Percoll-gradient separation of Leydig cells from postnatal rat testes

The characteristics of the fetal and adult populations of Leydig cells from postnatal rat testes were compared by Percoll gradient centrifugation. A single peak of hCG binding, due to the presence of fetal Leydig cells, was obtained after purification of intertubular cells from 8-day-old animals. Two peaks of specific hCG binding were obtained after purification of intertubular cells from 15-day-old rats: it was confirmed by autoradiographic techniques that the hCG was bound by adult-like Leydig cells in one peak and fetal Leydig cells in the other. Similarly, intertubular cell preparations from 21- and 25-day-old rats resolved into two peaks of hCG binding; adult-like Leydig cells were observed in the first peak, but fetal Leydig cells were rarely observed in the second of these peaks. These results demonstrate the separation of two Leydig cell populations from intertubular cells obtained from animals aged up to 15 days. Thereafter the pattern of the hCG binding profile is similar but is not due to the presence of the same cell types. Therefore these results emphasize the necessity for morphological identification of cell types to permit the correct interpretation of the corresponding biochemical data.     

Interstitial cell heterogeneity in rat testes. I. Purification of collagenase-dispersed Leydig cells by unit gravity sedimentation and demonstration of binding sites for gonadotropin in light cells versus enhanced steroidogenesis in heavier cells

Two testicular interstitial cell fractions, light and heavier, biochemically and morphologically distinct were obtained by a unit gravity sedimentation procedure. Binding sites for 125I-labeled human chorionic gonadotropin (hCG) were preferentially localized in the light cell fraction (apparent Kd = 2.02 X 10(-10) M; Bmax = 1.17 X 10(-5) nmol/2 X 10(6) cells). These cells did not synthesize testosterone in response to hCG, but the basal release of testosterone was higher than by cells in the heavier fraction (2.49 +/- 0.02 ng/2 X 10(6) cells in the light versus 0.22 +/- 0.00 ng/2 X 10(6) cells in the heavier fraction). The cells in the heavier fraction bound little or no hCG. The binding data from this fraction did not obey saturation kinetics, but testosterone levels were elevated 700-800% in the presence of hCG (i.e. basal value 0.22 +/- 0.00 ng/2 X 10(6) cells versus 1.81 +/- 0.04 ng/2 X 10(6) cells in hCG-stimulated cells). Electron microscopy revealed that heavier cells had features typical of Leydig cells such as large ovoid nucleus with peripherally located heterochromatin, numerous mitochondria with tubular cristae, some lipid droplets, extensively developed smooth endoplasmic reticulum, and well developed Golgi complex. The cells in the light fraction contained an ovoid nucleus with one or more deep infoldings, and their most notable cytoplasmic feature was the presence of numerous vacuoles of varying sizes and shapes. Based upon this and the investigation which follows (Bhalla, V.K., Flasch, M.V., Browne, E.S., Sohal, G.S., and Sharawy, M.M. (1987) J. Biol. Chem. 262, 5322-5332), we conclude that occupancy of high affinity hCG binding sites, generally assumed to be coupled to steroidogenesis, is not necessarily related to the elicitation of this biological response.     

Oxytocin in Leydig cells: an immunocytochemical study of Percoll-purified cells from rat testes

Summary Testicular interstitial cells, from rats aged 35 days, were dispersed with collagenase and separated through Percoll into 5 fractions (I–V); fraction I being the least dense. Measurement of basal testosterone production, histo-enzymological staining for 3-hydroxysteroid dehydrogenase activity and electron microscopy indicated that the majority of Ley dig cells were found in fraction IV (corresponding to a density of 1.076–1.097 g/ml). In addition, cells from this fraction responded to hCG treatment in a dose-dependent manner on day 0 and remained responsive after being cultured for 1 day. Immunostaining for oxytocin indicated that this fraction also contained the majority of the oxytocin-immunoreactive cells. On day 1 of culture, 56% of the cell population from fraction IV were positively stained for the steroidogenic enzyme and 75% immunoreactive for oxytocin. This overlap indicates that the Leydig cells were also the oxytocin immunoreactive cells.     

Characterization of insulin binding sites in cultured smooth muscle cells of rat aorta

Insulin receptors could be demonstrated in cultured smooth muscle cells of rat aorta. The specific binding of 125I-insulin was time-, temperature- and pH-dependent. The optimal temperature for our studies was 12 degrees C. At this temperature maximal specific binding was 0.5% of total counts at 120 min incubation. The pH-optimum for the binding process was between 7.5 and 8. Degradation of 125I-insulin at 12 degrees C was 14%, no degradation of binding sites could be measured at this temperature. Dissociation of 125I-insulin was rapid. 50% of the labeled hormone remained associated with the cells. Half-maximal inhibition of 125I-insulin binding was produced by insulin at 4 X 10(-11) mol/l. Scatchard-analysis gave curvilinear plots, that may suggest negative cooperativity. Specificity of binding was studied in competition experiments between 125I-insulin, insulin, proinsulin, insulin-like growth factors and human growth hormone. Half-maximal inhibition of 125I-insulin binding was produced by proinsulin at 2 X 10(-9) mol/l and by insulin-like growth factors at 9 X 10(-9) mol/l. Human growth hormone had no significant effect on the insulin binding.     

Distribution of concanavalin A binding sites on the surface of dissociated rat submandibular gland acinar cells.

The submandibular glands of 4-week-old rats were dissociated by a procedure involving digestions with collagenase and hyaluronidase, chelation of divalent cations and mechanical force. A suspension of single cells was obtained in low yield by centrifugation in a Ficoll-containing medium. Immediately after dissociation and after a culture period of 16-18 hr the dissociated cells were tested for agglutinability by concanavalin A (Con A). Using ferritin (tfer)-conjugated Con A the lectin binding by the isolated acinar cells was also studied. The dissociated cells were agglutinated by low concentrations of Con A and bound Fer-Con A molecules on their entire surface without any indication of polarization of the cell membrane. There was a considerable cell to cell variation in the amount of Fer-Con A binding which was, in general, sparse and patchy. The contact surfaces between agglutinated cells revealed a dense binding of Fer-Con A molecules irrespective of the types of cells participating in the agglutination reaction. Cells cultured for 16-18 hr were no longer agglutinated by Con A. As compared to the freshly dissociated cells the cultured acinar cells revealed a more uniform and denser binding of Fer-Con A molecules. Furthermore, there were more lectin molecules bound to the cell surface corresponding to the basal part of the cell, where the nucleus and most of the rough surface endoplasmic reticulum were located, than to the apical cell surface. It is suggested that the higher density of lectin-binding sites on the cell surface in the vicinity of the cisternae of the rough endoplasmic reticulum indicates insertion sites of newly synthesized membrane glycoproteins.     

Identification of specific binding sites for vasoactive intestinal peptide in rat testis Leydig cells and study of developmental changes

The interaction of vasoactive intestinal peptide (VIP) with isolated Leydig cells from rat testis was time- and temperature-dependent, as well as saturable and specific. Scatchard analysis suggested the presence of both high- and low-affinity binding sites with KD values of 1.7 and 43 nM, respectively, and receptor concentrations of 35 and 1394 fmol VIP bound/mg protein in mature (3- to 6-month old) rats. When considering pubertal (45-day old) rats, the affinities were similar but the binding capacities showed considerably lower values (25 and 193 fmol VIP bound/mg protein) indicating that VIP receptors are subject to developmental changes during animal maturation.     

Gonadotropin stimulation of cyclic adenosine monophosphate and testosterone production without detectable high-affinity binding sites in purified Leydig cells from rat testis.

Rat testicular interstitial cells were separated by three different gradient-density procedures and, with each, two biochemically and morphologically distinct cell fractions were isolated. The lighter density cells in fraction-I bound iodine 125-labeled human chorionic gonadotropin (hCG) with high-affinity (apparent equilibrium dissociation constant, Kd, approximately 10(-10) M) without producing either cyclic adenosine monophosphate or testosterone in response to hormone action. The heavier-density cells displayed morphologic features typical of Leydig cells and produced cyclic adenosine monophosphate and testosterone in the presence of hCG without detectable 125I-labeled hCG high-affinity binding. These cell fractions were further characterized by studies using deglycosylated hCG, a known antagonist to hCG action. Cell concentration-dependent studies with purified Leydig cells revealed that maximal testosterone production was achieved when lower cell concentrations (0.5 x 10(6) cells/250 microliters) were used for in vitro hCG stimulation assays. Under these conditions, the 125I-labeled hCG binding was barely detectable (2.24 fmol; 2,698 sites/cell). Furthermore, these studies revealed that the hCG-specific binding in Leydig cells is overestimated by the classic method for nonspecific binding correction using excess unlabeled hormone. An alternate method is presented.     

Distribution of IGF-I mRNA and IGF-I binding sites in the rat kidney.

In the present study we have investigated the distribution of IGF-I mRNA and IGF-I binding sites in the rat kidney. The distribution of IGF-I mRNA was investigated using a simple and sensitive non-radioactive in situ hybridisation technique based on probe labelling with digoxigenin labelled-UTP followed by detection with conventional immunocytochemical techniques. IGF-I mRNA was found predominantly in medullary collecting ducts and sparsely in cortical collecting duct cells. In addition IGF-I mRNA was expressed in scattered proximal tubular cells in the cortex and in cells confined to the glomerular tuft. IGF-I binding sites were studied using radiolabelled IGF-I and conventional autoradiographical techniques on tissue sections. It was found that IGF-I binding sites were widely distributed throughout the entire kidney and that the specific binding was highest in the inner medulla. These findings add further complexity to the understanding of IGF-I production and action on renal structures.     

Distribution of bombesin binding sites in the rat gastrointestinal tract

In an attempt to identify potential target sites for the satiety action of bombesin (BN), the distribution and pharmacological specificity of bombesin binding sites were examined in the rat gastrointestinal tract by in vitro autoradiography utilizing (125I-Tyr4) bombesin. Specific BN binding was localized to the circular muscle level of the gastric fundus and antrum, submucosal layer of the small intestine and longitudinal and circular muscle and submucosal layers of the colon. Pharmacological studies indicated that gastrin releasing peptide (GRP), Ac-GRP20-27 and BN-like compounds, litorin and ranatensin, inhibited the binding of (125I-Tyr4)BN with high affinity while compounds which lacked COOH-terminal homology with BN demonstrated a low affinity for BN binding sites. The wide distribution of BN binding sites in the gastrointestinal tract provides a number of potential sites for the mediation of the satiety action of BN.     

Effect of an acute exposure of rat testes to gamma rays on germ cells and on Sertoli and Leydig cell functions.

Germ cells and Sertoli and Leydig cell functions were studied from 7 to 180 days after an acute exposure of 2-month-old rat testes to 9 Gy of gamma rays. Body weight, testis and epididymal weights were recorded. Sertoli cell parameters (androgen-binding protein, ABP, in caput epididymis and plasma follicle stimulating hormone, FSH) and Leydig cell parameters (plasma luteinizing hormone, LH, testosterone and prostate and seminal vesicle weights) were determined together with the number of germ cells and Sertoli cells. Irradiation did not affect body weight but significantly reduced testicular and epididymal weights from day 7 and day 15 post-irradiation respectively. The cells killed by irradiation were mainly spermatogonia and preleptotene spermatocytes engaged in replicating their DNA at the time of exposure, but all spermatocytes seemed damaged as they gave abnormal descendent cells. By day 34, only elongated spermatids remained in a few tubules and thereafter very little regeneration of the seminiferous epithelium occurred, except for one rat which showed a better regeneration. Levels of ABP decreased by day 15 when the germ cell depletion had reached the pachytene spermatocytes, whereas FSH and LH levels rose when the number of elongated spermatids decreased. Levels of testosterone and the weight of the seminal vesicles did not change; occasionally, the prostate weight was slightly reduced. These results support our hypothesis that pachytene spermatocytes and elongated spermatids are involved in influencing some aspects of Sertoli cell function in the adult rat.     

Characterization of specific insulin binding sites on chromaffin cells from bovine adrenal medulla

1. Insulin receptors were investigated in isolated chromaffin cells from bovine adrenal medulla. 2. The cells were incubated with [125I]insulin in HEPES buffer, pH 7.8 at 15 degrees C for 180 min to obtain steady state binding. Specific binding was linearly related to the number of cells in the range 0.5-10 x 10(6) cells/ml. Insulin and proinsulin caused half maximal displacement of specifically bound tracer in concentrations of 0.18 and 2.46 nM, respectively. 3. Computer analysis of the binding data gave a linear Scatchard plot, consistent with a single class of non-interacting receptors with an affinity constant of 5.6 nM-1, the total number of receptors per cell being 1700. 4. The apparent MW of the insulin binding subunit of the receptor was 135,000, determined by affinity crosslinking and SDS gel electrophoresis under reducing conditions.     

Radioautographic and quantitative study of insulin binding sites in the rat brain

In the present study, we describe the specificity and the autoradiographic distribution of insulin binding sites in the rat central nervous system (CNS) after in vitro incubation of brain sections with [125I]-14A insulin. Increasing concentrations of unlabeled insulin produced a dose-dependent inhibition of [125I]-insulin binding which represented 92 +/- 2% displacement with 3 X 10(-5) M, whatever the brain sections tested. Half-maximum inhibition with native insulin was obtained with 2.2 X 10(-9) M, with 10(-7) M proinsulin whereas glucagon had no effect. Under our experimental conditions, no degradation of [125I]-insulin was observed. Autoradiograms obtained by apposition of LKB 3H-Ultrofilm showed a widespread distribution of [125I]-insulin in rat CNS. However, quantitative analysis of the autoradiograms with 10(-10) M of labeled insulin, showed a high number of [125I]-insulin binding sites in the choroid plexus, olfactory areas, in both cerebral and cerebellar cortices, the amygdaloid complex and in the septum. In the hippocampal formation, the dorsal dentate gyrus and various subfields of CA1, CA2 and CA3 were labeled. Moreover, arcuate, dorso- and ventromedial nuclei of the hypothalamus contained high concentrations of [125I]-insulin whereas a low density was observed in the mesencephalon. The metabolic role of insulin in the CNS is supported by the large distribution of insulin binding sites in the rat brain. However, the presence of high affinity binding sites in selective areas involved in perception and integrative processes as well as in the regulation of both feeding behavior and neuroendocrine functions, suggests a neuromodulatory role of insulin in the brain.     

Prolactin augments luteinizing hormone binding to rat Leydig cells in serum-free culture

Effect of prolactin on the testicular luteinizing hormone binding was studied in a serum-free culture system. By the collagenase digestion of decapsulated testes taken out from 25-day-old rats, Leydig cells were isolated and cultured for 7 days in DME/F12 (1:1) medium supplemented with insulin, transferrin, epidermal growth factor, and gentamicin. The cultured cells exhibited the 3β-hydroxysteroid dehydrogenase activity. Hill plots constructed from the data of competition experiment showed that the dissociation constant (K_d) was 0.33 × 10^–10M. The K_d value was approximately the same as the known value for the rat testicular homogenates. When the Leydig cells were cultured with ovine prolactin for the last 3 days of 7-day culture period, the binding of luteinizing hormone increased to 1.7-fold ofthat in the control group. From these results it is concluded that prolactin acts to up-regulate the binding of luteinizing hormone to rat testicular Leydig cells in serum-free culture     

Effect of annexin I on insulin secretion through surface binding sites in rat pancreatic islets

This study investigates the effect of extracellular annexin I (Anx I) on regulating insulin secretion in isolated rat pancreatic islets. Results show that Anx I stimulates insulin release in pancreatic islets regardless of the presence or absence of extracellular Ca2+. In particular, confocal microscopy shows that Anx I binds to the surface of islet cells in the absence of extracellular Ca2+. However, insulin secretion through Anx I significantly decreases in trypsin-treated islets. Likewise, there is minimal binding of Anx I to the surface of trypsin-treated islets. Anti-Anx I polyclonal antibody also inhibits the stimulating effect of Anx I on insulin secretion. These results indicate that Anx I is capable of binding to the cell surface receptor, in order to regulate the stimulation of insulin release in rat pancreatic islets.     

Gonadotropin receptor regulation in hypophysectomized rat Leydig cells.

The number of gonadotropin receptors decrease in the Leydig cells following hypophysectomy (hypox). The receptor number is reduced to 52, 48, 11, 10 and 12 % of the control 8 days following hypophysectomy in 40, 50, 60, 70 and 80 days old rats respectively. hCG injection (0.6 or 30 μg) produces a decrease in the receptor number in 58 days old hypox rat. Receptors remain almost undetectable between 24 to 72 hours following hCG injection. Desensitization to hCG is observed between 12 and 48 hours and full responsiveness to hCG is obtained at 60 hours following hCG injection (0.6 μg).The results demonstrate that LH is not a necessary condition for the presence of gonadotropin receptors in the Leydig cells and that hCG induces the “down regulation” of the receptors and the desensitized state as well in the hypox as in the intact animal. They also indicate that a variation in the number of gonadotropin receptors is probably not the major biochemical alteration esponsible for steroidogenic refractoriness in Leydig cells.