scholarly journals Testes of DAZL null sheep lack spermatogonia and maintain normal somatic cells

2019 ◽  
Author(s):  
Zachariah McLean ◽  
Sarah Jane Appleby ◽  
Jingwei Wei ◽  
Russell Grant Snell ◽  
Björn Oback
Keyword(s):  
Germ Cell ◽  
Somatic Cell ◽  
Germ Cells ◽  
Genetic Improvement ◽  
Rna Binding ◽  
Somatic Cells ◽  
Marker Genes ◽  
Specific Marker ◽  
Loss Of Function ◽  
Fetal Fibroblasts

AbstractMultiplying the germline would increase the number of offspring that can be produced from selected animals, accelerating genetic improvement for livestock breeding. This could be achieved by producing multiple chimaeric animals, each carrying a mix of donor and host germ cells in their gonads. However, such chimaeric germlines would produce offspring from both donor and host genotypes, limiting the rate of genetic improvement. To resolve this problem and produce chimaeras with absolute donor germline transmission, we have disrupted the RNA-binding protein DAZL and generated germ cell-deficient host animals. Using Cas9 mediated homology-directed repair (HDR), we introduced a DAZL loss-of-function mutation in male ovine fetal fibroblasts. Following manual single-cell isolation, 4/48 (8.3%) of donor cell strains were homozygously HDR-edited. Sequence-validated strains were used as nuclear donors for somatic cell cloning to generate three lambs, which died at birth. All DAZL-null male neonatal sheep lacked germ cells. Somatic cells within their testes were morphologically intact and expressed normal levels of somatic cell-specific marker genes, indicating that the germ cell niche remained intact. This extends the DAZL-mutant phenotype beyond mice into agriculturally relevant ruminants, providing a pathway for using absolute transmitters in rapid livestock improvement.

scholarly journals Müllerian Inhibiting Substance Is Required for Germ Cell Proliferation during Early Gonadal Differentiation in Medaka (Oryzias latipes)

Endocrinology ◽  
2007 ◽  
Vol 149 (4) ◽  
pp. 1813-1819 ◽  
Author(s):  
Eri Shiraishi ◽  
Norifumi Yoshinaga ◽  
Takeshi Miura ◽  
Hayato Yokoi ◽  
Yuko Wakamatsu ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Germ Cell ◽  
Germ Cells ◽  
Sex Differentiation ◽  
Somatic Cells ◽  
Oryzias Latipes ◽  
Cell Number ◽  
Gonadal Differentiation ◽  
Loss Of Function ◽  
Germ Cell Proliferation

Müllerian inhibiting substance (MIS) is a glycoprotein belonging to the TGF-β superfamily. In mammals, MIS is responsible for the regression of Müllerian ducts in the male fetus. However, the role of MIS in gonadal sex differentiation of teleost fish, which have no Müllerian ducts, has yet to be clarified. In the present study, we examined the expression pattern of mis and mis type 2 receptor (misr2) mRNAs and the function of MIS signaling in early gonadal differentiation in medaka (teleost, Oryzias latipes). In situ hybridization showed that both mis and misr2 mRNAs were expressed in the somatic cells surrounding the germ cells of both sexes during early sex differentiation. Loss-of-function of either MIS or MIS type II receptor (MISRII) in medaka resulted in suppression of germ cell proliferation during sex differentiation. These results were supported by cell proliferation assay using 5-bromo-2′-deoxyuridine labeling analysis. Treatment of tissue fragments containing germ cells with recombinant eel MIS significantly induced germ cell proliferation in both sexes compared with the untreated control. On the other hand, culture of tissue fragments from the MIS- or MISRII-defective embryos inhibited proliferation of germ cells in both sexes. Moreover, treatment with recombinant eel MIS in the MIS-defective embryos dose-dependently increased germ cell number in both sexes, whereas in the MISRII-defective embryos, it did not permit proliferation of germ cells. These results suggest that in medaka, MIS indirectly stimulates germ cell proliferation through MISRII, expressed in the somatic cells immediately after they reach the gonadal primordium.

scholarly journals Abnormal Meiosis Initiation in Germ Cell Caused by Aberrant Differentiation of Gonad Somatic Cell

2019 ◽  
Vol 2019 ◽  
pp. 1-8
Author(s):  
Min Chen ◽  
Min Chen ◽  
Suren Chen ◽  
Jingjing Zhou ◽  
Fangfang Dong ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
Germ Cell ◽  
Somatic Cell ◽  
Germ Cells ◽  
Somatic Cells ◽  
Ectopic Expression ◽  
Genital Ridge ◽  
Male And Female ◽  
Germ Cell Differentiation ◽  
Exact Function

The interaction between germ cell and somatic cell plays important roles in germ cell development. However, the exact function of gonad somatic cell in germ cell differentiation is unclear. In the present study, the function of gonad somatic cell in germ cell meiosis was examined by using mouse models with aberrant somatic cell differentiation. In Wt1R394W/R394W mice, the genital ridge is absent due to the apoptosis of coelomic epithelial cells. Interestingly, in both male and female Wt1R394W/R394W germ cells, STRA8 was detected at E12.5 and the scattered SYCP3 foci were observed at E13.5 which was consistent with control females. In Wt1-/flox; Cre-ERTM mice, Wt1 was inactivated by the injection of tamoxifen at E9.5 and the differentiation of Sertoli and granulosa cells was completely blocked. We found that most germ cells were located outside of genital ridge after Wt1 inactivation. STRA8, SYCP3, and γH2AX proteins were detected in germ cells of both male and female Wt1-/flox; Cre-ERTM gonads, whereas no thread-like SYCP3 signal was observed. Our study demonstrates that aberrant development of gonad somatic cells leads to ectopic expression of meiosis-associated genes in germ cells, but meiosis was arrested before prophase I. These results suggest that the proper differentiation of gonad somatic cells is essential for germ cell meiosis.

scholarly journals Single-Cell Atlas of Adult Testis in Protogynous Hermaphroditic Orange-Spotted Grouper, Epinephelus coioides

2021 ◽  
Vol 22 (22) ◽  
pp. 12607
Author(s):  
Xi Wu ◽  
Yang Yang ◽  
Chaoyue Zhong ◽  
Tong Wang ◽  
Yanhong Deng ◽  
...  
Keyword(s):  
Single Cell ◽  
Germ Cells ◽  
Expression Patterns ◽  
Somatic Cells ◽  
Human Testis ◽  
Marker Genes ◽  
Specific Marker ◽  
Epinephelus Coioides ◽  
Male Germ Cells ◽  
Adult Testis

Spermatogenesis is a process of self-renewal and differentiation in spermatogonial stem cells. During this process, germ cells and somatic cells interact intricately to ensure long-term fertility and accurate genome propagation. Spermatogenesis has been intensely investigated in mammals but remains poorly understood with regard to teleosts. Here, we performed single-cell RNA sequencing of ~9500 testicular cells from the male, orange-spotted grouper. In the adult testis, we divided the cells into nine clusters and defined ten cell types, as compared with human testis data, including cell populations with characteristics of male germ cells and somatic cells, each of which expressed specific marker genes. We also identified and profiled the expression patterns of four marker genes (calr, eef1a, s100a1, vasa) in both the ovary and adult testis. Our data provide a blueprint of male germ cells and supporting somatic cells. Moreover, the cell markers are candidates that could be used for further cell identification.

23 HYPOMETHYLATION OF DNA IN NUCLEAR TRANSFER EMBRYOS FROM PORCINE EMBRYONIC GERM CELLS

2007 ◽  
Vol 19 (1) ◽  
pp. 130
Author(s):  
K. S. Ahn ◽  
S. Y. Heo ◽  
J. Y. Won ◽  
H. Shim
Keyword(s):  
Dna Methylation ◽  
Somatic Cell ◽  
Germ Cells ◽  
Nuclear Transfer ◽  
Epigenetic Modification ◽  
Somatic Cells ◽  
Dna Demethylation ◽  
Term Survival ◽  
Cpg Sites ◽  
Fetal Fibroblasts

Epigenetic modification including genome-wide DNA demethylation is essential for normal embryonic development. Insufficient demethylation of the somatic cell genome may cause various anomalies and prenatal loss in the development of nuclear transfer embryos. Species-specific differences in the epigenetic status of cloned donor genomes have been reported. A level of DNA methylation in porcine somatic cell nuclear transfer (SCNT) embryos was similar to that of normally fertilized embryos, but hypermethylation of DNA in bovine SCNT embryos was commonly observed (Kang et al. 2001 J. Biol. Chem. 276, 39 980-39 984). Even in the same species, the source of the nuclear donor often affects later development of nuclear transfer embryos. In this study, appropriateness of porcine embryonic germ (EG) cells as karyoplasts for nuclear transfer with respect to epigenetic modification was investigated. These cells follow the methylation status of the primordial germ cells from which they originated, so they may contain a less methylated genome than somatic cells. The rates of blastocyst development were similar among embryos from EG cell nuclear transfer (EGCNT), SCNT, and intracytoplasmic sperm injection (ICSI) (16/62, 25.8% vs. 56/274, 20.4% vs. 16/74, 21.6%, respectively). Genomic DNA samples from EG cells (n = 3), fetal fibroblasts (n = 4), and blastocysts from EGCNT (n = 8), SCNT (n = 14), and ICSI (n = 6) were isolated and treated with sodium bisulfite. The satellite region (GenBank Z75640) that involves 9 selected CpG sites was amplified by PCR, and the rates of DNA methylation in each site were measured by pyrosequencing technique (Biotage AB, Uppsala, Sweden). The average methylation degrees of CpG sites in EG cells, fetal fibroblasts, and blastocysts from EGCNT, SCNT, and ICSI were 17.9, 37.7, 4.1, 9.8, and 8.9%, respectively. The genome of porcine EG cells was less methylated than that of somatic cells (P < 0.05), and DNA demethylation occurred in embryos from both EGCNT (P < 0.05) and SCNT (P < 0.01). However, the degree of DNA methylation in EGCNT embryos was approximately one-half that of SCNT (P < 0.01) and ICSI (P < 0.05) embryos; in SCNT and ICSI embryos, the genome was demethylated to the same degree. The present study demonstrated that porcine EG cell nuclear transfer results in hypomethylation of DNA in cloned embryos, yet leading to normal pre-implantation development. However, it would be interesting to further investigate whether such modification affects long-term survival of cloned embryos.

scholarly journals GC-1 spg cells are most similar to Leydig cells, a testis somatic cell-type, and not germ cells

2021 ◽  
Author(s):  
Andrew R Norman ◽  
Lauren Byrnes ◽  
Jeremy R Reiter
Keyword(s):  
Germ Cell ◽  
Somatic Cell ◽  
Cell Line ◽  
Germ Cells ◽  
Leydig Cells ◽  
Adult Mouse ◽  
Somatic Cells ◽  
Cell Type ◽  
Immortalized Cell ◽  
Somatic Cell Type

GC-1 spg is an immortalized cell line derived from an adult mouse testis and reported to be most similar to spermatocytes, a male germ cell-type. However, immunofluorescence indicates that GC-1 spg cells express WT1, a marker of testis somatic cells, and do not express markers of germ cells. Transcriptomic profiling indicate GC-1 cells are most similar to Leydig cells. Therefore, we conclude that GC-1 spg cells are most similar to testis somatic cells.

scholarly journals A Germ Cell-specific Gene, Prmt5, Works in Somatic Cell Reprogramming

2011 ◽  
Vol 286 (12) ◽  
pp. 10641-10648 ◽  
Author(s):  
Go Nagamatsu ◽  
Takeo Kosaka ◽  
Miyuri Kawasumi ◽  
Taisuke Kinoshita ◽  
Keiyo Takubo ◽  
...  
Keyword(s):  
Stem Cells ◽  
Germ Cell ◽  
Somatic Cell ◽  
Germ Cells ◽  
Pluripotent Stem Cells ◽  
Somatic Cells ◽  
Embryonic Stem ◽  
Cell Reprogramming ◽  
Specific Gene ◽  
Somatic Cell Reprogramming

Germ cells possess the unique ability to acquire totipotency during development in vivo as well as give rise to pluripotent stem cells under the appropriate conditions in vitro. Recent studies in which somatic cells were experimentally converted into pluripotent stem cells revealed that genes expressed in primordial germ cells (PGCs), such as Oct3/4, Sox2, and Lin28, are involved in this reprogramming. These findings suggest that PGCs may be useful for identifying factors that successfully and efficiently reprogram somatic cells into toti- and/or pluripotent stem cells. Here, we show that Blimp-1, Prdm14, and Prmt5, each of which is crucial for PGC development, have the potential to reprogram somatic cells into pluripotent stem cells. Among them, Prmt5 exhibited remarkable reprogramming of mouse embryonic fibroblasts into which Prmt5, Klf4, and Oct3/4 were introduced. The resulting cells exhibited pluripotent gene expression, teratoma formation, and germline transmission in chimeric mice, all of which were indistinguishable from those induced with embryonic stem cells. These data indicate that some of the factors that play essential roles in germ cell development are also active in somatic cell reprogramming.

scholarly journals Genetic control of programmed cell death in the Caenorhabditis elegans hermaphrodite germline

Development ◽  
1999 ◽  
Vol 126 (5) ◽  
pp. 1011-1022 ◽  
Author(s):  
T.L. Gumienny ◽  
E. Lambie ◽  
E. Hartwieg ◽  
H.R. Horvitz ◽  
M.O. Hengartner
Keyword(s):  
Cell Death ◽  
Caenorhabditis Elegans ◽  
Germ Cell ◽  
Somatic Cell ◽  
Cell Fate ◽  
Germ Cells ◽  
Mapk Pathway ◽  
Apoptotic Cell ◽  
C Elegans ◽  
Pathway Activation

Development of the nematode Caenorhabditis elegans is highly reproducible and the fate of every somatic cell has been reported. We describe here a previously uncharacterized cell fate in C. elegans: we show that germ cells, which in hermaphrodites can differentiate into sperm and oocytes, also undergo apoptotic cell death. In adult hermaphrodites, over 300 germ cells die, using the same apoptotic execution machinery (ced-3, ced-4 and ced-9) as the previously described 131 somatic cell deaths. However, this machinery is activated by a distinct pathway, as loss of egl-1 function, which inhibits somatic cell death, does not affect germ cell apoptosis. Germ cell death requires ras/MAPK pathway activation and is used to maintain germline homeostasis. We suggest that apoptosis eliminates excess germ cells that acted as nurse cells to provide cytoplasmic components to maturing oocytes.

scholarly journals Loss of Kallmann syndrome-associated gene WDR11 disrupts primordial germ cell development by affecting canonical and non-canonical Hedgehog signalling

2020 ◽  
Author(s):  
Jiyoung Lee ◽  
Yeonjoo Kim ◽  
Paris Ataliotis ◽  
Hyung-Goo Kim ◽  
Dae-Won Kim ◽  
...  
Keyword(s):  
Germ Cell ◽  
Germ Cells ◽  
Primary Cilia ◽  
Primordial Germ Cell ◽  
Kallmann Syndrome ◽  
Loss Of Function ◽  
Downstream Effectors ◽  
Cell Components ◽  
Underlying Mechanisms ◽  
And Migration

ABSTRACTMutations of WDR11 are associated with Kallmann syndrome (KS) and congenital hypogonadotrophic hypogonadism (CHH), typically caused by defective functions of gonadotrophin-releasing hormone (GnRH) neurones in the brain. We previously reported that Wdr11 knockout mice show profound infertility with significantly fewer germ cells present in the gonads. To understand the underlying mechanisms mediated by WDR11 in these processes, we investigated the effects of Wdr11 deletion on primordial germ cell (PGC) development. Using live-tracking of PGCs and primary co-cultures of genital ridges (GR), we demonstrated that Wdr11-deficient embryos contained reduced numbers of PGCs which had delayed migration due to significantly decreased proliferation and motility. We found primary cilia-dependent canonical Hedgehog (Hh) signalling was required for proliferation of the somatic mesenchymal cells of GR, while primary cilia-independent non-canonical Hh signalling mediated by Ptch2/Gas1 and downstream effectors Src and Creb was required for PGC proliferation and migration, which was disrupted by the loss of function mutations of WDR11. Therefore, canonical and non-canonical Hh signalling are differentially involved in the development of somatic and germ cell components of the gonads, and WDR11 is required for both of these pathways operating in parallel in GR and PGCs, respectively, during normal PGC development. Our study provides a mechanistic link between the development of GnRH neurones and germ cells mediated by WDR11, which may underlie some cases of KS/CHH and ciliopathies.

scholarly journals hnRNPH1 recruits PTBP2 and SRSF3 to cooperatively modulate alternative pre-mRNA splicing in germ cells and is essential for spermatogenesis and oogenesis

2021 ◽  
Author(s):  
Shuiqiao Yuan ◽  
Shenglei Feng ◽  
Jinmei Li ◽  
Hui Wen ◽  
Kuan Liu ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Germ Cell ◽  
Germ Cells ◽  
Rna Binding ◽  
Cell Development ◽  
Mrna Splicing ◽  
Conditional Knockout ◽  
Cell Junction ◽  
Germ Cell Development ◽  
Splicing Regulators

Abstract Coordinated regulation of alternative pre-mRNA splicing is essential for germ cell development. However, the molecular mechanism underlying that control alternative mRNA expression during germ cell development remains poorly understood. Herein, we showed that hnRNPH1, an RNA-binding protein, is highly expressed in the reproductive system and localized in the chromosomes of meiotic cells but excluded from the XY body in pachytene spermatocytes and recruits the splicing regulators PTBP2 and SRSF3 and cooperatively regulates the alternative splicing of the critical genes that are required for spermatogenesis. Conditional knockout Hnrnph1 in spermatogenic cells caused many abnormal splicing events that affect genes related to meiosis and communication between germ cells and Sertoli cells, characterized by asynapsis of chromosomes and impairment of germ-Sertoli communications, ultimately leading to male sterility. We further showed that hnRNPH1 could directly bind to SPO11 and recruit the splicing regulators PTBP2 and SRSF3 to regulate the alternative splicing of the target genes cooperatively. Strikingly, Hnrnph1 germline-specific mutant female mice were also infertile, and Hnrnph1-deficient oocytes exhibited a similar defective synapsis and cell-cell junction as shown in Hnrnph1-deficient male germ cells. Collectively, our data reveal an essential role for hnRNPH1 in regulating pre-mRNA splicing during spermatogenesis and oogenesis and support a molecular model whereby hnRNPH1 governs a network of alternative splicing events in germ cells via recruiting PTBP2 and SRSF3.

428 PRODUCTION OF GERM-LINEAGE CELL AND FUNCTIONAL GAMETES FROM MULTI-POTENT SPERMATOGONIAL STEM CELLS

2010 ◽  
Vol 22 (1) ◽  
pp. 371
Author(s):  
J. E. Lim ◽  
J. H. Eum ◽  
H. J. Kim ◽  
H. S. Lee ◽  
J. H. Kim ◽  
...  
Keyword(s):  
Stem Cells ◽  
Germ Cell ◽  
Germ Cells ◽  
Culture Medium ◽  
Genetically Modified ◽  
Spermatogonial Stem Cells ◽  
Specific Gene ◽  
Specific Marker ◽  
Male Gametes ◽  
Genetically Modified Mice

Multi-potent spermatogonial stem cells (mSSC), derived from uni-potent SSC, are a type of reprogrammed cells with similar characteristics to embryonic stem cells (ESC). Similar to ESC, mSSC are capable of differentiating into 3-germ layers in vitro and teratoma formation in vivo. Additionally, mSSC proliferate rapidly and can be transfected more easily than SSC. In contrast to previous reports, we have found that mSSC also have germ-cell-specific micro (mi)RNA and gene expression profiles. Therefore, the aims of this study were to compare the efficiency of mSSC v. ESC to differentiate into germ lineage and produce male gametes, as well as to develop a novel system for the production of genetically modified mice. Mouse mSSC were transfected with a lentiviral vector expressing green fluorescent protein (GFP) and testis-specific gene and maintained in the ESC-culture medium containing leukemia inhibitory factor (LIF). Embryonic bodies (EB) were formed after the cells were detached from the feeder cells. Bone morphogenetic protein (BMP)-4 (10 ng mL˜1) and retinoic acid (RA, 0.1 μM) were added to the ESC-culture medium for 3 days in order to induce differentiation into germ lineage cells. Then, these cells were changed to germ cell-culture medium (Stem-Pro™ containing GDNF; Invitrogen, Carlsbad, CA, USA) and cultured for 3 days. After 6 days, cultured cells were sorted by magnetic activating cell sorting system using specific marker for germ cells, CD-9. Isolated germ lineage cells were transplanted into a busulfan-treated mouse testis for the production of male germ cells. Three to 6 weeks later, the testis and epididymis were collected, and half of the sample was used to perform histological analysis and the other half for the production of intracytoplasmic sperm injection (ICSI)-derived embryos. The statistical significance of differences between the 2 groups was evaluated by Student’s t-test Immunocytochemical and flow cytometrical analysis performed 6 days after differentiation showed that the ratio of germ cell-specific markers in EB derived from mSSC was higher than those from ESC. Moreover, after 3 to 6 weeks of transplantation the testis produced sperms and germ cells expressing GFP. We have successfully produced embryos by ICSI and offspring by embryo transfer into uteri of poster mothers. These results demonstrate that mSSC can be easily differentiated into germ lineage cells compared with ESC and have the potential to generate functional gametes. Therefore, the differentiation and transgenesis of mSSC may be a useful model for production of genetically modified mice. This work was supported by a grant of the Korea Healthcare Technology R&D Project, Ministry for Health, Welfare & Family Affairs, Republic of Korea (A084923).

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