Search

The reproductive lifespan in humans is regulated by a delicate cyclical balance between follicular recruitment and atresia in the ovary. The majority of the small antral follicles present in the ovary are progressively lost through atresia without reaching dominance, but this process remains largely underexplored. In our study, we investigated the characteristics of atretic small antral follicles and proposed a classification system based on molecular changes observed in granulosa cells, theca cells, and extracellular matrix deposition. Our findings revealed that atresia spreads in the follicle with wave-like dynamics, initiating away from the cumulus granulosa cells. We also observed an enrichment of CD68+ macrophages in the antrum during the progression of follicular atresia. This work not only provides criteria for classifying three stages of follicular atresia in small antral follicles in the human ovary but also serves as a foundation for understanding follicular degeneration and ultimately preventing or treating premature ovarian failure. Understanding follicular remodeling in the ovary could provide a means to increase the number of usable follicles and delay the depletion of the follicular reserve, increasing the reproductive lifespan. [ABSTRACT FROM AUTHOR]

• This study describes ECM fertility biomarker candidates and characterizes their spatiotemporal deposition and 3D localization from prepuberty until menopause. • The dominant collagen type in human ovarian tissue at all age stages is collagen VI. • mTOR and Hippo pathways, which are involved in fundamental processes of female fertility, and their continuum interaction with ECM have been interrogated in silico and connected to ovarian ECM. • Proteins with cytoprotective and anti-oxidative activities increase with age, which challenges aging dogma and reorients future investigations. • Post-translational modifications correlate with age and have an impact on ovarian biomechanics and activity. Our modern era is witnessing an increasing infertility rate worldwide. Although some of the causes can be attributed to our modern lifestyle (e.g., persistent organic pollutants, late pregnancy), our knowledge of the human ovarian tissue has remained limited and insufficient to reverse the infertility statistics. Indeed, all efforts have been focused on the endocrine and cellular function in support of the cell theory that dates back to the 18th century, while the human ovarian matrisome is still under-described. Hereby, we unveil the extracellular side of the story during different periods of the ovary life, demonstrating that follicle survival and development, and ultimately fertility, would not be possible without its involvement. We examined the human ovarian matrisome and described its remodeling from prepuberty until menopause, creating the first ovarian proteomic codex. Here, we confidently identified and quantified 98 matrisome proteins present in the three ovary groups. Among them, 26 were expressed differently among age groups, delineating a peculiar matrisomal fingerprint at each stage. Such proteins could be potential biomarkers phenotyping ovarian ECM at each age phase of female reproductive life. Beyond proteomics, our study presents a unique approach to understanding the data and depicting the spatiotemporal ECM-intracellular signaling networks and remodeling with age through imaging, advanced text-mining based on natural language processing technology, machine learning, and data sonification. Our findings provide essential context for healthy ovarian physiology, identifying and characterizing disease states, and recapitulating physiological tissues or development in vitro. This comprehensive proteomics analysis represents the ovarian proteomic codex and contributes to an improved understanding of the critical roles that ECM plays throughout the ovarian life span. [ABSTRACT FROM AUTHOR]

Study Question: Can human theca cells (TCs) be differentiated in vitro?Summary Answer: It is possible to differentiate human TCs in vitro using a medium supplemented with growth factors and hormones.What Is Known Already: There are very few studies on the origin of TCs in mammalian ovaries. Precursor TCs have been described in neonatal mice ovaries, which can differentiate into TCs under the influence of factors from oocytes and granulosa cells (GCs). On the other hand, studies in large animal models have reported that stromal cells (SCs) isolated from the cortical ovarian layer can also differentiate into TCs.Study Design, Size, Duration: After obtaining informed consent, ovarian biopsies were taken from eight menopausal women (53-74 years of age) undergoing laparoscopic surgery for gynecologic disease not related to the ovaries. SCs were isolated from the ovarian cortex and in vitro cultured for 8 days in basic medium (BM) (G1), enriched with growth factors, FSH and LH in plastic (G2) or collagen substrate without (G3) or with (G4) a GC line.Participants/materials, Setting, Methods: To confirm TC differentiation, relative mRNA levels for LH receptor (Lhr), steroidogenic acute regulatory protein (Star), cholesterol side-chain cleavage enzyme (Cyp11a1), cytochrome P450 17A1 (Cyp17a1), hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 1 (Hsd3b1) and hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 2 (Hsd3b2) were assessed. Immunohistochemistry was also performed for their protein detection and a specific marker was identified for TCs (aminopeptidase-N, CD13), as were markers for theca and small luteal cells (dipeptidyl peptidase IV (CD26) and Notch homolog 1, translocation-associated (NOTCH1)). Finally, we analyzed cell ultrastructure before (Day 0) and after in vitro culture (Day 8), and dehydroepiandrosterone (DHEA) and progesterone levels in the medium using transmission electron microscopy (TEM) and ELISA, respectively.Main Results and the Role Of Chance: Results obtained from qPCR showed a significant increase (P < 0.05) in mRNA levels of Lhr in F2 (floating cells in G2) and G4, Cyp17a1 in G1 and F1 (floating cells in G1) and Hsd3b2 in G1, G2, G3 and G4. Immunohistochemistry confirmed expression of each enzyme involved in the steroidogenic pathway at the protein stage. However, apart from G1, all other groups exhibited a significant (P < 0.05) rise in the number of CD13-positive cells. There was also a significant increase (P < 0.05) in NOTCH1-positive cells in G3 and G4. Ultrastructure analyses by TEM showed a distinct difference between groups and also versus Day 0. A linear trend with time revealed a significant gain (q < 0.001) in DHEA concentrations in the medium during the culture period in G1, G2, G3 and G4. It also demonstrated a statistical increase (q < 0.001) in G2, G3 and G4 groups, but G1 remained the same throughout culture in terms of progesterone levels.Large Scale Data: N/A.Limitations, Reasons For Caution: Shorter periods of in vitro culture (e.g. 2, 4 and 6 days) could have led to increased concentrations of differentiated TCs in G2, G3 and G4. In addition, a group of cells cultured in BM and accompanied by COV434 cells would be necessary to understand their role in the differentiation process. Finally, while our results demonstrate that TCs can be differentiated in vitro from cells isolated from the cortical layer of postmenopausal ovaries, we do not know if these cells are differentiated from a subpopulation of precursor TCs present in ovarian cortex or ovarian SCs in general. It is therefore necessary to identify specific markers for precursor TCs in human ovaries to understand the origin of these cells.Wider Implications Of the Findings: This is a promising step toward understanding TC ontogenesis in the human ovary. Moreover, in vitro-generated human TCs can be used for studies on drug screening, as well as to understand TC-associated pathologies, such as androgen-secreting tumors and polycystic ovary syndrome.Study Funding/competing Interest(s): This study was supported by grants from the Fonds National de la Recherche Scientifique de Belgique (FNRS) (C.A.A. is an FRS-FNRS Research Associate; grant MIS #F4535 16 awarded to C.A.A.; grant 5/4/150/5 awarded to M.M.D.; grant ASP-RE314 awarded to P.A.) and Foundation Against Cancer (grant 2018-042 awarded to A.C.). The authors declare no competing interests. [ABSTRACT FROM AUTHOR]

Objective(s): We aimed to analyze if anti-apoptotic agent sphingosine-1-phosphate offers protection against in vitro follicle atresia during culture of human ovarian cortical samples.Study Design: A translational research study of ex-vivo and in-vitro models of human ovarian tissue.Material and Methods: Ovarian cortical tissue fragments (1 × 0.5 cm) were obtained from young patients (n = 15 mean age ± SD: 29.4 ± 2.5) undergoing laparoscopic excision of benign ovarian cysts. The samples were cultured for 4 days in 24-well format culture plate using conventional culture techniques. S1P was added to culture media at 200 and 400 μM concentrations. At the end of culture period the samples were processed for both histomorphological assessment and detection of apoptosis with immunohistochemistry and western blot methods using apoptosis marker cleaved caspase-3. In vitro estradiol (E2) and AMH productions of the samples were measured with ELISA. Follicle counts were expressed as the mean number of follicles per mm2.Results: The mean numbers of primordial and secondary follicles were 3.2 ± 0.4 and 0.7 ± 0.2 respectively, in the fresh fixed uncultured samples. After four days of culture their numbers were significantly decreased to 0.8 ± 0.2 (p < 0.01) and 0.1 ± 0.05 (p < 0.05) respectively, in the control samples cultured without S1P compared to fresh fixed samples. S1P treatment decreased follicle atresia and significantly higher number of primordials (2.3 ± 0.3, p < 0.01) and secondary follicles (0.5 ± 0.1, p < 0.05) survived in the samples after 4 day culture period compared to those cultured without S1P. In line with this there was dose-dependent decrease in the protein expression of cleaved caspase-3 on western blot and in the number of apoptotic follicles stained positive for cleaved caspase-3 on immunohistochemistry in the samples incubated with S1P at 200 and 400 μM concentrations. Furthermore, those samples incubated with S1P produced significantly higher amounts of E2 (2339 ± 321 vs. 1156 ± 125 pg/mL respectively, p < 0.01) compared to control samples.Conclusions: These results suggest that S1P promotes follicle survival in human ovarian cortical samples in vitro. [ABSTRACT FROM AUTHOR]

Purpose: The purpose of this study was to evaluate the effect of transportation at prolonged low temperatures on the survival of pre-antral follicles. Methods: Ovarian tissue was removed from six women with gender identity disorder. Tissues were stored in an icebox at 4 °C for 6 or 18 h prior to vitrification. After warming, ovarian tissues were cultured for 24 h and follicle survival was assessed via a viability/cytotoxicity kit. Morphological features and oxygen consumption rate (OCR) were evaluated by scanning electrochemical microscopy (SECM). Results: Survival rate of isolated primordial follicles was 95.7 and 100 %, and that of primary follicles was 91.7 and 81.8 % in the 6- and 18-h groups respectively. There was no difference in morphology between the 6- and 18-h storage groups. In comparison with OCR of vitrified-warmed follicles and OCR of 24-h culture after vitrified-warmed follicles, OCR of 24-h culture after vitrified-warmed primordial follicles was significantly higher in both 6-hour (0.02 ± 0.02 vs 0.07 ± 0.04, P < 0.05) and 18-h groups (0.02 ± 0.02 vs 0.11 ± 0.10, P < 0.05). Conclusions: This strongly suggests that prolonged transportation of ovarian tissue at low temperatures is useful when there are no available local systems for fertility preservation. [ABSTRACT FROM AUTHOR]

Background: Conflicting results of studies on mouse and human have either verified or refuted the presence of oogonia/primordial germ cells in the post-natal ovary. The aim of this study was to trace whether oogonia recognized by immunohistochemical methods in the first trimester human ovary were present also in peri- and post-natal ovaries. Methods: For this study, 82 human ovaries were collected: 25 from embryos from 5 to 10 weeks post conception (wpc), 2 at 18 wpc, 32 from 32 wpc to 2 years and 23 from 2 to 32 years. Of these, 80 ovaries were fixed and paraffin-embedded and 2 (8 year-old) ovaries were processed for plastic sections. Serial sections were prepared for immunohistochemical detection of markers for oogonia: tyrosine kinase receptor for stem cell factor (SCF)(C-KIT), stage-specific embryonic antigen-4 (SSEA4), homeobox gene transcription factor (NANOG), octamer binding transcription factor 4 (OCT4) and melanoma antigen-4 (Mage-A4), while noting that C-KIT also stains diplotene oocytes. Results: Almost all oogonia exclusively stained for SSEA4, NANOG, OCT4 and C-KIT, whereas MAGE-A4 only stained a small fraction. At birth only a few oogonia were stained. These disappeared before 2 years, leaving only diplotene oocytes stained for C-KIT. From 18 wpc to 2 years, the medulla contained conglomerates of healthy and degenerating oogonia and small follicles, waste baskets (WBs) and oogonia enclosed in growing follicles (FWB). Medulla of older ovaries contained groups of primordial, healthy follicles. Conclusions: We found no evidence for the presence of oogonia in the human ovary after their final clearing during the first 2 years. We suggest that perinatal medullary WB and FWB give rise to the groups of small, healthy follicles in the medulla. [ABSTRACT FROM AUTHOR]

The gonadotropins, follicle-stimulating hormone (FSH), and luteinizing hormone (LH), are of particular importance in ovarian physiology. However, FSH receptors and LH receptors are not expressed until the secondary follicle stage, indicating that initiation of follicular growth is independent of the gonadotropins. Among many intra-ovarian growth factors, many studies have shown that bone morphogenetic proteins (BMPs) play pivotal roles in regulating the early phases of follicular growth. The BMP system induces the gonadotropin system by modulating gonadotropin receptors in early-stage follicles. Interestingly, the BMP system also prevents precocious maturation of the follicle by suppressing luteinization. Signals provoked by the preovulatory LH surge eliminate BMPs, enabling luteinization to progress. Thus, the BMP system and the gonadotropin system seem to cooperate in regulating follicular development, maturation, and luteinization. [ABSTRACT FROM AUTHOR]

Objective: To determine the concentrations of stromal cell-derived factor-1 (SDF-1/CXCL12) and vascular endothelial growth factor (VEGF) in individual human preovulatory follicles in relation to their diameter or volume for clarifying the role of these molecules in folliculogenesis. Design: Prospective study. Setting: Research laboratory at Kansai Medical University. Patient(s): Twenty-seven women undergoing IVF. Intervention(s): Follicular fluid (FF) was collected from individual follicles. A total of 373 follicles were analyzed. Main Outcome Measure(s): The concentrations of SDF-1 and VEGF in FF and oocyte recovery rates. Result(s): The concentrations of SDF-1 and VEGF in follicles with a diameter ≤14 mm were significantly lower than those in follicles with a diameter ≥15 mm. The concentrations of SDF-1 and VEGF in FF increased with follicular diameters or volume, with concentrations peaking in follicles with a diameter of 18–20 mm or a volume of 3.6–5.0 mL. Furthermore, we found that there exists a positive correlation between the concentrations of SDF-1 and VEGF in FF from follicles ≤20 mm in diameter. The oocyte recovery rates increased with concentrations of SDF-1 and VEGF in FF. Conclusion(s): Our data suggest that SDF-1, as well as VEGF, may play an important role in follicular growth and development. [ABSTRACT FROM AUTHOR]

Objective: To investigate the relationship between clinical markers of ovarian reserve and the true ovarian reserve as determined by the ovarian primordial follicle number. Design: Prospective investigation. Setting: Academic medical center. Patient(s): Forty-two healthy women (aged 26-52 years) undergoing oophorectomy for benign gynecologic indications. Intervention(s): Transvaginal ultrasound examination for the determination of the ovarian antral follicle count (AFC) and serum measurements of clinical markers of ovarian reserve. All measurements were obtained within 2 weeks of surgery, irrespective of cycle day. Ovarian primordial follicle count was then determined using a validated fractionator/optical disector method. Main Outcome Measure(s): Univariate and partial correlations between ovarian reserve markers and ovarian primordial follicle count. Result(s): There were significant correlations between the ovarian primordial follicle count and AFC (r=0.78), anti-Müllerian hormone (AMH; r=0.72), FSH (r=-0.32), inhibin B (r=0.40), and chronological age (r=-0.80). After adjusting for age, significant correlations were identified between the ovarian primordial follicle count and AFC (r=0.53) and AMH (r=0.48). Conclusion(s): The ovarian AFC and serum levels of AMH correlate with the ovarian primordial follicle number even after adjustment for chronological age. [ABSTRACT FROM AUTHOR]

Abstract: WNT4 plays an important role in female sexual development and ovarian function. WNT4-deficiency leads disturbed development of the internal genitalia in mouse and human, and to a dramatic reduction of mouse oocytes. However, the expression and role of WNT4 in human ovaries is yet unknown. The expression of WNT4 mRNA and protein was studied in human adult and fetal ovaries (gestational ages 12–41 weeks), and the role of WNT4 in oocyte apoptosis was investigated in WNT4-deficient mice. WNT4 mRNA and protein were present in human ovaries throughout fetal development and in different follicular stages in adult ovaries. Compared with wild-type mice, WNT4-deficient mice had a markedly enhanced rate of oocyte apoptosis, with the highest values at gestational ages of 14.5 and 16.5 days post-coitum. The current results support a view that WNT4 may have a role in oocyte selection and follicle formation and maturation in human ovaries. [Copyright &y& Elsevier]

Dysfunction in thyroid regulation can cause menstrual and ovulatory disturbances, the mechanism of which is not clear. The distribution and activity of the thyroid-stimulating hormone (TSHR), and the thyroid hormone receptors (TR) α, α2 and β1 in human ovarian tissue and in granulosa cells was studied using immunohistochemistry, reverse-transcriptase polymerase chain reaction (RT-PCR), quantitative PCR and immunoassays. Strong immunostaining of TSHR, TRα1 and TRβ1 was observed in ovarian surface epithelium and in oocytes of primordial, primary and secondary follicles, with minimal staining in granulosa cells of secondary follicles. Granulosa cells of antral follicles expressed TSHR, TRα1 and TRβ1 proteins. Messenger RNA for all receptors was present in ovarian tissue. Mature human granulosa cells expressed transcripts for 5′ deiodinases types 2 and 3, but not type 1, indicating the possibility of conversion of peripheral thyroid hormone thyroxin (T4). Granulosa cells stimulated with TSH showed a significant increase in cAMP concentrations after 2 h of culture (P = 0.047), indicating activation through TSHR. Stimulation with T4 resulted in increased extracellular signal-regulated kinase 1 and 2 activation after 10, 30, 60 min and 24 h. These data demonstrate that TSH and thyroid hormone receptors may participate in the regulation of ovarian function. [ABSTRACT FROM AUTHOR]

This investigation compared conventional freezing of human ovarian tissue using either spontaneous or initiated ('seeded') ice formation. Biopsies of ovarian tissue were obtained from women with indications for chemotherapy or radiotherapy. Small pieces of experimental tissue were randomly distributed into three groups that were then subjected to different treatments prior to culture in vitro for 16 days: the control group, no treatment, cultured immediately after biopsy (group 1); cryopreservation/ thawing with spontaneous ice formation (group 2); and cryopreservation/thawing with initiated ice formation (group 3). Follicle viability and hormonal activity were then evaluated. There was no significant difference between groups regarding the concentration of oestradiol 17-β in the culture supernatant, whereas progesterone concentration was significantly (P < 0.05) higher in group 1 compared with group 2 or 3. There was a significant (P < 0.05) difference in primordial and primary follicle density between all of the groups (group 1 having the highest and group 2 having the lowest) and group 2 had significantly (P < 0.05) fewer normal grade follicles than the other two groups. For optimal cryopreservation of human ovarian tissue, the protocol of conventional freezing should therefore include a step of initiated ice formation. [ABSTRACT FROM AUTHOR]

Gonadotropin-releasing hormone (GnRH) plays a pivotal role in the physiology of reproduction in mammals. GnRH acts by binding to the GnRH receptor (GnRHR). In humans, only 1 conventional GnRH receptor subtype (type 1 GnRH receptor) has been found. In the human genome, 2 forms of GnRH have been identified, GnRH-I (mammal GnRH) and GnRH-II (chicken GnRH II). Both forms and their common receptor are expressed, apart from the hypothalamus, in various compartments of the human ovary. Gonadal steroids, gonadotropins, and GnRH itself controls the regulation of the GnRH/GnRHR system gene expression in the human ovary. The 2 types of GnRH acting paracrinally /autocrinally influence ovarian steroidogenesis, decrease the proliferation, and induce apoptosis of ovarian cells. In this review, the biology of GnRH/GnRHR system in humans, the potential roles of GnRH, and the direct effects of GnRH analogues in ovarian cells are discussed. [ABSTRACT FROM AUTHOR]

Abstract: Cryopreservation, which is the most important procedure in ovarian tissue banking, can be divided into two methods: conventional freezing and rapid freezing. In previous study, the higher effectiveness of rapid freezing in comparison with the conventional freezing for human oocytes and embryos was shown. Data on comparison of these two methods for human ovarian tissue are limited. The aim of this study was to compare conventional freezing and rapid freezing for human ovarian tissue. Ovarian tissue fragments from 14 patients were transported to the laboratory within 22–25h in a special, isolated transport box, which can maintain a stable temperature of between 5 and 8°C for 36h. Small pieces of ovarian tissue (1×1–1.5×0.7–1mm) were randomly distributed into four groups: Group 1: control, fresh pieces immediately after receiving transport box, Groups 2 and 3: experimental pieces after rapid freezing/warming, and Group 4: experimental pieces after conventional freezing/thawing. All pieces were cultured in vitro for 14 days. The viability of the tissue by in vitro production of hormones and development of follicles after culture was evaluated. The level of estradiol 17-β and progesterone was measured using heterogeneous competitive magnetic separation immunoassay. For histological analysis, the number of viable and damaged follicles was counted. After culture of fresh tissue pieces (Group 1), rapidly frozen/warmed pieces (Groups 2 and 3), and conventionally frozen/thawed pieces (Group 4), the supernatants showed estradiol 17-β concentrations of 358, 275, 331, and 345pg/ml, respectively, and progesterone concentrations of 3.02, 1.77, 1.99, and 2.01ng/ml, respectively. It was detected that 96%, 36%, 39%, and 84% follicles for Groups 1, 2, 3, and 4, respectively, were normal. For cryopreservation of human ovarian tissue, conventional freezing is more promising than rapid freezing. [Copyright &y& Elsevier]

Objective: To assess directly the dynamics of the formation of the ovarian reserve in the normal human ovary by evaluating the total number of follicles in developing ovaries when folliculogenesis occurs. Design: Histomorphometry-based follicle counts in complete serial tissue sections. Setting: Functional Anatomy Research Center, University of Milano. Patient(s): Thirteen fetuses, neonates, and one 8-month-old infant. Intervention(s): Fifteen ovaries were completely cut, obtaining serial sections. Ovarian volume, volume fractions, density and total number of follicles per ovary were calculated using quantitative morphometric methods. Main Outcome Measure(s): Age-related dynamics of the establishment of ovarian reserve in human developing ovary at the end of the organogenesis. Result(s): The ovarian reserve (100,000 follicles at 15 weeks of postconceptional age) increased progressively to 680,000 follicles at 34 weeks. At 8 months of postnatal age the pool was still about 680,000 primordial follicles. Conclusion(s): The consistence of the primordial follicle pool during organogenesis shows an exponential increase until month 8 of prenatal life and it is subsequently maintained without modifications at least until month 8 of postnatal life. [Copyright &y& Elsevier]

Objective: To study gene expression profiles of connective tissue components in polycystic ovaries using complementary deoxyribonucleic acid (cDNA) array technology. Design: Descriptive study of normal and polycystic human ovarian biopsy samples analyzed by cDNA array hybridizations. Setting: Experimental laboratory research. Patient(s): Eight women with polycystic ovary syndrome (PCOS) and two normally cycling women treated with electrocauterization and hysterectomy, respectively. Interventions: Ovarian biopsy samples. Main outcome measure(s): Expression levels of 588 genes involved in cellular invasion, extracellular matrix (ECM) turnover, and cell–ECM interactions in polycystic ovaries. Result(s): A majority of the 30 genes down-regulated in PCOS ovaries represented those related to cell adhesion and motility, as well as angiogenesis, followed by regulators of cell cycle and growth. The 14 up-regulated genes represented those regulating cell fate and development, growth factors, cytokines, chemokines, and cell–cell interactions. Of the 44 transcripts exhibiting marked changes in the cDNA array analysis, only one — proliferating cell nuclear antigen messenger ribonucleic acid (PCNA mRNA) — was systematically down-regulated; 2 transcripts, for CDC27HS protein and CD9 antigen, were down-regulated in 7 out of 8 PCOS samples. Conclusion(s): The present data suggest that gene expression profiling may become a useful tool to classify PCOS patients into subgroups with different etiologies. Genome-wide expression profiling using microarrays should be performed to better understand the metabolic derangement(s) in PCOS. [Copyright &y& Elsevier]

Objective: To document the expression of activated forms of caspase-3 in human granulosa cells. Design: Laboratory study. Setting: In vitro fertilization (IVF) laboratory of the Split University Hospital and laboratory of the Department of Anatomy, Histology, and Embryology. Patient(s): Ovarian tissues were obtained from women undergoing hysterectomy/ovariectomy for benign conditions and human granulosa cells were obtained from women undergoing oocyte retrieval for IVF. Intervention(s): Immunostaining of tissue sections and cell smears using antibody to active caspase-3 and terminal deoxynucleotidyl transferase (TdT) assay (TUNEL) for detection of internucleosomal DNA fragmentation. Main outcome measure(s): Microscopic evaluation to assess the presence and cellular co-localization of active caspase-3 and TUNEL-positive cells. Result(s): In human ovarian tissue, no apoptosis was observed in primordial and primary follicles. Apoptosis in granulosa cells was detected only in atretic antral follicles. Granulosa cells classified as apoptotic on the basis of their morphologic features contained a single condensed nucleus, multiple nuclear fragments, or apoptotic bodies. All apoptotic granulosa cells expressed active caspase-3, but only few contained fragmented DNA detected with the TUNEL method. The expression of active caspase-3 was also demonstrated in human granulosa cells of preovulatory follicles obtained from patients undergoing IVF. Conclusion(s): Caspase-3 dependent apoptosis occurs in human granulosa cells and activates when follicles begin to leave the resting pool. After initial formation of the antrum, activation of caspase-3 is a normal physiologic process of the follicle during atresia and luteinization. Higher numbers of granulosa cells positive with caspase-3 than cells positive with TUNEL suggest an earlier activation of caspase-3 compared with the DNA fragmentation detected by TUNEL assay and also a longer detection period of caspase-3 than DNA fragmentation in apoptotic granulosa cells. [Copyright &y& Elsevier]

RON is a member of the receptor tyrosine kinase gene family that includes the MET oncogene, whose germline mutations have been causally related to human tumorigenesis. In vitro, RON and MET receptors cross-talk, synergize in intracellular signaling, and cooperate in inducing morphogenic responses. Here we show that the RON and MET oncogenes were expressed in 55% and 56% of human ovarian carcinomas, respectively, and were significantly coexpressed in 42% (P < 0.001). In ovarian carcinoma samples and cell lines we did not find mutations in RON and MET gene kinase domain, nor coexpression of RON and MET receptor ligands (MSP and HGF, respectively). We show that motility and invasiveness of ovarian cancer cells coexpressing MET and RON receptors were elicited by HGF and, to a lesser extent, by MSP. More interestingly, invasion of both reconstituted basement membrane and collagen gel was greatly enhanced by the simultaneous addition of the two ligands. These data suggest that coexpression of the MET and RON receptors confer a selective advantage to ovarian cancer cells and might promote ovarian cancer progression. [Copyright &y& Elsevier]

The ovarian surface epithelium (OSE) is a key tissue in the pathogenesis of ovarian surface epithelial-stromal tumours and ovarian endometriosis, commonly encountered gynaecological diseases. Despite the high incidence of these diseases, experimental in vitro studies of OSE are few and so we used the scraping method with an enzymatic procedure to isolate human OSE and studied its characteristics in vitro. Nineteen normal ovaries were used. After incubation of the ovary for 40 min in collagenase type 1 solution (300 U/ml), the surface cells were removed by gentle scraping with a surgical blade. Cells obtained as a cluster after unit gravity sedimentation with 5% bovine serum albumin in medium 199 were cultured in medium 199 containing 15% fetal bovine serum. The viable cell number in a single ovary was 0.1-2.7 x 10(6). The outgrowth of cells started from a homogeneous population of single cells, and the cell population doubling time was between 7 and 10 days. Confluent monolayers were formed after 13-20 days and subcultured from one to three times. The monolayers mostly had a cobblestone appearance, and fusiform or polygonal cells were also observed. By cytochemistry, immunocytochemistry and scanning and transmission electron microscopy, the cells were shown to have characteristics of mesothelial OSE cells in short-term culture. This experimental approach was efficient in providing cultured human OSE, which can be utilized to investigate pathobiology and carcinogenesis. [ABSTRACT FROM AUTHOR]

Background: Normal pubertal ovary displays all stages of follicular development and a biased BAX/BCL2 protein ratio in favor of pro-apoptotic BAX protein comparable to the adult ovary. However, adolescents suffering malignant extra-gonadal disease show a limited follicle development after cytotoxic drug treatment and a reduced capacity of in vitro follicle growth. We evaluated the expression of pro- and anti-apoptotic members of the BCL2 gene family, the FAS/FAS-L proteins from the extrinsic apoptosis pathway, the germ-cell-specific marker VASA, the pluripotency marker OCT3/4, and markers of early and late apoptosis in the ovary of pubertal patients with malignant extra-gonadal disease, which received or not pre-surgery chemotherapy, entering a cryopreservation program. Results: Ovarian biopsies from 12 adolescent girls were screened for follicle count and expression of VASA, OCT3/4, BAX, BCL2, MCL1L and S, cleaved-BID, FAS/FAS-L and CASPASE 3 through immunohistochemistry, western blot and RT-PCR. All stages of folliculogenesis, from primordial to antral follicle, were present in all 12 patients analyzed. VASA and most of the screened apoptosis-related genes showed a pattern of immune-expression comparable to that previously reported. OCT3/4 showed a cytoplasmic localization in the great majority of the primordial follicles; however, in some cases the localization was nuclear. In addition, OCT3/4B showed a significant reduction compared to OCT3/4A. Unexpectedly, BCL2 was detected at all stages of folliculogenesis, associated to the Balbiani's body in the primordial follicles, regardless of whether patients had or had not received chemotherapy, ruling out the possibility that its expression is a protective response to chemotherapy. Conclusions: These findings reveal new information on the morphological status of the follicular reserve and the expression of apoptosis-related genes in histologically normal adolescent ovary from patients undergoing extragonadal cancer. The unexpected expression of apoptosis-inhibiting BCL2 protein, both in patients that had or had not received chemotherapy, opens a new avenue for thorough investigations. Moreover, the nuclear localization of OCT3/4 protein in primordial follicle-enclosed oocytes suggests a possible increased activity of ovarian stem cells in response to chemotherapy and/or extragonadal cancer. This new information can be essential for a better managing of in vitro culture of follicles that can be removed by filtration from preserved ovarian tissue, especially in girls that entered a cryopreservation program. [ABSTRACT FROM AUTHOR]