Culture Of Ovarian Tissue Research Articles

Effect of sequential medium on in vitro culture of goat ovarian cortical tissue

A sequential medium was evaluated on the survival, activation and growth rates of caprine preantral follicles submitted to a long-term culture period, aiming to establish an ideal in vitro culture system. Ovarian fragments were cultured for 16 days in α-MEM+ alone or supplemented with hormones (GH and/or FSH) added sequentially on different days of culture. Ovarian fragments were cultured in the first (days 0–8) and second (days 8–16) halves of the culture period, generating 10 treatments: α-MEM+/α-MEM+, FSH/FSH, FSH/GH, FSH/FSH+GH, GH/GH, GH/FSH, GH/FSH+GH, FSH+GH/FSH+GH, FSH+GH/FSH and FSH+GH/GH. Follicle morphology, viability and ultrastructure were analyzed. After day 1 of culture, FSH treatments maintained the percentage of normal follicles similar to the fresh control. At day 16 of culture, the treatment FSH/GH showed the highest (P<0.05) percentage of normal follicles. The ultrastructure of follicles was preserved in the fresh control and FSH/GH treatment. Follicles cultured with FSH/GH had a higher (P<0.05) viability than α-MEM+; however the viability was lower (P<0.05) when compared to the fresh control. The FSH/GH treatment showed the highest (P<0.05) percentage of follicular activation and secondary follicle formation and produced the largest (P<0.05) mean follicular diameter after 16 days of culture. In conclusion, a sequential medium supplemented with FSH followed by GH during a long-term culture maintains the survival, viability and ultrastructure of goat preantral follicles, and promotes activation and secondary follicles.

Freezing solution containing dimethylsulfoxide and fetal calf serum maintains survival and ultrastructure of goat preantral follicles after cryopreservation and in vitro culture of ovarian tissue

Goat ovarian cortex fragments were subjected to slow freezing in the presence of various solutions containing intracellular cryoprotectants, including 1.0M ethylene glycol (EG), propanediol (PROH), or dimethyl sulfoxide (DMSO), with or without sucrose and/or fetal calf serum (FCS). Histological examination revealed that only the DMSO-containing solutions were able to maintain a follicular ultrastructure similar to the morphology observed in the fresh control. Therefore, fragments previously cryopreserved in DMSO solutions (with and without sucrose and/or FCS) were cultured in vitro for 48h and then subjected to viability, histological, and ultrastructural analysis. No significant differences were observed among the percentages of morphologically normal follicles in cryopreserved ovarian tissue before in vitro culture (DMSO: 62.5%; DMSO + sucrose: 68.3%; DMSO + FCS: 60.0%; DMSO + sucrose + FCS: 60.0%) and after culture (DMSO: 60.8%; DMSO + sucrose: 64.2%; DMSO + FCS: 70.8%; DMSO + sucrose + FCS: 55.0%). Following in vitro culture, the viability analysis showed that only the freezing solution containing DMSO and FCS (75.6%) maintained a percentage of viable follicles similar to that observed after culture without cryopreservation (89.3%). As determined by ultrastructural analysis, morphologically normal preantral follicles were detected in the fresh control and in fragments cultured before and after cryopreservation with DMSO and FCS. Thus, a freezing solution containing DMSO and FCS, under the experimental conditions tested here, guaranteed the maintenance of viability and follicular ultrastructure after short-term in vitro culture.

Influence of vitrification techniques and solutions on the morphology and survival of preantral follicles after in vitro culture of caprine ovarian tissue

The objective was to compare the efficiency of various vitrification techniques and solutions for preserving morphology and viability of preantral caprine follicles enclosed in ovarian tissue. Fragments of ovarian cortex were cryopreserved by conventional vitrification (CV) in French straws, vitrification in macrotubes (MTV), or solid-surface vitrification (SSV). Six solutions containing 6 M ethylene glycol, with or without sucrose (SUC; 0.25 or 0.50 M) and/or 10% fetal calf serum (FCS) were tested (Experiment I). After 1 wk, samples were warmed and preantral follicles were examined histologically. To evaluate follicular viability (Experiment II), ovarian fragments were vitrified with the three techniques listed above, in a solution containing 0.25 M SUC and 10% FCS. After warming, follicles were assessed by the trypan blue dye exclusion test. In Experiment III, preantral follicles enclosed in ovarian tissue were vitrified using the protocol which yielded the highest percentage of viable preantral follicles (SSV with 0.25 M SUC and 10% SFB). After warming, the preantral follicles enclosed in ovarian tissue were cultured in vitro and then, were analyzed by histology and fluorescence microscopy (calcein-AM and ethidium homodimer-1). Every vitrification protocol significantly reduced the percentages of morphologically normal follicles relative to the control (88.0%); however, the addition of 0.25 M SUC and 10% FCS to the vitrification solution improved preservation of follicular morphology (67.4, 67.4, and 72.0% for CV, MTV, and SSV, respectively). Although follicular viability after SSV (80.7%) did not differ from that in fresh (non-vitrified) ovarian tissues (88.0%), after in vitro culture, percentages of viable follicles were significantly reduced (70.0%). Percentages of morphologically normal follicles after in vitro culture of vitrified ovarian tissue were similar (76.0%) to those in ovarian cortex fragments cultured without previous vitrification (83.2%). In conclusion, SSV using a solution containing 0.25 M SUC and 10% FCS, was the most efficient method for vitrifying caprine ovarian tissue.

In vitro development of primordial follicles after long-term culture of goat ovarian tissue

This study aims to investigate the effects of follicle stimulating hormone (FSH) and fibroblast growth factor-2 (FGF-2) on the survival and growth of caprine preantral follicles. Ovarian tissues were cultured for 1, 7, 14, 21 or 28 days in medium supplemented with FSH (FSH-2d or FSH-7d, i.e., with replacement of the culture medium every 2 or 7 days, respectively) or FSH + FGF-2 (replacement of the medium every 2 days). Non-cultured (control) and cultured ovarian fragments were processed for histological and ultrastructural analysis. After 28 days of culture, the media supplemented with FSH-2d was the most effective in maintaining the percentage of normal follicles and in promoting follicular growth. Furthermore, both treatments with FSH increased the percentage of the primary follicles. However, ultrastructural studies did not confirm follicular integrity from 14 days of culture onward. In conclusion, culturing tissue for up to 7 days in medium containing FSH alone or combined with FGF-2 maintains caprine preantral follicle integrity and promotes their growth in vitro.

In vitro culture of sheep lamb ovarian cortical tissue in a sequential culture medium

This study evaluates the effect of a sequential culture system on the follicular development of sheep lamb ovaries, aiming to establish an available in vitro culture system for ovarian culture. Lamb ovarian cortical fragments were cultured on a steel mesh with a nitrocellulose membrane pre-coated by type 1 collagen. Several culture media were used for the determinations, specifically, a control medium (alpha-MEM), a constant medium (control medium supplemented with 75 ng/mL human recombinant EGF, 200 mIU/mL sheep FSH, 100 ng/mL human recombinant GDF-9, and 100 ng/mL human recombinant bFGF), and a sequential medium (control medium supplemented with sequential growth factors added on different days). Ovarian tissues, both fresh and cultured, were processed for histological and apoptotic assays, while spent culture media were processed for hormone assays. It was found that the growth of lamb primordial follicles can be initiated during culture in vitro. Compared to the control medium, sequential culture medium significantly increased the percentage of secondary follicles in cultures, while the follicle and oocyte diameters of primary and secondary follicles were also observed to increase in this medium. The constant medium was found to increase the number and diameter of secondary follicles only 18 days after culture. After this same period of time, some normal antral follicles were found in the sequential medium, while a few abnormal antral-like follicles were found in the control medium. Moreover, sequential medium appeared to significantly increase estradiol and inhibin production, especially 10-18 days after culture. The highest percentage of normal follicles and the lowest apoptotic cell rates were observed in the sequential medium, suggesting that a sequential addition style of culture can improve follicle and tissue viability. The sequential addition of FSH, EGF, GDF-9,and bFGF can stimulate primordial follicle transmittal into the later development stages, even as far as the antral stage, improve the survival rate of follicles, and maintain follicular viability.

STAMP alters the growth of transformed and ovarian cancer cells

BackgroundSteroid receptors play major roles in the development, differentiation, and homeostasis of normal and malignant tissue. STAMP is a novel coregulator that not only enhances the ability of p160 coactivator family members TIF2 and SRC-1 to increase gene induction by many of the classical steroid receptors but also modulates the potency (or EC50) of agonists and the partial agonist activity of antisteroids. These modulatory activities of STAMP are not limited to gene induction but are also observed for receptor-mediated gene repression. However, a physiological role for STAMP remains unclear.MethodsThe growth rate of HEK293 cells stably transfected with STAMP plasmid and overexpressing STAMP protein is found to be decreased. We therefore asked whether different STAMP levels might also contribute to the abnormal growth rates of cancer cells. Panels of different stage human cancers were screened for altered levels of STAMP mRNA. Those cancers with the greatest apparent changes in STAMP mRNA were pursued in cultured cancer cell lines.ResultsHigher levels of STAMP are shown to have the physiologically relevant function of reducing the growth of HEK293 cells but, unexpectedly, in a steroid-independent manner. STAMP expression was examined in eight human cancer panels. More extensive studies of ovarian cancers suggested the presence of higher levels of STAMP mRNA. Lowering STAMP mRNA levels with siRNAs alters the proliferation of several ovarian cancer tissue culture lines in a cell line-specific manner. This cell line-specific effect of STAMP is not unique and is also seen for the conventional effects of STAMP on glucocorticoid receptor-regulated gene transactivation.ConclusionsThis study indicates that a physiological function of STAMP in several settings is to modify cell growth rates in a manner that can be independent of steroid hormones. Studies with eleven tissue culture cell lines of ovarian cancer revealed a cell line-dependent effect of reduced STAMP mRNA on cell growth rates. This cell-line dependency is also seen for STAMP effects on glucocorticoid receptor-mediated transactivation. These preliminary findings suggest that further studies of STAMP in ovarian cancer may yield insight into ovarian cancer proliferation and may be useful in the development of biomarker panels.

Current achievements and future research directions in ovarian tissue culture, in vitro follicle development and transplantation: implications for fertility preservation.

BACKGROUND Female cancer patients are offered 'banking' of gametes before starting fertility-threatening cancer therapy. Transplants of fresh and frozen ovarian tissue between healthy fertile and infertile women have demonstrated the utility of the tissue banked for restoration of endocrine and fertility function. Additional methods, like follicle culture and isolated follicle transplantation, are in development. METHODS Specialist reproductive medicine scientists and clinicians with complementary expertise in ovarian tissue culture and transplantation presented relevant published literature in their field of expertise and also unpublished promising data for discussion. As the major aims were to identify the current gaps prohibiting advancement, to share technical experience and to orient new research, contributors were allowed to provide their opinioned expert views on future research. RESULTS Normal healthy children have been born in cancer survivors after orthotopic transplantation of their cryopreserved ovarian tissue. Longevity of the graft might be optimized by using new vitrification techniques and by promoting rapid revascularization of the graft. For the in vitro culture of follicles, a successive battery of culture methods including the use of defined media, growth factors and three-dimensional extracellular matrix support might overcome growth arrest of the follicles. Molecular methods and immunoassay can evaluate stage of maturation and guide adequate differentiation. Large animals, including non-human primates, are essential working models. CONCLUSIONS Experiments on ovarian tissue from non-human primate models and from consenting fertile and infertile patients benefit from a multidisciplinary approach. The new discipline of oncofertility requires professionalization, multidisciplinarity and mobilization of funding for basic and translational research.

Vasoactive Intestinal Peptide Improves the Survival and Development of Caprine Preantral Follicles after in vitro Tissue Culture

The aim of this study was to evaluate the effect of vasoactive intestinal peptide (VIP)on the survival, activation and growth of goat preantral follicles after in vitro culture. The ovarian cortex was divided into small pieces and one fragment was immediately fixed (control). The remaining fragments were cultured in vitro for 1 or 7 days at 39°C and 5% CO₂, in supplemented minimum essential medium (MEM⁺) with or without different concentrations of VIP (1, 10, 50, 100 or 200 ng/ml). Noncultured (fresh control) and cultured ovarian fragments were processed for histological analysis and transmission electron microscopy. Follicles were classified as primordial or developing, and as normal or degenerated. Our findings indicate that when compared with control, addition of all concentrations of VIP except 200 ng/ml resulted in similar percentages of normal preantral follicles after 1 and 7 days of culture. Culture of ovarian cortex tissue for 1 and 7 days increased the percentage of follicular activation in all treatments when compared with control, except with 1 ng/ml of VIP after 1 day. However, no difference was observed between VIP-treated and MEM⁺-treated follicles. In addition, after 7 days of culture, the highest follicular and oocyte diameters were observed in follicles cultured with 10 ng/ml VIP relative to MEM⁺ alone. Transmission electron microscopy showed ultrastructural integrity of follicles after 7 days of culture in 10 ng/ml VIP. In conclusion, this study demonstrates that VIP maintains follicular integrity and stimulates caprine preantral follicle growth.

Re-vascularisation in human ovarian tissue after conventional freezing or vitrification and xenotransplantation

ObjectiveIn ovarian tissue grafts there is a massive loss of follicles during the ischaemic period until re-vascularisation is established. The aim of our study was to investigate the influence of different cryopreservation techniques on the ability for the re-vascularisation of ovarian tissue transplanted to SCID mice. Study designOvarian fragments from five patients were cut into pieces (∼0.5mm×1.0mm×1.0mm) and randomly distributed into three groups: fresh non-treated tissue (group A); tissue conventionally frozen in standard 0.5ml insemination straws with 1.5M ethylene glycol+0.1M sucrose, with thawing in a 40°C water bath and step-wise removal of cryoprotectants at room temperature in 0.5M, 0.25M and 0.15M sucrose with gentle agitation (group B); tissue vitrified in 2.62M dimethylsulphoxide+2.6M acetamide+1.31M propylene glycol+0.0075M polyethylene glycol, with warming by direct plunging of solid specimens with ovarian pieces into 20ml of 50% vitrification solution pre-warmed to 40°C and dilution of cryoprotectants in a decreasing concentration of vitrification solution (25%, 12.5%) at room temperature (group C). We used a xenograft model in which ovarian tissue pieces of all three groups were subcutaneously transplanted in SCID mice. The animals were sacrificed on the third day after ovarian tissue transplantation and then weekly during 1 month to obtain the ovarian tissue grafts. These samples were examined by immunohistochemical staining with the endothelial cell-specific marker platelet endothelial cell adhesion molecule-1 (PECAM-1) to determine angiogenesis. Histological observation of tissue after explantation was performed and quality and quantity of follicles were assessed. ResultsNo PECAM-1 staining was observed in all treatment groups prior to grafting. After warming and in vivo culture of ovarian tissue, the beginning of angiogenesis in pieces from all treatment groups on the third day was detected by PECAM-1 staining. After 4 weeks of in vivo culture the overall area of PECAM-1-positive blood vessels significantly increased (P<0.05), independent of the type of cryopreservation (groups B and C vs. group A). It was found that transplantation technique had negative influence on the integrity of follicles independent of the type of treatment during in vivo culture. The duration of in vivo culture has a negative, but not statistically significant, influence on follicle quality in long-cultured transplants inside each treatment group (P>0.5). ConclusionThe process of re-vascularisation of transplanted ovarian tissue is independent of the type of treatment and does not influence follicle quality.

325 VIABILITY AND GROWTH OF CATTLE PREANTRAL FOLLICLES AFTER IN VITRO CULTURE OF OVARIAN FRAGMENTS IN α-TOCOPHEROL

The development of culture systems to support the initiation of growth of primordial follicles is important to the study of the factors that control the earliest stages of folliculogenesis. The aims of the present study were to investigate the effects of α-tocopherol on survival, activation, and growth of cattle preantral follicles using histological and immunohistochemistry proliferating cell nuclear antigen (PCNA) studies. The ovarian cortex was divided into small fragments; one fragment was immediately fixed in Bouin (control). The other fragments were cultured for 2, 4, 6, or 8 d in culture plates with Minimum Essential Medium supplemented with insulin-transferrin-selenium (ITS), pyruvate, glutamine, hypoxanthine, BSA, and antibiotics (MEM+); and MEM+ plus α-tocopherol (5, 25, 50, 100, or 200 ng mL-1). Preantral follicles were classified according to their developmental stage (primordial, intermediate, primary, or secondary) and on the basis of morphological features (normal or degenerated). Pair-wise comparisons were done using Tukey’s procedure. Chi-square test was used to compare the percentage of follicles with PCNA-positive granulosa cells. All analyses were done with the SAS software (SAS Institute, Cary, NC, USA), and P &lt; 0.05 was considered significant. The results showed that, compared with non-cultured cortical tissue (Day 0), the culture of ovarian tissue significantly reduced (P &lt; 0.05) the percentage of normal follicles in all media tested, except for tissue cultured in the presence of 200 ng mL-1 of a-tocopherol. Furthermore, in all media tested, the percentage of primordial follicles was significantly reduced (P &lt; 0.05), with a concomitant increase in the percentage of developing follicles. The highest percentage of secondary follicles was observed after 6 days of culture in MEM plus 200 ng mL-1 of a-tocopherol. The PCNA analysis confirmed the viability of follicles cultured with 200 ng mL-1 of a-tocopherol after 6 d. After 8 days of in vitro culture, we observed severe follicular degeneration in all media tested, suggesting that other supplements are recommended for longer periods of culture. In conclusion, the results of the present study indicate that 200 ng mL-1 of a-tocopherol maintains the survival of cattle preantral follicles and promotes activation of primordial follicles after 6 days of in vitro culture. Financial support: L. A. Lisboa is a recipient of CAPES support; E. R. Andrade and A. A. Alfieri are recipients of PRODOC/CAPES fellowships.

The effect of nerve growth factor on the development of human primordial follicles in vitro culture

OBJECTIVE: To investigate the actions of NGF on human early follicle development. DESIGN: Case-controlled experiment. MATERIALS AND METHODS: Ovarian biopsies from 21 patients were cut into ovarian pieces(opt) of lmm thickness which distributed into 5 groups: groupI (non-culture group),groupII (NGF group),groupIII (basic culture group),groupIV (anti-NGF antibody group),groupV (control IgG group). After culture, all the tissue samples were fixed for histological analysis, immunohistochemistry analysis,TUNEL test. RESULTS: Here were 33.83%±31.73%, 53.51%±39.41%, 39.30±31.73% primordial follicles among groupII, groupIII and groupIV,significantly lower than the proportion of primordial of groupI,which was 82.16%±13.63%, P<0.01; the percentage of developing follicles in groupII was 54.14%±26.16%, significantly higher than that of groupI, groupIII and groupIV, which were 14.63%±15.00%, 23.39%±28.66%, 31.44±32.24% respectively, P<0.01. The percentage of abnormal follicles in groupI was 3.22%±7.48%, significantly lower than that of groupIII, groupII, groupIV, which were 23.1%±30.94%, 12.03%±11.75%, 29.26±28.61% respectively, P<0.05. The Positive Unit value of groupI, groupIII, groupII, groupIV were 6.63 ±3.77, 5.66 ± 4.74, 12.48 ± 6.61, 6.67±4.58, 6.92±6.66 respectively, the PU value of groupII was significantly higher than that of the other groups, p<0.05. The PCNA positive follicles in groupI, groupIII, groupII, groupIV, groupV were 28.45%, 37.25%, 59.79%, 22.72%, 33.33% respectively, the rate of positive follicles in groupII was higher than that of groupIII and groupIV, p<0.005. The apoptotic index of stroma cells of groupII, groupIII, groupIV, groupV were 0.06±0.06, 0.12±0.07, 0.14±0.09, 0.12±0.09 repectively. The apoptotic index of stroma cell in groupII was significantly lower than that of groupIII, groupIV, p<0.01. CONCLUSIONS: NGF can help to promote the transition of primordial-to-primary follicles and their development in vitro culture of human ovarian tissues; By decreasing the apoptosis and increasing proliferation of stroma cells, NGF can help to promote the survival and development of early follicles in vitro culture of human ovarian tissues.

Growth and differentiation factor-9 stimulates activation of goat primordial follicles in vitro and their progression to secondary follicles

The aim of the present study was to investigate the effects of growth and differentiation factor-9 (GDF-9) on the survival and activation of preantral follicles, as well as their subsequent progression to secondary follicles, using goat ovarian cortical culture in vitro. Pieces of ovarian cortex were cultured for 1 and 7 days in minimum essential medium (MEM) with or without different concentrations of GDF-9 (1-200 ng mL(-1)). On Day 0 and after 1 and 7 days of culture, cortical pieces were fixed for histological and transmission electron microscopy evaluation. Preantral follicles were classified according to their development stage (primordial, intermediate, primary and secondary) and on the basis of morphological features (normal or degenerated). In addition, follicular and oocyte diameters were determined before and after culture. The results showed that, compared with non-cultured cortical tissue (Day 0), the culture of ovarian tissue significantly reduced (P < 0.05) the percentage of normal follicles in all media tested, except for tissue cultured in the presence of 200 ng mL(-1) GDF-9. Furthermore, in all media tested, the percentage of primordial follicles was significantly reduced (P < 0.05), with a concomitant increase in the percentage of developing follicles. The highest percentage of secondary follicles was observed after 7 days of culture in MEM plus 200 ng mL(-1) GDF-9. At all concentrations of GDF-9 tested, follicular diameter increased significantly after 7 days of culture compared with non-cultured cortical tissue. In conclusion, the results of the present study indicate that 200 ng mL(-1) GDF-9 maintains the survival of preantral follicles and promotes activation of primordial follicles. Furthermore, GDF-9 stimulates the transition from primary to secondary follicles, maintaining ultrastructural integrity of the follicles.

High oxygen atmosphere improves human follicle development in organ cultures of ovarian cortical tissues in vitro

Obtaining mature human follicles from cultured ovarian tissue may be beneficial for clinical use for women who wish to preserve fertile competence. However, the methodology of culture such as culture condition and gas atmosphere has not been well established in humans. Therefore, we investigated the effect of oxygen concentration in organ culture in order to establish an ovarian tissue culture method. Ovarian tissue was obtained from 26-35-year-old women undergoing removal of a benign tumor (n = 12) or caesarean section (n = 16). The ovarian cortical tissues were cultured on a cell culture insert for 15 days under high (100%) and low (air, 20%) oxygen concentrations and then inspected for follicle development with light and electron microscopy. Estradiol and progesterone concentrations in the medium during culture were measured. The ultrastructure and the function of hormone secretion in the cultured tissues were well preserved after organ culture. The follicles developing under high oxygen were larger and more matured than those developing under low oxygen (P < 0.05). Human ovarian tissues can be cultured for 15 days under high oxygen concentration with the organ culture system used here. This technique could make it possible to utilize ovarian tissue for preservation of reproductive competence in cancer patients.

Interactions among Peroxisome Proliferator Activated Receptor-γ, Insulin Signaling Pathways, and Steroidogenic Acute Regulatory Protein in Human Ovarian Cells

Peroxisome proliferator activated receptor-gamma (PPAR-gamma) agonists thiazolidinediones (TZDs) are thought to ameliorate hyperandrogenism in polycystic ovary syndrome by reducing hyperinsulinemia. However, TZDs also exhibit direct effects in the human ovary. We examined interactions among PPAR-gamma, insulin signaling pathways, and steroidogenic acute regulatory (StAR) protein in human ovarian cells. Mixed human ovarian tissue culture that contained granulosa, theca, and stromal cells, and a culture of purified granulosa cells obtained during in vitro fertilization, were established as previously described. Cells were cultured in the presence or absence of insulin, with or without 25 or 50 microm rosiglitazone or pioglitazone. Expression of PPAR-gamma, insulin receptor, or insulin receptor substrate (IRS)-1 in both cell systems and of the StAR protein in granulosa cells was measured using immunoprecipitation and immunoblotting. Rosiglitazone stimulated expression of PPAR-gamma, insulin receptor alpha- and beta-subunits, and IRS-1 up to 168% (P < 0.05), 679% (P < 0.006), 290% (P < 0.037), and 323% (P < 0.01) of baseline, respectively. Pioglitazone stimulated expression of PPAR-gamma, insulin receptor alpha- and beta-subunits, and IRS-1 up to 222% (P < 0.01), 362% (P < 0.001), 402% (P < 0.029), and 492% (P < 0.03), respectively. Insulin alone stimulated expression of PPAR-gamma, alpha-subunit and beta-subunit of insulin receptor, and IRS-1 up to 174% (P < 0.001), 692% (P < 0.014), 275% (P < 0.024), and 431% (P < 0.01), respectively. In purified granulosa cell culture, rosiglitazone stimulated expression of StAR protein up to 540% (P < 0.007), and pioglitazone stimulated expression of StAR protein up to 670% (P < 0.007). Insulin alone stimulated expression of StAR protein up to 600% (P < 0.012). Insulin and TZDs independently stimulate expression of PPAR-gamma, insulin receptor, IRS-1, and StAR protein in human ovarian cells. Thus, PPAR-gamma, insulin receptor with its signaling pathways, and StAR protein constitute a novel human ovarian regulatory system with complex interactions among its components.

Healthy Early Preantral Follicle Can Be Obtained in a Culture of Frozen–Thawed Human Ovarian Tissue of 32 Weeks

The objective of this study was to report morphological and functional evidence of a well-preserved preantral follicle recovered from human frozen–thawed ovarian tissue in a long-term culture. The tissue was originally obtained from a 26-year-old woman with breast cancer. The ovarian cortex was collected by laparoscopy and frozen/thawed and cultured for 32 weeks in minimum essential medium α-MEM, supplemented with insulin transferrine selenite (ITS), human serum (HS), antibiotics, follicle-stimulating hormone (FSH). and N-acetyl cysteine (NAC). Thawed tissue samples were examined by light microscopy (LM), transmission electron microscopy (TEM), and real-time RT-PCR. LM examination of cortical pieces after 32 weeks of culture showed a healthy early preantral follicle; TEM and real-time PCR confirmed its good state of preservation. The synergy in action of NAC and FSH plays an important role in follicle growth of ovarian tissue cultures. For the first time a well-preserved preantral follicle was found in a culture of frozen–thawed human ovarian tissue.

P-725: Correlation of antimullerian hormone levels with primordial follicle count in human ovary

Previous studies indicated the anti mullerian hormone (AMH) is expressed in preantral follicles and it might be a marker of ovarian reserve. However, there is no direct evidence tying AMH production to primordial follicles in humans. In the human ovary, AMH expression begins at 36 weeks of gestation. Our objective was to investigate if AMH levels directly correlate with primordial follicle numbers in human adult ovarian tissue using an in vitro model. In vitro investigation of AMH production from adult ovarian cortical pieces and fetal ovarian tissue Ovarian cortical pieces from 9 cancer patients undergoing ovarian cryopreservation for fertility preservation and human fetal ovarian tissue from abortuses of 24 weeks gestation were included in the study. 2x2 mm fetal and 5x5 mm adult ovarian pieces were either fixed, paraffin embedded, and serially sectioned to determine baseline follicle density (primordial follicle/mm2) on every fifth section, or cultured in alpha-MEM medium supplemented with 20 % fetal bovine serum and 300-mIU/mL recombinant FSH for seven days. Half of the culture medium was removed every day and stored at - 80 0C until assayed for AMH levels by ELISA. Primordial follicle density was re-evaluated after the 7-day culture. Primordial follicle density was determined as the mean of follicle counts/mm2 in every fifth section. Mean ± [SE] age of the adult patients from whom the tissues were obtained was 27.5 ± 2.7 (range 18-36). The mean primordial follicle density at baseline and after culture was 29.5 ± 3.1 and 3.12 ± 1.3 in fetal ovaries, and 9.31 ± 2.6 and 5.83 ± 2.1 in adult patients, respectively. There were no growing follicles in fetal ovarian tissue before and after culture. In adult tissue 98.2% of follicles were at primordial stage before culture but no growing follicles were seen after the 7-day culture period. In fetal ovarian tissue, 100% of follicles were in primordial stage before and after culture. As expected, AMH was undetectable in fetal ovarian cultures. In adult ovarian tissue cultures, both AMH level on culture day 7 and age correlated with post-culture follicle counts (p<0.01, Fig-1). We provided the first evidence linking the in vitro AMH production to primordial follicle density in human ovarian tissue.

Effects of PKC beta inhibitor enzastaurin on parental and chemoresistant ovarian cancer cell lines

20037 Background: Enzastaurin (LY317615.HCl), a selective inhibitor of protein kinase C beta (PKCβ), inhibits induction of angiogenesis and apoptosis. Activation of PKCβ has been correlated with tumor cell proliferation and invasiveness. The impact of Enzastaurin was investigated in an ovarian cancer tissue culture model utilizing the parental cell line HEY and resistant subclones against different cytostatic drugs. Methods: The ovarian cancer cell line HEY was used to develop subclones with selective resistance against Cisplatin, Etoposide, Docetaxel and Paclitaxel, respectively. The ovarian cell line IGROV-1 with high expression of PKC beta served as control. All cell lines were stimulated with 5 μM Enzastaurin for up to four hours. Immunoblotting analyses determined the expression of GSK3β, a target of PKCβ. Proliferation and apoptosis induction after Enzastaurin treatment was analysed by a proliferation assay and DAPI staining. Results: All cell lines showed phosphorylation of GSK3β, the highest expression level was found in parental and Cisplatin-resistant HEY cells as well as in IGROV-1 cells. Phosphorylation decreased after stimulation with 5 μM Enzastaurin for 30 minutes, most obvious detectable in parental and Cisplatin-resistent HEY cells and also IGROV-1 cells. Proliferation and apoptosis assays demonstrated the Paclitaxel- and Docetaxel-resistant HEY cells as the most sensitive cell lines for Enzastaurin treatment. In contrast, Cisplatin-resistant HEY cells and IGROV-1 cells exhibited the highest resistance against Enzastaurin. Conclusion: The results indicate that ovarian cell lines with a high expression level of active PKCβ like Cisplatin-resistant HEY cells and IGROV-1 cells display also strong phosphorylation of GSK3β. Though in these cell lines the inhibitory effect of Enzastaurin is most prominent, the cell proliferation is hardly affected, nor apoptosis is accelerated when Enzastaurin concentrations up to 10–15 μM were used. Compared to the Cisplatin-resistant variants the normal HEY cells are more sensitive to the inhibitor. Taxane-resistant cells seem to respond to low concentrations of Enzastaurin. Therefore, Enzastaurin may become an effectful drug to treat ovarian carcinomas with resistance to Taxane-based chemotherapies. [Table: see text]

Effective method for in-vitro culture of cryopreserved human ovarian tissue

It is widely accepted that it is possible to successfully cryopreserve human ovarian tissue by direct plunging into liquid nitrogen using permeable cryoprotectants only, without disaccharides. This study aimed to search for and test a new method for in-vitro culture of vitrified tissue. Ovarian biopsies were obtained during operative laparoscopy. Pieces of ovarian tissue were vitrified and warmed. After warming, tissue pieces were randomly distributed into three groups for further culture: in 2 ml of culture medium which was regularly renewed (group 1), in 30 ml of culture medium without agitation (group 2) and in 30 ml of culture medium with agitation (group 3). During the 2-week and 6-week culture, the growth of follicles within the vitrified-warmed ovarian tissue pieces was investigated. After 2 weeks of culture, mean numbers of non-degenerated follicles per mm 2 of tissue were 1.5, 1.7 and 4.5 for groups 1, 2 and 3 respectively (groups 1 and 2 versus group 3, P < 0.05). Agitation during culture of ovarian tissue is beneficial, and can be used as a prognostic tool for future warming and autotransplantation of ovarian tissue.

Cryopreseved ovarian tissue transplantation into mammary gland

Cryopreseved ovarian tissue transplantation into mammary gland

Cryopreservation and culture of human ovarian cortical tissue containing early follicles.

Maturation of oocytes from primordial follicles after cryopreservation of ovarian tissue with subsequent in vitro fertilisation would be an important method of infertility treatment for young women who are to undergo premature ovarian failure due to chemotherapy or genetic causes. Cryopreservation of human ovarian tissue is already feasible. Human primordial follicles can be cultured regularly to secondary and occasionally to early antral stages, yet cultures have to be further optimised before meiotically competent oocytes can be achieved for final maturation in vitro. Maturation in vitro (IVM) of oocytes obtained by aspiration from small antral follicles is already a feasible technique resulting in pregnancies, with many healthy infants born.