Specification of PGCLCs from ESCs in vitro:
Germline specification occurs early during embryogenesis, when the primordial germ cells (PGCs) are relocated to the extraembryonic compartment. These germcell progenitors migrate to gonal ridges, undergo proliferation and meiosis to form haploid cells. To demonstrate meiosis occurs in vitro, four criteria need to be satisfied: (1) correct DNA content at individual metotic stages, (2)normal number and organization of chromosomes, (3) appropriate nuclear and chromosomal localization, (4) viable and fertile offspring generated from derived gametes.
Germline specification occurs early during embryogenesis, when the primordial germ cells (PGCs) are relocated to the extraembryonic compartment. These germcell progenitors migrate to gonal ridges, undergo proliferation and meiosis to form haploid cells. To demonstrate meiosis occurs in vitro, four criteria need to be satisfied: (1) correct DNA content at individual metotic stages, (2)normal number and organization of chromosomes, (3) appropriate nuclear and chromosomal localization, (4) viable and fertile offspring generated from derived gametes.
Mouse and human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) can undergo epiliblast-like developent program in vitro to induce epiblast-like cells (EpiLCs) that are can specify into PGCC-like cells (PGCLCs), from which they can produce haploid spermatozoa. In this study, they demonstrated a complete in vitro meiosis of murine ESC-derived PGCLCs to generate male spermatid like cells (SLCs) that are capable of producing healthy and fertile offspring via ICSC.
Blimp1-mVenus and Stella-ECFP double transgenic ESC line (BVSC) helped identify PGCs.Stra8-EGFP and Prm1-DsRed double transgenic ESC line (SGPD) identify meiotic spermatids. Both of these ESC lines have normal karyotype and produce completely ESC_derived live offspring that confirm their pluripotency. They first culture the SGPD and BVSC in serum-free and feeder-free conditions with addition of GSK3 inhibitor, MEK inhibitor, and LIF. Then these cells were exposed to ActA and bFGF to induce EpiLCs. Then the induced EpiLCs were continued to be cultured in differentiation medium containing EGF, SCF, LIF, BMP4, BMP8b. In this culture, EpiLCs differentiated into PGCLCs which were confirmed by expression of Blimp1-mVenus and Stella-ECFP after 4-6d. FACS helped isolate 11% of PGC cells from the total population based on PGC markers integrin b3 and SSEA1 (integrin b3+/SSEA1+). In addition, PGC genes were upregulated and somatic cell-related genes were downregulated in these cells, indicating in vitro PGCLC specification. Overall, they demonstrated that BGPD and BVSC ESCs were able to induce into PGCLCs.
Initiation of Meiosis in PGCLCs in vitro in Co-Culture:
Co-culturing PGCLCswith early postnatal testicular cells from KITw/KITw-v mice. It was known that high levels of CYP26B1 in fetal-stage gonadal somatic cells prohibits entry of male germ cells into meiosis by metabolizing endogenous retinoic acid (RA). So, in these early postnatal somatic testicular cells expressed low CYP26B1 levels, which were comparable with those of female fetal gonad at the stage of meiotic induction of PGCs at E13.5. Therefore, post-natal testicular cells of KITw/KITw-v mice provided a suitable environment for meiosis initiation when induced PGCLCs
were cultured with them. Three morphogenes (ActA, BMPs, and RA)-containing medium allowed the expression of Stra8-EGFP after 3 days, confirming the initiation of meiosis of these PGCLCs. In addition, they also observed the migration of PGCLCs toward Stra8-EGFP-expressing cells to form aggregation colonies within 6d. The Stra8-EGFP-positive colonies were DDX4+ (germ cells), GATA4+ (testis somatic cells), and SOX9+ (Sertoli cells), whereas PGC markers SSEA1 and OCT4 were undetectable. Therefore, PGC/SSC cells had differentiated into SGPD PGCLCs comprising of multiple testis cell types through meiosis. Moreover, BVSC cells expressed downregulated Blimp1-mVenus and Stella-ECFP expression, supporting the hypothesis.
Hormonal stimulation induces the formation of haploid SLCs in vitro:
On d7 of co-culture, they stop providing SGPD with ActA, BMPs, and RA, but instead adding FSh and testosterone (T), and BPE. In the presence of FSH/BPE/T, postmeiotic Prm1-DsRed-expressing cells became apparent on d10. haploid spermatid markers tp1,Prm1, acrosin and haprin were upregualted and 14% of these cells contained half DNA, conforming the haploid spermatid-like cell (SLC) formation. Therefore, all three hormones were required for progression of meiosis.
Chromosome synapsis and recombination in meiosis in vitro:
The meiosis progression was assessed via chromosomal synapsis and recombination which require the initiation and resolution of DNA double-strand breaks (DSBs). This SPO11 and RAD51 foci indicated the presence of DSBs in differentiating cells on d8. In addition, the broad distribution of gamma-H2AX on d8 reflected an association with DSBs and the its disappearance from the autosome region on d10 and accumulation on the unsynapsed sex chromosomes indicated the completion of synapsis. These events recapitulated the pachytene stage spermatocytes in vivo. The detection of SYCP1 and SYCP3 also indicated the progression of synapsis. After d8, 90% of spermatocytes were at the leptotene or zygotene stage of meiosis. On d10, 64% were at pachytene stage, indicating the completion of DSB repair and completion of synapsis. By d12, 50% entered diplotene. These results demonstrated that meiosis in vitro encompassed synapsis and recombination and was synchronized in the majority of germ cells. The expression levels of transcription factors during meiosis in these germ cells were correlated with in vivo meiosis. The observed conversion of PGCLCs to SLCs was only 10% (1 PGCLC is supposed to give 4 SLCs). On d12, Dividing Prm1-DsRed-positive cells were detected in co-cultures, indicating the formation of haploid cells.
Healthy fertile offspring produced by ICSC with in vitro derived SLCs:
Sorted SLCs contained a cap-shaped acrosome. Small and round cells were selected for sequencing and most of them 75% were haploid and have normal genome structure. The global transcription profile revealed similarities between in vitro SLCs and in vivo spermatids. ICSC was then used to fertilize oocytes with SGPD-derived SLCs, 80-85% developed into 2-cell stage embryos after activation. ICSC was also fertilize oocytes with BVSC-derived SLCs, 83% developed into 2-cell stage embryos. Bisulfite sequencing indicated a pattern associated with maternal and paternal genetic contributions. Embryos from BVSC-derived SLCs developed into live pups with Blimp1-mVenus and Stella-ECFP transgenes, normal karyotype, and normal weight gain. The proportion of high methylation sites (>80%) of the bVSC-derived pffspirng and control mice were all lower than that of sperm but higher than that of the oocyte.
The chromosomes pair up (a process called synapsis) to form a structure known as either a bivalent (two chromosomes) or a tetrad (four chromatids).
Prophase I is the stage of meiosis where the homologous chromosomes pair and exchange DNA (genetic recombination).
Prophase I comprised of five stages; 1) leptotene, 2) zygotene, 3) pachytene, 4) diplotene and 5) diakinesis.
1) Leptotene (Greek for "thin threads"): chromosomes begin to condense.
2) Zygotene (Greek for "paired threads"): chromosomes become closely paired.
3) Pachytene (Greek for "thick threads"): crossing over occurs.
4) Diplotene (Greek for "two threads"): homologous chromosomes begin to separate but remain attached by the chiasmata.
5) Diakinesis (Greek for "moving through"): chromosomes condense and separate until terminal chiasmata only connect the two chromosomes.
The chiasmata can be formed anywhere along the chromosome but during diakinesis, the chiasmatic connections are translated to the chromosomes' ends.
The homologous chromosomes are held together during synapsis by the synaptonemal complex.
Metaphase I: Bivalent chromosomes attach to the spindle and align at the metaphase plate.
The bivalents are randomly oriented with respect to the poles such that chromosomes (maternal or paternal or both) are evenly sorted.
Only the chiasmata hold the paired homologues together.
Anaphase I: Homologous chromosomes resolve the chiasmata and move (as a bivalent) to opposite ends of the cell.
Telophase I: Chromosomes arrive at the poles and nuclear envelopes form and cytokinesis occurs.
Meiosis II: The second part of meiosis (meiosis II) is very similar to mitotic division except that DNA synthesis does not occur between the two stages."
Blimp1-mVenus and Stella-ECFP double transgenic ESC line (BVSC) helped identify PGCs.Stra8-EGFP and Prm1-DsRed double transgenic ESC line (SGPD) identify meiotic spermatids. Both of these ESC lines have normal karyotype and produce completely ESC_derived live offspring that confirm their pluripotency. They first culture the SGPD and BVSC in serum-free and feeder-free conditions with addition of GSK3 inhibitor, MEK inhibitor, and LIF. Then these cells were exposed to ActA and bFGF to induce EpiLCs. Then the induced EpiLCs were continued to be cultured in differentiation medium containing EGF, SCF, LIF, BMP4, BMP8b. In this culture, EpiLCs differentiated into PGCLCs which were confirmed by expression of Blimp1-mVenus and Stella-ECFP after 4-6d. FACS helped isolate 11% of PGC cells from the total population based on PGC markers integrin b3 and SSEA1 (integrin b3+/SSEA1+). In addition, PGC genes were upregulated and somatic cell-related genes were downregulated in these cells, indicating in vitro PGCLC specification. Overall, they demonstrated that BGPD and BVSC ESCs were able to induce into PGCLCs.
Initiation of Meiosis in PGCLCs in vitro in Co-Culture:
Co-culturing PGCLCswith early postnatal testicular cells from KITw/KITw-v mice. It was known that high levels of CYP26B1 in fetal-stage gonadal somatic cells prohibits entry of male germ cells into meiosis by metabolizing endogenous retinoic acid (RA). So, in these early postnatal somatic testicular cells expressed low CYP26B1 levels, which were comparable with those of female fetal gonad at the stage of meiotic induction of PGCs at E13.5. Therefore, post-natal testicular cells of KITw/KITw-v mice provided a suitable environment for meiosis initiation when induced PGCLCs
were cultured with them. Three morphogenes (ActA, BMPs, and RA)-containing medium allowed the expression of Stra8-EGFP after 3 days, confirming the initiation of meiosis of these PGCLCs. In addition, they also observed the migration of PGCLCs toward Stra8-EGFP-expressing cells to form aggregation colonies within 6d. The Stra8-EGFP-positive colonies were DDX4+ (germ cells), GATA4+ (testis somatic cells), and SOX9+ (Sertoli cells), whereas PGC markers SSEA1 and OCT4 were undetectable. Therefore, PGC/SSC cells had differentiated into SGPD PGCLCs comprising of multiple testis cell types through meiosis. Moreover, BVSC cells expressed downregulated Blimp1-mVenus and Stella-ECFP expression, supporting the hypothesis.
Hormonal stimulation induces the formation of haploid SLCs in vitro:
On d7 of co-culture, they stop providing SGPD with ActA, BMPs, and RA, but instead adding FSh and testosterone (T), and BPE. In the presence of FSH/BPE/T, postmeiotic Prm1-DsRed-expressing cells became apparent on d10. haploid spermatid markers tp1,Prm1, acrosin and haprin were upregualted and 14% of these cells contained half DNA, conforming the haploid spermatid-like cell (SLC) formation. Therefore, all three hormones were required for progression of meiosis.
Chromosome synapsis and recombination in meiosis in vitro:
The meiosis progression was assessed via chromosomal synapsis and recombination which require the initiation and resolution of DNA double-strand breaks (DSBs). This SPO11 and RAD51 foci indicated the presence of DSBs in differentiating cells on d8. In addition, the broad distribution of gamma-H2AX on d8 reflected an association with DSBs and the its disappearance from the autosome region on d10 and accumulation on the unsynapsed sex chromosomes indicated the completion of synapsis. These events recapitulated the pachytene stage spermatocytes in vivo. The detection of SYCP1 and SYCP3 also indicated the progression of synapsis. After d8, 90% of spermatocytes were at the leptotene or zygotene stage of meiosis. On d10, 64% were at pachytene stage, indicating the completion of DSB repair and completion of synapsis. By d12, 50% entered diplotene. These results demonstrated that meiosis in vitro encompassed synapsis and recombination and was synchronized in the majority of germ cells. The expression levels of transcription factors during meiosis in these germ cells were correlated with in vivo meiosis. The observed conversion of PGCLCs to SLCs was only 10% (1 PGCLC is supposed to give 4 SLCs). On d12, Dividing Prm1-DsRed-positive cells were detected in co-cultures, indicating the formation of haploid cells.
Healthy fertile offspring produced by ICSC with in vitro derived SLCs:
Sorted SLCs contained a cap-shaped acrosome. Small and round cells were selected for sequencing and most of them 75% were haploid and have normal genome structure. The global transcription profile revealed similarities between in vitro SLCs and in vivo spermatids. ICSC was then used to fertilize oocytes with SGPD-derived SLCs, 80-85% developed into 2-cell stage embryos after activation. ICSC was also fertilize oocytes with BVSC-derived SLCs, 83% developed into 2-cell stage embryos. Bisulfite sequencing indicated a pattern associated with maternal and paternal genetic contributions. Embryos from BVSC-derived SLCs developed into live pups with Blimp1-mVenus and Stella-ECFP transgenes, normal karyotype, and normal weight gain. The proportion of high methylation sites (>80%) of the bVSC-derived pffspirng and control mice were all lower than that of sperm but higher than that of the oocyte.
"Meiosis
In the first part of meiosis (meiosis I) an unusual type of cell division produces two haploid cells that have chromosomes made up of two sister chromatids.The chromosomes pair up (a process called synapsis) to form a structure known as either a bivalent (two chromosomes) or a tetrad (four chromatids).
Prophase I is the stage of meiosis where the homologous chromosomes pair and exchange DNA (genetic recombination).
Prophase I comprised of five stages; 1) leptotene, 2) zygotene, 3) pachytene, 4) diplotene and 5) diakinesis.
1) Leptotene (Greek for "thin threads"): chromosomes begin to condense.
2) Zygotene (Greek for "paired threads"): chromosomes become closely paired.
3) Pachytene (Greek for "thick threads"): crossing over occurs.
4) Diplotene (Greek for "two threads"): homologous chromosomes begin to separate but remain attached by the chiasmata.
5) Diakinesis (Greek for "moving through"): chromosomes condense and separate until terminal chiasmata only connect the two chromosomes.
The chiasmata can be formed anywhere along the chromosome but during diakinesis, the chiasmatic connections are translated to the chromosomes' ends.
The homologous chromosomes are held together during synapsis by the synaptonemal complex.
Metaphase I: Bivalent chromosomes attach to the spindle and align at the metaphase plate.
The bivalents are randomly oriented with respect to the poles such that chromosomes (maternal or paternal or both) are evenly sorted.
Only the chiasmata hold the paired homologues together.
Anaphase I: Homologous chromosomes resolve the chiasmata and move (as a bivalent) to opposite ends of the cell.
Telophase I: Chromosomes arrive at the poles and nuclear envelopes form and cytokinesis occurs.
Meiosis II: The second part of meiosis (meiosis II) is very similar to mitotic division except that DNA synthesis does not occur between the two stages."
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