The pineal gland of the gerbil,Meriones unguiculatus

Summary Electron microscopy was employed in a study of the pineal gland of the Mongolian gerbil (Meriones unguiculatus). It was determined that the gerbil pineal gland contains pinealocytes and glial cells with the pinealocytes being the predominant cell type. The pinealocytes contain numerous organelles traditionally considered as being either synthetic or secretory in function such as an extensive Golgi region, smooth (SER) and rough (RER) endoplasmic reticulum, secretory vesicles and microtubules. Other cytoplasmic components are also present in the pinealocytes (synaptic ribbons, subsurface cisternae) for which no function has been assigned. Dense-cored vesicles are rare. Vacuolated pinealocytes are present and appear to be intimately associated with the formation of the pineal concertions. Evidence presented supports the proposal that the concretions form within the vacuoles. Once the concretions reach an enlarged state, the vacuolated pinealocytes break down and the concretions are thus extruded into the extracellular space where they apparently continue to increase in size. The morphology of the glial cells was interpreted as indicative of a high synthetic activity. The glial cells contain predominantly the rough variety of endoplasmic reticulum and form an expansion around the wide perivascular area.Supported by NSF grant PCM 77-05734     

Growth related changes in the content of heparin and 5-hydroxytryptamine of mast cells

Summary Fine structural changes of testicular interstitial cells of Leydig and secretory cells of seminal vesicles were studied in golden hamsters under different functional states of the pineal gland. Experiments were performed in the reproductive season (summer months). In the hamsters blinded for 8 weeks the testes and the seminal vesicles were markedly atrophic, and the Leydig cells and the secretory cells of seminal vesicles were extremely involuted. By contrast, both types of cells in the pinealectomized or superior cervical ganglionectomized hamsters exhibited cytological features suggestive of an enhanced secretory activity. This study shows that functional activity of Leydig cells as well as secretory cells of seminal vesicles in the hamster may be depressed or augmented by stimulating or inhibiting the pineal antigonadal function, respectively, without performing hypophysectomy or hormonal administration.Dedicated to Professor Shu Yeh on the occasion of his 70th birthday. This study was supported in part by a grant from the National Science Council, the Republic of China     

Effect of gossypol on pituitary reproductive axis: ultrastructural and biochemical studies

Oral administration of gossypol induced sterility in male rats by 10 weeks, at a dose of 15 mg/kg body weight/day. The pituitary FSH gonadotroph cells showed dilated endoplasmic reticulum and accumulation of secretory granules in the cytoplasm. LH cells were degranulated. The Leydig cells showed enhanced synthetic activity. There was no change in testis weight and testicular RNA, lipids and cholesterol in the treated group while significant increase was observed in DNA content. Testicular sialic acid content decreased significantly over controls. The Sertoli cells, spermatogonia, spermatocytes and early spermatids were not affected after the treatment. The weights of prostate, seminal vesicle were recorded normal and there were no ultrastructural variations. The levels of acid and alkaline phosphatase and RNA in prostatic tissue were insignificant as compared with controls. However, DNA content of prostate gland showed a significant increase. Sialic acid of seminal vesicle + coagulating gland were within the control range. A marked reduction in fructose values from the same organ was noted.     

Morphometric analysis of the pineal complex of the golden hamster over a 24-hour light:dark cycle: II. The deep pineal in untreated and optically enucleated animals

The deep pineal gland of golden hamsters was morphometrically analyzed and quantitatively compared with the superficial pineal under a 14:10 lighting regime and following blinding. The deep pineal comprised 6-10% of the total pineal parenchymal tissue. Pinealocytes of the deep gland were smaller than the cells of the superficial pineal and showed a greater percent volume of Golgi bodies, rough endoplasmic reticulum, and dense-cored vesicles. Twenty-four-hour rhythms in nucleoli and Golgi bodies were found in deep pinealocytes. These rhythms were out of phase with comparable rhythms in the superficial pineal gland, suggesting that distinct subpopulations of pinealocytes are present within the respective parts. Blinding resulted in decreased nuclear and nucleolar volume, while the amount of smooth endoplasmic reticulum, Golgi bodies, dense bodies, and dense-cored vesicles increased significantly. Marginal increases were seen in mitochondria and lipid droplets. The greater abundance of those organelles involved in synthesis and secretion suggests enhanced cellular activity after blinding. Many of the morphological responses are similar to alterations in the pinealocytes of the superficial pineal following optic enucleation.     

Genesis of unusual vesicles in rat periportal hepatocytes after administration of N-hydroxy-2-acetylaminofluorene

Administration of N-hydroxy-2-acetylaminofluorene (90 mumol/kg, iv) to rats results in damage to periportal hepatocytes. The most prominent ultrastructural changes are the appearance of numerous unusual vesicles of about 200 nm diameter and the proliferation of the endoplasmic reticulum to form fingerprint-like structures. In order to characterize these vesicles and to investigate their possible origin, their enzymatic activity was studied by ultrastructural enzyme histochemistry. The vesicles as well as the fingerprint-like structures exhibited glucose-6-phosphatase activity. Continuities between the vesicles and the membranes of both the RER and the SER were frequently seen. The vesicles lacked ATPase, 5'nucleotidase and catalase activity. From these results we conclude that the vesicles may be an unusual proliferation of the endoplasmic reticulum.     

Development of secretory activity in the seminal vesicle of the male migratory grasshopper,Melanoplus sanguinipes (fabr.) (Orthoptera : Acrididae)

This paper describes the ultrastructure of the seminal vesicle and the isoelectric focusing patterns of its secretion during sexual maturation and after allatectomy in Melanoplus sanguinipes (Fabr.) (Orthoptera : Acrididae). In epithelia from seminal vesicles of newly fledged males, the rough endoplasmic reticulum is well developed, and Golgi complexes are elaborate, which indicates the gland is metabolically active. The cells also contain large glycogen deposits and the lumen microvilli are well differentiated. These ultrastructural features are more dominant in 24-hr-old adults where the cytoplasm is clearly differentiated into basal and apical regions. Basally, the cytoplasm is dominated by rough endoplasmic reticulum, large Golgi complexes, glycogen deposits and numerous mitochondria, while the apical cytoplasm is filled with large secretory and/or lysosomal vesicles. Between days 3 and 7, the ultrastructural features change little other than the rough endoplasmic reticulum cisternae, which become vesicular. Analysis by isoelectric focusing shows that the amount of secretory protein increases with age until day 3, at which time the gland contains its full complement of secretion. In seminal vesicles from allatectomized insects, ultrastructural features of cells and isoelectric focusing patterns of the secretion arc identical to those from normal males.     

Comparison of components of the testis interstitium with testosterone secretion in hamster, rat, and guinea pig testes perfused in vitro

Components of the testis and cytoplasmic organelles in Leydig cells were quantified with morphometric techniques in hamster, rat, and guinea pig. Testosterone secretory capacity per gram of testis and per Leydig cell in response to luteinizing hormone (LH) (100 ng/ml) stimulation was determined in these three species from testes perfused in vitro. Numerous correlations were measured among structures, and between structures and testosterone secretion, to provide structural evidence of intratesticular control of Leydig cell function. Testosterone secretion per gm testis and per Leydig cell was significantly different in the three species: highest in the guinea pig, intermediate in the rat, and lowest in the hamster. The volume of seminiferous tubules per gm testis was negatively correlated, and the volumes of interstitium, Leydig cells, and lymphatic space per gm testis were positively correlated with testosterone secretion. No correlations were observed between volumes of blood vessels, elongated spindleshaped cells, or macrophages per gm testes and testosterone secretion. The average volume of a Leydig cell and the volume and surface area of smooth endoplasmic reticulum (SER) and peroxisomes per Leydig cell were positively correlated, and the volume of lysosomes and surface area of inner mitochondrial membrane per Leydig cell were negatively correlated with testosterone secretion. No correlations were observed between volume and surface area of rough endoplasmic reticulum (RER), Golgi apparatus, and lipid, and volume of ribosomes, cytoplasmic matrix, and the nucleus with testosterone secretion per Leydig cell. These results suggest that Leydig cell size is more important than number of Leydig cells in explaining the difference in testosterone-secreting capacity among the three species, and that this increase in average volume of a Leydig cell is associated specifically with increased volume and surface area of SER and peroxisomes. An important unresolved question is what is the role of peroxisomes in Leydig cell steroidogenesis.     

The epiphysis (pineal body) and the APUD system

Light and electron microscopic studies were conducted on 10 humans who died of the different cardiac diseases; and 20 guinea pigs pineal glands. Pinealocytes or secretory cells of the pineal gland have morphological likeness with the APUD system cells. They have a well-developed endoplasmic reticulum, Golgi complex, mitochondrial component and in cytoplasm dense-core vesicles are discovered. However the pinealocytes have a neuron-like structure and they are not separate cells as apudocytes, but they are a principal component of the pineal parenchyma in which pinealocytes are in tight interactions with glia, blood vessels and nerve terminations. Analysis of morphological and functional similarity and difference between pinealocytes and apudocytes allows to consider pineal gland as an APUD organ. A circadian rhythmicity of some secretory vesicles in pinealocytes of the guinea pig has been established.     

Testicular function and Leydig cell ultrastructure in long-term bilaterally cryptorchid rams

This study was conducted to investigate the effects of bilateral cryptorchidism induced in adult rams on testicular function and Leydig cell ultrastructure. The results indicated that long-term bilateral cryptorchidism resulted in decreased testicular size, degeneration of seminiferous tubules, elevated serum LH levels, maintenance of normal testosterone concentrations in peripheral and spermatic vein serum, impairment of the magnitude and duration of androgen response to exogenous luteinizing hormone (LH), a 13-fold reduction in total number of Leydig cells/paired testes, and a 3-fold hypertrophy in the average size of remaining Leydig cells. Based on quantitative morphometry, the hypertrophied Leydig cells exhibited significant increases in the volume of intracellular organelles, including the cell nucleus, mitochondria, smooth and rough endoplasmic reticulum, lysosome-like bodies and lipid vesicles. Quantitatively, the hypertrophy alone was not enough to offset the loss in number of Leydig cells and was insufficient to explain the maintenance of normal levels of testosterone in jugular and spermatic venous blood. The additional mechanisms responsible for production of normal serum testosterone levels in the cryptorchid ram remain to be elucidated.     

Ultrastructure of the pineal organ of the killifish,Fundulus heteroclitus,with special reference to the secretory function

Summary The pineal organ of the killifish, Fundulus heteroclitus, was investigated by electron microscopy under experimental conditions; its general and characteristic features are discussed with respect to the photosensory and secretory function. The strongly convoluted pineal epithelium is usually composed of photoreceptor, ganglion and supporting cells. In addition to the well-differentiated photosensory apparatus, the photoreceptor cell contains presumably immature dense-cored vesicles (140–220 nm in diameter) associated with a well-developed granular endoplasmic reticulum in the perinuclear region and the basal process. These dense-cored vesicles appear rather prominent in fish subjected to darkness. The ganglion cell shows the typical features of a nerve cell; granular endoplasmic reticulum, polysomes, mitochondria and Golgi apparatus are scattered in the electron-lucent cytoplasm around the spherical or oval nucleus. The dendrites of these cells divide into smaller branches and form many sensory synapses with the photoreceptor basal processes. Lipid droplets appear exclusively in the supporting cell, which also contains well-developed granular endoplasmic reticulum and Golgi apparatus. Cytoplasmic protrusions filled with compact dense-cored vesicles (90–220 nm in diameter) are found in dark-adapted fish. The origin of these cytoplasmic protrusions, however, remains unresolved. Thus, the pineal organ of the killifish contains two types of dense-cored vesicles which appear predominantly in darkness. The ultrastructural results suggest that the pineal organ of fish functions not only as a photoreceptor but also as a secretory organ.We thank Dr. Grace Pickford for the fishes.     

Histogenesis of human extraparenchymal Leydig cells

From 64 consecutive autopsies of patients with neither testicular nor hormonal pathology, 26 showed extraparenchymal Leydig cells, located mainly in the epididymis and in the spermatic cord. The ultrastructural study of these specimens plus those obtained from 2 patients affected with functional testicular tumors leads to the following conclusions: (1) The origin of ectopic Leydig cells is not interstitial Leydig cells having infiltrated the testicular nerves and migrated along them towards ectopic locations. (2) The ectopic Leydig cells are considered to develop from undifferentiated precursor cells, located extraparenchymally, mainly inside and beside the testicular nerves. These precursor cells are similar to those observed in the testicular interstitium and have an ovoid shape and some cytoplasmic projections. The cytoplasm contains vesicles of smooth endoplasmic reticulum, lysosomes, lipid droplets and abundant microfilament bundles. The transformation from these cells into mature Leydig cells implies a progressive differentiation of the cytoplasmic components involved in steroid biosynthesis.     

Ultrastructure of the pineal gland of the eastern chipmunk (Tamias striatus)

The ultrastructure of the pineal gland of the wild-captured eastern chipmunk (Tamias striatus) was examined. A homogenous population of pinealocytes was the characteristic cellular element of the chipmunk pineal gland. Often, pinealocytes showed a folliclelike arrangement. Mitochondria, Golgi apparatus, granular endoplasmic reticulum, lysosomes, centrioles, dense-core vesicles, clear vesicles, glycogen particles, and microtubules were consistent components of the pinealocyte cytoplasm. The extraordinary ultrastructural feature of the chipmunk pinealocyte was the presence of extremely large numbers of “synaptic” ribbons. The number of “synaptic” ribbons in this species exceeded by a factor of five to 30 times that found in any species previously reported. In addition to pinealocytes, the pineal parenchyma contained glial cells (oligodendrocytes and fibrous astrocytes). Capillaries of the pineal gland of the chipmunk consisted of a fenestrated endothelium. Adrenergic nerve terminals were relatively sparse.     

The pineal gland of the gerbil,Meriones unguiculatus

Summary By means of morphometric analytical procedures, a diurnal rhythm in the cellular volume of gerbil pinealocytes was determined. This rhythm has been attributed primarily to a change in the cytoplasmic volume of the pinealocytes which is low during the daylight hours and increases to reach a peak during the middle of the dark period. At the ultrastructural level, six cytoplasmic components of the pinealocytes were found to exhibit a rhythm: free cytoplasm, smooth endoplasmic reticulum (SER), rough endoplasmic reticulum (RER) and ribosomes, secretory vesicles, microtubules, and mitochondria. The presumptive secretory vesicles and the microtubules reached a peak in volume one hour before lights-off. It is suggested that lights-on and lights-off both signal a decrease in size and/or number of the secretory vesicles. The SER and RER/ribosomes reached their peak volume one hour after lights-off which is interpreted as indicating a peak in indoleamine synthesis and protein synthesis, respectively. The volume of free cytoplasm exhibits two peaks; one occurs one hour before lights-off while the second peak occurs in the middle of the dark phase. It is suggested that, although part of the secretory product of the pinealocyte may be present in dense-cored vesicles, other locations could include the free cytoplasm and clear secretory vesicles.Supported by NSF grant #PCM 77-05734     

Arecoline induces TNF-alpha production and Zonula Occludens-1 redistribution in mouse Sertoli TM4 cells

Background

Arecoline, a major alkaloid in Areca nut has the ability to induce oxidative stress. The effect of Areca nut, arecoline on reducing sperm quality and quantity were documented previously using several animal models. Junction disruption by down-regulation of the junction-adhesive protein via oxidative stress is an important route mediating abnormal spermatogenesis. Therefore, in this present study, we investigated the functional role of arecoline on junctional proteins.

Results

To analyze direct effects of arecoline on testis cells, confluent mouse testicular Sertoli cell line TM4 was exposed to arecoline. Arecoline decreased insoluble zonula occludens-1 (ZO-1) protein expression in TM4 cells, however, arecoline treatment increased TNF-alpha production in both TM4 and monocytic THP1 cells. In addition, ERK1/2 inhibitor PD98059 reversed arecoline effects on TNF-alpha and ZO-1.

Conclusions

Arecoline increases the production of TNF-alpha and induces protein redistribution of ZO-1. All these results explain the role of arecoline in male reproductive dysfunction, besides its cytotoxic induction.

Electronic supplementary material

The online version of this article (doi:10.1186/s12929-014-0093-z) contains supplementary material, which is available to authorized users.     

The effects of testosterone on the mice pinealocytes: a quantitative study.

In order to determine the effects of testosterone upon the function of the pineal gland we quantitated, under the electron microscope, the number of dense-core vesicles, lysosomes, lipid droplets and the fractional volume of the pineal gland components in adult male mice. We studied six groups of mice submitted to different treatments as follows: treated with testosterone propionate (0.1 mg/10 g body weight for 15 consecutive days), castrated and the respective control mice. In mice treated with testosterone we observed a decrease in the number and fractional volume of dense-core vesicles and an increase in the fractional volume of lysosomes and lipid droplets. In castrated mice, without treatment, we observed a decrease in the number of lysosomes and an increase in the number and fractional volume of dense-core vesicles. These results show that testosterone is closely related to the secretory process of mice pinealocytes, playing an inhibitory role upon the functional activity of the pineal gland.     

Effects of arecoline on testosterone release in rats

Arecoline is one of the major components of betel nuts, which have been consumed as chewing gum in Southeast Asia. In this study, the effects of arecoline on testosterone (T) secretion were explored. Male rats were injected with human chorionic gonadotropin (hCG, 5 IU/kg) or arecoline (1 microg/kg) plus hCG via a jugular catheter. Blood samples were collected at several time intervals subsequent to the challenge. Rat anterior pituitary was treated with gonadotropin-releasing hormone in vitro with or without arecoline, and then the concentrations of luteinizing hormone (LH) in the medium were measured. Rat Leydig cells were purified by Percoll density gradient centrifugation and incubated with arecoline, hCG, forskolin, 8-bromo-cAMP (8-Br-cAMP), nifedipine, nimodipine, or tetrandrine at 34 degrees C for 1 h. A single intravenous injection of arecoline resulted in an increase of the hCG-induced level of plasma T. Administration of arecoline (10(-8) to 10(-6) M) in vitro increased T production in Leydig cells. The stimulatory effect of arecoline on T release in vitro was enhanced by hCG (0.001 IU/ml), forskolin (10(-6) M), or 8-Br-cAMP (10(-5) M). By contrast, nifedipine, nimodipine, or tetrandrine inhibited the increased T concentrations induced by arecoline. Western blot showed that arecoline increases steroidogenic acute regulatory (StAR) protein expression compared with vehicle. These results suggested that arecoline stimulates testosterone production by acting directly on Leydig cells via mechanisms involving an activation of L-type calcium channels, increasing the activity of 17beta-hydroxysteroid dehydrogenase and enhancing the expression of StAR.     

Morphometric analysis of the pineal complex of the golden hamster over a 24-hour light:dark cycle: I. The superficial pineal in untreated and optically enucleated animals

Morphometric analysis of the superficial pineal gland of intact and blinded golden hamsters was conducted at both the light and electron microscopic level. The volume of the superficial gland was estimated to be 151 X 10(6) micron 3, comprising 90-94% of the total pineal parenchymal tissue. Analysis of structural rhythms in animals maintained under a 14:10 L:D cycle showed significant 24-hr variations in values for pinealocyte nuclei, nucleoli, rough and smooth endoplasmic reticulum, Golgi bodies, dense bodies, and dense-cored vesicles. Peak values for these structures generally occurred at the light:dark interface. These results provide morphological correlates for known rhythmic variations in the synthesis of pineal-gland products. Superficial pineals examined 8 weeks following optic enucleation exhibited a decrease in the volume of pinealocyte nuclei and cytoplasm, while nucleolar size and the amounts of smooth and rough endoplasmic reticulum, Golgi bodies, dense bodies and dense-cored vesicles were enhanced. The latter changes are interpreted as indications of increased synthetic activity by the superficial pineal gland in response to light deprivation.     

Localization of calcium in the secretory cells of the mammary glands in response to oxytocin

Distribution of Ca2+ ions, precipitated by means of pyroantimonate potassium, has been investigated electron microscopically in secretory cells of the mammary gland of lactating white mice. In the glandular cells, that are at the state of inhibition of secretory activity, the cytochemical reaction product is localized on the internal side of the basal, lateral and apical parts of the plasmolemma, in mitochondrial matrix, in cisterns and in the Golgi complex vesicles, in the nuclear areas, occupied by euchromatin. Oxytocin effect produces a certain complex of ultrastructural changes in the cell accompanied by redistribution of Ca2+ ions. Amount of precipitate in mitochondria decreases. It is revealed in the lumen of dilated canals of the granular endoplasmic reticulum, in the zone of decondensated nuclear chromatin, in the Golgi complex vesicles. The vesicles become larger and fuse with each other. The changes mentioned demonstrate increased synthetic and transport processes, occurring in the glandular epithelium of the mammary gland after oxytocin effect.     

Pineal sensitivity to nighttime swimming stress changes during the active season in Richardson's ground squirrels (Spermophilus richardsonii)

Melatonin synthesis in the pineal gland, which is primarily regulated by the environmental lighting regime, can also be influenced by other factors that elicit modifications in sympathetic tone. The objectives of this study were to determine if forced swimming alters the normal pattern of melatonin production in the pineal gland of the Richardson's ground squirrel (Spermophilus richardsonii). In early June, the squirrels were forced to swim for 10 min during the photophase or during the scotophase. In mid-July squirrels swam only during the scotophase. Animals were sacrificed 15, 30, or 60 min after the onset of swimming. Activities of pineal N-acetyltransferase (NAT) and hydroxyindole-O-methyltransferase (HIOMT) were assessed by radioenzyme assay, and pineal melatonin content was measured by radioimmunoassay. Daytime swimming elicited no major changes in enzyme activity or pineal melatonin. In June, swimming at night prevented the normal rises in NAT activity and pineal melatonin seen in nonswimming controls. In contrast, the pineals of squirrels that were tested 6 weeks later in mid-July did not appear to be as sensitive to nighttime swimming, as there were only minor differences in both NAT activity and melatonin content compared to controls. These results demonstrate that forced nighttime swimming, unlike several other aversive stimuli, can evoke changes in the normal pattern of pineal melatonin production in this species. Furthermore, the pineal's response to such stimuli may not be stable over the course of the active season.     

Ultrastructure of testicular biopsy from an XX male

Ultrastructural study of testicular biopsy specimens from an XX male showed hyalinized seminiferous tubules and tubules containing only mature Sertoli cells. These cells possessed large lipid inclusions as well as microfilament bundles which were perpendicular to the basement membrane and parallel to one another. The basal lamina was thickened and composed of several parallel layers with myofibroblast layers between them. The interstitium showed nodular to diffuse Leydig cell hyperplasia. Four types of Leydig cells were found: 1) normal Leydig cells with crystals of Reinke; 2) cells with abundant microcrystalline inclusions as well as microfilaments and concentric cisternae of smooth endoplasmic reticulum; 3) vacuolated cells containing numerous large lipid droplets; 4) immature Leydig cells. The different ultrastructural abnormalities found in the Sertoli and Leydig cells might be considered as the histological expression of a tubular-interstitial dysgenesis which is reflected in the high levels of gonadotropins and low levels of testosterone.