Male fertility restored by transplanting primordial germ cells into testes: a new way towards efficient transgenesis in chicken

Abstract

The ongoing progress in primordial germ cell derivation and cultivation is opening new ways in reproductive biotechnology. This study tested whether functional sperm cells can be matured from genetically manipulated primordial germ cells after transplantation in adult testes and used to restore fertility. We show that spermatogenesis can be restored after mCherry-expressing or GFP-expressing primordial germ cells are transplantated into the testes of sterilized G₀ roosters and that mCherry-positive or GFP-positive non-chimeric transgenic G₁ offspring can be efficiently produced. Compared with the existing approaches to primordial germ cell replacement, this new technique eliminates the germ line chimerism of G₀ roosters and is, therefore, faster, more efficient and requires fewer animals. Furthermore, this is the only animal model, where the fate of primordial germ cells in infertile recipients can be studied.

Figure 1

Schematic representation of PGC derivation, genetic modification, and transplantation into sterilized G₀ roosters. This has been followed by insemination of hens and analysis of G₁ offspring.

Figure 2

CB PGCs were derived from germinal crescent and stained for germline specific markers SSEA1 and chOLP. Expression was measured by flow cytometry.

Figure 3

Analysis of modified PGCs and sperm of recipient rooster. (a) CB PGCs were transfected with a mCherry expression plasmid consisting of mCherry under a chicken β-actin promoter and a puromycin resistance gene under a CAG promoter flanked by HS4 insulators. (b) Succesful selection and expression of mCherry was confirmed by fluorescence microscopy and (c) flow cytometry. (d) The DNA junction between the mCherry vector and adjacent chicken genomic sequence detected in the whole-genome sequence of parental PGC clone. The vector-derived attB sequence is in bold and the repetitive motifs in the chicken genomic DNA are underlined. (e) mCherry positivity in the spermiogenic epithelium of G₀ recipient rooster after orthotopic transplantation of mCherry-positive CB PGCs. (f) The mCherry reporter gene was detected by PCR in mCherry+ PGCs and semen samples of two recipient roosters.

Figure 4

Phenotypic and genetic analysis of G₁ offspring. (a) Freshly hatched G₁ offspring, one mCherry-negative chicken in the middle. (b) Distribution of mCherry-positive cells in the peripheral blood. FACS histogram of mCherry signal in red blood cells (blue) and white blood cells (red). (c) mCherry positivity visible in the beak and featherless skin of the mCherry-positive rooster (right) in the daylight. The mCherry-negative wt rooster shown as a control (left). (d) Photostability of mCherry shown as mCherry signal in adult feather. (e) The 195 bp PCR product of the CB-specific tpn allele amplified in DNA of G₁ offspring. The results of animals Nos 8 to 14 (from left to right) are shown, the non-CB chicken No. 11 is in the middle. Descendants of G₁ roosters Nos 769 and 795 are indicated. (f) Genomic relationship of inbred individual CB145 with itself, inbred individual CB151, the primordial germ cell line, the mCherry+ chicken Robin and a mCherry- chicken. The genomic relationship was calculated based on about 1.6 million maximum quality SNP detected in 78 chickens.

Figure 5

(a) mCherry-positive embryo in the middle of incubation. (b–e) mCherry positivity in embryo organs and tissues. From left to right: (b) liver, (c) spleen, (d) skeletal muscle, (e) heart.

Figure 6

Phenotypic and genetic analysis of eGFP transduction. (a) Freshly hatched G₁ offspring, one GFP-positive chicken right, wt chicken left. (b) PCR detection of eGFP transduction in the semen of roosters 649, 659, and 675 and in the GFP-positive G₁ chicken No. 146. The eGFP-specific PCR fragment is 525 bp in length.