Cumulus and granulosa cell markers of oocyte and embryo quality
2013; Elsevier BV; Volume: 99; Issue: 4 Linguagem: Inglês
10.1016/j.fertnstert.2013.01.129
ISSN1556-5653
AutoresAsli Uyar, Saioa Torrealday, Emre Seli,
Tópico(s)Sperm and Testicular Function
ResumoLack of an objective, accurate, and noninvasive embryo assessment strategy remains one of the major challenges encountered in in vitro fertilization. Cumulus and mural granulosa cells reflect the characteristics of the oocyte, providing a noninvasive means to assess oocyte quality. Specifically, transcriptomic profiling of follicular cells may help identify biomarkers of oocyte and embryo competence. Current transcriptomics technologies include quantitative reverse transcriptase–polymerase chain reaction (qRT-PCR) for analysis of individual genes and microarrays and high-throughput deep sequencing for whole genome expression profiling. Recently, using qRT-PCR and microarray technologies, a multitude of studies correlated changes in cumulus or granulosa cell gene expression with clinically relevant outcome parameters, including in vitro embryo development and pregnancy. While the initial findings are promising, a clinical benefit from the use of identified biomarker genes remains to be demonstrated in randomized controlled trials. Lack of an objective, accurate, and noninvasive embryo assessment strategy remains one of the major challenges encountered in in vitro fertilization. Cumulus and mural granulosa cells reflect the characteristics of the oocyte, providing a noninvasive means to assess oocyte quality. Specifically, transcriptomic profiling of follicular cells may help identify biomarkers of oocyte and embryo competence. Current transcriptomics technologies include quantitative reverse transcriptase–polymerase chain reaction (qRT-PCR) for analysis of individual genes and microarrays and high-throughput deep sequencing for whole genome expression profiling. Recently, using qRT-PCR and microarray technologies, a multitude of studies correlated changes in cumulus or granulosa cell gene expression with clinically relevant outcome parameters, including in vitro embryo development and pregnancy. While the initial findings are promising, a clinical benefit from the use of identified biomarker genes remains to be demonstrated in randomized controlled trials. Discuss: You can discuss this article with its authors and with other ASRM members at http://fertstertforum.com/uyara-cumulus-granulosa-cells-biomarkers-embryo-quality/ Discuss: You can discuss this article with its authors and with other ASRM members at http://fertstertforum.com/uyara-cumulus-granulosa-cells-biomarkers-embryo-quality/ Infertility affects approximately 15% of reproductive-age couples (1Report of the Meeting on the Prevention of Infertility at the Primary Health Care Level 12–16 December 1983, Geneva. 1984.Google Scholar) and in addition to medical consequences, has significant social and financial implications. Among the treatment modalities available to infertile couples, IVF offers the highest success rates of pregnancy and live-birth outcomes. A critical step in IVF treatment is assessment of oocyte and embryo competence to determine the most viable embryo(s) to be transferred. Currently, embryo assessment strategies rely primarily on embryo morphology and cleavage rate. While these methodologies have been successful in improving pregnancy rates and reducing multiple gestations, their precision is less than what is desired (2Bromer J.G. Seli E. Assessment of embryo viability in assisted reproductive technology: shortcomings of current approaches and the emerging role of metabolomics.Curr Opin Obstet Gynecol. 2008; 20: 234-241Crossref PubMed Scopus (107) Google Scholar). Consequently, many centers perform multiple embryo transfers (ETs) to increase the chances of success for a given cycle, at the expense of a significantly higher risk of multiple gestations. Multiple gestations, in turn, result in an increased risk of preterm birth and its complications, such as cerebral palsy, and infant death (reviewed in reference 2Bromer J.G. Seli E. Assessment of embryo viability in assisted reproductive technology: shortcomings of current approaches and the emerging role of metabolomics.Curr Opin Obstet Gynecol. 2008; 20: 234-241Crossref PubMed Scopus (107) Google Scholar). Therefore, the development of an objective and accurate test to assess oocyte and embryo viability remains one of the most significant contemporary goals of reproductive medicine. In an attempt to develop novel embryo assessment strategies that can be used alone or in combination with morphologic criteria, invasive and noninvasive methods have been applied (reviewed in reference 3Seli E. Robert C. Sirard M.A. OMICS in assisted reproduction: possibilities and pitfalls.Mol Hum Reprod. 2010; 16: 513-530Crossref PubMed Scopus (99) Google Scholar). These include the assessment of the genome using comparative genomic hybridization (CGH) arrays, single nucleotide polymorphism (SNP) arrays, quantitative real time–polymerase chain reaction (qPCR), transcriptomic analysis of cumulus/granulosa cells, and proteomic and metabolomic analysis of embryo culture media (reviewed in reference 3Seli E. Robert C. Sirard M.A. OMICS in assisted reproduction: possibilities and pitfalls.Mol Hum Reprod. 2010; 16: 513-530Crossref PubMed Scopus (99) Google Scholar). Among these approaches, transcriptomic analysis of cumulus/granulosa cells has been proposed as a noninvasive tool to assess oocyte quality and viability as a surrogate for the reproductive potential of embryos. In this review, we first describe the basic aspects of folliculogenesis and the interactions between the oocyte and the stromal cells of the follicle. Methods used to study cumulus/granulosa cell gene expression are then outlined followed by the review of the studies assessing transcriptional analysis of cumulus/granulosa cells in correlation with oocyte and embryo competence. Lastly, the variations in the findings of these studies are explored from a methodological perspective. Folliculogenesis requires a carefully orchestrated cross talk between the oocyte and the surrounding somatic cells. During fetal life, primordial germ cells (PGCs) migrate to the future gonad, undergo mitosis, and give rise to oogonia (4Gondos B. Bhiraleus P. Hobel C.J. Ultrastructural observations on germ cells in human fetal ovaries.Am J Obstet Gynecol. 1971; 110: 644-652Abstract Full Text PDF PubMed Scopus (98) Google Scholar). The oogonia are then transformed into oocytes as they enter the first meiotic division. Primordial follicles are formed perinatally as the oocytes arrested in prophase of the first meiotic division become enveloped by a single layer of flattened granulosa cells that are surrounded by a basement membrane (5Fritz M. Speroff L. The ovary—embryology and development.8th ed. Lippincott Williams & Wilkins, 2011Google Scholar) (Fig. 1). Thereafter, through an unknown selection mechanism, individual primordial follicles are recruited from this resting pool to undergo growth and differentiation (6McGee E.A. Hsueh A.J. Initial and cyclic recruitment of ovarian follicles.Endocr Rev. 2000; 21: 200-214Crossref PubMed Scopus (1149) Google Scholar). During this process, granulosa cells surrounding the oocyte become cuboidal and form the primary follicle (7Gougeon A. Chainy G.B. Morphometric studies of small follicles in ovaries of women at different ages.J Reprod Fertil. 1987; 81: 433-442Crossref PubMed Scopus (218) Google Scholar). Then granulosa cells proliferate and form multiple layers of somatic cells that surround the oocyte, resulting in the formation of a secondary follicle. This is followed by the formation of small, fluid-filled cavities within the follicle that coalesce to form the early antral (or tertiary) follicle (8Soyal S.M. Amleh A. Dean J. FIGalpha, a germ cell-specific transcription factor required for ovarian follicle formation.Development. 2000; 127: 4645-4654PubMed Google Scholar). In the absence of gonadotropin stimulation, these follicles become atretic and disappear from the ovary. However, once puberty ensues, pituitary follicle stimulating hormone (FSH) stimulates further follicular growth (5Fritz M. Speroff L. The ovary—embryology and development.8th ed. Lippincott Williams & Wilkins, 2011Google Scholar). Under the influence of FSH, the antrum continues to enlarge, resulting in the formation of a preovulatory (also called antral or late antral) follicle (Fig. 1). In the preovulatory follicle, the oocyte is surrounded by cumulus cells, a specialized type of granulosa cell, distinct from the mural granulosa cells that line the antrum (9Diaz F.J. Wigglesworth K. Eppig J.J. Oocytes determine cumulus cell lineage in mouse ovarian follicles.J Cell Sci. 2007; 120: 1330-1340Crossref Scopus (189) Google Scholar). The oocyte within the preovulatory follicle will remain arrested in prophase I until after the LH surge, which precedes ovulation (10Guzeloglu-Kayisli O. Lalioti M.D. Aydiner F. Sasson I. Ilbay O. Sakkas D. et al.Embryonic poly(A)-binding protein (EPAB) is required for oocyte maturation and female fertility in mice.Biochem J. 2012; 446: 47-58Crossref Scopus (44) Google Scholar). Several critical steps are necessary to activate and mature the preovulatory follicle. It is during this period that the cumulus cells undergo “cumulus expansion,” a process that requires cumulus cells to produce hyaluronic acid that is deposited into the extracellular space and further stabilized by secreted proteins (11Eppig J.J. Intercommunication between mammalian oocytes and companion somatic cells.Bioessays. 1991; 13: 569-574Crossref PubMed Scopus (346) Google Scholar, 12Richards J.S. Russell D.L. Ochsner S. Espey L.L. Ovulation: new dimensions and new regulators of the inflammatory-like response.Annu Rev Physiol. 2002; 64: 69-92Crossref PubMed Scopus (356) Google Scholar). This newly formed extracellular matrix binds the oocyte and cumulus cells together (11Eppig J.J. Intercommunication between mammalian oocytes and companion somatic cells.Bioessays. 1991; 13: 569-574Crossref PubMed Scopus (346) Google Scholar, 12Richards J.S. Russell D.L. Ochsner S. Espey L.L. Ovulation: new dimensions and new regulators of the inflammatory-like response.Annu Rev Physiol. 2002; 64: 69-92Crossref PubMed Scopus (356) Google Scholar). In the meantime, the oocyte resumes meiotic division and begins the process of maturation. The culmination of these processes is the formation of a mature cumulus-oocyte complex (COC) containing an oocyte arrested at the metaphase of the second meiotic division (MII) and ready for ovulation and subsequent fertilization (10Guzeloglu-Kayisli O. Lalioti M.D. Aydiner F. Sasson I. Ilbay O. Sakkas D. et al.Embryonic poly(A)-binding protein (EPAB) is required for oocyte maturation and female fertility in mice.Biochem J. 2012; 446: 47-58Crossref Scopus (44) Google Scholar). Follicular development, oocyte maturation, cumulus expansion, and ovulation rely on continuing cross talk between the oocyte and the somatic follicular cells. In this section, we will briefly review key aspects of these processes as they have implications for studies investigating cumulus/granulosa cell gene expression as a proxy for oocyte viability. Studies in murine models have shown that rodents deficient in oocytes, secondary to mutations or chemical ablation, are also deficient in follicles, suggesting that the oocyte plays a critical role in the growth and development of the surrounding follicle (13Hirshfield A.N. Relationship between the supply of primordial follicles and the onset of follicular growth in rats.Biol Reprod. 1994; 50: 421-428Crossref PubMed Scopus (95) Google Scholar). During folliculogenesis, factors exclusively or predominantly expressed by the oocyte are required for the progression of the follicle through different developmental stages. Among these, one of the earliest transcription factors implicated in postnatal oocyte-specific gene expression is FIGα (8Soyal S.M. Amleh A. Dean J. FIGalpha, a germ cell-specific transcription factor required for ovarian follicle formation.Development. 2000; 127: 4645-4654PubMed Google Scholar). FIGα is required for primordial follicle formation, and Figα-knockout mice are devoid of primordial follicles (8Soyal S.M. Amleh A. Dean J. FIGalpha, a germ cell-specific transcription factor required for ovarian follicle formation.Development. 2000; 127: 4645-4654PubMed Google Scholar). Another key factor expressed in the oocyte is NOBOX, an oocyte-specific homeobox gene product expressed in germ cell cysts and in the oocytes of primordial and growing follicles (14Suzumori N. Yan C. Matzuk M.M. Rajkovic A. Nobox is a homeobox-encoding gene preferentially expressed in primordial and growing oocytes.Mech Dev. 2002; 111: 137-141Crossref PubMed Scopus (136) Google Scholar). Nobox-knockout female mice are infertile and have atrophic ovaries that are devoid of oocytes at 6 weeks of age (15Rajkovic A. Pangas S.A. Ballow D. Suzumori N. Matzuk M.M. NOBOX deficiency disrupts early folliculogenesis and oocyte-specific gene expression.Science. 2004; 305: 1157-1159Crossref PubMed Scopus (413) Google Scholar). Interestingly, unlike the findings seen in Figα-knockout mice, germ cell proliferation and initial primordial follicle formation occur in the absence of NOBOX (15Rajkovic A. Pangas S.A. Ballow D. Suzumori N. Matzuk M.M. NOBOX deficiency disrupts early folliculogenesis and oocyte-specific gene expression.Science. 2004; 305: 1157-1159Crossref PubMed Scopus (413) Google Scholar). Thus the lack of NOBOX inhibits follicle growth beyond the primordial follicle stage and accelerates the loss of oocytes (15Rajkovic A. Pangas S.A. Ballow D. Suzumori N. Matzuk M.M. NOBOX deficiency disrupts early folliculogenesis and oocyte-specific gene expression.Science. 2004; 305: 1157-1159Crossref PubMed Scopus (413) Google Scholar). Another key mediator for folliculogenesis, growth differentiation factor-9 (GDF-9), is produced by the oocyte from the primary follicle stage until the time of ovulation (16McPherron A.C. Lee S.J. GDF-3 and GDF-9: two new members of the transforming growth factor-beta superfamily containing a novel pattern of cysteines.J Biol Chem. 1993; 268: 3444-3449Abstract Full Text PDF PubMed Google Scholar, 17McGrath S.A. Esquela A.F. Lee S.J. Oocyte-specific expression of growth/differentiation factor-9.Mol Endocrinol. 1995; 9: 131-136Crossref PubMed Google Scholar). In Gdf-9-knockout mice, follicular development is arrested at the primary follicle stage, despite the presence of numerous primordial and early primary follicles (18Dong J. Albertini D.F. Nishimori K. Kumar T.R. Lu N. Matzuk M.M. Growth differentiation factor-9 is required during early ovarian folliculogenesis.Nature. 1996; 383: 531-535Crossref PubMed Scopus (1287) Google Scholar). Additionally, an oocyte-specific homolog of GDF-9 called bone morphogenetic protein 15 (BMP-15) has been cloned on the X-chromosome (19Dube J.L. Wang P. Elvin J. Lyons K.M. Celeste A.J. Matzuk M.M. The bone morphogenetic protein 15 gene is X-linked and expressed in oocytes.Mol Endocrinol. 1998; 12: 1809-1817Crossref PubMed Google Scholar). Yan et al. showed a synergistic role for BMP-15 and GDF-9 in ovarian folliculogenesis (20Yan C. Wang P. DeMayo J. DeMayo F.J. Elvin J.A. Carino C. et al.Synergistic roles of bone morphogenetic protein 15 and growth differentiation factor 9 in ovarian function.Mol Endocrinol. 2001; 15: 854-866Crossref PubMed Scopus (531) Google Scholar). Despite having relatively normal ovarian histology, Bmp-15-knockout mice are subfertile, secondary to decreased ovulation and fertilization rates (20Yan C. Wang P. DeMayo J. DeMayo F.J. Elvin J.A. Carino C. et al.Synergistic roles of bone morphogenetic protein 15 and growth differentiation factor 9 in ovarian function.Mol Endocrinol. 2001; 15: 854-866Crossref PubMed Scopus (531) Google Scholar). Double homozygous knockout females for BMP-15 and GDF-9 (Bmp-15−/−; Gdf-9−/−) experience oocyte loss resembling the phenotype seen in the GDF-9-knockout mice. Interestingly, homozygous Bmp-15 and heterozygous Gdf-9-knockout (Bmp-15−/−; Gdf-9+/−) females have oocytes that fail to adhere to cumulus cells and often remain trapped in the corpus luteum (20Yan C. Wang P. DeMayo J. DeMayo F.J. Elvin J.A. Carino C. et al.Synergistic roles of bone morphogenetic protein 15 and growth differentiation factor 9 in ovarian function.Mol Endocrinol. 2001; 15: 854-866Crossref PubMed Scopus (531) Google Scholar). Gene expression during oocyte maturation, fertilization, and early embryo development until zygotic gene activation relies on translational activation of specific maternally derived mRNA that was attained during the first meiotic arrest (reviewed in reference 21Radford H.E. Meijer H.A. de Moor C.H. Translational control by cytoplasmic polyadenylation in Xenopus oocytes.Biochim Biophys Acta. 2008; 1779: 217-229Crossref Scopus (158) Google Scholar). Embryonic poly (A)-binding protein (EPAB), which is exclusively expressed in gametes and early embryos (22Seli E. Lalioti M.D. Flaherty S.M. Sakkas D. Terzi N. Steitz J.A. An embryonic poly(A)-binding protein (ePAB) is expressed in mouse oocytes and early preimplantation embryos.Proc Natl Acad Sci U S A. 2005; 102: 367-372Crossref PubMed Scopus (64) Google Scholar, 23Guzeloglu-Kayisli O. Pauli S. Demir H. Lalioti M.D. Sakkas D. Seli E. Identification and characterization of human embryonic poly(A) binding protein (EPAB).Mol Hum Reprod. 2008; 14: 581-588Crossref Scopus (35) Google Scholar), is the predominant poly (A)-binding protein that stabilizes maternal RNAs and promotes their translation (10Guzeloglu-Kayisli O. Lalioti M.D. Aydiner F. Sasson I. Ilbay O. Sakkas D. et al.Embryonic poly(A)-binding protein (EPAB) is required for oocyte maturation and female fertility in mice.Biochem J. 2012; 446: 47-58Crossref Scopus (44) Google Scholar). Epab-knockout female mice are infertile owing to the inability of the oocytes to undergo maturation. Interestingly, although EPAB is not expressed in the somatic cells of the follicle (22Seli E. Lalioti M.D. Flaherty S.M. Sakkas D. Terzi N. Steitz J.A. An embryonic poly(A)-binding protein (ePAB) is expressed in mouse oocytes and early preimplantation embryos.Proc Natl Acad Sci U S A. 2005; 102: 367-372Crossref PubMed Scopus (64) Google Scholar), follicular development beyond the secondary follicle stage is impaired. Furthermore, the follicles that reach the preovulatory stage fail to undergo cumulus expansion in Epab-knockout mice (10Guzeloglu-Kayisli O. Lalioti M.D. Aydiner F. Sasson I. Ilbay O. Sakkas D. et al.Embryonic poly(A)-binding protein (EPAB) is required for oocyte maturation and female fertility in mice.Biochem J. 2012; 446: 47-58Crossref Scopus (44) Google Scholar). The above-mentioned findings establish that follicular development is adversely affected by the absence of normal oogenesis. The oocyte-specific genes that are required for follicular development, and the stages at which follicular development is arrested in knockout mouse models of these genes are shown in Figure 1 (8Soyal S.M. Amleh A. Dean J. FIGalpha, a germ cell-specific transcription factor required for ovarian follicle formation.Development. 2000; 127: 4645-4654PubMed Google Scholar, 10Guzeloglu-Kayisli O. Lalioti M.D. Aydiner F. Sasson I. Ilbay O. Sakkas D. et al.Embryonic poly(A)-binding protein (EPAB) is required for oocyte maturation and female fertility in mice.Biochem J. 2012; 446: 47-58Crossref Scopus (44) Google Scholar, 15Rajkovic A. Pangas S.A. Ballow D. Suzumori N. Matzuk M.M. NOBOX deficiency disrupts early folliculogenesis and oocyte-specific gene expression.Science. 2004; 305: 1157-1159Crossref PubMed Scopus (413) Google Scholar, 18Dong J. Albertini D.F. Nishimori K. Kumar T.R. Lu N. Matzuk M.M. Growth differentiation factor-9 is required during early ovarian folliculogenesis.Nature. 1996; 383: 531-535Crossref PubMed Scopus (1287) Google Scholar, 20Yan C. Wang P. DeMayo J. DeMayo F.J. Elvin J.A. Carino C. et al.Synergistic roles of bone morphogenetic protein 15 and growth differentiation factor 9 in ovarian function.Mol Endocrinol. 2001; 15: 854-866Crossref PubMed Scopus (531) Google Scholar, 24Rankin T.L. O’Brien M. Lee E. Wigglesworth K. Eppig J. Dean J. Defective zonae pellucidae in Zp2-null mice disrupt folliculogenesis, fertility and development.Development. 2001; 128: 1119-1126PubMed Google Scholar, 25Pangas S.A. Choi Y. Ballow D.J. Zhao Y. Westphal H. Matzuk M.M. et al.Oogenesis requires germ cell-specific transcriptional regulators Sohlh1 and Lhx8.Proc Natl Acad Sci U S A. 2006; 103: 8090-8095Crossref PubMed Scopus (207) Google Scholar, 26Choi Y. Yuan D. Rajkovic A. Germ cell-specific transcriptional regulator sohlh2 is essential for early mouse folliculogenesis and oocyte-specific gene expression.Biol Reprod. 2008; 79: 1176-1182Crossref Scopus (99) Google Scholar). The growing oocyte derives most of its substrates for energy metabolism and biosynthesis from the surrounding somatic cells, stressing the importance of the intercommunication between these cells (27Heller D.T. Schultz R.M. Ribonucleoside metabolism by mouse oocytes: metabolic cooperativity between the fully grown oocyte and cumulus cells.J Exp Zool. 1980; 214: 355-364Crossref PubMed Scopus (76) Google Scholar, 28Brower P.T. Schultz R.M. Intercellular communication between granulosa cells and mouse oocytes: existence and possible nutritional role during oocyte growth.Dev Biol. 1982; 90: 144-153Crossref PubMed Scopus (233) Google Scholar). Cumulus cells communicate with each other and with the oocyte through specialized gap junctions that allow metabolic exchange and transport of signaling molecules (29Tanghe S. Van Soom A. Nauwynck H. Coryn M. de Kruif A. Minireview: functions of the cumulus oophorus during oocyte maturation, ovulation, and fertilization.Mol Reprod Dev. 2002; 61: 414-424Crossref PubMed Scopus (383) Google Scholar). The fundamental unit of the gap junctions is the connexon, which is a hexamer of proteins called connexins (Cxs) (30Hasegawa J. Yanaihara A. Iwasaki S. Mitsukawa K. Negishi M. Okai T. Reduction of connexin 43 in human cumulus cells yields good embryo competence during ICSI.J Assist Reprod Genet. 2007; 24: 463-466Crossref PubMed Scopus (34) Google Scholar). Cx43, is a major contributor to gap junctions in human cumulus cells and is essential for the developmental competence of human oocytes (30Hasegawa J. Yanaihara A. Iwasaki S. Mitsukawa K. Negishi M. Okai T. Reduction of connexin 43 in human cumulus cells yields good embryo competence during ICSI.J Assist Reprod Genet. 2007; 24: 463-466Crossref PubMed Scopus (34) Google Scholar). Reduction of Cx43 expression in the COC has been shown to initiate the resumption of meiosis by disrupting the gap junctions (30Hasegawa J. Yanaihara A. Iwasaki S. Mitsukawa K. Negishi M. Okai T. Reduction of connexin 43 in human cumulus cells yields good embryo competence during ICSI.J Assist Reprod Genet. 2007; 24: 463-466Crossref PubMed Scopus (34) Google Scholar). Other genes involved in gap junctions, such as Cx37 and Cx40, have also been detected on microarray studies and may play an additional role in this process (31Assou S. Haouzi D. De Vos J. Hamamah S. Human cumulus cells as biomarkers for embryo and pregnancy outcomes.Mol Hum Reprod. 2010; 16: 531-538Crossref PubMed Scopus (173) Google Scholar). Cumulus cells play a vital role in regulating oocyte maturation (32Dekel N. Beers W.H. Development of the rat oocyte in vitro: inhibition and induction of maturation in the presence or absence of the cumulus oophorus.Dev Biol. 1980; 75: 247-254Crossref PubMed Scopus (159) Google Scholar, 33Larsen W.J. Wert S.E. Brunner G.D. A dramatic loss of cumulus cell gap junctions is correlated with germinal vesicle breakdown in rat oocytes.Dev Biol. 1986; 113: 517-521Crossref PubMed Scopus (163) Google Scholar). During the meiotic arrest, cyclic guanosine monophosphate (cGMP) from cumulus cells passes into the oocyte through gap junctions and inhibits the hydrolysis of cyclic adenosine monophosphate (cAMP) by the phosphodiesterase PDE3A. This inhibition maintains a high concentration of cAMP in the oocyte and blocks meiotic progression. LH reverses the inhibitory signal by lowering cGMP levels in the somatic cells and by closing gap junctions between the oocyte and the somatic cells. The resulting decrease in oocyte cGMP relieves the inhibition of PDE3A, causes a decrease in oocyte cAMP and leads to the resumption of meiosis (34Norris R.P. Ratzan W.J. Freudzon M. Mehlmann L.M. Krall J. Movsesian M.A. et al.Cyclic GMP from the surrounding somatic cells regulates cyclic AMP and meiosis in the mouse oocyte.Development. 2009; 136: 1869-1878Crossref PubMed Scopus (353) Google Scholar). It is noteworthy that while the development of the follicle is regulated by bidirectional signals between the oocyte and surrounding somatic cells, the LH-induced maturation of the oocyte is under the control of signals from the follicular somatic cells (reviewed in reference 35Hsieh M. Zamah A.M. Conti M. Epidermal growth factor-like growth factors in the follicular fluid: role in oocyte development and maturation.Semin Reprod Med. 2009; 27: 52-61Crossref PubMed Scopus (90) Google Scholar). Cumulus expansion is another critical aspect in the final stages of follicular development. Oocytes obtained from follicles with impaired cumulus expansion have limited potential for implantation (36Veeck L.L. An atlas of human gametes and conceptuses: an illustrated reference of assisted reproductive technology. Parthenon Publishing Group, New York1999Crossref Google Scholar). GDF-9 secreted by the oocyte has been shown to play a key role in cumulus expansion (37Elvin J.A. Clark A.T. Wang P. Wolfman N.M. Matzuk M.M. Paracrine actions of growth differentiation factor-9 in the mammalian ovary.Mol Endocrinol. 1999; 13: 1035-1048Crossref PubMed Google Scholar). GDF-9 functions as an oocyte-secreted paracrine factor that regulates several key granulosa cell enzymes involved in cumulus cell expansion, thus creating a microenvironment optimal for oocyte developmental competence (38Pangas S.A. Matzuk M.M. The art and artifact of GDF9 activity: cumulus expansion and the cumulus expansion-enabling factor.Biol Reprod. 2005; 73: 582-585Crossref PubMed Scopus (83) Google Scholar). Cyclooxygenase 2 (COX2), gremlin1 (GREM1), hyaluronic acid synthase 2 (HAS2), and pentraxin 3 (PTX3) are all downstream GDF-9 target genes found in cumulus cells and have been evaluated as markers for oocyte developmental competence (39Cillo F. Brevini T.A. Antonini S. Paffoni A. Ragni G. Gandolfi F. Association between human oocyte developmental competence and expression levels of some cumulus genes.Reproduction. 2007; 134: 645-650Crossref PubMed Scopus (141) Google Scholar, 40McKenzie L.J. Pangas S.A. Carson S.A. Kovanci E. Cisneros P. Buster J.E. et al.Human cumulus granulosa cell gene expression: a predictor of fertilization and embryo selection in women undergoing IVF.Hum Reprod. 2004; 19: 2869-2874Crossref PubMed Scopus (310) Google Scholar). Evidence accumulating from several elegant studies using the model organisms described above strongly suggests that key events during oogenesis and folliculogenesis rely on interactions between the oocyte and the somatic cells. These data strongly suggest that cumulus and granulosa cells may be used to gain insight into the viability and reproductive potential of the oocytes. In the following sections we will introduce analytical methods that are applied for cumulus and granulosa cell analysis and review the findings from clinical studies. The survival and function of a cell relies on complex molecular pathways that are activated in a timely manner in response to changing biological needs. Since gene expression proceeds through transcription of the genetic code into messenger RNA (mRNA), relative quantities of individual mRNAs can be assessed as an approximation of the expression levels of corresponding genes and provide information on the well-being of a cell or tissue. The total RNA content of a cell is termed the transcriptome and includes mRNAs, ribosomal RNAs (rRNAs), transfer RNAs (tRNAs), and microRNAs (miRNAs). Transcriptomics studies enable qualitative and quantitative characterization of gene expression in a cell or tissue under physiologic or pathologic conditions. Within the context of IVF (3Seli E. Robert C. Sirard M.A. OMICS in assisted reproduction: possibilities and pitfalls.Mol Hum Reprod. 2010; 16: 513-530Crossref PubMed Scopus (99) Google Scholar), transcriptomics analysis enables monitoring of gene expression in somatic cells of the follicle, gametes, and embryos. Transcriptomics technology offers quantitative reverse transcription polymerase chain reaction (qRT-PCR), for analysis of individual genes, and microarrays and high-throughput deep sequencing techniques for whole genome transcriptomic profiling. The result of genome-wide transcriptome analysis is the generation of a list of genes that are differentially expressed between the experimental conditions. Analysis of cumulus and/or granulosa cells transcriptomics in association with oocyte maturation, fertilization, embryo competence, and pregnancy outcome may help identify novel diagnostic biomarkers as an alternative to conventional morphological criteria. Here we provide brief definitions of the transcriptomic technologies and related cumulus cell–specific procedures. The common initial step in transcriptomic experiments is sample collection and RNA isolation, where high-quality starting material is a prerequisite for reliable analysis. Sampling of somatic components of the follicle poses specific challenges. Compared with granulosa cells, mechanical separation of COCs from contaminating cells is easier. In addition, laser capture technology can be quite useful in minimizing the effect of regional differences in the cumulus physiology. Cumulus cells must also be separated from the at
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