Testicular blood flow and microcirculation in rats after treatment with ethane dimethyl sulfonate

The effects of ethane dimethyl sulfonate (EDS) on total testicular blood flow, microcirculation, and the testicular interstitial fluid volume (IFV) in rats were studied. In agreement with previous studies, treatment of control rats with human chorionic gonadotropin (hCG) induced an increase in IFV and total testicular blood flow as measured with radioactive microspheres. These effects of hCG were completely abolished in rats pretreated with EDS; in EDS-treated rats not receiving any hCG, there were decreases in IFV when compared with untreated control rats. Furthermore, the pulsatile pattern of testicular microcirculation registered with laser-Doppler flowmetry was abolished after EDS treatment, and this effect was not influenced by hCG treatment. The hCG-induced increase in IFV is associated with an increased accumulation of polymorphonuclear leukocytes locally in the testis, but this accumulation of leukocytes was not observed in rats pretreated with EDS. It was concluded from the present study that hCG-induced changes in total testicular blood flow and testicular microcirculation require functionally intact Leydig cells.     

Impaired gonadotrophin uptake in vivo by the cryptorchid rat testis

The specific testicular uptake in vivo of 125I-labelled hCG was compared in control adult rats and adult rats made bilaterally cryptorchid 5 weeks previously. Although a similar temporal pattern of uptake was observed in both groups, uptake of hCG by cryptorchid testes was reduced at all times after injection by up to 70%. The possible causes of this impairment were investigated. It could not be accounted for by differences in the rate of absorption or clearance of 125I-labelled hCG in the two groups. Therefore, because hCG-induced increase in the permeability of testicular capillaries is a crucial factor in determining hCG uptake by the testis, this change was compared in control and cryptorchid testes. Although hCG induced a characteristic increase in testicular capillary wall permeability in both groups, this change was temporally delayed in cryptorchid testes, and occurred after hCG values in the blood had fallen. Even when hCG had crossed the capillary wall into testicular interstitial fluid, its uptake into the testicular tissue was significantly lower in cryptorchid than in control testes. These changes probably account for the impairment of gonadotrophin uptake by the cryptorchid testis and have important implications with respect to the aetiology of Leydig cell changes in cryptorchidism.     

Gonadotrophin-induced accumulation of 'interstitial fluid' in the rat testis.

'Interstitial fluid' containing high levels of testosterone (60-250 ng/ml) was recovered from the testes of rats, the amounts increasing with increase in age and testis weight. Injection of 170 i.u. hCG/kg resulted 20 h later in significant increases in interstitial fluid and its testosterone content (300-800 ng/ml). In immature rats this effect of hCG was dose-dependent and time-related and the accumulated fluid contained high levels of potassium and phosphate; levels of sodium, calcium and protein were similar to those in serum. At 20 h after injection of hCG, other testicular changes were (1) increased 'adhesiveness', (2) reduced in-vitro binding of 125I-labelled hCG, and (3) an hCG-induced increase in the testis:blood ratio of hCG in vivo.     

Terbutaline treatment inhibits the hCG-induced increase in venular permeability in the rat testis

Treatment with hCG results in an increase in venular permeability in the rat testis. This change in vascular permeability can be detected by the carbon-labelling technique, by measurement of the volume of interstitial fluid and by quantification of the leucocyte migration into the interstitial space. Carbon-labelling, interstitial fluid volume and leucocyte migration were all reduced in rats treated with hCG+ terbutaline compared to the values in animals given hCG only. However, terbutaline treatment did not influence the hCG-induced increase in testosterone secretion. These observations suggest that the hCG-induced increase in vascular permeability in the testis can be reduced by a beta-adrenergic agonist.     

Treatment of rats with hCG induces inflammation-like changes in the testicular microcirculation

Adult rats were injected subcutaneously with 50 i.u. hCG and vascular permeability was compared to that in saline-treated control rats by two independent methods. At 4 h after hCG treatment the rats were injected intra-arterially (i.a.) with FITC-labelled macromolecular dextran (Mr 150,000) and the testicular microcirculation was studied in vivo by using a fluorescence microscope. Other rats were injected i.a. with a suspension of colloidal carbon and the location of leaking blood vessels was recorded in sections from the testes by light and electron microscopy. In hCG-treated animals leucocytes were found adhering to the endothelium in post-capillary venules and in these venular segments dextran was leaking into the interstitium. Carbon particles were deposited in the walls of post-capillary venules and leucocytes migrated through open interendothelial cell gaps in hCG-treated animals. In control animals leucocyte adhesion and migration were not observed, the injected dextran remained in the circulation and the blood vessels were not labelled by carbon. It is suggested that the hCG-induced increase in testicular interstitial fluid volume, like the tissue oedema in inflammation, is caused by a leucocyte-mediated increase in venular permeability.     

Luteinizing hormone receptor-mediated effects on initiation of spermatogenesis in gonadotropin-deficient (hpg) mice are replicated by testosterone

Testosterone (T) is an absolute requirement for spermatogenesis and is supplied by mature Leydig cells stimulated by LH. We previously showed in gonadotropin-deficient hpg mice that T alone initiates qualitatively complete spermatogenesis bypassing LH-dependent Leydig cell maturation and steroidogenesis. However, because maximal T effects do not restore testis weight or germ cell number to wild-type control levels, additional Leydig cell factors may be involved. We therefore examined 1). whether chronic hCG administration to restore Leydig cell maturation and steroidogenesis can restore quantitatively normal spermatogenesis and testis development and 2). whether nonandrogenic Leydig cell products are required to initiate spermatogenesis. Weanling hpg mice were administered hCG (0.1-100 IU i.p. injection three times weekly) or T (1-cm subdermal Silastic implant) for 6 weeks, after which stereological estimates of germinal cell populations, serum and testicular T content, and testis weight were evaluated. Human CG stimulated Leydig cell maturation and normalized testicular T content compared with T treatment where Leydig cells remained immature and inactive. The maximal hCG-induced increases in testis weight and serum T concentrations were similar to those for T treatment and produced complete spermatogenesis characterized by mature, basally located Sertoli cells (SCs) with tripartite nucleoli, condensed haploid sperm, and lumen development. Compared with T treatment, hCG increased spermatogonial numbers, but both hCG and T had similar effects on numbers of spermatocytes and round and elongated spermatids per testis as well as per SC. Nevertheless, testis weight and germ cell numbers per testis and per SC remained well below phenotypically normal controls, confirming the involvement of non-Leydig cell factors such as FSH for quantitative normalization of spermatogenesis. We conclude that hCG stimulation of Leydig cell maturation and steroidogenesis is not required, and that T alone mostly replicates the effects of hCG, to initiate spermatogenesis. Because T is both necessary and sufficient for initiation of spermatogenesis, it is likely that T is the main Leydig cell secretory product involved and that additional LH-dependent Leydig cell factors are not essential for induction of murine spermatogenesis.     

VEGF111b,a new member of VEGFxxxb isoforms and induced by mitomycin C,inhibits angiogenesis

Vascular endothelial growth factor (VEGF-A) stimulating angiogenesis is required for tumor growth and progression. The conventional VEGF-A isoforms have been considered as pro-angiogenic factors. Another family of VEGF-A isoforms generated by alternative splicing, termed VEGFxxxb isoforms, has anti-angiogenic property, exemplified by VEGF165b. Here, we identify a new number of VEGFxxx family-VEGF111b induced by mitomycin C, although not detected in mitomycin C-unexposed ovarian cancer cells. SKOV3 cells were transfected with pcDNA_3.1 empty vector, pcDNA_3.1-VEGF111b or pcDNA_3.1-VEGF165b to collect conditioned mediums respectively. VEGF111b overexpression inhibits proliferation, migration and tube formation of endothelial cell by inhibiting VEGF-R2 phosphorylation and its downstream signaling, similar to VEGF165b but slightly lower than VEGF165b. The anti-angiogenic property depends on the six amino acids of exon 8b of the VEGFxxxb isoforms. Our results show that VEGF111b is a novel potent anti-angiogenic agent that can target the VEGF-R2 and its signaling pathway to inhibit ovarian tumor growth.     

Effects of testosterone, estradiol, aromatase inhibitor, gonadotropin and prolactin on the response of mouse testes to acute gonadotropin stimulation

These studies determined the local acute responsiveness of the testis to intratesticular administration of human chorionic gonadotropin (hCG) under basal, stimulated (systemic hCG pre-treated), hypogonadotropic (steroid pre-treatment) and hyperprolactinemic conditions in male mice. In addition, testicular testosterone (T) levels were determined after intratesticular administration of the aromatase inhibitor, 4-hydroxyandrostenedione (4-OHA) or progesterone under basal or hCG-stimulated conditions. Intratesticular administration of 0.025, 0.25, 2.5 or 25 mIU hCG resulted in a dose-dependent (3- to 14-fold) increase in testicular T concentrations in hCG compared to vehicle-injected testes. Systemic (i.p.) pre-treatment with 5 IU hCG 24 h before prevented any further increases in the already elevated (10-fold basal) T levels after direct intratesticular hCG injection. Pretreatment with 250 micrograms testosterone propionate (TP) reduced basal testicular T concentrations, but resulted in increased responsiveness to intratesticular hCG administration. In contrast, estradiol benzoate (EB) pretreatment, which also reduced basal testicular T concentrations, did not affect the testicular responsiveness to hCG. Hyperprolactinemia reduced testicular responsiveness to intratesticular administration of 0.025, 0.25 or 2.5 mIU hCG, but basal levels of testicular T were elevated. One hour after intratesticular injections of an aromatase inhibitor, 4-OHA; (0.25 micrograms) testis, T levels were increased in males pre-treated with 5 IU hCG (i.p.) 24 h earlier. Higher doses of 4-OHA (2.5, 25 or 250 micrograms) resulted in significant, dose-related increases in basal testicular T levels which were attenuated by hCG-pre-treatment. Intratesticular administration of 20 micrograms progesterone increased testicular T concentrations 2.7-fold, but this effect was attenuated (1.5-fold) in hCG-pre-treated mice, suggesting that enzymatic lesions beyond progesterone may be involved in hCG-induced testicular desensitization. These results indicate that testicular responsiveness to hCG depends on the existing levels of gonadotropic stimulation. However, it is evident that estrogens and prolactin also influence the sensitivity of the testis to gonadotropin.     

Kinetic and autoradiographic studies on binding of hCG to the testicular LH receptors of neonatal rats

The properties of hCG binding to LH receptors of the neonatal (5-day-old) rat testis were analysed and compared with those of the adult testis. The equilibrium association constants (Ka) of hCG-binding were similar at both ages, 2-4 X 10(10) M-1. In contrast, kinetic binding studies revealed that the association and dissociation rate constants of hCG binding were more rapid in the neonatal testis. Likewise, it was observed that the progression from loose (easily dissociable) to tight (non-dissociable) binding was less complete in the young than in the adult testis. Autoradiography of 125I-labelled hCG binding to interstitial cell suspensions at the two ages showed that the gonadotrophin binding per Leydig cell was about 50% lower in the neonatal testis. Conversely, since the surface area of adult Leydig cells was about 4-fold larger, the receptor density appeared to be higher in the neonatal Leydig cells. The rapid recovery of LH receptors after hCG stimulation, typical of the neonatal cells, was due to rapid replenishment of binding in the cells initially occupied by the injected hormone, rather than to an hCG-induced increase of Leydig cell number. Finally, in-vivo experiments with cycloheximide revealed that the rapid recovery of LH receptors was dependent on protein synthesis. These differences in the kinetics of neonatal testicular LH receptor turnover may be involved in the unique functional features of the fetal-neonatal growth phase of rat testicular Leydig cells.     

VEGF189 stimulates endothelial cells proliferation and migration in vitro and up-regulates the expression of Flk-1/KDR mRNA

The vascular endothelial growth factor (VEGF) is a critical factor for development of the vascular system in physiological and pathological angiogenesis. This growth factor exists under at least three isoforms, VEGF120/121, VEGF164/165 and VEGF188/189 which are generated by alternative splicing. VEGF isoforms have different affinities for heparan sulphate as well as for VEGF receptors, and may play distinct roles in vascular development. The role of VEGF189 as an endothelial mitogen, however, remains controversial. VEGF189 is almost entirely bound to the cell surface or extracellular matrix, and is considered active after its cleavage and release from its extracellular binding site. In the present study, we demonstrate that VEGF189 induces endothelial cell proliferation and migration in vitro. The 30-60% increase observed with VEGF189 (10 ng/ml) in HUVEC proliferation was similar to that observed with VEGF165. However, the proliferative effect observed with VEGF189 appeared dependent on the origin of the endothelial cell, since the proliferation was clearly observed with HUVEC but not with BAEC or capillary endothelial cells from dermis (HMEC). The effect of VEGF189 on endothelial cell migration was also analyzed using the wound healing and the Boyden chamber assays. The migration effect was observed with BAEC which do not proliferate with VEGF189, suggesting that different mechanisms are involved in proliferation and migration. In addition, VEGF189 as well as VEGF165 induced a 2-fold increase of Flk-1/KDR expression in HUVEC, the receptor involved in proliferation and migration of endothelial cells. In the Matrigel plug assay in vivo, both VEGF189 and 165 (100 ng/ml) increased the infiltration of endothelial cells. These data suggest that VEGF189 induced endothelial cell migration and proliferation under certain circumstances.     

A physiological increase in LH may influence vascular permeability in the rat testis

Adult male rats were injected with different doses of hCG, or with 2.5 micrograms ovine LH subcutaneously, and other rats were mated with oestrous females. The animals were examined 4 h after treatments. Treatment with hCG resulted in a dose-dependent increase in leucocyte concentration in testicular blood vessels and in the number of blood vessels which could be labelled with intravenously injected carbon particles. Carbon leakage was not observed in control testes. Treatment with a low dose of ovine LH or inducing an endogenous LH peak by mating also resulted in leucocyte accumulation and vascular leakage of carbon in the testis. The magnitude of the response was considerably lower than after high doses of hCG. The physiological relevance of the discrete response observed after physiological LH stimulation is unknown but LH-induced changes in testicular microcirculation could be of interest for the understanding of the physiology and pathophysiology of the testis.     

Testicular steroidogenesis after human chorionic gonadotropin desensitization in rats.

When a single injection of 500 I.U. of human chorionic gonadotropin (hCG) is given to rats there is an initial acute rise of plasma testosterone and of testicular content for both cyclic AMP and testosterone. This response correlates with an increase in both lyase and 17 alpha-hydroxylase activities. Thereafter both plasma and testicular testosterone decline and do not increase after a second injection of hCG. During this period of desensitization, isolated Leydig cells were insensitive to the steroidogenic stimulatory effect of both hCG and dibutyryl cyclic AMP. The post-cyclic AMP block is not due to an alteration of the cyclic AMP-dependent protein kinase but it is correlated with a decrease in both lyase and 17 alpha-hydroxylase activities of the Leydig cell's microsomes. This decrease is not caused by the absence of the recently described cytosol activator of this enzyme because its addition did not restore the enzymatic activity. Within 60 to 96 h after hCG injection there was a spontaneous increase of both plasma and testicular testosterone and this parallels the recovery of lyase and 17 alpha-hydroxylase activities. These results suggest that both enzymatic activities are regulated, directly or indirectly, by hCG, and that this is partly responsible for the hCG-induced steroidogenic refractoriness of Leydig cells.     

CEA-related cell adhesion molecule 1: a potent angiogenic factor and a major effector of vascular endothelial growth factor

CEA-related cell adhesion molecule 1 (CEACAM1) exhibits angiogenic properties in in vitro and in vivo angiogenesis assays. CEACAM1 purified from granulocytes and endothelial cell media as well as recombinant CEACAM1 expressed in HEK293 cells stimulate proliferation, chemotaxis, and capillary-like tube formation of human microvascular endothelial cells. They increase vascularization of chick chorioallantoic membrane and potentiate the effects of vascular endothelial growth factor (VEGF)165. VEGF165 increases CEACAM1 expression both on the mRNA and the protein level. VEGF165-induced endothelial tube formation is blocked by a monoclonal CEACAM1 antibody. These data suggest that CEACAM1 is a major effector of VEGF in the early microvessel formation. Since CEACAM1 is expressed in tumor microvessels but not in large blood vessels, CEACAM1 may be a target for the inhibition of tumor angiogenesis.     

Effects of photoperiod and hCG on the regulation of testicular LH/hCG receptors in Syrian hamsters (Mesocricetus auratus)

The regulation of testicular LH/hCG receptors was studied in Syrian (golden) hamsters with testicular atrophy induced by exposure to short photoperiod (5L:19D) and in gonadally active hamsters kept in a long photoperiod (14L:10D). By 24 h after injection of hCG, long-photoperiod hamsters showed a dose-related decrease in the number of testicular LH/hCG receptors. At 48 and 72 h, there was a recovery from this 'down-regulation'. The recovery was much faster than has been reported for the rat and mouse, and it resulted in elevation of testicular LH/hCG receptor concentrations above basal values. Hamsters with short photoperiod-induced testicular atrophy showed an increase in testicular LH/hCG receptors after injection of hCG, except for animals injected with a very high dose. The hCG-induced increase in testicular LH/hCG binding in these animals was associated with reappearance of testosterone responses to subsequent hCG stimulation. Response of testicular LH/hCG receptors to hCG in prepubertal hamsters resembled that measured in animals with short photoperiod-induced gonadal atrophy.     

Rat testicular interstitial fluid contains mediators of vasopermeability

An intradermal injection of testicular interstitial fluid (IF) produced a marked increase in vasopermeability in a dose-dependent manner. Likewise bovine follicular fluid caused a smaller but significant response. The effect of IF was associated with accumulation of polymorphonuclear leucocytes (PMNs) inside the dermal venules and with their adherence to the venular endothelium. A minor but significant response was noticed after injecting anterior chamber fluid, but there was no response after an injection of amniotic fluid or serum intracutaneously. Destroying the Leydig cells with ethane dimethanesulphonate did not change the vasopermeability-increasing effect of IF, but after denaturation of IF proteins the effect was diminished by about 50%. Intravenous administration of hCG did not increase the ability of IF to cause the effect. These results suggest that rat testicular interstitial fluid contains mediators of vasopermeability, probably specific for the testis and also follicular fluid. The vasopermeability effect of IF does not seem to depend on the collecting time or on Leydig cells and is at least partly mediated by PMNs which are seen in the dermal venules shortly after an injection of IF.     

Differential effects of cyclic and static stretch on coronary microvascular endothelial cell receptors and vasculogenic/angiogenic responses

Mechanical stretch, an important growth stimulus, results not only from pulsatile blood flow and diastolic stretch of the ventricles [cyclic stretch (CS)] but also from tissue expansion during growth [constant static stretch (SS)]. We compared growth factor receptor expression and vasculogenic/angiogenic responses of rat coronary microvascular endothelial cells (ECs) by exposing cells to CS (10% elongation at 30 cycles/min) and SS (constant 10% elongation). Both CS and SS increased VEGF receptor (VEGF-R)2 protein levels and the extent of tube formation and branching. Moreover, both CS and SS enhanced VEGF-induced cell proliferation and tube formation, indicating that both types of stretch increase the sensitivity of ECs to VEGF. Blockade of VEGF-R2 prevented the increases in EC proliferation and aggregate tube length. However, CS but not SS enhanced EC Tie-2 protein and migration. CS affected a greater increase in tube length and branch formation than did SS. A unique finding was that SS but not CS increased VEGFR-1 in ECs. Our study is the first to distinguish between the effects of CS and SS on growth factor receptor expression and rat coronary microvascular EC proliferation, migration, and tube formation. In conclusion, EC angiogenic responses to these two types of stretch display both differences and similarities, but both CS and SS are dependent on VEGF-R2 signaling for their vasculogenic/angiogenic effects.     

Inhibition of in vitro human chorionic gonadotropin-stimulated testosterone production in testis and of ovulation in the rat by charcoal-treated rat testicular extract

Previously, we described the presence of a factor obtained from a 105,000 X g supernatant of rat testis that was found to inhibit human chorionic gonadotropin (hCG) binding to gonadal receptors. In the present study, similarly prepared testicular extract was tested for its effects on in vitro hCG-stimulated testosterone production by isolated testis interstitial cells and for its effect on spontaneous ovulation in the rat. Incubation of interstitial cells with charcoal-treated extract significantly inhibited the steroidogenic response to hCG in a dose-related manner. This inhibition was also apparent after heating the extract for 10 min at 100 degrees C. Preincubation of the cells with charcoal-treated extract resulted in an inhibitory effect that was not readily reversed by subsequent addition of hCG, revealing an element of irreversibility in the mechanism of inhibition. A single i.p. injection of testicular extract given between 1430-1630 h of proestrus inhibited spontaneous ovulation in the rat. This effect was also observed after heating the extract for 10 min at 100 degrees C; in contrast, no significant effect was obtained with the injection of a similar dose of liver extract. Administration of 5 IU hCG after pretreatment with the testicular extract did not reverse the inhibitory effect on ovulation, indicating that this effect was probably not exerted at the hypothalamus-pituitary level. It is concluded that the aqueous testicular extract contains a factor able to antagonize the physiological events mediated by luteinizing hormone (LH)/hCG, and that this factor is consistent with the presence of an LH/hCG-binding inhibitory activity in rat testis.     

Newly identified biologically active and proteolysis-resistant VEGF-A isoform VEGF111 is induced by genotoxic agents

Ultraviolet B and genotoxic drugs induce the expression of a vascular endothelial growth factor A (VEGF-A) splice variant (VEGF111) encoded by exons 1–4 and 8 in many cultured cells. Although not detected in a series of normal human and mouse tissue, VEGF111 expression is induced in MCF-7 xenografts in nude mice upon treatment by camptothecin. The skipping of exons that contain proteolytic cleavage sites and extracellular matrix–binding domains makes VEGF111 diffusible and resistant to proteolysis. Recombinant VEGF111 activates VEGF receptor 2 (VEGF-R2) and extracellularly regulated kinase 1/2 in human umbilical vascular endothelial cells and porcine aortic endothelial cells expressing VEGF-R2. The mitogenic and chemotactic activity and VEGF111's ability to promote vascular network formation during embyonic stem cell differentiation are similar to those of VEGF121 and 165. Tumors in nude mice formed by HEK293 cells expressing VEGF111 develop a more widespread network of numerous small vessels in the peritumoral tissue than those expressing other isoforms. Its potent angiogenic activity and remarkable resistance to proteolysis makes VEGF111 a potential adverse factor during chemotherapy but a beneficial therapeutic tool for ischemic diseases.     

Antiangiogenic and antitumor activities of peptide inhibiting the vascular endothelial growth factor binding to neuropilin-1

Neuropilin-1 (NRP-1), a non-tyrosine kinase receptor of vascular endothelial growth factor-165 (VEGF165), was found expressed on endothelial and some tumor cells. Since its overexpression is correlated with tumor angiogenesis and progression, the targeting of NRP-1 could be a potential anti-cancer strategy. To explore this hypothesis, we identified a peptide inhibiting the VEGF165 binding to NRP-1 and we tested whether it was able to inhibit tumor growth and angiogenesis. To prove the target of peptide action, we assessed its effects on binding of radiolabeled VEGF165 to recombinant receptors and to cultured cells expressing only VEGFR-2 (KDR) or NRP-1. Antiangiogenic activity of the peptide was tested in vitro in tubulogenesis assays and in vivo in nude mice xenotransplanted in fat-pad with breast cancer MDA-MB-231 cells. Tumor volumes, vascularity and proliferation indices were determined. The selected peptide, ATWLPPR, inhibited the VEGF165 binding to NRP-1 but not to tyrosine kinase receptors, VEGFR-1 (flt-1) and KDR; nor did it bind to heparin. It diminished the VEGF-induced human umbilical vein endothelial cell proliferation and tubular formation on Matrigel and in co-culture with fibroblasts. Administration of ATWLPPR to nude mice inhibited the growth of MDA-MB-231 xenografts, and reduced blood vessel density and endothelial cell area but did not alter the proliferation indices of the tumor. In conclusion, ATWLPPR, a previously identified KDR-interacting peptide, was shown to inhibit the VEGF165 interactions with NRP-1 but not with KDR and to decrease the tumor angiogenesis and growth, thus validating, in vivo, NRP-1 as a possible target for antiangiogenic and antitumor agents.