Oestrogen in the chick embryo can induce chromosomally male ZZ left gonad epithelial cells to form an ovarian cortex that can support oogenesis.

In chickens, the embryonic ovary differentiates into two distinct domains before meiosis: a steroidogenic core (the female medulla), overlain by the germ cell niche (the cortex). The differentiation of the medulla is a cell-autonomous process based on chromosomal sex identity (CASI). In order to address the extent to which cortex differentiation depends on intrinsic or extrinsic factors, we generated models of gonadal intersex by mixing ZW (female) and ZZ (male) cells in gonadal chimeras, or by altering oestrogen levels of ZW and ZZ embryos. We found that CASI does not apply to the embryonic cortex. Both ZW and ZZ cells can form the cortex and this can happen independently of the phenotypic sex of the medulla as long as oestrogen is provided. We also show that the cortex-promoting activity of oestrogen signalling is mediated via estrogen receptor alpha within the left gonad epithelium. However, the presence of a medulla with an 'intersex' or male phenotype may compromise germ cell progression into meiosis, causing cortical germ cells to remain in an immature state in the embryo.

Reviving rare chicken breeds using genetically engineered sterility in surrogate host birds.

In macrolecithal species, cryopreservation of the oocyte and zygote is not possible due to the large size and quantity of lipid deposited within the egg. For birds, this signifies that cryopreserving and regenerating a species from frozen cellular material are currently technically unfeasible. Diploid primordial germ cells (PGCs) are a potential means to freeze down the entire genome and reconstitute an avian species from frozen material. Here, we examine the use of genetically engineered (GE) sterile female layer chicken as surrogate hosts for the transplantation of cryopreserved avian PGCs from rare heritage breeds of chicken. We first amplified PGC numbers in culture before cryopreservation and subsequent transplantation into host GE embryos. We found that all hatched offspring from the chimera GE hens were derived from the donor rare heritage breed broiler PGCs, and using cryopreserved semen, we were able to produce pure offspring. Measurement of the mutation rate of PGCs in culture revealed that 2.7 × 10-10 de novo single-nucleotide variants (SNVs) were generated per cell division, which is comparable with other stem cell lineages. We also found that endogenous avian leukosis virus (ALV) retroviral insertions were not mobilized during in vitro propagation. Taken together, these results show that mutation rates are no higher than normal stem cells, essential if we are to conserve avian breeds. Thus, GE sterile avian surrogate hosts provide a viable platform to conserve and regenerate avian species using cryopreserved PGCs.

Efficient TALEN-mediated gene targeting of chicken primordial germ cells.

In this work we use TALE nucleases (TALENs) to target a reporter construct to the DDX4 (vasa) locus in chicken primordial germ cells (PGCs). Vasa is a key germ cell determinant in many animal species and is posited to control avian germ cell formation. We show that TALENs mediate homology-directed repair of the DDX4 locus on the Z sex chromosome at high (8.1%) efficiencies. Large genetic deletions of 30 kb encompassing the entire DDX4 locus were also created using a single TALEN pair. The targeted PGCs were germline competent and were used to produce DDX4 null offspring. In DDX4 knockout chickens, PGCs are initially formed but are lost during meiosis in the developing ovary, leading to adult female sterility. TALEN-mediated gene targeting in avian PGCs is therefore an efficient process.

Cell-autonomous sex differences in gene expression in chicken bone marrow-derived macrophages.

We have identified differences in gene expression in macrophages grown from the bone marrow of male and female chickens in recombinant chicken M-CSF (CSF1). Cells were profiled with or without treatment with bacterial LPS for 24 h. Approximately 600 transcripts were induced by prolonged LPS stimulation to an equal extent in the male and female macrophages. Many transcripts encoded on the Z chromosome were expressed ∼1.6-fold higher in males, reflecting a lack of dosage compensation in the homogametic sex. A smaller set of W chromosome-specific genes was expressed only in females. LPS signaling in mammals is associated with induction of type 1 IFN-responsive genes. Unexpectedly, because IFNs are encoded on the Z chromosome of chickens, unstimulated macrophages from the female birds expressed a set of known IFN-inducible genes at much higher levels than male cells under the same conditions. To confirm that these differences were not the consequence of the actions of gonadal hormones, we induced gonadal sex reversal to alter the hormonal environment of the developing chick and analyzed macrophages cultured from male, female, and female sex-reversed embryos. Gonadal sex reversal did not alter the sexually dimorphic expression of either sex-linked or IFN-responsive genes. We suggest that female birds compensate for the reduced dose of inducible IFN with a higher basal set point of IFN-responsive genes.

The Transcriptome of Chicken Migratory Primordial Germ Cells Reveals Intrinsic Sex Differences and Expression of Hallmark Germ Cell Genes.

Primordial germ cells (PGCs) are germline-restricted embryonic cells that form the functional gametes of the adult animal. The use of avian PGCs in biobanking and producing genetically modified birds has driven research on the in vitro propagation and manipulation of these embryonic cells. In avian species, PGCs are hypothesized to be sexually undetermined at an early embryonic stage and undergo differentiation into an oocyte or spermatogonial fate dictated by extrinsic factors present in the gonad. However, chicken male and female PGCs require different culture conditions, suggesting that there are sex-specific differences, even at early stages. To understand potential differences between male and female chicken PGCs during migratory stages, we studied the transcriptomes of circulatory stage male and female PGCs propagated in a serum-free medium. We found that in vitro cultured PGCs were transcriptionally similar to their in ovo counterparts, with differences in cell proliferation pathways. Our analysis also revealed sex-specific transcriptome differences between male and female cultured PGCs, with notable differences in Smad7 and NCAM2 expression. A comparison of chicken PGCs with pluripotent and somatic cell types identified a set of genes that are exclusive to germ cells, enriched in the germplasm, and associated with germ cell development.

Avian Primordial Germ Cells Are Bipotent for Male or Female Gametogenesis.

In birds, males are the homogametic sex (ZZ) and females are the heterogametic sex (ZW). Here, we investigate the role of chromosomal sex and germ cell competition on avian germ cell differentiation. We recently developed genetically sterile layer cockerels and hens for use as surrogate hosts for primordial germ cell (PGC) transplantation. Using in vitro propagated and cryopreserved PGCs from a pedigree Silkie broiler breed, we now demonstrate that sterile surrogate layer hosts injected with same sex PGCs have normal fertility and produced pure breed Silkie broiler offspring when directly mated to each other in Sire Dam Surrogate mating. We found that female sterile hosts carrying chromosomally male (ZZ) PGCs formed functional oocytes and eggs, which gave rise to 100% male offspring after fertilization. Unexpectedly, we also observed that chromosomally female (ZW) PGCs carried by male sterile hosts formed functional spermatozoa and produced viable offspring. These findings demonstrate that avian PGCs are not sexually restricted for functional gamete formation and provide new insights for the cryopreservation of poultry and other bird species using diploid stage germ cells.

Investigation of the Guinea fowl and domestic fowl hybrids as potential surrogate hosts for avian cryopreservation programmes.

In the last decade, avian gene preservation research has focused on the use of the early precursors of the reproductive cells, the primordial germ cells (PGCs). This is because avian PGCs have a unique migration route through the vascular system which offers easy accessibility. Furthermore, culturing of the cells in vitro, freezing/thawing, reintegration into a recipient embryo and the development of the germ cells can be carried out in well-defined laboratory circumstances. The efficient recovery of the donor genotype and the frequency of germline transmission from the surrogate host animals are still areas which need further development. Thus, the aim of the present study was to investigate an infertile interspecific hybrid (recipient) as an appropriate host for primordial germ cells from native poultry breeds. Guinea fowl × chicken hybrids were produced, the crossing was repeated inversely. The phenotype, the hatching time, the hatching rate, the sex ratio, the presence of own germ cells, the fertility and the phenotype of viable hybrids and the incidence of chromosomal abnormalities of dead hybrid embryos were described. 6.65% viable offspring was obtained with crossing of Guinea fowl females with domestic fowl males. Crossing of domestic fowl hens with Guinea fowl male resulted in lower fertility, 0.14% viable offspring. Based on the investigations, the observed offspring from the successful crossing were sterile male hybrids, thus an extreme form of Haldane's rule was manifested. The sterile hybrid male embryos were tested by injecting fluorescently labeled chicken PGCs. The integration rate of labeled PGCs was measured in 7.5-day, 14.5-day and 18.5-day old embryonic gonads. 50%, 5.3% and 2.4% of the injected hybrid embryos survived and 40%, 5.3% and 2.4% of the examined gonads contained fluorescent labeled donor PGCs. Therefore, these sterile hybrid males may be suitable recipients for male PGCs and possibly for female PGCs although with lower efficiency. This research work shows that the sterility of hybrids can be used in gene conservation to be a universal host for PGCs of different avian species.

Identification of disease markers by differential display: prion disease.

In order to identify molecular markers of prion disease in peripheral tissues, we used the differential display reverse-transcriptase polymerase chain reaction (DDRT-PCR) procedure to compare gene expression in spleens of infected and uninfected mice. In this study, we identified a novel erythroid-specific gene that was differentially expressed as a result of prion infection. We were able to demonstrate that a decrease in the expression levels of this transcript in hematopoietic tissues was a common feature of prion diseases. Our findings suggest a previously unknown role for the blood erythroid lineage in the development of prion diseases and should provide a new focus for research into diagnostic and therapeutic strategies.

Real-Time Sexing of Chicken Embryos and Compatibility with in ovo Protocols.

The chicken embryo is an established model system for studying early vertebrate development. One of the major advantages of this model is the facility to perform manipulations in ovo and then continue incubation and observe the effects on embryonic development. However, in common with other vertebrate models, there is a tendency to disregard the sex of the experimental chicken embryos, and this can lead to erroneous conclusions, a lack of reproducibility, and wasted efforts. That this neglect is untenable is emphasised by the recent demonstration that avian cells and tissues have an inherent sex identity and that male and female tissues respond differently to the same stimulus. These sexually dimorphic characteristics dictate that analyses and manipulations involving chicken embryos should always be performed using tissues/embryos of known sex. Current sexing protocols are unsuitable in many instances because of the time constraints imposed by most in ovo procedures. To address this lack, we have developed a real-time chicken sexing assay that is compatible with in ovo manipulations, reduces the number of embryos required, and conserves resources.

Gonadal asymmetry and sex determination in birds.

Although vertebrates display a superficial bilateral symmetry, most internal organs develop and locate with a consistent left:right asymmetry. There is still considerable debate as to when this process actually begins, but it seems that, at least for some species, the initial steps occur at a very early stage of development. In recent years, a number of model systems, including the chick embryo, have been utilised to increase our understanding of the molecular basis of this complex developmental process. While the basic elements of asymmetry are clearly conserved in chick development, the chick embryo also exhibits an additional unusual asymmetry in terms of the development of the gonads. In the female chick embryo, only 1 gonad and accessory structures fully develop, with the result that the adult hen has only 1 ovary and a single oviduct - both on the left side. With a small number of exceptions, this is a consistent feature of avian development. Here, we describe the morphological development and molecular basis of this unusual asymmetry, consider the implications for avian sex determination, and discuss the possible biological reasons why many birds have adopted a single-ovary system.