Developmental Expression of Alkaline
Phosphatase Genes; Reexpression in
Germ Cell Tumours and
in vitro Immortalized Germ Cells

Embryonic pluripotent stem cells (ESC) and induced pluripotent stem cells (iPSC) were reported capable of differentiating into primordial germ cell-like (PGCL) and functional gametes in vitro in the murine model (Hikabe et al. 2016 Nature 539, 299-303). The in vitro generation of primordial germ cells (PGC) and gametes from farm animals would greatly contribute to enhance animal production technologies and to the creation of adequate models for several disorders. The present study aimed at the generation of PGC in vitro (iPGC) from iPSC in cattle and their characterisation through pluripotency and germ cell markers. For that, bovine iPSC previously generated and characterised (Bressan et al. 2015 Reprod. Fertil. Dev. 27, 254) were submitted to in vitro differentiation into epiblast-like cells (EpiLC) and iPGC by the protocol adapted from mice (Hayashi et al. 2011 Cell 146, 519-532). The biPS cells were induced into EpiLC by culture in fibronectin-coated (16.7 µg mL−1) 6-well plates in N2B27 culture medium supplemented with 20 ng mL−1 activin A, 12 ng mL−1 basic fibroblast growth factor (bFGF), and 1% knockout serum replacement (KSR) for 48 h and further differentiated into iPGC by non-adherent culture (Agreewell plates, StemCell Technologies, Vancouver, BC, Canada) with GK15 medium (GMEM supplemented with 15% KSR, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, 0.1 mM 2-mercaptoethanol, 2 mm l-glutamine, and 1% antibiotics) in the presence of 500 ng mL−1 BMP4, 100 ng mL−1 SCF, 500 ng mL−1 BMP8b, and 50 ng mL−1 epidermal growth factor for 4 days. The cells were then characterised regarding morphology, detection of alkaline phosphatase, immunofluorescence for OCT4, DDX4, VASA, and c-Kit proteins, and transcripts of pluripotency-related genes OCT4 and SOX2, as well as of imprinted genes (H19, SNRPN) and imprinted-related (DNMT1, DNMT3B) genes were analysed through RT-qPCR and compared with constitutive genes GAPDH, NAT1, and ACTB. Alkaline phosphatase and immunofluorescence analysis were positive for all specific markers. Interestingly, although OCT4 and SOX2 expression was present in iPS, EpiLC, and iPGC, this last group presented greater OCT4 and lesser SOX2 transcript amounts compared with other groups, suggesting, as expected, that PGC are still pluripotent but may already be differentiating into germ-cell lineages. The expression of H19 was increased in iPGC, whereas the expression of SNRPN was decreased only in the fibroblast group, potentially indicating epigenetic reprogramming process in these cells. Expression of DNMT1 and DNMT3B was not different between pluripotent groups but subtly increased when compared with that in fibroblasts. The results obtained herein represent an important first step in the in vitro generation of PGC and gametes from domestic farm animals, an unprecedented and desirable tool for enhancing new reproductive technologies and providing new understanding of cellular reprogramming and pluripotent germ cell biology. Financially supported by FAPESP grants 2013/08135-2, 2013/13686-8, 2015/26818-5; CNPq 482163/2013-5.

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Epigenetic features of testicular germ cell tumours in relation to epigenetic characteristics of foetal germ cells

The International Journal of Developmental Biology ◽

10.1387/ijdb.130142ka ◽

2013 ◽

Vol 57 (2-3-4) ◽

pp. 309-317 ◽

Cited By ~ 31

Author(s):

Dina G. Kristensen ◽

Niels E. Skakkebk ◽

Ewa Rajpert-De Meyts ◽

Kristian Almstrup

Keyword(s):

Germ Cell ◽

Germ Cells ◽

Germ Cell Tumours ◽

Testicular Germ Cell ◽

Testicular Germ Cell Tumours

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Ligand–Receptor Interactions Elucidate Sex-Specific Pathways in the Trajectory From Primordial Germ Cells to Gonia During Human Development

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.661243 ◽

2021 ◽

Vol 9 ◽

Author(s):

Arend W. Overeem ◽

Yolanda W. Chang ◽

Jeroen Spruit ◽

Celine M. Roelse ◽

Susana M. Chuva De Sousa Lopes

Keyword(s):

Germ Cell ◽

Signaling Pathways ◽

Germ Cells ◽

Tyrosine Kinases ◽

Intracellular Localization ◽

Primordial Germ Cells ◽

Cell Lineage ◽

Systematic Analysis ◽

Receptor Interactions

The human germ cell lineage originates from primordial germ cells (PGCs), which are specified at approximately the third week of development. Our understanding of the signaling pathways that control this event has significantly increased in recent years and that has enabled the generation of PGC-like cells (PGCLCs) from pluripotent stem cells in vitro. However, the signaling pathways that drive the transition of PGCs into gonia (prospermatogonia in males or premeiotic oogonia in females) remain unclear, and we are presently unable to mimic this step in vitro in the absence of gonadal tissue. Therefore, we have analyzed single-cell transcriptomics data of human fetal gonads to map the molecular interactions during the sex-specific transition from PGCs to gonia. The CellPhoneDB algorithm was used to identify significant ligand–receptor interactions between germ cells and their sex-specific neighboring gonadal somatic cells, focusing on four major signaling pathways WNT, NOTCH, TGFβ/BMP, and receptor tyrosine kinases (RTK). Subsequently, the expression and intracellular localization of key effectors for these pathways were validated in human fetal gonads by immunostaining. This approach provided a systematic analysis of the signaling environment in developing human gonads and revealed sex-specific signaling pathways during human premeiotic germ cell development. This work serves as a foundation to understand the transition from PGCs to premeiotic oogonia or prospermatogonia and identifies sex-specific signaling pathways that are of interest in the step-by-step reconstitution of human gametogenesis in vitro.

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Interactome analyses reveal profound proteasome structural and functional rearrangements throughout mammalian spermatogenesis

10.1101/2021.06.18.447862 ◽

2021 ◽

Author(s):

Dusan Zivkovic ◽

Angelique Sanchez Dafun ◽

Thomas Menneteau ◽

Adrien Schahl ◽

Sandrine Lise ◽

...

Keyword(s):

Mass Spectrometry ◽

Germ Cell ◽

Germ Cells ◽

Protein Assembly ◽

Homologous Chromosomes ◽

High Proteolytic Activity ◽

Complex Processes ◽

Germ Cell Differentiation ◽

Mechanism Function

During spermatogenesis, spermatogonia undergo a series of mitotic and meiotic divisions on their path to spermatozoa. To achieve this, a succession of complex processes requiring high proteolytic activity are in part orchestrated by the proteasome. The spermatoproteasome (s20S) is a proteasome subtype specific to the developing gametes, in which the gamete-specific α4s subunit replaces the α4 isoform found in the constitutive proteasome (c20S). Although the s20S is conserved across species and was shown to be crucial for germ cell development, its mechanism, function and structure remain incompletely characterized. Here, we used advanced mass spectrometry (MS) methods to map the composition of proteasome complexes and their interactomes throughout spermatogenesis. We observed that the s20S becomes highly activated as germ cells enter meiosis, mainly through association with proteasome activators PA200 and 19S. Additionally, the proteasome population shifts from predominantly c20S (98%) to predominantly s20S (>82-92%) during differentiation, presumably due to the shift from α4 to α4s expression. We confirmed that s20S, but not c20S, interacts with components of the synaptonemal complex, the multi-protein assembly that connects homologous chromosomes during meiosis. In vitro, s20S preferentially bind to 19S, and displayed higher trypsin- and chymotrypsin-like activities, both with and without PA200 activation. Moreover, using MS methods to monitor protein dynamics, we identified significant differences in domain flexibility between α4 and α4s. We propose that these differences induced by α4s incorporation result in significant changes in the way the s20S interacts with its partners, and dictate its role in germ cell differentiation.

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Vascular endothelial growth factor regulates germ cell survival during establishment of spermatogenesis in the bovine testis

Reproduction ◽

10.1530/rep-09-0020 ◽

2009 ◽

Vol 138 (4) ◽

pp. 667-677 ◽

Cited By ~ 47

Author(s):

Kyle C Caires ◽

Jeanene de Avila ◽

Derek J McLean

Keyword(s):

Vascular Endothelial Growth Factor ◽

Growth Factor ◽

Germ Cell ◽

Cell Survival ◽

Germ Cells ◽

Pcr Analysis ◽

Vascular Endothelial ◽

Endothelial Growth Factor ◽

Testis Tissue

Vascular endothelial growth factor-A (VEGFA) is a hypoxia-inducible peptide essential for angiogenesis and targets nonvascular cells in a variety of tissues and cell types. The objective of the current study was to determine the function of VEGF during testis development in bulls. We used an explant tissue culture and treatment approach to test the hypothesis that VEGFA-164 could regulate the biological activity of bovine germ cells. We demonstrate that VEGFA, KDR, and FLT1 proteins are expressed in germ and somatic cells in the bovine testis. Treatment of bovine testis tissue with VEGFA in vitro resulted in significantly more germ cells following 5 days of culture when compared with controls. Quantitative real-time RT-PCR analysis determined that VEGF treatment stimulated an intracellular response that prevents germ cell death in bovine testis tissue explants, as indicated by increased expression of BCL2 relative to BAX and decreased expression of BNIP3 at 3, 6, and 24 h during culture. Blocking VEGF activity in vitro using antisera against KDR and VEGF significantly reduced the number of germ cells in VEGF-treated testis tissue to control levels at 120 h. Testis grafting provided in vivo evidence that bovine testis tissue treated with VEGFA for 5 days in culture contained significantly more differentiating germ cells compared with controls. These findings support the conclusion that VEGF supports germ cell survival and sperm production in bulls.

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Are cranial germ cell tumours really tumours of germ cells?

Neuropathology and Applied Neurobiology ◽

10.1111/j.1365-2990.2006.00797.x ◽

2006 ◽

Vol 32 (6) ◽

pp. 569-574 ◽

Cited By ~ 13

Author(s):

P. J. Scotting

Keyword(s):

Germ Cell ◽

Germ Cells ◽

Germ Cell Tumours

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Hepatocyte growth factor modulates in vitro survival and proliferation of germ cells during postnatal testis development

Journal of Endocrinology ◽

10.1677/joe.1.06528 ◽

2006 ◽

Vol 189 (1) ◽

pp. 137-146 ◽

Cited By ~ 11

Author(s):

A Catizone ◽

G Ricci ◽

J Del Bravo ◽

M Galdieri

Keyword(s):

Growth Factor ◽

Hepatocyte Growth Factor ◽

Germ Cell ◽

Germ Cells ◽

Cell Types ◽

Tyrosine Kinase Receptor ◽

Testis Development ◽

Pachytene Spermatocytes ◽

Hepatocyte Growth

The hepatocyte growth factor (HGF) is a pleiotropic cytokine that influences mitogenesis, motility and differentiation of many different cell types by its tyrosine kinase receptor c-Met. We previously demonstrated that the c-Met/HGF system is present and functionally active during postnatal testis development. We found also that spermatozoa express c-Met and that HGF has a positive effect on the maintenance of sperm motility. In the present paper, we extend our study on the germ cells at different stages of differentiation during the postnatal development of the testis. We demonstrate that c-met is present in rat spermatogonia, pachytene spermatocytes and round spermatids and that HGF significantly increases spermatogonial proliferation in 8- to 10-day-old pre-pubertal rats. At this age HGF does not affect Sertoli cells and peritubular myoid cells proliferation. In addition, we studied the effect of the factor on germ cell apoptosis and we show that HGF prevents the germ cell apoptotic process. We also studied the effect of HGF on 18- to 20-day-old and 28- to 30-day-old rat testes. At these ages also the factor significantly increases germ cell duplication and decreases the number of apoptotic cells. However, the effect on programmed cell death is higher in the 8- to 10-day-old rats and declines in the older animals. In conclusion, we report that rat germ cells (spermatogonia, pachytene spermatocytes and round spermatids) express c-met and that HGF modulates germ cell proliferating activity and apoptosis in vitro. These data indicate that the c-Met/HGF system is involved in male germ cell homeostasis and, consequently, has a role in male fertility.

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154 MOLECULAR CHARACTERIZATION OF CAT PRIMORDIAL GERM CELLS

Reproduction Fertility and Development ◽

10.1071/rdv28n2ab154 ◽

2016 ◽

Vol 28 (2) ◽

pp. 207

Author(s):

J. Galiguis ◽

C. E. Pope ◽

C. Dumas ◽

G. Wang ◽

R. A. MacLean ◽

...

Keyword(s):

Stem Cells ◽

Germ Cell ◽

Germ Cells ◽

Primordial Germ Cells ◽

Primordial Germ Cell ◽

Embryonic Stem ◽

Transcript Abundance ◽

Domestic Cat ◽

Sample Type

As precursors to germline stem cells and gametes, there are many potential applications for primordial germ cells (PGC). Primordial germ cell-like cells have been generated from mouse embryonic stem cells and induced pluripotent stem cells, which subsequently were used to produce functional spermatozoa, oocytes, and healthy offspring (Hayashi et al. 2012 Science 338(6109), 971–975). Applying this approach to generate sperm and oocytes of endangered species is an appealing prospect. Detection of molecular markers associated with PGC is essential to optimizing the process of PGC induction. In the current study, in vitro-derived domestic cat embryos were assessed at various developmental stages to characterise the expression of markers related to the specification process of cat PGC. In vivo-matured, IVF oocytes were cultured until Days 7, 9, and 12 post-insemination. Then, embryos were assessed by RT-qPCR to determine relative transcript abundance of the pluripotency markers NANOG, POU5F1, and SOX2; the epiblast marker DNMT3B; the primitive endoderm marker GATA4; the PGC marker PRDM14; and the germ cell marker VASA; RPS19 was used as the internal reference gene. To validate the qPCR results, fibroblasts served as the negative control cells, whereas spermatogonial stem cells (SSC) served as the positive control cells for GATA4, PRDM14, and VASA. Total mRNA were isolated using the Cells-to-cDNA™ II Kit (Ambion/Thermo Fisher Scientific, Waltham, MA, USA) from either pools of 2 to 6 embryos or ~25 000 fibroblasts/SSC. A minimum of 2 biological replicates for each sample type was analysed, with transcript abundance detected in 2 technical replicates by SYBR Green chemistry. Student’s t-tests were performed on the ΔCts for statistical analysis. PRDM14, specific to the germ cell lineage, was detected as early as Day 7, suggesting the presence of PGC precursor cells. Compared with their levels at Day 7, PRDM14 expression was 0.34-fold lower in SSC (P < 0.05), whereas expression of VASA and GATA4 were 1964-fold and 144-fold higher, respectively (P < 0.05). This seems to emphasise the relative importance of PRDM14 in pre-germ cell stages. In general, all genes analysed were up-regulated from Day 7 to Day 9. This up-regulation was statistically significant for SOX2 and GATA4 (P < 0.05). Relative to that at Day 9, all transcripts were relatively less abundant at Day 12 (P < 0.05 for NANOG, POU5F1, SOX2, DNMT3B, and PRDM14). The data suggest that PGC specification takes place near Day 9, with peak specification activity concluding by Day 12. Although much needs be explored about PGC specification in the cat before applying induction and in vitro germ cell production techniques, these findings represent the first step towards a new potential strategy for preserving endangered and threatened felids.

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Optimization of In Vitro Culture Conditions of Sturgeon Germ Cells for Purpose of Surrogate Production

Animals ◽

10.3390/ani9030106 ◽

2019 ◽

Vol 9 (3) ◽

pp. 106 ◽

Cited By ~ 2

Author(s):

Xuan Xie ◽

Ping Li ◽

Martin Pšenička ◽

Huan Ye ◽

Christoph Steinbach ◽

...

Keyword(s):

Germ Cell ◽

Germ Cells ◽

Somatic Cells ◽

Culture Conditions ◽

Specific Gene ◽

Acipenser Gueldenstaedtii ◽

Acipenser Ruthenus ◽

Before And After ◽

Optimal Culture Condition

To expand germ cell populations and provide a consistent supply for transplantation, we established basal culture conditions for sturgeon germ cells and subsequently increased their mitotic activity by eliminating gonad somatic cells, supplementing with growth factor, and replacing fetal bovine serum (FBS). The initial basal culture conditions were Leibovitz’s L-15 medium (pH 8.0) supplemented with 5% FBS (p < 0.001) at 21 °C. Proliferation of germ cells was significantly enhanced and maintained for longer periods by elimination of gonad somatic cells and culture under feeder-cell free conditions, with addition of leukemia inhibitory factor and glial-cell-derived neurotrophic factor (p < 0.001). A serum-free culture medium improved germ cell proliferation compared to the L-15 with FBS (p < 0.05). Morphology remained similar to that of fresh germ cells for at least 40 d culture. Germline-specific gene expression analysis revealed no significant changes to germ cells before and after culture. Sterlet Acipenser ruthenus germ cells cultured more than 40 days showed development after transplant into Russian sturgeon Acipenser gueldenstaedtii. Polymerase chain reaction showed 33.3% of recipient gonads to contain sterlet cells after four months. This study developed optimal culture condition for sturgeon germ cells. Germ cells after 40 d culture developed in recipient gonads. This study provided useful information for culture of sturgeon germ cells.

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