The developmental ability of human oocytes penetrated at the germinal vesicle stage after insemination in vitro

This study demonstrates that sperm penetration into the ooplasmoccurs at high frequency in germinal vesicle (GV)stage humanoocytes which failed to resume meiosis after ovulation inductionin cycles of ovarian hyperstimulation for in-vitro fertilization.The capacity of the immature human oocyte to prevent polyspermicpenetration at the cell surface level was suggested by the findingthat despite the presence of numerous spermatozoa within thezona pellucida and on the oocyte surface within 3 h after insemination,all normal-appearing GV stage oocytes examined in this studywere penetrated by a single spermatozoon. This notion was alsosupported by scanning confocal microscopic analysis of oocytesdouble-stained for DNA and cortical granules– which showedhighly localized regions of cortical granule-free cytoplasmin proximity to the penetrated spermatozoon. The developmentalability of these oocytes was assessed by culture in vitro. Theresults show that oocytes penetrated by a spermatozoon at theGV stage resume meiosis, develop the capacity to decondensesperm DNA, abstrict both first and second polar bodies, andform a male pronucleus from the spermatozoon which enters theoocyte prior to the resumption of meiotic maturation. Afterpenetration, sperm nuclei rapidly migrate to the centre of theoocyte and become juxtaposed with the germinal vesicle, suggestingthe presence of a cellular mechanism which permits directedmovement within the cytoplasm. The developmental ability ofthese oocytes and the normality of the resulting embryos arediscussed     

Preliminary findings in germinal vesicle transplantation of immature human oocytes

Transplanting a germinal vesicle (GV) from an aged woman's oocyte into a younger ooplasm has been proposed as a possible way to reduce the incidence of oocyte aneuploidy which is considered to be responsible for age-related infertility. In this study, we have assessed the efficiency of each step involved in nuclear transplantation-specifically cell survival, nuclear-cytoplasmic reconstitution, and the capacity of the reconstituted oocytes for in-vitro maturation. In addition, we have evaluated the fertilizability and karyotypic status of the manipulated oocytes by intracytoplasmic sperm injection (ICSI) and fluorescent in-situ hybridization technique respectively. Nuclear transplantation was accomplished with an overall efficiency of 73%. Due to the limited availability of materials, most nuclear transplantation procedures were performed between sibling oocytes. The maturation rate of 62% following reconstitution was comparable with that of control oocytes, as was the incidence of aneuploidy among the reconstituted oocytes. The ICSI results of the reconstituted oocytes yielded a survival rate of 77%, a fertilization rate of 52%, and a satisfactory early embryonic cleavage. Furthermore, in a limited number of observations where the nucleus of an aged oocyte was transferred into a younger ooplasm, there was an appropriate chromosomal segregation. These findings demonstrate that human oocytes reconstituted with GV nuclei are able to undergo maturation, fertilization, and early embryo cleavage, and maintain a normal ploidy. Although in-vitro maturation seems to be a limiting step, this technique would allow us to investigate further the nuclear-ooplasmic relationship during meiotic maturation.     

Regulation of epidermal growth factor receptor by androgens in human endometrial cells in culture

Women with polycystic ovaries (PCO) have a thicker endometrium than women with normal ovaries. This cannot be due to unopposed oestrogen, as it occurs in ovulatory cycles. Androgens may be involved, as these are raised in women with PCO. The effects of steroids are partly mediated by growth factors and their receptors. The aim of this study was to investigate the effect of androgens on epidermal growth factor (EGF) receptor in human endometrium. Endometrium was enzymatically dispersed and glands and stromal cells separated. Cells were incubated in Ham's F10 medium supplemented with 5% charcoal-stripped fetal calf serum and either androgens or vehicle. Specific binding of [125I]- labelled EGF was measured. Testosterone and dihydrotestosterone (DHT) (10 micromol/l) increased EGF receptor concentration (control 100 +/- 9%, testosterone 196 +/- 23% control; control 100 +/- 1%, DHT 244 +/- 6% control) but did not alter receptor affinity. The effect of testosterone was inhibited by the anti-androgen hydroxyflutamide, but not by the antioestrogen ICI182780 nor the aromatase inhibitor 4- hydroxyandrostenedione. EGF receptor levels were increased by androstenediol (control 100 +/- 2%, androstenediol 120 +/- 10% control) but not by androstanediol, dehydroepiandrosterone (DHA), DHA sulphate or androstenedione. Testosterone and DHT increased EGF receptor concentrations in glandular epithelium (control 100 +/- 24%, testosterone 147 +/- 5%, DHT 185 +/- 30% control). These data suggest that androgens may have an effect on the endometrium via an increase in EGF receptor concentrations.      

In vitro maturation of human oocytes and cumulus cells using a co-culture three-dimensional collagen gel system

BACKGROUND: Deficiencies remain in the ability of in vitro-matured human oocytes to acquire full developmental competence and give rise to a healthy pregnancy. A clear deficiency of current systems utilizing human oocytes has been the absence of cumulus cells. In the present study, a three-dimensional (3D) co-culture system exploiting an extracellular matrix was developed and compared to conventional methods for its ability to support maturation of human oocytes. METHODS AND RESULTS: Cumulus cells were embedded into a 3D collagen gel matrix with individual oocytes added to each gel. Oocytes from the same patient cultured in the gel matrix matured to metaphase II at rates similar to those of cumulus-free oocytes cultured in individual microdrops. Following maturation of oocytes and fixation of intact gels, chromatin and cytoskeletal elements were assessed in oocytes and cumulus cells. The activities of the key cell cycle kinases, maturation-promoting factor (MPF) and mitogen-activated protein kinase (MAPK), were compared in oocytes matured under the two culture conditions. Compared with denuded oocytes, co-cultured oocytes exhibited increased MAPK activity, but no difference in MPF levels. CONCLUSIONS: This work characterizes a novel and efficacious culture system that takes advantage of the unique properties of the extracellular matrix, a 3D microenvironment, and the presence of cumulus cells for maturing human oocytes in vitro.     

Vitrification of mouse germinal vesicle oocytes: effect of treatment temperature and egg yolk on chromatin and spindle normality and cumulus integrity

The success rates for cryopreservation of immature oocytes from several species including human remain low, in contrast to major improvements with mature oocytes. In this study, a new approach has been developed using a short exposure ultra-rapid freezing protocol, examining the effect of temperature and egg yolk (two factors which may be expected to influence membrane flexibility) on the cryostability of immature mouse oocytes and cumulus complexes. These two factors were tested in various patterns for their cryoprotective effect using ethylene glycol as the principal cryoprotectant. The results showed that 37 degrees C pre- and post-freeze exposure significantly improved both survival and normal spindle configuration after in-vitro maturation. Egg yolk was found to produce further beneficial effects on both the oocyte and cumulus cell integrity, with the best effects being obtained at 37 degrees C with inclusion of egg yolk both before and after the freezing. This protocol produced > 80% normal survival post-thaw with intact and attached cumulus complex, 84% maturation rate and 45% normal metaphase configuration. In summary, a unique combination of high survival and meiotic normality together with good preservation of the attached cumulus cell mass has been achieved using a simple new vitrification procedure.     

A confocal microscopy analysis of the spindle and chromosome configurations of human oocytes cryopreserved at the germinal vesicle and metaphase II stage

BACKGROUND: Routine oocyte cryopreservation remains an elusive technique in the wide range of assisted reproductive technologies available. This study examines the effect of a cryopreservation protocol on the spindle and chromosome configurations of human oocytes cryopreserved at the germinal vesicle (GV) and metaphase II (MII) stage. METHODS: GV oocytes were randomly assigned to one of three groups: (i) control oocytes matured in vitro to MII stage (n = 156); (ii) oocytes cryopreserved at the GV stage and then matured in vitro (n = 90); (iii) oocytes cryopreserved at the MII stage (n = 147). Following cryopreservation and in-vitro maturation, immunostaining of tubulin and chromatin was performed, before visualization using confocal microscopy. RESULTS: A statistically significant increase was observed in the survival rate in group 2 (73.3%, 66/90) compared to group 3 (55.7%, 82/147) (P < 0.007). Exposure of oocytes to the cryoprotective solutions without freezing had no effect on the structure of their second meiotic spindle. However, statistically significant differences were observed on both spindle and chromosome configurations of oocytes from group 2 (5.2 and 5.2% respectively) and group 3 (16.2 and 18.8% respectively) compared with group 1 oocytes (71.6 and 82.0% respectively) (P < 0.001 in all cases). CONCLUSIONS: The protocol followed results in high rates of survival and potential for in-vitro maturation, but has a deleterious effect on the organization of the meiotic spindle of human oocytes cryopreserved at both the GV and MII stages.     

The influence of epidermal growth factor on progesterone production by human granulosa--luteal cells in culture

Epidermal growth factor (EGF) stimulates progesterone productionby human granulosa—luteal cells in culture. The presentstudy investigated some of the parameters that affect the magnitudeof human granulosa—luteal cells' response to EGF. Cellsfrom pre-ovulatory follicles obtained 36 h post-human chorionicgonadotrophin (HCG) were cultured for 12 days with or withoutEGF (20 ng/ml). Medium was changed every 48 h and assayed forprogesterone by radio-immunoassay. DNA content of the culturedcells was determined fluorometrically. EGF was added every otherday to the culture medium, starting on either day 4, 6 or 8of culture, up to day 10, and compared with controls. When EGFwas initiated on day 4, the medium had significantly higherprogesterone concentration than control samples on days 6, 8,10 and 12 of culture (P < 0.01). When EGF was withheld untilday 6 or 8, progesterone concentrations were not significantlyhigher than control values. When EGF was added on day 4 anddiscontinued on day 8 or 10, progesterone concentrations werereduced significantly (P < 0.001) compared with the groupwhere EGF was added continuously from day 4 to 10. These datasuggest that: (i) human granulosa—luteal cells requirethe early exposure and continuous presence of EGF for the stimulatoryeffect on progesterone secretion, (ii) cells not exposed initiallyto EGF do not respond in a similar way, (iii) EGF is capableof maintaining progesterone production for a period > 12days. Therefore, normal luteal function may require the earlyand continuous presence of EGF.     

Developmental potential of murine germinal vesicle stage cumulus-oocyte complexes following exposure to dimethylsulphoxide or cryopreservation: loss of membrane integrity of cumulus cells after thawing

BACKGROUND: Cumulus cells of the cumulus-oocyte complex (COC) are important in oocyte maturation. Thus, in preserving immature oocytes it is prudent to also preserve their associated cumulus cells. The survival and function of oocytes and their associated cumulus cells was assessed following cryopreservation or exposure to cryoprotectant without freezing. METHODS: Immature COCs were collected from mice primed with pregnant mare's serum. COCs were either slow-cooled or exposed to 1.5 mol/l dimethylsulphoxide without freezing. Treated and fresh COCs were stained for membrane integrity or, after in-vitro maturation and IVF, were assessed for developmental capability. Development of cumulus-denuded fresh oocytes, as well as denuded and frozen-thawed oocytes co-cultured with fresh cumulus cells, was assessed. RESULTS: Slow-cooled oocytes had significantly reduced coverage by intact cumulus cells compared with fresh COCs. Cumulus cell association and developmental capability were not substantially affected by exposure to cryoprotectant without freezing. Denuded fresh oocytes and cryopreserved COCs had decreased developmental potential that was not overcome by co-culture with fresh cumulus cells. CONCLUSIONS: Loss of association between oocyte and cumulus cells was induced by cryopreservation, but not by treatment with cryoprotectant alone. The data indicate that direct physical contact between cumulus cells and the oocyte, throughout maturation, improves subsequent embryo development.     

Meiotic spindle configuration is differentially influenced by FSH and epidermal growth factor during in vitro maturation of mouse oocytes

BACKGROUND: To ascertain whether different hormonal treatment protocols could affect metaphase II (MII) spindle morphology, meiotic spindle organization was detected in prepubertal mouse oocytes matured under conditions allowing spontaneous, FSH- or epidermal growth factor (EGF)-dependent meiotic maturation. METHODS: Oocyte-cumulus complexes (OCCs) were matured either spontaneously (control; n=270) or in the presence of hypoxanthine (Hx) plus FSH (n=400) or EGF (n=370). Spindles were detected by immunofluorescence analysis. In vivo ovulated (IVO) oocytes were processed similarly. RESULTS: IVO oocytes displayed spindles underlying the oolemma and with focused poles marked by spots of gamma-tubulin, whereas the majority (89%) of control oocytes had barrel-shaped spindles, positioned away from the oolemma, and with gamma-tubulin distributed along microtubules. Similar configuration/localization was found in 85% of the oocytes matured in vitro in the presence of Hx and FSH. In the presence of Hx-EGF, 35% of the oocytes showed spindles with an IVO-like configuration, although gamma-tubulin was homogeneously distributed throughout microtubules. Independently of spindle shape, 52% of EGF-stimulated oocytes had spindles positioned near the oolemma, in comparison to just 24% of FSH-treated and 13% of control oocytes. CONCLUSIONS: These results indicate that FSH and EGF can differently affect meiotic spindle morphology, and that EGF might be a stronger contributor than FSH to the acquisition of oocyte competence.     

Fertilization and early embryology: Co-culture of human pronucleate oocytes with their cumulus cells

The aim of this prospective randomized work was to study thevalue of co-culturing human pronucleate oocytes with their cumuluscells. A total of 550 fertilized oocytes from 95 in-vitro fertilizationpatients were randomly divided into two groups on the day afterinsemination. Group A oocytes (n = 260) were left undisturbedwith their attached cumulus cells and group B oocytes (n = 290)were dissected from their cumulus cells. Both groups were incubatedand examined daily for 3 days. In group A, 78% (202/260) reachedthe 4-cell stage 48 h after retrieval compared to 69% (200/290)in group B. At 72 h after retrieval, 70% (141/202) had reachedthe 8-cell stage in group A compared to 56% (112/200) in groupB. The percentages of grade 1 embryos at 48 and 72 h after retrievalwere 70% (141/202) and 76% (107/141) in group A compared to50% (100/200) and 43% (48/112) in group B respectively. We concludedthat co-culture of human oocytes with their cumulus cells significantlydecreased their fragmentation and increased the number of embryosthat reached the 4-cell and 8-cell stages with regular blastomeres.The technique is simple and avoids the use of heterogeneouscells.     

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。     

Essential role of structural integrity and firm attachment of surface-anchored epidermal growth factor in adherent culture of neural stem cells

Surface immobilization of proteins provides various biomaterials that permit the control of cellular functions through protein-protein interactions. Our previous study demonstrated that human epidermal growth factor carrying a hexahistidine sequence at the C-terminus (hEGF-His) could be anchored to the Ni-chelated surface by coordination, providing the versatile substrate for the selective proliferation of neural stem cells. The present study was undertaken to gain deeper insights into the basis for such an outstanding property of the surface with coordinated hEGF-His. For this purpose, the structure of the coordinated hEGF-His was analyzed by multiple internal reflection-infrared absorption spectroscopy. In addition, stability of coordinate bonds was assessed under cell culture conditions using a spatially-restricted anchoring technique. These data were compared to the results obtained from surfaces with covalently immobilized and physically adsorbed hEGF-His. The results presented here demonstrate that coordinated hEGF-His remains its intact conformation and is firmly anchored to the surface during cell culture. These attributes are both crucial for establishing the adherent culture and hence selective expansion of neural stem cells.     

Gene expression and immunolocalization of heparin-binding epidermal growth factor-like growth factor and human epidermal growth factor receptors in human corpus luteum

BACKGROUND: The objective of this study was to elucidate gene expression and immunolocalization of heparin-binding epidermal growth factor-like growth factor (HB-EGF) and human epidermal growth factor receptor (HER) family in the human ovary during luteal growth and regression. METHODS: Ovaries obtained from pre-menopausal women were used for immunohistochemistry and semiquantitative RT-PCR analysis. RESULTS: Immunoreactive HB-EGF was not detected in follicles or oocyte, while HB-EGF became apparent in granulosa luteal cells in the early luteal phase, and most abundant in the mid-luteal phase, but less abundant in the late luteal phase. Immunostaining for HER1 was very weak in granulosa luteal cells in the early and mid-luteal phases, and was not detected in the late luteal phase. Immunoreactive HER4 was abundant in the early luteal phase and became less abundant in the mid-luteal phase, whereas it was negative in the late luteal phase. Semiquantitative RT-PCR analysis revealed that HB-EGF and HER1 mRNA levels were high in the mid-luteal phase, whereas HER4 mRNA expression was high in the early luteal phase. CONCLUSIONS: HB-EGF may play a vital role in regulating luteal growth in a juxtacrine manner and through activating HER4 signalling.     

The role of maternally derived epidermal growth factor and the epidermal growth factor receptor during organogenesis in the rat embryo

Epidermal growth factor (EGF) has been implicated in the control of embryonic development, but although the receptor is expressed from an early stage, there is little evidence of embryonic expression of EGF. In order to investigate the role of maternally derived EGF during organogenesis, rat embryos were explanted at d 9.5 and cultured in serum depleted of low molecular weight molecules (retenate) which was then supplemented with EGF. Serum depleted of low molecular weight molecules by prolonged filtration loses its capacity to support normal embryonic development, possibly due to the loss of growth promoting factors. The addition of EGF to retenate significantly improved embryonic development with a maximal effect at 8 ng/ml. The addition of an analogue of EGF, long EGF, to retenate also caused a significant increase in development, although at higher concentrations a decrease in its effect was observed, possibly due to down regulation of the EGF receptor. Therefore, embryos may be able to utilise maternally derived EGF during organogenesis. To test the effects of inhibiting the EGF receptor during organogenesis, d 9.5 embryos were cultured in the presence of tyrphostin 47, a specific EGF receptor inhibitor. Tyrphostin 47 caused a significant dose-dependent decrease in the development of embryos which was also observed when tyrphostin 47 was injected into the vitelline circulation at d 11.5 to bypass the effects of the yolk sac. These findings suggest that the EGF receptor is essential for normal organogenesis and may play a role in the control of proliferation and differentiation. Although EGF is not expressed in the rat embryo at this stage, maternally derived EGF may be the ligand for the embryonic EGF receptor.     

Effects of epidermal growth factor on lung maturation in fetal lambs

The ability of epidermal growth factor (EGF) to induce lung maturation was evaluated in fetal and neonatal lambs. EGF was infused (3-5 days) into one member of 10 fetal twin pairs, one member of 2 term twin pairs, and 2 singleton term lambs. All EGF-treated lambs had evidence of epithelial hyperplasia of the conducting airways typical of the EGF effect. With the exception of the most immature pair, the lungs of treated versus control lambs were judged more mature by morphologic criteria by use of light and electron microscopy. None of the 6 premature lambs treated with EGF and allowed to breath showed evidence of hyaline membrane disease, while 3 untreated control lambs developed typical hyaline membranes when delivered by cesarean section after maternal hypotension. All untreated control animals showed more severe clinical symptoms of respiratory distress than did the EGF-treated animals.     

Efficient proliferation and maturation of fetal liver cells in three-dimensional culture by stimulation of oncostatin M, epidermal growth factor, and dimethyl sulfoxide

For the purpose of applying fetal liver cells (FLCs) as a cell source to tissue-engineered bioartificial livers, three-dimensional (3-D) cultures of FLCs using a porous polymer scaffold, as well as monolayer cultures as a control, were simultaneously performed. To achieve efficient growth and differentiation, the FLCs were cultured in the growth medium for the first 3 weeks and then cultured in the differentiation medium for 3 more weeks. In these cultures, stimulating factors (oncostatin M (OSM), epidermal growth factor (EGF), hepatocyte growth factor (HGF), or dimethyl sulfoxide (DMSO)) were added to the media, and their effects were examined. When the growth medium containing OSM and EGF was used, EGF stimulated the growth of FLCs synergistically with OSM. For the differentiation of FLCs into mature hepatocytes, DMSO added to the differentiation medium remarkably enhanced albumin secretion in the 3-D and monolayer cultures, although HGF was effective only in the monolayer culture. Microscopic observation proved that FLCs exhibited hepatocyte-like morphology only in the media containing DMSO. In conclusion, successive supply of the growth medium containing EGF and OSM and the differentiation medium containing DMSO efficiently induced the growth of the 3-D cultured FLCs and their differentiation into mature hepatocytes.     

干细胞条件培养液促进小鼠卵母细胞体外成熟

目的:研究骨髓间充质干细胞(MSCs)条件培养液对小鼠未成熟卵母细胞体外成熟的作用.方法:分离、培养小鼠MSCs,获得MSCs条件培养液.收集3类生发泡期卵母细胞,分别在对照培养基和条件培养液中培养,观察卵母细胞成熟率,判断最佳时间点;FDA、Hoechst33258和PI联合染色评价细胞活力;荧光标记检测皮质颗粒分布、迁移及纺锤体复合物的形成情况.结果:条件培养液组3类生发泡期卵母细胞的成熟率高于对照培养基组;其中,完全/大部分裸露的生发泡卵母细胞和周围有疏松的颗粒细胞包裹的生发泡卵母细胞的最佳体外成熟时间为16h,有完整的数层颗粒细胞紧密包裹的生发泡卵母细胞的最佳体外成熟时间为24 h.体外成熟卵母细胞活力良好,皮质颗粒分布及纺锤体复合物形成与体内成熟卵母细胞一致.结论:MSCs条件培养液有利于小鼠体外成熟卵母细胞核、细胞质同步成熟,提高卵母细胞质量,是一种较好的体外成熟培养体系.