co-expression of oct-4 and survivin genes in human ovarian … · 2014. 3. 2. · ict of interests:...

Short Communication

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Licensee OA Publishing London 2013. Creative Commons Attribution Licence (CC-BY)

F : Varras M. Co-expression of Oct-4 and Survivin genes in human ovarian luteinized granulosa cell during in vitro fertilization or intracytoplasmic sperm injection and embryo transfer programs. OA Case Reports 2013 Jan 31;2(1):1.

Co-expression of Oct-4 and Survivin genes in human ovarian luteinized granulosa cells during in vitro fertilisation or

intracytoplasmic sperm injectionand embryo transfer programs

M Varras*

AbstractIntroductionThe adult human ovary is composed of various cell types. Pre-ovulatory f-ollicles contain two types of granulo-sa cells, the mural granulosa cells an-d cumulus cells. Mural granulosa cel-ls line the follicle wall and are respo-nsible for oestrogen production and follicular rupture. Cumulus cells are closely connected to the oocyte thro-ugh a gap junction network, which a-llows transfer of small molecules, gl-ucose metabolites and ions, and are responsible for the oocyte develop-ment. Paracrine interactions betwee-n the theca cells, granulosa cells and oocytes, are critical for normal follic-ular development. This paper discus-ses the co-expression of Oct-4 Survi-vin genes in human ovarian luteiniz-ed granulosa cells during in vitro fer-tilisation or intracytoplasmic sperm injection and embryo transfer progr-ams.Short CommunicationApoptotic and survival factors influ-ence the interaction between the th-eca cells, granulosa cells and oocytes. Apoptotic factors inducing follicular atresia are gonadotropin-releasing hormone (Gn-RH), androgens, tumo-ur suppressor protein P53, c-Myc, endothelins, protein bax, caspases, some members of the tumour necro-sis factor (TNF) family (TNF-alpha, Fas, FasL, TRAIL) and some membe-rs of the transforming growth factor

(TGF)-beta family (factor NODAL and anti-Müllerian hormone [AMH]). Sur-vival factors promoting follicular sur-vival by follicle-stimulating hormone (FSH) and luteinizing hormone (LH) include oestrogens, insulin and insul-in growth factors (IGFs), growth hor-mone (GH), survivin, stem cell factor (SCF), kinase Akt, interleukin 1b (I-L-1b), epidermal growth factor (EG-F), members of the bcl-2 family, TGF-alpha, some members of the TGF-be-ta family (activin, bone morphogene-tic protein [BMP]-4, BMP-7 and gro-wth differentiation factor-9 [GDF-9]), basic fibroblast growth factor (bFGF), KIT-ligand and c-KIT receptors. Anal-ysis of prognostic biomarkers in ova-rian follicular granulosa cells, for bet-ter reproductive outcomes during as-sisted reproduction technologies (A-RT), seems to be a very intriguing ap-proach. In a small sample of patients who under-went in vitro fertilisation (IVF) or intracytoplasmic sperm inje-ction (ICSI) for tubal or male factor infertility (20 cases), we examined t-he co-expression of Octamer–binding transcription factor 4 (Oct-4) and su-rvivin in luteinized granulosa cells u-sing quantitative competitive real-ti-me polymerase chain reaction (QC R-T-PCR) and found such events in 8 c-ases (8/20). Survivin messenger RNA (mRNA) was expressed in 19 cases (19/20), while Oct-4 mRNA was exp-ressed in only 9 cases (9/20). There was no significant correlation betwe-en the expression levels of Oct-4 and survivin in luteinized granulosa cells during ART.ConclusionWe conclude that the low or high lev-

els of mRNA survivin gene expressi-on are not related to the low or high levels of mRNA Oct-4 gene expressi-on in luteinized granulosa cells during ART.

IntroductionThe follicular granulosa cells of the human ovaries are somatic cells wh-ich lose their high proliferative pote-ntial as follicles grow and segregate, upon the formation of the fluid-filled follicular antrum, into two anatomic-ally and functionally distinct subtyp-es, the mural cells and the cumulus cells. The mural granulosa cells line the follicle wall, form a stratified epi-thelium with the basal lamina and a-re responsible for their oestrogen p-roduction and follicular rupture. The cumulus cells are closely connected to the oocyte through a gap junction network, which allows transfer of s-mall molecules, glucose metabolites and ions, and are responsible for oo-cyte development1-5. For normal foll-icular development, paracrine inter-actions are important between the t-heca cells, granulosa cells and oocyt-es. During ovarian follicular develop-ment, only a few follicles ovulate and more than 99% of follicles undergo degeneration in all follicular growth stages; this process is known as atre-sia4,6. Failure of the granulosa cells to synthesise active factors results in infertility. GDF-9 is expressed in the growing oocytes. It is involved in fol-licular development through the ex-pression of several genes in the cum-ulus cells such as HAS2, COX-2, PTG-ER2, PRDX2 and SMAD2/33,7,8. This short communication discusses the

* Corresponding authorEmail: [email protected]

Third Department of Obstetrics and Gynaecol-ogy, ‘Elena Venizelou’ General Maternity State Hospital, Athens, Greece

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co-expression of Oct-4 Survivin gen-es in human ovarian luteinized gran-ulosa cells during in vitro fertilisati-on or intracytoplasmic sperm inject-ion and embryo transfer programs.

Short CommunicationThis work conforms to the values la-id down in the Declaration of Helsin-ki (1964). The protocol of this study has been approved by the relevant e-thical committee related to our insti-tution in which it was performed. All subjects gave full informed consent to participate in this study.Stem cells are undifferentiated cells, capable of self-renewal that differen-tiate into one or more cell lineages a-nd maintain the tissues they produc-e9-11. Stem cells show a wide range of

differentiation potential. The zygote or fertilised egg is totipotent and has the potential to differentiate into all cells and tissues in the embryo and also into the extra-embryonic tissue-s9,10. Pluripotent stem cells have the capacity to give rise to cells derived from all three embryonic layers of t-he body: ectoderm, mesoderm and endoderm, as well as trophectoder-m. Pluripotent stem cells are the em-bryonic stem cells which are derived from the inner cell mass of the blast-ocysts and include oocytes and sper-ms. The induced pluripotent stem c-ells have many similarities with the embryonic stem cells (ESCs) at mol-ecular and functional levels due to a cocktail of transcription factors11,12. The adult stem cells are multipotent

and have the capability of producing a limited range of differentiated cell lineages. These cells are responsible for growth, homeostasis and repair of tissues appropriate to their locati-on9-11. Unipotent stem cells are capa-ble of differentiating into only one c-ell lineage. An example of unipotent stem cells is the epidermal stem cell-s. Oct-4, (also known as POU5F1) is a member of the Pit-Oct-Unc (POU) tr-anscription factor family. Oct-4 plays a significant role in the self-renewal and pluripotency of ESCs and is ther-efore used as a marker of such cell-s13-15. Oct-4 in co-operation with oth-er transcription factors such as SO-X2, FOXD3 and STAT3, regulates ma-ny genes via the consensus motif AT-GCAAAT16-19. It is possible that isola-tion of very small embryonic-like st-em cells with a pluripotent potential and positive-testing for Oct-4, may s-erve as a good source of pluripotent stem cells in adult tissues and may have a potential application in rege-nerative medicine15,20. In 2009, Kos-sowska-Tomaszczuk et al.21 pooled granulosa cells from all the studied patients who underwent ARTs and f-ound the expression of Oct-4 in a su-bpopulation of granulosa cells amo-ng the total sum of cells in their cult-ures. When the researchers prolong-ed the cultures of the luteinized gra-nulosa cells with supplementation of the leukaemia-inhibiting factor, they found that less differentiated granul-osa cells could differentiate in vitro into three distinct lineages which ar-e not normally found in healthy ova-ries: osteoblastic, chondrocytic and neuronal lineages21. Our study in lut-einized granulosa cells of women w-ho underwent IVF or ICSI, supports the findings of Kossowska-Tomaszc-zuk et al.21,22. We found Oct-4 expre-ssion in almost half of studied case-s22. Therefore, there is strong evide-nce that the follicular luteinized gra-nulosa cells consist of a subpopulati-on of cells with pluripotent and self-renewal capabilities21-23. Absence of of the Oct-4 gene expression in the r-

Figure 1: The co-expression of Oct-4 and survivin genes was examined according to their levels of mRNA in luteinized granulosa cells of eight women who underwent IVF or ICSI treatment and embryo transfer for infertility. The expression of the Oct-4 gene was normalised with the data for G6PD and the respective quantitative ratio was determined by the density of the target gene to the internal standard: Oct-4m RNA/G6PD mRNA. The expression of the survivin gene was normalised with the data for ABL and the respective quantitative ratio was determined by the density of the target gene to the internal standard: survivin mRNA/ABL mRNA. There was no statistically significant correlation between the expression levels of Oct-4 mRNA and survivin mRNA in luteinized granulosa cells during ART (p = 0.289, Spearman’s test).

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emaining half of the cases probably means that the productive journey of these cells towards oocytes has ended22,23. In addition, in our study, we did not find expression of the D-AZL gene in granulosa cells of wom-en who underwent IVF or ICSI22. T-his finding is in concordance with t-he previous findings reported by K-ossowska-Tomaszczuk et al.21, who did not observe characteristic mar-kers of germ cells, such as nanog, v-asa and stella, in the pluripotent st-em cells. Therefore, it is strongly s-upported that granulosa cells cann-ot be differentiated into germ cell-s21-23.Apoptosis is the process of progra-mmed cell death, and it is genetical-ly determined. It plays an important role in several physiological events, such as follicular atresia, luteolysis, placentation and in several patholo-gical conditions. Granulosa cell apo-ptosis is prevented by gonadotropi-ns and oestrogens, whereas, andro-gens promote granulosa cell-death by apoptosis24. Apoptotic and survi-val factors influence the interactio-ns between the theca cells, granulo-sa cells and oocytes. Some of the ap-optotic factors that induce follicular atresia include GnRH, androgens, tumour suppressor protein P53, c-Myc, endothelins, protein bax, casp-ases, some members of the TNF fa-mily (TNF-alpha, Fas, FasL, TRAIL) and some members of the TGF-beta family (factor NODAL and AMH). On the other hand, survival factors pro-mote follicular growth and protect granulosa apoptosis. Some of the s-urvival factors which promote folli-cular survival include oestrogens, i-nsulin and IGFs, GH, survivin, SCF, kinase Akt, IL-1b, EGF, members of the bcl-2 family, TGF-alpha, some members of the TGF-beta family (a-ctivin, BMP-4, BMP-7 and GDF-9), b-FGF, KIT-ligand and c-KIT receptor-s. The survival factors inhibit follic-ular atresia by FSH and LH25. Survi-vin is a member of the inhibitor-of-apoptosis gene family and is a cell s-

urvival factor which inhibits apopto-sis by targeting caspaces and directl-y inhibiting the activity of caspas-e-326. Survivin is a bifunctional prot-ein that also regulates cell division27. Survivin is found during embryonic and foetal development and is expr-essed in many human cancers. Addi-tionally, survivin is expressed in nor-mal tissues, such as the endometriu-m and the follicular granulosa cells of the ovaries28. It seems that surviv-in in granulosa cells regulates the ce-ll cycle and inhibits apoptosis29. The clinical significance of the expressio-n of apoptotic factors in granulosa c-ells during ART is controversial30,31. However, the expression of survival factors in granulosa cells seems very promising.There is a need of prognostic bioma-rkers for accurate oocyte and embr-yo selection in human ARTs for the f-ollowing reasons: to optimise the se-lected oocytes and embryos, to redu-ce the number of embryos transferr-ed into the uterus and to predict wh-ether IVF fertilisation will be succes-sful or whether there is a need for I-CSI unrelated to male factor infertili-ty. With useful biomarkers, we need to achieve the best balance between reducing the risk of multiple gestati-ons and maximising the probability of pregnancy. The current embryo s-election methods are based mainly on morphological and development-al criteria and include the speed of cell division, the regularity of cell di-vision and the degree of fragmentati-on. Also, the general use of ICSI has many disadvantages: it is a more co-mplicated technique compared to I-VF, it requires special equipment an-d skills and is more expensive. Addi-tionally, there is an increased risk of transmitting chromosomal anomali-es or imprinting disorders, although it is not clear whether these risks are due to the procedure or due to the f-actors causing male infertility32-38. In addition, in some countries like Italy, not all retrieved oocytes can be ferti-lised due to legal limitations39. In thi-

s respect, the analysis of granulosa c-ells seems to be a very intriguing ap-proach. Also, the predictive evaluati-on of prognostic biomarkers expres-sed in granulosa cells for better ART outcomes, is limited to the interval between oocyte retrieval and fertili-sation1,39,40. We investigated the cor-relations between the presence or a-bsence and the levels of Oct-4 gene expression in granulosa cells with infertility clinical parameters and as-sisted reproduction outcomes22. Als-o, we examined the expression of the survivin gene in such cells in correl-ation with clinical parameters41. The participating patients in the IVF gro-up gave written consent for some of the cumulus-mature oocyte comple-xes (CMOCs) to be used only for the study. Therefore, the CMOCs were r-andomly selected and manually den-uded separately using a fire-polished tip glass pipette. These granulosa ce-lls were analysed for each patient se-parately, but the corresponding mat-ure oocytes were not fertilised beca-use the IVF procedure required the presence of cumulus cells. Only som-e CMOCs were donated and the cum-ulus cells were taken only from mat-ure oocytes in each patient. In case of the ICSI method, the CMOCs were manually denuded from the granulo-sa cells using a fire-polished tip glass pipette. Granulosa cells from all the mature oocytes, from each patient, were collected together. ICSI was pe-rformed only in oocytes that were morphologically confirmed to be in metaphase II with the first polar bo-dy extruded (mature oocytes). In ca-se of the ICSI, the cumulus cells were taken only when they surrounded t-he mature oocytes and these were t-he ones that were analysed. Therefo-re, the populations of both groups were homogenous, as the cumulus c-ells in the ICSI group were derived o-nly from mature oocytes and not fro-m mature, immature and degenerat-ed oocytes. In this way, we examined the expression of Oct-4 and survivin mRNA in granulosa cells from each

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patient separately, in correlation wi-th duration of ovulation induc-tion, number of follicles aspirated, numb-er of oocytes retrieved, number of mature oocytes retrieved, embryo g-rade and clinical pregnancy. Women with endometriosis or polycystic ov-arian syndrome were not included in our study since endometriosis and a-ndrogens promote apoptosis24,30. O-ur population included only patients with male or tubal factor infertility. The median of Oct-4 mRNA/G6PD mRNA was 1.75 with the intra-quar-tile range between 0.10 and 98.21. The Oct-4 mRNA expression was sta-tistically significantly correlated wit-h the number of oocytes retrieved. We found that when the Oct-4 mRNA expression was higher, more than si-x oocytes were retrieved (p = 0.037, Wilcoxon rank-sum)22. We suggest t-hat the expression of Oct-4 mRNA in granulosa cells appears to play an i-mportant role in the regu-lation of f-ollicular growth during ART22,23. The possibility for stem cell contaminati-on during egg retrieval of the granul-osa cell collection and the possible Oct-4 expression should be excluded because of the absence of the DAZL gene expression, which is typically e-xpressed in gametes. Studying any c-linical significance of the expression of stem cell markers in luteinized gr-anulosa cells in ART, is a new field of research. To the best of our knowle-dge, our study was the first one of its kind; previous studies in the same fi-eld have not studied this before22. With regard to the expression of sur-vivin in granulosa cells, survivin mR-NA was detected in granulosa cells at 93.1%. The expression levels of s-urvivin were significantly lower in n-ormal women (male infertility facto-r) compared to women with tubal i-nfertility factor (p = 0.007). There w-as no additional statistically signific-ant correlation between levels of su-rvivin expression and estradiol leve-ls, dosage of FSH for ovulation indu-ction, number of dominant follicles aspirated, number of retrieved oocy-

tes, embryo grade or clinical pregna-ncy rates41. Therefore, survivin see-ms to play a protective role in ovarie-s with possible influenced perfusion due to ipsilateral salpingectomy. In c-ases with tubal inflammation or hyd-rosalpinges, survivin might try to in-hibit apoptosis in a paracrine enviro-nment25,41. Fujino et al. found that th-e gene expression levels of survivin i-n total pregnant patients were higher than those in total non-pregnant pat-ients and those in the male factor inf-ertility patients28.In addition to all these, we examined the co-expression of Oct-4 and survi-vin in luteinized granulosa cells of w-omen who underwent IVF or ISCI, in 20 cases, using QC RT-PCR. We found such an event in 8 cases (8/20). Sur-vivin mRNA was expressed in 19 cas-es (19/20), while Oct-4 mRNA was e-xpressed in 9 cases (9/20)42. The ex-pression of the Oct-4 gene was norm-alised with the data for G6PD and the respective quantitative ratio was det-ermined by the density of the target gene to the internal standard: Oct-4 mRNA/G6PD mRNA. On the other h-and, the expression of the survivin g-ene was normalised with the data for ABL and the respective quantitative ratio was determined by the density of the target gene to the internal sta-ndard: survivin mRNA/ABL mRNA. Guo et al.43 found that Oct-4 express-ion in mouse ESCs is required for pr-otection from apoptosis induced by different apoptosis-inducing stresse-s. The authors suggested that this pr-otective action of Oct-4 expression s-eems to be mediated through the ST-AT3-Survivin pathway43. To the best of our knowledge, study of the co-ex-pression of Oct-4 and survivin in lut-einized granulosa cells in ART, has n-ot been performed before. For the fi-rst time, we found that there is no st-atistically significant correlation bet-ween the expression levels of Oct-4 i-n luteinized granulosa cells during A-RT and the expression levels of surv-ivin (p = 0.289, Spearman’s test) (Fi-gure 1). So, while in the ESCs of mice,

the reduced activity of survivin is di-rectly related to the reduced activity of Oct-443, this does not seem to app-ly to luteinized granulosa cells of w-omen during ART42. It would be inte-resting to see whether further studi-es can validate the clinical significan-ce of the co-expression of survivin a-nd Oct-4 genes in luteinized follicul-ar granulosa cells in a larger number of patients who undergo ART.

ConclusionThere seems to be no statistically si-gnificant correlation between the ex-pression levels of Oct-4 and the exp-ression levels of survivin in luteiniz-ed granulosa cells during ART. Also, the low or high levels of survivin m-RNA expression are not directly rel-ated to the low or high levels of Oc-t-4 mRNA expression in these cells (-Figure 1). We suggest that further st-udies should investigate the co-expr-ession of Oct-4 and survivin using Western blot analysis and immunoh-istochemistry on granulosa cells, in order to overcome the possible limi-tations of our study and reinforce our findings.

Abbreviations listABL, Abelson murine leukaemia vir-us oncogene; AMH, anti-Müllerian h-ormone; ART, assisted reproduction technology, bFGF, basic fibroblast g-rowth factor; BMP, bone morphoge-netic protein; BMPR2, bone morpho-genetic protein receptor type II; CM-OCs, cumulus–mature oocyte compl-exes; DAZL, DAZ-like; EGF, epiderm-al growth factor; ESCs, embryonic st-em cells; FSH, follicle-stimulating ho-rmone; GDF-9, growth differentiatio-n factor 9; GH, growth hormone; Gn-RH, gonadotropin releasing hormon-e; G6PD, glucose- 6-phosphate dehy-drogenase; HAS2, hyaluronic acid s-ynthase 2; ICSI, intracytoplasmic sp-erm injection; IGFs, insulin growth f-actors; IL, inter-leukin; IVF, in vitro fertilisation; LH, luteinizing hormon-e; LHCGR, luteinizing hormone/cho-riogonadotrophin receptor; mRNA,

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messenger RNA; Oct-4, octamer-bin-ding transcription factor 4; PRDX2, peroxiredoxin 2; PTGER2, type 2 re-ceptor for prostaglandins; PTGS2 or COX-2, prostaglandin-endoperoxide synthase-2; PTX3, pentraxin 3; QC R-T-PCR, quantitative competitive rea-l-time polymerase chain reaction; S-CF, stem cell factor; SEMA3A, semap-horin 3A; SEMA6A, semaphorin 6A; SMAD2/3, SMAD family member 2 and 3; STAT3, signal transducer and activator of transcription 3; TGF, tra-nsforming growth factor; TNF, tumo-ur necrosis factor.

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human cumulus–oocyte complex gene-expression profile. Hum Reprod. 2006 Jul;21(7):1705–19.

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of male infertility. Lancet. 2001 Jun;357(9274):2068–9.

36. Oehninger S, Gosden RG. Should ICSI be the treatment of choice for all cases of in-vitro conception? No, not in light of the scientific data. Hum Reprod. 2002 Sep;17(9):2237–42.37. Devroey P, Van Steirteghem A. A review of ten years experience of ICSI. Hum Reprod Update. 2004 Jan–Feb;10(1):19–28.38. Jain T, Gupta RS. Trends in the use of intracytoplasmic sperm injection in the United States. N Engl J Med. 2007 Jul;357(3):251–7.

39. Cillo F, Brevini TA, Antonini S, Paffoni A, Ragni G, Gandolfi F. Association between human oocyte developmental competence and expression levels of some cumulus genes. Reproduction. 2007 Nov;134(5):645–50.40. Ragni G, Allegra A, Anserini P, Causio F, Ferraretti AP, Greco E, et al. The 2004 Italian legislation regulating assisted reproduction technology: a multicentre survey on the results of IVF cycles. Hum Reprod. 2005 Aug;20(8):2224–8.41. Varras M, Polonifi K, Mantzourani M,Stefanidis K, Papadopoulos Z, Akrivis Ch, et al. Expression of antiapoptosis gene survivin in luteinized ovarian granulosa cells of women undergoing IVF or ICSI and embryo transfer: clinical correlations. Reprod Biol Endocrinol. 2012 Sep;10:74.42. Varras M. Study of the expression levels of the genes Oct-4, DAZL and

Survivin in the human ovarian follicular luteinized granulosa cells of women who undergo ovulation induction treatment for in vitro fertilization (IVF) or micro-fertilisation (ICSI) and embryo-transfer: Clinical correlations. Thesis for Masters’ Degree (MSc) in Reproductive and Regenerative Medicine, Medical School, National and Kapodistrian University of Athens, Greece, 2012. Greek. 43. Guo Y, Mantel C, Hromas RA, Broxmeyer HE. Oct-4 is critical for survival/antiapoptosis of murine embry-onic stem cells subjected to stress: effects associated with Stat3/survivin. Stem Cells. 2008 Jan;26(1):30–4.

34. Orief Y, Dafopoulos K, Al-Hassani S. Should ICSI be used in non-male factor infertility? Reprod Biomed Online. 2004 Sep;9(3):348–56.35. Oehninger S. Place of intracyto-plasmic sperm injection in management

co-expression of oct-4 and survivin genes in human ovarian … · 2014. 3. 2. · ict of interests:...

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