Porcine oocyte maturation in vitro : role of cAMP and oocyte-secreted factors: a practical approach

Polyspermy or the penetration of more than one sperm cell remains a problem during porcine in vitro fertilization (IVF). After in vitro culture of porcine zygotes, only a low percentage of blastocysts develop and their quality is inferior to that of in vivo derived blastocysts. It is unknown whether the cytoplasmic maturation of the oocyte is sufficiently sustained in current in vitro maturation (IVM) procedures. The complex interplay between oocyte and cumulus cells during IVM is a key factor in this process. By focusing on this bidirectional communication, it is possible to control the coordination of cumulus expansion, and nuclear and cytoplasmic maturation during IVM to some extent. Therefore, this review focuses on the regulatory mechanisms between oocytes and cumulus cells to further the development of new in vitro embryo production (IVP) procedures, resulting in less polyspermy and improved oocyte developmental potential. Specifically, we focused on the involvement of cAMP in maturation regulation and function of oocyte-secreted factors (OSFs) in the bidirectional regulatory loop between oocyte and cumulus cells. Our studies suggest that maintaining high cAMP levels in the oocyte during the first half of IVM sustained improved oocyte maturation, resulting in an enhanced response after IVF and cumulus matrix disassembly. Recent research indicated that the addition of OSFs during IVM enhanced the developmental competence of small follicle-derived oocytes, which was stimulated by epidermal growth factor (EGF) via developing EGF-receptor signaling

Seminal Plasma Proteins

The ejaculated semen consists of two major components viz. sperm cells (spermatozoa) and the fluid part obtained after centrifugation called seminal plasma. The spermatozoa originate from the semniferous tubule and are suspended in the seminal plasma. The seminal plasma is composed of secretions contributed by the testis, epididymis, seminal vesicles, ampullae, prostate and bulbourethral glands. About 60-80 % of the ejaculated semen of the bull originates from these sources. Seminal plasma is a highly complex biological fluid containing proteins, amino acids, enzymes, fructose and other carbohydrates, lipids, major minerals and trace elements. Seminal plasma proteins partly originates from the blood plasma by exudation through the lumen of the male genital tract and partly are synthesized and secreted by various reproductive organs and are known as seminal plasma specific proteins. Several seminal plasma proteins of blood origin viz. prealbumin, albumin, globulin, transferring, α-antitrypsin, β-lipoprotein, β-glycoprotein, orsomucoid, kininogen, Peptide hormones, IgG, IgA and IgM have been identified and characterized. These proteins are involved in regulation of osmotic pressure and pH of seminal plasma, transport of ions, lipid and hormones. A major part of seminal plasma proteins originate from the testis, epididymis, vas deference, prostate, seminal vesicle and bulbourethral glands. The biosynthesis and secretion of these proteins is regulated by testosterone levels in the blood

Cortical granule exocytosis is mediated by alpha-SNAP and N-Ethilmaleimide sensitive factor in mouse eggs

Cortical granule exocytosis (CGE), also known as cortical reaction, is a calciumregulated secretion that represents a membrane fusion process during meiotic cell division of eggs. The molecular mechanism of membrane fusion during CGE is still poorly understood and is thought to be mediated by the SNARE pathway;nevertheless, it is unkown if SNAP (acronym for soluble NSF attachment protein) and NSF (acronym for N-ethilmaleimide sensitive factor), two key proteins in the SNARE pathway, mediate CGE in any egg model. In this paper, we documented the gene expression of α-SNAP, γ-SNAP and NSF in mouse oocytes. Western blot analysis showed that the expression of these proteins maintains a similar level during oocyte maturation and egg activation. Their localization was mainly observed at the cortical region of eggs, which is enriched in cortical granules. To evaluate the function of these proteins in CGE we set up a functional assay based on the quantification of cortical granules. Endogenous α-SNAP and NSF proteins were perturbed by microinjection of recombinant proteins or antibodies prior to CGE activation. The microinjection of wild type α-SNAP and the negative mutant of α-SNAP L294A in eggs inhibited CGE stimulated by strontium. NEM, an irreversibly inhibitor of NSF, and the microinjection ofthe negative mutant NSF D1EQ inhibited cortical reaction. The microinjection of anti-α-SNAP and anti-NSF antibodies was able to abolish CGE in activated eggs. The microinjection of anti-γ SNAP antibody had no effect on CGE. Our findings indicate, for the first time in any oocyte model, that α-SNAP, γ-SNAP, and NSF are expressed in mouse oocytes. We demonstrate that α-SNAP and NSF have an active role in CGE and propose a working model.Fil: de Paola, Maria Matilde. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Cienicas Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; Argentina. Universidad Nacional de Cuyo; ArgentinaFil: Bello, Oscar Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Cienicas Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; Argentina. University of Yale; Estados UnidosFil: Michaut, Marcela Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Cienicas Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; Argentina. Universidad Nacional de Cuyo. Facultad de Ciencias Exactas y Naturales; Argentin

Influence of sperm-oocyte coincubation period on porcine in vitro fertilization (IVF) efficiency

A major obstacle for successful in vitro production of porcine embryos is the polyspermic fertilization. One possibility to reduce polyspermic penetration is decreasing the number of spermatozoa added to the fertilization medium. Unfortunately, the lower rate of polyspermy is accompanied by a reduced penetration rate. A short gamete coincubation period of 10 min has been described to obtain fertilization rates similar to 6 h of coincubation and may improve IVF efficiency (number of monospermic fertilized oocytes/total number inseminated) depending on sperm-oocyte ratio (Gil, 2007, Theriogenology, 67(3), 620–626). Here we demonstrate that the optimal coincubation period in our IVF conditions is between 10 min and 6 h. In vitro matured oocytes (n = 600) were inseminated with frozen-thawed epididymal semen with 600 spermatozoa per oocyte and coincubated for 2, 4 and 6 h. At 2 and 4 h post insemination (hpi), oocytes were vortexed and transferred to fertilization medium without spermatozoa. At 6 hpi, presumed zygotes of all groups were washed three times in culture medium and cultured. At 22 hpi, zygotes were fixed overnight and stained with Hoechst 33,342 for the assessment of fertilization and polyspermy. The IVF efficiency was higher for the 4 h group (40 ± 5%) than the 2 and 6 h group (19 ± 8% and 17 ± 5%). Between 4 and 6 h of gamete coincubation, the increase in the number of polyspermic oocytes was relatively higher than the increase in penetration rate (+39% vs. +15%), resulting in a decline in efficiency. (This study was supported by Research Foundation-Flanders)

Occurrence of a Gynandromorphic Bombus bimaculatus (Hymenoptera: Apidae) in Southeastern Ohio

Herein, we introduce the first reported case of gynandromorphy in the bumblebee Bombus bimaculatus (Cresson) (Hymenoptera: Apidae), a relatively common North American species found east of the Mississippi River. The specimen was collected in Marietta, Ohio as part of a bee diversity assessment project for Washington County. Gynanders exhibit discrete male and female characters in a single individual. We discuss the potential causes of gynandromorphy exhibited by this specimen, which has differing antennal segments (12 and 13), facial maculation, abdominal hair coloration, and the presence of a corbicula – secondary sex characters that are characteristic for the genus Bombus

Sandfish hatchery techniques

Sandfish is arguably the most commercially valuable of the tropical species of sea cucumber that are processed into bΩche-de-mer. It is widely distributed throughout the Indo-Pacific, occurring in shallow inshore areas where it is easily accessible to coastal fishers. A-grade bΩche-de-mer processed from sandfish commands some of the highest prices on the international market. But these same attributes also make it vulnerable to overexploitation. Sadly, this has happened in most places where it occurs. While sandfish was an important component of bΩche-de-mer fisheries 20 to 30 years ago, its contribution to bΩche-de-mer exports is now relatively small, even trivial.Not surprisingly, there is widespread interest in restoring the production of sandfish, especially where it promises to deliver benefits to coastal fishing communities with few other options for earning livelihoods. it promises to deliver benefits to coastal fishing communities with few other options for earning livelihoods. This manual is designed to help government agencies and members of the private sector interested in implementing ways of increasing production of sandfish by outlining the basic methods for spawning and rearing juvenile sandfish. It builds on the pioneering work done in 1988 at the Tuticorin Research Centre of CMFRI (Central Marine Fisheries Research Institute) in India and is based largely on methods developed and applied by the WorldFish Center (formerly ICLARM) in Solomon Islands, Vietnam and New Caledonia.Sea cucumber, Hatching, Rearing, Aquaculture techniques, Brook stocks, Nursery ponds, Aquaculture techniques Holothurioidea