Human primordial germ cells (hPGCs), the precursors of sperm and eggs, originate during week 2-3 of early postimplantation development1. Using in vitro models of hPGC induction2–4, recent studies suggest striking mechanistic differences in specification of human and mouse PGCs5. This may partly be due to the divergence in their pluripotency networks, and early postimplantation development6–8. Since early human embryos are inaccessible for direct studies, we considered alternatives, including porcine embryos that, as in humans, develop as bilaminar embryonic discs. Here we show that porcine PGCs (pPGCs) originate from the posterior pre-primitive streak competent epiblast by sequential upregulation of SOX17 and BLIMP1 in response to WNT and BMP signalling. Together with human and monkey in vitro models simulating peri-gastrulation development, we show conserved principles for epiblast development for competency for PGC fate, followed by initiation of the epigenetic programme9–11, regulated by a balanced SOX17–BLIMP1 gene dosage. Our combinatorial approach using human, porcine and monkey in vivo and in vitro models, provides synthetic insights on early human development.

BackgroundEpigenetic reprogramming is critical for genome regulation during germ line development. Genome-wide demethylation in mouse primordial germ cells (PGC) is a unique reprogramming event essential for erasing epigenetic memory and preventing the transmission of epimutations to the next generation. In addition to DNA demethylation, PGC are subject to a major reprogramming of histone marks, and many of these changes are concurrent with a cell cycle arrest in the G2 phase. There is limited information on how well conserved these events are in mammals. Here we report on the dynamic reprogramming of DNA methylation at CpGs of imprinted loci and DNA repeats, and the global changes in H3K27me3 and H3K9me2 in the developing germ line of the domestic pig.ResultsOur results show loss of DNA methylation in PGC colonizing the genital ridges. Analysis of IGF2-H19 regulatory region showed a gradual demethylation between E22-E42. In contrast, DMR2 of IGF2R was already demethylated in male PGC by E22. In females, IGF2R demethylation was delayed until E29-31, and was de novo methylated by E42. DNA repeats were gradually demethylated from E25 to E29-31, and became de novo methylated by E42. Analysis of histone marks showed strong H3K27me3 staining in migratory PGC between E15 and E21. In contrast, H3K9me2 signal was low in PGC by E15 and completely erased by E21. Cell cycle analysis of gonadal PGC (E22-31) showed a typical pattern of cycling cells, however, migrating PGC (E17) showed an increased proportion of cells in G2.ConclusionsOur study demonstrates that epigenetic reprogramming occurs in pig migratory and gonadal PGC, and establishes the window of time for the occurrence of these events. Reprogramming of histone H3K9me2 and H3K27me3 detected between E15-E21 precedes the dynamic DNA demethylation at imprinted loci and DNA repeats between E22-E42. Our findings demonstrate that major epigenetic reprogramming in the pig germ line follows the overall dynamics shown in mice, suggesting that epigenetic reprogramming of germ cells is conserved in mammals. A better understanding of the sequential reprogramming of PGC in the pig will facilitate the derivation of embryonic germ cells in this species.

Spermatogonia are a potential source of adult pluripotent stem cells and can be used for testis germ cell transplantation. Markers for the isolation of these cells are of great importance for biomedical applications. Primordial germ cells and prepubertal spermatogonia in many species can be identified by their binding of Dolichos biflorus agglutinin (DBA). This lectin binds to two different types of glycans, which are a-linked N-acetylgalactosamine (GalNac) and b-linked GalNac, if this is part of the Sda or GM2 glycotopes. We used the MAB CT1, which is specific for the trisaccharides motif NeuAca2-3(GalNAcb1-4)Galb1-, which is common to both Sda and GM2 glycotopes, to further define the glycosylation of DBA binding germ cells. In porcine embryos, CT1 bound to migratory germ cells and gonocytes. CT1/DBA double staining showed that the mesonephros was CT1 negative but contained DBA-positive cells. Gonocytes in the female gonad became CT1 negative, while male gonocytes remained CT1 positive. In immunohistological double staining of cattle, pig, horse and llama testis, DBA and CT1 staining was generally colocalised in a subpopulation of spermatogonia. These spermatogonia were mainly single, sometimes paired or formed chains of up to four cells. Our data show that the Sda/GM2 glycotope is present in developing germ cells and spermatogonia in several species. Owing to the narrower specificity of the CT1 antibody, compared with DBA, the former is likely to be a useful tool for labelling and isolation of these cells.

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Contents Small community cattle farmers in the tropics are facing challenges to deliver quality products whilst under pressure to increase milk and beef yields per cow. These challenges could be partially met by crossbreeding Bos taurus with Bos indicus (F1) cattle utilizing embryo transfer (ET) technology. The Bos taurus infusion would increase milk production, whilst the Bos indicus influence can improve resistance to the harsh environment of the tropics. Here, individuals from existing herds can be used to produce F1 embryos which benefit from hybrid vigour. Resultant female offspring would in turn receive an F1 embryo on reaching breeding maturity. This approach would help to provide a cost‐effective, systematic approach to improve productivity in dairy and beef cattle in the tropics. However, full usage of ET, including in vitro applications, in the tropics will require improvements in procedures, resources and education.

Objective: This study was conducted to evaluate the effects of the provision of a protein-rich supplement on productive performance, and metabolic profile on grazing suckling female beef calves in tropical conditions during 150 d of experimentation.Methods: Fifty-six Nellore suckling female calves, and their respective dams were distributed in a completely randomised design and made to undergo two treatments as follows: UNS (without supplementation), and SUP (supplementation with 5 g/kg body weight [BW] of a protein supplement). Throughout the experiment, animal performance and metabolic profile were evaluated. Also, ureagenesis and gluconeogenesis were assessed for gene expression.Results: SUP female calves showed a higher voluntary intake (p≤0.03) of the diet components evaluated, digestibility of organic matter (p≤0.02) and microbial nitrogen production (MICN; p≤0.02) compared to UNS female calves. In its turn, serum urea nitrogen (p≤0.01) and insulin-like growth factor-1 (p≤0.03) levels and ureagenesis (p≤0.04) increased in SUP female calves compared to UNS female calves. Blood glucose and triglyceride levels were not affected by supplementation. The average daily gain (ADG) from SUP female calves was higher (p≤0.02) compared with UNS female calves. However, supplementation did not affect the body measures of the animals.Conclusion: In summary, provision of a protein-rich supplement improves the intake and nutrients digestibility, ADG and final BW and increases metabolic indicators of the protein status in grazing suckling female beef calves in tropical conditions.