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Introduction: In South Africa, access to higher education is viewed as a pathway to improved life chances. However, the transition from school to university is a stressful experience for students, marked by significant challenges. Although literature acknowledges these challenges, limited research has focused specifically on the role of hope during this period. Additionally, previous studies on hope have often utilized individualistic approaches, which may not fully capture the experience of students in collectivist cultures. This study addresses this gap by exploring hope from a culturally sensitive perspective within a collectivist context, aiming to understand how first-year South African university students experience hope during the transition to university., Methods: This study employed Interpretative Phenomenological Analysis (IPA) to explore the lived experiences of hope among first-year South African university students during their transition to higher education. Twenty-two students participated in semi-structured interviews, allowing for an in-depth examination of their personal and cultural perspectives on hope and the challenges they faced. The data were analyzed iteratively, with strategies implemented to enhance trustworthiness and credibility, ensuring a thorough interpretation of students' experiences., Results: The analysis yielded three main themes: (1) Affective and Social Duality: Students described mixed emotions and social challenges as they entered university, highlighting the dual nature of their experiences in adapting to a new environment. (2) Hope as a Multifaceted Concept: Hope was portrayed as a guiding force that helped students navigate periods of uncertainty. Participants described hope not just as a single idea but as a complex, evolving concept crucial to their resilience. (3) Beyond Academic Aspirations: Hope extended beyond academic success and was closely tied to personal fulfilment and the desire to contribute positively to society. This broader perspective on hope suggests that students' aspirations were not confined to individual achievement but also included a sense of collective and societal impact., Discussion: The study reveals that hope is a multidimensional construct that significantly influences students' transitional experiences, extending beyond academic goals to include personal growth and societal contributions. This finding challenges the traditional, individualistic approaches to studying hope by highlighting the cultural relevance of collectivist values in shaping students' experiences. The study underscores the need for culturally sensitive research and suggests that student support services should consider cultural contexts to address students' unique challenges and aspirations better. Future research is recommended to explore hope across various cultural backgrounds to gain a more comprehensive understanding of its role in student development., Competing Interests: The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Mason.)

Species lacking distinct secondary sex characteristics, such as differences in size or morphology, are often thought to experience lower levels of sex-specific selection in comparison to highly sexually dimorphic organisms. However, monomorphism in classic visible traits could be a result of genetic or physiological constraints that prevent the sexes from reaching divergent fitness optima. Additionally, biochemical and molecular work have revealed a variety of less easily observed phenotypes that nonetheless exhibit profound dimorphism. Sex-specific selection could act on these more subtle, less visible, traits. We investigate sex-specific selection in the polygynandrous dusky pipefish (Syngnathus floridae), which lacks distinct secondary sexual characteristics such as size, colour and morphological dimorphism. Using experimental breeding populations, we revealed that although males and females have similar opportunities for sexual selection, only males experience significant sexual selection pressures on body size. We also investigated patterns of sex-biased and sex-specific gene expression in gonads, livers and gills, and tested whether genes with highly divergent expression patterns between the sexes are more likely to be tissue-specific, and therefore relieved of genetic constraints. Sex bias in gene expression was widespread, although the reproductive organs had the most sex-biased and sex-specific genes. Sex-specific selection on gene expression in gills was primarily related to immune response, whereas the liver and gonads had a wide variety of cellular processes, as well as reproductive proteins, showing sex-biased expression. These sex-biased genes showed higher organ-specificity in their expression patterns, suggesting that pleiotropic constraints might have historically impacted the evolution of sex-specific expression patterns. Altogether, we find evidence for ongoing and historical sex-specific selection in the dusky pipefish., (© 2024 The Author(s). Molecular Ecology published by John Wiley & Sons Ltd.)

Although the nighttime ecology of organisms remains understudied, nocturnal surveys play an integral part in assessing fish assemblages and the selective forces shaping them. Eleuthera (Bahamas) contains an unusual population of lined seahorses ( Hippocampus erectus ) in an anchialine lake, possessing morphological characteristics distinct from those found in the ocean. Population surveys for seahorses and their potential predators were conducted at midnight and midday during wet and dry seasons, with belt transects perpendicular to the shoreline that increased in depth away from shore. Nocturnal surveys uncovered seahorse densities 259% higher than daytime transects on average. Sex ratios were consistently male-biased, and the frequency of animals from different reproductive categories varied significantly by time of day, with gravid males observed around the clock but females and nongravid males observed more often at night. Spatial and seasonal recruitment was detected for the first time in this species, with an increase in juveniles detected in the shallow ends of transects during dry season surveys. Juvenile recruitment is poorly understood across syngnathid fishes, so the detection of early recruits at night has broad implications for this fish family. Seahorses from all reproductive categories were perched significantly higher in the water column during the night regardless of their depth or season. Predator densities followed a similar pattern with higher densities observed at night, indicating that elevated nocturnal perch height may be a response to predator presence. However, the selective agents driving these nocturnal behaviors have yet to be identified. Considering H. erectus is listed on the IUCN Red List as "Vulnerable," the increase in nocturnal population size and the detection of juveniles has crucial implications for understanding their ecology, recruitment, and conservation., Competing Interests: The authors declare that there are no competing interests., (© 2023 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd.)

Neurodegenerative diseases are a major cause of death and disability worldwide and are influenced by many factors including age, genetics, and injuries. While these diseases are often thought to result from the accumulation and spread of aberrant proteins, recent studies have demonstrated that they can be shaped by the innate and adaptive immune system. Resident myeloid cells typically mount a sustained response to the degenerating CNS, but peripheral leukocytes such as T and B cells can also alter disease trajectories. Here, we review the sometimes-dichotomous roles played by immune cells during neurodegenerative diseases and explore how brain trauma can serve as a disease initiator or accelerant. We also offer insights into how failure to properly resolve a CNS injury might promote the development of a neurodegenerative disease., Competing Interests: Declaration of interests The authors declare no interests., (Published by Elsevier Ltd.)

Repetitive mild traumatic brain injuries (mTBI) disrupt CNS barriers, the erosion of which has been linked to long-term neurodegenerative and psychiatric conditions. Although much attention has been devoted to CNS vasculature following mTBI, little is known about the glia limitans superficialis - a barrier of surface-associated astrocytes that helps protect the CNS parenchyma and maintain homeostasis. Here, we identify the glia limitans superficialis as a crucial barrier surface whose breakdown after acute repeat mTBI facilitates increased cell death and recruitment of peripheral myelomonocytic cells. Using intravital microscopy, we show that brain-resident microglia fortify this structure after a single mTBI, yet they fail to do so following secondary injury, which triggers massive recruitment of myelomonocytic cells from the periphery that contribute to further destruction of the glia limitans superficialis but not cortical cell death. We demonstrate, instead, that reactive oxygen species (ROS) generated in response to repetitive head injury are largely responsible for enhanced cortical cell death, and therapeutic administration of the antioxidant glutathione markedly reduces this cell death, preserves the glia limitans, and prevents myelomonocytic cells from entering the brain parenchyma. Collectively, our findings underscore the importance of preserving the glia limitans superficialis after brain injury and offer a therapeutic means to protect this structure and the underlying cortex.

Severe traumatic brain injury (sTBI) survivors experience permanent functional disabilities due to significant volume loss and the brain's poor capacity to regenerate. Chondroitin sulfate glycosaminoglycans (CS-GAGs) are key regulators of growth factor signaling and neural stem cell homeostasis in the brain. However, the efficacy of engineered CS (eCS) matrices in mediating structural and functional recovery chronically after sTBI has not been investigated. We report that neurotrophic factor functionalized acellular eCS matrices implanted into the rat M1 region acutely after sTBI significantly enhanced cellular repair and gross motor function recovery when compared to controls 20 weeks after sTBI. Animals subjected to M2 region injuries followed by eCS matrix implantations demonstrated the significant recovery of "reach-to-grasp" function. This was attributed to enhanced volumetric vascularization, activity-regulated cytoskeleton (Arc) protein expression, and perilesional sensorimotor connectivity. These findings indicate that eCS matrices implanted acutely after sTBI can support complex cellular, vascular, and neuronal circuit repair chronically after sTBI., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

There are currently no effective treatments for moderate-to-severe traumatic brain injuries (TBIs). The paracrine functions of undifferentiated neural stem cells (NSCs) are believed to play a significant role in stimulating the repair and regeneration of injured brain tissue. We therefore hypothesized that fibroblast growth factor (FGF2) enriching chondroitin sulfate glycosaminoglycan (CS-GAG) matrices can maintain the undifferentiated state of neural stem cells (NSCs) and facilitate brain tissue repair subacutely post-TBI. Rats subjected to a controlled cortical impactor (CCI) induced TBI were intraparenchymally injected with CS-GAG matrices alone or with CS-GAG matrices containing PKH26GL labeled allogeneic NSCs. Nissl staining of brain tissue 4 weeks post-TBI demonstrated the significantly enhanced ( p < 0.05) tissue protection in CS-GAG treated animals when compared to TBI only control, and NSC only treated animals. CS-GAG-NSC treated animals demonstrated significantly enhanced ( p < 0.05) FGF2 retention, and maintenance of PKH26GL labeled NSCs as indicated by enhanced Sox1+ and Ki67+ cell presence over other differentiated cell types. Lastly, all treatment groups and sham controls exhibited a significantly ( p < 0.05) attenuated GFAP+ reactive astrocyte presence in the lesion site when compared to TBI only controls., Competing Interests: Notes The authors declare no competing financial interest.

Context: Polycystic ovary syndrome (PCOS) is an anovulatory disorder characterized by excess androgen production and increased LH secretion. Serum anti-Mullerian hormone (AMH) is also elevated in this disorder. Women with PCOS exhibit a positive correlation between AMH and LH levels and recent in vitro data demonstrate that LH can directly stimulate AMH production by granulosa cells from women with PCOS., Objective: The objective of the study was to directly test whether LH increases AMH production in women with PCOS in vivo by assessing responses after recombinant human chorionic gonadotropin (r-hCG) stimulation., Design: This was a prospective study., Setting: The study was conducted at a research center at an academic medical center., Participants: Women with PCOS (n = 28) and normal controls (n = 29) participated in the study., Interventions: Blood samples were obtained before and 24 hours after iv administration of 25 μg r-hCG., Main Outcome Measures: Basal and stimulated serum AMH, androstenedione, T, and 17-hydroxyprogesterone levels were measured., Results: Baseline AMH levels in women with PCOS were greater than in normal controls and correlated with levels of LH as well as androstenedione, T, and 17-hydroxyprogesterone. A rise of serum AMH levels was not observed after r-hCG administration in women with PCOS or normal ovaries., Conclusion: These findings are in contrast to in vitro evidence demonstrating that AMH secretion by granulosa cells of PCOS women in response to LH stimulation and suggest AMH regulation in vivo is complex and that the elevated serum AMH in women with PCOS is not a direct effect of the excess LH production characteristic of PCOS.

Background: Women with anovulatory polycystic ovary syndrome (PCOS) are generally insulin-resistant and as a consequence are often treated with the biguanide metformin. Results with metformin have, however, been variable with some studies demonstrating induction of regular cycles and an increase in ovulation, whereas others do not. Hence more understanding is needed regarding the mechanism of metformin's actions in ovarian granulosa cells especially in light of previous demonstrations of direct actions., Objective: The aim of this study was to investigate metformin's interaction with the FSH/cAMP/protein kinase A pathway, which is the primary signaling pathway controlling CYP19A1 (aromatase) expression in the ovary., Methods: The effect of metformin on FSH and forskolin-stimulated aromatase expression in human granulosa cells was measured by quantitative real-time PCR. Activity was assessed after transfection with a promoter II-luciferase construct, and by an RIA measuring conversion of androgen to estrogens. The effect on FSH receptor (FSHR) mRNA was assessed by quantitative PCR. Levels of phosphorylated cAMP response element binding protein (CREB) and CREB-regulated transcription coactivator 2 (CRTC2) were measured by Western blotting and cAMP by a bioluminescent assay., Results: Metformin markedly reduced FSH but not forskolin-stimulated aromatase expression and activity. This effect was exerted by inhibition of basal and ligand-induced up-regulation of FSHR expression. Metformin also reduced FSH-induced phosphorylation of CREB and hence CRE activity, which could potentially disrupt the CREB-CREB-binding protein-CRTC2 coactivator complex that binds to CRE in promoter II of the aromatase gene. This is mediated in an AMP-activated protein kinase-independent manner, and does not involve alteration of cAMP levels., Conclusion: These finding have implications for the use of metformin in the treatment of anovulation in women with PCOS.

Context: Altered hepatic cortisol-cortisone metabolism by type 1 11β-hydroxysteroid dehydrogenase (11βHSD1) has previously been linked with polycystic ovary (PCO) syndrome (PCOS)., Objectives: Our objectives were to establish whether ovarian 11βHSD activities are also altered in PCOS and to determine whether any changes in ovarian cortisol metabolism might reflect exposure to elevated concentrations of insulin or androgens., Design: Cortisol and cortisone concentrations were measured in follicular fluid aspirated from size-matched follicles dissected from normal, ovulatory, and anovulatory PCOs. Human granulosa-lutein cells, recovered during oocyte retrieval for assisted conception, were maintained in primary culture for 4 days, after which 11βHSD1 activities were measured as the net oxidation of [(3)H]cortisol (100 nmol/L) in the absence and presence of insulin (100 nmol/L) with or without metformin (1 μmol/L) or a range of androgens/oxy-androgen metabolites (0.01-10 μmol/L)., Results: Intrafollicular cortisol to cortisone ratios were elevated in anovulatory PCOs (2.1 ± 0.4, P < .05, n = 13) but did not differ between follicles from ovulatory PCOs (1.6 ± 0.1, n = 24) and normal ovaries (1.2 ± 0.1, n = 14). 11βHSD1 activities were lower in granulosa-lutein cells recovered from patients with PCOS compared with all other causes of infertility (median = 5.8 vs 14.9 pmol cortisone/4 h, respectively; P < .05). Cortisol oxidation was unaffected by insulin with or without metformin, dehydroepiandrosterone, and androstenedione, but was inhibited in a concentration-dependent manner by testosterone, 11β-hydroxyandrostenedione, and 7α- and 7β-hydroxy-dehydroepiandrosterone (P < .01)., Conclusions: There is decreased inactivation of cortisol in follicles from anovulatory PCOS. This may reflect inhibition of 11βHSD1 by androgens and their 7/11-oxy-metabolites, local concentrations of which are increased in PCOS, and may contribute to the block to folliculogenesis seen in PCOS.

Objective: To determine that anti-Müllerian hormone (AMH) has been shown to inhibits E(2) production in rodents and in luteinized granulosa cells (GC). We determined whether this occurs in human cells most highly expressing AMH (i.e., from small antral follicles) and whether this is an effect on aromatase promoter activity. We also investigated the effects of AMH on other factors determining FSH sensitivity., Design: Granulosa cells were exposed to AMH with and without gonadotropins for 48 hours., Setting: University laboratory., Patient(s): Not applicable., Intervention(s): None., Main Outcome Measure(s): Aromatase and FSH receptor messenger RNA expression measured using real time quantitative polymerase chain reaction (PCR). Aromatase promoter II activity measured using a luciferase assay. Estradiol, inhibin A and B, and vascular endothelial growth factor production were measured in the conditioned medium., Result(s): The AMH decreased gonadotropin-stimulated aromatase expression and decreased forskolin-stimulated aromatase in KGN cells and this effect was through a dose-dependent inhibition of promoter II. Surprisingly, AMH also reduced FSH receptor mRNA expression. High AMH doses had no effect on inhibin B, whereas a low dose stimulated production. There was no effect on inhibin A or vascular endothelial growth factor., Conclusion(s): The AMH inhibits factors affecting FSH sensitivity. As AMH levels decrease with follicle growth, this inhibition would be removed. The AMH overproduction in anovulatory polycystic ovaries (PCO) may therefore restrict folliculogenesis by an inhibitory effect on FSH sensitivity, thereby contributing to anovulation., (Crown Copyright © 2011. Published by Elsevier Inc. All rights reserved.)

Although not often discussed, the ovaries of women with polycystic ovary syndrome (PCOS) show all the hallmarks of increased TGF-β activity, with increased amounts of fibrous tissue and collagen in the ovarian capsule or tunica albuginea and ovarian stroma. Recent studies suggest that PCOS could have fetal origins. Genetic studies of PCOS have also found linkage with a microsatellite located in intron 55 of the extracellular matrix protein fibrillin 3. Fibrillins regulate TGF-β bioactivity in tissues by binding latent TGF-β binding proteins. We therefore examined expression of fibrillins 1-3, latent TGF-β binding proteins 1-4, and TGF-β 1-3 in bovine and human fetal ovaries at different stages of gestation and in adult ovaries. We also immunolocalized fibrillins 1 and 3. The results indicate that TGF-β pathways operate during ovarian fetal development, but most important, we show fibrillin 3 is present in the stromal compartments of fetal ovaries and is highly expressed at a critical stage early in developing human and bovine fetal ovaries when stroma is expanding and follicles are forming. These changes in expression of fibrillin 3 in the fetal ovary could lead to a predisposition to develop PCOS in later life.

Context: Hyperinsulinemia in polycystic ovary syndrome is widely treated with the insulin sensitizer metformin, which, in addition to its systemic effects, directly affects the ovarian insulin-stimulated steroidogenesis pathway., Objective: Our aim was to investigate the interaction of metformin with the other insulin-stimulated ovarian pathway, namely that leading to glucose uptake., Design: Human granulosa-luteal cells were cultured with metformin (10(-7) M), insulin (10 ng/ml) or metformin and insulin (met + ins) combined. Insulin receptor (IR) involvement was assessed by culture with an (anti)-insulin receptor (IR) antibody., Main Outcome Measures: The effect of metformin on insulin-receptor substrate proteins 1 and 2 (IRS-1 and -2) mRNA and protein expression was determined. The KGN granulosa-cell line was used to investigate the effect of insulin and metformin on Akt activation and glucose transporter-4 (Glut-4) expression. Glut-4 translocation from the cytosol to the membrane was determined in cytoplasmic and membrane-enriched fractions of protein lysates., Results: IRS-1 mRNA and protein increased with all treatments. In contrast, basal IRS-2 mRNA levels were barely detectable, but transcription was up-regulated by metformin. The anti-IR antibody reduced treatment-stimulated IRS-1 to basal levels and IRS-2 expression to an even greater extent than IRS-1, showing greater dependence on the IR than IRS-1. Metformin in the presence of insulin activated Akt and this was dependent on phosphoinositide-3 kinase, as was translocation of Glut-4 to the membrane. Metformin was able to substantially enhance the insulin-stimulated translocation of Glut-4 transporters from the cytosol to the membrane., Conclusion: This net increase in Glut-4 transporters in the plasma membrane has the potential to increase glucose uptake and metabolism by granulosa cells of the insulin-resistant polycystic ovary, thereby facilitating follicle growth.

Metformin is commonly used to treat women with polycystic ovary syndrome, but its precise mechanism of action is unclear, and it even appears to have direct ovarian effects. At the cellular level, it may act either via an insulin-dependent pathway or an independent pathway by activating AMP-activated protein kinase (AMPK). In the ovary, metformin directly decreased estradiol and progesterone production by human granulosa cells, and inhibition of progesterone production by metformin in rat granulosa cells caused an increase in phosphorylated AMPK (pAMPK). We investigated whether metformin activates AMPK in the human ovary by looking for changes in phosphorylation of AMPK and its downstream target acetyl CoA carboxylase (ACC). mRNA and protein for α1 and α2 AMPK subunits were present in all human ovarian tissue. Neither 100 nm nor 2 mm of metformin affected subunit expression. After 1 or 4 h, neither dose of metformin increased pAMPK or pACC, although after 1 h, the addition of insulin significantly enhanced pAMPK, whereas insulin alone had no effect on phosphorylation of either AMPK or ACC. The addition of compound C, an inhibitor of AMPK, negated the effect of metformin in the presence of insulin on pAMPK. This effect on AMPK was not due to a change in the ADP/ATP ratio measured by HPLC. In summary, the presence of insulin was required to cause a metformin-induced increase in pAMPK in these human ovarian cells. Although previous data suggest that metformin may act via an insulin-independent pathway, our results therefore imply that insulin may be required to initiate an effect.

Anti-Müllerian hormone (AMH) was initially thought to be produced solely by the foetal male during sexual differentiation to promote regression of the Müllerian ducts. Over the last decade, however, a new and interesting role has emerged for AMH in the ovary. In human ovaries, AMH is produced by granulosa cells from 36 weeks of gestation until menopause, with the highest expression being in small antral follicles. AMH production gradually declines as follicles grow; once follicles reach a size at which they are dominant, it has largely disappeared. Its removal from these larger follicles appears to be an important requirement for dominant follicle selection and progression to ovulation as AMH has an inhibitory role in the ovary, reducing both primordial follicle initiation and follicle sensitivity to FSH by inhibition of aromatase. It is for this reason that AMH is a focus of interest in polycystic ovary syndrome (PCOS). Serum levels are doubled, and granulosa cell production is greatly increased. Interestingly, there appear to be two groups of women with PCOS who can be distinguished by their AMH level: one group consists of those who have high levels which do not reduce with treatment and who respond less well to induction of ovulation, and a second group consists of those in whom the level is less elevated and reduces on treatment and who seem to respond rather better. Understanding the reason for the raised AMH in PCOS may give clues as to the mechanism of anovulation. To conclude, AMH appears to have a major inhibitory role during folliculogenesis, which may contribute to anovulation in PCOS.

Several studies have demonstrated an association between polycystic ovary syndrome (PCOS) and the dinucleotide repeat microsatellite marker D19S884, which is located in intron 55 of the fibrillin-3 (FBN3) gene. Fibrillins, including FBN1 and 2, interact with latent transforming growth factor (TGF)-beta-binding proteins (LTBP) and thereby control the bioactivity of TGFbetas. TGFbetas stimulate fibroblast replication and collagen production. The PCOS ovarian phenotype includes increased stromal collagen and expansion of the ovarian cortex, features feasibly influenced by abnormal fibrillin expression. To examine a possible role of fibrillins in PCOS, particularly FBN3, we undertook tagging and functional single nucleotide polymorphism (SNP) analysis (32 SNPs including 10 that generate non-synonymous amino acid changes) using DNA from 173 PCOS patients and 194 controls. No SNP showed a significant association with PCOS and alleles of most SNPs showed almost identical population frequencies between PCOS and control subjects. No significant differences were observed for microsatellite D19S884. In human PCO stroma/cortex (n = 4) and non-PCO ovarian stroma (n = 9), follicles (n = 3) and corpora lutea (n = 3) and in human ovarian cancer cell lines (KGN, SKOV-3, OVCAR-3, OVCAR-5), FBN1 mRNA levels were approximately 100 times greater than FBN2 and 200-1000-fold greater than FBN3. Expression of LTBP-1 mRNA was 3-fold greater than LTBP-2. We conclude that FBN3 appears to have little involvement in PCOS but cannot rule out that other markers in the region of chromosome 19p13.2 are associated with PCOS or that FBN3 expression occurs in other organs and that this may be influencing the PCOS phenotype.

Metformin treatment, now widely prescribed in polycystic ovary syndrome, is aimed at correcting the associated insulin resistance, but it has also been shown to directly inhibit ovarian steroidogenesis. The mechanisms, however, by which metformin inhibits estradiol production in human granulosa cells remains unknown. Granulosa luteal cells were incubated with metformin, insulin, or combined metformin and insulin treatment, and aromatase mRNA expression was quantified using real-time RT-PCR. Enzyme activity was assessed by the conversion of (3)H-androstenedione to estrone and estradiol. Metformin's effect on the expression of specific untranslated first exon aromatase promoters was analyzed using semiquantitative PCR. The involvement of MAPK kinase (MEK)/ERK pathway was investigated by immunoblotting for aromatase, phosphorylated, and total ERK-1,2 from cells cultured as above with/without the MEK inhibitor PD98059. Metformin significantly inhibited basal and insulin-stimulated aromatase mRNA expression, with parallel results from the aromatase activity assay and protein assessment. This suppression was via down-regulation of aromatase promoter II, I.3, and 1.4 expression and was reversed by the addition of PD98059. Involvement of the ERK signaling pathway was demonstrated by the significant increase in phosphorylated ERK-1,2 with the combined metformin and insulin treatment. We have shown for the first time in human granulosa cells that metformin signficantly attenuated basal and insulin-stimulated P450 aromatase mRNA expression and activity, via silencing of key promoters. This occurred by activation of MEK/ERK pathway, which negatively regulated aromatase production. This is an important consideration given metformin's widespread use in polycystic ovary syndrome and may further support a possible therapeutic indication in estrogen-dependent breast tumors.

Background: The ovarian follicular basal lamina underlies the epithelial membrana granulosa and maintains the avascular intra-follicular compartment. Additional layers of basal lamina occur in a number of pathologies, including pili annulati and diabetes. We previously found additional layers of follicular basal lamina in a significant percentage of healthy bovine follicles. We wished to determine if this phenomenon existed in humans, and if it was related to oocyte function in the bovine., Methods and Results: We examined follicles from human ovaries (n = 18) by electron microscopy and found that many follicles had additional layers of basal lamina. Oocytes (n = 222) from bovine follicles with normal or unusual basal laminas were isolated and their ability to undergo in vitro maturation, fertilization and culture to blastocyst was compared. Healthy bovine follicles with a single layer of basal lamina had oocytes with significantly (P < 0.01) greater developmental competence than healthy follicles with additional layers of follicular basal lamina (65% versus 28%)., Conclusions: These findings provide direct evidence that the phenotype of the follicular basal lamina is related to oocyte competence.

Histological studies have demonstrated that polycystic ovaries (PCO) contain increased numbers of preantral follicles with a specific increase in primary follicles. Polycystic ovary syndrome is associated with hyperandrogenism and pre- and postnatal androgenization of primates increases the pool of growing follicles producing changes resembling PCO. In vitro studies could test the hypothesis that androgens alter early folliculogenesis, but conventional culture techniques for small follicles are generally unsuitable in non-rodent species. Our objective was to develop and use a method to investigate the effects of testosterone on early folliculogenesis. We adapted an in ovo technique in which lamb cortical ovarian fragments were grafted onto the chorioallantoic membrane of fertilised chick eggs. Optimal experimental conditions for vascularisation and survival of tissue were determined and the model then used to investigate the effects of testosterone on follicle growth. Eggs were inoculated with testosterone at the time of implantation of the ovarian tissue, which was retrieved 5 days later. Tissue was sectioned and follicles staged and counted. There was no wholesale initiation of primordial follicle growth over the 5-day in ovo culture. Importantly, the proportion of primordial, primary and secondary follicles remained similar to those in unimplanted tissue. Testosterone increased the number of primary follicles by 50% compared with controls, an effect that was largely due to a reduction in atresia. In conclusion, incubation of ovarian cortex with testosterone reproduces the changes in early folliculogenesis reported in histological studies of PCO.

Pelvic ultrasonography is generally regarded as the gold standard for determination of pelvic maturity and hence the need for further weight gain in patients with anorexia nervosa. Many clinicians, however, have limited knowledge of this technique. Here, we describe the use of pelvic ultrasonography in anorexia nervosa and present an algorithm to assist the clinician, both with what questions to ask from the radiologist, and how to use the information provided to determine the morphology and hence maturity of the pelvic organs. We then show how this information can be used to assign the level of pelvic maturity a grade from 1 to 5. Finally, we demonstrate use of this system in two patients who progressively gained weight until pelvic maturity was achieved., (2006 John Wiley & Sons, Ltd and Eating Disorders Association)

There is evidence that certain phytoestrogens inhibit aromatase, the enzyme that converts androgens to oestrogens. Kinetic studies in cell-free preparations show that they may inhibit aromatase by competitive binding to the enzyme, but there is a paucity of studies investigating longer-term effects of phytoestrogens on the expression of steroidogenic enzymes. This study tested the hypothesis that phytoestrogens could reduce aromatase activity by down-regulation of its expression. Experiments were carried out on primary cultures of human granulosa-luteal (GL) cells after they had been exposed to phytoestrogens for 48 h. Aromatase activity was measured by the ability of cells to convert testosterone to estradiol over a 4h period and aromatase mRNA expression (mRNA(arom)) was subsequently measured from the same cells using quantitative real-time PCR. The compounds investigated were the flavones, apigenin and quercetin, and the isoflavones, genistein, biochanin A and daidzein at doses of 10 microM and 100 nM. Combinations of these compounds at the lower dose were also investigated. All compounds tested dose-dependently reduced mean mRNA(arom) compared with controls. Apigenin was the most potent inhibitor with significant inhibition of mRNA(arom) seen at both 10 microM and 100 nM, whilst other flavonoids (except biochanin A) only induced significant inhibition (p

Extracellular matrix regulates many cellular processes likely to be important for development and regression of corpora lutea. Therefore, we identified the types and components of the extracellular matrix of the human corpus luteum at different stages of the menstrual cycle. Two different types of extracellular matrix were identified by electron microscopy; subendothelial basal laminas and an interstitial matrix located as aggregates at irregular intervals between the non-vascular cells. No basal laminas were associated with luteal cells. At all stages, collagen type IV alpha1 and laminins alpha5, beta2 and gamma1 were localized by immunohistochemistry to subendothelial basal laminas, and collagen type IV alpha1 and laminins alpha2, alpha5, beta1 and beta2 localized in the interstitial matrix. Laminin alpha4 and beta1 chains occurred in the subendothelial basal lamina from mid-luteal stage to regression; at earlier stages, a punctate pattern of staining was observed. Therefore, human luteal subendothelial basal laminas potentially contain laminin 11 during early luteal development and, additionally, laminins 8, 9 and 10 at the mid-luteal phase. Laminin alpha1 and alpha3 chains were not detected in corpora lutea. Versican localized to the connective tissue extremities of the corpus luteum. Thus, during the formation of the human corpus luteum, remodelling of extracellular matrix does not result in basal laminas as present in the adrenal cortex or ovarian follicle. Instead, novel aggregates of interstitial matrix of collagen and laminin are deposited within the luteal parenchyma, and it remains to be seen whether this matrix is important for maintaining the luteal cell phenotype.

Background: We examined whether women with polycystic ovary syndrome (PCOS) have poorer health-related quality of life (HRQoL) than women in the general population and than patients with other medical conditions., Method: Women with PCOS were recruited from an outpatient clinic and a control group was recruited from a family planning clinic. Both groups completed the Short Form-36 (SF-36) and the Polycystic Ovary Syndrome Questionnaire (PCOSQ). SF-36 data from the Oxford Health and Lifestyle Survey were used to compare PCOS with other conditions., Results: Twenty-two women with PCOS and 96 control women took part. Women with PCOS scored lower in both summary scores of the SF-36 and in all domains of the PCOSQ. After adjusting for body mass index, the differences between the groups in the SF-36 disappeared, while those in the PCOSQ remained. When compared with asthma, epilepsy, diabetes, back pain, arthritis and coronary heart disease, our PCOS group had the same or better physical HRQoL but poorer psychological HRQoL. The PCOSQ showed good internal reliability, good concurrent validity and good discriminant validity., Conclusions: PCOS has a negative impact on HRQoL even when compared with other serious health conditions. The PCOSQ is reliable and valid for clinical use.

Background: We aimed to combine the generation of "artificial" antibodies with a rat pituitary bioassay as a new strategy to overcome 20 years of difficulties in the purification of gonadotrophin surge-attenuating factor (GnSAF)., Methods: A synthetic single-chain antibody (Tomlinson J) phage display library was bio-panned with partially purified GnSAF produced by cultured human granulosa/luteal cells. The initial screening with a simple binding immunoassay resulted in 8 clones that were further screened using our in-vitro rat monolayer bioassay for GnSAF. Initially the antibodies were screened as pooled phage forms and subsequently as individual, soluble, single-chain antibody (scAbs) forms. Then, in order to improve the stability of the scAbs for immunopurification purposes, and to widen the range of labelled secondary antibodies available, these were engineered into full-length human immunoglobulins. The immunoglobulin with the highest affinity for GnSAF and a previously described rat anti-GnSAF polyclonal antiserum was then used to immunopurify bioactive GnSAF protein. The two purified preparations were electrophoresed on 1-D gels and on 7 cm 2-D gels (pH 4-7). The candidate GnSAF protein bands and spots were then excised for peptide mass mapping., Results: Three of the scAbs recognised GnSAF bioactivity and subsequently one clone of the purified scAb-derived immunoglobulin demonstrated high affinity for GnSAF bioactivity, also binding the molecule in such as way as to block its bioactivity. When used for repeated immunopurification cycles and then Western blot, this antibody enabled the isolation of a GnSAF-bioactive protein band at around 66 kDa. Similar results were achieved using the rat anti-GnSAF polyclonal antiserum. The main candidate molecules identified from the immunopurified material by excision of 2-D gel protein spots was human serum albumin precursor and variants., Conclusion: This study demonstrates that the combination of bioassay and phage display technologies is a powerful tool in the study of uncharacterised proteins that defy conventional approaches. In addition, we conclude that these data support suggestions that GnSAF may be structurally related to serum albumin or very tightly bound to serum albumin.

Background: The role of prolactin in the regulation of ovarian folliculogenesis and corpus luteal function and in particular its relationship to atresia in these structures is as yet unclear. We established a model of apoptosis in which to examine the actions of prolactin., Method: Granulosa cells collected from IVF-flush were cultured at 0.1-0.3 x 10(6) cells/well in growth media for 48 h, placed into serum-free media for 24 h prior to dosing for 24 h. Dose responses to C2-ceramide and prolactin were performed. Cells were then treated with an apoptotic dose of C2-ceramide alone, prolactin (100 ng/ml) alone or a combination of the two. Cell death was assessed by Trypan Blue cell counting and MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Thiazolyl Blue] assay and apoptosis confirmed by morphological assessment and flow cytometry., Results: C2-ceramide (0-40 micro mol/l) induced a dose-dependent increase in cell death (63.8% increase at 40 micro mol/l) and, morphologically, cells exhibited classical features of apoptosis. Prolactin alone had no effect on metabolic activity or total cell number. On co- incubation, prolactin alone had no effect on cell death, whereas C2-ceramide induced an approximately 62.6% increase in apoptosis, which was inhibited in the presence of prolactin., Conclusions: Prolactin may contribute significantly to early corpus luteum formation and survival by acting as a potent antiapoptotic factor for human granulosa cells.

Purpose: To evaluate the characteristics of polycystic compared to normal ovaries using three-dimensional (3-D) power Doppler ultrasonography., Methods: We recruited 42 volunteers, all of whom were commencing IVF treatment. Each patient was examined in the cycle preceeding the start of drug therapy during the late follicular phase. If eight or more subcapsular follicles of 2-8 mm in diameter in one two-dimensional (2-D) plane were detected in either of the ovaries, the patient was categorized as having polycystic ovaries (PCO); otherwise the ovaries were considered normal. The parameters examined were volume of the ovary, vascularization index (VI), flow index (FI), vascularization flow index (VFI), and mean greyness (MG). In addition, the ovary was arbitrarily divided into cortex and stroma, and thereafter volume, VI, FI, VFI, and MG were calculated for these two regions., Results: Twenty-eight women had normal ovaries and 14 had PCO. The comparison between normal and PCO showed that as a group the PCO were larger, without any differences in VI, Fl, VFI, or MG. In patients with PCO, the right ovary was larger than the left one. In patients with normal ovaries, Fl was higher on the left side. Division into cortex and stroma revealed that there were no differences in cortical or stromal VI, FI, VFI, or MG between normal and PCO on either side., Conclusions: The ovaries defined as polycystic were larger than normal ovaries, but there was no difference in the echogenicity of the stroma between polycystic and normal ovaries. We were also unable to demonstrate that the polycystic ovarian stroma was more vascularized than the stroma in the normal ovaries.

We aimed to confirm the ovarian site of gonadotrophin surge-attenuating factor (GnSAF) production and produce granulosa/luteal cell-conditioned medium (G/LCM) containing GnSAF for purification studies. Blue dye affinity chromatography followed by pseudochromatofocusing of G/LCM yielded bioactive fractions at pH 5.74 and 5.77. The former had a major 60-66 kDa band with an internal amino acid sequence of EPQVYVHAP following tryptic digestion. A rat polyclonal antiserum (rPAb) raised against this band completely blocked in-vitro GnSAF bioactivity in human follicular fluid, serum and G/LCM. GnSAF bioactivity was localized to a 64 kDa band of serum-free G/LCM and following 2D gel electrophoresis, one of the spots recognized by Western blotting with the GnSAF rPAb had an N-terminal amino acid sequence of NH-XVPQGNAXXN. Neither amino acid sequence had significant homology with proteins in the human genome database. When ovarian tissues from spontaneously cycling women were cultured under serum-free conditions, neither theca- nor stroma-conditioned media contained GnSAF bioactivity. However, granulosa cell-conditioned medium significantly reduced GnRH-induced LH secretion, an effect that was reversed by incubation with the GnSAF rPAb. In conclusion, we have confirmed that human granulosa cells produce GnSAF within the ovary and have two candidate amino acid sequences for GnSAF. We have also demonstrated that serum-free granulosa cell culture constitutes the method of choice for the characterization of GnSAF since recovery of bioactivity is superior in the presence of fewer serum proteins.

The ovarian insulin-like growth factor (IGF)/IGF binding protein (IGFBP) system operates to permit maximal stimulation of steroidogenesis in the dominant follicle. In atretic follicles, the predominant IGFBPs are IGFBP-2 and IGFBP-4, which appear to be selectively cleaved in healthy follicles. We have recently demonstrated potent inhibition by IGFBP-4 of both theca and granulosa cell steroid production. The degree to which the inhibition occurred suggested that it was greater than might be expected by sequestration of IGF alone. Our study was designed to test this idea. Granulosa cells were harvested from follicles dissected intact from patients undergoing total abdominal hysterectomy and bilateral salpingoophorectomy. Granulosa cells were incubated with or without gonadotropins and IGFBP-4 in the presence or absence of either the IGF type I receptor blocker alphaIR3 or excess IGFBP-3 to remove the effects of endogenous IGF action. Steroid accumulation in the medium was assessed. IGFBP-4 continued to exert potent inhibitory effects when the action of endogenous IGF was removed from the system, demonstrating that its actions are independent of IGF binding. There was no effect on cell metabolism, and the effects on steroidogenesis were reversible after IGFBP-4 removal from the culture medium. No similar effects were seen with IGFBP-2. These reasults are the first evidence of IGF-independent IGFBP-4 actions and the first evidence of IGF-independent actions of any IGFBPs in the ovary.

Background: Women with polycystic ovaries (PCO) have a wide spectrum of presentation from anovulation and amenorrhoea to apparently regular, ovulatory menstrual cycles. We have recently reported a subtle defect in steroidogenic function in the luteal phase in the latter and an increase in the number of degenerating corpora lutea (CL) were observed in ovulatory PCO (ovPCO) during dissection. The possibility was therefore investigated of differences in structure or degeneration in CL formed during ovulatory cycles in women with PCO., Methods: This study compared the histology of the CL in ovPCO with that in the normal ovary. Corpora lutea were collected from nine normal ovaries (days 1-27 of the cycle) and from 13 women with ovPCO (days 5-38)., Results: Variations in the degree of regression, both in relation to onset of menses and between different areas within individual CL, were recorded in both groups. During development and regression no obvious differences were observed between either group apart from an apparent increase in luteal haemorrhage, which was more common and more extensive in CL from PCO., Conclusions: The findings suggest that possible luteal phase abnormalities of steroid secretion in women with ovulatory PCO are not associated with obvious morphological defects in the CL, however it is possible that the persistence of luteal structures seen in PCO was a consequence of increased luteal haemorrhage.

Background: We have previously demonstrated that follicles < or =11 mm diameter from women undergoing IVF contain higher concentrations of gonadotrophin surge attenuating factor (GnSAF) bioactivity than large follicles from the same ovaries., Methods: To determine whether this finding is relevant to spontaneous cycles, follicular fluid aspirated from 37 follicles between 3 and 25 mm in diameter from 14 pairs of ovaries from regularly cycling women undergoing total abdominal hysterectomy and bilateral salpingoophorectomy for benign gynaecological disease was pooled into size categories (3 + 4, 5 + 6, 7 + 8, 9 + 10, 11 + 12, 14 + 15, 18 and 25 mm). These pools were bioassayed for GnSAF and inhibin-A, inhibin-B and activin-A concentrations were determined., Results: Follicles of 5 + 6 mm diameter contained the highest concentrations of GnSAF bioactivity (reducing GnRH-induced LH secretion to 38 +/- 8% of control, P < 0.001), while those of 25 mm diameter contained one quarter of this concentration (reducing GnRH-induced LH secretion to 72 +/- 2% of control, P < 0.05). GnSAF bioactivity was closely related to follicle size (r = -0.836, P < 0.01), but not to inhibin-A, inhibin-B or activin-A concentrations., Conclusions: The finding that small follicles contain high concentrations of GnSAF bioactivity, which fall as folliculogenesis progresses during spontaneous cycles, support the hypothesis that GnSAF has a role in preventing the premature onset of the LH surge in women.

Polycystic ovary syndrome is the most common cause of anovulatory infertility. Anovulation in polycystic ovary syndrome is characterized by the failure of selection of a dominant follicle with arrest of follicle development at the 5-10 mm stage. In an attempt to elucidate the mechanism of anovulation associated with this disorder we have investigated at what follicle size human granulosa cells from normal and polycystic ovaries respond to LH. Granulosa cells were isolated from individual follicles from unstimulated human ovaries and cultured in vitro in serum-free medium 199 in the presence of LH or FSH. At the end of a 48-h incubation period, estradiol (E2) and progesterone (P) were determined in the granulosa cell-conditioned medium by RIA. In ovulatory subjects (with either normal ovaries or polycystic ovaries), granulosa cells responded to LH once follicles reached 9.5/10 mm. In contrast, granulosa cells from anovulatory women with polycystic ovaries responded to LH in smaller follicles of 4 mm. Granulosa cells from anovulatory women with polycystic ovaries were significantly more responsive to LH than granulosa cells from ovulatory women with normal ovaries or polycystic ovaries (E2, P < 0.0003; P, P < 0.03). The median (and range) fold increase in estradiol and progesterone production in response to LH in granulosa cell cultures from size-matched follicles 8 mm or smaller were E2, 1.0 (0.5-3.9) and P, 1.0 (0.3-2.5) in ovulatory women and E2, 1.4 (0.7-25.4) and P, 1.3 (0.3-7.0) in anovulatory women. Granulosa cells from anovulatory (but not ovulatory) women with polycystic ovaries prematurely respond to LH; this may be important in the mechanism of anovulation in this common endocrinopathy.

In experimental animal models, insulin-like growth factors (IGFs) have been found to be more potent stimulators of ovarian function than insulin. In human theca cells, however, insulin, IGF-I, and IGF-II have similar effects on androgen production. The relative effects of insulin and IGFs on human granulosa cell steroidogenesis is unknown. Furthermore, it is unclear whether effects of IGF-II on steroidogenesis are mediated by the type-I or type-II IGF receptor. The effects of insulin, IGF-I, and IGF-II on human granulosa cell steroidogenesis were compared in vitro. As expected, insulin, IGF-I, and IGF-II enhanced steroidogenesis. Previously, IGF-II has been shown to enhance granulosa cell steroid production after insulin preincubation. In this study, an effect of IGF-II, independent of insulin priming, also was observed. In granulosa cell cultures from small antral follicles (< or = 13 mm), insulin and IGF-I stimulated steroid production to a similar degree, whereas IGF-II was less effective. In contrast, IGFs were more effective than insulin (IGF-I > IGF-II > insulin) in granulosa cells isolated from preovulatory follicles. IGF-I and IGF-II actions were mediated via the type-1 IGF receptor. The increased responsiveness of mature granulosa cells to IGFs may be an important mechanism by which granulosa cells increase their steroidogenic output in the preovulatory follicle.

Several studies have now documented the existence of IGFBPs in follicular fluid and their correlation with the health of the follicle. In particular, increased levels of IGFBP-4 have been reported in androgen-dominant atretic follicles and those from polycystic ovaries. The aim of this study was to elucidate the role of IGFBP-4 in ovarian steroidogenesis. Granulosa cells and theca tissue were incubated with or without LH or FSH in the presence or absence of IGFBP-4 (0.5-50 ng/ml). Inhibition by IGFBP-4 of estradiol production in the presence of testosterone alone was seen in three of four experiments. IGFBP-4 completely inhibited FSH-stimulated estradiol production in three experiments and caused 67% inhibition in a fourth. Similar results were obtained for theca, in which concurrent incubation with IGFBP-4 completely negated the stimulatory effects of LH on androstenedione production. The mechanism by which IGFBP-4 exerts these potent effects and the possibility that this may by IGF-independent are currently being investigated.

The control of ovulation in mammalian species appears to be a highly robust process. The primary mechanism is believed to be competition amongst a group of developing follicles, mediated by a hormonal feedback loop involving in the first instance the pituitary. Successful follicles reach maturity and ovulate, the remainder atrophy and die. A model of this control process has been derived by Lacker and his group. Based on simple qualitative assumptions about the hormonal feedback loop, this is able to reflect many of the basic physiological features of ovulation in mammals. However, a fundamental hypothesis of Lacker's work is that all follicles are identical and respond to hormonal signals in precisely the same way. Not only is this improbable, but it also leads to several aspects of the model which are qualitatively unrealistic, most notable of these is its inability to accurately model the condition known as Polycystic Ovary Syndrome. This common malfunction of the ovulatory control mechanism accounts for up to three-quarters of cases of anovulatory infertility in humans and its understanding is therefore of considerable medical significance. In this paper we extend the analysis of Lacker's model to the case of non-identical follicles; this allows us to obtain behaviour much closer to that observed in PCOS patients and to draw some tentative conclusions about the mechanisms underlying this condition.

The IGFs are believed to play an important role in the regulation of steroidogenesis and follicular maturation in the human ovary. The activities of the IGFs are regulated by a family of binding proteins (IGFBPs) which are subject to a number of potential post-translational modifications. The aim of this study was to investigate both the production and modification of the IGFBPs in follicular fluid and in medium conditioned by granulosa cells and theca from individual follicles at different stages of maturation. In follicular fluid from healthy, dominant follicles there was an increase in the amount of IGFBP-2, -3 and -4 present as lower molecular weight forms (23 kDa, 29 kDa and 16.5 kDa respectively) in comparison to that seen in atretic follicles from the same ovary. Furthermore for IGFBP-4, this fragmentation was confirmed to be attributable to the presence of a specific protease which could be inhibited not only by the addition of metal ion chelators or serine protease inhibitors, but also by the addition of other recombinant unsaturated IGFBPs, particularly IGFBP-3. IGF-I did not modulate the activity of the IGFBP-4 protease in solution but was able to prevent the inhibition seen with IGFBP-3. Analysis of granulosa cell conditioned medium from the same series of healthy and atretic follicles revealed that IGFBP-2 and -4 were the predominant IGFBPs with no fragments seen on immunoblotting. In contrast, IGFBP-3 in conditioned medium from theca of atretic follicles was always found as an intact doublet, but was found partially fragmented (30 and 32 kDa) in medium conditioned by theca from healthy, dominant follicles with the proportion of IGFBP-3 in this lower molecular weight or fragmented doublet increasing with follicular maturation. A similar situation was also found for IGFBP-4 with the progressive increase in the amount of the 15 and 16.5 kDa fragments. IGFBP-2 was always found to be intact. Finally, IGFBP production from stroma explants was also examined. This revealed a wide variation in IGFBP pattern between different ovaries, although there was a remarkable degree of consistency between different stroma explant cultures from the same ovary. Immunoblotting for IGFBP-3 revealed that, where present, it existed as both an intact and a lower molecular weight doublet and that IGFBP-2 was again always found to be intact. In conclusion we have demonstrated alterations in the proteolytic modification of the IGFBPs which differ in the various follicular compartments and are closely linked to the stage of follicular development.

Expression of the messenger ribonucleic acids (mRNAs) for insulin-like growth factors (IGFs), their binding proteins (IGFBP1 through IGFBP-6), and receptors was examined in normal and polycystic human ovaries (PCO). Northern and dot blots and RT-PCR were used to evaluate mRNA levels. The IGF system was studied in fresh granulosa, stromal, and thecal samples and in thecal tissue after incubation with LH and GH. IGF-II expression was high in granulosa and thecal compartments, whereas IGF-I was only weakly detectable, suggesting the importance of IGF-II in the human ovarian IGF system. Northern blot analysis revealed IGFBP-2 and -4 mRNA in all ovarian compartments and IGFBP-5 mRNA in stroma and theca. IGFBP-2 mRNA was the most abundant IGFBP mRNA in the human ovary. No IGFBP-1 or -3 mRNA was detected in fresh ovarian tissues. IGFBP-6 and type 1 and 2 IGF receptor mRNA expression was detectable in all ovarian compartments only by RT-PCR. In cultured theca, the expression of IG-FBP-1, -3, and 4 was induced. Only IGFBP-5 expression showed some dependence on trophic hormone (LH) stimulation during theca incubation. Otherwise, GH and LH had no effect on IGF or IGFBP expression in thecal tissue. This study indicates that thecal tissue is an essential part of the IGF system in the human ovary. However, no differences were found between normal ovaries and PCO in IGF, IGFBP, or IGF receptor expression. Thus, our results (from a limited number of patients) together with recent in situ hybridization and immunohistochemistry data of others provide no evidence for a role for IGFs in the functional disturbances related to PCO. Interestingly, our data show that in cultured thecal samples, the IGF and IGFBP expression pattern is different from that in fresh tissue.

There is increasing evidence for an important regulatory role for the insulin-like growth factor (IGF) system in the human ovary. IGF-I and -II and IGF-binding proteins (IGFBPs)-1 to -4 have been identified by analysis of follicular fluid and granulosa cell-conditioned medium and by in situ hybridization and Northern and dot blot analyses of ovarian tissues. It has been suggested that abnormalities of intraovarian IGF-I or IGFBPs may play a part in the pathogenesis of polycystic ovary syndrome. The aim of this study was to identify production of IGF-I and -II and IGFBP-1 to -4 by unstimulated normal and polycystic ovaries. IGF-I and -II were measured by RIA after acid-gel exclusion chromatography in medium conditioned by incubation for 48 h with granulosa cells or explants of theca or stroma. Both IGF-I and -II were present in the low nanograms per mL range in theca- and stroma-conditioned medium (T+SCM). IGFBPs in T+SCM were initially analyzed by Western ligand blotting, which revealed that low mol wt IGFBPs were predominant, especially IGFBP-2 (35 kDa). There was a band corresponding to 26 kDa with smaller amounts of a 31-kDa band, but only a trace of IGFBP-3 (44 and 40 kDa, confirmed by immunoblot). We found no consistent differences between normal and polycystic ovary syndrome ovaries, and although there was a trend toward increased IGFBP accumulation in response to LH, this was not consistent. We were unable to detect IGFs or IGFBPs by Western ligand blotting in granulosa cell-conditioned medium. In further studies we attempted to measure IGFBP-3 by RIA using two different antisera (alpha-BP-3gl and 1287-2-14) that detect different epitopes of IGFBP-3 and allow the presence of proteolytic activity to be demonstrated. Results obtained using alpha-BP-3gl were lower than those using 1287-2-14, suggesting proteolysis of IGFBP-3 in the medium. There was no evidence of proteolysis of serum IGFBP-3 after incubation with conditioned medium, but in contrast, radiolabeled [125I]IGFBP-3 was cleaved after incubation with T+SCM. Immunoblotting revealed intact IGFBP-2 (35 kDa) and bands of various sizes between 16-33 kDa. Immunoreactive fragments of IGFBP-3 between 13-40 kDa were seen. In conclusion, T+SCM contained IGF-I and -II. IGFBP-2 and -4 were the predominant species of IGFBP in T+SCM. T+SCM also contained significant protease activity directed toward IGFBP-2 and -3. Proteolytic activity may be an important mechanism by which bioactive IGFs are made available to these tissues.

The mechanism of anovulation in polycystic ovary (PCO) syndrome remains unknown. As circulating concentrations of FSH are apparently normal, and in vivo, granulosa cells from anovulatory PCO are hyperresponsive to FSH, it has been suggested that the lack of follicular development in anovulatory PCO is caused by overexpression of a paracrine growth factor that inhibits steroidogenesis. Epidermal growth factor and the structurally homologous transforming growth factor-alpha (TGF alpha) are suitable candidates for this role, but although the production of the latter has been demonstrated in the ovary, no comparison has been performed between the levels in normal ovaries and PCO. We compared the levels of TGF alpha in follicular fluid and in granulosa cell- and theca- and stroma-conditioned media from normal ovaries and PCO. TGF alpha was present in the range of 0.2-200 ng/mL in follicular fluid. There was a significant inverse correlation of TGF alpha with follicle size, with no differences between follicles from normal ovaries and PCO. Granulosa cell-conditioned medium contained concentrations of TGF alpha ranging from 0.1-200 ng/1000 cells. There was a wide range of concentrations in theca- and stroma-conditioned media, with levels varying from 0.2-100 ng/mg tissue and no consistent effect of LH. There were no significant differences between the levels from normal ovaries or PCO in medium conditioned by any compartment of the ovary. We conclude that the failure of folliculogenesis in PCO syndrome is not likely to be due to overproduction of TGF alpha by the ovary.

Endogenous IGFBP-3 has been examined in the circulation and in four different extravascular fluids in normal healthy adults and in patients with psoriasis or arthritis. In all of these cases there was no apparent increase of IGFBP-3 protease activity in the circulation. In contrast, endogenous IGFBP-3 from normal skin interstititial fluid and synovial fluid from healthy adults was found to be predominantly in the 29 kDa proteolytically modified form. This indicated that in these extravascular fluids in normal healthy adults a protease was active which was similar, if not identical, to that found in the circulation in pregnancy and other conditions. This was confirmed by the fragmentation of recombinant IGFBP-3 when incubated with these fluids. When the skin interstitial fluid or synovial fluid were taken from abnormal tissues (psoriasis in the former and osteoarthritis or rheumatoid arthritis in the latter) there was a considerable reduction in the amount of endogenous IGFBP-3 in the 'clipped' form and a reduction in the protease activity. In psoriatic lesions, this reduction in IGFBP-3 protease activity was shown to be due to the presence of an inhibitor in the interstitial fluid but not in the circulation. In both peritoneal and follicular fluid, the ratio of intact to fragmented IGFBP-3 appeared to relate to the oestrogen status. In peritoneal fluid there was a decrease in intact IGFBP-3 during the late proliferative/early secretory phase of the endometrial cycle. In the ovary there was an increase in the amount of fragmented IGFBP-3 in the follicular fluid from the dominant follicle in comparison with atretic follicles from the same ovary. There is normally little proteo-lysis of IGFBP-3 in the circulation but this increases in many conditions where there is increased metabolic activity. The same enzyme(s) appear to be active in many extravascular fluids but under very different regulation. The activity in these extravascular fluids is normally high but can be decreased with local tissue inflammation; this decrease appears to be mediated by the induction of a local inhibitor.

The underlying cause of anovulation in polycystic ovary syndrome is unknown. Circulating levels of immuno- and bioactive FSH are within the normal range, and the follicles contain measurable levels of bioactive FSH. The aim of this study was to compare estradiol (E2) production in response to FSH by granulosa cells from normal ovaries with those from polycystic ovaries derived from both anovulatory (anovPCO) and ovulatory subjects (ovPCO). Intrafollicular levels of immunoactive FSH, E2, and androstenedione in follicles of less than 12 mm were also measured. Follicular fluid steroid concentrations were obtained from 41 pairs of normal ovaries and 23 pairs of polycystic ovaries (8 anovPCO and 15 ovPCO). In size-matched follicles from each group there were no significant differences in follicular fluid FSH or E2 concentrations, but androstenedione levels were significantly higher in 5- to 11-mm follicles from ovPCO than in corresponding follicles from normal ovaries. Dose responses to FSH were determined in granulosa cells derived from 9 pairs of normal ovaries, 7 anovPCO, and 8 ovPCO. Cells from anovPCO produced 6- to 10-fold more E2 in response to FSH than normal cells, although there was no significant difference in the ED50 values. The response in cells from ovPCO was reduced compared to normal, but this difference did not reach significance. In summary, as judged by their FSH and E2 contents, polycystic ovaries do not have a higher proportion of atretic follicles than normal. Indeed, cells from anovPCO are hyperesponsive to FSH in vitro. This could be explained by stimulation of aromatase in vivo by either paracrine or, more probably, by endocrine factors, of which insulin is an arguable candidate.