Examination of testicular lumicrine regulation of activins and immunoregulatory genes in the epididymal caput.

BACKGROUND: Immunoregulatory genes encoding activin A (Inhba) and B (Inhbb), and indolamine 2,3-dioxygenase-1 (Ido1) are highly expressed in the murine caput epididymidis, which also has a network of intraepithelial mononuclear phagocytes. This environment is postulated to promote immunological tolerance to epididymal sperm. The factors regulating the immunoregulatory agents in the epididymal caput are poorly understood. OBJECTIVES: This study aimed to investigate the potential role of testicular lumicrine factors in regulating activin and other immune-related genes in the caput epididymidis. MATERIALS AND METHODS: The efferent ducts in adult C57/Bl6 mice were exposed and ligated bilaterally. Serum and tissues were collected seven days later. Animals with bilateral sham ligation and animals with no ligations (collectively referred to as the "intact" group) were used as controls. RESULTS: Pressure-induced seminiferous epithelial damage due to intratubular fluid accumulation was observed in all ligated testes. Testicular inhibin was significantly increased and testosterone was elevated in some animals following bilateral ligation, but serum testosterone, serum LH, and serum inhibin were normal. Ligation caused epithelial regression in the initial segment, with similar but less severe effects in other caput segments. Activin A staining by immunohistochemistry in the epithelium was reduced in bilateral ligation, particularly in the initial segment, with moderately reduced staining intensity in the rest of the caput. Inhba expression within the caput was not significantly affected by bilateral ligation, but Inhbb was reduced by more than 60%. Transcripts encoding the macrophage-specific receptor Cx3cr1 were significantly reduced following bilateral ligation, but other immune cell markers, Ido1, and inflammatory genes were unaffected. CONCLUSION: These data indicate that testicular lumicrine secretion regulates several genes that are preferentially expressed in the initial segment, but has marginal effects on genes such as those encoding activin A and IDO1, which are expressed more widely in the caput.

The anti-Müllerian hormone prodomain is displaced from the hormone/prodomain complex upon bivalent binding to the hormone receptor.

Noncovalent complexes of transforming growth factor-ß family growth/differentiation factors with their prodomains are classified as latent or active, depending on whether the complexes can bind their respective receptors. For the anti-Müllerian hormone (AMH), the hormone-prodomain complex is active, and the prodomain is displaced upon binding to its type II receptor, AMH receptor type-2 (AMHR2), on the cell surface. However, the mechanism by which this displacement occurs is unclear. Here, we used ELISA assays to measure the dependence of prodomain displacement on AMH concentration and analyzed results with respect to the behavior expected for reversible binding in combination with ligand-induced receptor dimerization. We found that, in solution, the prodomain has a high affinity for the growth factor (GF) (Kd = 0.4 pM). Binding of the AMH complex to a single AMHR2 molecule does not affect this Kd and does not induce prodomain displacement, indicating that the receptor binding site in the AMH complex is fully accessible to AMHR2. However, recruitment of a second AMHR2 molecule to bind the ligand bivalently leads to a 1000-fold increase in the Kd for the AMH complex, resulting in rapid release of the prodomain. Displacement occurs only if the AMHR2 is presented on a surface, indicating that prodomain displacement is caused by a conformational change in the GF induced by bivalent binding to AMHR2. In addition, we demonstrate that the bone morphogenetic protein 7 prodomain is displaced from the complex with its GF by a similar process, suggesting that this may represent a general mechanism for receptor-mediated prodomain displacement in this ligand family.

Investigation of activin A in inflammatory responses of the testis and its role in the development of testicular fibrosis.

STUDY QUESTION: Does activin A contribute to testicular fibrosis under inflammatory conditions? SUMMARY ANSWER: Our results show that activin A and key fibrotic proteins are increased in human testicular biopsies with leukocytic infiltrates and impaired spermatogenesis and in murine experimental autoimmune orchitis (EAO) and that activin A stimulates fibrotic responses in peritubular cells (PTCs) and NIH 3T3 fibroblasts. WHAT IS KNOWN ALREADY: Fibrosis is a feature of EAO. Activin A, a regulator of fibrosis, was increased in testes of mice with EAO and its expression correlated with severity of the disease. STUDY DESIGN, SIZE, DURATION: This is a cross-sectional and longitudinal study of adult mice immunized with testicular homogenate (TH) in adjuvant to induce EAO, collected at 30 (n = 6), 50 (n = 6) and 80 (n = 5) days after first immunization. Age-matched mice injected with adjuvant alone (n = 14) and untreated mice (n = 15) were included as controls. TH-immunized mice with elevated endogenous follistatin, injected with a non-replicative recombinant adeno-associated viral vector carrying a gene cassette of follistatin (rAAV-FST315; n = 3) or vector with an empty cassette (empty vector controls; n = 2) 30 days prior to the first immunization, as well as appropriate adjuvant (n = 2) and untreated (n = 2) controls, were also examined.Human testicular biopsies showing focal inflammatory lesions associated with impaired spermatogenesis (n = 7) were included. Biopsies showing intact spermatogenesis without inflammation, from obstructive azoospermia patients, served as controls (n = 7).Mouse primary PTC and NIH 3T3 fibroblasts were stimulated with activin A and follistatin 288 (FST288) to investigate the effect of activin A on the expression of fibrotic markers. Production of activin A by mouse primary Sertoli cells (SCs) was also investigated. PARTICIPANTS/MATERIALS, SETTING, METHODS: Testicular RNA and protein extracts collected from mice at days 30, 50 and 80 after first immunization were used for analysis of fibrotic marker genes and proteins, respectively. Total collagen was assessed by hydroxyproline assay and fibronectin; collagen I, III and IV, α-smooth muscle actin (α-SMA) expression and phosphorylation of suppressor of mothers against decapentaplegic (SMAD) family member 2 were measured by western blot. Immunofluorescence was used to detect fibronectin. Fibronectin (Fn), αSMA (Acta2), collagen I (Col1a2), III (Col3a1) and IV (Col4a1) mRNA in PTC and NIH 3T3 cells treated with activin A and/or FST288 were measured by quantitative RT-PCR (qRT-PCR). Activin A in SC following tumour necrosis factor (TNF) or FST288 stimulation was measured by ELISA. Human testicular biopsies were analysed by qRT-PCR for PTPRC (CD45) and activin A (INHBA), hydroxyproline assay and immunofluorescence. MAIN RESULTS AND THE ROLE OF CHANCE: Production of activin A by SC was stimulated by 25 and 50 ng/ml TNF (P < 0.01, P < 0.001, respectively) as compared to untreated cells. INHBA mRNA was increased in human testicular biopsies with leukocytic infiltrates and impaired spermatogenesis, compared with control biopsies (P < 0.05), accompanied by increased total collagen (P < 0.01) and fibronectin deposition. Total testicular collagen (P < 0.0001) and fibronectin protein expression (P < 0.05) were also increased in EAO, and fibronectin expression was correlated with the severity of the disease (r = 0.9028). In animals pre-treated with rAAV-FST315 prior to immunization with TH, protein expression of fibronectin was comparable to control. Stimulation of PTC and NIH 3T3 cells with activin A increased fibronectin mRNA (P < 0.05) and the production of collagen I (P < 0.001; P < 0.01) and fibronectin (P < 0.05). Moreover, activin A also increased collagen IV mRNA (P < 0.05) in PTC, while αSMA mRNA (P < 0.01) and protein (P < 0.0001) were significantly increased by activin A in NIH 3T3 cells. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: A limited number of human testicular specimens was available for the study. Part of the study was performed in vitro, including NIH 3T3 cells as a surrogate for testicular fibroblasts. WIDER IMPLICATIONS OF THE FINDINGS: Resident fibroblasts and PTC may contribute to the progression of testicular fibrosis following inflammation, and activin A is implicated as a key mediator of this process. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the National Health and Medical Research Council of Australia, the Victorian Government's Operational Infrastructure Support Program and the International Research Training Group between Justus Liebig University (Giessen) and Monash University (Melbourne) (GRK 1871/1-2) on `Molecular pathogenesis on male reproductive disorders' funded by the Deutsche Forschungsgemeinschaft and Monash University. The authors declare no competing financial interests.

Weighting of orthostatic intolerance time measurements with standing difficulty score stratifies ME/CFS symptom severity and analyte detection.

BACKGROUND: Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is clinically defined and characterised by persistent disabling tiredness and exertional malaise, leading to functional impairment. METHODS: This study introduces the weighted standing time (WST) as a proxy for ME/CFS severity, and investigates its behaviour in an Australian cohort. WST was calculated from standing time and subjective standing difficulty data, collected via orthostatic intolerance assessments. The distribution of WST for healthy controls and ME/CFS patients was correlated with the clinical criteria, as well as pathology and cytokine markers. Included in the WST cytokine analyses were activins A and B, cytokines causally linked to inflammation, and previously demonstrated to separate ME/CFS from healthy controls. Forty-five ME/CFS patients were recruited from the CFS Discovery Clinic (Victoria) between 2011 and 2013. Seventeen healthy controls were recruited concurrently and identically assessed. RESULTS: WST distribution was significantly different between ME/CFS participants and controls, with six diagnostic criteria, five analytes and one cytokine also significantly different when comparing severity via WST. On direct comparison of ME/CFS to study controls, only serum activin B was significantly elevated, with no significant variation observed for a broad range of serum and urine markers, or other serum cytokines. CONCLUSIONS: The enhanced understanding of standing test behaviour to reflect orthostatic intolerance as a ME/CFS symptom, and the subsequent calculation of WST, will encourage the greater implementation of this simple test as a measure of ME/CFS diagnosis, and symptom severity, to the benefit of improved diagnosis and guidance for potential treatments.

Activin over-expression in the testis of mice lacking the inhibin α-subunit gene is associated with androgen deficiency and regression of the male reproductive tract.

Regionalised interaction of the activins, follistatin and inhibin was investigated in the male reproductive tract of mice lacking the inhibin α-subunit (Inha-/-). Serum and intratesticular activin B, although not activin A and follistatin, were increased in Inha-/- mice at 25 days of age, but all three proteins were elevated at 56 days. None of these proteins were altered within the epididymis and vas deferens at either age. At 25 days, histology of the epididymis and vas deferens was similar to wild-type. At 56 days, the testis contained extensive somatic cell tumours, leading to Leydig cell regression and testosterone deficiency. The epididymis and vas deferens showed epithelial regression and increased prominence of the interstitial stroma. Immunoregulatory and fibrotic gene expression in the epididymis and vas deferens were unchanged. Thus, absence of the inhibin α-subunit has marginal effects on activins in the epididymis and vas deferens, and regression of these tissues is associated with androgen deficiency.

A new 'total' activin B enzyme-linked immunosorbent assay (ELISA): development and validation for human samples.

BACKGROUND AND OBJECTIVE: There are currently no sensitive and specific assays for activin B that could be utilized to study human biological fluids. The aim of this project was to develop and validate a 'total' activin B ELISA for use with human biological fluids and establish concentrations of activin B in the circulation and fluids from the reproductive organs. DESIGN: The new ELISA was validated and then used to measure activin B levels in the circulation of healthy participants, IVF patients, pregnant women and in ovarian follicular fluid and seminal plasma. PATIENTS AND MEASUREMENTS: Healthy adult subjects (n = 143), subjects from an IVF clinic (n = 27) and pregnancy groups (n = 29) were sampled. RESULTS: The sensitivity of the assay was 0.019 ng/ml. Validation of the activin B ELISA showed good recovery (90.7 +/- 9.8%) and linearity in biological fluid and cell culture media and low cross-reactivity with related analytes (inhibin B = 0.077% and activin A = 0.0034%). There was a negative correlation between activin B concentration (r = -0.281, P < 0.011) and females with increasing age. Patients attending IVF clinics had significantly lower levels of activin B compared with gender-matched control subjects. Ovarian follicular fluid and seminal plasma had 50-80 fold higher levels of activin B (mean = 5.35 and 3.66 ng/ml respectively) than sera (mean = 0.071 ng/ml). CONCLUSIONS: This fully validated ELISA for activin B offers a tremendous utility for measuring this protein in a variety of normal physiological processes and in various clinical pathologies.

Nuclear and cytoplasmic expression of ERbeta1, ERbeta2, and ERbeta5 identifies distinct prognostic outcome for breast cancer patients.

PURPOSE: Previous conflicting results about the prognostic significance of estrogen receptor (ER)-beta in breast cancer may be explained by contribution of isoforms, of which five exist. Our aim was to elucidate the prognostic significance of ERbeta1, ERbeta2, and ERbeta5 by immunohistochemistry in a large cohort of breast carcinomas with long-term follow-up. EXPERIMENTAL DESIGN: Tissue microarrays were stained with ERbeta1, ERbeta2, and ERbeta5 antibodies and scored as percentage of positive tumor cells and using the Allred system. Nuclear and cytoplasmic staining was evaluated and correlated with histopathologic characteristics, overall survival (OS), and disease-free survival (DFS). RESULTS: Nuclear ERbeta2 and ERbeta5, but not ERbeta1, significantly correlated with OS (P = 0.006, P = 0.039, and P = 0.099, respectively), and ERbeta2 additionally with DFS (P = 0.013). ERbeta2 also predicted response to endocrine therapy (P = 0.036); correlated positively with ERalpha, progesterone receptor, androgen receptor, and BRCA1; and correlated inversely with metastasis and vascular invasion. Tumors coexpressing ERbeta2 and ERalpha had better OS and DFS. Cytoplasmic ERbeta2 expression, alone or combined with nuclear staining, predicted significantly worse OS. Notably, patients with only cytoplasmic ERbeta2 expression had significantly worse outcome (P = 0.0014). CONCLUSIONS: This is the first study elucidating the prognostic role of ERbeta1, ERbeta2, and ERbeta5 in a large breast cancer series. ERbeta2 is a powerful prognostic indicator in breast cancer, but nuclear and cytoplasmic expression differentially affect outcome. Measuring these in clinical breast cancer could provide a more comprehensive picture of patient outcome, complementing ERalpha.

Serum levels of retinol-binding protein 4 and adiponectin in women with polycystic ovary syndrome: associations with visceral fat but no evidence for fat mass-independent effects on pathogenesis in this condition.

CONTEXT: Insulin resistance, which associates with levels of retinol-binding protein 4 (RBP4) and adiponectin, is implicated in the development of polycystic ovary syndrome (PCOS). OBJECTIVE: The objective of the study was to explore the potential contribution of RBP4 and adiponectin in the etiology of PCOS and their relationships with specific fat depot measurements. DESIGN: This was a cross-sectional study. SETTING AND PARTICIPANTS: Serum RBP4 and adiponectin levels were compared between 50 PCOS cases and 28 female controls (including 22 body mass index/fat mass-matched pairs) and correlated with specific fat depot (including visceral) axial magnetic resonance imaging cross-sectional area measurements. All subjects were of U.K. British/Irish origin. MAIN OUTCOME MEASURE(S): Serum levels of RBP4 (automated immunonephelometric assay) and adiponectin [immunoassay: total and high molecular weight (HMW)]. Data are reported as geometric mean (sd, range) and optionally adjusted for fat mass and age. RESULTS: Between the 50 PCOS cases and 28 controls, serum RBP4 levels were indistinguishable [39.0 microg/ml (31.0, 49.0) vs. 41.6 microg/ml (32.7, 52.9), respectively, unadjusted P = 0.24; adjusted P = 0.55]. Total (and HMW) adiponectin levels were lower in PCOS cases [total adiponectin 19.9 microg/ml (14.2, 27.8) vs. 25.8 microg/ml (17.7, 37.7), respectively, unadjusted P = 2.4 x 10(-3); adjusted P = 0.10]. For the paired-sample analyzes, there were no differences in RBP4 (P = 0.09), total adiponectin (P = 0.06), HMW adiponectin (P =0.19), or HMW to total adiponectin ratio (P = 0.98). In PCOS cases, L4-visceral fat area was associated positively with RBP4 (r(2) = 0.34, P = 0.01) and negatively with HMW to total adiponectin ratio (r(2) = -0.44, P = 1.3 x 10(-3)). Controls showed similar relationships. CONCLUSIONS: Although associated with visceral fat, serum RBP4 and adiponectin levels do not play important, fat-mass-independent primary roles in the development of PCOS.

Development of a new antibody to the human inhibin/activin betaB subunit and its application to improved inhibin B ELISAs.

Inhibin B has emerged as a clinically useful analyte for studies of reproductive function in both men and women. The antibody to the betaB subunit (C5) used in current commercially available assays (DSL and OBI) was raised in this laboratory to a synthetic peptide from the betaB subunit. These assays require pre-treatment of samples with hydrogen peroxide to oxidise two methionines in the epitope to the sulfoxide for full immunoreactivity. It was also claimed that this antibody cross-reacted significantly with inhibin A leading to a 0.5% cross-reaction of inhibin A in the current generation of immunoassays. Both of the above immunoassays required overnight incubation with sample. The development of improved antibodies to the betaB subunit has proved difficult due to the conservation of the betaB subunit between species. We describe the development of new monoclonal antibodies to the betaB subunit, by immunisation of mice with recombinant X. laevis activin B using the RIMMS method of immunisation. The result has been the development of highly specific antibodies in a short time period. One of these antibodies 46A/F is shown to be a highly effective capture antibody in a human inhibin B ELISA, without any sample pre-treatment. The results of the validation of an improved inhibin B assay using 46A/F as the capture antibody are shown, with comparison to one of the commercially available inhibin B assays. Overall, the inhibin B assay is simplified and the performance improved by using this new antibody 46A/F. It was further shown that the cross-reaction of inhibin A in both the OBI and DSL inhibin B ELISAs is ten fold less than previously reported. This can be attributed to the poor quality of recombinant inhibin B available for use as standard in 1996. Although the present generation of inhibin B assays has proved adequate to enable the physiological function of inhibin to be determined and novel clinical applications found, the simplification of the assay made possible by the improved antibody should make possible a new generation of more rapid, sensitive, convenient and robust tools for routine use.

Evaluation of the relationship between follicular fluid oxidative stress, ovarian hormones, and response to gonadotropin stimulation.

OBJECTIVE: To investigate the relationship between oxidative stress and the underlying causes of infertility, preovulatory ovarian hormones, and ovarian response to gonadotropin stimulation in patients undergoing assisted reproductive techniques. DESIGN: Prospective, cross-sectional study. SETTING: Assisted conception unit, university hospital. PATIENT(S): One hundred thirty women presenting with infertility, of the following types: male factor (n = 56), unexplained (n = 36), tubal factor (n = 16), polycystic ovary syndrome (n = 15), and endometriosis (n = 7). INTERVENTION(S): Follicular fluid (FF) and peripheral blood samples were collected at oocyte retrieval. MAIN OUTCOME MEASURE(S): Blood and FF samples were analyzed for inhibin A, inhibin B, activin A, anti-Müllerian hormone, and E(2) by using ELISA. Total antioxidant capacity (TAC) was measured in plasma and FF by using a calorimetric microplate assay. RESULT(S): There was no significant relationship between plasma or FF TAC and the underlying etiology of infertility. There was a statistically significant positive association between FF E(2) levels and TAC (r = 0.26). Higher antral follicle count, delta E(2) (day 3 E(2) minus day 2 E(2)), preovulatory serum anti-Müllerian hormone, inhibin B, and E(2) were associated with good ovarian response, whereas higher FF E(2) was associated with a statistically significant poor response. No significant direct relationship was observed between TAC and ovarian response as well as between TAC or any of the parameters measured and pregnancy outcome. CONCLUSION(S): Oxidative stress has an impact on the production of granulosa cell steroid hormones, in particular E(2), which is an important predictor of ovarian response. The positive association between FF E(2) and total antioxidant capacity suggests that E(2) may play a role in the ovarian antioxidant-oxidant balance.

Human fetal testis Leydig cell disruption by exposure to the pesticide dieldrin at low concentrations.

BACKGROUND: Declining human reproductive health over the last 60 years has been proposed to be due to effects of environmental chemicals, especially endocrine disrupting compounds, on fetal development. We investigated whether a model pesticide, dieldrin, at concentrations within both maternal circulation and environmental ranges (1 pmol/l = 0.0004 p.p.b. = 380.9 pg/l), could disrupt the human fetal testis. METHODS: Human fetal testes were collected during the second trimester, a critical period of male sexual differentiation (development and masculinization). Testis explants were cultured for 24 h in the presence and absence of LH (10-1000 IU LH/l) and dieldrin (1 pmol and 1 nmol/l). Endocrine, immunohistological and proteome characteristics of the tissues were investigated. RESULTS: Exposure to dieldrin reduced LH-induced testosterone secretion (P < 0.05) and tissue protein concentrations of LH receptor and steroid acute regulatory protein (P < 0.05). Dieldrin altered proteins associated with cancer, apoptosis, transcription and development. Wnt-2b was reduced 3-fold and immunolocalized to Leydig and Sertoli cells. Dieldrin also reversed some LH-induced changes in protein expression, supporting the conclusion that Leydig cell function is at risk from environmental chemicals. CONCLUSIONS: Our findings indicate that exposure to very low, biologically relevant, concentrations of environmental chemicals could affect the fetal human Leydig cell, reducing testosterone secretion and potentially leading to subtle dysregulation of reproductive development and adult fecundity.

Rising follicle-stimulating hormone levels with age accelerate female reproductive failure.

Rising serum FSH levels is one of the earliest signs of human female reproductive aging. Whether or not elevated FSH remains a passive reflection of a diminishing ovarian follicle pool or actively contributes to declining female fertility with age has not been established. We therefore investigated female reproduction in mice expressing progressively rising serum levels of transgenic human FSH (Tg-FSH, 2.5-10 IU/liter) independently of follicle depletion. We show that serum LH and estradiol levels and uterine size remained normal in Tg-FSH females, whereas ovarian weight and corpora lutea number were significantly increased up to 1.3- and 5-fold, respectively. Furthermore, the monotrophic FSH rise produced a striking biphasic effect on female fertility. Tg-FSH females less than 22 wk old delivered increased litter sizes, then beyond 23 wk, litter sizes decreased rapidly culminating in premature infertility despite continued ovary follicle development, and increased ovulation and uterine embryo implantation sites as well as normal serum levels of anti-Mullerian hormone, a marker of ovarian follicle reserve. We found that rising circulating Tg-FSH produced premature infertility by increasing embryo-fetal resorption and parturition failure with age. Thus, our Tg-FSH mice present a novel paradigm to investigate selective contributions of elevated FSH to age-related female infertility, which revealed that rising FSH levels, despite no exhaustion of ovarian reserve, actively accelerates female reproductive aging primarily by postimplantation reduction of embryo-fetal survival.

Deregulation of the activin/follistatin system in hepatocarcinogenesis.

BACKGROUND/AIMS: Activins A and E negatively regulate hepatic cell number by inhibiting cell replication and inducing apoptosis. Follistatin and follistatin-like 3 bind activins and antagonise their biological activities. Aim of our study was to investigate, whether activins and follistatins may play a role in hepatocarcinogenesis. METHODS: Expression levels of follistatin, follistatin-like 3, and activin subunits beta(A) as well as beta(E) were investigated in chemically induced rat and human liver tumours by real-time PCR and immunohistochemistry. In addition, the effects of follistatin and activin A on DNA synthesis of normal as well as preneoplastic hepatocytes and hepatoma cells were analysed. RESULTS: Follistatin was overexpressed while both activin subunits were downregulated in the majority of rat and human liver tumours. Follistatin-like 3 expression was low in normal but enhanced in malignant rat liver. In human normal liver, in contrast, it was abundantly expressed but downregulated in liver cancer. Administration of follistatin to normal and preneoplastic hepatocytes stimulated DNA synthesis preferentially in preneoplastic rat hepatocytes, whereas activin A repressed it. CONCLUSIONS: The balanced expression of follistatins and activins becomes deregulated during hepatocarcinogenesis. The sensitivity of preneoplastic hepatocytes to activin signals suggests the activin/follistatin system as promising target for therapeutic intervention.

Meat and Livestock Association Plenary Lecture 2005. Oocyte signalling molecules and their effects on reproduction in ruminants.

Sheep (Ovis aries) are a highly diverse species, with more than 900 different breeds that vary significantly in their physiological characteristics, including ovulation rate and fecundity. From examination of inherited patterns of ovulation rate, several breeds have been identified with point mutations in two growth factor genes that are expressed in oocytes. Currently, five different point mutations have been identified in the BMP15 (GDF9b) gene and one in GDF9. Animals heterozygous for the GDF9 and/or the BMP15 mutations have higher ovulation rates than their wild-type counterparts. In contrast, those homozygous for any of the aforementioned BMP15 or GDF9 mutations are sterile owing to arrested follicular development. In bovine and ovine ovaries, GDF9 was expressed exclusively in oocytes throughout follicular growth from the primordial stage of development, whereas in sheep BMP15 was expressed exclusively in oocytes from the primary stage: no data for the ontogeny of BMP15 expression are currently available for cattle. In vitro, ovine growth differentiation factor 9 (oGDF9) has no effect on (3)H-thymidine incorporation by either bovine or ovine granulosa cells, whereas ovine bone morphogenetic protein 15 (oBMP15) has modest (1.2- to 1.6-fold; P < 0.05) stimulatory effects. Ovine GDF9 or oBMP15 alone inhibited progesterone production by bovine granulosa cells, whereas in ovine cells only oGDF9 was inhibitory. The effects of oGDF9 and oBMP15 together were often cooperative and not always the same as those observed for each factor alone. Active immunisation of ewes with BMP15 and/or GDF9 peptides affected ovarian follicular development and ovulation rate. Depending on the GDF9 and/or BMP15 vaccine formulation, ovulation rate was either increased or suppressed. A primary and single booster immunisation of ewes with a BMP15 peptide in a water-based adjuvant has led to 19-40% increases in lambs born per ewe lambing. Collectively, the evidence suggests that oocyte signalling molecules have profound effects on reproduction in mammals, including rodents, humans and ruminants. Moreover, in vivo manipulation of these oocyte signalling molecules provides new opportunities for the management of the fertility of ruminants.

Serum anti-mullerian hormone levels reflect the size of the primordial follicle pool in mice.

Reproductive aging is the decline of female fertility with age. It is caused by the decrease in the number of growing follicles, resulting from primordial follicle pool depletion. Recently, we have shown that anti-Müllerian hormone (AMH) is produced by growing follicles, and studies in women indicate that serum AMH levels decrease with age and correlate with antral follicle count. However, whether serum AMH levels correlate directly with the size of the primordial follicle pool cannot be determined in women. In this work, we describe studies in mice in which we determined the dynamics of ovarian follicles during aging. Furthermore, we describe the development of a mouse AMH ELISA, allowing us to measure AMH levels in mice, for the first time. We observed that serum AMH levels decline with increasing age, whereas expression of AMH in individual growing follicles, studied by immunohistochemistry, did not change with age. Thus, the decline in serum AMH correlates directly with the decline in the number of growing follicles (r = 0.86, P < 0.0001). We observed that the number of growing follicles correlated with the number of primordial follicles (r = 0.93, P < 0.0001). Similarly, we found a strong correlation between AMH levels and number of primordial follicles (r = 0.83, P < 0.0001). In conclusion, serum AMH levels reflect the size of the primordial follicle pool in aging mice. Therefore, AMH is an excellent marker to assess the quantitative aspect of ovarian reserve, which may be useful for women at risk for early ovarian aging such as survivors of childhood cancers.

Bovine follicle development is associated with divergent changes in activin-A, inhibin-A and follistatin and the relative abundance of different follistatin isoforms in follicular fluid.

The aim was to determine whether follicle growth in cattle is accompanied by changes in levels of inhibin-A (inh-A), activin-A (act-A) and different Mr isoforms of follistatin (FS) in bovine follicular fluid (bFF), reflecting differential roles of these proteins during folliculogenesis. Follicles (n = 146) from 2-20 mm diameter were dissected from ovaries of approximately 40 cattle. Immunoassays were used to measure total FS, act-A, inh-A, oestradiol (E) and progesterone (P) levels; immunoblotting was used to quantify the relative abundance of different FS isoforms. Follicle growth from 2-6 mm was associated with a 6-fold increase in inh-A and 30-fold increase in act-A; FS remained uniformly high from 2-10 mm. From 6-2 mm, inh-A remained high while act-A and FS fell 3-fold and 2-fold, respectively. Act-A/FS ratio increased 20-fold from 2-6 mm before falling slightly through to 20 mm. Act-A/inh-A ratio increased 6-fold from 2-6 mm before falling 2-fold from 6 to 17-20 mm. These findings imply a marked increase in relative activin 'tone' around the stage at which dominant follicle selection occurs. When larger follicles (13-20 mm) were subdivided according to E/P ratio, those with high ( > 5) E/P ratio had lower (2-fold; P < 0.001) levels of inh-A and act-A in comparison to follicles with low ( < 5) E/P ratio, but there were no significant differences in FS, act-A/inh-A ratio or act-A/FS ratio. Thus follicle size, but not oestrogenic status, has a major influence on the intrafollicular balance between act-A and its opposing factors, inh-A and FS. Six FS isoforms were detected in bFF (apparent Mr: 65, 41, 37, 35, 33 and 31 kDa) averaging 6, 13, 24, 26, 13 and 17% respectively of total FS. During growth from 2-20 mm the proportion of total FS represented by 65, 41 and 37 kDa isoforms increased approximately 2-fold while the proportion represented by the 33 and 31 kDa isoforms decreased by 3-fold and 1.6-fold, respectively. Treatment of bovine granulosa cells in vitro with FSH and IGF alone or in combination increased total FS secretion up to 12-fold but did not affect the relative abundance of the five different FS isoforms detected. While the functional significance of the intriguing shift in FS isoform abundance in bFF during follicle development remains to be established, we have shown that a marked increase in intrafollicular activin 'tone' accompanies bovine follicle growth from 3-6 mm, corresponding to the stage at which the FSH-dependent follicle selection mechanism operates in this species.

Follicular and hormonal dynamics during the estrous cycle in goats.

Transrectal ultrasonography of ovaries was performed daily in 6 goats for 3 consecutive estrous cycles. Blood samples collected daily were measured for concentrations of FSH, inhibin A, and estradiol-17beta. Follicular and hormonal data were analyzed for associations between the follicular waves and hormonal concentrations. During the interovulatory intervals, follicular growth and regression occurred in a wave like pattern (2-5 waves), and the predominant patterns were three and four follicular waves. In addition, there was no significant difference among the diameters of dominant follicles during the growth phase of the follicular waves. The number of 3 mm follicles peaked on days 0, 7, and 11 in interovulatory intervals that had three follicular waves and on days -1, 5, 11, and 15 in those that had four follicular waves. Plasma concentrations of FSH increased around the day of follicular wave emergence and declined with the growth of follicles. Circulating FSH increased again concomitant with regression of dominant follicles in the anovulatory wave, whereas FSH levels remained low in the ovulatory wave. Inhibin A was negatively correlated with FSH, while it was positively correlated with estradiol-17beta, suggesting that inhibin A is a product of healthy growing follicles and that it contributes to the suppression of FSH secretion. In conclusion, the growth of ovarian follicles in goats exhibits a wave-like pattern, and follicular dominance is less apparent in goats. Moreover, inhibin A may be a key hormone for regulation of the follicular wave through suppression of FSH secretion in goats.

Anti-müllerian hormone protein expression is reduced during the initial stages of follicle development in human polycystic ovaries.

CONTEXT: Polycystic ovary syndrome, the most common cause of anovulatory infertility, is characterized by disordered folliculogenesis, notably increased progression from the primordial to the primary stages. This ovarian phenotype is similar to that observed in mice lacking anti-müllerian hormone (AMH). OBJECTIVE: The objective of this study is to investigate whether AMH is involved in accelerating the transition of follicles from primordial to primary stages in polycystic ovaries. DESIGN: This study compares AMH expression in archive tissue from normal and polycystic ovaries. SETTING: This is a laboratory-based study. PATIENTS: Ovarian tissue from seven normoovulatory women and 16 women with polycystic ovaries (five of whom were anovulatory) was used in this study. Ovaries were classified by histology and with reference to menstrual cycle history and ultrasound. MAIN OUTCOME MEASURE: Presence and intensity of AMH expression in 1403 follicles was the main outcome measure. RESULTS: AMH was observed from the primordial stage onward. AMH immunostaining was observed in significantly fewer primordial (P = 0.007) and transitional follicles (P = 0.001) in ovaries from anovulatory women with polycystic ovaries compared with women with regular cycles and either normal or polycystic ovaries. AMH-negative follicles had fewer pregranulosa cells in the largest cross-section of the follicle at both the primordial (median, four and six for AMH-negative and -positive follicles, respectively; P < 0.0001) and transitional stages (median six and nine; P < 0.0007) in normal tissue, and fewer at the transitional stage (median, seven and 11; P < 0.0001) in tissue from anovulatory women with polycystic ovaries. This suggests that AMH expression is associated with granulosa cell mitosis. CONCLUSIONS: These findings indicate a relative deficiency of AMH in primordial and transitional follicles in ovaries from anovulatory women with polycystic ovaries. This may contribute to disordered early follicle development in polycystic ovary syndrome.

ERbeta isoform expression in colorectal carcinoma: an in vivo and in vitro study of clinicopathological and molecular correlates.

Colorectal carcinoma shows several sex-related differences with regard to incidence, response to chemotherapy and microsatellite instability. These differences may relate to differential expression of ERbeta1 (wild-type) as well as the truncated ERbeta2 and ERbeta5 splice variant isoforms, which have recently been detected in normal and malignant colorectal epithelium. This hypothesis was tested through the study of ERbeta isoform protein and/or mRNA expression amongst 91 primary colorectal carcinoma cases and 20 colorectal carcinoma cell lines. Study of the latter showed an absolute correlation between mRNA and protein expressions for ERbeta1 and ERbeta2. ERbeta1 and ERbeta2 protein expression was lost in 22% and 49%, respectively, of the primary colorectal carcinomas. By contrast, ERbeta5 expression was found in all primary colorectal carcinomas and all colorectal carcinoma cell lines studied. Lower ERbeta1 protein expression was associated with poorer differentiation, higher pT stage and absence of microsatellite instability. Higher ERbeta2 protein expression was associated with right-sided location and presence of lymph node metastases. Protein expression of ERbeta1 correlated positively with expression of the oestrogen-responsive protein trefoil factor 1 (TFF1). There was no correlation between ERbeta protein isoform expression and response to 5-fluorouracil therapy, tumour proliferation, or thymidylate synthase expression. These data suggest that ERbeta1 and/or ERbeta2 isoform expression may have prognostic value and may explain sex-related differences in microsatellite instability and colorectal carcinoma. The opposing associations shown by ERbeta1 and/or ERbeta2 in relation to colorectal carcinoma are in keeping with differential activities shown by the two isoforms.

Myostatin inhibits myogenesis and promotes adipogenesis in C3H 10T(1/2) mesenchymal multipotent cells.

Inactivating mutations of the mammalian myostatin gene are associated with increased muscle mass and decreased fat mass; conversely, myostatin transgenic mice that overexpress myostatin in the skeletal muscle have decreased muscle mass and increased fat mass. We investigated the effects of recombinant myostatin protein and antimyostatin antibody on myogenic and adipogenic differentiation of mesenchymal multipotent cells. Accordingly, 10T(1/2) cells were incubated with 5'-azacytidine for 3 d to induce differentiation and then treated with a recombinant protein for myostatin (Mst) carboxy terminal 113 amino acids or a polyclonal anti-Mst antibody for 3, 7, and 14 d. Cells were also cotransfected with a Mst cDNA plasmid expressing the full-length 375-amino acid protein (pcDNA-Mst375) and the silencer RNAs for either Mst (pSil-Mst) or a random sequence (pSil-RS) for 3 or 7 d, and Mst expression was determined. Adipogenesis was evaluated by quantitative image analysis of fat cells before and after oil-red-O staining, immunocytochemistry of adiponectin, and Western blot for CCAAT/enhancer binding protein-alpha. Myogenesis was estimated by quantitative image analysis-immunocytochemistry for MyoD (Myo differentiation protein), myogenin, and myosin heavy chain type II, or by Western blot for myogenin. 5'-Azacytidine-mediated differentiation induced endogenous full-length Mst expression. Recombinant Mst carboxy terminal 113 amino acids inhibited both early and late markers of myogenesis and stimulated both early and late markers of adipogenesis, whereas the antibody against Mst exerted the reverse effects. Myogenin levels at 7 d after transfection of pcDNA-Mst375 were reduced as expected and elevated by pSil-Mst, which blocked efficiently Mst375 expression. In conclusion, myostatin promotes the differentiation of multipotent mesenchymal cells into the adipogenic lineage and inhibits myogenesis.