Enriched gonadal migration of donor-derived gonadal primordial germ cells by immunomagnetic cell sorting in birds

This study was conducted to evaluate whether immunomagnetic treatment could improve the retrieval and migration capacity of avian gonadal primordial germ cells (gPGCs) collected from gonads in 5.5-day-old chick and 5-day-old quail embryos, respectively. Collected gPGCs were loaded into a magnetic-activated cell sorter (MACS) after being conjugated with specific gPGC antibodies and either MACS-treated or non-treated cells in each species were subsequently transferred to the recipient embryos. MACS treatment significantly (P < 0.05) increased the population ratio of gPGCs in gonadal cells retrieved (0.74 to 33.4% in the chicken and 2.68 to 45.1% in the quail). This was due to decreased number of non-gPGCs in total cell population. MACS treatment further enhanced gonadal migration of gPGCs transferred in both species (10% vs. 80-85% in the chicken and 10-15% vs. 70-80% in the quail). Increase in the number of microinjected cells up to 600 cells/embryo did not eliminate such promoting effect. In conclusion, MACS treatment greatly increased the population ratio of avian gPGCs in gonadal cells, resulting improved gonadal migration in recipient embryos.     

Characterisation and germline transmission of cultured avian primordial germ cells

Background

Avian primordial germ cells (PGCs) have significant potential to be used as a cell-based system for the study and preservation of avian germplasm, and the genetic modification of the avian genome. It was previously reported that PGCs from chicken embryos can be propagated in culture and contribute to the germ cell lineage of host birds.

Principal Findings

We confirm these results by demonstrating that PGCs from a different layer breed of chickens can be propagated for extended periods in vitro. We demonstrate that intracellular signalling through PI3K and MEK is necessary for PGC growth. We carried out an initial characterisation of these cells. We find that cultured PGCs contain large lipid vacuoles, are glycogen rich, and express the stem cell marker, SSEA-1. These cells also express the germ cell-specific proteins CVH and CDH. Unexpectedly, using RT-PCR we show that cultured PGCs express the pluripotency genes c-Myc, cKlf4, cPouV, cSox2, and cNanog. Finally, we demonstrate that the cultured PGCs will migrate to and colonise the forming gonad of host embryos. Male PGCs will colonise the female gonad and enter meiosis, but are lost from the gonad during sexual development. In male hosts, cultured PGCs form functional gametes as demonstrated by the generation of viable offspring.

Conclusions

The establishment of in vitro cultures of germline competent avian PGCs offers a unique system for the study of early germ cell differentiation and also a comparative system for mammalian germ cell development. Primary PGC lines will form the basis of an alternative technique for the preservation of avian germplasm and will be a valuable tool for transgenic technology, with both research and industrial applications.     

Improved Transfection Efficiency of Chicken Gonadal Primordial Germ Cells for the Production of Transgenic Poultry

Electroporation is a common method of DNA transfection for many types of eukaryotic cells, but has not been attempted in avian primordial germ cells (PGCs). DNA uptake in chicken primordial germ cells (PGCs) was tested using electroporation with and without dimethyl sulfoxide (DMSO). Gonadal tissue and chicken embryonic fibroblasts (CEFs) were isolated from 6-day-old embryos (stage 29), transfected with pCMV carrying the bacterial lacZ gene, and cultured for 24 h. Gonadal primordial germ cells (gPGCs) were purified from culture using a Ficoll gradient. The addition of DMSO significantly increased the transfection efficiency of gPGCs but had no effect on chicken embryonic fibroblasts. Electroporation of gPGCs resulted in an 80% transfection efficiency, compared with about 17% observed with liposomes. Approximately 200 transfected gPGCs were injected into 2.5-day-old (stage 17) recipient embryos and the eggs were incubated for an additional 3.5 days, 7.5 days or to ...     

Doubly uniparental inheritance of mitochondria as a model system for studying germ line formation

Background

Doubly Uniparental Inheritance (DUI) of mitochondria occurs when both mothers and fathers are capable of transmitting mitochondria to their offspring, in contrast to the typical Strictly Maternal Inheritance (SMI). DUI was found in some bivalve molluscs, in which two mitochondrial genomes are inherited, one through eggs, the other through sperm. During male embryo development, spermatozoon mitochondria aggregate in proximity of the first cleavage furrow and end up in the primordial germ cells, while they are dispersed in female embryos.

Methodology/Principal Findings

We used MitoTracker, microtubule staining and transmission electron microscopy to examine the mechanisms of this unusual distribution of sperm mitochondria in the DUI species Ruditapes philippinarum. Our results suggest that in male embryos the midbody deriving from the mitotic spindle of the first division concurs in positioning the aggregate of sperm mitochondria. Furthermore, an immunocytochemical analysis showed that the germ line determinant Vasa segregates close to the first cleavage furrow.

Conclusions/Significance

In DUI male embryos, spermatozoon mitochondria aggregate in a stable area on the animal-vegetal axis: in organisms with spiral segmentation this zone is not involved in cleavage, so the aggregation is maintained. Moreover, sperm mitochondria reach the same embryonic area in which also germ plasm is transferred. In 2-blastomere embryos, the segregation of sperm mitochondria in the same region with Vasa suggests their contribution in male germ line formation. In DUI male embryos, M-type mitochondria must be recognized by egg factors to be actively transferred in the germ line, where they become dominant replacing the Balbiani body mitochondria. The typical features of germ line assembly point to a common biological mechanism shared by DUI and SMI organisms. Although the molecular dynamics of the segregation of sperm mitochondria in DUI species are unknown, they could be a variation of the mechanism regulating the mitochondrial bottleneck in all metazoans.     

Birth of germline chimeras by transfer of chicken embryonic germ (EG) cells into recipient embryos

This study reports for the first time the production of chicken germline chimeras by transfer of embryonic germ (EG) cells into recipient embryos of different strain. EG cells were established by the subculture of gonadal tissue cells retrieved from stage 28 White Leghorn (WL) embryos with I/I gene. During primary culture (P(0)), gonadal primordial germ cells (gPGCs) in the stromal cells began to form colonies after 7 days in culture with significant (P < 0.0001) increase in cell population. Colonized gPGCs were then subcultured with chicken embryonic fibroblast monolayer for EG cell preparation. Prepared EG cells or gPGCs at P(0) were transferred to stage 17 Korean Ogol chicken (KOC) embryos with i/i gene. The recipient chickens were raised for 6 months to sexual maturity, then a testcross analysis by artificial insemination was conducted for evaluating germline chimerism. As results, transfer of EG cells and gPGCs yielded total 17 germline chimeras; 2 out of 15 (13.3%) and 15 of 176 sexually matured chickens (8.5%), respectively. The efficiency of germline transmission in the chimeras was 1.5-14.6% in EG cells, while 1.3-27.6% in gPGCs. In conclusion, chicken germline chimeras could be produced by the transfer of EG cells, as well as gPGCs, which might enormously contribute to establishing various innovative technologies in the field of avian transgenic research for bioreactor production.     

Generating Porcine Chimeras Using Inner Cell Mass Cells and Parthenogenetic Preimplantation Embryos

Background

The development and validation of stem cell therapies using induced pluripotent stem (iPS) cells can be optimized through translational research using pigs as large animal models, because pigs have the closest characteristics to humans among non-primate animals. As the recent investigations have been heading for establishment of the human iPS cells with naïve type characteristics, it is an indispensable challenge to develop naïve type porcine iPS cells. The pluripotency of the porcine iPS cells can be evaluated using their abilities to form chimeras. Here, we describe a simple aggregation method using parthenogenetic host embryos that offers a reliable and effective means of determining the chimera formation ability of pluripotent porcine cells.

Methodology/Significant Principal Findings

In this study, we show that a high yield of chimeric blastocysts can be achieved by aggregating the inner cell mass (ICM) from porcine blastocysts with parthenogenetic porcine embryos. ICMs cultured with morulae or 4–8 cell-stage parthenogenetic embryos derived from in vitro-matured (IVM) oocytes can aggregate to form chimeric blastocysts that can develop into chimeric fetuses after transfer. The rate of production of chimeric blastocysts after aggregation with host morulae (20/24, 83.3%) was similar to that after the injection of ICMs into morulae (24/29, 82.8%). We also found that 4–8 cell-stage embryos could be used; chimeric blastocysts were produced with a similar efficiency (17/26, 65.4%). After transfer into recipients, these blastocysts yielded chimeric fetuses at frequencies of 36.0% and 13.6%, respectively.

Conclusion/Significance

Our findings indicate that the aggregation method using parthenogenetic morulae or 4–8 cell-stage embryos offers a highly reproducible approach for producing chimeric fetuses from porcine pluripotent cells. This method provides a practical and highly accurate system for evaluating pluripotency of undifferentiated cells, such as iPS cells, based on their ability to form chimeras.     

Simple separation of chicken gonadal primordial germ cells with and without foreign genes

Simplicity is the key element of an inexpensive technique described that is superior in performance to previous methods. It can make it the rapid method of choice to obtain reasonable yields of purified primordial germ cells (PGCs) for immediate production of germline chimeric chickens with integrated foreign genes. After Ficoll centrifugation, the purity of PGCs from gonads was 80.9+/-0.08% (mechanical) compared with 86.1+/-0.19% (enzymatic). GFP gene and lacZ-transduced chicken gonadal primordial germ cells (gPGCs) examined 72h after transduction had a transfection efficiency of approximately 61% and approximately 64%, respectively. After 10 days of G418 selection, approximately 90 and 92% of pure gPGCs did not contain other cells following this Ficoll gradient centrifugation method of preparation.     

Improved germline transmission in chicken chimeras produced by transplantation of gonadal primordial germ cells into recipient embryos

In the avian species, germline chimera production could be possible by transfer of donor germ cells into the blood vessel of recipient embryos. This study was conducted to establish an efficient transfer system of chicken gonadal primordial germ cells (gPGCs) for producing the chimeras having a high capacity of germline transmission. Gonadal PGCs retrieved from 5.5-day-old embryos (stage 28) of Korean Ogol chicken (KOC with i/i gene) were transferred into the dorsal aorta of 2.5-day-old embryos (stage 17) of White Leghorn chicken (WL with I/I gene). Prospective evaluations of whether culture duration (0, 5, or 10 days) and subsequent Ficoll separation of gPGCs before transfer affected chimera production and germline transmission in the chimeras were made while retrospective analysis was conducted for examining the effect of chimera sexuality. A testcross analysis by artificial insemination of presumptive chimeras with adult KOC was performed for evaluating each treatment effect. First, comparison was made for evaluating whether experimental treatments could improve chimera production, but none of the treatments were significantly (P = 0.6831) influenced (5.1%-14.4%). Second, it was determined whether each treatment could enhance germline transmission in produced chimeras. More (P < 0.0001) progenies with black feathers (i/i) were produced in the germline chimeras derived from the transfer of 10-day-cultured gPGCs than from the transfer of 0- or 5-day-cultured gPGCs (0.6%-7.8% vs. 10.7%-49.7%). Ficoll separation was negatively affected (P < 0.0001), whereas there was no effect in chimera sexuality (P = 0.6011). In conclusion, improved germline transmission of more than a 45% transmission rate was found in chicken chimeras produced by transfer of 10-day-cultured gPGCs being separated without Ficoll treatment.     

Expression analysis of the NLRP gene family suggests a role in human preimplantation development

Background

The NLRP (Nucleotide-binding oligomerization domain, Leucine rich Repeat and Pyrin domain containing) family, also referred to as NALP family, is well known for its roles in apoptosis and inflammation. Several NLRPs have been indicated as being involved in reproduction as well.

Methodology

We studied, using the unique human gametes and embryo materials, the expression of the NLRP family in human gametes and preimplantation embryos at different developmental stages, and compared the expression levels between normal and abnormal embryos using real-time PCR.

Principal Findings

Among 14 members of the NLRP family, twelve were detected in human oocytes and preimplantation embryos, whereas seven were detected in spermatozoa. Eight NLRPs (NLRP4, 5, 8, 9, 11, 12, 13, and 14) showed a similar expression pattern: their expression levels were high in oocytes and then decreased progressively in embryos, resulting in a very low level in day 5 embryos. However, NLRP2 and NLRP7 showed a different expression pattern: their expression decreased from oocytes to the lowest level by day 3, but increased again by day 5. The expression levels of NLRP5, 9, and 12 were lower in day 1 abnormal embryos but higher in day3 and day5 arrested embryos, when compared with normal embryos at the same stages. NLRP7 was down-regulated in day 1 and day 5 abnormal embryos but over-expressed in day3 arrested embryos.

Conclusions

According to our results, different NLRPs possibly work in a stage-dependent manner during human preimplantation development.     

Production of quail (Coturnix japonica) germline chimeras by transfer of gonadal primordial germ cells into recipient embryos

The possibility of producing quail germline chimeras by the transfer of gonadal primordial germ cells (gPGCs) into recipient embryos was investigated. Japanese quail of the black (D: homozygous for the autosomal incomplete dominant gene D) and wild-type plumage (WP: d+/d+) strains were used as donors and recipients, respectively. Gonadal cells were retrieved from the gonads of 5-day-old D embryos, and gPGCs were enriched by magnetism-activated cell sorting. Fresh (noncultured) gPGCs or those isolated after culture for 3 days with gonadal stromal cells present in the mixed cell population were introduced into the dorsal aorta of 2-day-old recipient WP embryos. Hatchability of the recipient embryos was 23.7% (31/131) and 34.4% (31/90) for those transfused with cultured or noncultured gPGCs, respectively. Of the hatched quail, 28 acquired sexual maturity; among these animals, 7.1% (1/14) and 21.4% (3/14) of those that received cultured or noncultured gPGCs, respectively, were proved to be germline chimeras. The percentage of germline transmission to the donor-derived gametes in the chimeras that received cultured and noncultured gPGCs were 1.9 and 2.2-4.7%, respectively. In conclusion, quail gPGCs retrieved from 5-day-old embryos were thus transmitted in the germline after their transfer to quail embryos of a different strain. This property of the gPGCs was not adversely affected by culture for up to 3 days.     

Targeted expression of Cre recombinase provokes placental-specific DNA recombination in transgenic mice

Background

Inadequate placental development is associated with a high incidence of early embryonic lethality and serious pregnancy disorders in both humans and mice. However, the lack of well-defined trophoblast-specific gene regulatory elements has hampered investigations regarding the role of specific genes in placental development and fetal growth.

Principal Findings

By random assembly of placental enhancers from two previously characterized genes, trophoblast specific protein α (Tpbpa) and adenosine deaminase (Ada), we identified a chimeric Tpbpa/Ada enhancer that when combined with the basal Ada promoter provided the highest luciferase activity in cultured human trophoblast cells, in comparison with non-trophoblast cell lines. We used this chimeric enhancer arrangement to drive the expression of a Cre recombinase transgene in the placentas of transgenic mice. Cre transgene expression occurred throughout the placenta but not in maternal organs examined or in the fetus.

Significance

In conclusion, we have provided both in vitro and in vivo evidence for a novel genetic system to achieve placental transgene expression by the use of a chimeric Tpbpa/Ada enhancer driven transgene. The availability of this expression vector provides transgenic opportunities to direct the production of desired proteins to the placenta.     

Proteome analysis of chicken embryonic gonads: identification of major proteins from cultured gonadal primordial germ cells

The domestic chicken (Gallus gallus) is an important model for research in developmental biology because its embryonic development occurs in ovo. To examine the mechanism of embryonic germ cell development, we constructed proteome map of gonadal primordial germ cells (gPGCs) from chicken embryonic gonads. Embryonic gonads were collected from 500 embryos at 6 days of incubation, and the gPGCs were cultured in vitro until colony formed. After 7-10 days in culture, gPGC colonies were separated from gonadal stroma cells (GSCs). Soluble extracts of cultured gPGCs were then fractionated by two-dimensional gel electrophoresis (pH 4-7). A number of protein spots, including those that displayed significant expression levels, were then identified by use of matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry and LC-MS/MS. Of the 89 gPGC spots examined, 50 yielded mass spectra that matched avian proteins found in on-line databases. Proteome map of this type will serve as an important reference for germ cell biology and transgenic research.