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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.

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., Summary: Cell-autonomous sex identity does not apply to chicken ovarian cortex development. The gonadal epithelium responds to oestrogen via ERα and drives cortex formation independently of the medulla's phenotypic sex.

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.

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., Summary: TALE nucleases are used to target the DDX4 (vasa) locus in chicken primordial germ cells and generate DDX4 knockouts, which provide insights into DDX4 function in early chick development.

Biosecurity and sustainability in poultry production requires reliable germplasm conservation. Germplasm conservation in poultry is more challenging in comparison to other livestock species. Embryo cryopreservation is not feasible for egg-laying animals and chicken semen conservation has variable success for different chicken breeds. A potential solution is the cryopreservation of the committed diploid stem cell precursors to the gametes, the primordial germ cells (PGCs). Primordial germ cells are the lineage-restricted cells found at early embryonic stages in birds and form the sperm and eggs. We demonstrate here, using flocks of partially inbred lower fertility MHC restricted lines of chicken, that we can easily derive and cryopreserve a sufficient number of independent lines of male and female PGCs that would be sufficient to re-constitute a poultry breed. We demonstrate that germ line transmission can be attained from these PGCs using a commercial layer line of chickens as a surrogate host. This research is a major step in developing and demonstrating that cryopreserved PGCs could be used for the biobanking of specialised flocks of birds used in research settings. The prospective application of this technology to poultry production will further increase sustainability to meet current and future production needs.

R. sachalinensis plant extracts (Milsana was in ali trials as effective against U. necator as sulphur, even in years with very high disease pressure. The plant extract showed also positive effects in Botrytis control. Myco-Sin was in ali trials as effective in controlling P. viticola as the copper containing agent FW 450. The combination 01 Milsana, Myco-Sin and B. brevis revealed the potential 01 B. brevis lor control 01 B. cinerea in grape berries, and indicated additional positive effects towards the contro I 01 U. necator. The combined use 01 these biological control agents shows strong potential lor replacement 01 copper and sulphur in organic viticulture.