A PCR-amplified transgene fragment flanked by a single copy of a truncated inverted terminal repeat for recombinant adeno-associated virus production prevents unnecessary plasmid DNA packaging.

The application of recombinant adeno-associated viruses (rAAVs) for gene therapy faces certain challenges, including genome packaging of non-vector sequences. Inverted terminal repeats (ITRs) flanking the rAAV genome, comprising three inverted repeat regions (A, B, and C) and a non-inverted repeat region (D), contribute to non-vector genome packaging. We aimed to circumvent this issue by comparing the properties of rAAV containing DNA plasmids and PCR-amplified transgenes, including a single copy of the AD sequence (rAAV-pAD/L-AD, respectively), which is a truncated form of ITR, with those of wild-type ITR genome (single-stranded and self-complementary AAV; ssAAV and scAAV). The packaging efficiency of rAAV-pAD/L-AD was found to be comparable to that of scAAV, whereas the transduction efficiency of rAAV-pAD/L-AD was lower than that of ss/scAAV. Remarkably, rAAV-L-AD reduced the plasmid backbone packaging contamination compared to ss/scAAV. Furthermore, to confirm the functionality of this system, we generated a rAAV-L-AD harboring a short hairpin RNA targeting ATP5B (rAAV-L-AD-shATP5B) and found that it caused a significant decrease in ATP5B mRNA levels when transduced into HEK293EB cells, suggesting that it was functional. Thus, our system successfully packaged L-AD into capsids with minimal contamination of plasmid DNA, offering a novel functional packaging platform without causing plasmid backbone encapsidation.

Improved transduction of canine X-linked muscular dystrophy with rAAV9-microdystrophin via multipotent MSC pretreatment.

Duchenne muscular dystrophy (DMD) is a severe congenital disease associated with mutation of the dystrophin gene. Supplementation of dystrophin using recombinant adeno-associated virus (rAAV) has promise as a treatment for DMD, although vector-related general toxicities, such as liver injury, neurotoxicity, and germline transmission, have been suggested in association with the systemic delivery of high doses of rAAV. Here, we treated normal or dystrophic dogs with rAAV9 transduction in conjunction with multipotent mesenchymal stromal cell (MSC) injection to investigate the therapeutic effects of an rAAV expressing microdystrophin (µDys) under conditions of immune modulation. Bone-marrow-derived MSCs, rAAV-CMV-µDys, and a rAAV-CAG-luciferase (Luc) were injected into the jugular vein of a young dystrophic dog to induce systemic expression of µDys. One week after the first injection, the dog received a second intravenous injection of MSCs, and on the following day, rAAV was intravenously injected into the same dog. Systemic injection of rAAV9 with MSCs pretreatment improves gene transfer into normal and dystrophic dogs. Dystrophic phenotypes significantly improved in the rAAV-µDys-injected dystrophic dog, suggesting that an improved rAAV-µDys treatment including immune modulation induces successful long-term transgene expression to improve dystrophic phenotypes.

TDP-43 transports ribosomal protein mRNA to regulate axonal local translation in neuronal axons.

Mislocalization and abnormal deposition of TDP-43 into the cytoplasm (TDP-43 proteinopathy) is a hallmark in neurons of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). However, the pathogenic mechanism of the diseases linked to TDP-43 is largely unknown. We hypothesized that the failure of mRNA transport to neuronal axons by TDP-43 may contribute to neurodegeneration in ALS and FTLD, and sought to examine the function of TDP-43 by identifying its target mRNA for axonal transport. We found that mRNAs related to translational function including ribosomal proteins (RPs) were decreased by shRNA-based TDP-43 knock-down in neurites of cortical neurons. TDP-43 binds to and transports the RP mRNAs through their 5' untranslated region, which contains a common 5' terminal oligopyrimidine tract motif and a downstream GC-rich region. We showed by employing in vitro and in vivo models that the RP mRNAs were translated and incorporated into native ribosomes locally in axons to maintain functionality of axonal ribosomes, which is required for local protein synthesis in response to stimulation and stress to axons. We also found that RP mRNAs were reduced in the pyramidal tract of sporadic ALS cases harboring TDP-43 pathology. Our results elucidated a novel function of TDP-43 to control transport of RP mRNAs and local translation by ribosomes to maintain morphological integrity of neuronal axons, and proved the influence of this function of TDP-43 on neurodegeneration in ALS and FTLD associated with TDP-43 proteinopathy.

rAAV8 and rAAV9-Mediated Long-Term Muscle Transduction with Tacrolimus (FK506) in Non-Human Primates.

To establish an efficient, safe immunosuppressive regimen of adeno-associated vector (AAV)-mediated gene therapy for Duchenne muscular dystrophy (DMD), we evaluated the effect of tacrolimus (FK506) on skeletal muscle transduction with AAV8 and AAV9 vectors expressing the LacZ and microdystrophin (M3) genes labeled by FLAG. We utilized 3- to 4-year-old Macaca fascicularis, screened for neutralizing antibodies against AAV. 3 days before AAV injection and throughout the experiment, 0.06 mg/kg tacrolimus was intravenously administered. A viral suspension of 1 × 1013 viral genomes/muscle was intramuscularly injected bilaterally at the tibialis anterior and biceps brachii muscles, which were biopsied at 8, 16, 24, and 42 weeks after injection. Without tacrolimus, AAV8- and AAV9-mediated LacZ expression disappeared 8 and 16 weeks after transduction, respectively. With tacrolimus, AAV8/9-mediated LacZ expression persisted for at least 42 weeks after injection. At 42 weeks after AAV8CMVLacZ and AAV9CMVLacZ injection, nearly 50% and 17% of muscle fibers were positive for ß-galactosidase, respectively. AAV8/9-mediated M3-FLAG expression lasted for up to 42 weeks using tacrolimus. No significant generalized toxicity was observed in any monkey. These results indicate that tacrolimus administration regulated the immune response to transgenes and truncated microdystrophin in normal primates and may enhance the benefits of AAV-mediated gene therapy for DMD.

Gene suppressing therapy for Pelizaeus-Merzbacher disease using artificial microRNA.

Copy number increase or decrease of certain dosage-sensitive genes may cause genetic diseases with distinct phenotypes, conceptually termed genomic disorders. The most common cause of Pelizaeus-Merzbacher disease (PMD), an X-linked hypomyelinating leukodystrophy, is genomic duplication encompassing the entire proteolipid protein 1 (PLP1) gene. Although the exact molecular and cellular mechanisms underlying PLP1 duplication, which causes severe hypomyelination in the central nervous system, remain largely elusive, PLP1 overexpression is likely the fundamental cause of this devastating disease. Here, we investigated if adeno-associated virus-mediated (AAV-mediated) gene-specific suppression may serve as a potential cure for PMD by correcting quantitative aberrations in gene products. We developed an oligodendrocyte-specific Plp1 gene suppression therapy using artificial microRNA under the control of human CNP promoter in a self-complementary AAV (scAAV) platform. A single direct brain injection achieved widespread oligodendrocyte-specific Plp1 suppression in the white matter of WT mice. AAV treatment in Plp1-transgenic mice, a PLP1 duplication model, ameliorated cytoplasmic accumulation of Plp1, preserved mature oligodendrocytes from degradation, restored myelin structure and gene expression, and improved survival and neurological phenotypes. Together, our results provide evidence that AAV-mediated gene suppression therapy can serve as a potential cure for PMD resulting from PLP1 duplication and possibly for other genomic disorders.

Highly Efficient Ultracentrifugation-free Chromatographic Purification of Recombinant AAV Serotype 9.

Recombinant adeno-associated virus serotype 9 (rAAV9) can specifically transduce muscle and neuronal tissues; thus, rAAV9 can potentially be used in gene therapy. However, rAAV9 is the most challenging rAAV serotype to purify. Traditionally, rAAV9 has been purified by ultracentrifugation, which is not scalable. We recently described a chromatographic purification protocol for rAAV1; this protocol can achieve scalable purifications. In this study, we attempted to optimize this protocol for purifying rAAV9 preparations, and we developed a novel, effective method for high-yield purification of rAAV9 using quaternary ammonium anion exchangers and size-exclusion chromatography. The final purified rAAV9 contained mainly three capsid proteins, as observed by SDS-PAGE. Furthermore, negative-stain electron microscopy demonstrated that 96.1% ± 1.1% of rAAV9 particles carried the viral genome containing the EGFP transgene, indicating that impurities and empty capsids can be eliminated with our purification protocol. The final rAAV9 titer obtained by our protocol totaled 2.5 ± 0.4 × 1015 viral genomes produced from ∼3.2 × 109 HEK293EB cells. We confirmed that our protocol can also be applied to purify other varied AAV genome constructs. Our protocol can scale up production of pure rAAV9, in compliance with current good manufacturing practice, for clinical applications in human gene therapy.

Ultracentrifugation-free chromatography-mediated large-scale purification of recombinant adeno-associated virus serotype 1 (rAAV1).

Recombinant adeno-associated virus (rAAV) is an attractive tool for gene transfer and shows potential for use in human gene therapies. The current methods for the production and purification of rAAV from the transfected cell lysate are mainly based on cesium chloride and iodixanol density ultracentrifugation, although those are not scalable. Meanwhile, chromatography-based systems are more scalable. Therefore, in this study, we developed a novel method for the production and purification of rAAV serotype 1 (rAAV1) from serum-free culture supernatant based on ion-exchange and gel-filtration chromatography to obtain highly purified products with an ultracentrifugation-free technique towards Good Manufacturing Practice (GMP) production. The purified rAAV1 displayed three clear and sharp bands (VP1, VP2, and VP3) following sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and more than 90% of rAAV1 particles contained fully packaged viral genomes according to negative-stain electron micrographic analysis. Consequently, the resultant genomic titer of the purified rAAV1 was 3.63 × 10(13) v.g./ml (the total titer was 4.17 × 10(13) v.g.) from the 4 × 10(9) HEK293 cells. This novel chromatography-based method will facilitate scale-up of manufacturing for clinical applications in gene therapy.

Intra-amniotic rAAV-mediated microdystrophin gene transfer improves canine X-linked muscular dystrophy and may induce immune tolerance.

Duchenne muscular dystrophy (DMD) is a severe congenital disease due to mutations in the dystrophin gene. Supplementation of dystrophin using recombinant adenoassociated virus vector has promise as a treatment of DMD, although therapeutic benefit of the truncated dystrophin still remains to be elucidated. Besides, host immune responses against the vector as well as transgene products have been denoted in the clinical gene therapy studies. Here, we transduced dystrophic dogs fetuses to investigate the therapeutic effects of an AAV vector expressing microdystrophin under conditions of immune tolerance. rAAV-CMV-microdystrophin and a rAAV-CAG-luciferase were injected into the amniotic fluid surrounding fetuses. We also reinjected rAAV9-CMV-microdystrophin into the jugular vein of an infant dystrophic dog to induce systemic expression of microdystrophin. Gait and cardiac function significantly improved in the rAAV-microdystrophin-injected dystrophic dog, suggesting that an adequate treatment of rAAV-microdystrophin with immune modulation induces successful long-term transgene expression to analyze improved dystrophic phenotype.

Dystrophic mdx mice develop severe cardiac and respiratory dysfunction following genetic ablation of the anti-inflammatory cytokine IL-10.

Duchenne muscular dystrophy (DMD) is a progressive muscle-wasting disease that causes respiratory and cardiac failure. Inflammation is a key pathological characteristic of dystrophic muscle lesion formation, but its role and regulation in the disease time course has not been sufficiently examined. In the present study, we used IL-10(-/-)/mdx mice lacking both dystrophin and the anti-inflammatory cytokine, interleukin-10 (IL-10), to investigate whether a predisposition to inflammation affects the severity of DMD with advancing age. The IL-10 deficiency caused a profound DMD phenotype in the dystrophic heart such as muscle degeneration and extensive myofiber loss, but the limb muscle and diaphragm morphology of IL-10(-/) (-)/mdx mice was similar to that of mdx mice. Extensive infiltrates of pro-inflammatory M1 macrophages in regeneration of cardiotoxin-injured muscle, altered M1/M2 macrophage phenotype and increased pro-inflammatory cytokines/chemokines production were observed in the diaphragm and heart of IL-10(-/-)/mdx mice. We characterized the IL-10(-/-)/mdx mice as a dystrophic model with chronic inflammation and severe cardiorespiratory dysfunction, as evidenced by decreased percent fractional shortening (%FS) and ejection fraction percent (EF%) on echocardiography, reduced lower tidal volume on whole-body plethysmography. This study suggests that a predisposition to inflammation is an important indicator of DMD disease progression. Therefore, the development of anti-inflammatory strategies may help in slowing down the cardiorespiratory dysfunction on DMD.

Effect of the size of zona pellucida opening on hatching in the common marmoset monkey (Callithrix jacchus) embryo.

The use of the common marmoset monkey in biomedical research has increased recently, and further attention has been devoted to this model after the successful production of transgenic marmosets. To extend genetic engineering approaches to widespread biomedical research fields, efficient prolonged in vitro culturing of embryo development is necessary. We aimed to evaluate the effects of the size of the zona pellucida opening on promoting the hatching process in the marmoset embryo. Piezo-microdrilling of a 6-µm opening in eight embryos resulted in four partially hatched embryos and one hatched embryo after 5 days of culture. Piezo-microdrilling a 20-µm opening in 11 embryos resulted in nine partial hatchings and no hatched embryos. Piezo-scraping an 80-µm opening in six embryos resulted in no partially hatched embryos and five hatched embryos. These results suggest that an 80-µm opening, rather than 6-µm or 20-µm openings, is suitable to complete the hatching process in the marmoset embryo.

Ultrasound-guided non-surgical embryo collection in the common marmoset.

Experimental primate embryology has been hampered by limited access to embryos. In addition to surgical techniques, the less stressful non-surgical technique of uterine flushing has been developed but has had only limitedly used in recovering pre-implantation embryos from marmoset monkeys. In this study, we introduce the use of ultrasonography during marmoset non-surgical uterine flushing to make the cannulation easier, to further reduce stress, and to ensure thorough uterine flushing. We were able to cannulate in 99% of the transcervical cannulation attempts, repeat the flushing up to 17 times with the same animal, and recover up to 90% of the ovulation products. We also found that 8-cell or earlier stage embryos could be frequently obtained by non-surgical uterine flushing at 4 or 5 days after ovulation. The easiness and effectiveness of this novel ultrasound-guided technique will enable more research groups to study marmoset embryology and facilitate progress in this field.

Robust Long-term Transduction of Common Marmoset Neuromuscular Tissue With rAAV1 and rAAV9.

Profiles of recombinant adeno-associated virus (rAAV)-mediated transduction show interspecies differences for each AAV serotype. Robust long-term transgene expression is generally observed in rodents, whereas insufficient transduction is seen in animals with more advanced immune systems. Non-human primates, including the common marmoset, could provide appropriate models for neuromuscular diseases because of their higher brain functions and physiological resemblance to humans. Strategies to induce pathologies in the neuromuscular tissues of non-human primates by rAAV-mediated transduction are promising; however, transgene expression patterns with rAAV transduction have not been elucidated in marmosets. In this study, transduction of adult marmoset skeletal muscle with rAAV9 led to robust and persistent enhanced green fluorescent protein (EGFP) expression that was independent of the muscle fiber type, although lymphocyte infiltration was recognized. Systemic rAAV injection into pregnant marmosets led to transplacental fetal transduction. Surprisingly, the intraperitoneal injection of rAAV1 and rAAV9 into the neonatal marmoset resulted in systemic transduction and persistent transgene expression without lymphocyte infiltration. Skeletal and cardiac muscle were effectively transduced with rAAV1 and rAAV9, respectively. Interestingly, rAAV9 transduction led to intense EGFP signaling in the axons of the corpus callosum. These transduction protocols with rAAV will be useful for investigating gene functions in the neuromuscular tissues and developing gene therapy strategies.Molecular Therapy-Nucleic Acids (2013) 2, e95; doi:10.1038/mtna.2013.21; published online 28 May 2013.

Efficient embryo transfer in the common marmoset monkey (Callithrix jacchus) with a reduced transfer volume: a non-surgical approach with cryopreserved late-stage embryos.

Among primates, the common marmoset is suitable for primate embryology research. Its small body size, however, has delayed the technical development of efficient embryo transfer. Furthermore, three factors have been determined to adversely affect the performance of marmoset embryo transfer: nonsurgical approaches, the use of cryopreserved embryos, and the use of late-stage embryos. Here we performed embryo transfer under conditions that included the above three factors and using either a small (1 µl or less) or a large volume (2-3 µl) of medium. The pregnancy and birth rates were 50% (5/10) and 27% (3/11), respectively, when using the large volume, and 80% (8/10) and 75% (9/12), respectively, when using the small volume. The latter scores exceed those of previous reports using comparable conditions. Thus, it appears that these three previously considered factors could be overcome, and we propose that reducing the transfer volume to 1 µl or less is essential for successful marmoset embryo transfer.

Long-term engraftment of multipotent mesenchymal stromal cells that differentiate to form myogenic cells in dogs with Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is an incurable genetic disease with early mortality. Multipotent mesenchymal stromal cells (MSCs) are of interest because of their ability to differentiate to form myogenic cells in situ. In the present study, methods were developed to expand cultures of MSCs and to promote the myogenic differentiation of these cells, which were then used in a new approach for the treatment of DMD. MSC cultures enriched in CD271(+) cells grew better than CD271-depleted cultures. The transduction of CD271(+) MSCs with MyoD caused myogenic differentiation in vitro and the formation of myotubes expressing late myogenic markers. CD271(+) MSCs in the myogenic cell lineage transplanted into dog leukocyte antigen (DLA)-identical dogs formed clusters of muscle-like tissue. Intra-arterial injection of the CD271(+) MSCs resulted in engraftment at the site of the cardiotoxin (CTX)-injured muscle. Dogs affected by X-linked muscular dystrophy in Japan (CXMD(J)) treated with an intramuscular injection of CD271(+) MSCs similarly developed muscle-like tissue within 8-12 weeks in the absence of immunosuppression. In the newly formed tissues, developmental myosin heavy chain (dMyHC) and dystrophin were upregulated. These findings demonstrate that a cell transplantation strategy using CD271(+) MSCs may offer a promising treatment approach for patients with DMD.

[Effect of dexmedetomidine on intraoperative somatosensory evoked potential monitoring].

BACKGROUND: Intraoperative somatosensory-evoked potential (SEP) monitoring has become a part of neurosurgical procedures. In this study, we evaluated the effect of dexmedetomidine on SEP monitoring during neurosurgical anesthesia. METHODS: Eight patients scheduled for neurosurgery were studied. Anesthesia was maintained with continuous infusion of propofol at 2 microg x ml(-1) concentration using target controlled infusion (TCI). A loading dose of dexmedetomidine was infused at 6 microg x kg(-1) hr(-1) for 10 min and SEP was recorded. Infusion of dexmedetomidine was continued at 0.5 microg x kg(-1) x min(-1) for 10 min and SEP was recorded. We measured the change of amplitude and latency of SEP (N20-P25) and compared to baseline values. RESULTS: There was no statistically significant change in the cortical SEP amplitude (98.0 +/- 18.1% after a loading dose of dexmedetomidine and 112.7 +/- 16.8% at 0.5 microg x kg(-1) x min(-1) of dexmedetomidine, respectively) or latency (101.5 +/- 1.7% after a loading dose of dexmedetomidine and 101.9 +/- 1.7% at 0.5 microg x kg(-1) x min(-1) of dexmedetomidine, respectively). Mean blood pressure rose from 81 mmHg to 95 mmHg after initiation of dexmedetomidine infusion. Heart rate did not change. CONCLUSIONS: These findings suggest that dexmedetomidine has possibilities to produce an ideal environment as an anesthetic adjunct in patients requiring intrapoerative SEP monitoring.

Collection and culture of alveolar bone marrow multipotent mesenchymal stromal cells from older individuals.

In this work, we examined the culture condition of alveolar bone marrow multipotent mesenchymal stromal cells (ABMMSCs), aiming to apply regenerative therapy to older periodontitis patients. To better understand the character of cultured cells from alveolar bone marrow, the expression profiles of well-known genes and their responses to the induction of osteogenic, chondrogenic, or adipogenic differentiation were examined. Using alpha MEM-based culture, ABMMSCs could be obtained from older individuals than in previous reports. Interestingly, ABMMSCs expressing Klf4 were able to differentiate into osteoblasts. The prediction of differentiation potential by Klf4 could be a useful guide for further improvement of the culture conditions required to culture ABMMSCs derived from older individuals.

Collection and culture of primordial germ cells from cynomolgus monkeys (Macaca fascicularis).

Aim: To clarify the location of primordial germ cells (PGC) in an embryo of target-age and to examine the culture environment of the PCG. Methods: The days of ovulation and fertilization were estimated by measuring the serum concentration of estrogen. Pregnancy was confirmed by measurement of the serum concentration of the beta subunit of macaque chorionic gonadotropin and by ultrasonography. We also examined the location of PGC in the embryo at the time of retrieval. Results: Results showed that PGC in an embryo were in the hindguts at day 30 postfertilization, arrived at the genital ridges via mesenteries at approximately day 33 postfertilization, and colonized the gonads by day 36 postfertilization. Conclusions: In conclusion, embryos collected on day 33 postfertilization are more suitable for obtaining PGC from cynomolgus monkeys. The PGC collected from cynomolgus monkey fetuses were cultured under conditions for the derivation and culture of human embryonic germ cells; enzymatically dispersed single cells were cultured on a SIM thioguanine-resistant ouabain-resistant cells (STO) feeder layer with recombinant human leukemia inhibitory factor, recombinant human basic fibroblast growth factor and forskolin. The cells from genital ridges and mesenteries at day 33 postfertilization had alkaline phosphatase (ALP) activity in vitro for a maximum of 13 days. In contrast, ALP activity had been held for 2 months under the same culture condition when the cells were derived from the gonads at day 66 postfertilization. Derivation of an embryonic germ cell from a cynomolgus monkey was not achieved from these cultures. (Reprod Med Biol 2007; 6: 203-210).

[Effects of sevoflurane and propofol on evoked potentials during neurosurgical anesthesia].

BACKGROUND: The effect of anesthetics on somatosensory evoked potential (SEP) and auditory brain stem response (ABR) has been a subject of intense reseach over the last two decades. In fact, volatile anesthetics have been repeatedly shown to decrease cortical amplitude in a dose-dependent fashion but the information regarding the effect of propofol is incomplete. The purpose of this study was to compare the effects of sevoflurane and propofol on evoked potentials during comparable depth of anesthesia guided by bispectral index (BIS). METHODS: Forty four patients scheduled for neurosurgery were studied. Anesthesia was maintained with intravenous propofol using target controlled infusion (TCI). We measured the change of amplitude and latency of SEP(N20-P25), ABR (V wave) and visual evoked potential (VEP: P100) at three sets of sevoflurane (0%, 1%, 2%) or propofol concentrations (effect site concentration of 1.5, 2.0, 3.0 microug x ml(-1)). BIS monitor was used to measure relative depth of hypnosis. RESULTS: With increasing concentrations of sevoflurane (0, 1% and 2%), SEP showed dose-related reduction in its amplitude, ABR produced less marked changes and VEP showed a significant reduction at 1%. VEP at the propofol concentration of 3.0 microg x ml(-1) was decreased significantly compared with the amplitude at 1.5 microg x ml(-1) concentration. No significant change was observed with SEP and ABR during the change of propofol dosages. BIS values were almost the same with each anesthetics. CONCLUSIONS: VEP was most strongly affected with anesthetics, and ABR showed less marked influence of sevoflurane and propofol. Propofol based TIVA technique would induce less change in evoked potentials than sevoflurane.

Differential development of rabbit embryos following microinsemination with sperm and spermatids.

Microinsemination is the technique of delivering male germ cells directly into oocytes. The efficiency of fertilization after microinsemination and subsequent embryo development may vary with the animal species and male germ cells used. The present study was undertaken to observe the in vitro and in vivo developmental ability of rabbit embryos following microinsemination with male germ cells at different stages. First, we assessed their oocyte-activating capacity by injecting them into mouse and rabbit oocytes. The majority of mouse oocytes were activated irrespective of the type of rabbit male germ cell injected (61-77%), whereas rabbit oocytes were activated differently according to the type of male germ cells (89%, 75%, and 29% were activated by spermatozoa, elongated spermatids, and round spermatids, respectively; P < 0.05). After 120 hr in culture, 66%, 45%, and 13%, respectively, of these activated rabbit oocytes (pronuclear eggs) developed into blastocysts (P < 0.05). Additional electric pulse stimulation of round spermatid-injected oocytes increased the blastocyst rate to 43%. After 24 hr in culture, some four to eight cell embryos were transferred into the oviducts of pseudopregnant females. Normal pups were born from spermatozoa and elongated spermatids, but not from round spermatids. Karyotypic analysis at the morula/blastocyst stage revealed that the majority of round spermatid-derived embryos had abnormal ploidy (8 out of 12 embryos). Our study indicates that rabbit male germ cells acquire the ability to activate oocytes and to support subsequent embryo development as they undergo spermiogenesis. As these differential developmental patterns are similar to those reported for humans in vitro and in vivo, rabbits may provide an alternative small animal model for studying the biological nature and molecular basis of human microinsemination techniques, especially those using immature male germ cells.

Buffalo rat liver cells produce factors that support preimplantation development of mouse embryos cultured in vitro.

To examine the effects of buffalo rat liver (BRL) cells on the preimplantation development of mouse embryos in vitro, we first cultured two-cell mouse embryos alone in serum-free Dulbecco modified Eagle medium. As expected, the embryos did not develop to subsequent stages. However, when cocultured with BRL cells, the embryos developed to the blastocyst stage efficiently. Direct contact of embryos with BRL cells was not necessary for development: the medium conditioned by BRL cells contained soluble factors that supported the preimplantation development of mouse embryos. Embryos cultured with BRL-conditioned medium that was replaced at various intervals had a further increased rate of development to the blastocyst stage. This finding indicated that the activities of the factors were maintained only briefly. Seven proteins between 35 and 44 kDa that were detected in the medium were highly beneficial to the development of the embryos. Follistatin-related protein and pigment epithelium-derived factor are believed to be the factors supporting embryo development. The other five proteins also may improve the environment for the development of mouse embryos cultured in vitro.