Response of the human testis to long-term estrogen treatment: Morphology of Sertoli cells,Leydig cells and spermatogonial stem cells

Summary The present investigation is concerned with the morphological changes observed in human testicular tissue following prolonged estrogen administration. Testicular material obtained from 11 transsexual patients who had been submitted to long-term estrogen treatment prior to sex-reversal surgery was studied by means of light- and electron microscopy.The testes of all patients examined present a more or less uniform appearance: There are narrow seminiferous cords surrounded by an extensively thickened lamina propria. They contain Sertoli cells and spermatogonia exclusively. There is no evidence of typical Leydig cells.The persisting spermatogonia show the characteristic features of pale type-A spermatogonia, whereas dark type-A spermatogonia are almost completely eliminated from the epithelium. In view of the fact that spermatogonia that survived radiotherapy and treatment with various noxious agents have recently been regarded as the stem cells of the human testis, it is suggested that also the majority of those spermatogonial types that are less sensitive to disturbances of the endocrine balance may consist of stem cells. The present results, therefore, corroborate the concept that the stem cells of the human testis may be derived from pale type-A spermatogonia or the variants of this cell type.Sertoli cells display two types of ovoid nuclei. In contrast to untreated material the nuclei lie adjacent to the basal lamina, and organelles and telolysosomes are confined to the apical cytoplasm. The apico-basal differentiation of mature cells, therefore, is not observed. Moreover, typical organelles and inclusions of mature cells are absent, as are the junctional specializations. Thus, Sertoli cells have transformed into immature cells, resembling precursors prior to puberty.Fibroblast-like cells in the interstitial tissue, which display strongly lobulated nuclei, a well-developed smooth endoplasmic reticulum, lipid droplets, and numerous inclusions are assumed to represent dedifferentiated Leydig cells.Since after estrogen treatment serum testosterone and gonadotropin levels are known to be reduced, it appears that the morphological changes correlate well with the endocrine status.     

Pterostilbene exerts an anti-inflammatory effect via regulating endoplasmic reticulum stress in endothelial cells

Pterostilbene (PT), an analog of resveratrol, exerts a potent anti-inflammatory effect. However, the protective effects of PT against inflammation in endothelial cells have not been elucidated. Previous studies have confirmed that endoplasmic reticulum stress (ERS) plays an important role in regulating the pathological process of endothelial cell inflammation. In this study, we explored the effect of PT on the tumor necrosis factor-α (TNF-α)-induced inflammatory response in human umbilical vein endothelial cells (HUVECs) and elaborated the role of ERS in this process. TNF-α treatment significantly upregulated the levels of inflammation-related molecules in cell culture media, increased the adhesion of monocytes to HUVECs, and enhanced the expression of the MMP9 and ICAM proteins in HUVECs. Additionally, TNF-α potently increased ERS-related protein levels, such as GRP78 and p-eIF2α. However, PT treatment reversed the increased production of inflammatory cytokines and the adhesion of monocytes to HUVECs, as well as reduced the TNF-α-induced effects exerted by ERS-related molecules. Furthermore, thapsigargin (THA), an ERS inducer, attenuated the protective effect of PT against TNF-α-induced inflammation and ERS in HUVECs. Additionally, the downregulation of ERS signaling using siRNA targeting eIF2α and IRE1 not only inhibited ERS-related molecules but also simulated the therapeutic effects of PT on TNF-α-induced inflammation. In summary, PT treatment potently attenuates inflammation in vascular endothelial cells, which at least partly depends on the reduction of ERS.     

Nitric oxide/cGMP pathway components in the Leydig cells of the human testis

In this study we sought to determine whether the main components of the nitric oxide (NO) pathway are localized within the Leydig cells of the human testis and whether the soluble guanylyl cyclase (sGC), the enzyme that accounts for NO effects, is functionally active in these cells. Using an amplified immunocytochemical technique, immunoreactivity for nitric oxide synthase (NOS-I), sGC and cyclic guanosine monophosphate (cGMP) was detected within the cytoplasm of human Leydig cells. Distinct differences in staining intensity were found between individual Leydig cells, between cell groups and between Leydig cells of different patients. By means of a specific cGMP-RIA, a concentration-dependent increase in the quantity of cGMP was measured in primary cultures of human Leydig cells following exposure to the NO donor sodium nitroprusside. In addition, NOS-I immunoreactivity was seen in Sertoli cells, whereas cGMP and sGC immunoreactivity was found in Sertoli cells, some apically situated spermatids and residual bodies of seminiferous tubules. Dual-labelling studies and the staining of consecutive sections showed that there are several populations of Leydig cells in the human testis. Most cells were immunoreactive for NOS-I, sGC and cGMP, but smaller numbers of cells were unlabelled by any of the antibodies used, or labelled for NOS-I or cGMP alone, for sGC and cGMP, or for NOS-I and sGC. These results show that the Leydig cells possess both the enzyme by which NO is produced and the active enzyme which mediates the NO effects. There are different Leydig cell populations that probably reflect variations in their functional (steroidogenic) activity. Received: 27 March 1996 / Accepted: 14 July 1996     

The endoplasmic reticulum in regenerating liver cells

Summary Light-microscopic analysis of mouse liver homogenates six days after partial hepatectomy, showed a higher percentage of nuclei with adherent cytoplasm than homogenates from normal liver. This observation was true for animals with either a slow or rapid recovery of body weight after the operation. The phenomenon was not a function of the changes in the proportions of parenchymal and non-parenchymal tissue in the regenerating liver.Electron-microscopic analysis of random samples from normal and regenerating livers indicated an increase in the perinuclear rough endoplasmic reticulum, and a displacefment of the glycogen depots within the regenerating cells six days after partial hepatectomy.The marked resistance towards homogenization, shown by the cytoplasm of the regenerating cells, may have been due to the observed increase of perinuclear membranes. However, qualitative changes of the cell membranes and a general decrease of proteolytic activity connected with liver regeneration may also have contributed.     

G1对EA.hy926内皮细胞内质网应激的抑制作用

目的观察GPR30受体激动剂G1对高糖诱导的EA.hy926内皮细胞内质网应激(endoplasmic reticulum stress,ERS)的影响。方法选用EA.hy926内皮细胞为研究对象,分为3组:正常对照组(Con,17.51mmol/L葡萄糖)、高糖组(HG,33.3mmol/L葡萄糖)、高糖+G1组(HG+G1,HG+1umol/L G1),利用流式细胞术检测3组细胞凋亡率,Western blot法检测ERS相关分子Bip、IRE1、PERK及凋亡分子Bax、Bcl-2的表达变化,RT-PCR法检测Bip和CHOP的mRNA表达变化。结果 HG组与Con组比较,细胞凋亡率明显升高(P0.01),Bip、IRE1、PERK及凋亡分子Bax表达上调(P0.01,P0.05或P0.001),Bcl-2的表达下调(P0.01),Bip mRNA、CHOP mRNA表达上调(P0.001及P0.01);HG+G1组与HG组比较,细胞凋亡率明显降低(P0.05),Bip、IRE1、PERK及凋亡分子Bax表达下调(P0.05或P0.01),Bcl-2的表达上调(P0.05),Bip mRNA、CHOP mRNA表达下调(P0.001及P0.01)。结论 GPR30受体激动剂G1可抑制EA.hy926内皮细胞内质网应激。     

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).     

Ouabain enhances exocytosis through the regulation of calcium handling by the endoplasmic reticulum of chromaffin cells

The augmentation of neurotransmitter and hormone release produced by ouabain inhibition of plasmalemmal Na⁺/K⁺-ATPase (NKA) is well established. However, the mechanism underlying this action is still controversial. Here we have shown that in bovine adrenal chromaffin cells ouabain diminished the mobility of chromaffin vesicles, an indication of greater number of docked vesicles at subplasmalemmal exocytotic sites. On the other hand, ouabain augmented the number of vesicles undergoing exocytosis in response to a K⁺ pulse, rather than the quantal size of single vesicles. Furthermore, ouabain produced a tiny and slow Ca²⁺ release from the endoplasmic reticulum (ER) and gradually augmented the transient elevations of the cytosolic Ca²⁺ concentrations ([Ca²⁺]_c) triggered by K⁺ pulses. These effects were paralleled by gradual increments of the transient catecholamine release responses triggered by sequential K⁺ pulses applied to chromaffin cell populations treated with ouabain. Both, the increases of K⁺-elicited [Ca²⁺]_c and secretion in ouabain-treated cells were blocked by thapsigargin (THAPSI), 2-aminoethoxydiphenyl borate (2-APB) and caffeine. These results are compatible with the view that ouabain may enhance the ER Ca²⁺ load and facilitate the Ca²⁺-induced-Ca²⁺ release (CICR) component of the [Ca²⁺]_c signal generated during K⁺ depolarisation. This could explain the potentiating effects of ouabain on exocytosis.     

Folding of viral envelope glycoproteins in the endoplasmic reticulum

Viral glycoproteins fold and oligomerize in the endoplasmic reticulum of the host cell. They employ the cellular machinery and receive assistance from cellular folding factors. During the folding process, they are retained in the compartment and their structural quality is checked by the quality control system of the endoplasmic reticulum. A special characteristic that distinguishes viral fusion proteins from most cellular proteins is the extensive conformational change they undergo during fusion of the viral and cellular membrane. Many viral proteins fold in conjunction with and dependent on a viral partner protein, sometimes even synthesized from the same mRNA. Relevant for folding is that viral glycoproteins from the same or related virus families may consist of overlapping sets of domain modules. The consequences of these features for viral protein folding are at the heart of this review.     

Stimulation of interstitial cell growth after selective destruction of foetal Leydig cells in the testis of postnatal rats

Summary Five-day-old male rats received a single treatment of ethane dimethanesulphonate (EDS), and the response of the testis on days 6–10 and 21 was examined by light microscopy and morphometry, supplemented by measurement of peripheral testosterone levels. One day after treatment, foetal Leydig cells degenerated, showing fragmentation, condensation and nuclear pyknosis. Macrophages phagocytosed the foetal Leydig cells resulting in their disappearance by day 7. Destruction of foetal Leydig cells was followed by an arrest of testicular growth in comparison to testes of intact age-matched control rats. In testes of EDS-treated rats, gonocytes and spermatogonia also degenerated, forming pyknotic bodies within the seminiferous cords. In contrast, interstitial fibroblasts and mesenchymal cells showed proliferative activity, which on days 4 and 5 after treatment resulted in peritubular hyperplasia surrounding each seminiferous cord. Thereafter, on day 21 after EDS administration, the previously depressed serum testosterone levels became markedly elevated coincident with the development of many immature-type Leydig cells, of which the total volume per testis was similar to that of Leydig cells in control testes, despite a four- to five-fold difference in testicular volumes. The results indicate that, although EDS destroys the foetal Leydig cells and impairs spermatogenesis, the interstitial tissue exhibits increased cell growth. The latter probably occurs in response to altered gonadotrophic stimulation and/or disturbances in the interaction between the seminiferous cords and the interstitial tissue.     

Origin of regenerating Leydig cells in the testis of the adult rat

Summary Ethane dimethanesulphonate (EDS) was used as a specific cytotoxin to eliminate the Leydig cell population of the adult rat testis. Ultrastructural, morphometric and serum gonadotrophin and testosterone analysis was used to study the response of the intertubular tissue of the testis from 1 day to 10 weeks after EDS treatment. In control animals, the testis contained approximately 28 million Leydig cells and 8 million macrophages. Three to seven days after EDS treatment, Leydig cells were absent and serum testosterone was undetectable. Macrophage numbers increased three-fold by 3 days and returned to pretreatment values thereafter. At 2 and 3 weeks post-EDS, foetal-type Leydig cells (1–2 million per testis) appeared in proximity to perivascular and peritubular tissues, a feature also observed at 4 weeks when numerous such cells (15 million per testis) formed prominent clusters in perivascular and peritubular locations. Between 6 and 10 weeks after EDS treatment, the foetal-type Leydig cells were transformed morphologically into adult-type Leydig cells, they occupied central intertubular positions and their numbers were restored to pretreatment values. Regeneration of Leydig cells was reflected by elevated serum testosterone levels which returned towards the normal range. The results demonstrate the regenerative capacity of the testicular intertubular tissue and indicate a dual site of origin of Leydig cells which initially resemble foetal-type Leydig cells prior to establishing the adult-type Leydig cell population. The morphological pattern of Leydig cell regeneration suggests that in addition to gonadotrophic stimulation, local testicular factors from the seminiferous tubules may stimulate Leydig cell growth.     

Macrophages in the interstitial tissue of the rat testis

Summary Macrophages were identified in the intertubular tissue of the rat testis by loading animals with a particulate vital dye (trypan blue or India ink) and by localizing immunocytochemically a macrophage membrane antigen (MRC W3/25). Leydig cells were identified by the histochemical staining reaction for 3-hydroxysteroid dehydrogenase activity and by a monoclonal antibody. Macrophages were scattered in the interstitial tissue closely attached to and mixed with the Leydig cells. They were never found in the seminiferous tubules. The macrophages comprised about 25% of all the cells in the interstitium. Double staining with a vital dye and a marker antibody showed that all the phagocytosing cells were macrophages and that the Leydig cells did not take up vital dyes. Double staining for the demonstration of the 3-hydroxysteroid dehydrogenase activity and the macrophage antigen likewise revealed two distinctly different cell populations. Crude Leydig cell preparations obtained by collagenase treatment of the testis contained macrophages (12–14%). Macrophages were present throughout the postnatal prepuberal development of the testis. Their density was increased in the cryptorchid and irradiated testis.     

Microtubule-associated smooth endoplasmic reticulum in the frog's brain

Summary Electron microscopic studies of neural processes in the cerebellum, optic tectum, and cerebral hemisphere of the frog reveal a distinctive system of SER cisternae lying at intervals (commonly 1–2 m apart) perpendicular to the long axis of axons and dendrites, interconnected by tubular, longitudinally orientated SER elements, and in direct continuity with the outer membrane of mitochondria. The transverse cisternae are fenestrated, with a single mierotubule (or rarely, two) passing through the centre of each 50–75 nm fenestration. Extensions of the SER-microtubule complex may be located parasynaptically in axon terminals and dendrites. The SER of dendritic spines also appears to be continuous with the fenestrated cisternae.Possible roles for the specialized SER (particularly of the parasynaptic extensions), such as calcium ion sequestration and ATP or monoamine oxidase transport, are discussed.Thanks are due to Profs. E. G. Gray and J. Z. Young for helpful discussion and to Mrs. N. Morgan and Mr. R. Boddy for technical assistance.     

Indirubin derivatives protect against endoplasmic reticulum stress-induced cytotoxicity and down-regulate CHOP levels in HT22 cells

Indirubin and its derivatives have been reported to exhibit anti-cancer and anti-inflammatory activities. Recently, some of its derived analogs have been shown to have neuroprotective potential. Endoplasmic reticulum (ER) stress has been demonstrated to contribute to the pathogenesis of various neurodegenerative diseases, whereas the effects of indirubin derivatives on ER stress-induced cell death have not been addressed. In the present study, a series of 44 derivatives of indirubin was prepared to search for a novel class of neuroprotective agents against ER stress-induced neuronal death. The MTT reduction assay indicated that tunicamycin (TM), an inducer of ER stress, significantly decreased the viability of hippocampal neuronal HT22 cells. Among the compounds tested, eight showed significant inhibitory activity against TM-induced cell death. Western blot analysis showed that application of these analogs to the cells simultaneously with TM reduced the TM-induced expression of CHOP, an established mediator of ER stress. Our results suggest that the preventive effect of these indirubin derivatives against ER stress-induced neuronal death may be due, at least in part, to attenuation of the CHOP-dependent signaling system.     

Topography of Ca2+-sequestering endoplasmic reticulum in photoreceptors and pigmented glial cells in the compound eye of the honeybee drone

Summary The endoplasmic reticulum (ER) in the photoreceptors of the honeybee drone, Apis mellifera, is highly differentiated and morphologically more complex than suggested by previous studies. In addition to the prominent voluminous submicrovillar cisternae we describe a submitochondrial ER. It separates the mitochondria-containing periphery from the core of the cell. The cell core contains many fenestrated ER cisternae that are horizontally and periodically arranged. We show that all parts of the ER, except for a tubulovesicular portion but including the nuclear envelope, are continuous; all parts appear to accumulate Ca²⁺ actively and with high affinity. Our results confirm previous suggestions that the submicrovillar ER is the major intracellular Ca²⁺ -store in the photoreceptors. The submitochondrial ER is thought to participate in Ca²⁺-regulation in the mitochondrial microenvironment. Moreover, we describe for the first time an extensive, morphologically complex Ca²⁺-sequestering ER in the pigmented glial cells; it might participate in the regulation of the glycogen metabolism.     

Association of gap junctions with endoplasmic reticulum in rat parotid glands

Summary Examination of the parotid gland of the rat has shown specific associations of cisterns of the endoplasmic reticulum with gap junctions. About 20% of the junctions are so intimately associated with cisterns of the endoplasmic reticulum that in freeze fractured material the cisternal membranes remain attached to the junctional membrane faces, obscuring most of the junctional array except for a thin ring of telltale particles. This association was seen only in the parotid gland of the rat, but not that of the other species examined.     

Ultrastructural demonstration of hormone-induced movement of carotenoid droplets and endoplasmic reticulum in xanthophores of the goldfish,Carassius auratus L.

Summary The hormone-induced pigment dispersion in primary cultures of xanthophores of goldfish (Carassius auratus L.) has been shown to involve the dispersion of not only carotenoid droplets but also of smooth endoplasmic reticulum. The dispersion of these organelles is inhibited by cytochalasin B and is accompanied by thinning of the cell body, thickening of the processes, and also overall changes in cellular morphology (process extension) under certain conditions. Electron microscopic examination of heavy meromyosin treated glycerinated xanthophores in scales revealed the presence of actin filaments in these cells.This work was supported, in part, by grants AM-5384 and AM-13724 from U.S.P.H.S.     

Postnatal differentiation of Leydig cells in the African green monkey

At two years of age the interstitial tissue of Cercopithecus aethiops is composed principally of undifferentiated, fibroblast-like cells. Also present during this time are scattered differentiating Leydig cells, which are characterized by a large nucleus, numerous mitochondria, elements of smooth reticulum, and small cisternae of rough reticulum. A mean level of 1.69 ± 0.66 ng/ml of testosterone was found. At three years Leydig cells are much more numerous and developed; since all the elements of steroid secreting cells are present, even their morphology differs from that observed in mature cells. Lipid accumulation is characteristic during this period. A mean testosterone level of 2.28 ± 0.47 ng/ml was found. Mature Leydig cells are basically similar to that of other mammals, while they differ significantly from that of human Leydig cells.     

Specialized formations of endoplasmic reticulum in parenchyma cells ofMimosa pudica L.

Summary Parenchyma cells ofMimosa pudica display close associations between two or more cisternae of the endoplasmic reticulum. These associations form simplified types of lamellar bodies in which inner paired lamellae have lost their ribonucleoprotein granules and are separated by a dense layer.