Demonstration of neuropeptide Y and its precursor in plasma and follicular fluid.

The present investigation provides three lines of evidence for the presence of a pro-form of neuropeptide Y (NPY) in plasma and follicular fluid. First, by the demonstration of NPY-immunoreactive material of a size corresponding to the estimated mol wt of pro-NPY. Second, an antiserum specific for the C-terminal tyrosine amide of NPY and peptide YY does not react with this material. Third, it was possible to convert the pro-NPY extracted from plasma and follicular fluid using the protease, Endoproteinase-Lys C, to a NPY-immunoreactive form eluting slightly before NPY on a G-50 column. The size of the digested product was consistent with a cleavage of pro-NPY resulting in an immunoreactive species, NPY-Gly-Lys. Pro-NPY was also found in tissue culture media from the human neuroendocrine cell line SH-SY5Y. As in the case of plasma and follicular fluid, another NPY immunoreactive species eluted from a G-50 gel filtration column slightly before synthetic human NPY. Analysis of this material with an antibody directed against the tyrosine amide of NPY in combination with isoelectric focusing revealed that this peak consisted of at least two immunoreactive forms of NPY. In conclusion, at least three different forms of NPY immunoreactivity are likely to be present in plasma, follicular fluid, and cell tissue culture media; pro-NPY, a degradation form of pro-NPY, or a biosynthetic intermediate and NPY.

Insulin-like growth factor I (IGF-I) levels in follicular fluid from human preovulatory follicles: correlation with serum IGF-I levels.

Insulin-like growth factor I (IGF-I) levels were measured in both serum and fluid of preovulatory follicles (n = 156) in 43 women undergoing in vitro fertilization (IVF). The mean IGF-I level in follicular fluid (FF) was significantly lower than in serum (0.52 +/- 0.02 IU/L versus 0.66 +/- 0.23 IU/L), and FF levels were significantly correlated with individual serum IGF-I levels as well as with follicular size and FF volume but not with oocyte maturity, granulosa cell appearance, or IVF. This suggests that FF IGF-I levels cannot serve as a clinical indicator for the degree of oocyte/granulosa cell differentiation or a predictor for IVF. Serum IGF-I levels were inversely correlated with the number of human menopausal gonadotropin ampules administered during treatment, suggesting that IGF-I might enhance ovarian gonadotropic stimulation.

Differences in follicular morphology, steroidogenesis and oocyte maturation in naturally cyclic and PMSG/hCG-treated prepubertal gilts.

Ovaries were obtained from naturally cyclic pigs on Days 16-17, 18, 19, 20 and 21 of the oestrous cycle and on the basis of observed follicular characteristics were assigned as representative of the early (Group 1), mid- (Groups 2 and 3) or late (after LH; Group 4) follicular phase. Follicular development in cyclic gilts was compared with that in ovaries obtained from late prepubertal gilts 36 (Group 5) or 72 (Group 6) h after treatment with 750 i.u. PMSG alone, or with a combination of 500 i.u. hCG 72 h after PMSG and slaughter 30-40 h later (Group 7). After dissection of all follicles greater than 2 mm diameter, follicular diameter, follicular fluid volume, follicular fluid concentrations of progesterone, oestradiol and testosterone, and the stage of oocyte maturation were determined. Combined PMSG/hCG treatment of immature gilts resulted in a pattern of follicular development different from that in naturally cyclic gilts during the follicular phase. Overall exogenous gonadotrophin treatment also increased (P less than 0.001) the variability in follicular diameter and fluid volume. Comparisons between appropriate groups also established differences in the variability of both morphological (diameter and volume, Group 1 vs Group 5; P less than 0.05) and biochemical development (follicular fluid oestradiol, Group 3 vs Group 6 and Group 4 vs Group 7; both P less than 0.05). Such differences in both morphological and biochemical characteristics between cyclic and PMSG/hCG-treated gilts were particularly evident in the population of larger (greater than 6 mm) follicles. These results indicate that the pattern of follicular development in naturally cyclic and in PMSG/hCG-treated gilts is dissimilar and suggests that the ovaries of gonadotrophin-treated prepubertal gilts are functionally different from the ovaries of mature females.

Concentrations of insulin-like growth factor-I in blood and ovarian follicular fluid of cattle selected for twins.

Recent studies have implicated insulin-like growth factor I (IGF-I) as an intraovarian regulator of follicular growth and differentiation. Therefore, we investigated the possibility that cattle selected for twin births may have increased concentrations of IGF-I within the ovarian follicle and(or) in peripheral blood. The estrous cycles of 14 cows with histories of producing twins and 12 control monotocous cows were synchronized with 35 mg of prostaglandin F2 alpha (PGF2 alpha). Blood and follicular fluid were collected 48-50 h post-administration of PGF2 alpha (follicular phase of the estrous cycle). Concentrations of IGF-I were measured by RIA after acid-ethanol treatment of serum or follicular fluid. Twin-producing cows had a greater (p less than 0.05) number of large (greater than or equal to 4 mm) follicles and 47% greater (p less than 0.05) concentrations of IGF-I in peripheral blood than control cows. Cattle selected for high twinning frequency also had greater (p less than 0.05) concentrations of IGF-I (+/- SE) in the two largest follicles than control (unselected) cows (327 +/- 28 vs. 243 +/- 29 ng/ml). IGF-I concentrations in pooled small (1-3.9 mm) follicles were less (p less than 0.05) than in large follicles but did not differ between control and twin-producing cattle. In addition, the percentage of IGF-I concentrations measured in follicular fluid to that of serum was lower (p less than 0.05) in small follicles than in large follicles, and was greater (p less than 0.05) in large follicles of control (93.2 +/- 5.3%) than twin-producing (76.2 +/- 4.4%) cattle.(ABSTRACT TRUNCATED AT 250 WORDS)

Insulin-like growth factor binding proteins in follicular fluid from normal dominant and cohort follicles, polycystic and multicystic ovaries.

There is now considerable evidence that the insulin-like growth factors (IGFs) play an important role in the human ovary. It has also recently become apparent that the physiological activity of the IGFs is modulated by a number of specific binding proteins (IGFBPs). In order to understand the role of the IGFs in ovarian physiology, the presence and functions of these IGFBPs will need to be characterized. As an initial step towards this we have investigated the presence of the various binding proteins by Western ligand blotting and have measured the levels of one of them, IGFBP-1, in follicular fluid (FF) obtained from unstimulated dominant and cohort follicles in 19 normal women and in eight patients with polycystic and one with multicystic ovaries. In normal women, IGFBP-1 levels in dominant follicles were similar to matched serum levels but were significantly lower in cohort follicles. IGFBP-1 levels correlated with FF-volume (r = 0.58, P less than 0.001) and with paired serum levels (r = 0.63, P less than 0.001). In post-LH surge dominant follicles this relationship with serum levels no longer held and in three out of nine subjects FF levels were higher than in serum. Thus IGFBP-1 in normal human FF appears to be partly derived from the circulation but with additional local production in the larger developing dominant follicles. Western ligand blotting revealed five IGF-binding proteins in FF running parallel with those identified in serum, suggesting that the IGFBP species previously identified in serum may also be present in FF. The two bands in positions corresponding to the components of the large (150kDa) binding complex were, as in serum, the predominant forms and in most FF samples these were even more prominent than in the accompanying serum sample. This contrasts with previous studies in lymph which suggested that the 150kDa complex was largely retained in the circulation. All three small IGFBPs varied considerably between FF samples even within an individual; each IGFBP varied independently of the other IGFBPs. Our results demonstrate that at least four discrete IGFBPs are present in FF and suggest that each may be produced independently within the ovary.

Alpha 2-macroglobulin and tissue inhibitor of metalloproteinases: collagenase inhibitors in human preovulatory ovaries.

Extensive remodeling of the follicular extracellular matrix occurs during the process of ovulation. This remodeling involves the breakdown of collagen, which is regulated, in part, by the action of the metalloproteinase collagenase and its associated inhibitors. In the present study, follicular metalloproteinase inhibitors were characterized to determine whether they were serum-borne or of ovarian origin, possibly a tissue-derived inhibitor known as tissue inhibitor of metalloproteinase (TIMP). Human follicular fluid and granulosa cells were obtained from preovulatory follicles of patients in an in vitro fertilization program. Chromatographic separation of follicular fluid on Sepharose 6B resulted in two peaks of inhibitory activity. The large molecular radius (Mr) inhibitor was similar in size to the serum-borne metalloproteinase inhibitor alpha 2-macroglobulin (i.e. Mr 700,000) whereas the small Mr inhibitor approximated the size of TIMP (i.e. Mr 29,000). Incubation of aliquots from either of the two peaks of inhibitor activity or an alpha 2-macroglobulin standard with an antibody to alpha 2-macroglobulin decreased the inhibitory activity in both the large Mr peak and the alpha 2-macroglobulin standard by 86.6 +/- 1.7% and 71.5 +/- 7.7% (n = 4, P less than 0.005), respectively, implying cross-reactivity with the alpha 2-macroglobulin antibody. The inhibitory activity in the small Mr peak, however, was unchanged. Northern analysis of total granulosa cell RNA demonstrated TIMP messenger RNA (mRNA) in all eight granulosa cell samples examined whereas alpha 2-macroglobulin mRNA was virtually undetectable. A positive correlation (r = 0.85, P less than 0.01) was observed between the levels of TIMP mRNA and the ratio of the follicular estradiol-progesterone concentration. However, inhibitor activity in the follicular fluid was not correlated with the levels of TIMP mRNA (r = 0.05). These findings confirm the presence of alpha 2-macroglobulin in follicular fluid and demonstrate that human preovulatory granulosa cells contain mRNA for TIMP, an inhibitor that regulates metalloproteinases such as collagenase, gelatinase, and proteoglycanase. Additionally, the expression of TIMP mRNA is steroid related and may be hormonally regulated. It is proposed that TIMP produced in the granulosa cell compartment in conjunction with alpha 2-macroglobulin from the serum may act to control the site and extent of ovarian connective tissue remodeling.

The effect of prostaglandin synthetase inhibitors on human preovulatory follicular fluid prostaglandin, thromboxane, and leukotriene concentrations.

This study evaluates the eicosanoid concentration in luteinized unruptured follicles (LUFs) on the ovaries of patients who had been treated with inhibitors of prostaglandin synthetase. Indomethacin, bromfenac, or azapropazone (or a placebo) was administered orally to 41 women during the periovulatory period. Follicular development was monitored by serial ultrasound examinations, and the onset of ovulation was regulated by an injection of hCG. Follicular fluid was aspirated during sterilization by minilaparotomy, which was performed just before the expected time of ovulation. Prostaglandin E2 and PGF2 alpha levels in the fluid were significantly reduced by indomethacin and bromfenac compared to those after placebo treatment. Bromfenac also reduced the follicular fluid leukotriene B4 level. Therefore, the development of luteinized unruptured follicles after treatment with nonsteroidal antiinflammatory drugs appears to be associated with a significant decrease in the synthesis of ovarian eicosanoids.

Action kinetics of inhibin in superfused pituitary cells depend on gonadotropin-releasing hormone treatment.

We examined the effects of partly purified inhibin from porcine follicular fluid on FSH and LH release in superfused rat pituitary cell cultures exposed to different GnRH stimuli. Pituitary cells from immature male rats were cultured in chemically defined medium. After 4 days of static culture in the absence of inhibin preparation and GnRH, the cell monolayers were superfused for approximately 10 h at a constant speed (0.15 or 0.25 ml/min) with medium with or without inhibin preparation (1 micrograms/ml). During the superfusion, some cultures were stimulated with GnRH (10 nM) continuously or intermittently (1 min/0.5 h or 6 min/1 h). In the basal condition (no GnRH), inhibin suppressed FSH release after 5 h of exposure (P less than 0.01), whereas LH secretion was not affected. In cultures treated with GnRH pulses (of either frequency), the inhibitory effects on the GnRH-stimulated FSH and LH release were statistically significant (P less than 0.01) after 2 h of exposure, became more pronounced in the next several hours, then remained stable until the end of the experiment. In cultures exposed to GnRH continuously, the suppressing effects of inhibin preparation became significant (P less than 0.01) after 3 h of exposure and were maximal at 4 h (52% and 61% of control values for FSH and LH, respectively). Later, the suppressing effect became less pronounce due to the decreasing rate of gonadotropin secretion in control (no inhibin) cultures exposed continuously to GnRH. The magnitude of FSH and LH suppression after 9 h of exposure to the inhibin preparation was statistically different (P less than 0.05) for different GnRH treatments and was more pronounced with GnRH pulses (24-27% and 54-57% of control values for FSH and LH, respectively) than with cultures exposed to GnRH continuously (77% and 89% of control values for FSH and LH, respectively) or in the absence of GnRH (50% and 92% of control values for FSH and LH, respectively). We conclude that both the kinetics and magnitude of action of the inhibin preparation on FSH and LH release can differ significantly depending on the presence or absence of GnRH as well as on the mode of GnRH stimulation. Of particular importance is the observation that suppressive effects of inhibin preparation decline in cultures that have been desensitized to GnRH after prolonged continuous GnRH exposures. These differences stress the role of GnRH-inhibin interactions in the regulation of gonadotropin secretion and emphasize the importance of the mode of GnRH stimulation in studies concerning inhibin action on pituitary cells in vitro.

Isolation of a protein from human ovarian follicular fluid which exerts major stimulatory effects on in vitro steroid production of testicular, ovarian, and adrenal cells.

We describe the isolation of a steroidogenesis-inducing protein (SIP) from human ovarian follicular fluid and its effects on in vitro steroid synthesis of testicular, ovarian, and adrenal cells. After heating at 60 C, precipitation with 80% ammonium sulfate and dialysis, the human ovarian follicular fluid proteins were fractionated by gel chromatography on Sephacryl S-200. The bioactivity was eluted in the mol wt region between 40 and 60 K. SIP was further purified by affinity chromatography on blue Sepharose (CL-6B), preparative isoelectrofocusing on sucrose density gradients, anion exchange chromatography on Mono Q by using fast protein liquid chromatography system and gel chromatography on Superose 12. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified SIP under both reducing and nonreducing conditions revealed a single band with an approximate mol wt of 60 K. SIP exhibited a pI value of 4.8, was heat sensitive, and lost activity upon lyophilization. SIP copurified with albumin in various isolation procedures and was distinct from human serum albumin but may represent a modified form of human albumin. SIP stimulated testosterone production by testicular pieces, interstitial cells, or purified Leydig cells from rats under different in vitro conditions. The SIP also stimulated basal and human CG stimulated in vitro progesterone production of human ovarian granulosa-lutein cells and corticosterone production of rat adrenal cells. The effects of SIP on testicular, ovarian, and adrenal cells were evident in the presence of maximal concentrations of tropic hormones. The steroid-free spent media from human granulosa-lutein cell cultures also stimulated testosterone production by rat interstitial cells, suggesting that granulosa cells may be the cellular source of SIP. In conclusion, human ovarian cells secrete a hitherto unknown albumin-like protein which enhances both basal and tropic hormone stimulated steroidogenesis in gonadal and adrenal cells. This protein may play a significant role in ovarian function by modifying steroidogenesis in the preovulatory follicle.

Inhibition of progesterone synthesis and cAMP accumulation in small bovine luteal cells by a low molecular weight component of bovine follicular fluid.

Follicular fluid from large follicles of cows was extracted with charcoal and filtered through an Amicon XM-50 membrane. The XM-50 filtrate was further fractionated on a column of Fractogel TSK HW-40 (s) using Krebs-Ringer-phosphate buffer (1/100th dilution), pH 7.2, as an eluant. Two fractions (1 and 2) were obtained. Inhibition of progesterone secretion by small luteal cells was associated with the XM-50 filtrate and Fraction 2. Whole follicular fluid, the XM-50 retentate and Fraction 1 had no significant inhibitory activity. Fraction 2, which contained about 1/100,000th of the original follicular fluid proteins, inhibited the LH- or (Bu)2cAMP-induced progesterone production during a 2-h incubation. This inhibition was dose-dependent. Fraction 2 also inhibited LH-induced cAMP accumulation, but did not affect the conversion of pregnenolone to progesterone or the basal progesterone production. The molecular weight of the inhibitory factor was estimated to be less than 10,000 and its ability to inhibit steroidogenesis was lost after digestion with protease but retained after heating for 60 min at 75 degrees C. These results demonstrate that bovine follicular fluid contains a heat-stable factor likely to be a polypeptide and which suppresses the steroidogenic response of small luteal cells to LH. The action of this inhibitory factor could involve both an inhibition of the LH-induced synthesis of cAMP and an inhibition of the action of cAMP.

Demonstration of a nonsteroidal factor in human follicular fluid that attenuates the self-priming action of gonadotropin-releasing hormone on pituitary gonadotropes.

The self-priming effect of gonadotropin-releasing hormone (GnRH) on luteinizing hormone (LH) release can be demonstrated in vitro by perfusing pituitary tissue with a continuous GnRH stimulus. A characteristic biphasic response is produced. We have used this system to investigate whether or not human follicular fluid (hFF) contains a nonsteroidal substance that can attenuate the GnRH-induced LH secretion in perfused rat pituitary glands. Steroid-extracted hFF, added to the perfusing medium, attenuated the self-priming action of GnRH in a dose-dependent manner. This was not abolished by selectively depleting the inhibin content of hFF by 97% on an immunoaffinity column. Furthermore, the biological activity of the substance was resistant to heating and was removed by dialysis. It is concluded that hFF contains a nonsteroidal factor, distinct from inhibin, that can attenuate the self-priming action of GnRH on pituitary gonadotropes.

Purification and characterization of lutropin receptor from membranes of pig follicular fluid.

Membranes derived from free floating granulosa cells in porcine ovarian follicular fluid were used as a starting material for structural characterization of both LH/hCG and FSH receptors. The receptors were highly hormone-specific and showed single classes of high-affinity binding sites (Kd = 19-74 pM). Their molecular weights as determined by affinity cross-linking with their respective 125I-ligands were similarly 70,000. The membrane-localized receptors could be solubilized with reduced Triton X-100 in the presence of 20% glycerol with good retention of hormone binding activity. The Triton extracts of membranes also showed hormone specificity and equilibrium binding constants similar to the membrane receptors (Kd = 32-48 pM). Affinity chromatography on divinylsulfonyl-Sepharose-oLH columns was utilized to purify the solubilized LH/hCG receptor to a specific activity of 2000 pmol/mg of protein. The purified receptor exhibited a high specificity for hCG and hLH but not for hFSH nor bTSH. The purified receptor was iodinated and visualized to be composed of a major protein of Mr approximately 70,000 and other minor proteins of molecular weights ranging from 14,000 to 40,000. Except for the Mr 14,000 protein, all other protein species bound to the concanavalin A-Sepharose column. The data suggest that the ovarian LH/hCG and FSH receptors are structurally similar and consist of a single polypeptide chain, as recently documented for the LH/hCG receptor (Loosefelt et al., 1989; McFarland et al., 1989).

Estrogens, androgens, and progestins in follicular fluid from preovulatory follicles: identification and quantification by gas chromatography/mass spectrometry associated with stable isotope dilution.

The synthesis and use of stable isotope-labeled analogs of various steroids have made it possible to undertake a study of follicular fluid (FF) aspirated from mature and preovulatory follicles. Our previous results have been brought together here in order to review quantitative work done by gas chromatography-mass spectrometry. A positive gradient between peripheral plasma and FF concentrations of a steroid suggests the possibility of ovarian biosynthesis. This is particularly relevant to the catecholestrogens, 19-norsteroids, and some corticosteroids.

Characterization of a monoclonal antibody specific for prostatic secretory protein of 94 amino acids (PSP94) and development of a two-site binding enzyme immunoassay for PSP94.

Purified prostatic secretory protein of 94 amino acids (PSP94) was used to generate polyclonal rabbit anti-PSP94 IgG and a murine monoclonal antibody 6B6 (mAB6B6). Both antibodies were highly specific for PSP94 by immunoblotting. Immunohistochemical studies demonstrated the specificity of mAB6B6 for human prostatic epithelial tissue. A two-site binding enzyme immunoassay for the detection of PSP94 was developed using a combination of the antibodies. The sandwich-ELISA yielded satisfactory results when mAB6B6 complexed to peroxidase conjugated goat anti-mouse-IgG (Fc) was incubated simultaneously with the sample. The assay has a dynamic range of 2-30 micrograms/l. This immunoassay was employed to measure PSP94 in male human sera (8 +/- 4 micrograms/l), female human sera (5.7 +/- 3.4 micrograms/l), follicular fluid (3.9 +/- 2.9 micrograms/l) and human seminal plasma (1.02 +/- 0.8 g/l).

Effect of human follicular fluids from pregnant and nonpregnant patients on the development of mouse zygotes in vitro.

Mouse zygotes were cultured in medium containing follicular fluid from patients who had follicles containing oocytes which fertilized, did not fertilize, or were atretic and who did or did not become pregnant after in vitro fertilization and embryo replacement. The inhibitory effect was least with the follicular fluid from follicles in which the oocytes subsequently fertilized, greater when the oocytes did not fertilize, and most inhibitory when the follicle contained an atretic oocyte. More mouse zygotes developed to blastocysts when culture medium was supplemented with follicular fluid from patients who became pregnant compared to those who did not become pregnant. There was no difference in pregnancy outcome when an oocyte which subsequently fertilized was obtained from the follicle. These results indicate that follicles contain a substance(s) which inhibits mouse zygote development in vitro and that the inhibitory activity is related to the developmental potential of the oocyte in the follicle.

Localization and hormonal regulation of ovarian production of histamine in the sheep.

Concentrations of histamine were measured within the follicular wall, follicular fluid and ovarian interstitium throughout the periovulatory period in sheep. Histamine within follicular tissue declined after the onset of the preovulatory surge of luteinizing hormone (LH) and remained low until after ovulation, when levels then increased markedly. Alterations in histamine within the follicular wall were not reflected by corresponding changes within follicular fluid or ovarian interstitium. Release of histamine from tissue during short-term incubation was greatest for follicles obtained after ovulation, which was not influenced by presence of LH in the incubation medium. Luteinizing hormone caused depletion of stores of histamine from the wall of follicles collected before the preovulatory surge of LH. Histamine could act as a paracrine mediator in the follicular mechanisms of ovulation and(or) luteinization.

[Oocyte fertilizability and hormonal environment of human preovulatory follicles in hyperstimulated cycles in an in vitro fertilization program: Are mature preovulatory follicles undergoing atresia?].

Steroid hormones in preovulatory follicular fluid, maturity of the oocyte-corona-cumulus complex (OCCC), and oocyte fertilizability were studied in 55 hyperstimulated cycles in 40 patients who were indicated for in vitro fertilization and embryo transfer due to tubal infertility. Aspirated follicles were categorized into four groups according to the degree of oocyte maturation and fertilizability, i.e. follicle (Fol)-1 (non-fertilized, intermediate maturation), Fol-2 (fertilized, intermediate maturation), Fol-3 (fertilized, full maturation), and Fol-4 (non-fertilized, full maturation). Estradiol (E2), progesterone (P), and delta 4- androstenedione (delta 4A) concentrations were highest in Fol-3, being lower in Fol-2 and Fol-1 sequentially. Fol-3 contained significantly more E2 and P than did Fol-1, showing that the degree of oocyte maturation and fertilizability correlate with concentrations of E2 and P in the follicles. A comparison of Fol-3 and Fol-4 revealed significantly higher concentrations of delta 4A in Fol-4, whereas differences in E2 and P concentrations were not significant although Fol-4 values were lower. This comparison indicated that Fol-4 were already undergoing atresia at the time of oocyte retrieval, containing morphologically mature OCCC which had lost fertilizability. This study shows that steroid hormones in the follicular fluid reflect oocyte viability and fertilizability, and suggests that preovulatory follicles with mature oocytes can also undergo atresia.

Follicular fluid Lidocaine levels during transvaginal oocyte retrieval.

Lidocaine has been shown to have adverse effects on mouse oocyte fertilization and embryo development. We have demonstrated the presence of pharmacologic levels of lidocaine in human serum and follicular fluid obtained during ultrasound guided transvaginal oocyte retrieval. The significance of this finding is unclear, as four of the eight patients studied became pregnant, including the patient with the highest follicular fluid lidocaine levels. Further evaluation of the effect of lidocaine on human embryos is warranted.

Chemical characterization of angiotensin I in porcine follicular fluid.

In the search for novel neuropeptides in porcine follicular fluid (pff) using smooth muscle contractile activity as a response parameter, a substance with a marked activity was isolated in a pure form. By amino acid analysis and sequential study, this substance has been chemically revealed to be angiotensin I. A much smaller amount of additional activity was isolated and found to be angiotensin II, as determined by radioimmunoassay. Radioimmunoassays for angiotensin I and II confirmed that the amount of angiotensin I determined was much greater than that of angiotensin II. A comparative study of the extractions, however, indicated a large amount of angiotensin I had been generated from angiotensinogen by endogenous renin in the follicular fluid which could be activated during extraction and ultrafiltration at a low pH. These findings are consistent with the previous reports that described a high concentration of prorenin in the follicular fluid, acid activation of prorenin to renin and the subsequent generation of angiotensin I from endogenous angiotensinogen.

Steroid determinations in human ovarian follicular fluid using capillary gas chromatography.

A method is presented based on capillary GLC using both a thermionic and a flame ionization detector to simultaneously analyse all major unconjugated steroids in ovarian follicular fluids (FF). Although specificity can not always be guaranteed for the smaller concentrations of androstenedione and cortisol, accuracy and reproducibility are excellent for the major progestagens and estrogens (progesterone, 17- and 16 alpha-hydroxyprogesterone, pregnenolone, 20 alpha-dihydroprogesterone, estradiol and estrone). Above all the analysis is performed with relatively cheap instrumentation and products. Apart from the "profiles" of unconjugated steroids, a semi-quantitative analysis of steroid conjugates is possible if a preliminary group separation with disposable anion exchanger columns is included.