Steroidogenic and maturation-inducing potency of native gonadotropic hormones in female chub mackerel, Scomber japonicus.

BACKGROUND: The gonadotropins (GtHs), follicle-stimulating hormone (FSH) and luteinizing hormone (LH) are produced in the pituitary gland and regulates gametogenesis through production of gonadal steroids. However, respective roles of two GtHs in the teleosts are still incompletely characterized due to technical difficulties in the purification of native GtHs. METHODS: Native FSH and LH were purified from the pituitaries of adult chub mackerel, Scomber japonicus by anion-exchange chromatography and immunoblotting using specific antisera. The steroidogenic potency of the intact chub mackerel FSH (cmFSH) and LH (cmLH) were evaluated in mid- and late-vitellogenic stage follicles by measuring the level of gonadal steroids, estradiol-17beta (Ε2) and 17,20beta-dihydroxy-4-pregnen-3-one (17,20beta-P). In addition, we evaluated the maturation-inducing potency of the GtHs on same stage follicles. RESULTS: Both cmFSH and cmLH significantly stimulated E2 production in mid-vitellogenic stage follicles. In contrast, only LH significantly stimulated the production of 17,20beta-P in late-vitellogenic stage follicles. Similarly, cmLH induced final oocyte maturation (FOM) in late-vitellogenic stage follicles. CONCLUSIONS: Present results indicate that both FSH and LH may regulate vitellogenic processes, whereas only LH initiates FOM in chub mackerel.

Hormone storage granules in the beef anterior pituitary. I. Isolation, ultrastructure, and some biochemical properties.

A method is described for isolation of relatively large quantities of large and small hormone storage granules from the beef adenohypophysis. The hormone storage granules are highly purified, as indicated by ultrastructural and biochemical criteria. The average size of large granules is 400 mmicro and of small granules is 220 mmicro. The large granules contain growth hormone and prolactin; the small granules contain high concentrations of follicle-stimulating, luteinizing, and thyroid-stimulating hormones. An alkaline protease with a pH optimum of 8.3 is associated with the small granule fraction.

Impact of pituitary FSH purification on in vitro early folliculogenesis in goats.

Porcine pituitary follicle stimulating hormone (pFSH) is known to regulate the production of growth factors that have an essential role in early foliculogenesis. However, the effects of different preparations of pFSH on the survival and development of caprine follicles are not yet known. The aim of this study was to evaluate the effects of different pFSH (Stimufol and Folltropin) on the in vitro survival and growth of caprine preantral follicles. Pieces of caprine ovarian tissues were cultured for either one or seven days in a supplemented Minimum Essential Medium, alone or containing either Stimufol (50 ng/mL) or Folltropin (10, 50, 100 and 1000 ng/mL). Fresh control ovarian tissues as well as cultured tissued were processed for histological and ultrastructural studies. The results showed that after seven days, only Stimufol maintained follicular morphology similar to control. Moreover, follicular degeneration was higher in medium alone or with Folltropin at 50, 100 and 1000 ng/mL. However, at day seven, the percentage of growing follicles was higher in 100 ng/mL of Folltropin than Stimufol. In conclusion, FSH preparations affect differently the performance of in vitro culture of caprine preantral follicles. Stimufol was better to preserve follicular morphology while Folltropin was more efficient to promote follicular growth.

Purification and characterization of follicle-stimulating hormone from pituitary glands of sea bass (Dicentrarchus labrax).

Follicle-stimulating hormone (FSH) was purified from pituitaries of sea bass (Dicentrarchus labrax), and its biochemical and biological properties were studied. Sea bass FSH (sbsFSH) was purified by ethanol extraction-precipitation (40-85%), followed by anion-exchange chromatography on a LKB Ultropac TSK-DEAE column using a linear gradient of ammonium bicarbonate (50-1000 mM) and reverse phase chromatography on a RESOURCE 15RPC column with a linear gradient of acetonitrile (0-50%), using a FPLC system. The molecular mass of the purified sbsFSH, estimated by mass spectrometry, was of 28.5 kDa for the dimer, 12.6 kDa for the glycoprotein alpha (GPalpha) and 13.6 kDa for FSHbeta subunits. After separation by SDS-PAGE under reducing condition, the intact sbsFSH was dissociated in the respective subunits (GPalpha and FSHbeta). Subunit identity was confirmed by immunological detection and N-terminal amino acid sequencing. Deglycosylation treatment with N-glycosidase F, decreased the molecular mass of both subunits. Intact sbsFSH activated the sea bass FSH receptor stably expressed in the cell line HEK 293, in a dose dependent manner. Purified sbsFSH showed gonadotropic activity, by stimulating the release of estradiol-17beta (E2) from sea bass ovary and testosterone (T) and 11-ketotestosterone (11KT) from testicular tissue cultured in vitro, in a dose and time dependent manner. These results showed that the purified sbsFSH is a heterodimeric hormone, composed of two distinct glycoprotein subunits (GPalpha and FSHbeta), and has biological activity judged by its ability to stimulate its receptor in a specific manner and to promote steroid release from gonadal tissue fragments.

Expression of recombinant human follicle-stimulating hormone in the mammary gland of transgenic mice.

In this article, we describe the use of a caprine beta-lactoglobulin (betaLG) expression cassette previously obtained by our group to target the expression of the human follicle-stimulating hormone (hFSH) to the mammary gland of transgenic mice. The hFSH is a pituitary glycoprotein composed of two subunits (alpha and beta). At present, this protein is obtained from mammalian cellular fermentors, and it is extensively used in the treatment of human infertility. Four lines of double (hFSHalpha/beta) transgenic mice that stably transmitted the transgenes were obtained, and hFSHalpha and hFSHbeta mRNA was detected by reverse transcriptase-polymerase chain reaction in the mammary gland of lactating females from all four transgenic lines. The hFSH protein was present in the mammary gland of the lactating females, but could not be detected in the milk by Western blot, probably as a result of low levels of transgene expression.

Fractionation of follicle stimulating hormone charge isoforms in their native form by preparative electrophoresis technology.

Complex glycoprotein biopharmaceuticals, such as follicle stimulating hormone (FSH), erythropoietin and tissue plasminogen activator consist of a range of charge isoforms due to the extent of sialic acid capping of the glycoprotein glycans. Sialic acid occupies the terminal position on the oligosaccharide chain, masking the penultimate sugar residue, galactose from recognition and uptake by the hepatocyte asialoglycoprotein receptor. It is therefore well established that the more acidic charge isoforms of glycoprotein biopharmaceuticals have higher in vivo potencies than those of less acidic isoforms due to their longer serum half-life. Current strategies for manipulating glycoprotein charge isoform profile involve cell engineering or altering bioprocesss parameters to optimise expression of more acidic or basic isoforms, rather than downstream separation of isoforms. A method for the purification of a discrete range of bioactive recombinant human FSH (rhFSH) charge isoforms based on Gradiflowtrade mark preparative electrophoresis technology is described. Gradiflowtrade mark electrophoresis is scaleable, and incorporation into glycoprotein biopharmaceutical production bioprocesses as a potential final step facilitates the production of biopharmaceutical preparations of improved in vivo potency.

Sepharose-linked concanavalin A in the purification and characterization of glycoprotein hormones of the bovine pituitary.

Affinity chromatography on concanavalin A-Sepharose is a time saving step in both large and small scale isolations of the bovine pituitary glycoprotein hormones. After ion-exchange chromatography, the final yield of purified lutropin is 40-50% of material in starting concentrates and of purified thyrotropin is approximately 20%. The final products have the same electrophoretic and immunological properties and amino acid compositions as previous preparations. Less than 3% of the immunoreactive lutropin, follitropin and thyrotropin are present as non-glycosylated forms in either crude pituitary extracts or concentrates. Thyrotropin and follitropin elute from the immobilized lectin as a single fraction, whereas lutropin separates into two glycosylated fractions. Gel filtration of both crude extracts and the glycoprotein fractions shows that less than 5% of the immunoreactivity of the hormones is present as material of apparently high molecular weight. Substantial alpha subunit immunoreactivity, however, is in three fractions (as found by others in human pituitary extracts) corresponding to "high molecular weight material" (7%), intact hormones (46%) and free subunit (47%).

Comparative analytical characterization of two commercial human follicle-stimulating hormones extracted from human urine.

OBJECTIVE: Pharmaceutical preparations of human menopausal gonadotrophin (hMG), urine-derived follicle-stimulating hormone (u-FSH) and highly purified u-FSH (u-FSH-HP) have been available since the early 1960s and the mid 1980s and 1990s, respectively. Another commercial preparation of u-FSH-HP, Folyrmon P, was launched in Japan in 1999. The aim of this study is to assess the purity of Folyrmon P and to compare it with Fertinorm-P, another commercial preparation of u-FSH-HP that has been available since 1993. METHODS: Folyrmon P and Fertinorm-P were assessed for total protein content, biological activity, immunological activity, specific activity, purity and levels of protein contamination. RESULTS: Folyrmon P, which is extracted from the urine of post-menopausal women, has a specific activity of between 4000 and 5000 IU/mg, while Fertinorm-P, which is also manufactured from the urine of post-menopausal women, has a specific activity of at least 10,000 IU/mg. It has been well documented that commercially available hMG and u-FSH preparations can contain a number of urine-derived protein contaminants. This also proves to be the case for Folyrmon P, in which contaminant proteins other than FSH were shown to be present. It was also demonstrated that both preparations, Folyrmon P and Fertinorm-P, contained high levels of oxidized FSH. CONCLUSIONS: The low specific activity and high level of contaminants in Folyrmon P indicate that this u-FSH is not highly purified. Overall, Fertinorm-P, with higher specific activity and lower levels of contaminant proteins, appears to be of higher quality compared with Folyrmon P.

Production, purification, and characterization of recombinant hFSH glycoforms for functional studies.

Previously, our laboratory demonstrated the existence of a ß-subunit glycosylation-deficient human FSH glycoform, hFSH(21). A third variant, hFSH(18), has recently been detected in FSH glycoforms isolated from purified pituitary hLH preparations. Human FSH(21) abundance in individual female pituitaries progressively decreased with increasing age. Hypo-glycosylated glycoform preparations are significantly more active than fully-glycosylated hFSH preparations. The purpose of this study was to produce, purify and chemically characterize both glycoform variants expressed by a mammalian cell line. Recombinant hFSH was expressed in a stable GH3 cell line and isolated from serum-free cell culture medium by sequential, hydrophobic and immunoaffinity chromatography. FSH glycoform fractions were separated by Superdex 75 gel-filtration. Western blot analysis revealed the presence of both hFSH(18) and hFSH(21) glycoforms in the low molecular weight fraction, however, their electrophoretic mobilities differed from those associated with the corresponding pituitary hFSH variants. Edman degradation of FSH(21/18)-derived ß-subunit before and after peptide-N-glycanase F digestion confirmed that it possessed a mixture of both mono-glycosylated FSHß subunits, as both Asn(7) and Asn(24) were partially glycosylated. FSH receptor-binding assays confirmed our previous observations that hFSH(21/18) exhibits greater receptor-binding affinity and occupies more FSH binding sites when compared to fully-glycosylated hFSH(24). Thus, the age-related reduction in hypo-glycosylated hFSH significantly reduces circulating levels of FSH biological activity that may further compromise reproductive function. Taken together, the ability to express and isolate recombinant hFSH glycoforms opens the way to study functional differences between them both in vivo and in vitro.

Electrophoretic purification of radioiodinated follicle-stimulating hormone for radioligand receptor assay and radioimmunoassay.

A method is described for electrophoretic purification of [125I]human (h) FSH after radioiodination that improves radioligand binding to FSH membrane receptors. Lactoperoxidase-iodinated hFSH was separated from reaction products by electrophoresis on 7.5% polyacrylamide tube gels (PAGE). Material eluted from 3-mm gel slices was analyzed for incorporation of 125I and binding to antibody (RIA) or receptor (RRA), and by sodium dodecyl sulfate-PAGE for protein composition. Sodium dodecyl sulfate-PAGE analysis of individual PAGE fractions demonstrated that iodinated proteins, both higher and lower in apparent mol wt than intact FSH, were separated by PAGE, but not by gel filtration chromatography (Sephadex G-25). PAGE purification of radioligand resulted in significantly greater (compared to gel filtration) RRA sensitivity and specificity. Maximum binding of PAGE-purified [125I]hFSH to excess calf tests membrane receptors was 45%, with a specific activity of approximately 26 microCi/micrograms, as determined by the method of self-displacement. Maximum binding to excess hFSH antisera (NIH anti-hFSH 4) was 80-85%. This allowed a useful final dilution of 1:120,000, thereby facilitating development of a sensitive and specific RIA with this antiserum. These data indicate that PAGE separation of intact [125I]hFSH from other iodinated proteins results in improved radioligand binding, assay sensitivity, and assay specificity. In addition, PAGE-purified lactoperoxidase-iodinated hFSH is suitable for use in both RIA and RRA.

Follicle-stimulating hormone (FSH) immunoactivity in porcine follicular fluid is not pituitary FSH.

Two inhibitors of FSH binding to receptor have been isolated from porcine follicular fluid and shown to have in vitro biological activity. These inhibitors were distinct separable entities with opposite biological effects (agonist and antagonist) on cultured FSH-responsive Sertoli cells. In light of the fact that the agonist-containing fraction (P4) inhibited [125I]human (h) FSH binding to anti-hFSH antiserum as well as to receptor, characterization of this factor was undertaken to determine its relationship to pituitary FSH. The P4 fraction was further purified by affinity chromatography, which removed a major protein from immunoreactive components. Western blotting of sodium dodecyl sulfate-polyacrylamide gels using polyclonal (anti-hFSH) and monoclonal (anti-hFSH beta) antibodies revealed a major immunoreactive band at 55,000 mol wt (Mr). When electrophoresed under reducing conditions, major immunoreactive proteins at 58,000 and 45,000 Mr were identified. These bands were also observed in extracts from bovine testes and raw porcine follicular fluid after electrophoresis and Western blotting. Whereas the monoclonal antibody used to characterize this inhibitor does not recognize porcine pituitary FSH, the Mr of the immunoreactive proteins are greater than that of pituitary FSH, and the immunoreactive bands do not reduce to subunits, as observed for pituitary FSH under reducing conditions, we conclude that gonadal extracts contain FSH-immunoreactive proteins that are immunologically and biochemically distinguishable from pituitary FSH. While the physiological role of these proteins remains to be determined, their presence in gonadal extracts or fluids vitiates assessment of FSH within the gonad by RIA using antiserum against hFSH.

Studies on the isolation and chemical-physical properties of porcine follicle-stimulating hormone.

Methods are described for isolating highly purified FSH from porcine pituitary glands. The biological activity of pure material was 22 NIH-FSH-P2 U/mg, as judged by the ovarian weight augmentation bioassay. Virtually no immunoreactive LH was indicated by RIA, and only one component was detected by both gel electrophoresis and immunoprecipitation. Amino acid and carbohydrate analyses of the highly purified FSH indicated the presence of 1-tryptophan, a high content of aspartic and glutamic acids, and 6% sialic acid. Molecular weights of untreated and guanidine-treated porcine FSH were estimated from hydrodynamic measurements of Stokes radii and sedimentation coefficients. A highly specific and sensitive RIA was also developed for this hormone.

Rapid and easy separation of the subunits of bovine and human glycoprotein hormones by use of high performance liquid chromatography.

A single method of reverse phase high performance liquid chromatography is used to separate the subunits of human and bovine glycoprotein hormones. This rapid and easy method is applicable for the separation and detection of subunits from as little as 10 micrograms hormone or the isolation of subunits from as much as 100 mg hormone. Separation is achieved by chromatography on a Vydac 218TP1010 column with a linear (60-min) gradient of 0.1 M sodium phosphate, pH 6.8, plus 1 mM sodium azide to a solvent containing 50% acetonitrile and 50% 0.1 M sodium phosphate, pH 6.8, plus 1 mM sodium azide. Although in some cases the interaction between the hydrophobic support and the hormone is sufficient for dissociation, preincubation of the hormone with guanidine hydrochloride ensures optimum dissociation and improves resolution of the subunits. The subunits isolated by high performance liquid chromatography are functional in that they will reassociate with their counterpart subunits.

Isolation and characterization of follicle-stimulating hormone and luteinizing hormone and its subunits from snapping turtle (Chelydra serpentina) pituitaries.

Highly purified luteinizing hormone and follicle-stimulating hormone have been isolated from extracts of snapping turtle (Chelydra serpentina) pituitaries. Both hormones are potent in non-mammalian gonadotropin bioassays (1.8 X NIH-LH-S1 and 30 X NIH-FSH-S1). The materials have been characterized by polyacrylamide gel electrophoresis, amino terminal group analysis, amino acid and carbohydrate content, and, in the case of turtle luteinizing hormone, ultracentrifugation. The luteinizing hormone was shown to dissociate and subunits were prepared by the countercurrent distribution technique and characterized. Biological activity of the hormone could be regenerated by recombination of the subunits. In addition, it was shown that the snapping turtle luteinizing hormone subunits could be combined with subunits from ovine luteinizing hormone with generation of significant biological activity. Comparisons in properties of the turtle gonadotropins have been made with ovine gonadotropins, showing, in many cases, similarities in properties, suggesting structural features which have been conserved during evolution.

Effect of neuraminidase treatment on the biological activity of highly purified ovine FSH and LH in hypophysectomized immature male and female rats.

Highly purified ovine FSH and LH were treated with neuraminidase to remove sialic acid and the desialylated derivatives were examined for biological activity in hypophysectomized immature male and female rats. The male rats were hypophysectomized at 22 days of age and beginning on day 25 were injected sc twice daily for 4 days with native or neuraminidase-treated FSH (total dose, 15 or 60 mug) or LH (12 mug). The ventral prostates, seminal vesicles, and testes were then removed and weighed, and serum testosterone levels were measured by radioimmunoassay. The female rats were hypophysectomized on day 28 and beginning on day 35 were injected sc twice daily for 4 days with native or neuraminidase-treated FSH (8 mug) or saline. On the morning of day 39, the rats were given an ovulating dose of gonadotropin (8 mug native or neuraminidase-treated FSH, or 1.28 mug native or neuraminidase-treated LH) or 1.0 ml saline iv via tail vein. Twenty-four hours later ova were counted in the oviducts the ovaries were weighed, and serum levels of progesterone and 20alpha-dihydroprogesterone were determined by radioimmunoassay. Treatment of ovine LH with neuraminidase did not diminish the ability of this hormone to increase prostate and testes weights and serum testosterone levels. Desialylation also did not decrease the ability of LH to induce ovulation. Although native ovine FSH significantly increased the weights of the ventral prostate, seminal vesicles, and testes, and elevated plasma testosterone levels, the desialylated derivative was essentially inactive. Neuraminidase treatment also eliminated the ability of ovine FSH to increase ovarian weight, to induce ovulation, and to elevate serum progesterone and 20alpha-dihydroprogesterone. These results indicate that the LH-like activity of ovine FSH is an intrinsic property of the FSH molecule.

Purification and characterization of the gonadotropin secreted by cultured horse trophoblast cells.

Equine chorionic gonadotropin (eCG) secreted by horse trophoblast cells cultivated in vitro was isolated and its chemical, immunochemical, and biological characteristics were compared to those of the gonadotropin secreted in vivo and isolated from PMS. It was also compared with eCG isolated from the tissue of origin, the endometrial cups. The gonadotropin secreted in vitro had a smaller molecular size, contained appreciably less carbohydrate, and showed different amino-terminal residues from that secreted in vivo. In addition, there were significant differences in amino acid composition between eCG secreted in vivo and that secreted in vitro. The reactivity of eCG (isolated from the medium of cultured trophoblast cells) in homologous eCG (isolated from PMS) and equine LH and FSH RIAs suggested close antigenic similarities to eCG isolated from PMS. However, compared to serum-derived eCG, eCG secreted in vitro showed reduced activity in both LH (13%) and FSH (9-24%) bioassays and in LH and FSH radioreceptor assays (40-50%). The differences in bioassay potencies may be accounted for in part by the lower sialic acid content of the gonadotropin secreted in vitro. With respect to both bioactivity and chemical composition, the eCG secreted in vitro more closely resembled the eCG isolated from endometrial cups than that isolated from serum.

Isolation and physicochemical characterization of human follicle-stimulating hormone isoforms.

Twenty hFSH isoforms were isolated from human pituitary extracts, 15 of which were highly pure. The mild purification procedure, which used a FSH RRA to monitor FSH activity, involved an initial fractionation of pituitary extracts by gel filtration and isoelectric focusing. Six pI regions (mean pI values, 3.63, 3.88, 4.07, 4.23, 4.84, and 5.13) of human (h) FSH were obtained and further fractionated on ion exchange and gel filtration HPLC. Recoveries of FSH radioreceptor activity at each stage were greater than 80%. Fifteen isoform preparations were judged as near homogeneous by HPLC-gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, exhibiting elution and migration behaviors consistent with the known mol wt of intact hFSH and its subunits. The remaining 5 isoform preparations contained a higher mol wt component that is probably hFSH related, as this component was detected after iodination, immunoprecipitation with hFSH antiserum, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Similar amino acid compositions were obtained for the 20 hFSH isoforms, except for evidence of some oxidative degradation of serine, threonine, and tyrosine and decreased levels of glutamic acid, possibly due to carboxy-terminal heterogeneity of the beta-subunit. An average amino acid composition value for all isoforms was comparable to that of 2 other highly purified hFSH preparations. Using the First International Standard for pituitary hFSH (83/575) as standard, radioreceptor activities were obtained ranging from 7,800-56,300 IU/mg protein. It is concluded that a mild purification procedure for the isolation of hFSH isoforms has been developed which gives high recoveries and has enabled the isolation of 15 isoforms in high purity suitable for further physicochemical and biological characterization.

Biosynthesis and secretion of follicle-stimulating hormone beta-subunit from ovine pituitary cultures: effect of 17 beta-estradiol treatment.

An assay was developed to detect tritium-labeled ovine FSH beta-subunit [( 3H]oFSH beta) secreted from primary ovine pituitary cultures. This procedure used affinity-enriched antibodies raised against reduced and carbamylmethylated oFSH beta (RCM-oFSH beta) in a two-cycle immunoextraction procedure. A discrete species with an apparent mol wt of 21,000 was detected in sodium dodecyl sulfate electrophoretic patterns of immunoextracts from culture medium. This species was identified as RCM-[3H]oFSH beta by its comigration with highly purified RCM-oFSH beta, its reduction in culture media after cultures were treated with 17 beta-estradiol, which normally decreases radioimmunoassayable oFSH; and its displacement from the extracting antibodies by excess unlabeled RCM-oFSH beta. The assay was used in a pulse-chase study to determine that [3H]oFSH beta is secreted within 1-2 h of its synthesis. Prior treatment of cultures with 17 beta-estradiol did not change this timing of secretion.

Capacity of immunologically purified FSH to stimulate cyclic AMP accumulation and steroidogenesis in Graafian follicles and to induce ovum maturation and ovulation in the rat.

Reference preparations of ovine follicle-stimulating hormone (NIH-FSH-S8 and S9; 10-50 mug/ml) induced ovum maturation and stimulated cyclic AMP formation, as well as progesterone and 17beta-estradiol secretion, by rat Graafian follicles in vitro. These actions of NIH-FSH were retained after immunoabsorption of any contaminating luteinizing hormone (LH) present in the preparations, by treatment with an antiserum to the beta-subunit of purified ovine LH (anti-betaLH). In contrast, the corresponding biological actions of NIH-LH-S18 (0.5-10 mug/ml) were abolished by treatment with this anti-betaLH serum. A highly purified FSH preparation (64-96 CD, 0.25 mug/ml) also triggered oocytic meiosis and increased follicular progesterone secretion in vitro. Intraperitoneal (ip) administration of anti-betaLH-treated NIH-FSH-S9 (50 mug/rat at 1430 h) consistently induced ovulation in proestrous rats in which the endogenous gonadotropin surge had been blocked by ip injection of either Nembutal (1345 h) or antiserum to the LH-releasing hormone (1200 h). Injection (ip) of anti-betaLH serum on its own into proestrous rats at 1200 h prevented ovum maturation and follicular rupture. We conclude that currently available reference preparations of ovine FSH possess the capacity to stimulate follicular adenylate cyclase, steroidogenesis, and ovum maturation in vitro, as well as ovulation in vivo, in the rat, and that this capacity cannot be attributed to contamination with material immunochemically identical with LH. However, it is inferred that the physiological triggering of ovulation and related events in this species depends principally on LH.

Gonadotropin and prolactin levels in follicular fluid of human ova successfully fertilized in vitro.

Follicular fluid (FF) and oocytes were obtained from 94 follicles of 36 women for fertilization in vitro. Ovulation was induced with human menopausal gonadotropin, and follicular aspiration was performed 36 h after an ovulatory injection of hCG. The concentrations of immunoreactive hCG, FSH, and PRL were correlated with the degree of maturation of the oocyte-corona-cumulus complex mass (OCCC), fertilization, rate of cleavage, and the incidence of pregnancy after embryo transfer. Immature OCCC were derived from follicles that contained significantly lower levels of FSH than those from which intermediate and mature OCCC were derived (5.2 +/- 0.6 vs. 11.1 +/- 1.2 mlU/ml; P less than 0.05). FF from oocytes that were successfully fertilized contained higher levels of both hCG and FSH than FF surrounding oocytes that did not fertilize (136.7 +/- 8.7 vs. 108.5 +/- 10.3 mlU/ml hCG; 10.55 +/- 0.6 vs. 5.3 +/- 0.8 mIU FSH, respectively). There was no correlation between early embryonic growth rate and FF concentrations of FSH, hCG, and PRL. Ova reaching the two-cell stage 40 h after fertilization in vitro were associated with the same FF concentrations of FSH, hCG, and PRL as those that cleaved to the four-cell stage. The PRL concentration in FF was significantly higher in mature fertilized ova and in fertilized ova that were associated with a successful pregnancy. It is suggested that the intrafollicular concentration of FSH is associated with the degree of mucification of the OCCC, but FF levels of both FSH and hCG are associated with successful fertilization. High levels of PRL in FF were associated with successful pregnancy and may imply a role of this hormone in oocyte maturation.