Intrafollicular paraoxonase 1 activity and the steroidogenic potential of the first post-partum dominant follicle in dairy cows.

Cows experiencing high levels of inflammation and specific metabolic conditions tend to have slower follicular growth and lower serum and follicular concentrations of oestradiol (E2). Paraoxonase1 (PON1) activity decreases during inflammatory processes. Therefore, the aim of this study was to evaluate the association between serum and intrafollicular (FF) PON1 activity and the serum and intrafollicular levels of E2 and progesterone (P4), as well as the mRNA expression of genes related to steroidogenesis, metabolism and inflammation in the first post-partum dominant follicle of Holstein cows. No correlation was found between PON1 activity, the expression of the analysed genes and levels of follicular E2 and P4, except for a negative correlation between serum E2 and follicular PO1 activity. Also, no correlation was found between serum and follicular PON1 during the first post-partum follicular wave.

Pro-nerve growth factor in the ovary and human granulosa cells.

BACKGROUND: Pro-nerve growth factor must be cleaved to generate mature NGF, which was suggested to be a factor involved in ovarian physiology and pathology. Extracellular proNGF can induce cell death in many tissues. Whether extracellular proNGF exists in the ovary and may play a role in the death of follicular cells or atresia was unknown. MATERIALS AND METHODS: Immunohistochemistry of human and rhesus monkey ovarian sections was performed. IVF-derived follicular fluid and human granulosa cells were studied by RT-PCR, qPCR, Western blotting, ATP- and caspase-assays. RESULTS AND CONCLUSION: Immunohistochemistry of ovarian sections identified proNGF in granulosa cells and Western blotting of human isolated granulosa cells confirmed the presence of proNGF. Ovarian granulosa cells thus produce proNGF. Recombinant human proNGF even at high concentrations did not affect the levels of ATP or the activity of caspase 3/7, indicating that in granulosa cells proNGF does not induce death. In contrast, mature NGF, which was detected previously in follicular fluid, may be a trophic molecule for granulosa cells with unexpected functions. We found that in contrast to proNGF, NGF increased the levels of the transcription factor early growth response 1 and of the enzyme choline acetyl-transferase. A mechanism for the generation of mature NGF from proNGF in the follicular fluid may be extracellular enzymatic cleavage. The enzyme MMP7 is known to cleave proNGF and was identified in follicular fluid and as a product of granulosa cells. Thus the generation of NGF in the ovarian follicle may depend on MMP7.

Pregnancy-associated plasma protein A in human ovarian follicles and its association with intrafollicular hormone levels.

OBJECTIVE: To evaluate follicular fluid (FF) levels of pregnancy-associated plasma protein A (PAPP-A) in relation to levels of intrafollicular hormones. Furthermore, immunostaining of human follicles of varying diameters was studied for PAPP-A, antimüllerian hormone (AMH), and aromatase, and the biological activity of PAPP-A in FF was evaluated. DESIGN: Laboratory investigation. SETTING: University hospital. PATIENT(S): A total of 43 women with a total of 80 samples were obtained from three different size-groups of antral follicles collected before and after the LH surge. INTERVENTION(S): ELISA measurement of steroids, PAPP-A, and AMH, immunohistochemistry of PAPP-A, AMH, and aromatase on follicles of different diameter, and proteolytic activity of PAPP-A toward insulin-like growth factor (IGF)-binding protein 4 (IGFBP-4). MAIN OUTCOME MEASURE(S): Association between FF levels of PAPP-A and measured ovarian hormones, PAPP-A activity in FF, localization of PAPP-A, AMH, and aromatase in antral follicles. RESULT(S): A highly significant association between FF levels of PAPP-A and all measured hormones were obtained with positive associations toward E2 and P, whereas AMH, T, and A showed strong negative associations. PAPP-A proteolytic activity toward IGFBP-4 was detected in human FF. PAPP-A immunostaining shifted from being primarily present in theca cells of small antral follicles to being expressed in granulosa cells (GCs) of preovulatory follicles. In contrast, AMH expression became reduced with increasing follicular diameter. Aromatase expression was highly specifically localized to GCs of preovulatory follicles. CONCLUSION(S): The results suggest that PAPP-A is specifically involved in the regulation of steroidogenesis in human antral follicles. Local regulation of IGF-II activity may represent a mechanism by which PAPP-A exerts this function and highlights the importance of IGF signaling during follicular development.

Do alterations in follicular fluid proteases contribute to human infertility?

PURPOSE: Cathepsin L and ADAMTS-1 are known to play critical roles in follicular rupture, ovulation, and fertility in mice. Similar studies in humans are limited; however, both are known to increase during the periovulatory period. No studies have examined either protease in the follicular fluid of women with unexplained infertility or infertility related to advanced maternal age (AMA). We sought to determine if alterations in cathepsin L and/or ADAMTS-1 existed in these infertile populations. METHODS: Patients undergoing in vitro fertilization (IVF) for unexplained infertility or AMA-related infertility were prospectively recruited for the study; patients with tubal or male factor infertility were recruited as controls. Follicular fluid was collected to determine gene expression (via quantitative polymerase chain reaction), enzyme concentrations (via enzyme-linked immunosorbent assays), and enzymatic activities (via fluorogenic enzyme cleavage assay or Western blot analysis) of cathepsin L and ADAMTS-1. RESULTS: The analysis included a total of 42 patients (14 per group). We found no statistically significant difference in gene expression, enzyme concentration, or enzymatic activity of cathepsin L or ADAMTS-1 in unexplained infertility or AMA-related infertility as compared to controls. We also found no statistically significant difference in expression or concentration with advancing age. CONCLUSIONS: Cathepsin L and ADAMTS-1 are not altered in women with unexplained infertility or AMA-related infertility undergoing IVF, and they do not decline with advancing age. It is possible that differences exist in natural cycles, contributing to infertility; however, our findings do not support a role for protease alterations as a common cause of infertility.

Evidence that increased ovarian aromatase activity and expression account for higher estradiol levels in African American compared with Caucasian women.

CONTEXT: Serum estradiol levels are significantly higher across the menstrual cycle in African American (AAW) compared with Caucasian women (CW) in the presence of similar FSH levels, yet the mechanism underlying this disparity is unknown. OBJECTIVE: The objective of the study was to determine whether higher estradiol levels in AAW are due to increased granulosa cell aromatase mRNA expression and activity. DESIGN: The design of the study included daily blood sampling and dominant follicle aspirations at an academic medical center during a natural menstrual cycle. SUBJECTS: Healthy, normal cycling AAW (n = 15) and CW (n = 14) aged 19-34 years participated in the study. MAIN OUTCOME MEASURES: Hormone levels in peripheral blood and follicular fluid (FF) aspirates and aromatase and FSH receptor mRNA expression in granulosa cells were measured. RESULTS: AAW had higher FF estradiol [1713.0 (1144.5-2032.5) vs 994.5 (647.3-1426.5) ng/mL; median (interquartile range); P < .001] and estrone [76.9 (36.6-173.4) vs 28.8 (22.5-42.1) ng/mL; P < .001] levels than CW, independent of follicle size. AAW also had lower FF androstenedione to estrone (7 ± 1.8 vs 15.8 ± 4.1; mean ± SE; P = .04) and T to estradiol (0.01 ± 0.002 vs 0.02 ± 0.005; P = .03) ratios, indicating enhanced ovarian aromatase activity. There was a 5-fold increase in granulosa cell aromatase mRNA expression in AAW compared with CW (P < .001) with no difference in expression of FSH receptor. FSH, inhibin A, inhibin B, and AMH levels were not different in AAW and CW. CONCLUSIONS: Increased ovarian aromatase mRNA expression, higher FF estradiol levels, and decreased FF androgen to estrogen ratios in AAW compared with CW provide compelling evidence that racial differences in ovarian aromatase activity contribute to higher levels of estradiol in AAW across the menstrual cycle. The absence of differences in FSH, FSH receptor expression, and AMH suggest that population-specific genetic variation in CYP19, the gene encoding aromatase, or in factors affecting its expression should be sought.

Effects of growth differentiation factor-9 and FSH on in vitro development, viability and mRNA expression in bovine preantral follicles.

The present study investigated the role of growth differentiation factor (GDF)-9 and FSH, alone or in combination, on the growth, viability and mRNA expression of FSH receptor, proliferating cell nuclear antigen (PCNA) and proteoglycan-related factors (i.e., hyaluronan synthase (HAS) 1, HAS2, versican, perlecan) in bovine secondary follicles before and after in vitro culture. After 12 days culture, sequential FSH (100 ng mL⁻¹) from Days 0 to 6 and 500 ng mL⁻¹ from Days 7 to 12) increased follicular diameter and resulted in increased antrum formation (P<0.05). Alone, 200 ng mL⁻¹ GDF-9 significantly reduced HAS1 mRNA levels, but increased versican and perlecan mRNA levels in whole follicles, which included the oocyte, theca and granulosa cells. Together, FSH and GDF-9 increased HAS2 and versican (VCAN) mRNA levels, but decreased PCNA mRNA expression, compared with levels in follicles cultured in α-minimum essential medium supplemented with 3.0 mg mL⁻¹ bovine serum albumin, 10 µg mL⁻¹ insulin, 5.5 µg mL⁻¹ transferrin, 5 ng mL⁻¹ selenium, 2 mM glutamine, 2mM hypoxanthine and 50 µg mL⁻¹ ascorbic acid (α-MEM⁺). Comparisons of uncultured (0.2 mm) and α-MEM⁺ cultured follicles revealed that HAS1 mRNA expression was higher, whereas VCAN expression was lower, in cultured follicles (P<0.05). Expression of HAS1, VCAN and perlecan (HSPG2) was higher in cultured than in vivo-grown (0.3 mm) follicles. In conclusion, FSH and/or GDF-9 promote follicular growth and antrum formation. Moreover, GDF-9 stimulates expression of versican and perlecan and interacts positively with FSH to increase HAS2 expression.

Intrafollicular levels of matrix metalloproteinases-2 and -9 in patients with polycystic ovaries are not associated with pregnancy rate during IVF cycle.

This study aimed to detect the levels of matrix metalloproteinases (MMP)-2 and -9, using enzyme-linked immunosorbent assays, in the follicular fluid of 35 patients with polycystic ovaries, compare them with the levels found in 35 normally ovulating women enrolled in their first in vitro fertilization (IVF) cycle and then correlate them with pregnancy rates in these two groups. Levels of MMP-9 were found significantly increased in women with polycystic ovaries when compared with the controls, while MMP-2 levels were higher in women with polycystic ovaries without reaching statistical significance. The two groups did not differ in age, in the number of embryos transferred or in pregnancy rates. In conclusion, the results indicated an increased gelatinolytic activity in patients with polycystic ovaries after ovarian stimulation for IVF treatment without detecting any association between levels of MMP-2 and 9 and IVF pregnancy rates.

Inactivation of glucocorticoids by 11beta-hydroxysteroid dehydrogenase enzymes increases during the meiotic maturation of porcine oocytes.

Recent reports have shown that glucocorticoids can modulate oocyte maturation in both teleost fish and mammals. Within potential target cells, the actions of physiological glucocorticoids are modulated by 11beta-hydroxysteroid dehydrogenase (HSD11B) isoenzymes that catalyse the interconversion of cortisol and cortisone. Hence, the objective of this study was to establish whether HSD11B enzymes mediate cortisol-cortisone metabolism in porcine oocytes and, if so, whether the rate of glucocorticoid metabolism changes during oocyte maturation. Enzyme activities were measured in cumulus-oocyte complexes (COCs) and denuded oocytes (DOs) using radiometric conversion assays. While COCs and DOs oxidised cortisol to inert cortisone, there was no detectable regeneration of cortisol from cortisone. The rate of cortisol oxidation was higher in expanded COCs than in compact COCs containing germinal vesicle (GV) stage oocytes (111+/-6 vs 2041+/-115 fmol cortisone/oocyte.24 h; P<0.001). Likewise, HSD11B activities were 17+/-1 fold higher in DOs from expanded COCs than in those from compact COCs (P<0.001). When GV stage oocytes were subject to a 48 h in vitro maturation protocol, the enzyme activities were significantly increased from 146+/-18 to 1857+/-276 fmol cortisone/oocyte.24 h in GV versus MII stage oocytes respectively (P<0.001). Cortisol metabolism was inhibited by established pharmacological inhibitors of HSD11B (glycyrrhetinic acid and carbenoxolone), and by porcine follicular and ovarian cyst fluid. We conclude that an HSD11B enzyme (or enzymes) functions within porcine oocytes to oxidise cortisol, and that this enzymatic inactivation of cortisol increases during oocyte maturation.

Granulosa cell aromatase enzyme activity: effects of follicular fluid from patients with polycystic ovary syndrome, using aromatase conversion and [11C]vorozole-binding assays.

The local regulation of ovarian aromatase enzyme in polycystic ovary syndrome (PCOS) was studied with aromatase conversion and [11C]vorozole-binding assays to analyze aromatase activity, substrate-enzyme affinity and number of aromatase binding sites in non-cultured human granulosa cells (GC) incubated with different sources and preparations of follicular fluid (FF). Incubation with FF from women stimulated in in vitro fertilization cycles with follicle-stimulating hormone yielded higher conversion activity than with FF from healthy women and PCOS patients, paralleled with higher substrate affinity (lower Kd) than with FF from healthy women. In PCOS women, charcoal-pretreated FF yielded higher conversion, whereas the ether-pretreated FF yielded lower conversion activity, than with untreated PCOS FF. Both preparations of FF yielded higher affinity to substrate (lower Kd values) and the ether-pretreated FF a lower number of binding sites (Bmax). It seems that steroids with the presence of proteins in PCOS FF reduced aromatase conversion activity through decreased substrate affinity, whereas FF preparations devoid of proteins reduced the aromatase conversion activity mainly through blocking of aromatase active sites. Identification of specific agents responsible for this rapid regulation of aromatase function might help to understand normal regulation of the menstrual cycle and supposed imbalances of inhibitors/activators in PCOS.

[Intrafollicular levels of sexual steroids and their relation with the antioxidant enzymes on the oocyte quality in an in vitro fertilization program]. / Las concentraciones intrafoliculares de esteroides sexuales y su correlación con las enzimas antioxidantes en la calidad de los ovocitos en un programa de fertilización in vitro.

OBJECTIVE: To establish a correlation between intrafollicular superoxide dismutase enzyme concentrations, activity with oestradiol levels, and the effects on oocyte quality and maturity. STUDY: Prospective, descriptive and observational. MATERIAL AND METHODS: Forty-one patients underwent IVF-ET program. The ovarian stimulation protocol was made with recombinant FSH and GnRH antagonists. All follicles were aspirated one by one, and the follicular fluid was stored at a -20 degrees C room temperature. We retrieved 120 follicular fluids and performed the measurement of oestradiol and superoxide dismutase enzyme on each follicular fluid and its correlation with fertilization and cleavage rates. Statistical analysis was carried out with ANOVA, t Student, chi2 and P Pearson tests. RESULTS: Patients' mean age was of 33.74 +/- 5.04 years, the mean of enzyme activity was of 76.89%, and the mean concentration of superoxide dismutase enzyme was of 68.71 UI/L. According to oocyte quality or maturity, no statistical differences were observed when comparing oestradiol levels with superoxide dismutase enzyme concentrations. But when we analized both variables, we observed a positive correlation in metaphase 2 oocytes (p = 0.236). When we correlated the superoxide dismutase enzyme activity with oestradiol concentrations in relation to oocyte quality, a positive correlation in good quality oocytes was observed too (p = 0.218). We perceived a strong correlation between SOD concentrations and oestradiol intrafollicular measurements in good quality oocytes. COMMENTS: Oocyte maturity and development are conditionated by a close relationship between SOD and intrafollicular oestradiol.

Matrix metalloproteinase-2 and -9 secretion by the equine ovary during follicular growth and prior to ovulation.

Profound hormonally controlled tissue remodelling occurs in the equine ovary for follicle growth and development, and also for the alteration in follicle shape directed towards the ovulation fossa, the site where ovulation occurs. The aim of this study was to examine the spatial and temporal regulation of matrix metalloproteinases (MMP)-2 and MMP-9, important enzymes in tissue remodelling, during follicle growth, and ovulation. Using gelatin substrate zymography, we measured these MMPs in follicular fluid of large anovulatory follicles collected during spring transition, early dominant follicles (> 23 mm), and at oestrus in follicles approximately 3 days prior to ovulation, and post-hCG treatment when ovulation was predicted in approximately 4 h. The most abundant activity detected in follicular fluid was MMP-2, although there were no changes in secretion or activation in association with ovulation. The activity of MMP-9 was detected in lower amounts, with no changes prior to ovulation, although it decreased significantly (P < 0.05) post-hCG treatment. At oestrus, when different regions of the ovary were maintained in explant culture for 24 h, there were no significant changes in either MMP-2 or MMP-9 secretion by stromal tissues collected at the ovarian fossa, adjacent to the preovulatory follicle but away from the fossa, and a further site remote from the preovulatory follicle. Over this same time period, follicular progesterone (P < 0.01) and oestradiol (P < 0.05) increased significantly, although oestradiol tended to decrease after hCG administration. These findings indicate that MMP-2 and MMP-9 are not key acute regulators for the changes in follicle shape immediately prior to ovulation.

Matrix metalloproteinases 2 and 9 in follicular fluids of patients undergoing controlled ovarian stimulation for ICSI/ET.

The objective of this study was to detect the level of matrix metalloproteinases 2 and 9 in the follicular fluid of patients undergoing controlled ovarian stimulation and to correlate these levels with hormonal and clinical parameters. Follicular fluids were collected from thirty patients participating in an intracytoplasmic sperm injection/embryo transfer (ICSI/ET) program in "Otmar Bauer" Assisted Reproduction Center, Alexandroupolis, Greece. Follicular fluid was collected at oocyte retrieval and MMP-2 and MMP-9 were identified by gelatin zymography. Gelatin zymography of the follicular fluids revealed bands consistent with proMMP-2 and proMMP-9. ProMMP-2 was the predominant band in the majority of samples. The levels of MMPs varied considerably among the samples. A significant positive correlation between MMP-2 and the peak estradiol levels was found.

Decreased expression of tissue inhibitor of matrix metalloproteinases in follicular fluid from women with polycystic ovaries compared with normally ovulating patients undergoing in vitro fertilization.

OBJECTIVE: To compare activity of matrix metalloproteinases (MMP) and expression of their tissue-specific inhibitor (TIMP) in the follicular fluid of normally ovulating women and women with the polycystic ovary syndrome (PCOS). DESIGN: Prospective study. SETTING: IVF unit and endocrine research unit. PATIENT(S): Fourteen patients undergoing IVF treatment (seven with normal ovulation and seven with PCOS). MAIN OUTCOME MEASURE(S): Activity of MMP-2 and MMP-9 and expression of MMP-1, TIMP-1, and TIMP-2 was measured in follicular fluid of the leading follicles by using gel zymography and immunoblot analysis. RESULT(S): The activity of MMP-2 and MMP-9 and expression of MMP-1 was similar in follicular fluid of normally ovulating patients and patients with PCOS. Significantly lower expression of TIMP-1 was found in follicular fluid of patients with PCOS women compared with normally ovulating patients. CONCLUSION(S): Because MMPs and TIMPs play a role in the physical and chemical structure of the follicular compartment, the decreased expression of TIMP in patients with PCOS may be part of a compensatory process to overcome the physical properties of the thick ovarian capsule.

Ovarian modulators of 11beta-hydroxysteroid dehydrogenase (11beta HSD) activity in follicular fluid from bovine and porcine large antral follicles and spontaneous ovarian cysts.

In the ovary, cortisol is oxidized to cortisone by 11beta-hydroxysteroid dehydrogenase (11betaHSD). The present study investigated whether follicular fluid (FF) from large antral follicles and spontaneous ovarian cysts, isolated from bovine and porcine ovaries, contained modulators of 11betaHSD activity. Whereas FF from antral follicles had no significant effect over 1 h on NADP+-dependent 11betaHSD activity in rat kidney homogenates, enzyme activity was inhibited by FF from bovine and porcine ovarian cysts (80.5% +/- 2.3% and 72.8% +/- 3.4% of control, respectively). Following C18 reverse-phase chromatography, the hydrophilic fractions of FF from bovine and porcine antral follicles stimulated NADP+-dependent 11betaHSD activities (111.5% +/- 21.6% and 55.2% +/- 5.7% respectively). Hydrophobic compounds inhibited NADP+-dependent cortisol oxidation by 58.2% +/- 5.1% (bovine) and 45.7% +/- 2.0% (porcine). In both species, FF from ovarian cysts appeared to contain less of the hydrophilic stimuli to 11betaHSD activity and more of the hydrophobic inhibitors. The FF from antral follicles and ovarian cysts, and the C18 fractions thereof, had no significant effect on NAD+-dependent cortisol oxidation. The ovarian modulators of NADP+-dependent 11betaHSD activities did not coelute with cortisol, cortisone, estradiol, testosterone, progesterone, pregnenolone, and cholesterol. However, the 11betaHSD stimuli in porcine FF from both antral follicles and cysts coeluted with prostaglandin (PG) E2 and PGF2alpha. We conclude that large antral follicles and spontaneous ovarian cysts, in both the cow and the pig, contain ovarian modulators of the NADP+-dependent 11betaHSD activity. Moreover, FF from spontaneous ovarian cysts, because of decreased content of the 11betaHSD stimulus accompanied by increased content of the 11betaHSD inhibitors, exerts a net inhibitory effect on 11betaHSD activity.

Matrix metalloproteinases-2 and -9 activities in bovine follicular fluid of different-sized follicles: relationship to intra-follicular inhibin and steroid concentrations.

Matrix metalloproteinases (MMPs) play very important roles in extracellular matrix (ECM) remodeling during ovarian follicular development, ovulation and atresia. The aim of the present study was to determine the content of gelatinases in follicular fluid in various sized bovine follicles. Bovine ovaries were collected from local slaughterhouse and follicular fluid from follicles of 2 to over 25 mm in diameter was collected. Gelatinase activity within the follicular fluid was analyzed by gelatin zymography. The concentration of inhibin in the follicular fluid was also measured by immunoblot analysis. The proMMP-2 and alpha-subunit (alphaN) inhibin was detected in all follicles regardless of their size. The abundance of proMMP-2 varied with follicular size, while alphaN inhibin increased significantly (P<0.01) in follicles of 10-14 and 15-20 mm in size. There was a positive and negative correlation between estradiol (E(2)) and progesterone (P(4)) concentrations with abundance of proMMP-2, respectively. Follicles of diameter over 25 mm had greater proMMP-9 activity than other follicles. These same follicles had significantly (P<0.01) lower inhibin levels than follicles of 10-14 and 15-20 mm in size. In conclusion, these results suggest a significant role of these proteases in growth and development of bovine follicle, particularly proMMP-2 and active MMP-2 activities in the follicular fluid could serve as markers of follicular health while abundance of proMMP-9 may possibly denote a follicular cyst.

Lactate dehydrogenase estimation in follicular fluid: correlation with patient age, follicle size and super ovulation in ART cycles.

OBJECTIVE: To quantify the lactate dehydrogenase (LDH) level in human follicular fluids, and to define its relationship with follicle size, patient age, serum estradiol (E(2)) level, and the amount of gonadotropins administered during superovulation in ART cycles. STUDY DESIGN: In this prospective study, 21 women undergoing ART treatment were selected. Follicular fluid from the largest follicle of both ovaries was collected from each patient on the day of oocyte aspiration and analyzed for LDH. Serum oestradiol was estimated on the day of hCG administration. Relationship between LDH level and (1) patient age, (2) follicle size, (3) follicle stimulating hormone administered during superovulation period and (4) serum oestradiol was studied. RESULT(S): LDH activity increased with chronological age of the patient. As follicular size (diameter) increased, increase in the LDH concentration in follicular fluid was observed. Serum estradiol level did not show any relationship with LDH activity. Similarly, administration of various doses of follicle stimulating hormone during superovulation did not show any correlation with LDH level. CONCLUSION(S): Follicular fluid LDH level has shown association with patient age and the follicle size, however, no significant association was found with other parameters studied.

Angiotensin converting enzyme in bovine ovarian follicular fluid and its relationship with oestradiol and progesterone.

CONTENT: The purpose of the present study was to identify angiotensin converting enzyme (ACE) in bovine ovarian follicular fluid and to relate the ACE activity to the phase of the oestrous cycle, pregnancy, and the follicular fluid concentrations of oestradiol and progesterone. The ACE activity was similar to that found in bovine serum and was completely inhibited by the specific ACE inhibitor captopril. The 50% inhibitory concentration (IC50) was 1.4 x 10(-8) mol/l (range 0.8 x 10(-8) to 5.0 x 10(-8) mol/l; n=6), which is similar to that found in bovine and human serum. The ACE activity did not differ in the pre-ovulatory and luteal phase, pregnancy or cystic follicles. It correlated with the follicular fluid concentration of progesterone in cycling cows (rho=0.476; p < 0.005; n=36), but did not correlate with the diameter of the follicles, the follicular fluid concentration of oestradiol or the ratio between the oestradiol and progesterone concentrations. The demonstration of ACE in bovine ovarian follicular fluid provides further evidence for the presence of a local renin-angiotensin system in the bovine ovary.

Presence of angiotensin-converting enzyme in follicular fluids of porcine ovaries and its possible involvement in the intrafollicular breakdown of bradykinin.

The follicular fluid of porcine ovaries contains a metalloenzyme capable of hydrolyzing the synthetic substrate, benzyloxycarbonyl-Val-Lys-Met-MCA. This enzyme was purified by ammonium sulfate fractionation followed by column chromatography on DEAE-cellulose, CM-cellulose, Zn(2+)-chelating Cellulofine, and Diol-300 gel-filtration columns. The molecular weight of the purified enzyme was estimated to be 170,000 by SDS-PAGE and 400,000 by gel-filtration analysis, suggesting that the native enzyme is a dimer of the 170-kDa subunit polypeptide. The enzyme activity was drastically enhanced by the presence of chloride ion, and strongly inhibited by captopril and bradykinin potentiator B. A 9-residue peptide containing a processing site of human amyloid precursor protein was degraded by its dipeptidyl carboxypeptidase activity. Furthermore, the purified protein was recognized by specific antibody raised against human angiotensin-converting enzyme. The enzyme rapidly degraded bradykinin in vitro. These results indicate that benzyloxycarbonyl-Val-Lys-Met-MCA-hydrolyzing enzyme is a porcine angiotensin-converting enzyme, and that the enzyme may play a role in bradykinin turnover within the follicles of porcine ovaries.

Ovarian modulators of 11beta-hydroxysteroid dehydrogenase (11betaHSD) activity in follicular fluid from gonadotrophin-stimulated assisted conception cycles.

In the ovary, cortisol-cortisone interconversion is catalysed by isoforms of 11beta-hydroxysteroid dehydrogenase (11betaHSD). The objective of this study was to establish whether human follicular fluid (hFF), obtained after controlled ovarian hyperstimulation, contains paracrine modulators of 11betaHSD activity. Of 274 hFF samples tested for effects in rat kidney homogenates, 206 hFF samples significantly inhibited NADP(+)-dependent oxidation of cortisol within 1 h (by 11-67% of control 11betaHSD activity), whereas 42 hFF samples significantly stimulated 11betaHSD activity (16-210% increase relative to control). Although charcoal-stripping of hFF prevented the inhibition and potentiated the stimulation of NADP(+)-dependent cortisol oxidation in a renal homogenate, effects of individual hFF samples on NADP(+)-dependent cortisol oxidation were independent of intrafollicular progesterone concentrations. Hydrophilic fractions of hFF samples, isolated by C18 column chromatography, stimulated both the NADP(+)-dependent oxidation of cortisol (by 55+/-5%, n=98) and the NADPH-dependent reduction of cortisone (by 86+/-22%, n= 5). In contrast, the hydrophobic fractions of hFF (eluted at 65-85% methanol) inhibited both NADP(+)-dependent 11beta-dehydrogenase and NADPH-dependent 11-ketosteroid reductase activities (by 63+/-2% and 74+/-4%, respectively). None of the C18 column fractions of 50 hFF samples had any significant effect on NAD(+)-dependent 11beta-dehydrogenase activities. The hydrophobic inhibitors of NADP(H)-dependent cortisol-cortisone metabolism did not co-elute with several candidate compounds (prostaglandins E(2) and F(2alpha), cortisol, cortisone, oestradiol, testosterone, progesterone, pregnenolone or cholesterol). Hence, hFF aspirated from women undergoing controlled ovarian hyperstimulation for assisted conception contains both hydrophilic stimuli and hydrophobic inhibitors of glucocorticoid metabolism which appear to be selective for the NADP(H)-dependent, type 1 isoform of 11betaHSD.

Localization of the renin-angiotensin system in the bovine ovary: cyclic variation of the angiotensin II receptor expression.

Angiotensin (Ang) II may modulate reproductive function in the bovine ovary. Therefore, expression and localization of a local ovarian renin-angiotensin system (RAS) were investigated by elucidating the influence of the estrus cycle, pregnancy, and the presence of follicular cysts. Receptor analysis and autoradiography were used to characterize and localize Ang II receptors. Cyclic variations in the density of ovarian Ang II receptors were found with a higher value in estrus than in diestrus. The density in ovaries with follicular cysts was in the same order of magnitude as in estrus. The Ang II receptor type 2 (AT(2)) dominated in all three groups. Autoradiography showed that the majority of antral follicles and follicular cysts had intense AT(2) receptor binding in the theca externa. Binding was less intense in the theca interna, whereas there was no binding in the granulosa layer. In the corpora lutea, the AT(2) receptor was dominant in the capsule and in connective tissue infoldings, whereas no binding was observed in the luteal tissue. The type 1 Ang II receptor (AT(1)) was dominant in the stroma and showed no cyclic changes. Angiotensin-converting enzyme (ACE) activity was detected in all aspirated follicular fluids and homogenates of ovarian tissue. Autoradiography showed that most of the ACE was localized on endothelial cells. Renin immunoreactivity was found in granulosa and thecal cells of antral follicles and in luteal cells. Furthermore, solitary cells in the stroma, presumably macrophages, displayed intense staining. Our finding of cyclic changes support the concept of an active and regulated RAS in the bovine ovary.