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Reproductive biology. Potential egg stem cells reignite debate
Science. 2012 Mar 2;335(6072):1029-30.
Reproductive biologist Jonathan Tilly of Massachusetts General Hospital has challenged the conventional wisdom that female mammals are born with all the egg cells, or oocytes, they will ever have, arguing that in mice—and perhaps also in humans—there must be an ongoing source of new eggs. In 2005, he proposed that bone marrow was a source of eggs in mice. (That idea was discredited a year later.) But this week online in Nature Medicine, Tilly and colleagues report isolating rare cells in ovarian tissue from adult women that can grow in lab dishes and form immature oocytes. This latest claim is earning some cautious acceptance.
Oocyte formation by mitotically active germ cells purified from ovaries of reproductive-age women
Nat Med. 2012 Feb 26;18(3):413-21. doi: 10.1038/nm.2669.
White YA, Woods DC, Takai Y, Ishihara O, Seki H, Tilly JL. Source Vincent Center for Reproductive Biology, Massachusetts General Hospital Vincent Department of Obstetrics and Gynecology, Massachusetts General Hospital, Boston, Massachusetts, USA. Abstract Germline stem cells that produce oocytes in vitro and fertilization-competent eggs in vivo have been identified in and isolated from adult mouse ovaries. Here we describe and validate a fluorescence-activated cell sorting-based protocol that can be used with adult mouse ovaries and human ovarian cortical tissue to purify rare mitotically active cells that have a gene expression profile that is consistent with primitive germ cells. Once established in vitro, these cells can be expanded for months and can spontaneously generate 35- to 50-μm oocytes, as determined by morphology, gene expression and haploid (1n) status. Injection of the human germline cells, engineered to stably express GFP, into human ovarian cortical biopsies leads to formation of follicles containing GFP-positive oocytes 1-2 weeks after xenotransplantation into immunodeficient female mice. Thus, ovaries of reproductive-age women, similar to adult mice, possess rare mitotically active germ cells that can be propagated in vitro as well as generate oocytes in vitro and in vivo. Comment in
Nat Med. 2012 Mar;18(3):353-4.
Retransplantation of cryopreserved ovarian tissue: the first live birth in Germany
Dtsch Arztebl Int. 2012 Jan;109(1-2):8-13. Epub 2012 Jan 9.
Müller A, Keller K, Wacker J, Dittrich R, Keck G, Montag M, Van der Ven H, Wachter D, Beckmann MW, Distler W.
Abstract BACKGROUND: Cryopreserved ovarian tissue can be retransplanted to restore fertility after radiation or chemotherapy. To date, 15 live births after retransplantation have been reported worldwide. We report the first pregnancy and the first live birth after retransplantation in Germany. CASE REPORT: A 25-year-old female patient received initial chemotherapy and radiation of the mediastinum for Hodgkin's lymphoma in 2003 and suffered a relapse two years later. Ovarian tissue was laparoscopically removed and cryopreserved, and she was then treated with high-dose chemotherapy and stem cell transplantation. She remained in remission for 5 years and she could not conceive during this time. The cryopreserved ovarian tissue was thawed and laparoscopically retransplanted into a peritoneal pouch in the ovarian fossa of the right pelvic wall. Three months later, her menopausal symptoms resolved, and she had her first spontaneous menstruation. Six months after retransplantation, after two normal menstrual cycles, low-dose follicle stimulating hormone (FSH) treatment induced the appearance of a dominant follicle in the tissue graft. Ovulation was then induced with human chorionic gonadotropin (HCG), whereupon the patient conceived naturally. After an uncomplicated pregnancy, she bore a healthy child by Caesarean section on 10 October 2011. Histological examination of biopsy specimens revealed that the ovarian tissue of the graft contained follicles in various stages of development, while the original ovaries contained only structures without any reproductive potential. CONCLUSION: This was the first live birth after retransplantation of cryopreserved ovarian tissue in Germany and also the first case with histological confirmation that the oocyte from which the patient conceived could only have come from the retransplanted tissue. In general, young women who will be undergoing chemotherapy and/or radiotherapy for cancer must be informed and counseled about the available options for fertility preservation.
Recent advances in oocyte and ovarian tissue cryopreservation and transplantation
Best Pract Res Clin Obstet Gynaecol. 2012 Jan 31. [Epub ahead of print] Rodriguez-Wallberg KA, Oktay K. Source Karolinska Institute, Department of Clinical Science, Intervention and Technology, Division of Obstetrics and Gynecology K57, Karolinska University Hospital Huddinge SE-141 86 Stockholm, Sweden; Karolinska University Hospital Huddinge, Fertility Unit, Stockholm, Sweden.
Options for preserving fertility in women include well-established methods such as fertility-sparing surgery, shielding to reduce radiation damage to reproductive organs, and emergency in-vitro fertilisation after controlled ovarian stimulation, with the aim of freezing embryos. The practice of transfering frozen or thawed embryos has been in place for over 25 years, and today is a routine clinical treatment in fertility clinics. Oocytes may also be frozen unfertilised for later thawing and fertilisation by intracytoplasmic sperm injection in vitro. In recent years, oocyte cryopreservation methods have further developed, reaching promising standards. More than 1000 children are born worldwide after fertilisation of frozen and thawed oocytes. Nevertheless, this technique is still considered experimental. In this chapter, we focus on options for fertility preservation still in development that can be offered to women. These include freezing of oocytes and ovarian cortex and the transplantation of ovarian tissue. Copyright © 2012 Elsevier Ltd. All rights reserved.
Predictive value of oocyte morphology in human IVF: a systematic review of the literature
Hum Reprod Update. 2011 Jan-Feb;17(1):34-45. Epub 2010 Jul 16.
Rienzi L, Vajta G, Ubaldi F. Source G.EN.E.R.A Centre for Reproductive Medicine, Clinica Valle Giulia, Via G. De Notaris 2, 00197 Rome, Italy. firstname.lastname@example.org Abstract BACKGROUND: Non-invasive selection of developmentally competent human oocytes may increase the overall efficiency of human assisted reproduction and is regarded as crucial in countries where legal, social or religious factors restrict the production of supernumerary embryos. The purpose of this study was to summarize the predictive value for IVF success of morphological features of the oocyte that can be obtained by light or polarized microscopic investigations. METHODS: Studies about oocyte morphology and IVF/ICSI outcomes were identified by using a systematic literature search. RESULTS: Fifty relevant articles were identified: 33 analysed a single feature, 9 observed multiple features and investigated the effect of these features individually, 8 summarized the effect of individual features. Investigated structures were the following: meiotic spindle (15 papers), zona pellucida (15 papers), vacuoles or refractile bodies (14 papers), polar body shape (12 papers), oocyte shape (10 papers), dark cytoplasm or diffuse granulation (12 papers), perivitelline space (11 papers), central cytoplasmic granulation (8 papers), cumulus-oocyte complex (6 papers) and cytoplasm viscosity and membrane resistance characteristics (2 papers). None of these features were unanimously evaluated to have prognostic value for further developmental competence of oocytes. CONCLUSIONS: No clear tendency in recent publications to a general increase in predictive value of morphological features was found. These contradicting data underline the importance of more intensive and coordinated research to reach a consensus and fully exploit the predictive potential of morphological examination of human oocytes.
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3001337/figure/DMQ029F3/ Figure 3 Metaphase II human oocyte observed using polarized light microscopy
CD9 tetraspanin generates fusion competent sites on the egg membrane for mammalian fertilization
Proc Natl Acad Sci U S A. 2011 Jun 20. [Epub ahead of print]
Jégou A, Ziyyat A, Barraud-Lange V, Perez E, Wolf JP, Pincet F, Gourier C. Source Laboratoire de Physique Statistique, Ecole Normale Supérieure de Paris, Université Pierre et Marie Curie, Université Paris Diderot, Centre National de la Recherche Scientifique, 24 rue Lhomond, 75005 Paris, France.
CD9 tetraspanin is the only egg membrane protein known to be essential for fertilization. To investigate its role, we have measured, on a unique acrosome reacted sperm brought in contact with an egg, the adhesion probability and strength with a sensitivity of a single molecule attachment. Probing the binding events at different locations of wild-type egg we described different modes of interaction. Here, we show that more gamete adhesion events occur on Cd9 null eggs but that the strongest interaction mode disappears. We propose that sperm-egg fusion is a direct consequence of CD9 controlled sperm-egg adhesion properties. CD9 generates adhesion sites responsible for the strongest of the observed gamete interaction. These strong adhesion sites impose, during the whole interaction lifetime, a tight proximity of the gamete membranes, which is a requirement for fusion to take place. The CD9-induced adhesion sites would be the actual location where fusion occurs.
PMID: 21690351 http://www.ncbi.nlm.nih.gov/pubmed/21690351
Defining ovarian reserve to better understand ovarian aging
Reprod Biol Endocrinol. 2011 Feb 7;9:23.
Gleicher N, Weghofer A, Barad DH. Source Center for Human Reproduction-New York, NY, USA. email@example.com
Abstract Though a widely utilized term and clinical concept, ovarian reserve (OR) has been only inadequately defined. Based on Medline and PubMed searches we here define OR in its various components, review genetic control of OR, with special emphasis on the FMR1 gene, and discuss whether diminished OR (DOR) is treatable. What is generally referred to as OR reflects only a small portion of total OR (TOR), a pool of growing (recruited) follicles (GFs) at different stages of maturation. Functional OR (FOR) depends on size of the follicle pool at menarche and the follicle recruitment rate. Both vary between individuals and, at least partially, are under genetic control. The FMR1 gene plays a role in defining FOR at all ages. Infertility treatments have in the past almost exclusively only centered on the last two weeks of folliculogenesis, the gonadotropin-sensitive phase. Expansions of treatments into earlier stages of maturation will offer opportunity to significantly improve ovarian stimulation protocols, especially in women with DOR. Dehydroepiandrosterone (DHEA) may represent a first such intervention. Data generated in DHEA-supplemented women, indeed, suggest a new ovarian aging concept, based on aging of ovarian environments and not, as currently is believed, aging oocytes.
PMID: 21299886 http://www.ncbi.nlm.nih.gov/pubmed/21299886
Downregulation of both gene expression and activity of Hsp27 improved maturation of mouse oocyte in vitro
Liu JJ, Ma X, Cai LB, Cui YG, Liu JY. Reprod Biol Endocrinol. 2010 May 14;8:47.
BACKGROUND: Heat shock protein 27 (Hsp27), a member of the small heat shock protein family, is an apoptosis regulator. Our previous proteomic study showed that Hsp27 mainly expressed in human oocyte, and that Hsp27 expression was downregulated in the ovaries derived from women with the polycystic ovary syndrome (PCOS), a well known endocrinal disorder with abnormal apoptotic activity and folliculogenesis. However, the exact effects of Hsp27 downregulation on oocyte development have not yet been clarified.
CONCLUSIONS: Downregulation of Hsp27 improved the maturation of mouse oocytes, while increased early stage of apoptosis in oocytes by inducing the activation of extrinsic, caspase 8-mediated pathway.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
(good annexin and cleaved caspase immunofluorecence images)
Three-dimensional in vitro follicle growth: overview of culture models, biomaterials, design parameters and future directions
Reprod Biol Endocrinol. 2010 Oct 14;8(1):119.
Desai N, Alex A, Abdelhafez F, Calabro A, Goldfarb J, Fleischman A, Falcone T.
Abstract ABSTRACT: In vitro ovarian follicle culture is a new frontier in assisted reproductive technology with tremendous potential, especially for fertility preservation. Folliculogenesis within the ovary is a complex process requiring interaction between somatic cell components and the oocyte. Conventional two-dimensional culture on tissue culture substrata impedes spherical growth and preservation of the spatial arrangements between oocyte and surrounding granulosa cells. Granulosa cell attachment and migration can leave the oocyte naked and unable to complete the maturation process. Recognition of the importance of spatial arrangements between cells has spurred research in to three-dimensional culture system. Such systems may be vital when dealing with human primordial follicles that may require as long as three months in culture. In the present work we review pertinent aspects of in vitro follicle maturation, with an emphasis on tissue-engineering solutions for maintaining the follicular unit during the culture interval. We focus primarily on presenting the various 3-dimensional culture systems that have been applied for in vitro maturation of follicle:oocyte complexes. We also try to present an overview of outcomes with various biomaterials and animal models and also the limitations of the existing systems.
Loss of maternal ATRX results in centromere instability and aneuploidy in the mammalian oocyte and pre-implantation embryo
PLoS Genet. 2010 Sep 23;6(9). pii: e1001137.
Baumann C, Viveiros MM, De La Fuente R.
Female Germ Cell Biology Group, Department of Clinical Studies, University of Pennsylvania, Kennett Square, Pennsylvania, United States of America. Abstract
The α-thalassemia/mental retardation X-linked protein (ATRX) is a chromatin-remodeling factor known to regulate DNA methylation at repetitive sequences of the human genome. We have previously demonstrated that ATRX binds to pericentric heterochromatin domains in mouse oocytes at the metaphase II stage where it is involved in mediating chromosome alignment at the meiotic spindle. However, the role of ATRX in the functional differentiation of chromatin structure during meiosis is not known. To test ATRX function in the germ line, we developed an oocyte-specific transgenic RNAi knockdown mouse model. Our results demonstrate that ATRX is required for heterochromatin formation and maintenance of chromosome stability during meiosis. During prophase I arrest, ATRX is necessary to recruit the transcriptional regulator DAXX (death domain associated protein) to pericentric heterochromatin. At the metaphase II stage, transgenic ATRX-RNAi oocytes exhibit abnormal chromosome morphology associated with reduced phosphorylation of histone 3 at serine 10 as well as chromosome segregation defects leading to aneuploidy and severely reduced fertility. Notably, a large proportion of ATRX-depleted oocytes and 1-cell stage embryos exhibit chromosome fragments and centromeric DNA-containing micronuclei. Our results provide novel evidence indicating that ATRX is required for centromere stability and the epigenetic control of heterochromatin function during meiosis and the transition to the first mitosis.
Loss of maternal ATRX results in centromere instability and aneuploidy in the mammalian oocyte and pre-implantation embryo
Baumann C, Viveiros MM, De La Fuente R. PLoS Genet. 2010 Sep 23;6(9). pii: e1001137.
Abstract The α-thalassemia/mental retardation X-linked protein (ATRX) is a chromatin-remodeling factor known to regulate DNA methylation at repetitive sequences of the human genome. We have previously demonstrated that ATRX binds to pericentric heterochromatin domains in mouse oocytes at the metaphase II stage where it is involved in mediating chromosome alignment at the meiotic spindle. However, the role of ATRX in the functional differentiation of chromatin structure during meiosis is not known. To test ATRX function in the germ line, we developed an oocyte-specific transgenic RNAi knockdown mouse model. Our results demonstrate that ATRX is required for heterochromatin formation and maintenance of chromosome stability during meiosis. During prophase I arrest, ATRX is necessary to recruit the transcriptional regulator DAXX (death domain associated protein) to pericentric heterochromatin. At the metaphase II stage, transgenic ATRX-RNAi oocytes exhibit abnormal chromosome morphology associated with reduced phosphorylation of histone 3 at serine 10 as well as chromosome segregation defects leading to aneuploidy and severely reduced fertility. Notably, a large proportion of ATRX-depleted oocytes and 1-cell stage embryos exhibit chromosome fragments and centromeric DNA-containing micronuclei. Our results provide novel evidence indicating that ATRX is required for centromere stability and the epigenetic control of heterochromatin function during meiosis and the transition to the first mitosis.
PMID: 20885787 http://www.ncbi.nlm.nih.gov/pubmed/20885787
Chromatin configurations in the germinal vesicle of mammalian oocytes
Mol Hum Reprod. 2009 Jan;15(1):1-9. Epub 2008 Nov 18.
Tan JH, Wang HL, Sun XS, Liu Y, Sui HS, Zhang J.
Laboratory for Animal Reproduction and Embryology, College of Animal Science and Veterinary Medicine, Shandong Agricultural University, Tai-an City, Shandong Province 271018, Peolple's Republic of China. firstname.lastname@example.org
Abstract In all the studied mammalian species, chromatin in the germinal vesicle (GV) is initially decondensed with the nucleolus not surrounded by heterochromatin (the NSN configuration). During oocyte growth, the GV chromatin condenses into perinucleolar rings (the SN configuration) or other corresponding configurations with or without the perinucleolar rings, depending on species. During oocyte maturation, the GV chromatin is synchronized in a less condensed state before germinal vesicle breakdown (GVBD) in species that has been minutely studied. Oocytes may also take on a SN/corresponding configuration during early atresia, but they undergo GVBD at the advanced stage of atresia. As not all the species show the SN configuration while in all the species, gene transcription always stops at the late stage of oocyte growth, it is suggested that not the formation of perinucleolar rings but a thorough condensation of GV chromatin is essential for transcriptional repression. The GV chromatin configuration is highly correlated with oocyte competence; oocytes must end the NSN configuration before they gain the full meiotic competence, and they must take on the SN/corresponding configurations and stop gene transcription before they acquire the competence for early embryonic development. While factors inhibiting follicle atresia tend to synchronize oocytes in a chromatin configuration toward maturation, factors inducing follicle atresia tend to synchronize oocytes in a chromatin configuration reminiscent of early atresia. Furthermore, although condensation of GV chromatin is associated with transcriptional repression, both processes may not be associated with histone deacetylation during oocyte growth.
PMID: 19019837 http://www.ncbi.nlm.nih.gov/pubmed/19019837
Germline stem cells and neo-oogenesis in the adult human ovary
Dev Biol. 2007 Jun 1;306(1):112-20. Epub 2007 Mar 12. Liu Y, Wu C, Lyu Q, Yang D, Albertini DF, Keefe DL, Liu L.
College of Life Sciences, Sun Yat-Sen University, Guangzhou 510275, China. Abstract It remains unclear whether neo-oogenesis occurs in postnatal ovaries of mammals, based on studies in mice. We thought to test whether adult human ovaries contain germline stem cells (GSCs) and undergo neo-oogenesis. Rather than using genetic manipulation which is unethical in humans, we took the approach of analyzing the expression of meiotic marker genes and genes for germ cell proliferation, which are required for neo-oogenesis, in adult human ovaries covering an age range from 28 to 53 years old, compared to testis and fetal ovaries served as positive controls. We show that active meiosis, neo-oogenesis and GSCs are unlikely to exist in normal, adult, human ovaries. No early meiotic-specific or oogenesis-associated mRNAs for SPO11, PRDM9, SCP1, TERT and NOBOX were detectable in adult human ovaries using RT-PCR, compared to fetal ovary and adult testis controls. These findings are further corroborated by the absence of early meiocytes and proliferating germ cells in adult human ovarian cortex probed with markers for meiosis (SCP3), oogonium (OCT3/4, c-KIT), and cell cycle progression (Ki-67, PCNA), in contrast to fetal ovary controls. If postnatal oogenesis is confirmed in mice, then this species would represent an exception to the rule that neo-oogenesis does not occur in adults.
How eggs arrest at metaphase II: MPF stabilisation plus APC/C inhibition equals Cytostatic Factor http://www.celldiv.com/content/2/1/4
- (Henneguy, 1887) is a cytoplasmic structure present in young immature oocytes in mammals and many other species
- transient structure composed of a large number of mitochondria, Golgi complexes, endoplasmic reticulum.
- ill-defined electron-dense granulofibrillar material that congregates at a perinuclear location
- as the oocyte matures it moves to a peripheral location in the oocyte cortex (in some species corresponding to the future vegetal pole) and disperses (reviewed by Kloc et al., 2004).
- in some species contribute to the assembly and transportation of the germ plasm determinants to the cortex of the mature oocyte
- In mammals ( “inductive”, “regulative” or “epigenetic” mode of germ cell formation) do not have germ plasm determinants, the function remains largely obscure may include
- the controlled amplification of mitochondria or the creation of an asymmetry or polarity in the oocyte.
Henneguy F. (1887). Note sur la vesicle de Balbiani. C. R. Hebd. Seances Soc. Biol. Ses. Fil. 39, 69.
Kloc M., Bilinski S. and Etkin L.D. (2004). The Balbiani body and germ cell determinants: 150 years later. Curr. Top. Dev. Biol. 59, 1-36.
Egg coat proteins activate calcium entry into mouse sperm via CATSPER channels
Xia J, Ren D. Biol Reprod. 2009 Jun;80(6):1092-8. Epub 2009 Feb 11. PMID: 19211808
ZP2 and ZP3 traffic independently within oocytes prior to assembly into the extracellular zona pellucida
Hoodbhoy T, Avilés M, Baibakov B, Epifano O, Jiménez-Movilla M, Gauthier L, Dean J. Mol Cell Biol. 2006 Nov;26(21):7991-8. PMID: 17047254