Complete in vitro generation of fertile oocytes from mouse primordial germ cells

Morohaku, Kanako; Tanimoto, Ren; Sasaki, Keisuke; Kawahara-Miki, Ryouka; Kono, Toru; Hayashi, Kozaburo; Hirao, Yuji; Obata, Yayoi

doi:10.1073/pnas.1603817113

Cited by 160 publications

(157 citation statements)

References 42 publications

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“…Following improvements in the technique and after alterations to the culture medium several mouse embryos and offspring have been obtained using oocytes that have been IVG then combined with IVM and IVF (O'Brien et al, 2003). More recently, in vitro systems that support the complete development of mouse oocytes starting from induced pluripotent stem cells (Hikabe et al, 2016) and from primordial germ cells (Morohaku et al, 2016) have been reported and these systems have resulted in competent oocytes that produce embryos and live young.…”

Section: Discussionmentioning

confidence: 99%

“…The most immature stage of follicles (primordial) can be grown in a two-step culture system to produce oocytes that can be fertilized, and live offspring have been produced from these in vitro grown (IVG) oocytes (Eppig and O'Brien, 1996;O'Brien et al, 2003). Recent reports have shown that the process of mouse oocyte development can be recapitulated in vitro starting from primordial germ cells and induced pluripotent stem cells (Hikabe et al, 2016;Morohaku et al, 2016) but little is known about the potential for human IVG oocytes.…”

Section: Introductionmentioning

confidence: 99%

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Metaphase II oocytes from human unilaminar follicles grown in a multi-step culture system

McLaughlin

Albertini

Wallace

et al. 2018

MHR: Basic Science of Reproductive Medicine

250

281

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STUDY QUESTION: Can complete oocyte development be achieved from human ovarian tissue containing primordial/unilaminar follicles and grown in vitro in a multi-step culture to meiotic maturation demonstrated by the formation of polar bodies and a Metaphase II spindle?SUMMARY ANSWER: Development of human oocytes from primordial/unilaminar stages to resumption of meiosis (Metaphase II) and emission of a polar body was achieved within a serum free multi-step culture system. WHAT IS KNOWN ALREADY:Complete development of oocytes in vitro has been achieved in mouse, where in vitro grown (IVG) oocytes from primordial follicles have resulted in the production of live offspring. Human oocytes have been grown in vitro from the secondary/multi-laminar stage to obtain fully grown oocytes capable of meiotic maturation. However, there are no reports of a culture system supporting complete growth from the earliest stages of human follicle development through to Metaphase II.STUDY DESIGN, SIZE, DURATION: Ovarian cortical biopsies were obtained with informed consent from women undergoing elective caesarean section (mean age: 30.7 ± 1.7; range: 25-39 years, n = 10).PARTICIPANTS/MATERIALS, SETTING, METHODS: Laboratory setting. Ovarian biopsies were dissected into thin strips, and after removal of growing follicles were cultured in serum free medium for 8 days (Step 1). At the end of this period secondary/multi-laminar follicles were dissected from the strips and intact follicles 100-150 μm in diameter were selected for further culture. Isolated follicles were cultured individually in serum free medium in the presence of 100 ng/ml of human recombinant Activin A (Step 2). Individual follicles were monitored and after 8 days, cumulus oocyte complexes (COCs) were retrieved by gentle pressure on the cultured follicles. Complexes with complete cumulus and adherent mural granulosa cells were selected and cultured in the presence of Activin A and FSH on membranes for a further 4 days (Step 3). At the end of Step 3, complexes containing oocytes >100 μm diameter were selected for IVM in SAGE medium (Step 4) then fixed for analysis. MAIN RESULTS AND THE ROLE OF CHANCE:Pieces of human ovarian cortex cultured in serum free medium for 8 days (Step 1) supported early follicle growth and 87 secondary follicles of diameter 120 ± 6 μm (mean ± SEM) could be dissected for further culture. After a further 8 days, 54 of the 87 follicles had reached the antral stage of development. COCs were retrieved by gentle pressure from the cultured follicles and those with adherent mural granulosa cells (n = 48) were selected and cultured for a further 4 days (Step 3). At the end ofStep 3, 32 complexes contained oocytes >100 μm diameter were selected for IVM (Step 4). Nine of these complexes contained polar bodies within 24 h and all polar bodies were abnormally large. Confocal immuno-histochemical analysis showed the presence of a Metaphase II spindle confirming that these IVG oocytes had resumed meiosis but their developmental potential is unknown. LIMITATIONS, R...

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Metaphase II oocytes from human unilaminar follicles grown in a multi-step culture system

McLaughlin

Albertini

Wallace

et al. 2018

MHR: Basic Science of Reproductive Medicine

250

281

View full text Add to dashboard Cite

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“…Notably, the system produced MII oocytes, followed by fertilized eggs, from mitotic PGCs in the fetal ovaries, thereby for the first time demonstrating the successful completion of meiosis in a female germ line in culture (Fig. 2) (Morohaku et al 2016). Apart from the completion of meiosis, the culture system provides two important components that are requisite to achieving a robust production of oocytes in culture.…”

Section: Reconstitution Of the Female Germ Line: Oogenesis In Organ Cmentioning

confidence: 94%

“…The GV oocytes were then cultured in a medium containing LH and EGF. Importantly, the resultant MII oocytes from the culture were capable of fertilization and full-term development (Morohaku et al 2016).…”

Section: Proof Onlymentioning

confidence: 99%

Stem cells, in vitro gametogenesis and male fertility

Nagamatsu

Hayashi

2017

Reproduction

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Reconstitution in culture of biological processes, such as differentiation and organization, is a key challenge in regenerative medicine, and one in which stem cell technology plays a central role. Pluripotent stem cells and spermatogonial stem cells are useful materials for reconstitution of germ cell development in vitro, as they are capable of differentiating into gametes. Reconstitution of germ cell development, termed in vitro gametogenesis, will provide an experimental platform for a better understanding of germ cell development, as well as an alternative source of gametes for reproduction, with the potential to cure infertility. Since germ cells are the cells for 'the next generation', both the culture system and its products must be carefully evaluated. In this issue, we summarize the progress in in vitro gametogenesis, most of which has been made using mouse models, as well as the future challenges in this field. Reproduction (2017) 154 F79-F91

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“…Recently, we successfully demonstrated the complete in vitro generation of fertile mouse oocytes from PGCs for the first time 63. Such an in vitro system is expected to unravel the mechanisms of oogenesis and preserve female gametes.…”

Section: Introductionmentioning

confidence: 99%

Differentiation of Mouse Primordial Germ Cells into Functional Oocytes In Vitro

2017

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Various complex molecular events in oogenesis cannot be observed in vivo. As a bioengineering technique for female reproductive tissues, in vitro culture systems for female germ cells have been used to analyze oogenesis and preserve germ cells for over 20 years. Recently, we have established a new methodological approach for the culture of primordial germ cells (PGCs) and successfully obtained offspring. Our PGC culture system will be useful to clarify unresolved mechanisms of fertility and sterility from the beginning of mammalian oogenesis, before meiosis. This review summarizes the history of culture methods for mammalian germ cells, our current in vitro system, and future prospects for the culture of germ cells.

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Complete in vitro generation of fertile oocytes from mouse primordial germ cells

Cited by 160 publications

References 42 publications

Metaphase II oocytes from human unilaminar follicles grown in a multi-step culture system

Metaphase II oocytes from human unilaminar follicles grown in a multi-step culture system

Stem cells, in vitro gametogenesis and male fertility

Differentiation of Mouse Primordial Germ Cells into Functional Oocytes In Vitro

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