Inositol hexakisphosphate kinase 1 maintains hemostasis in mice by regulating platelet polyphosphate levels.

Polyphosphate (polyP), a polymer of orthophosphate moieties released from the dense granules of activated platelets, is a procoagulant agent. Inositol pyrophosphates, another group of phosphate-rich molecules, consist of mono- and diphosphates substituted on an inositol ring. Diphosphoinositol pentakisphosphate (IP7), the most abundant inositol pyrophosphate, is synthesized on phosphorylation of inositol hexakisphosphate (IP6) by IP6 kinases, of which there are 3 mammalian isoforms (IP6K1/2/3) and a single yeast isoform. Yeast lacking IP6 kinase are devoid of polyP, suggesting a role for IP6 kinase in maintaining polyP levels. We theorized that the molecular link between IP6 kinase and polyP is conserved in mammals and investigated whether polyP-dependent platelet function is altered in IP6K1 knockout (Ip6k1(-/-)) mice. We observe a significant reduction in platelet polyP levels in Ip6k1(-/-) mice, along with slower platelet aggregation and lengthened plasma clotting time. Incorporation of polyP into fibrin clots was reduced in Ip6k1(-/-) mice, thereby altering clot ultrastructure, which was rescued on the addition of exogenous polyP. In vivo assays revealed longer tail bleeding time and resistance to thromboembolism in Ip6k1(-/-) mice. Taken together, our data suggest a novel role for IP6K1 in regulation of mammalian hemostasis via its control of platelet polyP levels.

Effect of vitrification on meiotic maturation and expression of genes in immature goat cumulus oocyte complexes.

The aim of the study was to evaluate meiotic maturation, and expression of genes coding for oocyte secreted factors (GDF9, BMP15, TGFBR1, and BPR2) and apoptosis (BCL2, BAX and P53) after vitrification of immature goat cumulus oocyte complexes (COCs) and in vitro maturation. COCs were vitrified in a solution containing ethylene glycol, dimethyl sulfoxide and sucrose using either a conventional straw (CS), open pulled straw (OPS), cryoloop (CL), hemistraw (HS) or cryotop (CT). Freshly collected COCs (Control), COCs exposed to vitrification and dilution solutions without cryopreservation (EC) and vitrified-warmed COCs were matured in vitro for 27h. The viability of vitrified-warmed COCs 2 h post warming and in vitro maturation was similar for CL, HS and CT. The proportion of oocytes that extruded a 1st polar body and reached TI/MII was significantly higher with CT and HS followed by CL, OPS and CS. Gene expression of GDF9, BMP15, BMPR2, BAX and P53 were comparable to control levels for OPS, CL, HS and CT. The gene expression pattern in CS vitrified COCs was by contrast changed in that GDF9, BMP15, TGFBR1 and BAX were up regulated and BMPR2, BCL2 and P53 down regulated. In conclusion immature goat COCs vitrified using CT and HS showed that viability, maturation rates and expression of genes coding for oocyte secreted factors and apoptosis were similar to non-vitrified controls.

Meiotic maturation of vitrified immature chousingha (Tetracerus quadricornis) oocytes recovered postmortem.

The ability to recover and cryopreserve oocytes from postmortem ovaries of endangered or wildlife species holds tremendous potential for conservation using assisted reproductive technologies. The objective of this study was to assess the in vitro meiotic maturation of chousingha (four-horned antelope) oocytes following vitrification using open pulled straw (OPS) method. The average number of oocytes recovered per ovary was 65.6. The proportion of oocytes that matured was significantly lower in vitrified oocytes (29.4%) when compared with fresh oocytes (69.3%). The study provides evidence that it is possible to cryopreserve immature oocytes by vitrification collected from the ovaries of chousingha at postmortem and also demonstrates that these cryopreserved oocytes retain their potential to undergo in vitro meiotic maturation.

Developmental competence of oocytes recovered from postmortem ovaries of the endangered Indian blackbuck (Antilope cervicapra).

The ability to rescue gametes from endangered or wildlife species and to subsequently produce viable embryos holds tremendous potential as a means to increase the population size of endangered or wildlife species. The objective of this study was to assess the meiotic and developmental competence of oocytes recovered from postmortem ovaries of the Indian blackbuck. Oocytes collected from the ovaries of dead blackbucks were allowed to mature in vitro and then tested for developmental potential by activation with ionomycin followed by treatment with 6-dimethylaminopurine. The average number of oocytes recovered per ovary was 10.9, and recovery of the oocytes did not depend on the presence or absence of the corpus luteum, on the side, size and weight of the ovaries or on the type of oocytes recovered. The proportion of good quality oocytes showing cumulus expansion and extrusion of the first polar body were 79.3% and 46.1% when cultured with gonadotropins. In vitro maturation studies indicated that the proportion of oocytes that reached MII stage was significantly higher when good quality oocytes (68%) were used compared with fair quality oocytes (48%) when cultured in the presence of gonadotropins. Furthermore, fifty eight percent of the in vitro matured oocytes cleaved, and thirteen percent of the cleaved oocytes developed into blastocysts. These findings suggest that the oocytes recovered from postmortem ovaries of the blackbuck can be utilized for production of embryos.

Meiotic maturation of oocytes recovered from the ovaries of Indian big cats at postmortem.

In order to increase the available sources of genetic material for endangered members of the great cat family, this study was designed to assess the meiotic competence of oocytes recovered from postmortem ovaries of the Indian leopard, tiger and lion. The average number of oocytes that were recovered per ovary was 11.0 ± 5.0, 11.0 ± 3.5 and 21.3 ± 8.8 for tiger, lion and leopard, respectively. The proportion of culture grade oocytes for tiger, lion and leopard were 72.7, 78.8 and 71.9%, respectively. The culture grade oocytes were matured in tissue culture medium 199 modified with sodium bicarbonate supplemented with 0.3% BSA (fatty acid-free) (w/v), 10 µg/ml FSH, 6 IU/ml LH, 1 µg/ml 17ß-estradiol, 0.36 mM pyruvate, 2.2 mM calcium lactate, 2.0 mM L-glutamine, 100 IU/ml penicillin and 0.1 mg/ml streptomycin in an incubator with 5% CO2 under humidified air at 38.5°C for 36 h. After in vitro maturation, 56.3, 53.8 and 58.7% of the tiger, lion and leopard oocytes, respectively, were matured. The proportion of oocytes that extruded first polar body was significantly higher when the oocytes were collected from the animals of less than 15 yr of age compared to above 15 yr. These findings suggest that the oocytes recovered from ovaries of tiger, lion and leopard immediately postmortem can be successfully matured to MII stage.