Effects of tumor necrosis factor-alpha on luteinizing hormone-stimulated prorenin production by bovine ovarian thecal cells in vitro.

Investigations have been carried out to determine if the cytokine tumor necrosis factor-alpha (TNF alpha), a putative intraovarian regulator, plays a role in the regulation of the ovarian prorenin-renin-angiotensin system. Addition of TNF alpha to cultured bovine thecal cells resulted in a dose-dependent inhibition of LH- or 8-bromo-cAMP-stimulated production of prorenin and renin by the cells in a noncytotoxic manner. No clear inhibitory effect on progesterone production was noted. There was no inhibition of LH- or forskolin-stimulated cAMP formation by TNF alpha. The time-course experiment with TNF alpha revealed that the synthesis, rather than the secretion, of prorenin was inhibited. Also, it was evident that to observe a maximal inhibitory effect, it was necessary to add TNF alpha either before or together with LH. With the increasing delay in the addition of TNF alpha relative to the time of addition of LH, the extent of inhibition gradually decreased, and TNF alpha added 6 h after the addition of LH failed to produce any inhibitory effect. The results obtained permit us to conclude that TNF alpha can counterregulate LH-stimulated prorenin production by thecal cells in culture. The TNF alpha-induced lesion appears to be located at an early step of the biosynthetic pathway of prorenin, which is distal to the activation of LH receptor-coupled adenylate cyclase. Thus, this cytokine appears to be an important intraovarian regulator of prorenin production, a process that is under the stimulatory control of the pituitary gonadotropin.

Characterization of angiotensin-II receptor subtype on bovine thecal cells and its regulation by luteinizing hormone.

In this study the primary objectives were to localize angiotensin-II (AII) receptors on specific ovarian cells and determine whether these receptors are regulated by LH. AII receptor analysis, carried out using membrane fractions prepared from isolated thecal, granulosa, and luteal cells from bovine ovary, revealed that [125I]AII-binding sites were present only on thecal cells. The Kd and binding capacity were determined to be 0.29 +/- 0.08 nM and 66.9 +/- 8.1 fmol/mg membrane protein (mean +/- SEM from three experiments, each with duplicate determinations), respectively. None of the peptides unrelated to AII affected the binding of [125I]AII. Unlabeled AII, saralasin, and AIII were equipotent (IC50, approximately 5 nM for all three peptides) in competing with the radioligand. However, the binding affinity for AI was less by almost 2 log units. Using AII receptor subtype-specific nonpeptide antagonists, Losartan [a selective antagonist for the type 1 AII (AT1) receptor] and PD 123319 [a selective antagonist for the type 2 AII (AT2) receptor], AII receptors on thecal cells could be classified pharmacologically as AT2-type receptors. The IC50 determined from the competitive binding inhibition experiments for the various unlabeled competing substances were 5 nM, 20 nM, 200 nM, and 200 microM with respect to AII, p-amino-phenylalanine-AII, PD123319, and Losartan, respectively. Thecal cells cultured in a serum-free medium also expressed AII receptors, which could be up-regulated by LH or 8-bromo-cAMP in a dose-dependent manner. The number of AII receptors on thecal cells nearly doubled when the cells were cultured in the presence of 100 ng/ml LH, with little change in their Kd value. This increase in the number of AII receptors was inhibitable by a protein synthesis inhibitor, cycloheximide. In summary, we have demonstrated that in the bovine ovary, AII receptors belonging to AT2 subclass are predominantly expressed on thecal cells, and these receptors can be up-regulated by LH via a cAMP-dependent mechanism. Thus, the bovine thecal cells in primary culture can potentially become a useful in vitro system to study the mechanism of regulation of AII receptor induction as well as the so far unknown function of this class of receptor.

Inhibitory effects of a tumor-promoting phorbol ester on luteinizing hormone-stimulated renin and prorenin production by cultured bovine theca cells.

In this report, we have described a serum free culture method for culturing bovine theca cells in vitro. These cultured cells could be stimulated with LH to produce renin and prorenin. Though intracellular prorenin as well as renin was increased by LH, no renin was found to be released into the extracellular medium. The extracellular medium contained prorenin exclusively. The stimulatory effect of LH could be mimicked by 8-bromo-cAMP (8-Br-cAMP) and forskolin, suggesting cAMP to be the second messenger involved. Induction of renin and prorenin production by LH in cultured theca cells was dependent upon de novo protein synthesis, since the action of LH could be completely blocked by the protein synthesis inhibitor, cycloheximide. The stimulatory effects of LH, 8-Br-cAMP, or forskolin were also blocked by 4 beta-phorbol 12-myristate 13-acetate (PMA), via putative activation of protein kinase C. However, to completely block the stimulatory effect of LH or 8-Br-cAMP, it was necessary to add PMA either before LH/8-Br-cAMP was added or simultaneously with the agonists. With progressive delay in addition of PMA after LH/8-Br-cAMP was added, the extent of inhibition decreased gradually and if PMA was added anytime after 4 h of LH/8-Br-cAMP addition, no inhibitory effect could be observed. Thus we have shown that renin/prorenin production by theca cells in vitro can be directly regulated by LH in a cAMP-dependent manner. Prorenin is preferentially released from the cells. Activation of protein kinase C appears to inhibit some very early steps in the induction of prorenin/renin production which however is distal to cAMP formation.

The relationship between prorenin levels in follicular fluid and follicular atresia in bovine ovaries.

Bovine follicles having a higher concentration of progesterone than estradiol in the follicular fluid can be considered as atretic. Since we observed previously that there was an inverse relationship between the follicular fluid estradiol to progesterone (E/P) ratio and the prorenin level, we have proposed that a high prorenin level may be associated with follicular atresia. The aim of the present study was to corroborate this hypothesis by including additional indices to distinguish unambiguously between atretic and nonatretic follicles and to compare the prorenin levels in these two groups of follicles. The present study included examination of more than 200 follicles in the follicular fluid of which we have measured steroid and prorenin levels. The results obtained show a highly significant negative correlation between the prorenin level on the one hand and the E/P ratio, estrogen to total androgen ratio, or estradiol concentration on the other hand. As a further criterion for atresia, we have examined the histological characteristics of the follicles by light and electron microscopy and have found that 90% of histologically characterized atretic follicles had an E/P ratio less than 1 and an average prorenin level four to five times higher than nonatretic follicles. Finally, when we determined the FSH-stimulated cAMP response and the aromatase activity, in terms of the ability to convert exogenous androgen to estrogen in granulosa cells isolated from individual follicles, we observed a markedly higher prorenin level in the fluid of follicles whose granulosa cells responded poorly to FSH and showed a low aromatase activity, compared to follicles whose granulosa cells responded strongly to FSH and contained high aromatase activity. In summary, follicles that were classified as atretic on the basis of a number of biochemical and histological parameters contained significantly higher prorenin levels in their follicular fluid than nonatretic ones. Thus, a high follicular fluid prorenin level is a valid indicator for follicular atresia in bovine ovaries. However, the reason for this increase in follicular fluid prorenin level and whether this increase is a cause or a consequence of atresia remains to be determined.

Human seminal fluid contains significant quantities of prorenin: its correlation with the sperm density.

The relevance of the tissue prorenin-renin-angiotensin system (PRAS) to male reproduction has been suggested by several investigators in the past. Although the presence of angiotensin converting enzyme in semen has been demonstrated, unequivocal evidence for the presence of prorenin and renin in the semen is not yet available. We have used a specific immunoradiometric assay based on an antibody directed against the pro-segment of the prorenin molecule to demonstrate that significant quantities of prorenin are present in human semen samples. Although semen is a rich source of proteases and protease inhibitors, the assay used by us, unlike the usual enzymatic renin assay, is not affected by such proteases, and their inhibitors. Furthermore, Western blotting data clearly demonstrated that prorenin is present in semen as a 48 kDa protein. In a majority of semen samples, the prorenin content was found to be several fold greater than that measured in EDTA-plasma samples. Interestingly, the level of prorenin was found to be directly proportional to the sperm density in semen samples. Our results suggest that seminal prorenin is produced locally within the male reproductive system, although its exact origin is yet to be defined, that a complete prorenin-renin-angiotensin system exists in human semen and that this system may be relevant to sperm function.

The effect of microtubule-disrupting drugs on morphology, progesterone and prorenin secretion of bovine cultured ovarian theca cells.

The effect of three microtubule-disrupting drugs (vinblastine, colchicine and nocodazole) on basal and LH-stimulated secretion of progesterone and prorenin by bovine theca cells was studied. Microtubules were visualized immunocytochemically using a monoclonal antibody against the alpha-subunit of tubulin and a secondary antibody conjugated with rhodamine. Progesterone and prorenin secretion were detected by adequate radioimmunoassays. Theca cells treated with LH alone or with a combination of LH, colchicine and vinblastine, showed round shape and disorganization of microtubules which were more obvious than after treatment with the two disrupting drugs alone. Nocodazole or LH treatment alone resulted in the formation of numerous cell processes, conspicuously different in shape from those in the controls. All three drugs increased basal progesterone secretion independently on the dose, but were without effect on basal prorenin secretion. LH-stimulated progesterone secretion was significantly suppressed by each of the three drugs while LH-stimulated prorenin secretion was decreased only by high doses of vinblastine and nocodazole. Colchicine in all three doses used did not exert any significant effect. The results obtained suggest that microtubules are involved in progesterone but not in prorenin secretion.

Paracrine regulation of the bovine ovarian prorenin-renin-angiotensin-system.

In the bovine ovary, prorenin production by theca cells is known to be regulated by LH. In the present study the aim was to evaluate whether LH-stimulated prorenin production could be further modulated by intraovarian factors in vitro. Theca cells were isolated from bovine ovaries by enzymatic dispersion, purified over Percoll gradient and cultured under serum-free conditions with LH/8Br-cAMP in the absence or presence of different steroids and growth factors and the amount of prorenin secreted into the medium was measured. None of the steroids used (androstendione, estradiol, progesterone) influenced the basal or LH-stimulated prorenin production. In contrast, cytokines and growth factors, like TNF alpha, TGF alpha, TGF beta and bFGF proved to be important regulators of prorenin synthesis. Whereas TNF alpha, TGF alpha and bFGF significantly reduced the LH- and 8Br-cAMP-induced prorenin synthesis at a site distal to cAMP formation, addition of TGF beta led to a further increase in the amount of prorenin secreted into the medium. None of the agonists had an influence on prorenin production by itself. The observed effects of cytokines and growth factors seemed to be confined to prorenin production only, since cell number, cell viability and steroidogenic response were not at all influenced by the agonists. We conclude that, although LH appears to be the primary regulator of ovarian prorenin production, several paracrine/autocrine intraovarian factors may be involved in "finely tuning" the secretion of prorenin, which is necessary for maintaining the differentiated state of the follicle.

Isogeneic MSC application in a rat model of acute renal allograft rejection modulates immune response but does not prolong allograft survival.

Application of mesenchymal stromal cells (MSCs) has been proposed for solid organ transplantation based on their potent immuno-modulatory effects in vitro and in vivo. We investigated the potential of MSCs to improve acceptance of kidney transplants in an MHC-incompatible rat model including isogeneic kidney transplantation (RTx) as control. MSCs were administered i.v. or i.a. at time of transplantation. No immunosuppression was applied. Renal function was monitored by serum-creatinine, histopathology, immunochemistry for graft infiltrating cells and expressions of inflammatory genes. We demonstrated the short-term beneficial effects of MSC injection. In the long term, however, MSC-related life-threatening/shortening events (thrombotic microangiopathy, infarctions, infections) were evident despite decreased T- and B-cell infiltration, lower interstitial inflammation and downregulated inflammatory genes particularly after i.a. MSC injection. We conclude that i.a. MSC administration provides efficient immunomodulation after allogeneic RTx, although timing and co-treatment strategies need further fine-tuning to develop the full potential of powerful cell therapy in solid organ transplantation.

Expression of osteopontin (OPN) mRNA in bovine ovarian follicles and corpora lutea.

The matricellular protein osteopontin (OPN) plays a role in various physiological processes, including angiogenesis and tissue remodelling. As these processes are essential for the maintenance of ovarian physiology, the aim of the study was to investigate the expression of OPN (mRNA) in ovarian cells and to evaluate whether it can be regulated by gonadotrophins. Using conventional RT-PCR and real-time PCR, we have detected and quantified OPN mRNA as well as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA expression in bovine granulosa, theca and luteal cells. In all cells examined, both genes were found in equal amounts and no striking variations in the expression could be observed between granulosa, theca and luteal cells. Furthermore, no effect on either OPN or GAPDH mRNA expression was evident after culturing ovarian cells in the presence of gonadotrophic hormones, although the cells were still highly responsive in terms of cAMP formation. Although neither variations between different cell types nor a regulation of OPN mRNA expression by gonadotrophic hormones could be detected, the high and unambiguous mRNA expression in steroidogenic cells suggests that OPN should be added to the growing list of intraovarian factors which may be involved in ovarian physiology.

Stimulation of nitric oxide-cyclic guanosine monophosphate pathway in bovine ovarian theca cells by tumor necrosis factor alpha (TNF alpha). Is this pathway implicated in the TNF alpha-induced inhibition of luteinizing hormone-stimulated prorenin production?

Gonadal function is known to be controlled by many factors, including locally acting cytokines like tumor necrosis factor alpha (TNF alpha). One of the ways this cytokine acts is via the nitric oxide (NO)-cGMP pathway. Since we have shown that in the ovary theca cells are a target of TNF alpha's action, it was of interest to determine whether TNF alpha stimulates the NO-cGMP pathway in these cells and whether such a mechanism can be implicated in the observed TNF alpha-mediated inhibition of LH-stimulated prorenin synthesis and secretion. Treatment of isolated theca cells with TNF alpha resulted in a dose- and time-dependent increase in cGMP production. This increase was not detectable until 6 h after the addition of TNF alpha and was totally abolished by the protein synthesis inhibitor cycloheximide. Addition of either L-N6-nitroarginine methyl ester (L-NAME), an inhibitor of all three NO synthase (NOS) isoforms or 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine (AMT), a specific inhibitor of the inducible isoform of the enzyme, likewise reversed the action of TNF alpha on cGMP formation. Finally, addition of 1H-[1,2,4]oxadiazolo [4,3-a] quinoxalin 1-one (ODQ), an inhibitor of NO-sensitive soluble guanylate cyclase, resulted in a concentration-dependent reduction of TNF alpha-stimulated cGMP formation. In contrast, the TNF alpha-mediated inhibition of LH-stimulated prorenin secretion was not affected by either L-NAME, AMT, or ODQ. Also the addition of stimulators of soluble guanylate cyclase, sodium nitroprusside, and S-nitroso-N-acetylpenicillamine, or 8 bromo-cGMP had no effect on the action of LH on theca cells. We conclude that although TNF alpha is able to stimulate cGMP formation in theca cells by inducing the expression of inducible NOS, the mechanism underlying the TNF alpha-mediated inhibition of LH-stimulated prorenin production is independent of its ability to induce cGMP formation.

Follicular maturation and atresia--possible role of intraovarian regulatory factors.

In addition to the gonadotrophins, factors known to be present or produced within the ovary are also involved in the regulation of follicular development and atresia. One such modulator of ovarian function is the prorenin-renin-angiotensin system (PRAS) which seems to be associated with atresia. Although it is primarily under gonadotrophic control, the expression of the ovarian PRAS is also shown here to be subject to modulation by intraovarian factors. Addition of granulosa cell conditioned medium to bovine theca cells inhibited the LH-stimulated prorenin secretion in a dose-dependent manner, which leads to the assumption that granulosa cells secrete a factor(s) that, even in the presence of LH, keeps prorenin secretion low. Since growth factors and cytokines are known to be produced by granulosa cells, we have evaluated the effects of tumour necrosis factor alpha, basic fibroblast growth factor and transforming growth factor alpha on the LH- and 8Br-cAMP-induced prorenin secretion, and observed that all agonists inhibited the LH- and 8Br-cAMP-stimulated prorenin secretion in a dose-dependent manner. In contrast, addition of transforming growth factor beta, a secretory product of theca cells, further augmented gonadotrophin-induced prorenin synthesis. In conclusion, we assume that in the presence of healthy granulosa cells, suppression of the ovarian PRAS by granulosa cell derived factors may prevent a follicle from undergoing atresia, whereas in the presence of atretic granulosa cells, theca cell derived factors may enhance the expression of the PRAS, resulting in follicular atresia.