Human FF Research Articles

Human follicular fluid elicits select dose- and age-dependent effects on mouse oocytes and cumulus-oocyte complexes in a heterologous in vitro maturation assay.

Follicular fluid (FF) is a primary microenvironment of the oocyte within an antral follicle. Although several studies have defined the composition of human FF in normal physiology and determined how it is altered in disease states, the direct impacts of human FF on the oocyte are not well understood. The difficulty of obtaining suitable numbers of human oocytes for research makes addressing such a question challenging. Therefore, we used a heterologous model in which we cultured mouse oocytes in human FF. To determine whether FF has dose-dependent effects on gamete quality, we performed in vitro maturation of denuded oocytes from reproductively young mice (6-12 weeks) in 10%, 50%, or 100% FF from participants of mid-reproductive age (32-36 years). FF impacted meiotic competence in a dose-dependent manner, with concentrations >10% inhibiting meiotic progression and resulting in spindle and chromosome alignment defects. We previously demonstrated that human FF acquires a fibro-inflammatory cytokine signature with age. Thus, to determine whether exposure to an aging FF microenvironment contributes to the age-dependent decrease in gamete quality, we matured denuded oocytes and cumulus-oocyte complexes (COCs) in FF from reproductively young (28-30 years) and old (40-42 years) participants. FF decreased meiotic progression of COCs, but not oocytes, from reproductively young and old (9-12 months) mice in an age-dependent manner. Moreover, FF had modest age-dependent impacts on mitochondrial aggregation in denuded oocytes and cumulus layer expansion dynamics in COCs, which may influence fertilization or early embryo development. Overall, these findings demonstrate that acute human FF exposure can impact select markers of mouse oocyte quality in both dose- and age-dependent manners.

FGFR2 fusion proteins drive oncogenic transformation of mouse liver organoids towards cholangiocarcinoma

About 15% of intrahepatic cholangiocarcinomas (iCCAs) express fibroblast growth factor receptor 2 (FGFR2) fusion proteins (FFs), usually alongside mutational inactivation of TP53, CDKN2A or BAP1. In FFs, FGFR2 residues 1-768 fuse to sequences encoded by a diverse array of partner genes (>60) causing oncogenic FF activation. While FGFR-specific tyrosine kinase inhibitors (F-TKI) provide clinical benefit in FF+ iCCA, responses are partial and/or limited by resistance mechanisms, such as the V565F substitution in the FGFR2 gatekeeper residue. Improving on FF targeting in iCCA therefore remains a critical unmet need. Herein, we aimed to generate a murine model of FF-driven iCCA and use this to uncover actionable FF-associated dependencies. Four iCCA FFs carrying different fusion sequences were expressed in Tp53-/- mouse liver organoids. Tumorigenic properties of genetically modified liver organoids were assessed by transplantation into immuno-deficient mice. Cellular models derived from neoplastic lesions were exploited for pre-clinical studies. Transplantation of FF-expressing liver organoids yielded tumors diagnosed as CCA based on histological, phenotypic and transcriptomic analyses. The penetrance of this tumorigenic phenotype was influenced by FF identity. Tumor organoids and 2D cell lines derived from CCA lesions were addicted to FF signaling via Ras-Erk, regardless of FF identity or V565F mutation. Dual blockade of FF and the Ras-Erk pathway by concomitant pharmacological inhibition of FFs and Mek1/2 provided greater therapeutic efficacy than single agent F-TKI invitro and invivo. FF-driven iCCA pathogenesis was successfully modeled on a Tp53-/- murine background, revealing biological heterogeneity among structurally different FFs. Double blockade of FF-ERK signaling deserves consideration for precision-based approaches against human FF+ iCCA. Intrahepatic cholangiocarcinoma (iCCA) is a rare cancer that is difficult to treat. A subtype of iCCA is caused by genomic alterations that generate oncogenic drivers known as FGFR2 fusions. Patients with FGFR2 fusions respond to FGFR inhibitors, but clinical responses are often of modest duration. We used animal and cellular models to show that FGFR2 fusions require the activity of a downstream effector named Mek1/2. We found that dual blockade of FGFR2 fusions and Mek1/2 was more effective than isolated inhibition of FGFR2 fusions, pointing to the potential clinical utility of dual FGFR2-MEK1/2 blockade in patients with iCCA.

Apolipoprotein B is regulated by gonadotropins and constitutes a predictive biomarker of IVF outcomes.

BackgroundFollicular fluid (FF) is an important micro-environment influencing oocyte growth, its development competence, and embryo viability. The FF content analysis allows to identify new relevant biomarkers, which could be predictive of in vitro fertilization (IVF) outcomes. Inside ovarian follicle, the amount of FF components from granulosa cells (GC) secretion, could be regulated by gonadotropins, which play a major role in follicle development.MethodsThis prospective study included 61 female undergoing IVF or Intra-cytoplasmic sperm injection (ICSI) procedure. Apolipoprotein B (APOB) concentrations in follicular fluid and APOB gene and protein expression in granulosa cells from reproductively aged women undergoing an in vitro fertilization program were measured. The statistical analyses were performed according to a quartile model based on the amount of APOB level found in FF.ResultsAmounts of APOB were detected in human FF samples (mean ± SD: 244.6 ± 185.9 ng/ml). The odds of obtaining an oocyte in the follicle and a fertilized oocyte increased significantly when APOB level in FF was higher than 112 ng/ml [i.e., including in Quartile Q 2, Q3 and Q4] (p = 0.001; p < 0.001, respectively). The probabilities of obtaining an embryo and a top quality embryo on day 2, were significantly higher if APOB levels were within the ranges of 112 and 330 ng/ml (i.e. in Q2 and Q3) or 112 and 230 ng/ml (i.e. in Q2), respectively (p < 0.001; p = 0.047, respectively). In addition, our experiments in vitro indicated that APOB gene and protein expression, along with APOB content into culture were significantly under-expressed in GC upon stimulation with gonadotropins (follicular stimulating hormone: FSH and/or human chorionic gonadotropin: hCG).ConclusionWe are reporting a positive and statistically significant associations between APOB and oocyte retrieval, oocyte fertilization, and embryo quality. Using an experimental study component, the authors report significant reduced APOB expression and content for luteinized granulosa cells cultured in the presence of gonadotropins.Electronic supplementary materialThe online version of this article (doi:10.1186/s12958-016-0150-4) contains supplementary material, which is available to authorized users.

Day 5 blastocyst has higher viability compared to day 6 blastocyst as vitrified: clinical outcome of 1074 cases

OBJECTIVE: The purpose of the study was to examine whether the viability of the blastocyst-stage embryos vitrified using EM-grid after artificial shrinkage on day 5 and day 6.DESIGN: Retrospective clinical study.MATERIALS AND METHODS: Approval for this study was obtained from the Institutional Review Board of the Maria Infertility Hospital. This retrospective study was performed for 1074 patients participated in thawing-ET program from January 2004 to October 2006. The embryos derived from conventional IVF or ICSI were cocultured with cumulus cells in a 20ul YS (Yoon Sanhyun) medium containing 20% hFF. After transferring the embryos into the uterus on day 3 or 5, the surplus embryos developed to the expanded blastocyst stage were vitrified on day 5 or 6 using EM-grid following artificial collapse of blastocoel. The solution for vitrification was composed of DPBS containing 20% (v/v) hFF, 40% (v/v) ethylene glycol, 18% (w/v) Ficoll, and 0.3 M sucrose. For thawing of vitrified blastocysts, a two-step cryoprotectant dilution method was used in which the EM grids stored in LN2 were directly transferred into a 500ul drop of 0.5 mol/L sucrose solution (prepared in DPBS containing 20% human FF) at room temperature as soon as possible and then were transferred in to 500ul drops containing 20% human FF in DPBS for 5minutes at room temperature. The embryos were washed three times in culture medium and co-cultured with cumulus cells in 20ul of YS medium containing 10% human FF. Thawed blastocyst were overnight cultured and transferred on the next day. The viability was assessed based on the survival, hatching embryo, implantation, and clinical pregnancy (x2 test).RESULTS: The survival rate of blastosysts that were thawed had no difference between day 5 (95.0%, 1324/1393) and day 6 (91.5% 1409/1539). However the clinical pregnancy rate (56.4%, 288/511) and implantation rate (30.8%, 404/1393) of day 5 blastocysts were significantly higher than those on day 6(41.0%, 231/563; 20.4%, 286/1539).CONCLUSIONS: The survival and hatching rates of the blastocysts vitrified on day 6 were similar to those on day 5. However the clinical pregnancy and implantation rates of day 6 blastocysts were significantly lower than those on day 5. These results suggest that the viability of day 5 blastocysts was superior to that of day 6 blastocysts. Therefore, further efforts to improve culture conditions are necessary so that a great number of embryos can normally develop to the expanded blastocyst stage on day 5. OBJECTIVE: The purpose of the study was to examine whether the viability of the blastocyst-stage embryos vitrified using EM-grid after artificial shrinkage on day 5 and day 6. DESIGN: Retrospective clinical study. MATERIALS AND METHODS: Approval for this study was obtained from the Institutional Review Board of the Maria Infertility Hospital. This retrospective study was performed for 1074 patients participated in thawing-ET program from January 2004 to October 2006. The embryos derived from conventional IVF or ICSI were cocultured with cumulus cells in a 20ul YS (Yoon Sanhyun) medium containing 20% hFF. After transferring the embryos into the uterus on day 3 or 5, the surplus embryos developed to the expanded blastocyst stage were vitrified on day 5 or 6 using EM-grid following artificial collapse of blastocoel. The solution for vitrification was composed of DPBS containing 20% (v/v) hFF, 40% (v/v) ethylene glycol, 18% (w/v) Ficoll, and 0.3 M sucrose. For thawing of vitrified blastocysts, a two-step cryoprotectant dilution method was used in which the EM grids stored in LN2 were directly transferred into a 500ul drop of 0.5 mol/L sucrose solution (prepared in DPBS containing 20% human FF) at room temperature as soon as possible and then were transferred in to 500ul drops containing 20% human FF in DPBS for 5minutes at room temperature. The embryos were washed three times in culture medium and co-cultured with cumulus cells in 20ul of YS medium containing 10% human FF. Thawed blastocyst were overnight cultured and transferred on the next day. The viability was assessed based on the survival, hatching embryo, implantation, and clinical pregnancy (x2 test). RESULTS: The survival rate of blastosysts that were thawed had no difference between day 5 (95.0%, 1324/1393) and day 6 (91.5% 1409/1539). However the clinical pregnancy rate (56.4%, 288/511) and implantation rate (30.8%, 404/1393) of day 5 blastocysts were significantly higher than those on day 6(41.0%, 231/563; 20.4%, 286/1539). CONCLUSIONS: The survival and hatching rates of the blastocysts vitrified on day 6 were similar to those on day 5. However the clinical pregnancy and implantation rates of day 6 blastocysts were significantly lower than those on day 5. These results suggest that the viability of day 5 blastocysts was superior to that of day 6 blastocysts. Therefore, further efforts to improve culture conditions are necessary so that a great number of embryos can normally develop to the expanded blastocyst stage on day 5.

The high throughput screening of neuropeptide FF2 receptor ligands from Korean herbal plant extracts

We have screened 356 libraries of Korean herbal plant extracts to find potential anti-obesity drugs. We employed the recently developed fluorescence polarization high throughput screening (FP HTS) assays of human neuropeptide FF (NPFF) receptors in 384-well microtiter plates. The primary hits were cherry-picked from the libraries and further analyzed by secondary displacement curve assays, in vitro GTPγS binding assays and cell-based CRE luciferase reporter assays. Agonists of NPFF receptors showed biphasic affinity curves while the antagonist, BIBP 3226, gave a monophasic affinity curve in competitive binding assays. We isolated and characterized two agonists of human NPFF2 receptor, PC 314 with K i of 1.42 μM, and PC 315 with K i of 2.17 μM from Schizandra chinensis. PC 314 and PC 315 have been characterized as benzoylgomisin Q (M.W. 552) and gomisin G (M.W. 536). We report that PC 314 and PC 315 are the first non-peptide, natural compounds, which bind to human NPFF2 receptors with good affinity. PC 314 and PC 315 inhibit forskolin-stimulated luciferase expression when CHO cells are co-transfected with NPFF2 receptor and CRE reporter vector. They possess the pharmacological and functional profiles of full agonists. The FP HTS system provides a specific, sensitive and reproducible methodology for studying and screening NPFF receptor ligands.

Functional properties of PFR(Tic)amide and BIBP3226 at human neuropeptide FF2 receptors

The functional characteristics of two putative neuropeptide FF (NPFF) antagonists, BIBP3226 and PFR(Tic)amide, on the human neuropeptide FF receptor subtype 2 (hNPFF2) were investigated. Surprisingly, PFR(Tic)amide was shown to exhibit agonist properties in the [ 35 S ]guanosine-5′- O-(3-thio)triphosphate ([ 35 S ]GTPγS) binding assay. The efficacy of PFR(Tic)amide was significantly greater than that of (1DMe)Y8Fa, a stable analog of NPFF, and PFR(Tic)amide can therefore be classified as a ‘super-agonist’. BIBP3226 did act as a reversible competitive antagonist on the hNPFF2 receptor. However, high concentrations of BIBP3226 also non-specifically increased [ 35 S ]GTPγS binding. The usefulness of BIBP3226 as an antagonist tool on the NPFF receptor is thus limited.

Calcium requirements for human sperm function in vitro

Objective To determine extracellular calcium (Ca 2+) requirements for the maintenance of human sperm function in vitro. Design Prospective study. Setting Basic research laboratory. Patient(s) Normozoospermic volunteers provided fresh semen samples; follicular fluid (human FF) and oocytes were collected from women undergoing IVF-ET. Intervention(s) Spermatozoa were incubated for ≤18 hours in media containing different CaCl 2 concentrations (maximum, 2.5 mM [control]). Main outcome measure(s) Protein tyrosine phosphorylation patterns, development of hyperactivated motility, induction of the acrosome reaction (AR) in response to human FF, and sperm interaction with homologous zona pellucida (ZP). Result(s) Cells maintained for 18 hours in medium containing ≥0.1 mM of Ca 2+ were able to undergo the AR when exposed to human FF in the presence of 2.5 mM of Ca 2+. Calcium concentrations of ≥0.22 mM were sufficient to reach protein tyrosine phosphorylation levels and hyperactivated motility values similar to those of controls. Higher Ca 2+ concentrations (≥0.58 mM) were required to produce maximum human FF–induced AR in previously capacitated cells and to obtain an adequate sperm–ZP binding. Conclusion(s) Different steps of the fertilization process have distinctive Ca 2+ requirements. Whereas 0.22 mM of Ca 2+ is sufficient for the development of some capacitation-related events, human FF–induced AR and sperm–ZP interaction require 0.58 mM of this cation.

Prolactin releasing peptide has high affinity and efficacy at neuropeptide FF2 receptors.

Neuropeptide FF (NPFF) and prolactin-releasing peptide (PrRP) are two members of the RFamide peptide family. In this study we investigated whether these RFamide peptides, which have common structural features in their C-terminal RFamide motif and share several physiologically important functions, could exert their effects through the same set of receptors. The affinity and functional activity of several related RFamide peptides were determined at the human neuropeptide FF receptor subtype 2 (hNPFF2) and the human prolactin-releasing peptide (hPrRP) receptors. The full-length human prolactin releasing peptide 31 (hPrRP31) had significantly higher efficacy compared with NPFF and its stable analog, (1DMe)Y8Fa, at the hNPFF2 receptor. In contrast, NPFF and (1DMe)Y8Fa were not efficacious at the hPrRP receptor. Our study indicated a generally relatively low level of discrimination for RFamide peptides at the NPFF receptor, whereas the hPrRP receptor clearly preferred PrRP or very closely related peptides. The seemingly promiscuous binding of the RFamide peptides to the NPFF receptor was further confirmed by receptor autoradiography. PrRP may thus signal through the NPFF receptors in vivo.

Analysis of human neuropeptide FF gene expression.

As an initial step to study the function of the gene encoding the human neuropeptide FF (NPFF), we cloned a 4.7-kb sequence from the promoter region. Primer extension and 5'-rapid amplification of cDNA ends revealed multiple transcription initiation sites. Northern blot analysis of the mRNA expression revealed a specific signal only in poly(A) + RNA from medulla and spinal cord. Chimeric luciferase reporter gene constructs were transiently transfected in A549, U-251 MG, SK-N-SH, SK-N-AS and PC12 cells. The promoter activity was directly comparable with the level of endogenous NPFF mRNA as determined by real-time quantitative RT-PCR. The highest promoter activity was measured when a region from - 552 to - 830 bp of the 5'-flanking region was fused to the constructs, and a potential silencer element was localized between nucleotides -220 and -551. A twofold increase in NPFF mRNA was observed after 72 h of nerve growth factor stimulation of PC12 cells and the region between - 61 and - 214 bp of the 5'-flanking region was found to be responsive to this stimulation. We postulate that control of human NPFF gene expression is the result of both positive and negative regulatory elements and the use of multiple transcription initiation sites.

Antisense oligonucleotides to human SQA-neuropeptide FF decrease morphine tolerance and dependence in mice

Neuropeptide FF (Phe–Leu–Phe–Gln–Pro–Gln–Arg–Phe–NH2) is able to modulate opioid analgesia. Intracerebroventricular treatment for 5 days with antisense-oligodeoxynucleotides complementary to the sequence of human SQA-neuropeptide FF (Ser–Gln–Ala–Phe–Leu–Phe–Gln–Pro–Gln–Arg–Phe–NH2) precursor gene or by mismatch-oligodeoxynucleotides did not change the antinociceptive activity of morphine in the mouse tail flick test. In contrast, antisense- but not mismatch-oligodeoxynucleotides attenuated significantly the tolerance to the analgesic activity of morphine and the withdrawal syndrome precipitated by naloxone in morphine-treated mice. These treatments with oligodeoxynucleotides did not modify neuropeptide FF-immunoreactivity content in whole brain but repeated injections of an agonist of neuropeptide FF receptors increased the intensity of morphine tolerance. These results demonstrate the important role of neuropeptide FF in opioid pharmacodependence.

Biochemical, cellular and pharmacological activities of a human neuropeptide FF-related peptide

We report on the biochemical, cellular and pharmacological activities of SQA-neuropeptide FF (Ser-Gln-Ala-Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe–NH 2), a peptide sequence contained in the human neuropeptide FF (neuropeptide FF, Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe–NH 2) precursor. Quantitative autoradiography revealed that, in the superficial layers of the rat spinal cord, SQA-neuropeptide FF displayed the same high affinity for [ 125 I ]1DMe ([ 125 I ] d-Tyr-Leu-(NMe)Phe-Gln-Pro-Gln-Arg-Phe–NH 2) binding sites ( K i=0.33 nM) as did neuropeptide FF ( K i=0.38 nM). In acutely dissociated mouse dorsal root ganglion neurones, SQA-neuropeptide FF reduced by 40% the depolarisation-induced rise in intracellular Ca 2+ as measured with the Ca 2+ indicator, Fluo-3. In mice, 1DMe and SQA-neuropeptide FF dose-dependently inhibited the antinociceptive effect of intracerebroventricular (i.c.v.) injections of morphine, but SQA-neuropeptide FF was less potent than 1DMe. Furthermore, SQA-neuropeptide FF, as well as 1DMe, produced marked hypothermia following third ventricle injections in mice. These data demonstrate that the human peptide, SQA-neuropeptide FF, exhibits biochemical and pharmacological properties similar to those of neuropeptide FF or neuropeptide FF analogues, and belongs to the neuropeptide FF family.

Immunoassays for inhibin and its subunits Further applications of the synthetic peptide approach

We describe the preparation of a new rat monoclonal antibody (CRC1) to the N-terminal sequence of the 43 kDa subunit of human ovarian inhibin, and its use together with other anti-peptide monoclonal antibodies, in two-site immunoassays for the detection of inhibin-related material in biological fluids. The Fab fraction of a mouse monoclonal antibody (R1) to the N-terminal portion of the 20 kDa α subunit, coupled to alkaline phosphate, was used for detection, and either CRC1 or a monoclonal antibody (E4) to the β-A subunit were used as capture antibodies. The E4/R1 combination, expected to measure dimeric bioactive inhibin, could detect less than 2 pg/ml of recombinant inhibin in diluent, gave good recovery of activity spiked into human blood, and could measure significant levels of immunoreactivity in sera from women undergoing ovulation induction, and in some normal women. Sera from post-menopausal women contained undetectable levels. Apparent inhibin levels in human follicular fluid were increased six-fold by pretreatment with 8 M urea, suggesting masking of epitopes in this fluid. Activin cross-reactivity in the assay was 0.05%. The R1/CRC1 assay, expected to measure only large molecular weight forms of inhibin or its α subunit, could detect immunoreactivity in human FF diluted 50,000-fold, and in all sera tested, although the levels in the hyperovulated women were higher. By contrast to the E4/R1 assay much of the immunoreactivity was labile during the clotting process, or subsequent assay, and reliable measurements on blood with this assay will require special sample collection procedures. These results demonstrate the value of anti-peptide monoclonal antibodies in the study of inhibin, and the results obtained with CRC1 show that antibodies useful for immunoassays can sometimes be obtained without the purified target molecule being available for immunization or screening.

CD8+ lymphocyte subsets in human follicular fluid

In conclusion, we have shown that cells of the CD8 + CD11b + phenotype, characteristic of antigen-unspecific cytotoxic and suppressor cells, are the principal CD8 + subset detected in human FF

Precursors of a-Inhibin Modulate Follicle-Stimulating Hormone Receptor Binding and Biological Activity*

Although several forms of monomeric alpha-inhibin have been isolated from follicular fluid, no biological function has yet been ascribed to these posttranslationally processed forms of the alpha-subunit precursor protein. Moreover, previous studies of a FSH receptor binding competitor (FRBC) isolated and characterized from porcine follicular fluid (pFF) suggested certain biochemical similarities between this protein and alpha-inhibin precursors. We, therefore, investigated the hypothesis that alpha-inhibin and/or its precursors might represent autocrine and/or paracrine modulators of FSH action in the ovary, accounting for some of this FRBC activity and thereby exerting some degree of regulation over follicular maturation. Three separate sources of alpha-inhibin proteins were investigated for FRBC activity, including pFF, human FF (hFF), and a 293 cell line into which the full-length human alpha-inhibin cDNA had been stably transfected. Conditioned medium from these transfected cells contained several forms of alpha-inhibin precursors as well as mature alpha-inhibin, but no beta-subunit or intact inhibin. alpha-Inhibin proteins from all three sources, purified by a variety of methods, including immunoaffinity chromatography on an anti-alpha-inhibin column, inhibited FSH binding to both natural tissue FSH receptors as well as recombinant rat FSH receptors expressed in 293 cells. Furthermore, dimeric inhibin and activin, medium from untransfected 293 cells, and non-alpha-inhibin-containing purification fractions were inactive in either assay. In addition, purified recombinant alpha-inhibin proteins were partial in vitro FSH antagonists in a bioassay in which cAMP generation from 293 cells expressing the recombinant FSH receptor is used as an index of FSH biological activity. These same fractions of hFF containing FRBC activity did not bind to LH receptors, thereby demonstrating receptor specificity for this activity. Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting with alpha-inhibin or FRBC antisera, a 57,000 mol wt protein was identified in FRBC-active fractions from all three sources, suggesting that the active moiety was the full-length alpha-inhibin precursor protein or a large mol wt fragment, but not mature alpha-inhibin. Lastly, all FRBC activity from all three sources was extracted by an alpha-inhibin immunoaffinity column and was recoverable upon elution. These results demonstrate that proteins derived from the alpha-inhibin precursor modulate FSH binding to its receptor as well as its biological activity.(ABSTRACT TRUNCATED AT 400 WORDS)

Platelet-derived growth factor is detected in human blastocyst culture medium but not in human follicular fluid—a preliminary report

The content of PDGF in human blastocyst culture medium (n = 8), serum (n = 12), and FF (n = 17) from natural IVF cycles was determined by an RIA specific for PDGF B-chain. The blastocysts were cultured under serum-free conditions throughout development. The findings show that PDGF B-chain is released into the culture medium of human blastocysts and that serum is positive, whereas FF is negative for PDGF.

DEVELOPMENT OF A TWO-SITE IMMUNORADIOMETRIC ASSAY FOR DIMERIC INHIBIN USING ANTIBODIES AGAINST CHEMICALLY SYNTHESIZED FRAGMENTS OF THE α AND β SUBUNIT

A two-site (liquid-phase) immunoradiometric assay (IRMA) for dimeric inhibin has been developed using antibodies raised against synthetic peptide sequences corresponding to the N-terminus (1-32) of the alpha subunit and the C-terminal region (82-114) of the beta A subunit of Mr approximately 30,000 human inhibin. Highly-purified Mr 32,000 bovine inhibin (standard) gave a dilution curve parallel to those for bovine follicular fluid (bFF), human (h)FF and rat ovary extract. Whilst the assay detected both Mr 56,000 and 32,000 inhibin forms in bFF, little reaction with higher Mr forms was evident. Cross-reaction of 'free' inhibin subunit (Mr 25,000 form) and recombinant human activin A in the IRMA were minimal (less than 0.1 and less than 2% respectively). Although the detection limit of the IRMA (approximately 50 pg/tube) was similar to that of several reported radioimmunoassays (RIA), the IRMA was unable to detect dimeric inhibin in jugular or utero-ovarian vein plasma of heifers. Similarly, when assayed by IRMA, bFF, hFF and rat ovary extract contained 8-58 times less inhibin than was indicated by RIA. These observations are consistent with earlier evidence that the ovary secretes a substantial excess of 'free' inhibin alpha subunit and that this material reaches the peripheral circulation. Surprisingly, however, the inhibin contents of bFF, hFF and rat ovary extract determined by in vitro bioassay were 8-23 times greater than the corresponding IRMA values, being similar to those derived by RIA.(ABSTRACT TRUNCATED AT 250 WORDS)

Steroidogenesis-Stimulating Activity in the Gonads: Comparison of Rat Testicular Fluid with Bovine and Human Ovarian Follicular Fluids1

The steroidogenesis-stimulating activities of ovarian follicular fluid from bovine (bFF) and human (hFF) sources, were compared with those of adult rat testicular fluid (rTF) using an in vitro bioassay system based on stimulation of testosterone production by purified adult rat Leydig cells during a 20-h incubation. Rat TF and bFF were charcoal-treated to remove steroids prior to assay, and the major active fraction of hFF was collected after gel-permeation chromatography. All three fluid samples stimulated both basal and maximal hCG-stimulated testosterone production, although the resulting log dose-response lines of bFF and the hFF extracts were not parallel with those of rTF. Both rTF and bFF were active over a similar dose range (5.2-150 microliters and 9.7-150 microliters, respectively) and both had a more than additive interaction with hCG on testosterone production. The stimulatory activity of the hFF extract was considerably greater than that of either rTF or bFF in the absence of hCG, but hFF extract had only an additive effect with hCG in stimulating testosterone production. Moreover, unlike rTF activity, which was inhibited by co-incubation with the protein synthesis inhibitor, cycloheximide, the activity of the hFF extract was not affected by cycloheximide. The factors responsible for activity in all three fluids were of a large molecular size (greater than 30 kDa), as determined by ultrafiltration or gel-permeation chromatography. However, in contrast to both rTF and bFF, hFF extract activity was removed by charcoal extraction. Human FF extract was inactivated by heat (100 degrees C, 30 min), whereas rTF activity was partially (70%) heat-labile and bFF was not affected by heart.(ABSTRACT TRUNCATED AT 250 WORDS)

Human Follicular Fluid Contains a Follicle-Stimulating Hormone (FSH) Receptor Binding Inhibitor Which Has FSH Agonist Activity, is Immunologically Similar to FSH, But Can Be Distinguished from FSH*

We have previously reported the presence in human follicular fluid (hFF) of a high (greater than 5000) mol wt FSH receptor binding inhibitor (FSH-BI). This hFF FSH-BI was further purified by removal of material insoluble in acidified acetone (pH 4.1) but soluble in diethyl ether (pH 10.5), followed by molecular sieving through Sephacryl S-100. FSH-BI activity eluted from S-100 with an elution volume similar to that of hFSH, but could be distinguished from hFSH on the basis of a differential sensitivity to acid inactivation. Human FSH was inactivated in acetone at pH 4.1 (1 h, 25 C), whereas hFF FSH-BI retained activity under these conditions. Human FF FSH-BI also demonstrated FSH-like agonist activity, defined as the ability to stimulate basal levels of estradiol synthesis in cultured rat Sertoli cells. Human FSH-BI strongly cross-reacted to a commercially available monoclonal antibody used to measure serum hFSH. Indeed, recovery of FSH immunologic activity was significantly greater (134-fold on a mass basis) after partial purification, indicating that antibody recognition sites were apparently masked in unfractionated hFF. In summary, large mol wt hFSH-BI has been partially purified from hFF and found to be similar in size to pituitary hFSH and to have FSH-like agonist activity in vitro. Although distinguishable from pituitary hFSH on the basis of stability to acid, hFSH-BI appears immunologically related to pituitary hFSH so that measurements of hFSH levels in hFF using immunologic techniques should be interpreted with caution.

Antisera to porcine follicular fluid in monkeys: neutralization of human and pig inhibin activity in vivo and in vitro.

Repetitious administration of charcoal-stripped porcine follicular fluid (pFF) to four intact cycling females and six long term castrate rhesus monkeys was associated with a gradual onset of resistence to the FSH-suppressing effects of pFF. After a treatment hiatus of 6 or more months, the pFFinduced selective suppression of FSH in circulation was again demonstrable, but only transiently; that is, within 10–14 weeks after the resumption of therapy, even large doses of pFF were no longer effective in reducing serum FSH in these monkeys. One monkey was studied through seven regimens over 4 yr. Accordingly, our series of investigations was divided into three parts. First, we observed that these changing FSH patterns were apparently due to cyclic fluctuations of pFF antibodies in conjunction with the intermittent schedule of pFF administration. The amnestic response to booster injections occurred within 7–14 days, followed by a gradual decline in the anti-pFF titer during succeeding months. Second, we tested whether these anti-pFF sera would neutralize inhibin activities (selective FSH suppression) in rat pituitary cell cultures. Indeed, the anti-pFF sera were effective in blocking the FSH-suppressing activity of pFF. Again, each successive episode of pFF administration was correlated with an enhancement of the anti-pFF titer, indicated by greater potency of the monkey antisera in the in vitro setting. Thirdly, we assessed whether the monkey's anti-pFF sera would neutralize the inhibin activity in human FF (hFF); thereby establishing cross-reactivity with human inhibin. Clearly, the mixture of anti-pFF serum along with charcoal-stripped hFF prevented the selective suppression of FSH secretion that occurred when control monkey serum was used instead. These findings indicate that porcine inhibin present in charcoalstripped pFF is highly immunogenic in monkeys; further, some of the antibodies generated cross-react with the active inhibin component in hFF. Indeed, the neutralization of inhibin activity in hFF by anti-pFF monkey sera suggests some significant structural similarities between human and pig inhibins; yet, concurrently, the ready immunogenicity of pFF administered to monkeys indicates clear recognition of the antigen as an intrinsically foreign protein. These anti-pFF monkey sera may be useful in the establishment of RIA for porcine and/or human inhibins as well as the preparation of improved reagents for studying the physiological significance of this substance (s) in follicular fluid and circulation.