Expression of cytosolic phospholipase A2 in equine endometrium during the oestrous cycle and early pregnancy.

Phospholipase A2 (PLA2) is a key enzyme for biosynthesis of PGF2α. Real-time RT-PCR and immunohistochemistry were used to determine transcription and cellular distribution of cytosolic PLA2 (cPLA2) in the equine endometrium during the oestrous cycle and early pregnancy. Endometrial biopsy and blood samples were collected from cycling mares on days (d) 8, 11, 15 and 18 (oestrus) (Day 0 = Day of ovulation; n = 5 for each day) and from pregnant mares (n = 4) on d15. Except for mares on d18 and some cyclic mares (n = 2) on d15 with low progesterone (P4) concentrations (< 3.18 nm), P4 concentrations were high. Cytosolic PLA2 was mainly localized in the luminal epithelium and stroma was negative. Cytosolic PLA2 expression was negatively correlated with P4 concentration (r = -0.75, P < 0.001) and differed according to the stage of the oestrous cycle (P < 0.05). Cytosolic PLA2 expression was high during oestrus and declined to basal levels on d8 (P < 0.05). Thereafter, there was a trend for increased cPLA2 expression as luteal phase progressed. However, as P4 dropped below 3.18 nm on d15, cPLA2 expression increased (P < 0.05). In pregnant mares, cPLA2 expression was not different from cyclic mares on d15 with high P4 concentrations. However, it was lower than cyclic mares with low P4 concentrations (p < 0.05). In conclusion, cPLA2 isoform, a member of the PLA2 family that controls PGF2α secretion, is highly expressed in the endometrium at the expected time of luteolysis. Progesterone may be an inhibitor for cPLA2 expression. During pregnancy, cPLA2 expression may be sufficient to initiate the cascade for PGF2α secretion and does not play a direct role in maternal recognition of pregnancy.

Age-dependent expression of 5alpha-reductase and androgen receptors mRNA by the canine prostate.

The growth of the prostate gland is androgen-dependent. Testosterone is converted to the most potent dihydrotestosterone (DHT) by 5alpha-reductase within the prostate. Androgen interacts with androgen receptors (AR) to regulate normal growth of the prostate and has also been implicated in both the progression of benign prostate hyperplasia and prostate cancer. This study was conducted to compare the mRNA expression of AR and 5alpha-reductase by the prostate gland from three age categories: immature, young-mature and old dogs. Quantitative gene expression was assessed by the real-time PCR and the results were expressed as a relative mRNA expression of the target gene. This study revealed that there was no significant difference in the mRNA expression of the AR gene by the prostate gland of immature, young and old dogs. In contrast, there is a highly significant (P<0.001) down-regulation in 5alpha-reductase gene by the prostate of young and old dogs as compared with immature dogs. However, there is no significant difference in mRNA expression of the 5alpha-reductase gene by the prostate gland from young and old dogs. This differential expression of AR and 5alpha-reductase genes, which are involved in the regulation of androgen effect on prostate gland, might reflect an age-dependent growth requirement of the gland for androgens.

Development-related expression of KGF and FGF-10 mRNA in the canine prostate gland.

Keratinocyte growth factor (KGF) and fibroblast growth factor 10 (FGF-10) are stromal-derived growth factors that interact with their epithelial FGFR2 receptors to mediate stromal--epithelial cell interaction within the prostate gland. This study was conducted to compare the development-related mRNA expression of KGF, FGF-10 and their receptor FGFR2 in immature and mature canine prostate glands. In addition, their expression levels were correlated with the differentiation of stromal cells using vimentin as a mesenchymal cell marker. Quantitative mRNA expression was assessed by real-time polymerase chain reaction (PCR) and the results were expressed as relative mRNA expression of the target gene, which was normalized to the GAPDH reference gene. mRNA analysis revealed a differential expression of KGF, FGF-10 and FGFR2 receptor by the prostate glands of immature and mature dogs. The results showed a 7.3- and 9-fold decrease in mRNA expression of KGF and FGF-10 by mature and immature prostate glands respectively. However, there was no significant change in FGFR2 receptor mRNA expression by mature or immature prostate glands. This downregulation of KGF and FGF-10 expression was associated with a 15-fold decrease in vimentin expression. These results indicate that KGF and FGF-10 expression varied according to the differentiation status of stromal cells and might reflect differential developmental requirements of immature and mature canine prostate glands.

Activin-A differentially regulates steroidogenesis by sheep granulosa cells.

Intra-ovarian factors, such as activin, are implicated in multiple aspects of follicular development in mammalian ovaries. This study was conducted to investigate a possible effect of activin-A on steroidogenesis in sheep granulosa cells in vitro. Sheep granulosa cells were obtained from medium antral follicles and cultured in a chemically defined RPMI -1640. Oestradiol and progesterone production, secreted by the cultured cells, was evaluated by enzyme-linked immunosorbent assay. In order to determine the dose effect of activin-A on steroidogenesis, granulosa cells were cultured in the presence of increasing concentrations of activin-A (0, 0.5, 5 and 50 ng ml(-1)) for 48 hours. The results revealed that activin-A exerts a differential effect on steroidogenesis in granulosa cells in such a way that it significantly (P < 0.05) suppressed progesterone production and enhanced oestradiol production. These results were confirmed by the time effect of activin-A on oestradiol and progesterone production in granulosa cells. In the absence of activin-A treatment, granulosa cells showed enhanced capacity to produce progesterone, but not oestradiol, as the time progressed from 12 to 48 hours. Treatment of sheep granulosa cells with 25 ng ml(-1)activin-A for 12, 24 and 48 hours significantly stimulated oestradiol production but inhibited progesterone production. These results suggest that activin-A is a local regulator of sheep folliculogenesis that might act to support differentiation in granulosa cells and suppress luteinisation.

Effect of activin-a on goat granulosa cell steroidogenesis.

Growth factors are said to play a significant role in the development of ovarian follicles. We wished to measure the content of one growth factor, activin-A in goat ovarian follicles, and study its effect on goat granulosa cells steroidogenesis. The follicular fluid content of activin-A from small, medium and large antral follicles was determined by two-site enzyme immunoassay. The results showed that activin-A concentration in the follicular fluid increased as the size of the follicle increased and, thus, may act as a local regulator of follicle development. To examine this possibility, the effect of increasing concentration of activin-A (0, 1, 10, 100 ng/mL) on differentiated goat granulosa cells steroidogenesis was evaluated in vitro for 48 hours in a chemically defined medium. Activin-A treatment resulted in a significant inhibition of progesterone production concomitant with a significant stimulation of estradiol production. These results were confirmed by time-effect of 50 ng/mL activin-A on goat granulosa cells steroidogenesis for 24, 48 and 72 hours. Granulosa cells displayed differential steroidogenic responses to activin-A, estradiol production becoming enhanced and progesterone production suppressed. Based on these findings, it appears that activin-A is a local regulator of goat granulosa cell steroidogenesis, and may act to promote granulosa cell differentiation and inhibit its luteinization.

Gossypol arrests human benign prostatic hyperplastic cell growth at G0/G1 phase of the cell cycle.

Recently we demonstrated that gossypol (GP), a male antifertility agent, is a potent inhibitor of malignant human prostate cancer cell growth that acts by arresting cells in G0/G1 phase and that this inhibitory effect may be mediated by transforming growth factor-beta 1 (TGF-beta 1). In this study we examined the effect of GP on the growth of prostatic cells from human benign prostatic hyperplasia (BPH) patients in vitro. Consistent with its inhibitory effect on the growth of malignant human prostate cancer cells, GP also acts as a potent inhibitor of cultured human BPH cell growth as assessed by thymidine incorporation assay. These results were confirmed by flow cytometric analysis which revealed that treatment of human BPH cells with increasing concentrations of GP resulted in a dose-dependent accumulation of cells in the G0/G1 phase with a concomitant decrease in cells progressing to the S and G2/M phases. Since inhibition of prostate cancer cells by GP appears to be mediated by TGF-beta 1, we also investigated the effect of GP on TGF-beta 1 gene expression in BPH cells. The results show that GP treatment resulted in a marked elevation of TGF-beta 1 gene expression indicating that TGF-beta 1 might be involved at least in part in the inhibitory pathway that is initiated by GP.

Gossypol induces spermidine/spermine N1-acetyltransferase in canine prostate epithelial cells.

Gossypol is an antisteroidogenic compound naturally found in cottonseed. Gossypol has been shown to inhibit steroidogenesis in the canine prostate and may inhibit canine prostate growth. Its mechanism of action, however, is largely unknown. Our laboratory has previously demonstrated that in vivo administration of gossypol to male dogs can reduce circulating levels of testosterone and estradiol. Gossypol also showed an ability to reduce prostate weights. To search for genes regulated by gossypol in the canine prostate, differential display RT-PCR was performed on total RNAs isolated from control and gossypol-treated male dogs. Gossypol was demonstrated to induce expression of spermidine/spermine-N1-acetyl-transferase (SSAT), the major catabolic enzyme for polyamines. This induction was confirmed by Northern hybridization analysis of total RNA isolated from prostates of mature dogs treated with gossypol for 2 months. Gossypol was also shown to inhibit the progression of cells into the S phase mediated by spermidine. Our findings support the notion that gossypol can inhibit prostate cell proliferation and may be a potential therapeutic agent for use in controlling overgrowth of the prostate.

Differential effect of keratinocyte growth factor (KGF) on aromatase activity in cultured canine prostatic epithelial cells.

We have recently cloned the mRNA encoding KGF from canine prostate and produced recombinant canine KGF (rcKGF) which specifically acts on cultured canine prostatic epithelial cells (CCPECs) which possess KGF receptors (Canatan et al., 1996; DNA Cell Biol. 15:247). In the present study, the effect of rcKGF on aromatase activity in CCPECs from young (6-month-old) and mature (3-year-old) dogs was examined. Release of 3H2O from labeled substrate was used as the indicator of aromatase activity. CCPECs were pulsed with [1-beta-3H]-androstenedione (1 microCi/ml, 6 hr). The amounts of 3H2O released into culture medium were measured (dpm) and total cellular proteins were determined. Aromatase activity was expressed as 3H2O dpm/mg cellular protein (mean +/- SEM). The basal level of aromatase activity in CCPECs from mature dogs was approximately 4 times higher (p < 0.05) than that in cells from young dogs. Aromatase activity in CCPECs from mature dogs increased in a dose-dependent manner upon treatment with rcKGF. Interestingly, rcKGF, at any of the concentrations tested, had no significant effect on aromatase activity in CCPECs from young dogs. These results are the first to indicate that aromatase activity is affected by KGF in mature CCPECs, suggesting that KGF may be involved indirectly in the etiology of benign prostatic hyperplasia by increasing aromatase activity and thus increasing aromatization of androgens. Aromatase induction by KGF may explain, at least in part, the increased aromatization of androgens observed in aged dogs. The exact mechanism of how KGF induces aromatase activity in CCPECs is needed to be addressed further.

Inhibition of human prostate cancer cells growth by gossypol is associated with stimulation of transforming growth factor-beta.

Gossypol (GP), an antifertility agent in males, is also capable of inhibiting the proliferation of a wide range of cancer cells in vivo and in vitro. Thus, in this study we investigated the effect of GP on the growth of human androgen-independent prostate cancer cell line (PC3). The results showed that GP acts as a potent inhibitor of PC3 cells as determined by thymidine incorporation assay and flow cytometric analysis. Flow cytometry revealed that treatment of PC3 cells with GP resulted in a dose- and time-dependent accumulation of cells in the GO/GI phase with a concomitant decrease in cells progressing to the S and G2/M phase. These data support our thymidine incorporation results which indicated that GP is a potent inhibitor of PC3 cells. By ribonuclease protection assay, we also investigated the effect of GP on transforming growth factor-beta 1 (TGF-beta 1) gene expression in PC3 cells. Interestingly, the stimulatory effect of GP on TGF-beta 1 gene expression correlates well with its inhibitory effect on PC3 cell DNA synthesis and its ability to arrest cells in GO/G1 phase. Based on these data, it can be concluded that GP is a potent inhibitor of prostate cancer cell growth that acts by arresting cells in GO/G1 phase and that this inhibitory effect may be mediated by TGF-beta 1.

Experimentally-induced prostatic hyperplasia in young beagles: a model to evaluate the chemotherapeutic effects of gossypol.

This study investigated the effect of gossypol on hyperplastic canine prostates induced with long-term administration of androgen and estrogen. Twelve 16-week-old male beagle dogs were divided evenly (n = 3) into 4 treatment groups: (1) CONTROL: vehicle only; (2) Gossypol-Treated: 20 mg/kg gossypol acetic acid; (3) Steroid-Induced: 4 mg/kg testosterone and 40 micrograms/kg estradiol-17 beta; (4) Gossypol-Treated and Steroid-Induced: 4 mg/kg testosterone, 40 micrograms/kg estradiol-17 beta and 20 mg/kg gossypol. The subjects received treatments every other day for 1 month. The beagles treated with steroids developed an acute enlargement (approximately 10-fold) of the prostate as compared to control. The prostatic acini were underdeveloped and characterized by simple squamous to low cuboidal epithelium in the control subjects while acini in steroid-induced subjects were characterized by simple tall columnar epithelium. The subjects treated with gossypol had prostates histologically similar to controls with the exception of loosened periurethral connective tissue. Serum testosterone and estradiol-17 beta levels imply that gossypol can reduce steroid hormonal levels. Mean serum testosterone levels in gossypol-treated subjects were reduced approximately 50% from controls. Serum biochemistry profiles indicate that steroid and/or gossypol treatments were not toxic at the doses and duration used in this study. These observations imply that gossypol and steroid hormones can interact to alter prostate development and gossypol metabolism and/or clearance.

Keratinocyte growth factor (KGF/FGF-7) has a paracrine role in canine prostate: molecular cloning of mRNA encoding canine KGF.

cDNA encoding the canine keratinocyte growth factor (KGF) was cloned from normal canine prostate tissue. The authentic canine KGF cDNA sequence, 686 bp in length, spans the protein-coding region and 88 bp of the 5' and 19 bp of the 3' untranslated regions of canine KGF. The predicted amino acid sequence of canine KGF is composed of 194 amino acid residues. Canine KGF exhibits highest homology with the human KGF cDNA and amino acid sequences (95.8% and 97.4%, respectively), while it demonstrates the lowest homology with the rat sequences at 88.0% and 92.3%, respectively. The degrees of homology with mouse cDNA and amino acid sequences are 91.8% and 95.9%, respectively. By using RNase protection assay, KGF was shown to be expressed in normal prostate tissues of both mature and young (5-month-old) dogs. In vitro, the recombinant canine KGF has mitogenic activity on cultured canine epithelial cells, whereas it has no effect on cultured canine prostatic stromal cells. This novel canine KGF cDNA may be a valuable tool in the study of human benign prostatic hyperplasia using the canine prostatic as a model.

Detection of keratinocyte growth factor (KGF) messenger ribonucleic acid and immunolocalization of KGF in the canine testis.

Keratinocyte growth factor (KGF) was originally discovered in human embryonic lung fibroblasts and is a member of the fibroblast growth factor (FGF) family. Members of the FGF family have been shown to regulate testicular function. However, the recently discovered KGF has not been studied in the testis. KGF has been detected in many other tissues, including the prostate, an organ whose development and function have been associated with presence of the testis. In this study, KGF mRNA was detected in the whole testis using reverse transcription polymerase chain reaction (RT-PCR). The 575-bp KGF-specific product was detected along with a 594-bp ß-actin-specific product. To identify the cell types in which KGF mRNA was predominantly expressed, interstitial cells were physically separated from seminiferous tubules. The interstitial cells were then sorted on a discontinuous Percoll gradient and total cellular mRNAs isolated. Using RT-PCR and Southern hybridization with specific cDNA probes, the KGF mRNA was detected in interstitial cells. KGF expression levels were then evaluated semiquantitatively with a competitive RT-PCR assay. KGF expression levels were highest in interstitial cells that equilibrated between 20 and 30% Percoll. Enriched Leydig cells and seminiferous tubules expressed low levels of KGF. Finally, immunohistochemical analysis was performed on canine testes using a rabbit anti-KGF polyclonal antibody. The KGF protein was localized predominantly to peritubular cells of the canine testis. These results suggest that KGF is synthesized in the canine testis.