares bovinas fueron evaluados por retro-transcripción seguida de la reacción en cadena de la polimerasa (RT-PCR). Resultados. La presencia de los transcriptos de ARNm de los genes GDF9, BMP15, FGF2, TGFβ-R1, BMPR-IB y FGFR2 fue detectada por RT-PCR en todas las células estudiadas. Es la primera vez que se reporta en ovocitos bovinos la expresión de los receptores TGFβ-R1 y BMPR-IB. Conclusiones. La presencia de transcriptos de factores de crecimiento y sus receptores en las células estudiadas, indica que estos factores podrían actuar como reguladores paracrinos y autocrinos de la foliculogénesis.

Calpain System in meat tenderization: A molecular approach / El sistema proteolítico calpaina en la tenderización de la carne: Un enfoque molecular

ABSTRACT Tenderness is considered the most important meat quality trait regarding its eating quality. Post mortem meat tenderization is primarily the result of calpain mediated degradation of key proteins within muscles fibers. The calpain system originally comprised three molecules: two Ca2+-dependent proteases and a specific inhibitor. Numerous studies have shown that the calpain system plays a central role in postmortem proteolysis and meat tenderization. The objective of this review is to describe the last biochemical and molecular findings in connection with this proteolytic system and their relation with meat tenderness in bovine. Findings of DNA polymorphisms and mRNA and protein expression are described as tools to predict meat tenderness. Understanding the molecular basis of meat tenderization may be useful particularly to the meat industry and may allow amendment of pre-slaughter handling practices and postmortem treatments that improves meat quality.

RESUMEN La terneza de la carne es considerada como el atributo de mayor importancia en el concepto de calidad de carne. El proceso de tenderización de la carne post mortem es principalmente el resultado de la degradación de proteínas clave de las fibras musculares, mediado por las proteasas del sistema calpaína. Este sistema proteico está compuesto por tres moléculas: dos proteasas calcio-dependientes y su inhibidor específico. Numerosos estudios han demostrado que el sistema calpaína desempeña un papel central en la proteólisis postmortem y en la tenderización de la carne. El objetivo de esta revisión es describir los últimos descubrimientos bioquímicos y moleculares de este sistema proteolítico y su relación con la terneza de la carne bovina. Se describen los hallazgos de polimorfismos de ADN y de expresión de ARNm y proteínas, como herramientas para predecir la terneza de la carne. La comprensión de las bases moleculares de la tenderización de la carne puede ser de utilidad para la industria cárnica, permitiendo la modificación de las prácticas de manipulación antes del sacrificio y los tratamientos post mortem, mejorando la calidad de la carne bovina.

In vivo effect of growth hormone on the expression of connexin-43 in bovine ovarian follicles.

This study assessed the in vivo effects of recombinant growth hormone (rGH) administration on the expression of connexin-43 (Cx43) in bovine ovarian follicles. Two independent experiments were carried out using either estrous unsynchronized or synchronized multiparous Aberdeen Angus cows. rGH-treated animals were inoculated with a single dose of hormone (500 mg, intramuscular) while control animals were inoculated with hormone diluent. Five and 14 days after treatment (Experiments 1 and 2, respectively), ovarian Cx43 and apoptosis expression were assessed using immunohistochemistry. In both experiments primary, secondary, and tertiary follicles from rGH-treated and control groups distinctly expressed Cx43 protein. Primordial and atretic follicles were Cx43-negative. Interestingly, the number of Cx43 dots per granulosa cell did not show significant variation at different folliculogenesis stages neither in the rGH-treated nor in the control group. In unsynchronized animals, Cx43-positive follicles per total number of follicles ratio showed an interaction between stage of folliculogenesis and treatment due to significant differences between treatment groups in the early secondary follicle stage. In synchronized animals, there were significant differences between treatment groups and folliculogenesis stage. In both experiments, atretic follicles showed apoptosis-related DNA-fragmentation as determined by terminal uridin nick end labeling (TUNEL) assay. Tertiary follicles presented moderate TUNEL staining. Our results show significant increment in the number of ovarian follicles expressing the gap junction subunit Cx43 after in vivo rGH treatment. Therefore, we conclude that growth hormone can modulate in vivo gap junction assembly at early stages of folliculogenesis.

Immunolocalization of bovine sperm protease BSp120 by light and electron microscopy during capacitation and the acrosome reaction: its role in in vitro fertilization.

Mammalian fertilization involves various steps in which the participation of specific enzymes has been demonstrated by numerous studies. Acrosin is one of the most widely acrosomal protease in mammalian spermatozoa studied, including bovine; however, other proteases have also been described. A new trypsin-like serine protease named bovine serine protease of 120 kDa (BSp120) and its pre-cursor BSp66 (66 kDa) were identified in bovine spermatozoa. Cytological and ultrastructural immunolocalization studies on BSp120 were performed in live and fixed cells. Immunoflorescence assays with specific polyclonal antibodies revealed localization of BSp120 on the sperm head, with a signal homogeneously distributed over the acrosome resembling a horseshoe. After the acrosome reaction, sperm showed a patchy pattern in the acrosomal cap. Immune electron microscopy analysis indicated that BSp120 is located over the head plasma membrane of capacitated spermatozoa and acrosome reacting spermatozoa. To assess BSp120 function in sperm-oocyte interaction, in vitro fertilization studies were conducted. Oocytes were incubated with spermatozoa pre-treated with anti-BSp120, anti-guinea pig acrosin, and anti-BSp120 plus anti-guinea pig acrosin. Pre-treatment of bovine spermatozoa with antibodies towards each protein did not significantly modify fertilization rates. However, when both anti-acrosin and anti-BSp120 antibodies were simultaneously added, there was a significant decrease in the fertilization rate, suggesting that both enzymes may be required for fertilization. Altogether, the results from the present study described the localization of BSp120 over the acrosome of bovine sperm, and suggest its involvement in fertilization.