Steroidogenesis-inducing Protein Research Articles

The stimulation of rat Leydig cell steroidogenesis by human ovarian steroidogenesis-inducing protein (SIP) may not require endogenous cAMP.

Recently a protein from ovarian follicular fluid was isolated which stimulates steroid production in different cells (Khan et al., 1990). The present study was performed to further characterize the short term effects of this steroidogensis-inducing protein (SIP) on steroid production in isolated rat Leydig cells and to compare the effects with LH/hCG and LHRH. SIP stimulated testosterone production in a dose-dependent manner. In Leydig cells isolated from adult rats the degree of stimulation was much higher than that obtained with hCG, dibutyryl cAMP (db cAMP) or LHRH. Moreover, the stimulated steroid production in the presence of hCG or db cAMP was further enhanced by SIP. The time courses of hCG and SIP action on testosterone production were comparable and maximal stimulation of steroid production was obtained within one hour. In contrast to hCG, SIP did not stimulate cAMP production. An antagonist of LHRH action was unable to block the effects of SIP on Leydig cells indicating that SIP does not act via LHRH receptors. Neither SIP nor LH could further stimulate the steroid production in the presence of 22-R-OH-cholesterol, illustrating that both stimulators control the availability of cholesterol as substrate. An inhibitor of mitochondrial cholesterol side chain cleavage (CSCC), aminoglutethemide, completely blocked the stimulatory effects of SIP and LH/hCG. Thus the effects of SIP on steroid production are not the result of conversion of contaminating steroids in the SIP-preparation. SIP and LH/hCG actions were also blocked when the cells were incubated in the presence of cycloheximide.(ABSTRACT TRUNCATED AT 250 WORDS)

A DING phosphatase in Thermus thermophilus

Phosphate transport in bacteria occurs via a phosphate specific transporter system (PSTS) that belongs to the ABC family of transporters, a multisubunit system, containing an alkaline phosphatase. DING proteins were characterized due to the N-terminal amino acid sequence DINGG GATL, which is highly conserved in animal and plant isolates, but more variable in microbes. Most prokaryotic homologues of the DING proteins often have some structural homology to phosphatases or periplasmic phosphate-binding proteins. In E. coli, the product of the inducible gene DinG, possesses ATP hydrolyzing helicase enzymic activity. An alkaline phosphorolytic enzyme of the PSTS system was purified to homogeneity from the thermophilic bacterium Thermus thermophilus. N-terminal sequence analysis of this protein revealed the same high degree of similarity to DING proteins especially to the human synovial stimulatory protein P205, the steroidogenesis-inducing protein and to the phosphate ABC transporter, periplasmic phosphate-binding protein, putative (P. fluorescens Pf-5). The enzyme had a molecular mass of 40 kDa on SDS/PAGE, exhibiting optimal phosphatase activity at pH 12.3 and 70 degrees C. The enzyme possessed characteristics of a DING protein, such as ATPase, ds endonuclease and 3' phosphodiesterase (3'-exonuclease) activities and binding to linear dsDNA, displaying helicase activity on supercoiled DNA. Purification and biochemical characterization of a T. thermophilus DING protein was achieved. The biochemical properties, N-terminal sequence similarities of this protein implied that the enzyme belongs to the PSTS family and might be involved in the DNA repair mechanism of this microorganism.

A protein signal triggers sexual reproduction in Brachionus plicatilis (Rotifera)

The defining feature of the life cycle in monogonont rotifers such as Brachionus plicatilis (Muller) is alternation of asexual and sexual reproduction (mixis). Why sex is maintained in such life cycles is an important unsolved evolutionary question and one especially amenable to experimental analysis. Mixis is induced by a chemical signal produced by the rotifers which accumulates to threshold levels at high population densities. The chemical features of this signal were characterized using size exclusion, enzymatic degradation, protease protection assays, selective binding to anion ion exchange and C3 reversed phase HPLC columns, and the sequence of 17 N-terminal amino acids. These studies were carried out over two years beginning in 2003 using B. plicatilis Russian strain. When rotifer-conditioned medium was treated with proteinase K, its mixis-inducing ability was reduced by 70%. Proteinase K was added to medium auto-conditioned by 1 female ml−1 where typically 17% of daughters became mictic and mixis was reduced to 1%. A cocktail of protease inhibitors added to conditioned medium significantly reduced degradation of the mixis signal by natural proteases. Conditioned medium subjected to ultrafiltration retained mixis-inducing activity in the >10 kDa fraction, but the <10 kDa fraction had no significant activity. The putative mixis signal bound to an anion exchange column, eluting off at 0.72 M NaCl. These fractions were further separated on a C3 reversed phase HPLC column and mixis-inducing activity was associated with a 39 kDa protein. Seventeen amino acids from the N-terminus have strong similarity to a steroidogenesis-inducing protein isolated from human ovarian follicular fluid. The 39 kDa protein is an excellent candidate for the rotifer mixis induction signal.

Steroidogenesis-inducing protein (SIP) stimulates endometrial matrix metalloproteinase-3 (MMP-3) expression and antagonizes progesterone-mediated suppression of this enzyme.

Objective: The regulation of ectopic cell growth and tissue invasion during establishment of endometriosis is complex and poorly understood. Although endometriosis is largely dependent on steroid action, a recently identified ovarian protein may also play a role in this disease. SIP, isolated from human follicular fluid, stimulates mitochondrial conversion of cholesterol to pregnenelone and is a potent stimulator of cell proliferation and DNA synthesis. The release of SIP into the peritoneal environment at ovulation could play an important role(s) in enhancing ectopic endometrial growth. In the current study, we investigated the ability of SIP to stimulate expression of MMP-3, a stromal-specific enzyme implicated in numerous invasive diseases, including endometriosis. We have also determined the effect of SIP on MMP-3 regulation relative to estradiol (E), progesterone (P) and interleukin-1α (IL-1α), an inflammatory cytokine associated with MMP expression and endometriosis. Design: Endometrial stromal cells were isolated from proliferative phase (days 10–12) biopsies from normal women with no history of endometriosis or uterine dysfunction. Cells were isolated by established methods and maintained in monolayers on Type-1 collagen in DMEM/F-12 media with 3% serum, steroids, IL-1α or SIP. Materials/Methods: SIP has homology with p205, a protein isolated from human synovial fluid. Using anti-p205 antibody coupled sepharose beads and an immunoaffinity column, SIP was extracted from human follicular fluid from women without endometriosis. Following elution from the column, the p205 bound fraction was found to contain SIP activity and gave a single band at 48 kD after SDS-PAGE. Isolated stromal cells were maintained in vitro in the presence of E (1 nM) or E plus P (E = 1 nM, P = 500 nM) for 0-96 hours prior to 24 hr exposure to IL-1α (100 pg/mL) or SIP (50 μg/mL). Conditioned media were analyzed for MMP-3 protein by established Western procedures. Results: Treatment of isolated endometrial stromal cells with E is associated with MMP-3 expression while treatment with E and P suppressed MMP-3. Simultaneous addition of SIP to either steroid treatment resulted in a significant stimulation of MMP-3 expression. Pretreatment of stromal cells with the combination of E and P prevented induction of MMP-3 by IL-1α, but a similar pretreatment of cells with E and P was unable to prevent MMP-3 stimulation by SIP. Conclusions: These results indicate that SIP, a follicular fluid protein, can stimulate the expression of endometrial MMP-3 and, unlike IL-1α, the stimulatory effects of SIP are not blocked by prior P exposure. Since endometrial MMP expression has been implicated in the development of endometriosis, the introduction of SIP into the peritoneal fluid at ovulation could contribute to a local environment which enhances establishment and/or progression of endometriosis. Supported By: Supported through cooperative agreement (U54-HD-37321) as part of the Specialized Cooperative Centers Program in Reproduction Research and by a grant from the Endometriosis Association.

Regulation of DNA synthesis in leydig cells obtained from neonatal pig testes

Three distinct waves of Leydig cell development are found in the pig testes, which occur during fetal, perinatal, and prepubertal periods. Proliferation of Leydig cells is primarily regulated by luteinizing hormone (LH); however, effects of LH on proliferation of immature rat Leydig cells are mediated by specific growth factors and cytokines such as transforming growth factor-α (TGFα), insulin-like growth factor-1 (IGF-1), interleukin-1β (IL-1β), steroidogenesis-inducing protein (SIP), and TGFβ. The objective of the present study was to identify growth factors that could possibly be involved in the proliferation of Leydig cells in the neonatal pig testis. Leydig cells were isolated from 3- to 5-d-old pig testes, cultured for 48 hr in serum-free media, washed, and treated with hCG and/or IGF-1, epidermal growth factor (EGF), IL-1β, SIP, and TGFβ for 18 hr. Tritiated thymidine incorporation into DNA was assessed over a subsequent 4-hr period. Incorporation of [ 3H]-thymidine was stimulated by hCG treatment with a 2.3-fold increase over control cultures. SIP also induced a significant increase (P < 0.0001) in the incorporation of [ 3H]thymidine into Leydig cell DNA. Similarly, EGF and IGF-1 also increased DNA synthesis in neonatal porcine Leydig cells, whereas IL-1β had no effect. TGFβ had very little, if any, effect on DNA synthesis when added alone, but inhibited the stimulatory effects of other mitogens (SIP, hCG, EGF/TGFα, and IGF-1). Our results indicate that these growth factors may play a role in the LH/hCG-dependent proliferation of Leydig cells during the perinatal period of development.

Transforming growth factor-β inhibits DNA synthesis in immature rat Leydig cells in vitro

TGFβ isoforms and its receptors are present in the testis and regulate in vitro function of various testicular cells. We have investigated the effects of TGFβ on basal and mitogen stimulated in vitro proliferation of immature rat Leydig cells. Leydig cells were cultured with TGFβ1, either alone or in combination with hCG, steroidogenesis-inducing protein (SIP), interleukin-1β (IL-1β), insulin or TGFα, and the incorporation of [ 3H]thymidine into DNA was determined. TGFβ1 blocked the stimulatory effects of hCG, SIP, IL-1β, insulin and TGF-α on DNA synthesis. Since G 1- to S-phase transition depends upon cyclins and their associated kinases (cdks), we investigated the effects of TGFβ on cdks. Immunoreactive levels of cdc2 (or cdk1) and cdk2 were significantly decreased in Leydig cells treated with TGFβ1. We conclude that TGFβ1 inhibits proliferation of immature rat Leydig cells and this effect may be mediated, at least in part, through down-regulation of cdc2 and cdk2 synthesis.

Mitogenic activity of steroidogenesis-inducing protein (SIP) during hypoxic stress of human ovarian carcinoma cells

Steroidogenesis-inducing protein (SIP) is a novel growth factor isolated from human ovarian follicular fluid. While the steroidogenic and mitogenic effects appear to be restricted towards gonadal cell types, we have recently demonstrated that SIP is also a potent mitogen for cell lines derived from ovarian surface epithelial carcinomas. Here, we demonstrate that SIP reverses hypoxia-induced cell proliferation arrest of the human ovarian carcinoma cell line SKA, as determined by flow cytometry and cell proliferation assays. Concomitant with this reversal of proliferation arrest is an increase in expression of cyclins D and E and a reduction in expression of the cyclin-dependent kinase inhibitor p27. Pretreatment of hypoxic SKA cells with SIP is also shown to increase Taxol sensitivity of these cells by two-fold. These studies further characterize the mitogenic activity of SIP at the molecular level and suggest that this protein may be an effective biological response modifier for ovarian carcinoma cells.

Steroidogenesis-Inducing Protein, Isolated from Human Ovarian Follicular Fluid, Is a Potent Mitogen for Cell Lines Derived from Ovarian Surface Epithelial Carcinomas

The majority of human ovarian cancers originate from the surface epithelial (OSE) cells that surround the ovary. The incidence of OSE cancer is correlated with the number of ovulations that occur during fertile life. OSE cells remain quiescent but undergo rapid mitotic activity after ovulation to repair the wound. This increase in mitotic activity following each ovulation may give rise to mutations that make the OSE susceptible to malignant transformation. Steroidogenesis-inducing protein (SIP), a protein isolated from human follicular fluid obtained from hyperstimulated ovaries, is a potent mitogen for several gonadal cells. To investigate the possibility that SIP may be involved in the proliferation of OSE cells, we have studied its effects on DNA synthesis in seven cell lines derived from OSE carcinomas (HEY, MLS, SKA, OW-1, SAU, NIH:OVCAR-3, and Caov-3). The cells were cultured in serum-free medium in the presence of SIP for 18 hr, followed by incubation with [3H]thymidine for 4 hr. The radioactivity incorporated into the DNA was measured. SIP stimulated DNA synthesis in six of the cell lines. HEY, SKA, MLS, and OVCAR3 were most responsive to SIP. Interactions between SIP and other growth factors and cytokines known to be present in follicular fluid (EGF/TGFα, TGFβ, FGF, IGF-1, IL-1β, and TNFα) were also investigated in HEY and SKA cells. EGF/TGFα and IGF-1 potentiated the effects of SIP. TGFβ had no effect on SIP, and/or EGF/TGFα stimulated DNA synthesis. Other growth factors which were tested in this study had no effect on DNA synthesis in SKA cells. Dibutyryl cyclic-AMP blocked the effects of SIP on DNA synthesis. We conclude that SIP is a potent mitogen for OSE cell lines and together with TGFα and IGF-1 may be involved in the proliferation of normal OSE cells after ovulation. Since SIP is obtained from the preovulatory follicle, it may represent a link between the number of ovulations and the increased incidence of OSE cancers.

Steroidogenesis-inducing protein stimulates protein-tyrosine kinase activity in rat Leydig cells.

Steroidogenesis-inducing protein (SIP) isolated from human ovarian follicular fluid stimulates steroid production in Leydig cells, human luteal cells, and rat adrenal cells. In addition, SIP is a potent mitogen that stimulates the proliferation of Leydig cells from immature rats to a greater extent than do LH/hCG and other known growth factors. We have shown previously that the actions of SIP on Leydig cells are independent of the adenyl cyclase-cAMP pathway. In the present study we have explored the possibility that SIP, like many growth factors, may exert its effects by activation of tyrosine kinase(s). Stimulation of Leydig cells isolated from immature rats with SIP resulted in an increase in the tyrosine phosphorylation of proteins that were detected with phosphotyrosine-specific antibodies. The phosphorylation of a 90-kilodalton (kDa) protein band, a 65-kDa protein band, and a doublet at 140 kDa was apparent after 5 min. After 30 min, additional SIP-induced phosphotyrosine proteins were detected at 42, 44, 50, 80, 100, and 150 kDa. In addition to phosphorylation at tyrosine residues, all of the proteins isolated from SIP-stimulated cells were phosphorylated at threonine and serine residues. SIP-induced phosphoproteins recovered with phosphotyrosine-specific antibodies were found to have associated protein-tyrosine kinase activity. The major substrate for this kinase activity in vitro was a 140-kDa protein, similar to one of the major phosphotyrosine-containing proteins induced by SIP treatment of intact cells. These observations suggest that SIP influences gonadal cell steroidogenesis and proliferation, presumably by activating cellular protein-tyrosine kinase(s) as part of a phosphorylation-based signalling pathway.

Steroidogenesis-inducing protein stimulates protein-tyrosine kinase activity in rat Leydig cells

Steroidogenesis-inducing protein stimulates protein-tyrosine kinase activity in rat Leydig cells

Steroidogenesis-inducing protein interacts with transforming growth factor-β to stimulate DNA synthesis in rat granulosa cells

We have examined the effects of Steroidogenesis-inducing protein (SIP), previously isolated from human follicular fluid, on the synthesis of DNA by granulosa cells isolated from diethylstilbestrol-primed immature rats. SIP alone had no effect but in conjunction with transforming growth factor-β (TGF-β) there was an increase in [ 3H]thymidine incorporation into granulosa cell DNA. The increase in [ 3H]thymidine into DNA was due to an increase in the number of labeled granulosa cells as assessed by autoradiography. Epidermal growth factor (EGF) and transforming growth factor-α (TGF-α) interfered with the ability of SIP and TGF-β to promote DNA synthesis. Previously, we reported that the growth-promoting action of follicle-stimulating hormone (FSH) on rat granulosa cells in vitro was dependent on TGF-β, and EGF inhibited the actions of FSH plus TGF-β on [ 3H]thymidine incorporation into DNA. Since the dependency of SIP on its interactions with TGF-β and the ability of EGF to interfere with the process were similar to the properties reported for FSH, this raised the possibility that the actions of SIP were mediated through the accumulation of intracellular cAMP. However, when the hypothesis was tested, SIP had no effect on cAMP levels in the presence or absence of TGF-β, under conditions in which FSH stimulated cAMP accumulation. In conclusion, DNA synthesis in rat granulosa cells is dependent on the presence of TGF-β. In the presence of TGF-β, FSH or SIP, acting through cAMP-dependent and cAMP-independent mechanisms respectively, can recruit more cells to enter the cell cycle and initiate DNA synthesis. While FSH plays a major role in regulating granulosa cell proliferation, our results indicate that an intragonadal peptide, SIP, can promote growth as effectively as FSH.

Interleukin-1 stimulates deoxyribonucleic acid synthesis in immature rat Leydig cells in vitro.

The number of interstitial macrophages in the testis fluctuates according to age, increasing gradually during prepubertal development to reach 15-20% in the interstitial compartment in the adult rat. These macrophages are in close morphological association with Leydig cells. Macrophage products, interleukin-1 (IL-1) and tumor necrosis factor alpha stimulate and/or inhibit steroid production in cultures of Leydig cells. We have studied the effects of macrophage products on DNA synthesis in rat Leydig cells to investigate a possible paracrine role of testicular macrophage products on the proliferation of Leydig cells. Leydig cells isolated from 10-, 20-, and 70-day-old rats were cultured for 48 h in serum-free medium, washed, and treated with different cytokines for 18 h. The medium was then removed, fresh medium containing 0.5 microCi [3H]thymidine was added, and cells were incubated for 4 h prior to determining the incorporation of [3H]thymidine into DNA. Human recombinant IL-1 beta caused a dose-dependent stimulation in the incorporation of [3H]thymidine into DNA in the Leydig cells from 10- and 20-day-old rats but had no effect on DNA synthesis in interstitial cells from adult rats. Maximum stimulation of DNA synthesis in immature Leydig cells was observed with 1-2 ng/ml IL-1 beta. Autoradiography after incubation with [3H]thymidine showed a dramatic increase in the number of labeled Leydig cells after treatment with IL-1 beta (19.27 +/- 3.77% vs. 1.44 +/- 0.52% in control cultures) indicating that IL-1 beta recruited more cells to enter the cell cycle and initiate DNA synthesis. Human recombinant IL-1 alpha and tumor necrosis factor alpha also caused significant stimulation of DNA synthesis in Leydig cells but these cytokines were much less potent (1-10%) than IL-1 beta. IL-1 beta enhanced the effects of maximally effective concentrations of growth-promoting agents previously known to stimulate DNA synthesis in immature rat Leydig cells, i.e. human CG, steroidogenesis-inducing protein, and transforming growth factor alpha plus insulin. On the basis of these results it is concluded that IL-1 may play an important role in the proliferation of Leydig cells during prepubertal development in immature rats.

Effects of steroidogenesis inducing protein (SIP) on steroid production in MA-10 mouse Leydig tumor cells: utilization of a non-cAMP second messenger pathway

We have investigated the effects of steroidogenesis inducing protein (SIP) (Endocrinology (1990) 126, 3043–3052) on steroid production in MA-10 mouse Leydig tumor cells. Our results indicate that SIP results in the stimulation of progesterone production in MA-10 cells to the same extent obtained when maximal doses of luteinizing hormone (LH), human chorionic gonadotropin (hCG) and dibutyryl cAMP (dbcAMP) are used. It was also observed that the increased progesterone production in response to SIP was not accompanied by an increase in intracellular cAMP levels as was seen following hCG stimulation. In addition, stimulation of progesterone production using maximal doses of LH, hCG and dbcAMP could be further increased by the addition of SIP to the incubation medium also indicating that this steroidogenic activity was acting through a differential signal transducing system than these hormones. That SIP was not acting through the cAMP second messenger pathway was also demonstrated by its lack of sensitivity to the neutralizing effects of a monoclonal antibody to LH as well as by its insensitivity to the protein kinase A inhibitor HA 1004 while both of these treatments significantly decreased LH and hCG stimulated steroid production. Lastly, SIP was unable to elicit the induction of several mitochondrial proteins which have previously been shown to be synthesized in MA-10 cells in response to LH, hCG and dbcAMP. Our results indicate that SIP stimulates the production of high levels of steroids through a signal transduction pathway which is distinct from that employed by trophic hormone stimulation in Leydig cells.

Steroidogenesis-inducing protein promotes deoxyribonucleic acid synthesis in Leydig cells from immature rats.

A protein [steroidogenesis-inducing protein (SIP)] has been isolated from human ovarian follicular fluid and shown previously to stimulate steroidogenesis in Leydig cells, adrenal cells, and early luteal cells. Since proteins and peptides known to regulate steroidogenesis, such as gonadotropins and growth factors, also influence the growth of gonadal cells, the present study was designed to assess the effects of SIP on the synthesis of DNA by Leydig cells in vitro. Leydig cells were isolated from 10- and 20-day-old rats and cultured in serum-free medium for 48 h. The cells were then treated with the test materials for 18 h. Incorporation of [3H]thymidine into DNA was measured during the final 4 h of the culture. SIP significantly stimulated DNA synthesis in Leydig cells in a dose- and time-dependent manner, and the response to SIP was higher than that obtained with maximal concentrations of LH/hCG. The stimulatory effects of SIP were significantly enhanced when the cell cultures were preincubated in the presence of low levels of ovine LH (2 ng/ml). Cultures treated with SIP, followed by incubation with [3H]thymidine, contained 22 times as many labeled cells as control cultures, as assessed by autoradiography. The cells that were labeled were identified morphologically as Leydig cells. Insulin/insulin-like growth factor-I and/or transforming growth factor-alpha alone stimulated DNA synthesis and enhanced the effects of SIP on DNA synthesis. Dramatic changes in the morphology of cultured Leydig cells treated with SIP were observed; cells became flattened and developed extended projections which connected adjacent cells. LH/hCG, insulin, and transforming growth factor-alpha did not induce effects comparable to those of SIP on the morphology of Leydig cells. The effects of SIP on the synthesis of DNA and the morphology of Leydig cells were blocked in the presence of cycloheximide. It is concluded that SIP not only stimulates steroid production in Leydig cells, as shown previously, but also stimulates DNA synthesis and induces morphological changes in these cells. The latter properties of SIP combined with the magnitude of the responses elicited identify SIP as a unique gonadal protein.

Steroidogenesis-inducing protein promotes deoxyribonucleic acid synthesis in Leydig cells from immature rats

Steroidogenesis-inducing protein promotes deoxyribonucleic acid synthesis in Leydig cells from immature rats

Isolation of a Protein from Human Ovarian Follicular Fluid which Exerts Major Stimulatory Effects onin VitroSteroid Production of Testicular, Ovarian, and Adrenal Cells*

We describe the isolation of a steroidogenesis-inducing protein (SIP) from human ovarian follicular fluid and its effects on in vitro steroid synthesis of testicular, ovarian, and adrenal cells. After heating at 60 C, precipitation with 80% ammonium sulfate and dialysis, the human ovarian follicular fluid proteins were fractionated by gel chromatography on Sephacryl S-200. The bioactivity was eluted in the mol wt region between 40 and 60 K. SIP was further purified by affinity chromatography on blue Sepharose (CL-6B), preparative isoelectrofocusing on sucrose density gradients, anion exchange chromatography on Mono Q by using fast protein liquid chromatography system and gel chromatography on Superose 12. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified SIP under both reducing and nonreducing conditions revealed a single band with an approximate mol wt of 60 K. SIP exhibited a pI value of 4.8, was heat sensitive, and lost activity upon lyophilization. SIP copurified with albumin in various isolation procedures and was distinct from human serum albumin but may represent a modified form of human albumin. SIP stimulated testosterone production by testicular pieces, interstitial cells, or purified Leydig cells from rats under different in vitro conditions. The SIP also stimulated basal and human CG stimulated in vitro progesterone production of human ovarian granulosa-lutein cells and corticosterone production of rat adrenal cells. The effects of SIP on testicular, ovarian, and adrenal cells were evident in the presence of maximal concentrations of tropic hormones. The steroid-free spent media from human granulosa-lutein cell cultures also stimulated testosterone production by rat interstitial cells, suggesting that granulosa cells may be the cellular source of SIP. In conclusion, human ovarian cells secrete a hitherto unknown albumin-like protein which enhances both basal and tropic hormone stimulated steroidogenesis in gonadal and adrenal cells. This protein may play a significant role in ovarian function by modifying steroidogenesis in the preovulatory follicle.