Adrián Mutto

Expression Profile of Genes as Indicators of Developmental Competence and Quality of In Vitro Fertilization and Somatic Cell Nuclear Transfer Bovine Embryos

Reproductive biotechnologies such as in vitro fertilization (IVF) and somatic cell nuclear transfer (SCNT) enable improved reproductive efficiency of animals. However, the birth rate of in vitro-derived embryos still lags behind that of their in vivo counterparts. Thus, it is critical to develop an accurate evaluation and prediction system of embryo competence, both for commercial purposes and for scientific research. Previous works have demonstrated that in vitro culture systems induce alterations in the relative abundance (RA) of diverse transcripts and thus compromise embryo quality. The aim of this work was to analyze the RA of a set of genes involved in cellular stress (heat shock protein 70-kDa, HSP70), endoplasmic reticulum (ER) stress (immunoglobulin heavy chain binding protein, Bip; proteasome subunit β5, PSMB5) and apoptosis (BCL-2 associated X protein, Bax; cysteine aspartate protease-3, Caspase-3) in bovine blastocysts produced by IVF or SCNT and compare it with that of their in vivo counterparts. Poly (A) + mRNA was isolated from three pools of 10 blastocysts per treatment and analyzed by real-time RT-PCR. The RA of three of the stress indicators analyzed (Bax, PSMB5 and Bip) was significantly increased in SCNT embryos as compared with that of in vivo-derived blastocysts. No significant differences were found in the RA of HSP70 and Caspase-3 gene transcripts. This study could potentially complement morphological analyses in the development of an effective and accurate technique for the diagnosis of embryo quality, ultimately aiding to improve the efficiency of assisted reproductive techniques (ART).

Detection of recombinant human lactoferrin and lysozyme produced in a bitransgenic cow

Lactoferrin and lysozyme are 2 glycoproteins with great antimicrobial activity, being part of the nonspecific defensive system of human milk, though their use in commercial products is difficult because human milk is a limited source. Therefore, many investigations have been carried out to produce those proteins in biological systems, such as bacteria, yeasts, or plants. Mammals seem to be more suitable as expression systems for human proteins, however, especially for those that are glycosylated. In the present study, we developed a bicistronic commercial vector containing a goat β-casein promoter and an internal ribosome entry site fragment between the human lactoferrin and human lysozyme genes to allow the introduction of both genes into bovine adult fibroblasts in a single transfection. Embryos were obtained by somatic cell nuclear transfer, and, after 6 transferences to recipients, 3 pregnancies and 1 viable bitransgenic calf were obtained. The presence of the vector was confirmed by fluorescent in situ hybridization of skin cells. At 13 mo of life and after artificial induction of lactation, both recombinant proteins were found in the colostrum and milk of the bitransgenic calf. Human lactoferrin concentration in the colostrum was 0.0098 mg/mL and that in milk was 0.011 mg/mL; human lysozyme concentration in the colostrum was 0.0022 mg/mL and that in milk was 0.0024 mg/mL. The molar concentration of both human proteins revealed no differences in protein production of the internal ribosome entry site upstream and downstream protein. The enzymatic activity of lysozyme in the transgenic milk was comparable to that of human milk, being 6 and 10 times higher than that of bovine lysozyme present in milk. This work represents an important step to obtain multiple proteins or enhance single protein production by using animal pharming and fewer regulatory and antibiotic-resistant foreign sequences, allowing the design of humanized milk with added biological value for newborn nutrition and development. Transgenic animals can offer a unique opportunity to the dairy industry, providing starting materials suitable to develop specific products with high added value.

Extracellular cAMP activates molecular signalling pathways associated with sperm capacitation in bovines

STUDY QUESTION Is extracellular cAMP involved in the regulation of signalling pathways in bovine sperm capacitation? SUMMARY ANSWER Extracellular cAMP induces sperm capacitation through the activation of different signalling pathways that involve phospholipase C (PLC), PKC/ERK1-2 signalling and an increase in sperm Ca2+ levels, as well as soluble AC and cAMP/protein kinase A (PKA) signalling. WHAT IS KNOWN ALREADY In order to fertilize the oocyte, ejaculated spermatozoa must undergo a series of changes in the female reproductive tract, known as capacitation. This correlates with a number of membrane and metabolic modifications that include an increased influx of bicarbonate and Ca2+, activation of a soluble adenylyl cyclase (sAC) to produce cAMP, PKA activation, protein tyrosine phosphorylation and the development of hyperactivated motility. We previously reported that cAMP efflux by Multidrug Resistance Protein 4 (MRP4) occurs during sperm capacitation and the pharmacological blockade of this inhibits the process. Moreover, the supplementation of incubation media with cAMP abolishes the inhibition and leads to sperm capacitation, suggesting that extracellular cAMP regulates crucial signalling cascades involved in this process. STUDY DESIGN, SIZE, DURATION Bovine sperm were selected by the wool glass column method, and washed by centrifugation in BSA-Free Tyrodes Albumin Lactate Pyruvate (sp-TALP). Pellets were resuspended then diluted for each treatment. For in vitro capacitation, 10 to 15 × 106 SPZ/ml were incubated in 0.3% BSA sp-TALP at 38.5°C for 45 min under different experimental conditions. To evaluate the role of extracellular cAMP on different events associated with sperm capacitation, 10 nM cAMP was added to the incubation medium as well as different inhibitors of enzymes associated with signalling transduction pathways: U73122 (PLC inhibitor, 10 μM), Gö6983 (PKC inhibitor, 10 μM), PD98059 (ERK-1/2 inhibitor, 30 μM), H89 and KT (PKA inhibitors, 50 μM and 100 nM, respectively), KH7 (sAC inhibitor, 10 μM), BAPTA-AM (intracellular Ca2+ chelator, 50 μM), EGTA (10 μM) and Probenecid (MRPs general inhibitor, 500 μM). In addition, assays for binding to oviductal epithelial cells and IVF were carried out to test the effect of cAMP compared with other known capacitant agents such as heparin (60 μg/ml) and bicarbonate (40 mM). PARTICIPANTS/MATERIALS, SETTING, METHODS Straws of frozen bovine semen (20-25 × 106 spermatozoa/ml) were kindly provided by Las Lilas, CIALE and CIAVT Artificial Insemination Centers. The methods used in this work include western blot, immunohistochemistry, flow cytometry, computer-assisted semen analysis, live imaging of Ca2+ and fluorescence scanning. At least three independent assays with bull samples of proven fertility were carried. MAIN RESULTS AND THE ROLE OF CHANCE In the present study, we elucidate the molecular events induced by extracellular cAMP. Our results showed that external cAMP induces sperm capacitation, depending upon the action of PLC. Downstream, this enzyme increased ERK1-2 activation through PKC and elicited a rise in sperm Ca2+ levels (P < 0.01). Moreover, extracellular cAMP-induced capacitation also depended on the activity of sAC and PKA, and increased tyrosine phosphorylation, indicating that the nucleotide exerts a broad range of responses. In addition, extracellular cAMP-induced sperm hyperactivation and concomitantly increased the proportion of spermatozoa with high mitochondrial activity (P < 0.01). Finally, cAMP increased the in vitro fertilization rate compared to control conditions (P < 0.001). LARGE SCALE DATA None. LIMITATIONS, REASONS FOR CAUTION This is an in vitro study performed with bovine cryopreserved spermatozoa. Studies in other species and with fresh samples are needed to extrapolate these data. WIDER IMPLICATIONS OF THE FINDINGS These findings strongly suggest an important role of extracellular cAMP in the regulation of the signalling pathways involved in the acquisition of bull sperm fertilizing capability. The data presented here indicate that not only a rise, but also a regulation of cAMP levels is necessary to ensure sperm fertilizing ability. Thus, exclusion of the nucleotide to the extracellular space might be essential to guarantee the achievement of a cAMP tone, needed for all capacitation-associated events to take place. Moreover, the ability of cAMP to trigger such broad and complex signalling events allows us to hypothesize that cAMP is a self-produced autocrine/paracrine factor, and supports the emerging paradigm that spermatozoa do not compete but, in fact, communicate with each other. A precise understanding of the functional competence of mammalian spermatozoa is essential to generate clinical advances in the treatment of infertility and the development of novel contraceptive strategies. STUDY FUNDING AND COMPETING INTEREST(S) This work was supported by Consejo Nacional de Investigaciones Científicas y Técnicas [PIP0 496 to S.P.-M.], Agencia Nacional de Promoción Científica y Tecológica [PICT 2012-1195 and PICT2014-2325 to S.P.-M., and PICT 2013-2050 to C.D.], Boehringer Ingelheim Funds, and the Swedish Farmers Foundation [SLF-H13300339 to J.M.]. The authors declare there are no conflicts of interests.

Intensive Neonatal Care of the First Bitransgenic Bovine Clone for Human Lysozyme and Lactoferrin Production

Somatic cell nuclear transfer associated with transgenesis allows the production of animals with beneficial properties that cannot be obtained by conventional breeding programs. Unfortunately these biotechnologies are characterized by a very low efficiency being extremely important to optimize the results of each step from skin biopsy and cloning, until the birth of calf. Animals obtained by cloning have special requirements after birth that must be considered to ensure their survival capacity. Taken into account that there is not much information regarding neonatology of cloned animals, new cases represent a great challenge, and each clinical finding and treatment report should be considered of great value. In this work, we describe all facilities and medical procedures used to ensure the survival of the first bitransgenic bovine clone for human lysozyme and lactoferrin obtained by Somatic Cell Nuclear Transfer (SCNT), born under clinical patterns of Large Offspring Syndrome (LOS). We summarize all maneuvers performed from cesarean section and primary neonatal evaluation, to intensive cares such as sepsis management, internal medium evaluation and correction, and total parental nutrition over a total period of 80 days.

Reproductive Characteristics in Old and Young Subfertile Mares: Are They Really Different?

Abstract Physiological and pathological mechanisms that determine subfertility (pregnancy failure, irregular cycles, and abnormalities in reproductive tract) in old mares (OM) have being studied by many authors. However, some young mares also share this reproduction condition although no previous reports have being published. We decided to investigate reproductive parameters of young subfertile mares (YSM) in order to understand the basis of their reproductive behavior. Forty‐nine subfertile mares were classified and separated into 2 groups: YSM (3–10 years old; n = 28) and OM (13–23 years old; n = 21). Different number of cycles (1–8) was used for data analysis on the embryo recovery rate (ERR), interovulatory interval (IOI), multiple ovulation rate (MOR) and plasmatic progesterone. Embryo quality was evaluated by gene expression through RNAm analysis. Effluent samples were taken for bacteriological and cytological evaluation and endometrial biopsies were performed to evaluate the presence of inflammatory cells and endometrial progesterone receptors (PR). There was no significant differences in ERR (P = .1230) on the percentage of each embryonic stage found on the different days of flushing (P > .05); on embryo gene expression (P > .05); on MOR (P = .1218); and on plasmatic progesterone at day 8 PO (P = .1230). However, differences were found on the percentage of positive cytologies (P = .0122) and bacteriological cultures (P = .0023); the amount of mononuclear cells in biopsies (P < .05) and distribution of PR on endometrial localization. In conclusion, YSM share some physiopathological mechanisms with OM that could explain their reproductive performance’s similarities. HighlightsWe compare reproductive behavior between two age groups of subfertile mares.Young subfertile mares have similar reproductive behavior to old mares.In old mares, we found additional alterations related to uterine function which could be responsible for embryonic loss.

239 USE OF CYCLIC ADENOSINE MONOPHOSPHATE MODULATORS IN IN VITRO PRODUCTION OF BOVINE EMBRYOS

In vitro embryo production systems (IVP) try to emulate and enhance molecular events that occur in in vivo reproductive systems in order to increase, not only the number of embryos generated, but also their quality. Despite advances, IVP processes are still inefficient compared with in vivo systems. Several studies have attributed this deficiency to a lack of oocyte competence due to spontaneous premature resumption of meiotic maturation in the oocyte following the removal from its follicular environment. Therefore, our objective was to increase oocyte competence avoiding premature resumption of meiosis by using cyclic adenosine monophosphate modulators. Cumulus-oocyte complexes (COC) were obtained from ovaries of slaughterhouses, washed, and randomly allocated in 2 culture systems. Oocytes in the control group (IVM) were cultured for a period of 24 h in basal medium TCM-199 with EGF (1 µg mL–1) supplemented with rhFSH (25 mIU mL–1). Oocytes in the biphasic in vitro maturation (b-IVM) group were cultured for 2 h in a basal medium supplemented with a phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX, 500 µM), and an activator of adenylate cyclase (forskolin, 100 µM). Subsequently, COC were washed and cultured in basal medium supplemented with cilostamide (20 µM) and rhFSH (25 mIU mL–1) for 24 h. Maturation rates were analysed and IVF was performed with a dose of 1 × 106 sperm cells mL–1 in IVF-SOF medium. The presumptive zygotes were cultured in continuous-single-culture medium (Irvine) supplemented with 8 mg mL–1 of BSA until they reached the blastocyst stage. No significant differences in maturation, cleavage, and cryotolerance were observed between b-IVM and IVM groups (P > 0.05; Table 1). This study showed that b-IVM produced a significant increase in IVP compared with the control (IVM) at Days 7 and 8 (P < 0.01). Blastocyst hatching rate was significant (P < 0.05) for both treatment and day of analysis. The b-IVM group yielded an increase of 10 and 7.5% at Days 7 and 8, respectively, of IVP. The biphasic maturation showed an improvement in quality regarding the control group, in the timing analysis of production, and hatching percentages, and these results show that the use of cyclic adenosine monophosphate modulators in the oocyte maturation process enhances oocyte competence, which is reflected in increased productivity and embryo quality. We propose this treatment as an alternative to the standard protocols currently used in IVP of bovine embryos. Table 1.Effect of treatment on maturation, cleavage, and cryotolerance