Preparation of monospecific anti-PAG antibodies for cattle pregnancy detection: use of synthetic peptides to improve specificity

Immunological methods involving pregnancy associated glycoproteins (PAG) are used for cattle pregnancy detection. The faults of these methods could be overcomed by using antibodies specific for each member of the PAG family. In order to differentiate between very similar proteins, preparation of antibodies specific for peptides is a method of choice. In this work, we summarize a series of considerations regarding peptide design and choose free access NCBI, Antigenicity Plot, EMBOSS Antigenic and Expasy tools to apply them. We design peptides specific for different reported PAG members and obtain the corresponding polyclonal antibodies for five of them.Fil: Ruiz Álvarez, Jimena Inés. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario (IBR-CONICET); ArgentinaFil: Teijeiro, Juan Manuel. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario (IBR-CONICET); ArgentinaFil: Marini, Patricia Estela. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario (IBR-CONICET); Argentina Borrar seleccionado

Improved farrowing rate using intrauterine insemination in sows

Intrauterine insemination (IUI), a technique that uses a lower number of spermatozoa than conventional artificial insemination (CAI), could contribute to improve reproductive efficiency of boars. However, since some field trial reports show suboptimal performance for IUI, it is necessary to continue evaluating and standardizing this technique. In this work, the use of fixed reduced sperm amounts and doses volumes for IUI respect to CAI using the same semen samples was assessed. The results show an increase in the farrowing rate using IUI vs CAI (84.80 ± 0.36 vs 71.44 ± 2.63, P<0.05). Parameters such as litter size, live piglets/litter, stillborn or mummified fetuses were analyzed as well and showed non-significant differences between techniques. Statistical positive correlation analyses showed a positive correlation between live piglets/litter and stillborn piglets and between stillborn and total number of piglets, only for CAI. In addition, the economic analysis showed a positive impact on the productivity of the farm, and possibly of the region, by lowering costs using IUI instead of CAI. In conclusion, the intrauterine insemination had a positive impact on the reproductive performance and on the economic parameters of porcine production.Fil: Cane, Fernando. Medax; ArgentinaFil: Pereyra, Norma. Medax; ArgentinaFil: Cane, Valentina. Medax; ArgentinaFil: Marini, Patricia Estela. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Laboratorio de Medicina Reproductiva; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Teijeiro, Juan Manuel. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Laboratorio de Medicina Reproductiva; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; Argentin

Use of Assisted Reproduction Techniques in Male Pathologies: Molecule Based Sperm Selection Methods

Infertility is a widespread problem that is found in over 15% of couples. In 50 % of the cases, the male factor is the key. In order to improve pregnancy rates in assisted reproductive techniques (ART), new methods to select sperm subpopulations are required. These methods are under constant evaluation, and their possible genetic and epigenetic effects on the offspring must be considered. New concepts such as DNA fragmentation and sperm apoptosis are being introduced in andrology and are considered to develop new techniques. Recently, molecule-based methods have been developed for the purpose of overcoming deficiencies in classical selection methods that involve sperm morphology and movement. One of these methods is physiologic intracytoplasmic sperm injection (PICSI), which selects spermatozoa through their property to bind to hyaluronic acid, a component of the cell-oocyte complexes that acts as a natural selector of spermatozoa. But the top rated method is based on the property of the protein annex in A5 (ANXA V) to bind to phosphatidylserine (PS), a marker of apoptosis. Magnetic-Activated Cell Sorting (MACS) conjugated with ANXA V (MACS-ANXA V) is used to remove apoptotic sperm with exposed PS from a semen sample. The use of MACS-ANX V seems to produce good quality sperm when it is evaluated by in vitro assays. But when MACS-ANXA V is used previous to ART, controversies persist regarding the benefits of sperm selection in reproductive outcomes. In this review, we consider the use of MACS-ANXA V, particularly in cases of male pathologies.Fil: Marini, Patricia Estela. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Laboratorio de Medicina Reproductiva; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Munuce, María José. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Laboratorio de Medicina Reproductiva; ArgentinaFil: Caille, Adriana María. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Laboratorio de Medicina Reproductiva; ArgentinaFil: Zumoffen, Carlos María. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Laboratorio de Medicina Reproductiva; ArgentinaFil: Teijeiro, Juan Manuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Laboratorio de Medicina Reproductiva; Argentin

Deleted in malignant brain tumor 1 (DMBT1) expression pattern in normal cervix and at different stages of squamous intraepithelial lesions

Background: Cervical cancer is one of the most frequently occurring malignancies in women worldwide, with high mortality rates. Cervical Squamous Cell Carcinoma (SCC) presents previous states of non-invasive precursor lesions, and early stage Low-Grade Squamous Intraepithelial Lesions (LSIL) regress to normal or Atypical Squamous Cells of Undetermined Significance (ASCUS) in approximately 50% of cases. Deleted in Malignant Brain Tumors 1 (DMBT1) is a tumour suppression glycoprotein, which absence is considered a malignancy marker in many epithelial cancers. Objective: To analyse DMBT1’s presence and localization in SCC and precursor lesions. Method: Immunohistochemistry for DMBT1 was performed in cervix biopsies classified as normal, LSIL, HSIL and SCC. Results: DMBT1 was detected at the supranuclear and sometimes infranuclear regions of the endocervix monolayer epithelial cells in normal and HSIL biopsies. In LSIL samples the detection of DMBT1 in endocervix was variable between patients. Also variable was DMBT1 staining in cells of glandular epithelium. The glycoprotein was not detected in the stratified epithelium of the exocervix, regardless of the lesion grade; nor in dysplastic cells. Conclusion: The absence of DMBT1 from endocervix only in some samples of LSIL is promising as a candidate for possible lesion regression potential marker.Fil: Valero, Andrés. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; ArgentinaFil: Roldán, María Lorena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Ruiz, Maria Fernanda. Universidad Nacional de Rosario. Facultad de Ciencias Médicas; ArgentinaFil: Teijeiro, Juan Manuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnol.conicet - Rosario. Unidad de Direccion; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; ArgentinaFil: Marquez, Susana Beatriz. Universidad Nacional de Rosario. Facultad de Ciencias Médicas; ArgentinaFil: Marini, Patricia Estela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentin

First evidence of the interaction between deleted in malignant brain tumor 1 and galectin-3 in the mammalian oviduct

The oviduct supports the transport and final maturation of gametes, and harbors fertilization and early embryo development. The oviductal epithelium is responsible for providing the correct environment for these processes. Deleted in malignant brain tumor 1 (DMBT1) is expressed by multiple organisms and several cell types, and the interaction of the rabbit ortholog of DMBT1 with galectin-3 (gal-3) modulates the polarity of epithelial cells. This interaction has not yet been shown in locations other than rabbit kidney and human-cultured endothelial cells. DMBT1 and gal-3 also protect epithelial layers from pathogens and trauma, and are innate immunity components. DMBT1 has been detected in the porcine oviduct, and gal-3 has been reported in the Fallopian tube and in the cow oviduct. Interaction between both proteins would show a probable physiological function in the female reproductive tract. This work describes the presence and co-localization of DMBT1 and gal-3 mainly in the apical region of the epithelial cells of the Fallopian tube and the porcine oviduct, and co-immunoprecipitation in membrane-enriched epithelial cell extracts from the porcine oviduct. The findings strongly support a functional interaction in the mammalian oviduct, suggestive of a role on epithelial protection and homeostasis, which might be related to epithelium–gamete interaction.Fil: Roldán, María Lorena. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario (IBR-CONICET); Argentina.Fil: Marini, Patricia Estela. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario (IBR-CONICET); Argentina

Annexin A2 and S100A10 in the mammalian oviduct

In many mammals, upon entry into the female reproductive tract, a subpopulation of sperm is stored in the oviduct forming a functional reservoir. In the oviducts of pig and cow, Annexin A2 (AnxA2) has been linked to the binding of sperm. This protein may exist as a monomer or bound to S100A10 and both forms are associated with different biological functions. S100A10 has not yet been reported in the oviduct. The objective of this work is to analyze for the presence of S100A10 in the oviduct and to advance the study of AnxA2 and S100A10 in this organ. This work shows the presence of both proteins, AnxA2 and S100A10, in the oviduct of human, pig, cow, cat, dog and rabbit. At least in pig, AnxA2 is found devoid of S100A10 in the outer surface of the apical plasma membrane of oviductal epithelial cells, indicating that it binds to sperm as a monomer or in association with proteins different from S100A10. In the apical cytoplasm of pig oviductal epithelial cells, AnxA2 is associated with S100A10. In primary culture of porcine oviductal cells, the expression of ANXA2 is increased by progesterone, while the expression of S100A10 is increased by progesterone and estradiol. The widespread detection of both proteins in the oviduct of mammals indicates a probable conserved function in this organ. In summary, S100A10 and AnxA2 are widespread in the mammalian oviduct but AnxA2 binds sperm in vivo devoid of S100A10 and may be related to reservoir formation.Fil: Teijeiro, Juan Manuel. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Laboratorio de Medicina Reproductiva (UNR-CONICET); ArgentinaFil: Roldán, María Lorena. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario (UNR-CONICET); ArgentinaFil: Marini, Patricia Estela. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Laboratorio de Medicina Reproductiva (UNR-CONICET); ArgentinaFil: Marini, Patricia Estela. Universidad Nacional de Rosario. Consejo de Investigaciones; ArgentinaFil: Marini, Patricia Estela. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario (UNR-CONICET); Argentin

Hormone-regulated PKA activity in porcine oviductal epithelial cells

The oviduct is a dynamic organ that suffers changes during the oestrous cycle and modulates gamete and embryo physiology. We analyse the possible existence of Protein kinase A (PKA)-dependent hormone-regulated pathways in porcine ampulla and primary cell cultures by 2D-electrophoresis/Western blot using anti-phospho PKA substrate antibodies. Differential phosphorylation was observed for ten proteins that were identified by mass spectrometry. The results were validated for five of the proteins: Annexin A5, Calumenin, Glyoxalase I and II and Enolase I. Immunofluorescence analyses show that Calumenin, Glyoxalase II and Enolase I change their localisation in the oviductal epithelium through the oestrus cycle. The results demonstrate the existence of PKA hormone-regulated pathways in the ampulla epithelium during the oestrus cycle.Fil: Teijeiro, Juan Manuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Laboratorio de Medicina Reproductiva; ArgentinaFil: Marini, Patricia Estela. Consejo Nacional de Investigaciones Científicas y Técnicas Centro Científico Tecnológico - CONICET -Rosario. Instituto de Biologia Molecular y Celular de Rosario; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Laboratorio de Medicina Reproductiva; Argentina. Universidad Nacional de Rosario. Consejo de Investigaciones de la Universidad de Rosario; Argentin

S100A7 in the Fallopian tube: a comparative study

The oviduct is a dynamic organ in which final gamete maturation, fertilization and early embryo development take place. It is considered to be a sterile site; however the mechanism for sterility maintenance is still unknown. S100A7 is an anti-microbial peptide that has been reported in human reproductive tissues such as prostate, testicle, ovary, normal cervical epithelium and sperm. The current work reports the presence of S100A7 in the Fallopian tube and its localization at the apical surface of epithelial cells. For comparison, porcine S100A7 was used for antibody development and search for peptide in reproductive tissues. Although present in boar seminal vesicles and seminal plasma, S100A7 was not detected on female porcine organs. Also, in contrast with the human protein, porcine S100A7 did not show anti-microbial activity under the conditions tested. Phylogenetic analyses showed high divergence of porcine S100A7 from human, primate, bovine, ovine and equine sequences, being the murine sequence at a most distant branch. The differences in sequence homology, Escherichia coli-cidal activity, detectable presence and localization of S100A7 from human and pig, suggest that there are possible different functions in each organism.Fil: Teijeiro, Juan Manuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Marini, Patricia Estela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentin

Use of Annexin V based Sperm Selection in Assisted Reproduction

Innovative methods to select sperm subpopulations with the best fertilizing ability are needed in assisted reproductive techniques (ART) in order to improve fertilization and pregnancy rates, while also considering possible epigenetic effects on the offspring. Molecular based selection methods are searched for, under the premise that they could be an improvement over classical selection by morphology and movement. One of these methods sustains the elimination of sperm that can bind to annexin A5 (ANX V), coupled to paramagnetic beads, through the phosphatidyl-serine exposed on their membranes upon apoptosis. Although reports accumulate about the use of this method, controversy persists as to the benefits of ANX V based sperm selection in ART. In this review we consider the arguments in favour and against this method and conclude that to the moment the evidence does not support MACS regular use in ART.Fil: Munuce, María José. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Laboratorio de Medicina Reproductiva; Argentina.Fil: Teijeiro, Juan Manuel. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Laboratorio de Medicina Reproductiva; Argentina.Fil: Caille, Adriana María. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Laboratorio de Medicina Reproductiva; Argentina.Fil: Zumoffen, Carlos. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Laboratorio de Medicina Reproductiva; Argentina.Fil: Marini, Patricia Estela. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Laboratorio de Medicina Reproductiva; Argentina

Use of Assisted Reproduction Techniques in Male Pathologies: Molecule Based Sperm Selection Methods

Infertility is a widespread problem that is found in over 15% of couples. In 50 % of the cases, the male factor is the key. In order to improve pregnancy rates in assisted reproductive techniques (ART), new methods to select sperm subpopulations are required. These methods are under constant evaluation, and their possible genetic and epigenetic effects on the offspring must be considered. New concepts such as DNA fragmentation and sperm apoptosis are being introduced in andrology and are considered to develop new techniques. Recently, molecule-based methods have been developed for the purpose of overcoming deficiencies in classical selection methods that involve sperm morphology and movement. One of these methods is physiologic intracytoplasmic sperm injection (PICSI), which selects spermatozoa through their property to bind to hyaluronic acid, a component of the cell-oocyte complexes that acts as a natural selector of spermatozoa. But the top rated method is based on the property of the protein annex in A5 (ANXA V) to bind to phosphatidylserine (PS), a marker of apoptosis. Magnetic-Activated Cell Sorting (MACS) conjugated with ANXA V (MACS-ANXA V) is used to remove apoptotic sperm with exposed PS from a semen sample. The use of MACS-ANX V seems to produce good quality sperm when it is evaluated by in vitro assays. But when MACS-ANXA V is used previous to ART, controversies persist regarding the benefits of sperm selection in reproductive outcomes. In this review, we consider the use of MACS-ANXA V, particularly in cases of male pathologies.Fil: Teijeiro, Juan Manuel. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Laboratorio de Medicina Reproductiva; Argentina.Fil: Zumoffen, Carlos. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Laboratorio de Medicina Reproductiva; Argentina.Fil: Caille, Adriana María. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Laboratorio de Medicina Reproductiva; Argentina.Fil: Munuce, María José. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Laboratorio de Medicina Reproductiva; Argentina.Fil: Marini, Patricia Estela. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Laboratorio de Medicina Reproductiva; Argentina.Fil: Marini, Patricia Estela. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina.Fil: Teijeiro, Juan Manuel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina.Fil: Teijeiro, Juan Manuel. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina