Fibroblast growth factor 18 regulates steroidogenesis in fetal bovine ovarian tissue in vitro.

In cattle and other species, the fetal ovary is steroidogenically active before follicular development commences, and there is evidence that estradiol and progesterone inhibit follicle formation and activation. Estradiol levels decline sharply around the time of follicle formation. In the present study, we hypothesized that FGF10 and FGF18, which inhibit estradiol secretion from granulosa cells of antral follicles, also regulate fetal ovarian steroid production. Fetuses were collected at local abattoirs, and age determined by crown-rump length measurements. Real-time polymerase chain reaction assays with RNA extracted from whole ovaries revealed that the abundance of CYP19A1 messenger RNA (mRNA) decreased from 60 to 90 days of gestation, which is consistent with the decline in estradiol secretion previously observed. Immunohistochemistry revealed the presence of FGF18 in ovigerous cords in early gestation and in oocytes later in fetal age (≥150 days). The abundance of FGF18 mRNA increased after Day 90 gestation. Addition of recombinant FGF18 to fetal ovarian pieces inhibited estradiol and progesterone secretion in vitro, whereas FGF10 was without effect. Consistent with these results, FGF18 decreased levels of mRNA for CYP19A1 and CYP11A1 in ovarian pieces in vitro. These data suggest that FGF18 may be an intraovarian factor that regulates steroidogenesis in fetal ovaries.

Janus particles: design, preparation, and biomedical applications.

Janus particles with an anisotropic structure have emerged as a focus of intensive research due to their diverse composition and surface chemistry, which show excellent performance in various fields, especially in biomedical applications. In this review, we briefly introduce the structures, composition, and properties of Janus particles, followed by a summary of their biomedical applications. Then we review several design strategies including morphology, particle size, composition, and surface modification, that will affect the performance of Janus particles. Subsequently, we explore the synthetic methodologies of Janus particles, with an emphasis on the most prevalent synthetic method (surface nucleation and seeded growth). Following this, we highlight Janus particles in biomedical applications, especially in drug delivery, bio-imaging, and bio-sensing. Finally, we will consider the current challenges the materials face with perspectives in the future directions.

Genetic and genomic analyses of embryo production in dairy cattle.

The Canadian dairy industry has been using invivo and invitro assisted reproductive technologies to produce embryos. Technological improvements have helped increase the number and quality of embryos produced, but genetic and genomic tools for improving these traits have yet to be assessed for the Canadian Holstein population. Genetic parameters and a genome-wide association study were performed in Canadian Holstein for the total number of embryos (NE) and the number of viable embryos (VE). Results showed potential for genetic selection for both NE and VE, with heritability estimates (± s.e.) of approximately 0.15±0.01. Genetic correlations between the number of embryos produced using different procedures (invivo and invitro) suggested that a similar number of embryos should be expected from a donor regardless of the procedure used. A region on chromosome 11 of the bovine genome was found to be significantly associated with the number of embryos, indicating a potential regulatory role of this region on embryo production. Overall, these findings are of interest for the Canadian dairy industry because they provide useful information for breeders that are interested in producing embryos from the elite donors in their herds or in the population using assisted reproductive technologies.

Expression of fibroblast growth factor 22 (FGF22) and its receptor, FGFR1B, during development and regression of bovine corpus luteum.

The aim of this study was to determine the expression of fibroblast growth factor 22 (FGF22) in the bovine corpus luteum (CL) and to investigate the effects of in vivo total or partial cloprostenol-induced luteolysis on the mRNA abundance of FGF22 and its receptor, FGFR1B. Corpora lutea at different stages of development were then dissected from abattoir ovaries (n = 10/stage); a portion of the tissue samples was fixed in paraformaldehyde and the remaining samples were homogenized and subjected to total RNA extraction. To assess mRNA abundance of target genes during induced luteolysis, nineteen cows were synchronized and then randomly assigned to a Latin square design as follows: Control; 2 administrations of prostaglandin F2α (PGF2α, total luteolysis; 2 × 250 µg of cloprostenol sodium) and 1/6PGF2α (partial luteolysis; 83.33 µg of cloprostenol sodium). FGF22 and FGFR1B expression levels were measured by RT-qPCR, and FGF22 protein expression was detected by immunohistochemistry. In summary, FGF22 mRNA was detected at all stages of CL development, and FGF22 protein was also detected in luteal tissue. FGF22 mRNA expression was lower at stage IV than at stage III (P < 0.05), and the same pattern was observed in luteal immunoreactivity. Furthermore, cloprostenol-induced luteolysis, both total and partial, increased FGFR1B mRNA abundance in luteal tissue (P < 0.05), but did not affect FGF22 mRNA abundance. In conclusion, these data suggest a potential role for the FGF22-FGFR1B system during development and regression of bovine CL.

Genome-wide association study and in silico functional analysis of the number of embryos produced by Holstein donors.

Superovulation or ovum pick-up and in vitro fertilization are technologies used to produce an increased number of embryos from elite females. Embryo production traits have been shown to be heritable, but the genes that cause this variability have not yet been assessed. The main objectives of this study were to perform a genome-wide association study (GWAS) to find single nucleotide polymorphisms (SNP) associated with embryo production traits and to identify candidate genes affecting the number of embryos produced by Holstein donors in Canada that may provide insight into the regulation of embryo production. Breeding values were estimated and de-regressed for all donors and sires using a data set of 150,971 records of superovulation or ovum pick-up and in vitro fertilization. A total of 11,607 animals were genotyped, but of that number only 5,118 were genotyped with at least a 50K SNP panel and had a de-regressed estimated breeding value reliability of at least 10%. For the GWAS, 606,406 imputed SNP on 29 autosomal chromosomes were considered after applying quality control measures. A single-SNP univariate mixed linear animal model was used to perform the GWAS, and a 5% false discovery rate was applied to adjust for multiple testing. We found 36 and 14 significant SNP associated with the total number of embryos and the number of viable embryos, respectively, with most of them located on chromosome 11. Using these significant SNP, positional genes located within 10,000 bp upstream and downstream of the SNP were retrieved. Thirteen genes were harboring or near the significant SNP for the total number of embryos, 4 of them also being near the significant SNP for viable embryos. Some of these genes (CRB2, DENND1A, MAD1L1, NDUFA8, PTGS1) could be considered as potential positional candidate genes related to the number of embryos produced by a donor. This list will need to be validated in an independent population to confirm the role of the genes for embryo production.

Genetic analysis for quality of frozen embryos produced by Holstein cattle donors in Canada.

The number of embryos produced by Holstein donors has been shown to be heritable, so it could be possible to genetically select for this trait to improve the efficiency of the assisted reproductive technology (ART) in dairy cattle. Another important parameter to consider for achieving good results from ART is embryo quality because embryos of good quality have more chance of producing live offspring. The possibility of using genetic selection for increasing the quality of embryo produced from ART has yet to be assessed. The objective of this study was, therefore, to perform a genetic analysis of embryo quality of Holstein donors in Canada using data recorded by Holstein Canada. The data set used was missing quality score data for embryos transferred fresh into a recipient, so the analyses were only performed for frozen embryos. With most traits in the Canadian dairy industry being evaluated with linear models, embryo quality was also evaluated with this class of models. However, considering the categorical nature of embryo quality, a threshold model was also evaluated. Embryo quality data were analyzed with either a univariate linear animal model or a univariate binomial threshold animal model. Genetic parameters estimated from the different models were comparable. A low heritability was found for the donor (0.04 ± <0.01) and the service sire (0.02 ± <0.01), but the repeatability estimate for the donor was higher (0.17), indicating that it was worthwhile to use a repeated records model. Overall, considering the low genetic parameters estimated, slow genetic progress is expected for the quality of frozen embryos produced by Canadian Holstein donors. Rank correlations were calculated between breeding values estimated from different models. High correlations were found between all models, indicating that no substantial re-ranking of the animals is expected from the different models. So, even though a threshold model is better suited for the analysis of categorical data, a linear model could be used for the analysis of embryo quality because it is less computationally demanding.

Evidence that fibroblast growth factor 10 plays a role in follicle selection in cattle.

There is evidence that regulation of follicle selection in cattle involves locally produced growth factors. In the present study, we investigated the expression of members of the fibroblast growth factor (FGF) 7 family during follicle deviation. The largest and second largest follicles were recovered during the second day of a synchronised follicle wave and the future dominant and future subordinate follicles were identified based on diameter and cytochrome P450, family 19, subfamily A, polypeptide 1 (CYP19A1) mRNA levels in granulosa cells. Theca cells of the future dominant follicle contained less mRNA encoding FGF7 and FGF10 compared with those from the future subordinate follicle 2.5 days after ovulation, before a significant difference between the diameters of the future dominant and future subordinate follicles could be observed, but FGF22 mRNA levels did not change. Levels of mRNA encoding FGF receptors FGFR1B and FGFR2B in theca and granulosa cells, respectively, were lower in the future dominant follicle compared with the future subordinate follicle. Addition of FGF10 to granulosa cells in vitro significantly decreased oestradiol secretion, as well as CYP19A1, FSH receptor (FSHR) and insulin-like growth factor 1 receptor (IGF1R) mRNA abundance, whereas FGF22 had no effect. We conclude that FGF10 and FGFR2B expression is increased in the future subordinate follicle before morphological deviation, which may contribute to follicle selection.

Short communication: Genetic correlations between number of embryos produced using in vivo and in vitro techniques in heifer and cow donors.

Multiple embryos can be produced from a heifer or cow donors using an in vivo or an in vitro technique. Comparisons of the number of embryos produced by the same donors as heifers and cows and using different techniques are limited. The main objectives of this study were to assess the genetic correlation between the number of embryos produced by Holstein donors using an in vivo and in vitro technique as a heifer and as a cow. The data set used was recorded by Holstein Canada and included all successful superovulations or ovum pickup and in vitro fertilization procedures performed on Holstein donors for more than 20yr. The type of technique used was known for all records and the status of the donor at recovery was retrieved from calving records. Bivariate repeatability animal model analyses were performed for both the total number of embryos (NE) and the number of viable embryos (VE) recovered per procedure. Logarithmic transformation was performed on the traits to normalize the data. Heritability estimates for the donor varied between 0.14 (0.02) and 0.19 (0.03) over all analyses, indicating that the number of embryos produced by a donor is influenced by the genetic potential of the donor. Genetic correlations between records produced in vivo and in vitro were moderately high and positive (NE=0.85±0.07; VE=0.63±0.09), suggesting that donors with high genetic potential for in vivo superovulation tend also to have high potential to produce multiple embryos in vitro. Similarly, the moderately high genetic correlations (NE=0.79±0.05; VE=0.72±0.05) found between heifer and cow records indicate that a donor tends to produce a comparable number of embryos as a heifer or as a cow. The estimated repeatabilities (0.23 to 0.35) indicated that the number of embryos recovered should be somewhat repeatable in the same donor over time. On the other hand, the service sires seem not to play an important role on the total number of embryos produced by a donor no matter the technique used or the status of the donor at recovery.

Localization of angiotensin receptor type 2 in fetal bovine ovaries.

In the ovary, angiotensin II (ANGII) acts through the type 2 receptor (AGTR2) to induce ovulation and may play a role in follicle atresia. In this study, we determined the expression of AGTR2 mRNA and protein during follicle formation in the bovine ovary. Female fetuses at different gestational ages (60, 75, 90, 120, 150 and 210 days) were used for immunolocalization of AGTR2. At day 60, AGTR2 was localized to the cytoplasm of oogonia; from days 75 to 150, during follicle formation and development to secondary stage, AGTR2 immunostaining was weak and irregular, but from day 210 staining became evident in granulosa cells of preantral follicles and in both granulosa and theca cells of small antral follicles. These data differ from those in pigs, in which AGTR2 protein is detected in preantral follicles throughout gestation. Abundance of AGTR2 mRNA in whole ovaries did not change with fetal age. In conclusion, AGTR2 protein is expressed in ovigerous cords in fetal bovine ovaries but not in preantal follicles until the formation of antral follicles. These data suggest important species-specific differences in the expression of AGTR2 in fetal ovaries from polyovulatory and monovulatory animals.

Genetic analysis of superovulatory response of Holstein cows in Canada.

Superovulation of dairy cattle is frequently used in Canada. The cost of this protocol is high, and so is the variability of the outcome. Knowing the superovulatory potential of a donor cow could influence the breeder's decision to superovulate it or not. The main objective of this study was to perform a genetic analysis for superovulatory response of Holstein cows in Canada using data recorded by Holstein Canada, and to investigate if these data could be used for genetic evaluation. Data contained the total number of embryos and the number of viable embryos from every successful flushing performed across Canada. After editing, 137,446 records of superovulation performed between 1992 and 2014 were analyzed. A univariate repeatability animal model analysis was performed for both total number of embryos and number of viable embryos. Because both data and residuals did not follow a normal distribution, records were subject to either logarithmic or Anscombe transformation. Using logarithmic transformation, heritability estimates (SE) of 0.15 (0.01) and 0.14 (0.01) were found for total number of embryos and number of viable embryos, respectively. Using Anscombe transformation, heritability estimates (SE) of 0.17 (0.01) and 0.14 (0.01) were found for total number of embryos and number of viable embryos, respectively. The genetic correlation between the 2 traits was estimated at 0.97 using logarithmic transformation and 0.95 using Anscombe transformation. Breeding values were estimated for 54,463 cows, and 3,513 sires. Only estimated breeding values of sires having a reliability higher than 40% were considered for estimated breeding values correlations with other routinely evaluated traits. The results showed that selection for a higher response to superovulation would lead to a slight decrease in milk production, but an improvement for functional traits, including all reproduction traits. In all cases, the estimated correlations are either low or modest. We conclude that genetic selection for increased superovulatory response in donors is possible; daughters of sires with high estimated breeding values for superovulatory response will tend to yield more embryos, whereas the additive effect of service sire seems not to contribute to the variability of the 2 superovulation traits and was not significantly correlated with the additive effect of the donor.

Expression of fibroblast growth factor 10 and cognate receptors in the developing bovine ovary.

In the mammalian ovary, FGF10 is expressed in oocytes and theca cells and is a candidate for paracrine signaling to the developing granulosa cells. To gain insight into the participation of FGF10 in the regulation of fetal folliculogenesis, we assessed mRNA expression patterns of FGF10 and its receptors, FGFR1B and FGFR2B, in relation to fetal follicle dynamics and localized FGF10 protein in bovine fetal ovaries at different ages. Primordial, primary, secondary, and antral follicles were first observed on Days 75, 90, 150, and 210 of gestation, respectively. The levels of GDF9 and BMP15 mRNA, markers for primordial and primary follicles, respectively, increased during fetal ovary development in a consistent manner with fetal follicle dynamics. CYP17A1 mRNA abundance increased from Day 60 to Day 75 and then from Day 120 to Day 150, coinciding with the appearance of secondary follicles. FGF10 mRNA abundance increased from Day 90, and this increase was temporally associated with increases in FGFR1B mRNA abundance and in the population of primary follicles. In contrast, FGFR2B mRNA expression was highest on Day 60 and decreased thereafter. FGF10 protein was localized to oogonia and oocytes and surrounding granulosa cells at all fetal ages. The present data suggest a role for FGF10 in the control of fetal folliculogenesis in cattle.

Bioassay for follicle stimulating activity of equine gonadotropic hormone in mare serum using frozen/thawed transiently transfected reporter cells.

The objective was to establish a cell line-based bioassay for FSH in horse serum for screening samples with high eCG bioactivity. A cell line (HEK293) was transiently cotransfected with an FSH reporter expression plasmid and a cAMP-responsive ß-galactosidase reporter plasmid. Cells were bulk frozen, and thawed for assay purposes. This assay was specific for FSH, with no cross-reaction with LH or insulin-like growth factor-1. Standard curves (eCG) and serum samples from pregnant mares passed parallel line bioassay validity tests (linearity and parallelism). Estimates of bioactivity with this bioassay were highly correlated with estimates obtained with the Steelman-Pohley hCG augmentation assay. The colorimetric end point permitted the use of this assay as a rapid screen for FSH bioactivity without the need for animal use or complex cell culture facilities.

Fibroblast growth factor-2 regulation of Sprouty and NR4A genes in bovine ovarian granulosa cells.

Fibroblast growth factors (FGFs) alter ovarian function, at least in part by inhibiting steroid hormone secretion and affecting survival of granulosa cells. The mechanism of action of FGFs in ovarian follicle cells is largely unknown; in the present study we identified the major pathways used by FGF2 in non-luteinizing granulosa cells cultured under serum-free conditions. FGF2 increased abundance of mRNA encoding SPRY1, 2, and 4, but not SPRY3. Common pathways employed by FGF2 in the regulation of SPRY1, 2, and 4, as demonstrated by immunoblot and inhibitor studies, included ERK1/2 and Akt signaling. In contrast, PKC activation was necessary for FGF2-stimulated expression of SPRY1 and 4, but not for SPRY2. Intracellular calcium flux is critical and sufficient for SPRY2 expression, but not for SPRY1 and 4. We also identified the orphan nuclear receptor NR4A1 as a potential early response gene in FGF2 signaling, whose expression, like that of SPRY2, is critically dependent on calcium signaling. Together, these data identify FGF2-target genes in follicular granulosa cells, and demonstrate alternative pathway use for the differential control of SPRY genes.

Follicular somatic cell factors and follicle development.

Considerable attention is currently paid to oocyte-derived secreted factors that act upon cumulus and granulosa cells. Also important for follicle development are somatic cell-derived secreted factors. This is illustrated by the ability of granulosa cell-derived Kit ligand (KITL) to promote primordial follicle activation, and the loss of follicle development that accompanies KITL gene disruption. This review summarises our current understanding of somatic cell factors during both preantral and antral follicle growth, involving not only signalling from granulosa cells to the oocyte, but also signalling between granulosa and theca cells. Principal granulosa cell-derived factors include activin, anti-Müllerian hormone (AMH), bone morphogenetic proteins (BMPs) and fibroblast growth factors (FGFs). Theca cells also secrete BMPs and FGFs. The interplay between these factors is equally important for follicle growth as the activity of oocyte-derived factors.

Regulation and action of fibroblast growth factor 17 in bovine follicles.

Fibroblast growth factor 17 (FGF17) is a member of the FGF8 subfamily that appears to be relevant to folliculogenesis and oogenesis, as the prototype member FGF8 is an oocyte-derived protein that signals to cumulus cells. FGF8 has structural and receptor-binding similarities to FGF17, whose expression in the ovary has not been reported. In this study, we demonstrate localization of FGF17 protein to the oocyte of preantral follicles, and to the oocyte and granulosa cells of antral follicles. Real-time PCR demonstrated the presence of mRNA in oocytes and, to a lesser extent, in granulosa and theca cells. FGF17 mRNA abundance was low in granulosa and theca cells from healthy follicles and increased significantly in atretic follicles. Addition of FSH or IGF-I to granulosa cells in vitro decreased FGF17 mRNA abundance, and treatment with FGF17 inhibited estradiol and progesterone secretion from granulosa cells in relation to control cultures without these additives. We conclude that FGF17 is a potential mediator of granulosa cell differentiation.

Expression of fibroblast growth factor receptors during development and regression of the bovine corpus luteum.

There is evidence that fibroblast growth factors (FGFs) are involved in the regulation of growth and regression of the corpus luteum (CL). However, the expression pattern of most FGF receptors (FGFRs) during CL lifespan is still unknown. The objective of the present study was to determine the pattern of expression of 'B' and 'C' splice variants of FGFRs in the bovine CL. Bovine CL were collected from an abattoir and classed as corpora hemorrhagica (Stage I), developing (Stage II), developed (Stage III) or regressed (Stage IV) CL. Expression of FGFR mRNA was measured by semiquantitative reverse transcription-polymerase chain reaction and FGFR protein was localised by immunohistochemistry. Expression of mRNA encoding the 'B' and 'C' spliced forms of FGFR1 and FGFR2 was readily detectable in the bovine CL and was accompanied by protein localisation. FGFR1C and FGFR2C mRNA expression did not vary throughout CL lifespan, whereas FGFR1B was upregulated in the developed (Stage III) CL. FGFR3B, FGFR3C and FGFR4 expression was inconsistent in the bovine CL. The present data indicate that FGFR1 and FGFR2 splice variants are the main receptors for FGF action in the bovine CL.

Expression of fibroblast growth factor 10 and its receptor, fibroblast growth factor receptor 2B, in the bovine corpus luteum.

There is evidence that several fibroblast growth factors (FGFs) are involved in growth and development of the corpus luteum (CL), but many FGFs have not been investigated in this tissue, including FGF10. The objective of this study was to determine if FGF10 and its receptor (FGFR2B) are expressed in the CL. Bovine CL were collected from an abattoir and classed as corpus hemorrhagica (stage I), developing (stage II), developed (stage III), and regressed (stage IV) CL. Expression of FGF10 and FGFR2B mRNA was measured by reverse transcription-polymerase chain reaction (RT-PCR). Both genes were expressed in bovine CL, and FGF10 expression did not differ between stages of CL development. FGF10 protein was localized to large and small luteal cells by immunohistochemistry. FGFR2B expression was approximately threefold higher in regressed compared to developing and developed CL (P < 0.05). To determine if FGF10 and FGFR2B expression is regulated during functional luteolysis, cattle were injected with PGF2alpha and CL collected at 0, 0.5, 2, 4, 12, 24, 48, and 64 hr thereafter (n = 5 CL/time point), and mRNA abundance was measured by real-time RT-PCR. FGF10 mRNA expression did not change during functional luteolysis, whereas FGFR2B mRNA abundance decreased significantly at 2, 4, and 12 hr after PGF2alpha, and returned to pretreatment levels for the period 24-64 hr post-PGF2alpha. These data suggest a potential role for FGFR2B signaling during structural luteolysis in bovine CL.

Expression and function of fibroblast growth factor 10 and its receptor, fibroblast growth factor receptor 2B, in bovine follicles.

Some fibroblast growth factors (FGFs) affect ovarian follicle cell growth and/or differentiation. Whereas many FGFs activate several FGF receptors, FGF7 and FGF10 primarily activate only one, FGFR2B. As FGF7 is produced by bovine theca cells and acts on granulosa cells, we tested the hypothesis that FGF10 may also play a role in folliculogenesis in cattle. Reverse transcription-polymerase chain reaction demonstrated the presence of FGF10 mRNA in the oocytes and theca cells of the antral follicles, as well as in the preantral follicles. FGF10 protein was detected by immunohistochemistry in the oocytes of the preantral and antral follicles, and in the granulosa and theca cells of the antral follicles. FGF10 expression in theca cells changed during follicle development; mRNA abundance decreased with increasing follicular estradiol concentration in healthy follicles, and was lowest in highly atretic follicles. Culturing of granulosa cells in serum-free medium revealed FSH regulation of FGF10 receptor expression. The addition of FGF10 to cultured granulosa cells decreased the level of estradiol but did not alter cell proliferation. These data support a role for FGF10 in signaling to granulosa cells from theca cells and/or the oocyte.

Expression of LH receptor mRNA splice variants in bovine granulosa cells: changes with follicle size and regulation by FSH in vitro.

In cattle, most evidence suggests that granulosa cells express LH receptors (LHR) after (or as) the follicle becomes dominant, however there is some suggestion that granulosa cells from smaller pre-dominant follicles may express several LHR mRNA splice variants. The objective of this study was to measure LHR expression in bovine follicles of defined size and steroidogenic ability, and in granulosa cells from small follicles (<6 mm diameter) undergoing differentiation in vitro. Semiquantitative RT-PCR demonstrated that LHR mRNA was undetectable in granulosa cells of follicles <7 mm diameter (nondominant follicles), and increased with follicle diameter in follicles >7 mm diameter. Splice variants with deletions of exon 10 and part of exon 11 were detected as previously described, and we detected a novel splice variant with a deletion of exon 3. Cultured granulosa cells contained LHR mRNA, but with significantly greater amounts of variants with deletions of exon 10 and/or exon 11 compared with cells from dominant follicles. FSH increased the abundance of some but not all LHR mRNA splice variants in cultured granulosa cells. The addition of LH to cultured cells did not increase progesterone secretion, despite the presence of LHR mRNA. Collectively, these data suggest that granulosa cells do not acquire functional LHR until follicle dominance occurs.

Control of oestradiol secretion and of cytochrome P450 aromatase messenger ribonucleic acid accumulation by FSH involves different intracellular pathways in oestrogenic bovine granulosa cells in vitro.

The objective of this study was to determine the major intracellular signalling pathways used by FSH and insulin to stimulate cytochrome P450 aromatase (Cyp19) mRNA and oestradiol accumulation in oestrogenic bovine granulosa cells in vitro. Bovine granulosa cells from small follicles (2-4 mm diameter) were cultured for 6 days under non-luteinizing conditions in the presence of insulin at 100 ng/ml, or insulin (10 ng/ml) and FSH (1 ng/ml). On day 4 of culture, specific inhibitors of phosphatidylinositol 3-kinase (PI3K; LY-294002), protein kinase C (PKC; GF-109203X), protein kinase A (PKA; H-89) or mitogen-activated protein (MAP) kinase activation (PD-98059) were added. The addition of PI3K and PKC inhibitors, but not of PKA inhibitor, significantly decreased insulin-stimulated Cyp19 mRNA levels and oestradiol accumulation (P < 0.001). The PKA inhibitor significantly decreased FSH-stimulated Cyp19 mRNA abundance and oestradiol secretion, whereas PI3K and PKC inhibitors decreased oestradiol secretion without affecting Cyp19 mRNA accumulation. Inhibition of MAP kinase pathway significantly increased Cyp19 mRNA abundance in insulin- and FSH-stimulated cells. P450scc mRNA levels and progesterone secretion were not affected by any inhibitor in either experiment. Although FSH stimulates Cyp19 expression predominantly through PKA, oestradiol secretion is altered by PI3K and PKC pathways independently of Cyp19 mRNA levels. In addition, we suggest that Cyp19 is under tonic inhibition mediated through a MAP kinase pathway.