Identification of a novel Xenopus laevis poly (A) binding protein

Poly (A) binding proteins are intimately implicated in controlling a number of events in mRNA metabolism from nuclear polyadenylation to cytoplasmic translation and stability. The known poly(A) binding proteins can be divided into three distinct structural groups (prototypes PABP1, PABPN1/PABP2 and Nab2p) and two functional families, showing that similar functions can be accomplished by differing structural units. This has prompted us to perform a screen for novel poly(A) binding proteins using Xenopus laevis. A novel poly(A) binding protein of 32 kDa (p32) was identified. Sequence analysis showed that p32 has about 50% identity to the known nuclear poly(A) binding proteins (PABPN1) but is more closely related to a group of mammalian proteins of unknown function. The expression of Xenopus laevis ePABP2 is restricted to early embryos. Accordingly, we propose that p32 is the founder member of a novel class of poly(A) binding proteins named ePABP2.     

Large-scale in vitro embryo production and pregnancy rates from Bos taurus, Bos indicus, and indicus-taurus dairy cows using sexed sperm

Herein we describe a large-scale commercial program for in vitro production of embryos from dairy Bos taurus, Bos indicus, and indicus-taurus donors, using sexed sperm. From 5,407 OPU, we compared the number of recovered oocytes (n = 90,086), viable oocytes (n = 64,826), and embryos produced in vitro from Gir (Bos indicus, n = 617), Holstein (Bos taurus, n = 180), 1/4 Holstein × 3/4 Gir (n = 44), and 1/2 Holstein-Gir (n = 37) crossbred cows, and the pregnancy rate of recipient cows. Viable oocytes were in vitro matured (24 h at 38.8 °C, 5% CO₂ in air) and fertilized by incubating them for 18 to 20 h with frozen-thawed sexed sperm (X-chromosome bearing) from Gir (n = 8) or Holstein (n = 7) sires (2 × 10⁶ sperm/dose). Embryos were cultured in similar conditions of temperature and atmosphere as for IVM, with variable intervals of culture (between Days 2 and 5) completed in a portable incubator. All embryos were transferred fresh, after 24 to 72 h of transportation (up to 2,000 km). On average, 16.7 ± 6.3 oocytes (mean ± SEM) were obtained per OPU procedure and 72.0% were considered viable. Total and viable oocytes per OPU procedure were 17.1 ± 4.5 and 12.1 ± 3.9 for Gir cows, 11.4 ± 3.9 and 8.0 ± 2.7 for Holstein cows, 20.4 ± 5.8 and 16.8 ± 5.0 for 1/4 Holstein × 3/4 Gir, and 31.4 ± 5.6 and 24.3 ± 4.7 for 1/2 Holstein-Gir crossbred females (P < 0.01). The mean number of embryos produced by OPU/IVF and the pregnancy rates were 3.2 (12,243/ 3,778) and 40% for Gir cows, 2.1 (2,426/1,138) and 36% for Holstein cows, 3.9 (1,033/267) and 37% for 1/4 Holstein × 3/4 Gir, and 5.5 (1,222/224), and 37% for 1/2 Holstein-Gir. In conclusion, we compared oocyte yield from two levels of indicus-taurus breeds and demonstrated the efficiency of sexed sperm for in vitro embryo production. Culturing embryos during long distance transportation was successful, with potential for international movement of embryos.     

A comparison between in vitro fertilization and microinjection of immobilized spermatozoa from bulls producing spermatozoa with defects.

The objectives of this study were to compare the fertilization rate of bovine in vitro matured oocytes by in vitro fertilization (IVF) and by microinjection of a single spermatozoon (MI) and to relate these rates with fertility reported for these bulls in artificial breeding. Bull A (Holstein) had a nonreturn rate of 75%. Semen from this bull is routinely used in our standard IVF procedure. Bull B (Ayrshire), used regularly in artificial breeding and related to bull D, had a nonreturn rate of 69.2%. Bull C (Brown Swiss), with a chromosomal translocation and trisomy, achieved a nonreturn rate of 42%. Bull D (Ayrshire) produced nonmotile spermatozoa (SPZ) and had an abnormality described as "tail stump defect." No pregnancies sired by bull D have been reported. Oocytes were either fertilized in vitro by capacitated SPZ or by microinjection of a single immobilized SPZ into the ooplasm. SPZ were treated with 0.1 microM A23187 and used for IVF. For microinjection SPZ were cocultured for 5 h with bovine oviduct epithelial cells (BOEC) and then immobilized by freezing and thawing twice without cryoprotectant. A single batch of killed SPZ (stored at -25 degrees C) was used for all microinjections. All oocytes were cultured in Medium 199 for 22 h at 39 degrees C and subsequently fixed, stained, and examined for evidence of fertilization (i.e., female and male pronucleus formation, SPZ decondensation). Fertilization rates following IVF with semen from bulls A, B, C, and D were 80%, 54%, 1%, and 2%, and following microinjection were 39%, 22%, 21%, and 34%, respectively.     

In vitro production of cattle × buffalo hybrid embryos using cattle oocytes and African buffalo (Syncerus caffer caffer) epididymal sperm

Interspecies hybridization of bovids occurs between domestic cattle and at least three other species; American bison (Bison bison), yak (Bos grunniens) and banteng (Bos banteng). Birth of a cattle × buffalo (Bubalus bubalis) hybrid has reportedly occurred in Russia and in China, but these reports were not authenticated. Such hybrids could be important in improving livestock production and management of diseases that impede production in tropical Africa. This study investigated hybridization between cattle and its closest African wild bovid relative, the African buffalo (Syncerus caffer caffer). In an attempt to produce cattle × buffalo hybrid embryos in vitro, matured cattle oocytes were subjected to a standard in vitro fertilization (IVF) procedure with either homologous cattle (n = 1166 oocytes) or heterologous African buffalo (n = 1202 oocytes) frozen-thawed epididymal sperm. After IVF, 67.2% of the oocytes inseminated with the homologous cattle sperm cleaved. In contrast, fertilization with buffalo sperm resulted in only a 4.6% cleavage rate. The cleavage intervals were also slower in hybrid embryos than in the IVF-derived cattle embryos. Of the cleaved homologous cattle embryos 52.2% progressed to the morula stage compared with 12.7% for the buffalo hybrid embryos. No hybrid embryos developed beyond the early morula stage, while 40.1% of the cleaved cattle × cattle embryos developed to the blastocyst stage. Transfer of buffalo hybrid IVF embryos to domestic cattle surrogates resulted in no pregnancies at 60 days post-transfer. This study indicates that interspecies fertilization of cattle oocytes with African buffalo epididymal sperm can occur in vitro, and that a barrier to hybridization occurs in the early stages of embryonic development. Chromosomal disparity is likely the cause of the fertilization abnormalities, abnormal development and subsequent arrest impairing the formation of hybrid embryos beyond the early morula stage. Transfer of the buffalo hybrid embryos did not rescue the embryos from development arrest.     

Injection of isolated centriolar complex to induce mitosis in starfish eggs

When centriolar complex isolated from starfish spermatozoa was injected into starfish immature oocytes (fully grown, germinal vesicle stage) followed by treating the latter with 1-methyladenine, mitotic events such as condensation and division of chromosomes of the female pronucleus and cytokinesis following completion of meiosis were observed. No cortical reaction detected in the oocytes. Essentially the same was noted for mature oocytes (pronuclear stage) into which the centriolar complex had been injected. The oocytes that had received sperm tail fraction or buffer alone did not initiate cleavage. It would thus appear that sperm centriolar complex is significantly essential to the initiation of cleavage.     

Application of a microfluidic sperm sorter to the in-vitro fertilization of porcine oocytes reduced the incidence of polyspermic penetration

The objective of this study was to use a microfluidic sperm sorter (MFSS), designed to isolate motile human spermatozoa with laminar flows (no centrifugation), for porcine IVF. Boar spermatozoa were diluted at 1 × 10⁸ cells/mL with a diluent containing 20% seminal fluid and flowed with modified TCM-199 (mM199, with 5 mM caffeine) to introduce motile sperm into the exit chamber for IVF. In Experiment 1, after flowing for 5 min, sperm concentration varied significantly among specific sites within the MFSS collecting chamber (range, 0.8 ± 0.5 × 10⁴ to 575.0 ± 56.3 × 10⁴ cells/mL; mean ± SEM). In Experiment 2, when porcine IVM oocytes were placed at three locations in the MFSS exit chamber (where only motile spermatozoa accumulated) and subsequently cultured in caffeine-free mM199 for 8 h, sperm penetration rate was not significantly different among places (86.1 ± 10.5 to 100%), but the monospermic penetration rate was lower (P < 0.05) in oocytes 3.5 mm from the exit position (12.5 ± 4.8%) than those at 7.5 mm (53.1 ± 6.0%) or further (41.9 ± 2.8%) from the exit. In Experiment 3, the normal fertilization index (ratio of monospermic oocytes to number of oocytes examined) 8 h after insemination was higher (P < 0.05) in the MFSS-IVF system (0.375 ± 0.040) than both standard IVF and transient IVF (0.222 ± 0.028 and 0.189 ± 0.027, respectively, with co-culture for 8 h and for 5 min). Developmental competence of fertilized oocytes (blastocyst formation) was higher (P < 0.05) in the MFSS-IVF system (40.9 ± 2.3%) than in either standard or transient IVF (22.6 ± 1.4 and 33.7 ± 3.5%). In conclusion, brief co-culture of porcine oocytes with spermatozoa gradually accumulated in the MFSS chamber improved the efficiency of producing monospermic fertilized embryos and blastocysts. Furthermore, efficiencies were significantly affected by oocyte location within the chamber.     

Expression of Bcl-2 and Bax proteins in relation to quality of bovine oocytes and embryos produced in vitro

The mechanisms underlying the visual assessment and selection of immature oocytes resulting in optimum embryonic development following in vitro maturation, fertilization and culture (in vitro maturation (IVM)/in vitro fertilization (IVF)/in vitro embryo culture (IVC)) are unknown. Also, the reasons for the more frequent occurrence of cytoplasmic fragmentation in in vitro produced bovine embryos, resulting in poor survival following cryopreservation and decreased pregnancy rates following embryo transfer are not clear. The objectives of this study are: (1) to investigate whether differences in the quality of immature oocytes and embryo fragmentation are associated with apoptosis; and (2) to study the pattern of Bcl-2 and Bax expression in oocytes and embryos to help elucidate their potential roles in the regulation of apoptosis during development. Bovine oocytes were obtained from slaughterhouse ovaries and divided into four grades (grades I–IV) based on their morphology. Oocytes of different grades were cultured in serum-free medium for 48 h. Embryos were produced only from grade I oocytes (highest quality) via IVM, IVF and IVC procedures. The morphological analysis of apoptosis in oocytes and embryos was carried out using propidium iodide staining and terminal deoxynucleotidyl transferase mediated dUTP nick end labeling. The expression of Bcl-2 and Bax in oocytes and embryos of different qualities and stages was determined using western blotting. The results showed that the number of morphologically abnormal oocytes with shrinkage and/or fragmentation of the ooplasm, which are typical features of apoptosis, was significantly higher in grade IV oocytes (denuded oocytes, the lowest quality) than in grade I oocytes after 48 h in vitro culture (P<0.05). DNA fragmentation, a hallmark of the biochemical changes seen in apoptotic cell death, was observed in morphologically fragmented oocytes and embryos. The expression of Bcl-2 was high in good quality oocytes and embryos, low in fragmented embryos, and hardly detectable in denuded oocytes. In contrast, the expression of Bax was found in all types of oocytes and embryos with the highest expression in the denuded oocytes. This implies that the ratio of Bcl-2 to Bax may be used to gauge the tendency of oocytes and embryos towards either survival or apoptosis. Overall, our results show that apoptosis appears to be an underlying mechanism of bovine oocyte degeneration and embryo fragmentation. Interactions between the Bcl-2 family of proteins may play a critical role in pre-implantation embryo development. These findings could have important implications for improving IVF and related techniques.     

Differing sperm ability to penetrate the oocyte in vivo and in vitro as revealed using colloidal preparations

The penetration ability of boar (Sus scrofa domestica) spermatozoa exposed to viscous preparations under in vivo and in vitro fertilization conditions has been examined. Experiments involving induced ovulation in prepubertal animals and surgical insemination directly into the oviduct isthmus revealed an advantage of colloidal preparations. Based on within-animal comparisons, the incidence of penetration was 100% using both spermatozoa suspended in a viscous preparation of plant extracts and spermatozoa suspended in a control medium. However, percentages of monospermy were 22.2% in 54 oocytes inseminated with the control suspension compared with 62.5% in 48 oocytes inseminated with the colloidal preparation. An in vitro study involving 355 oocytes from slaughterhouse ovaries inseminated with in vitro-capacitated spermatozoa gave similar percentages of penetrated oocytes for both the control and colloidal suspensions. In this case, however, the percentage of monospermy was 32.7% in the control group compared with 10.6% for spermatozoa suspended in the colloidal preparation. Higher mean numbers of sperm inside the oocytes and higher numbers of sperm bound to the zona pellucida were also observed with the colloidal suspensions. In vitro motility and viability for spermatozoa in the colloidal suspensions were enhanced compared with that of the control group. Lower sperm membrane lipid disorder and reactive oxygen species generation were also observed in the viscous solution. These findings suggest that viscous fluids can enhance the ability of sperm to move, bind, and penetrate the oocyte in vitro, although this influence may be masked in vivo due to the already high viscosity in the oviductal fluid close to the time of ovulation.     

Effect of bovine viral diarrhoea virus biotypes on adherence of sperm to oocytes during in-vitro fertilization in cattle

Bovine viral diarrhoea virus (BVDV), a member of the Pestivirus genus, is one of the most important pathogens of dairy cattle; it can cause several clinical syndromes, ranging from subclinical to severe disease. The objectives of the current studies were to assess the effects of two biotypes of BVDV on sperm attachment to the zona pellucida (ZP) of oocytes and on fertilization rate in bovine in vitro fertilization (IVF). In two experiments, sperm at two concentrations (10⁵ and 10⁶/mL) and oocytes were incubated with 10⁶ TCID₅₀/mL cythopatic (CP) or noncythopatic (NCP) BVDV. In the first experiment, with the lower sperm concentration (10⁵/mL), male and female gametes were infected with CP or NCP BVDV, whereas in the second experiment, the sperm concentration was 10⁶/mL, and sperm and oocytes were also infected with CP or NCP BVDV. The number of sperm attached to the ZP and the fertilization rate were evaluated with fluorescence microscopy on the ZP of fertile and infertile oocytes. In the first experiment, compared to the control group (n = 97), oocytes infected with CP BVDV and incubated at the lower (10⁵/mL) sperm concentration positively affected sperm attachment (n = 123) to the ZP of fertile oocytes (P < 0.05). In comparison with the control group (n = 115), sperm infected with CP BVDV negatively affected sperm binding (n = 93) to the ZP of infertile oocytes (P < 0.05). In the second experiment (10⁶ sperm/mL), for both fertile and infertile oocyte groups, sperm attachment in the control group was very high and deemed uncountable. However, in treated groups, the number of sperm attached to the ZP was countable. Only sperm infected with CP BVDV negatively affected sperm binding capacity (n = 81) to the ZP of fertile oocytes (P < 0.05). Although CP and NCP BVDV significantly reduced the fertilization rate of oocytes incubated with a higher sperm concentration, with the lower sperm concentration, only NCP BVDV significantly diminished fertilization rate with contaminated sperm and oocytes (P < 0.05). In conclusion, this study supported the detrimental impacts of sperm or ooctyes infected with CP or NCP BVDV on sperm attachment to the ZP of bovine oocytes and on fertilization rate during bovine IVF.     

Cryogenic changes in proteases and antiprotease activities of buffalo (Bubalus bubalis) and cattle (Bos taurus) semen

The postthaw motility and fertility of buffalo and cattle semen is reduced when they are cryopreserved for artificial insemination. In the present study, an attempt was made to characterize the cryogenic changes in proteases and antiprotease activities (APA) of buffalo and cattle semen because these proteolysis regulators have been reported to be associated with sperm motility and fertility. Buffalo sperm demonstrated at least two major proteases of 45 and 42 kDa and three minor proteases of 95, 52, and 33 kDa. Similarly, cattle sperm demonstrated three major proteases of 62, 45, and 42 kDa and two minor proteases of 85 and 78 kDa. Buffalo seminal plasma demonstrated at least three major proteases of 78, 68, and 62 kDa and one minor protease of 98 kDa and cattle seminal plasma demonstrated one major protease of 68 kDa and two minor proteases of 78 and 75 kDa. Except for the 45 kDa protease, most of the previously mentioned proteases were found to be metalloproteinases. Compared with fresh sperm, cryopreserved buffalo and cattle sperm demonstrated a major protease band of 52/49 kDa and the activity of this protease reduced progressively with the duration of cryopreservation. On the contrary, compared with the fresh seminal plasma, cryopreserved buffalo and cattle semen extenders displayed the presence of a new protease band of 45 kDa and demonstrated that this protease activity was leaked from buffalo and cattle cryopreserved spermatozoa. Buffalo and cattle seminal plasmas displayed at least two major APA of 86 and 26 kDa. Compared with buffalo, cattle seminal plasma demonstrated significantly greater APA. Thus, the present study demonstrated the presence of an array of proteases and APA in buffalo and cattle semen and the activities of which changed during cryopreservation. The leakage of the specific protease activity and changes in the proteases and APA might be attributed to reduced motility and fertility of cryopreserved semen in these species.     

Capacitation-associated protein tyrosine phosphorylation starts early in buffalo (Bubalus bubalis) spermatozoa as compared to cattle

A comparative study was conducted on protein tyrosine phosphorylation events of capacitating sperm of two ruminant species, cattle and buffalo. Ejaculated cattle and buffalo bull spermatozoa were suspended separately in sp-TALP medium at 50 × 10⁶ mL⁻¹ and incubated at 38.5 °C with 5% CO₂ in air in the absence or presence of heparin for a period of 6 h. The extent of sperm capacitation after various periods of incubations was assessed by lysophosphatidyl choline-induced acrosome reaction followed by a triple-staining technique and capacitation-associated tyrosine-phosphorylated proteins were detected by immunoblotting technique using a monoclonal antiphosphotyrosine antibody. In the same media, over a time-period, a significant increase in capacitation percentage was observed even in control group of buffalo spermatozoa as compared to a non-significant increase in that of cattle sperm. In both cattle and buffalo spermatozoa, at 6 h, four proteins of molecular weight 49, 45, 32, and 20 kDa (designated as p49, p45, p32, and p20) were tyrosine phosphorylated. However, in buffalo, two additional proteins of 38 and 30 kDa were also tyrosine phosphorylated. In a time-course study, p20 appeared as early as at 0 h in capacitated buffalo spermatozoa as compared to 4 h in cattle. Further, in heparin-treated buffalo spermatozoa, with a time-dependent increase in tyrosine phosphorylation of some proteins, there was time-dependent dephosphorylation of some other proteins that was never seen in heparin-treated cattle sperm. Thus, the present findings revealed that though buffalo sperm takes more time than cattle for capacitation but its associated protein tyrosine phosphorylation event starts very early as compared to cattle.     

Effect of reactive oxygen species on capacitation and associated protein tyrosine phosphorylation in buffalo (Bubalus bubalis) spermatozoa

In the present study, the effect of two particular reactive oxygen species (ROS), superoxide anion (O(2)(-)) and hydrogen peroxide (H(2)O(2)) on buffalo (Bubalus bubalis) sperm capacitation and associated protein tyrosine phosphorylation was studied. Ejaculated buffalo spermatozoa were suspended in sp-TALP medium at 50 x 10(6)/mL and incubated at 38.5 degrees C for 6h with or without heparin (10(g/mL; a positive control), or xanthine (X; 0.5mM)-xanthine oxidase (XO; 0.05 U/mL)-catalase (C; 2100 U/mL) system that generates O(2)(-) or NADPH (5mM) that stimulates the endogenous O(2)(-) production or H(2)O(2) (50 microM). The specific effect of O(2)(-), H(2)O(2) and NADPH on buffalo sperm capacitation and protein tyrosine phosphorylation was assessed by the addition of superoxide dismutase (SOD), catalase and diphenylene iodonium (DPI), respectively, to the incubation medium. Each of X+XO+C system, NADPH and H(2)O(2) induced a significantly higher percentage (P<0.05) of capacitation in buffalo spermatozoa compared to control. However, DPI inhibited this NADPH-induced capacitation and protein tyrosine phosphorylation and suggested for existence of an oxidase in buffalo spermatozoa. Using immunoblotting technique, at least seven tyrosine-phosphorylated proteins (20, 32, 38, 45, 49, 78 and 95 kDa) were detected in capacitated buffalo spermatozoa. Out of these, the tyrosine phosphorylation of p95 was induced extensively by both O(2)(-) as well as exogenous source of H(2)O(2) and using specific activators and inhibitors of signaling pathways, it was found this induction was regulated through a cAMP-dependent PKA pathway. Further, immunofluorescent localization study revealed that these ROS-induced tyrosine-phosphorylated proteins are mostly distributed in the midpiece and principal piece regions of the flagellum of capacitated spermatozoa and suggested for increased molecular activity in flagellum during capacitation. Thus, the study revealed that both O(2)(-) and H(2)O(2) promote capacitation and associated protein tyrosine phosphorylation in buffalo spermatozoa and unlike human and bovine, a different subset of sperm proteins were tyrosine-phosphorylated during heparin- and ROS-induced capacitation and regulation of these ROS-induced processes were mediated through a cAMP/PKA signaling pathway.     

NuMA expression and function in mouse oocytes and early embryos

Nuclear mitotic apparatus protein (NuMA), originally described as a nuclear protein, is an essential component in the formation and maintenance of mitotic spindle poles. In this study, we analyze the expression pattern and function of NuMA in mouse oocytes and early embryos. In germinal vesicle-stage occytes, NuMA was detected both at the centrosome and in the nucleus. However, after nuclear maturation and extrusion of the first polar body, NuMA was concentrated at the broad meiotic spindle poles and at cytasters (centers of cytoplasmic microtubule asters) of mature metaphase II oocytes. Cold-induced depolymerization of microtubules appeared to disassociate NuMA foci from the cytoplasmic cytasters. During fertilization, NuMA was relocated into the reformed male and female pronuclei. Microinjection of anti-NuMA antibody into 1 of 2 cells of 2-cell-stage embryos inhibited normal cell division. These results suggest that NuMA might play an important role in cell division during early embryonic mitosis.