Cytoplasmic granule formation in mouse pancreatic acinar cells. Evidence for formation of immature granules (condensing vacuoles) by aggregation and fusion of progranules of unit size,and for reductions in membrane surface area and immature granule volume during granule maturation

We used a computer-assisted morphometry approach to analyze quantitatively the process of cytoplasmic granule formation in mouse pancreatic acinar cells stimulated with pilocarpine to induce secretion. Our findings suggest that each condensing vacuole/immature granule of pancreatic acinar cells is formed by the progressive aggregation of 106 to 128 unit progranules of narrowly fixed volume, define a range of 7.7 to 9.2 for the factor of volume condensation between the largest immature granules and the mature unit granule, and predict that the formation of a single mature unit granule by the aggregation and fusion of unit progranules involves a net reduction of at least 95% in the amount of membrane surface area associated with these structures.     

Non-granulated peripolar cells exist in the rat glomerulus

Summary The peripolar cell is a unique cell type in the mammalian glomerulus. Peripolar cells are said to be identifiable during light microscopy by their cytoplasmic granules and by their position at the vascular pole; and during scanning electron microscopy by their distinctive surface morphology. We used both techniques to count peripolar cells in 6 normal rat kidneys. Scanning microscopy revealed that 55(±5)% of glomeruli contained at least one peripolar cell whereas light microscopy revealed granulated peripolar cells in only 4(±2)% of glomeruli. Vascular poles which contained peripolar cells previously identified by scanning were then examined by light and by transmission electron microscopy. Serial sections through these peripolar cells demonstrated the absence of cytoplasmic granules. Our observations suggest that the majority of peripolar cells in the rat contain no granules.     

Hyperplasia of jejunal smooth muscle in the myenterically denervated rat

Summary Application of a cationic surfactant, benzalkonium chloride, to the serosa of rat jejunum results in an increase in thickness of both longitudinal and circular smooth muscle layers. The increase in thickness is due primarily to an increase in the number of smooth muscle cells (hyperplasia). Little cellular hypertrophy was observed. The time sequence of surfactant-induced effects on the muscle layers was determined. Within 24 h, total destruction of the longitudinal muscle and partial destruction of the circular muscle was evident. The myenteric plexus was also necrotic; however, the submucosal plexus remained intact. By 48 h after surfactant treatment, the smooth muscle cells remaining in the circular muscle layer had begun to divide, as indicated by the presence of mitotic figures and incorporation of ³H-thymidine. A repopulation of the longitudinal muscle layer began at this time, apparently the result of migration of cells arising in the circular muscle layer. By 5 days post-treatment, both muscle layers had regenerated to their original states. The myenteric plexus was totally absent. The denervated smooth muscle cells proceeded to divide until approximately day 15, resulting in hyperplasia of both muscle layers. Between 15 and 105 days, the number of muscle cells in the circular layer progressively declined, eventually returned to the value seen in control tissue. In contrast, the number of smooth muscle cells in the longitudinal layer remained elevated through the period of study (165 days). We hypothesize that the smooth muscle hyperplasia observed after serosal benzalkonium chloride application results from loss of the myenteric nerves.     

Ultrastructural characteristics of rat peritoneal mast cells undergoing differential release of serotonin without histamine and without degranulation

Summary Rat mast cells pretreated with the tricyclic antidepressant drug amitriptyline and stimulated with compound 48/80 secreted 60% of the total serotonin present in the cells, but only 15% of histamine, another amine stored in the same granules. Ultrastructural studies demonstrated that mast cells undergoing such differential release do not exhibit classical degranulation by compound sequential exocytosis. However, there were changes in granule shape and size, as well as alterations in many morphometric parameters consistent with secretion. Storage granules lost their homogeneity, exhibited greatly reorganized matrix and were surrounded by clear spaces which were often associated with small (0.1–0.01 m) cytoplasmic vesicles, some of which contained electron-dense material. Secretory granules often had bud-like protrusions or were fused together in series. Quantitative autoradiography localized ³H-serotonin outside the storage granules, close to small vesicles, while staining with ruthenium red demonstrated that vesicular structures associated with differential release were not endocytotic. These results suggest that amitriptyline may inhibit regular exocytosis and permit at least serotonin to be moved selectively from storage granules to the cytosol or small vesicles from which it is eventually released.     

Mast cells and fibrosis in compartments of lymph nodes of normal,gnotobiotic, and athymic rats

Summary Reports vary on the amount and distribution of mast cells in lymph nodes. We analysed the mast-cell population in compartments of nodes of diverse sites, from euthymic and athymic animals of various ages. Nodal mast cells were few in young animals, occurring mostly in medullary sinuses. Aging is often accompanied by a moderate increase of nodal nast cells. In compartments of a few nodes of some aged athymic and euthymic animals, the mast cells were greatly increased in the extrafollicular zone overlying medulla directly. In certain cases, this great increase was accompanied by pronounced mast-cell degranulation and by fibrosis in the mast cell-rich extrafollicular zone. It is suggested that the mast cells of medullary sinuses relate to non-immunological events, while those of the lymphoid parenchyma relate to elements that can induce humoral immune responses or are somehow involved in nodal processes of such responses. It is further suggested that an occasional emergence, with aging, of a deficiency of particular humoral immune responses may induce an excessive increase of cortical mast cells, and that activities of the resulting dense mast-cell population contribute to the onset of fibrosis.Abbreviations AUC above unit center - AUP above unit periphery - AM above medulla (directly above medulla) Funded by the Medical Research Council of Canada     

Cellular changes in rat parathyroids provoked by progesterone and testosterone

Summary Male rats kept on a standard diet were treated either with progesterone or testosterone by a single intramuscular injection of preparations which are slowly absorbed and metabolized. The rats were anaesthetized 24 h after application of the hormones, perfused with glutaraldehyde, and the parathyroid glands prepared for electron microscopy. Morphometric analysis revealed that both progesterone and testosterone provoked (1) an increment in nuclear and cell volume and a concomitant increment in cell surface area, and (2) an increment in surface area of rough endoplasmic reticulum by 42% and 49%, and of the Golgi complex by 85% and 63%, respectively. Previously, we had found that oestradiol treatment led to a similar response in parathyroid cells. The conclusion is thus drawn that male and female sex hormones induce membrane synthesis resulting in an enhanced capacity for parathyroid hormone secretion since RER and Golgi complex are concerned with this secretion. It is considered probable that sex hormones have the ability fundamentally to modulate secretory activity in parathyroid cells.     

Endocytosis of native and glycosylated bovine serum albumin by duct cells of the rat parotid gland

Summary The ability of duct cells of the rat parotid gland to internalize bovine serum albumin (BSA) and several glycosylated albumins (glucosamide, galactosamide, fucosamide, lactosyl, p-aminophenyl-N-acetyl-D-glucosamide, p-aminophenyl-N-acetyl-D-mannopyranoside, p-aminophenyl-N-acetyl-D-galactosamide) was investigated. The various BSA preparations were infused into the gland via the main excretory duct, after which the tissues were fixed and prepared for light and electron microscopy. Immunolocalization of native BSA, as well as the glycosylated BSAs, was performed on thin sections, using an unlabeled antibody to BSA followed by protein A-colloidal gold. Gold particles were present over the lumina of both intercalated ducts and striated ducts, and over small endocytic structures and large vacuoles in the apical cytoplasm of both duct cell types. Endocytosis of the glycosylated BSAs by duct cells was greater than native BSA. Fucosylamide-BSA and mannopyranoside-BSA were taken up to a greater extent than the other glycosylated BSAs. Uptake by intercalated duct cells was greater than by striated duct cells, was independent of the concentration of the glycosylated BSA, and was reduced by an excess of the corresponding sugar. Striated duct cells showed some damage by the glycosylated BSAs that was concentration-dependent, and which was reduced in the presence of an excess of the corresponding sugar. These results suggest that endocytosis by salivary gland duct cells may involve specific recognition of carbohydrate residues and that the endocytosis of acinar secretory proteins observed in certain conditions may be due to increased and/or altered protein glycosylation.     

Suramin inhibits binding and degradation of platelet-derived growth factor in arterial smooth muscle cells but does not interfere with autocrine stimulation of DNA synthesis

Summary During in vitro culture arterial smooth muscle cells of adult rats are able to produce a platelet-derived growth factor (PDGF)-like protein and to promote their own growth in an autocrine manner. Here, this process has been studied using suramin, a polyanionic drug that has been reported to interfere with the cellular binding of several growth factors. Our results indicate that suramin speeds up the transition of the cells from a contractile to a synthetic phenotype early in primary culture. It inhibits the binding of PDGF to the cells, displaces PDGF bound to the cell surface, and slows down the degradation of PDGF internalized by the cells. It reduces the specific activities of the lysosomal enzymes acid phosphatase, -N-ace-tylglucosaminidase and -glucuronidase, and gives rise to an accumulation of lysosomes with myelin-like inlcusions. It blocks PDGF- and serum-induced DNA synthesis and cellular proliferation in secondary cultures, but lacks a distinct inhibitory effect on DNA synthesis in primary cultures under serum-free conditions. The results suggest that the PDGF-like protein produced by the smooth muscle cells under the latter conditions may bind to its receptor and exert its autocrine effect intracellularly, without prior release into the pericellular space.     

Chloroquine and monensin inhibit induction of DNA synthesis in rat arterial smooth muscle cells stimulated with platelet-derived growth factor

Summary The weak base chloroquine and the Na⁺/H⁺ ionophore monensin were used to study the role of lysosomes in the induction of DNA synthesis by platelet-derived growth factor (PDGF) in rat arterial smooth muscle cells cultivated in vitro. The results show that PDGF initiates DNA synthesis in a defined, serum-free medium. This indicates that a single factor may control, directly or indirectly, the transition from the G0 to the G1 phase, the progress through the G1 phase, and the entrance into the S phase of the cell cycle. It is further demonstrated that PDGF has to be present throughout most of the prereplicative period (12–16 h) to induce DNA synthesis in the maximum number of cells, suggesting that one or more processes need to be stimulated continually or successively to push the cell into the S phase. Chloroquine and monensin inhibit induction of DNA replication by PDGF, with maximum effect at 50 M and 5 M, respectively. To be fully active, the drugs have to be added within 4–8 h after the growth factor, but a partial inhibition persists if they are added at any time during the prereplicative period. Both drugs reduce PDGF-stimulated RNA and protein synthesis, and suppress degradation of [³H]leucine-labeled cellular protein and [¹²⁵I]-labeled PDGF. Fine-structurally, they give rise to an accumulation of lysosomes or prelysosomal vacuoles with inclusions of incompletely degraded material. These findings suggest that the mitogenic effect of PDGF is dependent on a normal function of lysosomes during the prereplicative phase, especially its first half (0–8 h).     

Evidence for a coupling of prolactin secretion and synthesis by rat pituitary cells in culture

The relationship between the release and the synthesis of prolactin by rat pituitary cells in culture was studied using a microtubule-disrupting drug, vinblastine. (1) Prolactin secretion was inhibited by vinblastine in short-term incubations. Vinblastine did not act via the dopamine pathway, since a potent anti-dopaminergic drug, fluphenazine, was unable to reverse the inhibiting action of the antimicrotubular agent. (2) Continuous treatment by vinblastine induced a progressive decrease of the rate of incorporation of [³H]leucine in prolactin. The half-inhibition time was about 2 days. This inhibition of prolactin synthesis was selective, since total protein synthesis remained unaffected. (3) Measurements of radioimmunoassayable prolactin showed that the inhibition of hormone release by vinblastine led to a transient increase of the intracellular content of prolactin. The phase of over-accumulation was followed by a progressive reduction of the total (cell + medium) prolactin. This result is in agreement with the observed inhibition of de novo synthesis of prolactin and indicates that a degradation process takes place in pituitary cells in culture. In conclusion, the use of vinblastine allows us to demonstrate that the rate of prolactin synthesis is dependent upon the secretory status of the cell.     

Appearance of B and H blood group antigens in the developing cochlear hair cells

Summary The presence of human blood-group antigens was analyzed in the rat cochlea during its postnatal development, using anti-A, anti-B and anti-H antibodies. At no stage was reactivity with anti-A antibody observed. With the anti-H antibody, a strong reactivity was observed from 1 to 9 days after birth within hair cells and some other surface epithelial cells of the cochlear duct. After postnatal day 9, only a faint reactivity persisted in a few non-sensory cells. With the anti-B antibody, only hair cells were selectively labeled. At early stages (postnatal day 1 and 3), the reactivity was intense and observed both around the cell surface and within the supranuclear region of cytoplasm. Later on, the reactivity decreased; it was limited at postnatal day 9 to a reactive spot below the cuticular plate. Results are compared with a preliminary finding describing the first appearance of B and H antigens in the organ of Corti at a prenatal stage, and with data concerning other sensory and neural structures. The appearance and progressive disappearance of B and H antigens on sensory and non-sensory cells can be correlated with significant events in the development of the cochlea. The transient expression of B and H antigens in cochlear sensory cells may correspond to developmental changes in their surface glycoconjugates.     

Effects of endogenous and exogenous cholesterol on the ultrastructure and steroid secretion of undifferentiated rat adrenocortical cells in primary culture

Summary The present study was undertaken to define the effects of lipoprotein-derived cholesterol and endogenous, de novo synthesized cholesterol on the ultrastructure and function of undifferentiated rat adrenocortical cells [lipoprotein (HDL₃ and LDL) receptor-negative, zona glomerulosa-like adrenocortical cells] in primary culture. For this purpose human plasma high density lipoprotein (HDL₃) or low density lipoprotein (LDL) was added to culture medium devoid of cholesterol. Steroid secretion remained at the low basal level even after addition of lipoproteins, and the amount of intracellular lipid droplets did not increase. When mevinolin (0.96 µg/ml), an inhibitor of cholesterol synthesis, was added to the culture medium, a low secretion of corticosterone was measured both in serum-free and serum-containing media. Ultrastructurally, lipid droplets disappeared after treatment with mevinolin in both media used. At this concentration of mevinolin cell proliferation was similar to that in the controls, but at higher concentrations (4.8 or 9.6 µg/ml) proliferation was inhibited to 42% and 26% in serum-free medium, and 20% and 12% in serum-supplemented medium, respectively. This study demonstrates that cell proliferation and synthesis of corticosterone by undifferentiated rat adrenocortical cells is identical in the absence or presence of exogenous lipoprotein cholesterol. Inhibition of de novo cholesterol synthesis by mevinolin over a period of 7 days does not inhibit corticosterone secretion or proliferation of cells but decreases the amount of intracellular lipid droplets, thus suggesting utilization of intracellular cholesterol esters. However, higher concentrations of mevinolin inhibit proliferation of cells both in serum-free and serum-containing media.     

Neuronal influence on B and H human blood-group antigen expression in rat cochlear cultures

Summary The presence of B and H human blood-group antigens was analyzed by immunocytochemistry in rat cochleas developing either in vivo or in vitro. Developing animals, on embryonic day (E) 18 and postnatal day (P) 3, were used for in vivo studies. For in vitro studies, cochleas were removed at E18 and placed for 3 or 8 days in organotypic culture either directly or after partial spiral ganglion removal. Results from epithelial regions from cochleas developing in vivo were similar to those observed in corresponding areas of direct organotypic cultures where the innervation from spiral ganglion neurons was present. Antibodies to human blood group antigens, anti B and anti AB, selectively labeled hair cells. The intensity of labeling was weak at E18, but increased at P3 in vivo or after 3–8 days in organotypic culture. Anti H antibodies showed weak labeling of the apical surface of hair cells and other epithelial cells at E18; this labeling also increased at P3 or after 3–8 days in culture. In contrast, the non-innervated regions from organotypic cultures, where ganglia were partially removed, exhibited an epithelial disorganization and no hair cell labeling with any of the antibodies studied. The present findings suggest that human blood-group antigen expression on developing cochlear hair cells of rats may be related to afferent nerve fiber influence.     

Morphology and function of capillary networks in subregions of the rat tuber cinereum

Summary The differentiated cytology, cytochemistry, and functions within subdivisions of the tuber cinereum prompted this morphometric and physiological investigation of capillaries in the median eminence and arcuate nucleus of albino rats. Morphometric studies established that the external zone of the median eminence had 3–5 times the number and surface area of true and sinusoidal capillaries than the internal or subependymal median eminence zones, or either of two subdivisions examined in the arcuate nucleus. Type-I true capillaries, around which Virchow-Robin spaces comprise 1% of arcuate tissue area, were situated proximally to the median eminence border. This finding is consistent with a premise that confluent pericapillary spaces enable infiltration of arcuate neurons by factors from capillary blood from the median eminence or Virchow-Robin spaces. Physiologically, the rate of penetration across the median eminence capillaries by blood-borne [¹⁴C]-aminoisobutyric acid (a neutral amino acid used as a capillary permeability tracer) was 142 times greater than for capillaries in the distal arcuate nucleus within 12 s of tracer administration. A new finding was that the proximal arcuate nucleus had a permeability x surface area product of 69 l g^–1 min^–1, 34 times greater than that in more distal aspects of the tuber where blood-brain barrier properties exist. We also found that the microcirculatory transit time of a plasma space marker, [¹⁴C]sucrose, was considerably longer (1.2 s) in the median eminence and proximal arcuate nucleus than in the distal arcuate or ventromedial nucleus (0.4 s). By virtue of its high capillary permeability and extensive blood-tissue surface area, including the wide Virchow-Robin spaces, the median eminence external zone could be a gateway for flooding other tuberal compartments with blood-borne factors. This effect may be compounded by capillary bed specializations in the proximal arcuate nucleus where Type-I true capillaries, Type-III sinusoids, and pericapillary spaces are confluent with those in the median eminence. The results indicate that the proximal arcuate parenchyma could be exposed to circulating neuroactive substances on a moment-to-moment basis.Dedicated to Dr. Milton W. Brightman of Bethesda, Maryland, USA on the occasion of his 67th birthday and tribute as Craigie scholar at the First Craigie Conference on Brain Capillaries, Toronto, Ontario, June 24, 1990     

The volume and ionic composition of cells in incubated slices of rat renal cortex, medulla and papilla

The apparent extracellular space in incubated slices of rat renal cortex, medulla and papilla has been measured using three differently sized marker molecules, mannitol, sucrose and inulin. Cellular volumes have been estimated by following the efflux of from equilibrated slices. Sucrose appears to be the most accurate extracellular marker in each of the regions examined, in that the sum of its volume of distribution plus cellular volume approximates most closely to the total slice fluid volume. Inulin has the same volume of distribution as sucrose in cortical slices, but under-penetrates medullary and papillary tissue. Mannitol overestimates the extracellular space in all three regions, although its larger volume of distribution, relative to that of sucrose, was not statistically significant in papillary slices. When cell volume and composition are estimated (a) using sucrose as extracellular marker and (b) making appropriate allowance for the presence of bound tissue electrolytes, it is found that cells in each region have low Na⁺ and high K⁺ concentrations and contents. When papillary slices are incubated in medium of very high osmolality (NaCl plus urea, 2000 mosmol/kg H₂O) there is a moderate (approx. 23%) decrease in cell volume and an increase in cell fluid Na⁺ and Cl⁻ concentrations equal to approx. 50% of the increase in the extracellular concentrations. Cell K⁺ concentrations remain unchanged. The results show that cells in renal slices are able to maintain high K⁺-to-Na⁺ ratios when incubated in isosmotic (cortex) or moderately hyperosmotic media (medulla and papilla), and suggest that regulation of papillary cell volume following hyperosmotic shock can only partly be ascribed to uptake of extracellular electrolytes.     

Effects of mevinolin,an inhibitor of cholesterol synthesis,on the morphology and function of differentiating and differentiated rat adrenocortical cells in primary culture

Summary Mevinolin, an inhibitor of cholesterol synthesis, was used to study the effect of endogenous cholesterol synthesis on the morphology and function of differentiating and differentiated fetal rat adrenocortical cells grown in primary culture. Upon adrenocorticotrophic hormone (ACTH) stimulation under conditions in which endogenous cholesterol synthesis was inhibited but exogenous (lipoprotein) cholesterol was available, the cells differentiated normally from glomerulosa-like to fasciculata-like cells; the steroid hormone secretion was maximally induced. Under conditions in which cholesterol synthesis was maximally inhibited by mevinolin and the cells had no access to exogenous cholesterol, the cells did not differentiate into fasciculata-like cells; the ACTH-induced steroid response was highly suppressed under these conditions. The addition of either human low-density lipoprotein (LDL) or high-density lipoprotein (HDL₃) to the culture medium restored the ACTH-induced differentiation and steroid secretion. Thus, in the absence of exogenous cholesterol, endogenous cholesterol synthesis was a prerequisite for differentiation. In cultures grown in the presence of exogenous cholesterol and ACTH with mevinolin-inhibited cholesterol synthesis and high steroid output, an increase in cytoplasmic lipids was evident, suggesting upregulation of LDL and HDL receptors. The results also demonstrated that induction of phenotypic differentiation from glomerulosalike into fasciculata-like cells can proceed in the presence of a cholesterol synthesis inhibitor like mevinolin; this differentiation in the absence of endogenous cholesterol synthesis is accompanied by the appearance of cytoplasmic cholesterol ester droplets, typical of fasciculata cells.     

Peptidergic innervation of rat lymphoid tissue and lung: Relation to mast cells and sensitivity to capsaicin and immunization

Summary The peptidergic innervation of lymphoid tissue and the lung in relation to mast cells was studied in rat. The sensitivity of neuropeptide-containing nerves to capsaicin treatment and immunization was also examined. Measurements of the content of neurokinin A and calcitonin gene-related peptide revealed that the lung contained the highest content of both neuropeptides; lymph nodes had intermediate levels, whereas the spleen had the lowest content. Immuhohistochemistry showed that the calcitonin gene-related peptide- and neurokinin A-immunoreactive nerves in lymph nodes were mainly found around blood vessels, whereas in the lung the nerves were present within the lining respiratory epithelium, bronchial smooth muscle, around blood vessels and close to lymphoid aggregates. Combined immunohistochemistry for serotonin (5-hydroxytryptamine), as a marker for mast cells, and tachykinins or calcitonin gene-related peptide revealed that a close association was often present between the nerves and 5-hydroxytryptamine-positive cells in the bronchi of the lung, while 5-hydroxytryptamine-positive cells were not observed in lymph nodes. The neurokinin A and calcitonin gene-related peptide content in lymph nodes, spleen and lung, but not the content of neuropeptide Y, was markedly decreased by capsaicin treatment, suggesting a sensory origin for the two former peptides. Aerosol immunization increased the levels of calcitonin gene-related peptide in the lung, whereas the content in mediastinal lymph nodes was not affected. These data demonstrate a peptidergic innervation mainly of blood vessels in lymphoid tissue and a close relation between sensory nerves and mast cells as well as lymphoid aggregates in the bronchi of the lung. This further suggests that the sensory innervation of lymph nodes is mainly related to regulation of vascular tone and lymph flow. Furthermore, at the site of immunization, i.e., in the airway mucosa, sensory nerve mediators may interact both with mast cells and lymphoid cells.     

Immunolocalization of albumin and transferrin in germ cells and Sertoli cells during rat gonadal morphogenesis and postnatal development of the testis

The localization of albumin and transferrin was examined immunohistochemically in germ cells and Sertoli cells during rat gonadal morphogenesis and postnatal development of the testis. These proteins appeared as early as the 13th day of gestation in migrating primordial germ cells before Sertoli cell differentiation. In the fetal testis, strong immunoreactivity was only detected in the gonocytes. In the prepubertal testis, spermatogonia, primary spermatocytes, and some Sertoli cells accumulate albumin and transferrin. At puberty, different patterns of immunostaining of the germ cells were observed at the various stages of the cycle of the seminiferous epithelium. Diplotene spermatocytes at stage XIII, spermatocytes in division at stage XIV, and round spermatids at stages IV–VIII showed maximal staining. Labeling was evident in the cytoplasm of adult Sertoli cells. Albumin and transferrin staining patterns paralleled each other during ontogenesis.     

Expression of thyrotropin-releasing hormone receptors in rat testis and their role in isolated Leydig cells

Thyrotropin-releasing hormone (TRH) was initially discovered as a neuropeptide synthesized in the hypothalamus. Receptors for this hormone include TRH-receptor-1 (TRH-R1) and -2 (TRH-R2). Previous studies have shown that TRH-R1 and TRH-R2 are localized exclusively in adult Leydig cells (ALCs). We have investigated TRH-R1 and TRH-R2 expression in the testes of postnatal 8-, 14-, 21- 35-, 60-, and 90-day-old rats and in ethane dimethane sulfonate (EDS)-treated adult rats by using Western blotting, immunohistochemistry, and immunofluorescence. The effects of TRH on testosterone secretion of primary cultured ALCs from 90-day-old rats and DNA synthesis in Leydig cells from 21-day-old rats have also been examined. Western blotting and immunohistochemistry demonstrated that TRH-R1 and TRH-R2 were expressed in fetal Leydig cells (in 8-day-old rats) and in all stages of adult-type Leydig cells during development. Immunofluorescence double-staining revealed that newly regenerated Leydig cells in post-EDS 21-day rats expressed TRH-R1 and TRH-R2 on their first reappearance. Incubation with various doses of TRH affected testosterone secretion of primary cultured ALCs. Low concentrations of TRH (0.001, 0.01, and 0.1 ng/ml) inhibited basal and human chorionic gonadotrophin (hCG)-stimulated testosterone secretion of isolated ALCs, whereas relatively high doses of TRH (1 and 10 ng/ml) increased hCG-stimulated testosterone secretion. As detected by a 5-bromo-2′-deoxyuridine incorporation test, the DNA synthesis of Leydig cells from 21-day-old rats was promoted by low TRH concentrations. Thus, we have clarified the effect of TRH on testicular function: TRH might regulate the development of Leydig cells before maturation and the secretion of testosterone after maturation. This research was supported by grants from the National Natural Science Foundation of China (nos. 39870109 and 30370750).