Nociceptin is upregulated by FSH signaling in Sertoli cells in murine testes

In postnatal testes, follicle-stimulating hormone (FSH) acts on somatic Sertoli cells to activate gene expression directly via an intracellular signaling pathway composed of cAMP, cAMP-dependent protein kinase (PKA), and cAMP-response element-binding protein (CREB), and promotes germ cell development indirectly. Yet, the paracrine factors mediating the FSH effects to germ cells remained elusive. Here we show that nociceptin, known as a neuropeptide, is upregulated by FSH through cAMP/PKA/CREB pathway in Sertoli cells in murine testes. Chromatin immunoprecipitation from Sertoli cells shows that CREB phosphorylated at Ser133 associates with prepronociceptin gene encoding nociceptin. Analyses with Sertoli cells and testes demonstrates that both prepronociceptin mRNA and the nociceptin peptide are induced after FSH signaling is activated. In addition, the nociceptin peptide is induced in testes after 9days post partum following FSH surge. Thus, our findings may identify nociceptin as a novel paracrine mediator of the FSH effects in the regulation of spermatogenesis.     

Synergistic effects of cyclic AMP and Ca2+ ionophore A23187 on de novo synthesis of histidine decarboxylase in mastocytoma P-815 cells.

In the preceding paper (Kawai, H. et al. (1992) Biochim. Biophys. Acta 1133, 172-178), we reported that in mastocytoma P-815 cells dexamethasone and 12-O-tetradecanoylphorbol-13-acetate (TPA) synergistically enhanced the de novo synthesis of L-histidine decarboxylase (HDC). Here we found that Ca2+ acted synergistically with cAMP in the induction of HDC mRNA and HDC activity in mastocytoma P-815 cells, and that the mechanism underlying the enzyme induction by Ca2+ plus cAMP was distinguishable from that by dexamethasone plus TPA. Ca2+ ionophore A23187, itself having no significant activity, markedly enhanced the induction of HDC activity by N6,O2'-dibutyryl cAMP (db cAMP) or cAMP-inducible prostaglandins such as PGE1, PGE2 and PGI2 in the presence of the phosphodiesterase inhibitor, Ro201724. However, A23187 had little effect on increases in HDC activity induced by other known stimulants, such as TPA, dexamethasone and sodium butyrate. These results suggest that A23187 has a specific effect on the induction of HDC activity due to an increased level of cAMP. The finding that both A23187 and cAMP enhanced HDC activity suggests that both Ca2+/calmodulin and cyclic nucleotide dependent protein kinase play essential roles in the process of enhancement of HDC activity. To examine this possibility, we studied the effects of W-7, an inhibitor of calmodulin, removal of extracellular Ca2+, and H-8, an inhibitor of cAMP-dependent protein kinase, on the enhancing activity of A23187 plus db cAMP. The enhancement of HDC activity by A23187 plus db cAMP was inhibited by W-7, removal of extracellular Ca2+, and H-8. The increase in HDC activity was due to the de novo synthesis of the enzyme, since it was suppressed by the addition of cycloheximide or actinomycin D, and was well correlated with the marked accumulation of a 2.7 kilobase HDC mRNA. Furthermore, the mechanism underlying the induction of HDC by db cAMP plus A23187 is distinguishable from that in the case of dexamethasone plus TPA, since preexposure to dexamethasone plus TPA for 12 h, for a plateau level to be reached, did not affect the subsequent increase in HDC activity due to db cAMP plus A23187.     

Protein phosphorylation in intact coronary endothelial cells by bradykinin

In cultured coronary endothelial cells obtained from guinea pig hearts, bradykinin (10(-6) M) stimulated the 32Pi-incorporation into 5 substrate proteins with molecular weights corresponding to 27, 32, 60, 86 and 100 kDa. The time course of phosphorylation of the 60, 86 and 100 kDa proteins was rapid (within 30 s), but transient (max. within 1-2 min.), while the 32Pi incorporation into the 27 and 32 kDa protein was delayed but increased within 10 minutes. Ca+(+)-ionophore A 23187 (10(-5) M) and 12-O-tetradecanoylphorbol-13-acetate (TPA) (10(-5) M) both mimicked the effects of the bradykinin induced phosphorylation pattern. While A 23187 enhanced the phosphorylation of the 27, 60 and 100 kDa substrates, TPA increased the 32Pi-incorporation into the 32 and 86 kDa proteins. Furthermore the time course of protein phosphorylation elicited by A 23187 and TPA showed marked similarities to those obtained with bradykinin. Our findings are consistent with the view, that stimulation of coronary endothelial bradykinin-receptors activates both Ca+(+)-dependent protein kinases and protein kinase C.     

Granulosa cells from pig follicles of different sizes demonstrate maturational differences in their steroidogenic responses to FSH, calcium ionophore A23187, and phorbol diester

Cultures of granulosa cells from small (less than 3 mm), medium (3-6 mm), or large (8-10 mm) pig follicles were treated as follows: (1) basal controls, (2) cyclic adenosine 3',5'-monophosphate (cAMP) pathway agonists (pig FSH: 100 ng/ml; forskolin: 10 microM; dibutyryl cAMP; 1 mM), (3) calcium ionophore A23187 (0.005-1 micrograms), or (4) phorbol 12-myristate 13-acetate (TPA; 0.05-4 ng/ml). The combination of A23187 or TPA together with cAMP agonists was also examined in cultures of granulosa cells from follicles of different sizes. All substances were added at the time of culture, and oestradiol and progesterone were measured in the culture media after 48 h. All cAMP agonists were most potent in their stimulation of steroidogenesis (as a % of control) in cells from small follicles (P less than 0.05) with the exception of forskolin, which increased oestradiol in cells from large follicles to a greater extent than in cells of small follicles (P less than 0.05) (cells from medium follicles demonstrated less stimulation than those from small follicles except in progesterone production, for which FSH was equipotent). With the exception of forskolin, however, granulosa from large follicles showed little (oestradiol) or no stimulation (progesterone) with cAMP agonists. Under basal conditions, A23187 inhibited progesterone in all groups (P less than 0.05), and oestradiol production was reduced in granulosa cells from small follicles (P less than 0.05), unchanged in cells from medium follicles, and significantly stimulated in cells from large follicles. A23187 inhibited the enhanced production of both hormones after administration of cAMP agonists from cells of small and medium follicles (P less than 0.05), with inhibition significantly greater in cells of small follicles compared with medium. In cells from large follicles challenged with cAMP agonists, A23187 inhibited progesterone but stimulated oestradiol production; substitution of TPA (a protein kinase C stimulator) for A23187 gave identical results under basal or FSH-treated cultures of granulosa cells from small-, medium- or large-sized follicles. Our results suggest that TPA, A23187 and cAMP agonists modulate steroidogenesis differently in pig granulosa cells, depending on the stage of maturation of the follicle. Oestradiol production in granulosa cells from large preovulatory follicles may come under the stimulatory control of regulators of protein kinase C as in follicles near ovulation.     

Temporal patterns of protein phosphorylation after angiotensin II, A23187 and/or 12-O-tetradecanoylphorbol 13-acetate in adrenal glomerulosa cells.

The temporal patterns of protein phosphorylation in the adrenal glomerulosa cell were analysed by two-dimensional electrophoresis after stimulation with 10 nM-angiotensin II or various agents [10 nM-12-O-tetradecanoylphorbol 13-acetate (TPA), 50 nM-A23187, 1 microM-nitrendipine], administered singly or in combination. These patterns were compared with the temporal patterns of aldosterone secretion induced by the same agonists and antagonists. After 1 and 30 min of stimulation with angiotensin II, different patterns of protein phosphorylation were observed. A comparison of these patterns reveals that: the phosphorylation of only one protein was persistently enhanced during the continuous incubation with angiotensin II; the phosphorylation of five proteins was transiently enhanced (at 1 min but not 30 min); and the phosphorylation of three proteins did not occur at 1 min but was seen at 30 min. Addition of the phorbol ester TPA alone, which at 30 min is without effect in enhancing aldosterone production, has no effect on protein phosphorylation. The combined addition of TPA and the Ca2+ ionophore, A23187, which, like angiotensin II, evokes a sustained increase in aldosterone production, reproduced the temporal patterns of protein phosphorylation seen after angiotensin II action. Manipulations (A23187 alone, angiotensin II plus nitrendipine) which evoke only a transient rise in aldosterone production rate induce a transient rise in cellular protein phosphorylation. The 1 min patterns of phosphorylation seen after A23187 or combined angiotensin II and nitrendipine (a Ca2+ channel antagonist) are similar to those observed after 1 min of angiotensin II stimulation. These results suggest that, when angiotensin II acts, the initial cellular response is mediated by a different mechanism than that responsible for the sustained response.     

Alteration of cell membrane proteoglycans impairs FSH receptor/Gs coupling and ERK activation through PP2A-dependent mechanisms in immature rat Sertoli cells

Background

During the pre-pubertal life, the cessation of Sertoli cell proliferation and the onset of differentiation are associated with a shift in the FSH-mediated signaling leading to inhibition of the ERK-mitogenic pathway and to a concomitant sensitization of cAMP/PKA pathway.

Methods

To highlight the role of cell proteoglycans (PGs) in the shift of FSH signaling, both FSH-induced cAMP production and ERK1/2 inactivation were studied in untreated and sodium chlorate PG-depleted cultured Sertoli cells from 20 day-old rats.

Results

Depletion of cell membrane PGs by sodium chlorate reduced FSH-, but not cholera toxin-stimulated cAMP production as well as basal ERK phosphorylation through an okadaic acid (OA)-sensitive mechanism. Involvement of PP2A was further substantiated by a marked decrease in membrane- associated PP2A activity under SC conditions and by the OA-induced restoration of PKA-dependent ERK inactivation in SC-treated cells.

Conclusions

In 20-day-old rat Sertoli cells, transmembrane cell PGs, through tethering/activation of PP2A activity exerts regulatory control on both FSH receptor/Gs coupling and ERK phosphorylation.

General significance

Besides their antiproliferative roles, cell PGs such as syndecan-1, could be involved in the increase in cAMP response to FSH occurring in Sertoli cells at the time of transition between proliferative and differentiated states.     

Upregulation of heat shock protein 32 in Sertoli cells alleviates the impairments caused by heat shock-induced apoptosis in mouse testis

Heat stress results in apoptosis in testicular germ cells. A small heat shock protein (hsp), hsp32, is induced by heat stress in the testis, but little is known about its definitive function in physiological processes. To clarify the underlying role of hsp32, hsp32 expression and related signals in the heat shock pathway were analysed in mouse testes and Sertoli cells after heat stress in vivo and in vitro; meanwhile, expression of hsp32 was silenced only in the Sertoli cells using three different small interfering RNAs (siRNAs) to further verify the functional role of hsp32 in Sertoli cells, and hsp32-derived carbon monoxide (CO) contents in cultured media were analysed to clarify whether hsp32-derived CO involve in the apoptosis regulation mechanisms. The results from the in vivo experiment showed that the high expression levels of hsp32 (P?<?0.05) were observed whether chronic, moderate or acute, transient heat exposure. The in vitro experiment showed that acute, transient heat exposure resulted in increases in Sertoli cells apoptosis (P?<?0.01), the expression of hsp32 and caspase-3 activity; hsp32-siRNA knockdown of hsp32 expression resulted in upregulated apoptosis (P?<?0.01) and caspase-3 activity (P?<?0.01) in the Sertoli cells and hyperthermia increases CO (P?<?0.01) release by Sertoli cells. The results suggested that upregulating hsp32 in Sertoli cells inhibits caspase-3 activity and alleviates heat-induced impairments in mouse testis; hsp32-derived CO may involve in the regulation mechanism.     

Follicle-stimulating hormone-induced lactate secretion by cultured Sertoli cells does not require extracellular calcium

Sertoli cells of the testis secrete lactate in response to follicle-stimulating hormone (FSH). It is thought that the developing germ cells use lactate as an energy substrate preferentially over glucose. However, the biochemical mechanism(s) involved in the regulation of lactate secretion in response to FSH are unknown. The purpose of this study was to determine if extracellular calcium was important for the actions of FSH during this response. It was found that the FSH-induced increase in lactate production by Sertoli cells was not dependent upon the presence of extracellular calcium. However, A23187 (a calcium ionophore) stimulated lactate secretion in the presence of extracellular calcium. When FSH and A23187 were tested together at maximal concentrations, more lactate was secreted than when either FSH or A23187 was tested alone. Neither verapamil, nifedipine nor diltiazem (calcium channel "blockers") were able to inhibit the ability of FSH to increase lactate secretion. These results indicate that FSH-induced secretion of lactate by cultured Sertoli cells is not dependent upon extracellular calcium.     

Identification and characterization of a novel protein from Sertoli cells, PASS1, that associates with mammalian small stress protein hsp27

hsp27 is involved in development of tolerance to stress, possibly by its involvement in molecular chaperoning, maintenance of glutathione status, and/or modulation of microfilament structure and function. We hypothesize that hsp27 function depends on specific association with other proteins. To discover proteins that associate with hsp27, we made a differentiated rat Sertoli cell cDNA expression library and screened it using the yeast two-hybrid system. We obtained a cDNA coding for a novel protein of 428 amino acids that we have named PASS1 (protein associated with small stress proteins 1). BLAST searches did not reveal major similarity of PASS1 to any known protein, but the cDNA sequence matched several mouse EST clones and shares 34% homology with a Caenorhabditis elegans genomic sequence. In vitro, bacterially expressed glutathione S-transferase-PASS1 fusion protein bound to hsp27, and hsp27 was co-immunoprecipitated with c-Myc-tagged PASS1 overexpressed in several cell lines. The region of PASS1 responsible for association with hsp27 was identified as existing predominantly between amino acids 108 and 208 of PASS1. Northern hybridization and Western blot analysis demonstrated that PASS1 is expressed in several tissues, with the highest expression occurring in testis, primarily in Sertoli cells. The presence of a 1.4-kilobase PASS1 mRNA in kidney as well as the 1. 8-kilobase mRNA seen in other tissues suggests that alternate splicing may occur in this organ. Ectopic expression of PASS1 in two cultured cell lines was observed to inhibit the ability of hsp27 to protect cells against heat shock, indicating that PASS1 does interact with hsp27 in the live cell.     

Signal transduction pathways in FSH regulation of rat Sertoli cell proliferation

The final number of Sertoli cells reached during the proliferative periods determines sperm production capacity in adulthood. It is well known that FSH is the major Sertoli cell mitogen; however, little is known about the signal transduction pathways that regulate the proliferation of Sertoli cells. The hypothesis of this investigation was that FSH regulates proliferation through a PI3K/Akt/mTORC1 pathway, and additionally, AMPK-dependent mechanisms counteract FSH proliferative effects. The present study was performed in 8-day-old rat Sertoli cell cultures. The results presented herein show that FSH, in addition to increasing p-Akt, p-mTOR, and p-p70S6K levels, increases p-PRAS40 levels, probably contributing to improving mTORC1 signaling. Furthermore, the decrease in FSH-stimulated p-Akt, p-mTOR, p-p70S6K, and p-PRAS40 levels in the presence of wortmannin emphasizes the participation of PI3K in FSH signaling. Additionally, the inhibition of FSH-stimulated Sertoli cell proliferation by the effect of wortmannin and rapamycin point to the relevance of the PI3K/Akt/mTORC1 signaling pathway in the mitotic activity of FSH. On the other hand, by activating AMPK, several interesting observations were made. Activation of AMPK produced an increase in Raptor phosphorylation, a decrease in p70S6K phosphorylation, and a decrease in FSH-stimulated Sertoli cell proliferation. The decrease in FSH-stimulated cell proliferation was accompanied by an increased expression of the cyclin-dependent kinase inhibitors (CDKIs) p19INK4d, p21Cip1, and p27Kip1. In summary, it is concluded that FSH regulates Sertoli cell proliferation with the participation of a PI3K/Akt/mTORC1 pathway and that AMPK activation may be involved in the detention of proliferation by, at least in part, a decrease in mTORC1 signaling and an increase in CDKI expression.     

Regulation of tissue inhibitor of metalloproteinases-1 in rat Sertoli cells: induction by germ cell residual bodies, interleukin-1alpha, and second messengers

In the testis, FSH has been shown to induce the expression and secretion of tissue inhibitor of metalloproteinases-1 (TIMP-1) from Sertoli cells in vitro. This study was performed to elucidate further the cellular origin of testicular TIMP-1 and its expression by hormonal and paracrine factors. This is the first report on the expression of testicular TIMP-1 in vivo. TIMP-1 mRNA in whole testis was decreased after hypophysectomy and strongly increased by the injection of FSH-S17 to hypophysectomized rats. Primary cultures of both peritubular and Sertoli cells showed basal expression of TIMP-1 mRNA. In contrast, we were unable to detect TIMP-1 mRNA in Leydig cells, freshly isolated immature germ cells (primary spermatocytes and spermatids), or residual bodies. We further show that treatment of Sertoli cells with 8-(4-chlorophenyl)thio-cAMP (8-CPTcAMP) in combination with 12-O-tetradecanoylphorbol 13-acetate (TPA) or Ca(2+) inducers (calcium ionophore A23187 or thapsigargin) had additive (TPA) and synergistic effects (Ca(2+)) on the level of TIMP-1 mRNA and secreted protein. We also show that both the level of TIMP-1 mRNA and secreted protein from Sertoli cells were strongly increased by residual bodies, as well as by the cytokine interleukin-1alpha. TIMP-1 was not up-regulated by either 8-CPTcAMP or interleukin-1alpha in peritubular cells. In contrast to the regulated secretory fraction of TIMP-1, we also detected constitutively expressed immunoreactive TIMP-1 in the nucleus of Sertoli cells, suggesting a role of nuclear TIMP-1 in these cells. In conclusion, our data show that secretion of TIMP-1 from Sertoli cells is highly regulated by hormonal and local processes in the testis, indicating that TIMP-1 is of physiological importance during both testicular development and spermatogenesis.     

A novel hypothalamic peptide, pituitary adenylate cyclase activating peptide, modulates Sertoli cell function in vitro.

Pituitary adenylate cyclase-activating peptide (PACAP), a novel hypothalamic peptide that has been shown to exist in several tissues including the testis, was examined for its effects on cultured rat Sertoli cells. PACAP stimulates cAMP accumulation in Sertoli cells cultured from 15-day-old rats in the presence or absence of methylisobutylxanthine, a phosphodiesterase inhibitor, and in the presence of pertussis toxin, a blocker of the adenylate cyclase inhibitory pathway. Maximal stimulation, which is 20-40% of that attainable with FSH, occurs at PACAP concentrations of 10 nM: the ED50 is approximately 100 pM. The ability of PACAP to stimulate Sertoli cell cAMP declines with increasing age of donor animals (15-60 days of age) in a fashion similar to the FSH effect. PACAP stimulation of Sertoli cell cAMP accumulation is additive with submaximal, but not maximal, concentrations of FSH or forskolin. PACAP also stimulates the secretion of lactate, estradiol, and inhibin in a concentration-dependent manner. The stimulation of Sertoli cell cAMP accumulation by PACAP is not altered by a vasoactive intestinal peptide antagonist, and vasoactive intestinal peptide alone does not stimulate cAMP accumulation, indicating that PACAP is not acting via vasoactive intestinal peptide receptors. Further experiments are needed to determine whether PACAP is synthesized within the testis and if so, in which cell types; however, the present data clearly demonstrate that PACAP can modulate Sertoli cell function in vitro.