Liver development is restored by blastocyst complementation of HHEX knockout in mice and pigs.

BACKGROUND: There are over 17,000 patients in the US waiting to receive liver transplants, and these numbers are increasing dramatically. Significant effort is being made to obtain functional hepatocytes and liver tissue that can for therapeutic use in patients. Blastocyst complementation is a challenging, innovative technology that could fundamentally change the future of organ transplantation. It requires the knockout (KO) of genes essential for cell or organ development in early stage host embryos followed by injection of donor pluripotent stem cells (PSCs) into host blastocysts to generate chimeric offspring in which progeny of the donor cells populate the open niche to develop functional tissues and organs. METHODS: The HHEX gene is necessary for proper liver development. We engineered loss of HHEX gene expression in early mouse and pig embryos and performed intraspecies blastocyst complementation of HHEX KO embryos with eGFP-labeled PSCs in order to rescue the loss of liver development. RESULTS: Loss of HHEX gene expression resulted in embryonic lethality at day 10.5 in mice and produced characteristics of lethality at day 18 in pigs, with absence of liver tissue in both species. Analyses of mouse and pig HHEX KO fetuses confirmed significant loss of liver-specific gene and protein expression. Intraspecies blastocyst complementation restored liver formation and liver-specific proteins in both mouse and pig. Livers in complemented chimeric fetuses in both species were comprised of eGFP-labeled donor-derived cells and survived beyond the previously observed time of HHEX KO embryonic lethality. CONCLUSIONS: This work demonstrates that loss of liver development in the HHEX KO can be rescued via blastocyst complementation in both mice and pigs. This complementation strategy is the first step towards generating interspecies chimeras for the goal of producing human liver cells, tissues, and potentially complete organs for clinical transplantation.

Cardiac adaptations in SCNT newborn cloned calves during the first month of life assessed by echocardiography.

The aim of this study was to compare cardiac morphology in newborn and month-old control and cloned calves. A total of 10 in vivo-derived (IVD) control (five Holstein, five Hereford) and seven cloned (five Holstein, two Hereford) calves were subjected to echocardiographic examination, including 2D, M-mode, spectral and color flow Doppler evaluation at Day 1 (mean 26.3 h) and Day 30 (mean 29.2 days) after birth. Echocardiographic measurements were compared between control and cloned calves, and between Hereford and Holstein control calves of the same age. At Day 1 and at Day 30 after birth, left ventricular free wall (LVFW) and interventricular septal (IVS) thicknesses were greater in Holstein calves than Hereford calves. Several indices of myocardial wall thickness were increased in cloned versus control calves at Day 1 after birth, and included systolic LVFW thickness, systolic right ventricular free wall (RVFW) thickness, diastolic LVFW thickness, diastolic RVFW thickness and diastolic IVS thickness (p < 0.05). Differences between cloned and non-cloned calves were no longer evident at Day 30 after birth. The apparent disappearance of the cloning effect on cardiac structures may reflect the influence of placenta on fetal cardiac morphology, suggestive of a placental hemodynamic role in fetal cardiac muscle development. Differences seen in clones at birth spontaneously resolved by Day 30 of age, after organ development recovery from cardiovascular abnormalities of presumed placental origin. Echocardiographic measurements should provide useful data for research and clinical evaluation of high-risk neonatal calves of both breeds and from clones of the same breed.

Purification of binder of sperm protein 1 (BSP1) and its effects on bovine in vitro embryo development after fertilization with ejaculated and epididymal sperm.

The present study evaluated functional aspects of binder of sperm 1 (BSP1) in the bovine species. In a first experiment, cumulus-oocyte complexes (n = 1274) were incubated with frozen-thawed ejaculated sperm (18 hours) in Fert-TALP medium containing: heparin, 10, 20, or 40 µg/mL BSP1. Heparin followed by gelatin affinity chromatography was used for purification of BSP1 from bovine seminal vesicle fluid. With ejaculated sperm, cleavage rates were similar when Fert-TALP medium was incubated with heparin (74.1 ± 2.7%), 10 µg/mL BSP1 (77.8 ± 3.1%), or 20 µg/mL BSP1 (74 ± 2.0%). Day-7 blastocyst rates were equivalent after incubations with heparin (40.8 ± 5.0%) and 10 µg/mL BSP1 (34.1 ± 4.4%), but reduced after 20 µg/mL BSP1 (22.4 ± 2.9%) and 40 µg/mL BSP1 (19.3 ± 4.1%; P < 0.05). In the second experiment, cumulus-oocyte complexes (n = 1213) were incubated with frozen-thawed cauda epididymal sperm (18 hours) in Fert-TALP medium containing: no heparin, heparin, 10, 20, or 40 µg/mL. Cleavage and blastocyst rates were similar after treatments with heparin (68.5 ± 1.3% and 24.7 ± 3.2%, respectively) or without heparin (65.5 ± 1.8% and 27.3 ± 1.6%, respectively). Cleavage was higher after treatment with any BSP1 concentrations (74.2 ± 2.7%-79.0 ± 1.1%) than without heparin (P < 0.05). Also, cleavage was better after Fert-TALP medium incubation with 40 µg/mL BSP1 (79.0 ± 1.1%) than with heparin (68.5 ± 1.3%; P < 0.05). Embryo development was higher (P < 0.05) after treatment with 20 µg/mL BSP1 (35.6 ± 2.5%) and 40 µg/mL (41.1 ± 2%) than after incubations with heparin (24.7 ± 3.2%) or without heparin (27.3 ± 1.6%). Interestingly, BSP1 did not cause reductions in blastocyst rates after fertilization with epididymal sperm, as observed with ejaculated sperm. On the basis of immunocytochemistry, there was BSP1 binding to frozen-thawed ejaculated but not to epididymal sperm. Also, anti-BSP1 reaction remained on ejaculated sperm (as expected) and appeared on epididymal sperm after incubation with purified BSP1. Acrosome reaction of ejaculated and epididymal sperm was induced after incubation with purified BSP1 as well, indicating an effect of BSP1 on capacitation. In conclusion, purified BSP1 from bull seminal vesicles was able to bind to and induce capacitation of ejaculated and epididymal sperm. Also, BSP1 added to fertilization media and allowed proper cleavage and embryo development, with the effects being modulated by previous exposure or not of spermatozoa to seminal plasma.

Solid-surface vitrification and in-straw dilution after warming of in vitro-produced bovine embryos.

Three experiments were designed to test a solid-surface vitrification system for bovine in vitro-produced embryos and to develop a simple method of in-straw dilution after warming, which can be potentially used for direct transfer in the field. Experiment 1 evaluated embryo survival rates (i.e. re-expansion and hatching) after vitrification and warming in three different solutions: VS1 (20% ethylene glycol (EG) + 20% propanediol (PROH) + 0.25 m trehalose (Tr)), VS2 (20% EG + 1M Tr) or VS3 (30% EG + 0.75 m Tr). Re-expansion and hatching rates were higher (p < 0.05) for embryos vitrified in VS3 (72.2 ± 1.9 and 58.2 ± 0.8) than VS1 (64.4 ± 0.9 and 37.2 ± 2.5) or VS2 (68.5 ± 1.5 and 49.6 ± 1.0; p < 0.05). Experiment 2 was designed to compare two methods of vitrification: glass micropipettes or solid surface, using the VS1 or VS3 solutions. No significant differences were detected between the two methods; but re-expansion and hatching rates were higher (p < 0.05) with VS3 (73.5 ± 3.1 and 47.1 ± 2.1) than VS1 (63.3 ± 3.3 and 39.7 ± 2.8). In experiment 3, embryos were vitrified by solid surface in VS1 or VS3 solutions and cryoprotectants were diluted in-straw after warming in a TCM 199, 0.25 m sucrose solution or holding media. Survival rates of embryos vitrified in VS3 did not differ between those exposed to 0.25 m sucrose (74.7 ± 1.3 and 57.2 ± 2.2) or holding (77.3 ± 1.4 and 58.0 ± 2.5) medium after warming; however, survival rates of embryos vitrified in VS1 were higher (p < 0.05) in those exposed to 0.25 m sucrose (67.7 ± 2.3 and 47.0 ± 1.7) than holding medium (54.5 ± 1.0 and 27.7 ± 3.1). In conclusion, solid-surface vitrification using simplified EG-based solutions and in-straw dilution with holding media may be a practical alternative for cryopreservation and direct transfer of in vitro-produced bovine embryos.

Fertilization rates and in vitro embryo production using sexed or non-sexed semen selected with a silane-coated silica colloid or Percoll.

The aim of this study was to evaluate sperm fertilization rates and in vitro embryo development rates for sexed and non-sexed semen selected using a silane-coated silica colloid method (Isolate) or Percoll. Frozen/thawed, sexed and unsexed semen samples from four Holstein bulls were randomly allocated to one of two different density gradient selection methods. Sperm quality (motility, concentration, morphology and membrane integrity) were evaluated and compared before and after sperm selection. Sperm motility and morphology improved (P < 0.005) after the sperm selection process with no differences between the two methods. For non-sexed semen, Percoll gradient increased the mean (± SEM) percentage of sperm recovered (57.3 ± 2.8) compared to Isolate (46.0 ± 1.8; P < 0.01). However, membrane integrity was higher after Isolate than Percoll (sexed semen: 41.0 ± 0.6 vs. 38.8 ± 0.8 and non-sexed semen 60.8 ± 1.6 vs. 58.8 ± 0.5; P < 0.05). The percentage of blastocysts produced was higher when either sexed or non-sexed semen was selected by Isolate (14.0 ± 1.0; 22.0 ± 1.1) than by Percoll (10.5 ± 1.5; 17.0 ± 2.1, respectively; P < 0.05). In summary, Isolate was a more effective method for the recovery of high quality sperm for in vitro fertilization embryo production.