Vasoactive intestinal peptide stimulates testosterone production by cultured fetal rat testicular cells

As a part of a series of studies on the regulation of testicular steroidogenesis in the fetal rat, we found that VIP stimulated fetal testicular cAMP production at a dose of 10⁻⁹ mol/l, while a dose as low as 10⁻¹² mol/l stimulated testosterone production. RT-PCR analysis could not reveal either VIP mRNA in fetal tissues or VIP1 receptor mRNA in the fetal or newborn testes, while VIP2 receptor mRNA was detected in fetal testes as early as E15.5. The testicular VIP content was unmeasurable by our radioimmunoassay method (<1 fmol/testis), while the circulating levels of VIP were 82.9±1.1 pmol/l at E17.5 and decreased with advanced fetal ages. In conclusion, our results suggest that VIP, from and extratesticular source, may regulate fetal testicular steroidogenesis through type 2 receptors as early as E15.5.     

Cytochalasin B stimulates insulin-like growth factor-binding protein-1 production by Hep G2 cells.

Hep G2 cells were used to study the early sequence of events regulating production of insulin-like growth factor-binding protein-1 (IGFBP-1). Cytochalasin B (100 microM) specifically inhibited 2-deoxyglucose uptake by Hep G2 cells and stimulated IGFBP-1 production 2-fold. Insulin (300 nM) did not stimulate hexose uptake but inhibited IGFBP-1 production more than 50%. A change in IGFBP-1 secretion was observed as early as 2 h after a 15-min or 2-h pulse exposure to either effector. In contrast to IGFBP-1, albumin production was diminished in the presence of cytochalasin B and increased by insulin. From these results we conclude that IGFBP-1 synthesis is (i) stimulated by transient inhibition of cellular glucose uptake and further stimulated by long-term glucose deprivation, and (ii) inhibited by transient exposure to insulin with further inhibition on long-term exposure. These effects are consistent with the dynamic regulation of IGFBP-1 by nutritional status.     

Epidermal growth factor stimulates testosterone production of human Leydig cells in vitro.

Epidermal growth factor (EGF) and insulin-like growth factor I (IGF-I) have been shown to regulate Leydig cell steroidogenesis in several species. We have investigated the effects, if any, of EGF and IGF-I on in vitro testosterone production of human Leydig cells. Interstitial cells or Percoll purified Leydig cells were isolated from the testes obtained from patients (n = 9) undergoing orchidectomies for treatment of prostate cancer and were cultured for different time periods with hCG, dibutyryl cAMP, EGF and IGF-I. Testosterone in the culture media was measured by radioimmunoassay. While EGF had a stimulatory effect on basal testosterone production of isolated interstitial cells or purified Leydig cells, IGF-I was ineffective. When the interstitial cells were cultured in the presence of hCG or EGF for 3, 6 or 24 h, the stimulatory effects of EGF on testosterone production were only evident after 24 h. On the other hand, hCG stimulated testosterone production at all time points (i.e after 3, 6, 24 h of incubation). When added in the presence of maximal concentrations of hCG and dibutyryl cAMP, EGF did not further enhance steroidogenesis. On the other hand, IGF-I potentiated the effects of hCG on testosterone production. These studies suggest that EGF and IGF-I may play a regulatory role in steroidogenic function of the human testes.     

Growth factors regulate immunoreactive insulin-like growth factor-I production by cultured porcine granulosa cells

The effects of various growth factors on the production of immunoreactive insulin-like growth factor I (iIGF-I) in short term (3-day) cultures of porcine granulosa cells was investigated. Epidermal growth factor (EGF) was shown to be a potent dose-dependent stimulator of iIGF-I production, achieving a 3.6-fold stimulation at a dose of 10 ng/ml. Transforming growth factor-alpha (10 ng EGF equivalents/ml) was also stimulatory. Platelet-derived growth factor (10 ng/ml) had no effect of its own, but enhanced EGF-stimulated iIGF-I production. The acidic and basic fibroblast growth factors (100 ng/ml) had no effect alone or in combination with EGF. Transforming growth factor-beta (10 ng/ml) had no effect of its own, but inhibited EGF-stimulated iIGF-I production. The interactive effects of EGF and FSH (200 ng/ml) on iIGF-I production were investigated in short term and longer term (7-day) cultures. In short term cultures under conditions optimized for EGF-dependent iIGF-I production, FSH had no effect of its own and inhibited EGF action. Conversely, in longer term cultures optimized for FSH-dependent iIGF-I production, EGF had no effect of its own and inhibited FSH action. Thus IGF production by cultured porcine granulosa cells is regulated in a complex manner and is highly dependent on the culture conditions. Our results suggest that IGF production in the ovary may also be regulated in a complex manner which is dependent on the developmental state of the follicle.     

Epidermal growth factor stimulates production of insulin-like growth factor-binding protein-1 in human granulosa-luteal cells.

Insulin-like growth factor-I (IGF-I) enhances and epidermal growth factor (EGF) inhibits gonadotrophin-induced aromatization in granulosa cells. Our previous studies have shown that human ovarian granulosa cells synthesize insulin-like growth factor-binding protein-1 (IGFBP-1) which inhibits IGF-stimulated DNA synthesis. The present study addresses the effect of EGF and gonadotrophins in the regulation of IGFBP-1 release by human granulosa cells cultured in serum-free medium. At concentrations of 1-100 micrograms/l EGF was found to stimulate IGFBP-1 secretion. This was not due to cell proliferation, as the viable cell count remained unaffected. Growth hormone and gonadotrophins had no effect on IGFBP-1 secretion when added alone to culture medium. These results suggest that EGF regulates IGFBP-1 secretion in human granulosa-luteal cells.     

Lack of melatonin action on testosterone production by superfused rat interstitial cells

The direct effect of increasing doses of melatonin (10(-8) to 10(-5) M) on testosterone production by superfused rat interstitial cells was studied. A constant basal testosterone output was observed for approximately 3 h after the initial high release. A continuous hypothalamo-pituitary stimulation induced a rapid testosterone response reaching peak values in 40-60 min, then decreasing progressively. Basal testosterone release was not modified by 20 or 140 min melatonin infusions. Furthermore, melatonin induced no alteration of the stimulative testosterone response when directly infusing the cells. This study demonstrates that melatonin in vitro has no direct effect on testosterone production by adult rat interstitial cells. It would seem, therefore, that the well known inhibitory influence of melatonin on rat reproductive function is not produced by a direct effect on Leydig cells.     

Prolactin restores plasma testosterone levels and stimulates testicular growth in hamsters exposed to short day-length.

In male hamsters, light deprivation was reported to reduce both testis weight and prolactin (PRL) levels. Therefore, we decided to examine the effects of PRL on testicular function in hamsters exposed to short day-length. Adult hamsters were exposed to 5 h of light per day to induce gonadal atrophy, and, starting two months later, were injected daily with 300 mug PRL, 20 mug LH or 150 mug FSH daily for 2 1/2 weeks. In animals treated with PRL the concentration of testosterone in peripheral plasma, as well as the weights of the testes and the accessory reproductive glands, were significantly greater than in the controls. Treatment with LH or FSH had no effect on any of the parameters examined. It is suggested that changes in the rate of PRL release may mediate the effects of light on testicular function in the hamster and possibly, in other species.     

Thyroid hormone stimulates the production of insulin-like growth factor I (IGF-I) by immature rat Sertoli cells

The effects of thyroid hormone on insulin-like growth factor I (IGF-I) production by Sertoli cells isolated from immature rats have been investigated. In Sertoli cells from hypothyroid rats the production of IGF-I was significantly lower than in controls and was greatly stimulated by the administration of triiodothyronine (T3) in vivo. The in vitro addition of physiological doses of T3 (1 nmol/l) significantly increased the production of IGF-I by cultured Sertoli cells indicating a direct action of the hormone on local IGF-I production. Our results suggest the involvement of IGF-I in the thyroid hormone-dependent maturation of testicular function.     

Production of insulin-like growth factors by ovarian granulosa cells

Evidence for ovarian secretion of somatomedins or insulin-like growth factors (IGF's) was generated by two approaches. First, porcine granulosa cells were shown to produce IGF's and an IGF-binding protein under serum-free conditions in vitro. The ovarian IGF's were recognized in two competitive binding assays specific for IGF's, a RIA using antibodies to human IGF-I and a radioreceptor assay using rat liver plasma membranes. IGF secretion was maintained for at least 10 days in culture. Second, ovarian production of IGF's in vivo was suggested by studies which showed that IGF levels in follicular fluid from preovulatory follicles were significantly greater than those in either serum or immature follicles. In contrast, similar low levels of insulin were observed in the follicles and serum. In conjunction with previous evidence of IGF action on granulosa cells, the present studies suggest the possibility of an autocrine role of IGF's in regulating follicular growth and development.     

Independent control of the production of insulin-like growth factor I and its binding protein by cultured testicular cells

The production of insulin-like growth factor I (IGF-I) and IGF-I binding protein (BP) was investigated in Sertoli, Leydig and peritubular cells derived from the immature rat testis and cultured in vitro. It is demonstrated that all these cells secrete not only IGF-I but also IGF-I BP. In Sertoli cells follitropin (FSH) and other agonists which increase intracellular cAMP stimulate IGF-I secretion but inhibit IGF-I BP release. The response of the BP is pronounced and very sensitive which makes it a new and useful parameter of FSH action. The calcium ionophore A23187 markedly decreases IGF-I BP production in Sertoli cells without noticeable effect on IGF-I itself. This effect can only partially be mimicked by a phorbol ester suggesting that intracellular calcium itself may play a major role in the control of IGF-I BP secretion. Peritubular cells produce high amounts of IGF-I and low amounts of IGF-I BP. Androgens do not affect the production of IGF-I or its BP neither by monocultures nor by cocultures of peritubular and Sertoli cells. In Leydig cells, lutropin (LH) and cAMP stimulate both IGF-I and IGF-I BP secretion. The production of IGF-I by Leydig-Sertoli cocultures clearly exceeds that expected from the monocultures suggesting that cell-cell interactions may also play a role in the control of testicular IGF-I production. The observation that the production of IGF-I and its activity are tightly and independently controlled supports the contention that this growth factor plays an important role in the paracrine and autocrine control of testicular function. Whether IGF-I BP increases or decreases the effects of IGF-I in the testis remains to be investigated.     

Transforming growth factor-beta stimulates production of insulin-like growth factor-binding protein-3 by human skin fibroblasts.

Human neonatal fibroblasts in monolayer culture produce insulin-like growth factor-binding protein-3 (IGFBP-3), the IGF-binding subunit of the circulating 140-kDa IGFBP complex. We now report that transforming growth factor-beta (TGF beta) is a potent stimulator of IGFBP-3 production by fibroblasts. After 72-h incubation with 1 ng/ml TGF beta, the levels of IGFBP-3 in conditioned medium were increased 5.8 +/- 1.2-fold (mean +/- SE; n = 9). Half-maximal stimulation of IGFBP-3 production was seen at 0.4 +/- 0.05 ng/ml TGF beta (n = 4). Coincubation of fibroblasts with TGF beta and either IGF-I or IGF-II at 50 ng/ml enhanced IGFBP-3 production 1.5- to 2-fold compared to TGF beta alone. As previously reported, fetal calf serum (FCS) stimulated IGFBP-3 production 5- to 6-fold; 1 ng/ml TGF beta increased the stimulated production of IGFBP-3 by FCS a further 2.5- to 3.5-fold. Acidification of FCS before addition enhanced the stimulation of IGFBP-3 compared to that caused by untreated FCS, but decreased further potentiation by TGF beta. This effect of acidified FCS was reversed by a neutralizing antibody to TGF beta. Similarly, the stimulation of IGFBP-3 levels by human serum or conditioned serum-free fibroblast medium was significantly increased by acidification of serum or medium before addition and was reversed by TGF beta antibody. These observations are consistent with acid-mediated activation of latent TGF beta added in serum or secreted by fibroblasts. Since IGFBP-3 is known to regulate IGF activity in fibroblasts, these results raise the possibility that TGF beta may modulate IGF actions in these cells by stimulating the production of IGFBP-3.     

Effects of evodiamine on the secretion of testosterone in rat testicular interstitial cells

Evodiamine, a bioactive component isolated from the Chinese medicine Wu-chu-yu, exhibits vasodilative and antianoxic action. Although evodiamine indeed has many biological effects, its effects on the endocrine system are not clear. The present study explored the effects of evodiamine on testosterone secretion in vitro. Rat collagenase-dispersed testicular interstitial cells (TICs) were incubated with evodiamine (0 to 10(-4) mol/L) in the presence or absence of human chorionic gonadotropin (hCG), forskolin, 8-bromo-adenosine 3':5'-cyclic monophosphate (8-Br-cAMP), or steroidogenic precursors (including 25-hydroxycholesterol, pregnenolone, progesterone, 17alpha-hydroxyprogesterone, and androstenedione) at 34 degrees C for 1 hour. The testosterone concentration in the media samples was measured by radioimmunoassay. Evodiamine 10(-4) mol/L was effective to reduce both basal and hCG-stimulated testosterone secretion in rat TICs after 1, 2, or 4 hours of incubation. The stimulatory effect of forskolin on testosterone release in TICs was prevented by administration of evodiamine. Evodiamine 10(-4) mol/L also decreased 8-Br-cAMP- and androstenedione-stimulated testosterone secretion. These results suggest that evodiamine reduces testosterone secretion in rat TICs via a mechanism involving reduced activity of cAMP-related pathways and 17beta-hydroxysteroid dehydrogenase (17beta-HSD).     

The albumin fraction of rat testicular fluid stimulates steroid production by isolated Leydig cells

Rat testicular fluid (rTF), but not rat serum (rS) or plasma (rP) can further increase within 4 h maximally luteinizing hormone (LH)-stimulated or 22 R-hydroxycholesterol-supported pregnenolone production by immature rat Leydig cells in vitro. This effect of rTF is dose dependent (0.05-1.2% protein, w/v) with an increase up to 4-fold. The objective of the present study was to isolate and characterize the bioactive factor(s) in rTF. After sequential ammonium sulfate fractionation, gel filtration chromatography on Superose 12 and affinity chromatography on concanavalin A-Sepharose it was shown that the albumin fraction was a major biologically active fraction in rTF. The relative specific activity in these fractions was never greater than 1.3-1.4, which is in agreement with the purification factor required to obtain pure albumin from rTF. Commercially obtained albumin fractions from human, bovine and rat sera, up to 99% purity, also increased Leydig cell steroid production more than 3-fold when added in concentrations between 0.1 and 1% w/v in combination with LH or 22R-hydroxycholesterol. Other proteins such as hemoglobin and ovalbumin were not effective in stimulation of steroid production. Bovine serum albumin (bSA, fraction V) at concentrations of 0.25 and 1.0% (w/v), had no or minor effects on LH-stimulated steroid production by rat granulosa cells or adrenocorticotrophic hormone (ACTH)-stimulated steroid production by rat adrenal cells. These findings indicate that albumin itself or minor compounds copurified with albumin represent the main biologically active component in rTF for short-term stimulation of Leydig cell steroid production. Since bioactivity could not be demonstrated in serum which contains similar amounts of albumin as rTF, inhibitory compounds may be present in rat serum.     

Transforming growth factor-beta stimulates vascular endothelial growth factor production by folliculostellate pituitary cells

TGF-beta isoforms are expressed in the anterior pituitary and modulate the growth and function of endocrine pituitary cells. Recently, TGF-beta has been shown to stimulate growth and basic fibroblast growth factor secretion in nonendocrine folliculostellate (FS) pituitary cells. We therefore studied whether the production of FS cell-derived vascular endothelial growth factor (VEGF), the most important regulator of vascular permeability and angiogenesis, is affected by TGF-beta. We observed by RT-PCR that TtT/GF cells, which are FS mouse pituitary tumor cells, synthesize TGF-beta1, -beta2, and -beta3. They also express TGF-beta receptors types 1 and 2, as well as Smad2, Smad3, and Smad4 proteins, which are essential for TGF-betabinding and signaling. Stimulation of TtT/GF cells with either TGF-beta1 or TGF-beta3 induced a rapid translocation of Smad2 into the cell nuclei. Both TGF-beta isoforms dose dependently stimulated VEGF production in TtT/GF cells, but not in lactosomatotroph GH3 cells. Time-course studies and suppression of TGF-beta-induced VEGF production by cycloheximide suggest that TGF-beta induces de novo synthesis of VEGF in folliculostellate cells, which is completely blocked by dexamethasone. In primary rat pituitary cell cultures, TGF-beta1 and -beta3 stimulated VEGF production. TGF-beta stimulation of VEGF production by folliculostellate cells could modulate intrapituitary vascular permeability and integrity as well as angiogenesis in an auto-/paracrine manner.     

Insulin and insulin-like growth factors (IGFs) stimulate production of IGF-binding proteins by ovarian granulosa cells.

Ligand blot analysis of granulosa cell (GC)-conditioned culture medium revealed several easily measurable insulin-like growth factor (IGF)-binding proteins (IGFBPs), including IGFBP-3 [40-44 kilodaltons (kDa)] and IGFBP-2 (34 kDa). In the present study, IGF-I, in a dose-dependent manner, significantly stimulated the production of these IGFBPs. Insulin, but not IGF-II, mimicked IGF-I's action on IGFBP-3 and -2 production, but was less potent. The synthetic IGF, long R3-IGF-I, which has very low affinity for IGFBPs and only slightly reduced affinity for the IGF-I (type I) receptor, had significantly greater potency in stimulating IGFBP-3 and -2 production compared to IGF-I. Des-(1-3)-IGF-I had similar effects. IGF-I, IGF-II, and the IGF-I analogs, but not insulin, also induced production of an unidentified 30-kDa IGFBP not normally detectable in these cultures. However, in the presence of epidermal growth factor (which was without independent effect on the 30-kDa IGFBP), insulin also induced this 30-kDa IGFBP. By Northern analysis the expression of IGFBP-3 mRNA was found to be significantly stimulated by IGF-I. In summary, insulin stimulated IGFBP-3 and -2 production in a manner that mimics that of IGF-I and the more potent long R3-IGF-I. However, its low potency suggested that IGFBP production is regulated via the IGF-I (type I) receptor. The much higher potency of long R3-IGF-I compared to that of IGF-I suggests that the IGFBPs themselves modulate the action of IGFs by sequestering exogenous IGFs. Thus, one cellular response to IGF stimulation is the production of IGFBPs, which, in turn, reduce or negate the biological activity of the IGFs. The effects of insulin-like peptides are exerted at least in part by increasing levels of mRNA for specific BPs.     

Multiple myeloma cells are protected against dexamethasone-induced apoptosis by insulin-like growth factors

Multiple myeloma cell lines express functional receptors for insulin-like growth factors (IGFs) and several cell types that make up the bone marrow microenvironment produce these cytokines. This suggests that IGFs may play a role in survival and/or expansion of the malignant clone within the marrow in patients with multiple myeloma. We tested the effects of these growth factors on myeloma cells challenged with dexamethasone. Dye exclusion and MTT assays demonstrated that both IGF-I and IGF-II protected the 8226 and dox-40 myeloma cell lines and three primary myeloma cultures from dexamethasone-induced cytotoxicity in a dose-dependent fashion. Morphologic studies of target cells and their nuclei as well as DNA electrophoresis confirmed the IGFs afforded protection against dexamethasone-induced apoptosis. Insulin also protected but was less impressive and required much higher concentrations. IGFs also protected against cycloheximide-induced apoptosis but were ineffective against serum starvation, topoisomerase II inhibitors, or anti-fas antibodies. IGF-induced protection against dexamethasone was not associated with any alteration in quantitative or qualitative expression of BCL-2, BAX or BCL-X proteins. These data indicate that insulin-like growth factors may play a role in maintenance of the malignant clone in patients with myeloma by protecting tumour cells from apoptotic death.     

Stimulation of prostaglandin E and testosterone production in rat interstitial cells by a gonadotropin-releasing hormone agonist

The early direct effects of a GnRH agonist analog [D-Ala6]des-Gly10-GnRH N ethylamide (GnRHa) on rat testicular interstitial cells include increased production of prostaglandin E (PGE) and testosterone (T) at 3 h (ED50 values of 0.5 and 0.75 nM, respectively). On the other hand, LH action on testicular function, which is mediated by increased cAMP, involves an increase in T production at 30 min followed by increased PGE formation at 3 h. GnRHa at concentrations of 10(-12)-10(-8) M had no effect on basal or LH-stimulated cAMP production during a 4-h incubation test. The stimulatory effect of GnRHa on PGE, but not on T production, was abolished by the prostaglandin synthesis inhibitor indomethacin (1.5 microns). We conclude that cAMP does not play a role in mediating the direct testicular effects of GnRH on PGE and T production; that PGE is not involved in mediating GnRH-induced T production; and, finally, that increased PGE and T production might be involved in mediating the direct inhibitory and stimulatory testicular effects of GnRH and its agonist analogs.     

Structural basis for the inhibition of insulin-like growth factors by insulin-like growth factor-binding proteins

Insulin-like growth factor-binding proteins (IGFBPs) control bioavailability, activity, and distribution of insulin-like growth factor (IGF)1 and -2 through high-affinity IGFBP/IGF complexes. IGF-binding sites are found on N- and C-terminal fragments of IGFBPs, the two conserved domains of IGFBPs. The relative contributions of these domains to IGFBP/IGF complexation has been difficult to analyze, in part, because of the lack of appropriate three-dimensional structures. To analyze the effects of N- and C-terminal domain interactions, we determined several x-ray structures: first, of a ternary complex of N- and C-terminal domain fragments of IGFBP4 and IGF1 and second, of a "hybrid" ternary complex using the C-terminal domain fragment of IGFBP1 instead of IGFBP4. We also solved the binary complex of the N-terminal domains of IGFBP4 and IGF1, again to analyze C- and N-terminal domain interactions by comparison with the ternary complexes. The structures reveal the mechanisms of IGF signaling regulation via IGFBP binding. This finding supports research into the design of IGFBP variants as therapeutic IGF inhibitors for diseases of IGF disregulation. In IGFBP4, residues 1-38 form a rigid disulphide bond ladder-like structure, and the first five N-terminal residues bind to IGF and partially mask IGF residues responsible for the type 1 IGF receptor binding. A high-affinity IGF1-binding site is located in a globular structure between residues 39 and 82. Although the C-terminal domains do not form stable binary complexes with either IGF1 or the N-terminal domain of IGFBP4, in the ternary complex, the C-terminal domain contacts both and contributes to blocking of the IGF1 receptor-binding region of IGF1.     

Fibroblast growth factor inhibits luteinizing hormone-stimulated androgen production by cultured rat testicular cells

The effect of fibroblast growth factor (FGF) on LH-stimulated testosterone production was investigated using primary cultures of rat testicular cells. Testicular cells obtained from neonatal rats (8-9 days of age) were maintained in culture for 3 days and then challenged with LH with or without basic FGF. After 3 additional days of culture, the media were collected for steroid RIA. LH treatment of cultured cells stimulated testosterone production in a dose-dependent fashion whereas FGF alone did not affect androgen biosynthesis. In contrast, cotreatment with FGF caused a dose-dependent decrease of LH-stimulated testosterone production, with an IC50 value of 1.1 X 10(-9) M (as calculated from three separate experiments). The inhibitory effect of FGF was evident 24 h after the initiation of treatment and this effect was reversible 1 day after the cessation of FGF treatment. The inhibition of LH-induced testosterone production by FGF (maximal inhibition greater than 90%) was accompanied by a 12-fold increase in progesterone levels, suggesting that the inhibitory effect of FGF was distal to the step of progesterone formation. FGF also inhibited forskolin (10(-5) M)- and (Bu)2cAMP (5 X 10(-4) M)-stimulated testosterone production. Furthermore, FGF inhibited the conversion of exogenously added androgen precursors (progesterone and 17 alpha-hydroxyprogesterone) to testosterone in LH-stimulated cultures indicating that FGF might inhibit 17 alpha-hydroxylase activity. The concept of a direct testicular action of FGF was further supported by the demonstration of high affinity (Kd: 3.9 X 10(-10) M; n = 3 experiments) and low capacity (46,900 sites per cell) FGF receptors in cultured testis cells. The binding of [125I]FGF was inhibited by basic and acidic FGF but not by several other growth factors. In conclusion, we suggest that FGF binds to testicular cells and inhibits LH-stimulated testosterone production by inhibiting, at least partially, 17 alpha-hydroxylase enzyme activities. Because FGF has been purified from testis extracts, this growth factor may have intratesticular paracrine or autocrine functions.