Joni Macdonald

Efficient genetic modification and germ-line transmission of primordial germ cells using piggyBac and Tol2 transposons

The derivation of germ-line competent avian primordial germ cells establishes a cell-based model system for the investigation of germ cell differentiation and the production of genetically modified animals. Current methods to modify primordial germ cells using DNA or retroviral vectors are inefficient and prone to epigenetic silencing. Here, we validate the use of transposable elements for the genetic manipulation of primordial germ cells. We demonstrate that chicken primordial germ cells can be modified in vitro using transposable elements. Both piggyBac and Tol2 transposons efficiently transpose primordial germ cells. Tol2 transposon integration sites were spread throughout both the macro- and microchromosomes of the chicken genome and were more prevalent in gene transcriptional units and intronic regions, consistent with transposon integrations observed in other species. We determined that the presence of insulator elements was not required for reporter gene expression from the integrated transposon. We further demonstrate that a gene-trap cassette carried in the Tol2 transposon can trap and mutate endogenous transcripts in primordial germ cells. Finally, we observed that modified primordial germ cells form functional gametes as demonstrated by the generation of transgenic offspring that correctly expressed a reporter gene carried in the transposon. Transposable elements are therefore efficient vectors for the genetic manipulation of primordial germ cells and the chicken genome.

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.

Prolonged exposure to acetaminophen reduces testosterone production by the human fetal testis in a xenograft model

Prolonged exposure to therapeutic doses of acetaminophen reduces testosterone production by human fetal testis xenografts in mice. A risk of prolonged acetaminophen use The analgesic acetaminophen is one of the most commonly used medications worldwide. Although it has a good safety profile, previous human studies have found an association between the use of acetaminophen in pregnancy and cryptorchidism in male offspring. Now, van den Driesche et al. confirmed and refined this observation in a xenograft model using human fetal testicular tissue. The authors found that a single therapeutic dose of acetaminophen had no effect on testicular development, but continued dosing of acetaminophen for a week at clinically relevant doses reduced fetal testosterone and markers of androgen exposure. Further work will be needed to determine a safe dose and duration of acetaminophen exposure, but the current findings suggest a need for caution. Most common male reproductive disorders are linked to lower testosterone exposure in fetal life, although the factors responsible for suppressing fetal testosterone remain largely unknown. Protracted use of acetaminophen during pregnancy is associated with increased risk of cryptorchidism in sons, but effects on fetal testosterone production have not been demonstrated. We used a validated xenograft model to expose human fetal testes to clinically relevant doses and regimens of acetaminophen. Exposure to a therapeutic dose of acetaminophen for 7 days significantly reduced plasma testosterone (45% reduction; P = 0.025) and seminal vesicle weight (a biomarker of androgen exposure; 18% reduction; P = 0.005) in castrate host mice bearing human fetal testis xenografts, whereas acetaminophen exposure for just 1 day did not alter either parameter. Plasma acetaminophen concentrations (at 1 hour after the final dose) in exposed host mice were substantially below those reported in humans after a therapeutic oral dose. Subsequent in utero exposure studies in rats indicated that the acetaminophen-induced reduction in testosterone likely results from reduced expression of key steroidogenic enzymes (Cyp11a1, Cyp17a1). Our results suggest that protracted use of acetaminophen (1 week) may suppress fetal testosterone production, which could have adverse consequences. Further studies are required to establish the dose-response and treatment-duration relationships to delineate the maximum dose and treatment period without this adverse effect.

Tuning thermal properties and microphase separation in aliphatic polyester ABA copolymers

Four alkyl substituted β-lactones were investigated as monomers in ring opening polymerisation to produce a family of poly(3-hydroxyalkanoate)s. Homopolymers were synthesised using a robust aluminium salen catalyst, resulting in polymers with low dispersity (Đ < 1.1) and predictable molecular weights. ABA triblock copolymers were prepared using poly(L-lactic acid) as the A block and the aforementioned poly(3-hydroxyalkanoate) as the B block via a sequential addition method. Characterisation of these copolymers determined they were well controlled with low dispersities and predictable molecular weight. DSC analysis determined copolymers prepared from β-butyrolactone or β-valerolactone yielded polymers with tunable and predictable thermal properties. Copolymers prepared from β-heptanolactone yielded a microphase separated material as indicated by SAXS, with two distinct Tgs. The polymers could be readily cast into flexible films and their improved tensile properties were explored.

Intratubular germ cell neoplasia of the human testis: heterogeneous protein expression and relation to invasive potential

Testicular germ cell cancer develops from premalignant intratubular germ cell neoplasia, unclassified cells that are believed to arise from failure of normal maturation of fetal germ cells from gonocytes (OCT4+/MAGEA4−) into pre-spermatogonia (OCT4−/MAGEA4+). Intratubular germ cell neoplasia cell subpopulations based on stage of germ cell differentiation have been described, however the importance of these subpopulations in terms of invasive potential has not been reported. We hypothesized that cells expressing an immature (OCT4+/MAGEA4−) germ cell profile would exhibit an increased proliferation rate compared with those with a mature profile (OCT4+/MAGEA4+). Therefore, we performed triple immunofluorescence and stereology to quantify the different intratubular germ cell neoplasia cell subpopulations, based on expression of germ cell (OCT4, PLAP, AP2γ, MAGEA4, VASA) and proliferation (Ki67) markers, in testis sections from patients with preinvasive disease, seminoma, and non-seminoma. We compared these subpopulations with normal human fetal testis and with seminoma cells. Heterogeneity of protein expression was demonstrated in intratubular germ cell neoplasia cells with respect to gonocyte and spermatogonial markers. It included an embryonic/fetal germ cell subpopulation lacking expression of the definitive intratubular germ cell neoplasia marker OCT4, that did not correspond to a physiological (fetal) germ cell subpopulation. OCT4+/MAGEA4− cells showed a significantly increased rate of proliferation compared with the OCT4+/MAGEA4+ population (12.8 versus 3.4%, P<0.0001) irrespective of histological tumor type, reflected in the predominance of OCT4+/MAGEA4− cells in the invasive tumor component. Surprisingly, OCT4+/MAGEA4− cells in patients with preinvasive disease showed significantly higher proliferation compared to those with seminoma or non-seminoma (18.1 versus 10.2 versus 7.2%, P<0.05, respectively). In conclusion, this study has demonstrated that OCT4+/MAGEA4− cells are the most frequent and most proliferative cell population in tubules containing intratubular germ cell neoplasia, which appears to be an important factor in determining invasive potential of intratubular germ cell neoplasia to seminomas.

The Roslin Institute Transgenic Chicken Facility: developments in chicken transgenesis

Abstracts from the UC Davis Transgenic Animal Research Conference XIs from the UC Davis Transgenic Animal Research Conference XI

DMRT1 repression using a novel approach to genetic manipulation induces testicular dysgenesis in human fetal gonads

Abstract STUDY QUESTION Does loss of DMRT1 in human fetal testis alter testicular development and result in testicular dysgenesis? SUMMARY ANSWER DMRT1 repression in human fetal testis alters the expression of key testicular and ovarian determining genes, and leads to focal testicular dysgenesis. WHAT IS KNOWN ALREADY Testicular dysgenesis syndrome (TDS) is associated with common testicular disorders in young men, but its etiology is unknown. DMRT1 has been shown to play a role in the regulation of sex differentiation in the vertebrate gonad. Downregulation of DMRT1 in male mice results in trans-differentiation of Sertoli cells into granulosa (FOXL2+) cells resulting in an ovarian gonadal phenotype. STUDY DESIGN, SIZE, DURATION To determine the effect of DMRT1 repression on human fetal testes, we developed a novel system for genetic manipulation, which utilizes a Lentivral delivered miRNA during short-term in vitro culture (2 weeks). A long-term (4–6 weeks) ex vivo xenograft model was used to determine the subsequent effects of DMRT1 repression on testicular development and maintenance. We included first and second-trimester testis tissue (8–20 weeks gestation; n = 12) in the study. PARTICIPANTS/MATERIALS, SETTING, METHODS Human fetal testes were cultured in vitro and exposed to either of two DMRT1 miRNAs (miR536, miR641), or to scrambled control miRNA, for 24 h. This was followed by a further 14 days of culture (n = 3–4), or xenografting (n = 5) into immunocompromised mice for 4–6 weeks. Tissues were analyzed by histology, immunohistochemistry, immunofluorescence and quantitative RT-PCR. Endpoints included histological evaluation of seminiferous cord integrity, mRNA expression of testicular, ovarian and germ cell genes, and assessment of cell number and protein expression for proliferation, apoptosis and pluripotency factors. Statistical analysis was performed using a linear mixed effect model. MAIN RESULTS AND THE ROLE OF CHANCE DMRT1 repression (miR536/miR641) resulted in a loss of DMRT1 protein expression in a sub-population of Sertoli cells of first trimester (8–11 weeks gestation) human fetal testis; however, this did not affect the completion of seminiferous cord formation or morphological appearance. In second-trimester testis (12–20 weeks gestation), DMRT1 repression (miR536/miR641) resulted in disruption of seminiferous cords with absence of DMRT1 protein expression in Sertoli (SOX9+) cells. No differences in proliferation (Ki67+) were observed and apoptotic cells (CC3+) were rare. Expression of the Sertoli cell associated gene, SOX8, was significantly reduced (miR536, 34% reduction, P = 0.031; miR641 36% reduction, P = 0.026), whilst SOX9 expression was unaffected. Changes in expression of AMH (miR536, 100% increase, P = 0.033), CYP26B1 (miR641, 38% reduction, P = 0.05) and PTGDS (miR642, 30% reduction, P = 0.0076) were also observed. Amongst granulosa cell associated genes, there was a significant downregulation in R-spondin 1 expression (miR536, 76% reduction, P < 0.0001; miR641, 49% reduction, P = 0.046); however, there were no changes in expression of the granulosa cell marker, FOXL2. Analysis of germ cell associated genes demonstrated a significant increase in the expression of the pluripotency gene OCT4 (miR536, 233%, P < 0.001). We used the xenograft system to investigate the longer-term effects of seminiferous cord disruption via DMRT1 repression. As was evident in vitro for second-trimester samples, DMRT1 repression resulted in focal testicular dysgenesis similar to that described in adults with TDS. These dysgenetic areas were devoid of germ cells, whilst expression of FOXL2 within the dysgenetic areas, indicated trans-differentiation from a male (Sertoli cell) to female (granulosa cell) phenotype. LIMITATIONS, REASONS FOR CAUTION Human fetal testis tissue is a limited resource; however, we were able to demonstrate significant effects of DMRT1 repression on the expression of germ and somatic cell genes, in addition to the induction of focal testicular dysgenesis, using these limited samples. In vitro culture may not reflect all aspects of human fetal testis development and function; however, the concurrent use of the xenograft model which represents a more physiological system supports the validity of the in vitro findings. WIDER IMPLICATIONS OF THE FINDINGS Our findings have important implications for understanding the role of DMRT1 in human testis development and in the origin of testicular dysgenesis. In addition, we provide validation of a novel system that can be used to determine the effects of repression of genes that have been implicated in gonadal development and associated human reproductive disorders. STUDY FUNDING/COMPETING INTEREST(S) This project was funded by a Wellcome Trust Intermediate Clinical Fellowship (Grant No. 098522) awarded to RTM. LBS was supported by MRC Programme Grant MR/N002970/1. RAA was supported by MRC Programme Grant G1100357/1. RMS was supported by MRC Programme Grant G33253. This work was undertaken in the MRC Centre for Reproductive Health which is funded by the MRC Centre grant MR/N022556/1. The funding bodies had no input into the conduct of the research or the production of this manuscript. The authors have declared no conflicts of interest.

17-P042 Regulation of growth and proliferation of primordial germ cells

Primordial germ cells (PGCs) are the precursors of the germ cell lineage. A method for the prolonged invitro culture of chicken PGCs, which retain the ability to colonise the gonad when introduced into developing embryos, has been described (van de Lavoir etal., Nature 441 (2006) 766–769). The invitro culture of PGCs provides a useful tool for the creation of transgenic chickens and investigation of germ cell self-renewal and differentiation. By investigating and manipulating the described culture conditions we seek to define the signalling pathways involved in both PGC proliferation and survival. We have found that the level of FGF-2 is critical for PGC culture. Several other growth factors, identified as critical for mouse germ cell growth and proliferation, are currently being investigated. Cultured PGCs can also be manipulated to de-differentiate into cells resembling embryonic stem (ES) cells. This has been shown in both mammalian and avian systems. Studies carried out in mice have identified genes involved in pluripotency (oct4, nanog, sox2, c-myc and klf-4) that are differentially expressed between the two cell types. We have extended these studies to characterise the expression levels of these genes in avian PGCs and avian ES cells. A comparative analysis of these results suggests that qualitative differences exist between the germ cell lineages of chicken and mice.