脑源性神经营养因子及其受体在人卵巢的表达与卵泡发育的关系

目的：研究脑源性神经营养因子(BDNF)及其受体——酪氨酸蛋白激酶受体B(TrkB)在人卵巢中的表达。初步探讨其与卵泡发育的关系。方法：取排卵前期人卵巢组织和来自体外受精．胚胎移植(IVF-ET)周期的取卵日卵泡液、人卵巢黄素化壁层颗粒细胞(hMGLCs)、人卵巢黄素化卵丘颗粒细胞(hCGLCs)、未受精卵母细胞或未成熟卵母细胞。用免疫组织化学SABC法研究BDNF在人卵巢组织中不同发育阶段卵泡的表达，酶联免疫吸附实验(ELISA)方法检测BDNF在人卵泡液，hMGLCs和hCGLCs无血清培养上清液中的表达。用免疫组织化学SABC法研究TrkB在人卵巢组织不同发育阶段卵泡的表达，免疫细胞化学SABC法研究TrkB在hMGLCs，hCGLCs和未受精或未成熟卵母细胞的表达，Western blotting方法检测TrkB在hMGLCs和hCGLCs的表达及其差异。结果：人卵泡液中存在BDNF，BDNF表达于初级卵泡和次级卵泡的颗粒细胞及窦状卵泡的卵丘颗粒细胞。TrkB表达于初级卵泡和次级卵泡的颗粒细胞、窦状细胞的卵丘颗粒细胞、壁层颗粒细胞及卵母细胞，壁层颗粒细胞表达量高于卵丘颗粒细胞。结论：在人卵巢BDNF及其受体TrkB可能(通过旁分泌和自分泌的方式)参与颗粒细胞功能的完成和卵母细胞发育的调控。     

Reduction of progesterone receptor expression in human cumulus cells at the time of oocyte collection during IVF is associated with good embryo quality

BACKGROUND: It has been reported that the progesterone receptor (PR) level is transiently increased within the follicle by LH stimulation and controls cumulus cells in follicles and oocyte maturation. The purpose of this study was to predict developmental competence of human oocytes during IVF via analysis of PR in cumulus cells surrounding mature oocytes. METHODS: Prior to oocyte retrieval, the follicular diameter was measured and follicular fluid was collected from each mature follicle. Cumulus cells were manually separated from the oocyte-cumulus complex under a microscope. PR and PR mRNA were assessed by immunohistochemistry and real-time quantitative polymerase chain reaction (RT-PCR) measurement in human cumulus cells. RESULTS: Immunoreactive PR-A was mainly localized in the cytoplasm and PR-B was localized in the nuclei. There was no significant relationship between PR expression and follicular diameter, follicular fluid concentration of steroids, or LH. There was no significant relationship between expression of PRs and fertilization or cleavage rate. However, PR expression was lower in the good morphology group (blastomeres > or =7 cells with fragmentation > or =5% on day 3) when compared to the other groups (P<0.05). CONCLUSIONS: These results suggest that follicular LH or steroids do not affect PR expression, and full reduction of total PR expression on cumulus cells at the time of oocyte collection is associated with good morphology in human oocytes.     

Oocyte control of granulosa cell development: how and why

Interaction between oocytes and granulosa cells is complex and involves both gap junctions and paracrine signalling factors. Oocyte development in antral follicles is highly dependent on communication with cumulus cells, a subset of granulosa cells that is intimately associated with oocytes. Cumulus cells express characteristics distinct from the mural granulosa cells of preovulatory follicles. The thesis of this paper is that, without the influence of oocytes, the pathway of granulosa cell differentiation in antral follicles leads to the establishment of the mural granulosa cell phenotype. Oocytes in antral follicles abrogate that pathway of granulosa cell differentiation and promote the development of the cumulus cell phenotype. Oocytes may do this in order to control their own microenvironment by regulating differentiation of the supporting cells that are in direct communication with them. Possibly, some aspects of the mural granulosa cell phenotype are antagonistic to, or insufficient for, supporting the final stages of oocyte development. We present evidence that oocytes control their environment by suppressing differentiation of the mural granulosa cell phenotype and promoting differentiation of the cumulus cell phenotype. They achieve this suppression via the secretion of labile paracrine signalling factors. Errors in this regulatory mechanism, whether instigated by defects in the production of oocyte-derived ligands or granulosa cell responses to them, may result in the production of oocytes unable to undergo embryo development or that undergo abnormal follicular development.      

Fertilization and early embryology: The effect of testosterone on the maturation and developmental capacity of murine oocytes in vitro

A dose-dependent inhibition of meiotic maturation and embryonicdevelopment was observed in both cumulus-enclosed and cumulus-denudedmurine oocytes following incubation in the presence of 10, 20,40, 60 and 80 µM testosterone for 18 h in vitro. Maturationto metaphase II was enhanced in cumulus-enclosed oocytes followingmaturation in the presence of human pre-ovulatory mural granulosacells. However, maturation of cumulus-denuded oocytes was enhancedonly when oocytes were cultured on a monolayer of human polycysticovarian granulosa cells. The presence of cumulus cells had asignificantly positive effect on both oocyte maturation (P =0.002) and embryonic development (P < 0.001). In addition,the presence of follicular cells during maturation improvedthe number of fertilized oocytes reaching the blastocyst stage.These data indicate that the exposure of immature murine oocytesto testosterone during maturation significantly reduces theirability to mature and undergo normal embryonic development.     

Characterization of an immortalized human granulosa cell line (COV434)

We have investigated the biological characteristics of an immortalized granulosa cell line (COV434), which may be used to study follicular and oocyte maturation in vitro. Granulosa cell function was defined as consisting of three distinct properties: (i) production of 17beta-oestradiol in response to follicle stimulating hormone (FSH); (ii) presence of specific molecular markers of apoptosis enabling the induction of follicular atresia; and (iii) capacity to form intercellular connections with cells surrounding an oocyte. The addition of FSH to the culture medium supplemented with 10% fetal calf serum and 4-androstene-3,17-dione resulted in proliferation of the COV434 granulosa cells and in an increased synthesis of 17beta-oestradiol, indicating the presence of the FSH receptor and cytochrome P450 aromatase in these cells. The receptor for luteinizing hormone (LH) was undetectable. Similar expression of various apoptosis-associated genes was found in COV434 granulosa cells and in granulosa cells of patients stimulated with gonadotrophins for in-vitro fertilization, thus indicating that the immortalized COV434 granulosa cells were able to sustain apoptosis. Multiple intercellular connections were formed during co-culture of COV434 granulosa cells with cumulus cells containing an immature oocyte but not with cumulus cells devoid of an oocyte. Detailed morphological analysis of the intercellular connections with scanning electron microscopy and confocal light microscopy demonstrated the presence of long slender structures. It is concluded that the immortalized human granulosa cell line COV434 may be useful for experimental studies on follicular development.     

The clinical role of interleukin-6 and interleukin-6 soluble receptor in human follicular fluids

In order to investigate the role of interleukin-6 (IL-6) and interleukin-6 soluble receptor (sR) in human ovulation, we evaluated the concentrations in human follicular fluid and analyzed the correlation of IL-6 and IL-6 sR with oocyte maturation. The oocytes were obtained from the follicular fluid of 45 women undergoing in vitro fertilization and embryo transfer. The concentrations of IL-6 and IL-6 sR in follicular fluid were measured by ELISA. In addition, granulosa cells obtained from the follicular fluid were cultured and treated with forskolin and 12-o-tetradecanoylphorbol 13-acetate for 24–48 h. The concentration of IL-6 was significantly higher in the follicular fluid than in the serum (P<0.01). In contrast, the concentration of IL-6 sR was significantly lower in the follicular fluid than in the serum (P<0.001). The concentrations of IL-6 and IL-6 sR were significantly higher in the follicular fluid containing mature oocytes than in fluid containing immature oocytes (P<0.05). The production of IL-6 was markedly increased over the basal level after 24 h of treatment with forskolin(P<0.001) and 48 h of treatment (P<0.01) with cultured granulosa cells. Our data suggest that IL-6 and IL-6 sR may play an important role in follicular growth and development in human preovulatory processes. It is possible that IL-6 in particular may be regulated by cAMP. IL-6 and IL-6 sR might also be valuable biochemical markers in the evaluation of oocyte maturation. Received: 6 July 2002 / Accepted: 18 December 2002 Correspondence to Y. Kawano     

Human ovarian granulosa cells and follicular fluid indices: the relationship to oocyte maturity and fertilization in vitro

The study investigates the correlation between oocyte maturity and fertilization and a variety of hormonal parameters in follicular fluid and ovarian granulosa cells. A methodology for purification of granulosa cells from contaminating blood cells is also established. A total of 63 follicular aspirates were collected at oocyte retrieval from 30 women superovulated using the long luteinizing hormone- releasing hormone (LHRH analogue)/human menopausal gonadotrophin regimen. Oestradiol, progesterone, testosterone and human chorionic gonadotrophin (HCG) were quantified in follicular fluid and granulosa cells were immunostained for human chorionic gonadotrophin. Immunopurification of granulosa cells from contaminating blood cells was performed. HCG in follicular fluid was significantly high in follicles yielding immature (grade 3) oocytes (P=0.002); there was no correlation with fertilization. Aspirates from follicles containing mature (grade 1) oocytes and oocytes that subsequently fertilized had significantly more granulosa cells immunobound to HCG (P < 0.001, P=0.02). Moreover, the immunomagnetic purification technique provided >98% pure population of granulosa cells. The data demonstrate that HCG in follicular fluid and on granulosa cells may help to predict oocyte maturity and fertilization. Furthermore, immunomagnetic beads provide a reliable procedure for the purification of ovarian granulosa cells.      

The synthesis and fate of glycodelin in human ovary during folliculogenesis

The ontogeny of glycodelin in human ovarian follicles during folliculogenesis was studied. Glycodelin immunoreactivity began to be detected in the granulosa cells and thecal cells of late secondary follicles. Immunoreactivity was also found in both the luteinized granulosa cells and cumulus cells obtained from women undergoing the assisted reproduction treatment. However, only the luteinized granulosa cells, and not the cumulus cells, expressed glycodelin mRNA. Results also showed that the cumulus cells took up radiolabelled glycodelin and partially deglycosylated some of it. Glycodelin (and a partially deglycolsylated form of glycoldelin) appeared to complex with two cytoplasmic or membrane components of the cumulus cells. The data also demonstrated that ZIF-1, a glycoprotein isolated from human follicular fluid, was immunologically similar to glycodelin. In conclusion, we suggest that glycodelin is synthesized in the granulosa cells of ovarian follicles at late secondary follicle stage. It then may be released into the follicular fluid from where it is taken up and partially modified by the cumulus cells.     

Andrology: Assessment of human sperm functional changes after in-vitro coincubation with cells retrieved from the human female reproductive tract

Human spermatozoa must undergo functional changes prior to fertilization;however, the site of this physiological event is still unclear.To evaluate the influence of the female reproductive tract onsperm fertilizing capacity, fertile sperm samples were coincubatedwith endometrial, oviductal, granulosa and cumulus cells, follicularfluid and maternal serum. Sperm penetration into the zona-freehamster ova and motion parameters were measured daily for 72h. Compared to control samples, endometrial and oviductal cellcultures did not alter sperm fertilizing capacity or their movementcharacteristics. Sperm coincubated with follicular fluid, granulosaor cumulus cells exhibited a significantly higher ability topenetrate zona-free hamster ova for up to 48 h. Sperm motilityincreased at 4 h in the presence of follicular fluid and serum.At 24 h sperm velocity and amplitude of lateral head displacementsignificantly declined in sperm samples exposed to serum, andvelocity also declined in follicular fluid and with coincubationusing ovarian follicle cells. Sperm motility and velocity decreasedat 48 h in the presence of serum, follicular fluid, cumulusor granulosa cells. Our findings may suggest that specific secretoryfactors produced in the human pre-ovulatory ovarian follicleenhance human sperm fertilizing capacity.     

Expression of CD44 in human cumulus and mural granulosa cells of individual patients in in-vitro fertilization programmes

CD44 is a polymorphic and polyfunctional transmembrane glycoprotein widely expressed in many types of cells. Here, the expression of this protein on human membrana granulosa was studied by two techniques. Using confocal laser scanning microscopy (CLSM) with the mouse monoclonal antibody to human CD44 (clone G44-26), cells immunoreactive for CD44 were observed in both cumulus and mural granulosa cell masses. On the other hand, using monoclonal antibody to human CD44v9, goat polyclonal antibody to human CD44v3-10 and the clone G44-26, no immunoreactivity for CD44v9 and/or CD44v3-10 was observed in either cell group by flow cytometry. In the flow cytometric analysis of 32 patients, the incidence of CD44 expression in cumulus cells (62.6+/-1.3%) was significantly higher than that in mural granulosa cells (38.5+/-3.2%) (P<0.0001). In the comparison of CD44 expression by flow cytometry according to the maturation of each cumulus-oocyte complex, the incidence of CD44 expression of cumulus cells was significantly higher in the mature group than in the immature group (P<0.05). In a flow cytometric analysis, patients with endometriosis showed a significantly lower incidence of CD44 expression in cumulus cells compared to the infertility of unknown origin group (P<0.05), and compared to both the male infertility group and the unknown origin group in mural granulosa cells (P<0.01). These findings suggest that the standard form of CD44 is expressed in human membrana granulosa with polarity and may play an important role in oocyte maturation.     

Morphological analysis of resumption of meiosis observed in oocytes of mouse vesicular follicles

Resumption of meiosis observed in vesicular follicles of immature 32-34 day-old mice was classified morphologically, and the morphological mechanism to induce resumption of meiosis was examined. Resumption of meiosis was observed in 22.1% of vesicular follicles, and the dominant stage of the meiotic division was the first or second metaphase. Cumulus-enclosed and cumulus-free oocytes were separated from ovaries by dissection of ovaries. All of the cumulus-enclosed oocytes contained germinal vesicle, however 57.9% of cumulus-free oocytes did not, suggesting that the disappearance of cumulus cells around oocytes precedes the induction of resumption of meiosis. About 80-90% of the oocytes with germinal vesicle formed first polar body after 15 hrs of incubation. After incubation of oocytes showing resumption of meiosis, but without the first polar body, 36% of the oocytes extruded the first polar body indicating that resumption of meiosis is associated with progression to the second metaphase. Morphological characteristics of the surrounding structures of oocytes showing resumption of meiosis was classified into three categories; extensive proliferation of granulosa cells, degeneration of cumulus cells and their disappearance around oocytes, and detachment of cumulus cell-oocyte complexes from granulosa cell layer. It was suggested that cell-to-cell interaction among cumulus cells, oocytes and granulosa cells was impaired at the time of resumption of meiosis. Granulosa cells secrete an inhibitor of the resumption of meiosis. The resumption of meiosis after the surge of luteinizing hormone follows cumulus dispersion which make oocytes independent from the influence of granulosa cells by the disruption of communication.(ABSTRACT TRUNCATED AT 250 WORDS)     

人卵丘颗粒细胞和壁层颗粒细胞体外培养方法比较

目的:建立有效分离、提纯及培养人卵丘颗粒细胞的方法,并与人壁层颗粒细胞的体外培养相比较。方法:收集卵胞浆内单精子显微注射取卵时的卵泡液和直接机械分离卵母细胞所得的卵丘颗粒细胞复合物,直接将卵丘颗粒细胞复合物接种于培养皿中培养。用密度梯度离心法分离卵泡液中的人壁层颗粒细胞。用免疫荧光法检测卵泡刺激素受体(follicle stimulating hormone receptor,FSHR)的表达;用CCK-8法检测细胞生长曲线;用ELISA检测这2种颗粒细胞分泌雌激素的能力。结果:直接法所得人卵丘颗粒细胞培养24 h后可贴壁,体外培养生长状态与人壁层颗粒细胞相似;免疫荧光检测显示两者均表达FSHR;CCK-8实验结果表明,两者体外培养生长曲线相似;ELISA结果显示两者分泌雌激素能力相当。结论:利用机械切割获得人卵子周围卵丘颗粒细胞复合物直接培养的方法操作简单,获得的人卵丘颗粒细胞具有与人壁层颗粒细胞相似的生长状态、生长曲线以及雌激素分泌能力,可作为人颗粒细胞亚群体外培养方法的补充。     

Developmental incompetency of denuded mouse oocytes undergoing maturation in vitro is ooplasmic in nature and is associated with aberrant Oct-4 expression

BACKGROUND: Germinal vesicle (GV) oocytes constitute a potential resource but their developmental competence is questionable especially when surrounding cumulus cells are removed. The intercellular factors/mechanisms underlying such poor embryonic competence may originate at a nuclear and/or ooplasmic level. METHODS: Immature or mature oocytes were obtained from three mouse strains following pregnant mare serum gonadotropin (PMSG) or PMSG+ human chorionic gonadotropin (hCG) treatment. Immature oocytes were denuded of cumulus cells prior to in vitro maturation. Pronuclear (PN) transfer was used to examine nuclear-ooplasmic interplay on resultant embryonic development and Oct-4 immuno-staining patterns. RESULTS: Embryos arising from ooplasts of in vivo matured oocytes displayed significant increases in blastocyst formation rates and total blastomere numbers when compared to those created from ooplasts of denuded oocytes. Oct-4 staining was more pronounced and restricted to the inner cell mass (ICM) in blastocysts arising from the ooplasm of in vivo matured zygotes than in those created from denuded oocytes. CONCLUSIONS: Developmental defect(s) appear to develop primarily in the ooplasm of oocytes that are denuded of their cumulus cells prior to in vitro maturation. Such oocytes result in embryos with poor developmental competence. These defects result in anomalies in cell number and Oct-4 expression during the morula-blastocyst developmental transition.     

A novel mechanism of vascular endothelial growth factor, leptin and transforming growth factor-beta2 sequestration in a subpopulation of human ovarian follicle cells

This study describes the occurrence of a highly specialized subpopulation of granulosa and cumulus oophorus cells that accumulate and sequester specific growth factors by a novel mechanism. These cells are characterized by multiple balloon-like processes tethered to the cell by means of a slender stalk of plasma membrane. Time-lapse analyses demonstrate that these tethered structures (TS) form in minutes and frequently detach from the cell with the bulbous portion remaining motile on the cell surface. Serial section reconstruction of transmission electron microscopic images shows a specific and stable intracellular organization in which an apparent secretory compartment composed of densely packed vacuoles, vesicles, and cisternae is separated by a thick filamentous network from a nuclear compartment containing mitochondria, polyribosomes, lipid inclusions, and rough- surfaced endoplasmic reticulum. Immunofluorescent analysis performed during the formation of these structures showed a progressive accumulation of vascular endothelial growth factor, leptin, and transforming growth factor-beta2 in the bulbous region. TS were identified in newly aspirated masses of granulosa and cumulus oophorus, and their production persists for months in culture. Observations of TS- forming cells made over several days of culture indicates that their production is episodic and factor release from these cells may be pulsatile. The findings suggest that a novel method of growth factor storage and release by an apparent apocrine-like mechanism occurs in the human ovarian follicle. The results are discussed with respect to possible roles in pre- and post-ovulatory follicular development.      

The vascular character of ovarian follicular granulosa cells: phenotypic and functional evidence for an endothelial-like cell population

Ovarian follicular granulosa cells express temporally and spatially distinct functions throughout the follicle cycle. During the entire cycle, granulosa cells exhibit an unusually broad range of activities including the secretion of steroid hormones, enzymes, growth factors and cytokines. To date, the identity(ies) of these cells (lineage/cell type) remains unknown. We demonstrate expression of the Tie, Tek, cKit, Flt-1, CD-31 and vWF proteins and the ability to rapidly internalize acetylated low density lipoprotein among mural and cumulus subpopulations of human and murine follicular granulosa cells. In addition, we provide evidence that human and murine granulosa cells can engage in tube-forming activity in vitro. To the best of our knowledge, the six phenotypic and two functional markers examined during this study, as a group, are associated only with endothelial or endothelial-like cells. In total, the findings suggest that some granulosa cells may have the potential to actively participate in the vascularization of the corpus luteum, by way of an inherent capacity which is likely to be a characteristic of their unique identity and lineage. This inherent capacity of granulosa cells to behave and respond, at least to some extent, like endothelial cells may be of possible importance in the aetiology of certain follicular pathologies.     

Human sperm chemotaxis: both the oocyte and its surrounding cumulus cells secrete sperm chemoattractants

BACKGROUND: Human sperm chemotaxis to pre-ovulatory follicular fluid is well established in vitro. However, it is not known whether the female's oocyte-cumulus complex secretes sperm chemoattractants subsequent to ovulation (for enabling sperm chemotaxis within the Fallopian tube) and, if so, which of these cell types--the oocyte or the cumulus oophorus--is the physiological origin of the secreted chemoattractant. METHODS: By employing a directionality-based chemotaxis assay, we examined whether media conditioned with either individual, mature (metaphase II) human oocytes or the surrounding cumulus cells attract human sperm by chemotaxis. RESULTS: We observed sperm chemotaxis to each of these media, suggesting that both the oocyte and the cumulus cells secrete sperm chemoattractants. CONCLUSIONS: These observations suggest that sperm chemoattractants are secreted not only prior to ovulation within the follicle, as earlier studies have demonstrated, but also after oocyte maturation outside the follicle, and that there are two chemoattractant origins: the mature oocyte and the surrounding cumulus cells.     

Altered composition of the cumulus-oocyte complex matrix during in vitro maturation of oocytes

BACKGROUND: In vitro maturation (IVM) of mammalian oocytes has potential health benefits for patients undergoing assisted reproduction as an alternative to gonadotrophin treatment. This procedure is also useful for studying the process of oocyte and early embryo development. However, oocytes undergoing IVM have much lower competence than in vivo matured oocytes. Efforts to optimize IVM success have focused on replicating in vivo timing, hormonal milieu and cumulus cell responses associated with maturing oocytes. We have previously identified two extracellular matrix proteins, the protease Adamts1 and hyaluronan-binding proteoglycan Versican, produced by mural granulosa cells that selectively incorporate into the periovulatory cumulus-oocyte complex (COC). METHODS: Murine COC were cultured in the presence of epidermal growth factor and/or FSH. mRNA and protein were measured by real time PCR and Western blot and compared to in vivo derived COC. RESULTS: COCs from mice that underwent IVM for 6 or 20 h in the presence of epidermal growth factor, FSH or in combination had a > 10-fold reduction in mRNA (P < 0.05) for Adamts1 and Vcan when compared with in vivo matured COCs. Hyaluronan synthase 2 expression was up-regulated up to 8-fold (P < 0.05) over the unstimulated control, demonstrating successful induction of cumulus gene expression by the IVM conditions. While in vivo matured COCs showed abundant levels of these proteins, COCs that underwent IVM had neither detectable Adamts1, nor intact or Adamts1-cleaved Vcan. Human cumulus and granulosa cells matured in vivo contained abundant mRNA for Adamts1 and Vcan, demonstrating the potential relevance to human IVM. CONCLUSION: These results indicate that extensively altered COC matrix composition is present during IVM and may contribute to the observed poorer competence of the derived oocytes.     

Expression of receptors for insulin-like growth factor-I and transforming growth factor-beta in human follicles

The in-vitro growth of immature oocytes in early follicles from cryopreserved human ovarian tissues is a new concept in in-vitro fertilization programmes for the treatment of infertile and cancer patients. To better understand the regulatory mechanism of follicular development, immunohistochemistry was used to study the expression of insulin-like growth factor (IGF) type I receptor (IGF-IR) and transforming growth factor-beta (TGFbeta) type I (TbetaR-I) and type II (TbetaR-II) receptors in fresh and frozen ovarian tissues from 14 women. Immunoreactivities for IGF-IR and TbetaR-I were present simultaneously in the oocytes of primordial, pre-antral and antral follicles. Staining for both IGF-IR and TbetaR-I was also observed in granulosa cells of primordial, pre-antral and antral follicles. IGF-IR and TbetaR-I also stained in thecal cells of pre-antral and antral follicles. Stromal cells in surrounding ovarian tissue expressed IGF-IR and TbetaR-I at various follicular stages. Unlike TbetaR-I, TbetaR-II was expressed only in the oocytes of primordial and primary follicles, and with weak staining intensity in thecal cells. No significant staining for TbetaR-II was found in oocytes and granulosa cells of antral follicles. There was no difference in staining patterns for IGF-IR, TbetaR-I and TbetaR-II between fresh and frozen ovarian tissues, indicating that cryopreservation might not significantly alter the immunoreactivities of these receptors in frozen ovarian tissue. The results suggest that IGF-I and TGFbeta may participate in the regulation of follicular growth by binding to their receptors through an autocrine or paracrine mechanism. IGF-I and TGFbeta may be useful in regulating the in-vitro or in-vivo maturation of oocytes not only in later follicles but also very early follicles, from cryopreserved ovarian tissues for clinical use in the future.