Silver staining in pinealocyte nuclei: Observations and quantitative analysis during the rat oestrous cycle

Summary Morphology, distribution and number of different argyrophilic aggregates appearing in pinealocyte nuclei have been studied during the rat oestrous cycle. Using the Ag-NOR reaction we have found two types of argyrophilic aggregates in pinealocyte nuclei. One of them, corresponding to the fibrillar centres and the surrounding fibrillar component, appears in the nucleoli. The silver grains are more loosely packed in the dense fibrillar component than in the fibrillar centres. A decrease in the number of these nucleolar argyrophilic aggregates was obtained at oestrous. A second type of silver grain aggregate was observed in the pinealocyte nucleoplasm. We call them Ag-granule clusters because they are similar to the interchromatin granule clusters and constitute the only silver deposit forming aggregates apart from the nucleolus. The number of Ag-granule clusters is significantly smaller at oestrous and meta-oestrous than at dioestrous and pro-oestrous.     

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.     

Proliferative changes in the genital tissue of female mice during the oestrous cycle

Changes in proliferation and number of epithelial cells of the murine genital tract, during the oestrous cycle, have been studied. A total of 47 animals in the prooestrous, metoestrous and dioestrous phases of the cycle were staged retrospectively on the basis of the genital tract histology. The average duration of the oestrous cycle in these animals was 4 days, and half of this period was occupied by the prooestrous/oestrous phases. Significant cycles of growth were observed in the luminal uterine epithelium and in the basal epithelium of the cervix-vagina. Most of this growth occurred during the pro-oestrous phase, which lasted approximately 1 day. During this time the numbers of luminal epithelial cells in the uterus and suprabasal cells in the cervix-vagina increased 2-3 fold. This pattern of growth appeared partly synchronous and corresponded to the period when serum oestrogen levels are at their highest. A corresponding and rapid reduction in the numbers of uterine luminal epithelial cells and suprabasal cells in the cervix and vagina was noted during the early metoestrous phase; and this occured during the period when serum oestrogens are at their lowest levels. No significant periodicity in the proliferation and numbers of the uterine gland epithelial cells was noted during the cycle. The kinetic role and function of the gland cells is discussed in relation to these data.     

Proliferative changes in the genital tissue of female mice during the oestrous cycle

Abstract. Changes in proliferation and number of epithelial cells of the murine genital tract, during the oestrous cycle, have been studied. A total of 47 animals in the pro-oestrous, metoestrous and dioestrous phases of the cycle were staged retrospectively on the basis of the genital tract histology. The average duration of the oestrous cycle in these aninals was 4 days, and half of this period was occupied by the pro-oestrous/oestrous phases. Significant cycles of growth were observed in the luminal uterine epithelium and in the basal epithelium of the cervix-vagina. Most of this growth occurred during the pro-oestrous phase, which lasted approximately 1 day. During this time the numbers of luminal epithelial cells in the uterus and suprabasal cells in the cervix-vagina increased 2–3 fold. This pattern of growth appeared partly synchronous and corresponded to the period when serum oestrogen levels are at their highest.
A corresponding and rapid reduction in the numbers of uterine luminal epithelial cells and suprabasal cells in the cervix and vagina was noted during the early metoestrous phase; and this occured during the period when serum oestrogens are at their lowest levels. No significant periodicity in the proliferation and numbers of the uterine gland epithelial cells was noted during the cycle. The kinetic role and function of the gland cells is discussed in relation to these data.     

Influence of the oestrous cycle on L-glutamate and L-aspartate transport in rat brain synaptosomes.

Oestrous cycle and sex differences in sodium-dependent transport of L-[3H]glutamate and L-[3H]aspartate were investigated employing well washed synaptosomes prepared from rat brain cortex. Transport was best analysed on the basis of two components, a high and low affinity transport site. Oestrous cycle and sex differences were observed for both substrates. The high affinity transporter displayed highest affinity for glutamate transport in synaptosomes from female rats during proestrous and oestrous. This differed significantly from glutamate transport during dioestrous and in male rats. High affinity aspartate transport displayed highest affinity during oestrous and differed significantly from transport during dioestrous. Maximal velocity of high affinity glutamate transport was higher in synaptosomes from females during dioestrous compared with oestrous and lower in synaptosomes from male rats when compared with female rats in dioestrous and metoestrous. The low affinity sodium-dependent glutamate transporter displayed a 10-fold higher affinity for glutamate during proestrous than during the other three phases of oestrous and in male rats. Exogenously applied oestradiol and progesterone to synaptosomes from male rats showed no effect on glutamate or aspartate transport. No acute effect of oestradiol or progesterone on glutamate currents in oocytes expressing EAAT1 or EAAT2 subtype of glutamate transporter was observed. These results suggest hormonal regulation of high and low affinity sodium-dependent excitatory amino acid transporters over the four day oestrous cycle in synaptosomes from rat cortex. This regulation is unlikely to be due to a direct effect of oestradiol or progesterone on glutamate transporters.     

Characteristics of a prompt morphodynamic response of the pineal gland to an acute ACTH injection

The morphodynamic response of the pineal gland in the period 5-30 min after a single ACTH injection was studied. In 5 min a saline injection, functioning as a control, elicited a prompt release of the pineal active compounds advocated to be contained in clear vesicles and structures traditionally labeled lipid droplets. In the subsequent time period, 15 min, a rise in the relative volume of the granular endoplasmic reticulum (GER) and the Golgi apparatus, as well as prosecretory granules, was interpreted as a sign of an increased pinealocyte activity which leads to the restoration of the pineal gland endocrine potential. A decline in the increased mass of this membranous elaborative compartment in 30 min could be understood as an illustration of the stabilisation of the pinealocyte stress-evoked response. The ACTH injection did not apparently change the pattern of the morphodynamic pinealocyte response promoted by the saline injection in 5 min. However, starting from 15 min, a significant increase of the mass of GER and the Golgi apparatus was found in both pinealocyte populations. The augmented presence of prosecretory granules and lipid droplets, in comparison with the saline-injected animals in the respective time period, as well as the absence of a clearly observable evidence of the pinealocyte secretory activity pointed out that the ACTH stimulative action on the pinealocyte membranous elaborative compartment rather facilitated the pinealocyte endocrine recovery than supported their current secretory activity. From the functional viewpoint, the results obtained suggest that a stress-induced ACTH burst could modulate the post-secretory restitution of the endocrine potential of previously stress-activated pinealocytes.     

Ultrastructural changes in the rat pineal gland after sympathetic denervation. Quantitative study

Ultrastructural changes in the rat pineal gland were studied quantitatively 7 and 60 days after the sympathetic denervation by bilateral excission or decentralization of superior cervical ganglia. The surface occupied by pineal parenchymal cells decreased in rats of experimental groups with respect to the control group. Furthermore, profile areas of the cytoplasm, nucleus and nucleolus of the pinealocytes were also diminished. Cytoplasmic lipid droplets in the pinealocytes were markedly decreased in number and size in experimental rats. As demonstrated by the Kruskal-Wallis H test, statistically significant differences were found between rats of the control and operated groups. Rats treated by superior cervical ganglionectomy or decentralization showed morphological changes indicating a hypofunctional pineal gland, although differences were found between both groups.     

Immunocytochemical and circadian biochemical analysis of neuroactive amino acids in the pineal gland of the rat: effect of superior cervical ganglionectomy

Summary Semiquantitative immunocytochemistry by immuno-gold techniques revealed differences in the spatial distribution of glutamate, glutamine, and taurine within the pineal gland, with greatest labeling over pinealocytes, glia, and endothelia, respectively. At the subcellular level, glutamate labeling tended to be highest over pinealocyte synaptic ribbons and mitochondria, and lowest over lipid inclusions. Pineal levels of glutamate, glutamine and taurine, as measured by high performance liquid chromatography, did not vary over a light: dark cycle. Superior cervical sympathetic denervation, which abolishes pineal melatonin synthesis, resulted in a nearly 50% reduction in pineal glutamate levels, but had no effect on levels of glutamine and taurine. Other amino acids (alanine, arginine, aspartate, serine) were reduced by 23%–33% following sympathectomy. These data suggest an important role for glutamate in pinealocyte function(s) possibly related to the noradrenergic innervation of the gland.     

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.     

Quantitative variations in nucleolar components within pineal gland cells during the rat estrous cycle

Quantitative changes in the size of pinealocyte nucleoli have been reported in various studies on this cell type. However, the significance of quantitative changes in the nucleolar components is unknown. The present study is an attempt to analyze ultrastructural and morphometric modifications occurring in the pinealocyte nucleolar components during the estrous cycle in female rats. The fibrillar centers showed an increase during estrus consistent with a decrease in pinealocyte nucleolar activity and melatonin pineal levels. The fibrillar components and granular components tended to display a reciprocal relationship. An increase in the dense fibrillar component took place at metaestrus and diestrus when melatonin synthesis increased in pinealocytes. Maximum values of granular and interstitial components were found at the proestrus phase before the day of ovulation.     

Morphology and histochemistry of ovarian changes in the field rat, Millardia meltada

A study has been made of the morphological and histochemical changes of the ovary of the field rat, Millardia meltada during its oestrous cycle and pregnancy. The follicular growth and atresia, ovulation and formation of corpora lutea occur throughout the year except severe winter months (December and January). Fluctuations in the follicular development occur on different days of the oestrous cycle and pregnancy. The granulosa cells show a progressive increase in their size in successive stages of follicle growth. The granulosae of normal follicles show some sparsely scattered lipid bodies which consist of phospholipids. Theca interna cells during follicular growth develop diffuse lipoproteins and lipid droplets consisting of triglycerides, phospholipids and cholesterol and/or its esters. The luteal cells of corpora lutea are formed by the granulosa cells as the theca interna cells degenerate and disappear. The fibroblast-like cells of thecal origin, alongwith the blood vessels, invade the luteal cell mass. The luteal cells during metoestrus, dioestrus and first half of pregnancy show abundant diffuse lipoproteins and a few lipid droplets composed mainly of phospholipids and some triglycerides, which are indicative of active steroidogenesis. The details of degenerative histological and histochemical alterations of corpora lutea during oestrous cycle and pregnancy are also described and discussed. Morphological and histochemical changes of follicular atresia are described. The granulosa cells of atretic follicle degenerate and disappear leaving behind theca interna cells which form patches of interstitial gland cells during the reproductive activity of the present rat. Interstitial gland cells show diffusely distributed sudanophilic lipoproteins and lipid droplets consisting of triglycerides, cholesterol and/or its esters and some phospholipids, which are indicative of steroidogenesis. The functional significance of histological and histochemical changes, which occur in various components of the ovary during oestrous cycle and pregnancy, has been discussed.     

Expression of cellCAM-105 in the apical surface of rat uterine epithelium is controlled by ovarian steroid hormones

Affinity-purified antibodies to cellCAM-105, an adhesive cell surface glycoprotein, were used in immunohistochemical investigations of rat uteri at various functional stages: (i) the oestrous, pro-oestrous, metoestrous, and dioestrous stages of the oestrous cycle, (ii) Days 1-8 of normal pregnancy, (iii) delayed implantation, (iv) 18 h after oestrogen reactivation from delay of implantation, and (v) juvenile rats, and normal ovariectomized adults, respectively, before and after experimental injection of progesterone and/or oestrogen. CellCAM-105 was present in the apical zones of the luminal and glandular epithelium cells in a stage-specific and hormone-dependent manner. The results indicate that: (1) steroid hormones are essential for the expression of cellCAM-105 in the uterine epithelial cells; (2) progesterone induces cellCAM-105 expression in the glandular epithelium, and oestrogen induces cellCAM-105 expression in the luminal epithelium; (3) progesterone induces down-regulation of cellCAM-105 from the surface of the uterine luminal epithelium of juvenile rats; (4) cellCAM-105 is absent in the luminal epithelial cells but present in the glandular epithelial cells of the rat uterus at the time of blastocyst implantation.     

Morphology of the pig's pineal gland from 8th to 16th week of foetal life

Pineal glands of the pig from the 8th to the 16th week of foetal life were examined by means of both light and electron microscopy, and changes in the pineal structure were observed. The pinealocyte structure manifests the secretory activity of the pineal gland at the time of examination.     

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.     

Effect of photoperiod on pineal gland volume and pinealocyte size in the Chinese hamster, Cricetulus griseus

Male adult (200-day-old) Chinese hamsters (Cricetulus griseus) raised from weaning under either LD 16:8 or LD 8:16 were used. The pineal gland of the Chinese hamster consists of superficial (major) and deep (minor) components and a continuous, or interrupted, narrow parenchymal stalk interposed between them. The volume of the superficial pineal including the parenchymal stalk is greater under LD 16:8 than under LD 8:16. Under both photoperiods, pinealocytes in the superficial pineal have larger nuclei and more abundant cytoplasm than those in the deep pineal. Nuclei in the superficial pineal appear pale and usually have irregular profiles, whereas those in the deep pineal appear dark and have round profiles. In the superficial pineal, pinealocyte nuclei are larger, paler, and more irregular; and, in addition, nuclear density is lower under LD 16:8 than under LD 8:16. Similar, but less prominent, photoperiod-induced changes occur in the volume of the deep pineal, the size of pinealocytes, and pinealocyte nuclear morphology in the deep pineal. The results indicate that the development and differentiation of pinealocytes in both pineal portions may be advanced under long photoperiods and delayed under short photoperiods, although pinealocytes in the deep pineal may remain not fully differentiated even in adults. Since testicular weights and body weights are similar under both photoperiods, the photoperiod may exert marked influences on the development of the pineal gland without affecting reproductive activity and growth rates of animals.     

Effect of stress on the course of oestrous cycle and the release of luteinizing hormone; the role of endorphin in these processes

It has been postulated that stress induces discorrelations of the hypothalamo-pituitary and pituitary gonadal axis. In our experiments on the effect of stress on the reproductive physiology in rats and sheep we applied mild electrical footshocking of short or prolonged duration. Foot-shocking applied with some breaks during 9 h within one a day (15th day of the oestrous cycle) induced in ewes acceleration of the release of LH. Prolonged footshocking applied with some breaks during 3 days in cycling sheep caused disturbances in the circadian rhythm of the cortisol secretion, disturbances in the release of LH and led to the blockade of ovulation. Disturbances in the course of oestrous cycle occurred not only during the current cycle but also during two subsequent cycles. Rats exposed to relatively long-term stressful situation (24 h) during dioestrous displayed marked changes in the length of this phase in three subsequent post-stress oestrous cycles. To follow the neurohormonal background of the stress-induced disturbances in LH release and in the course of oestrous cycle in sheep the concentrations of beta-endorphin (beta-END) in the infundibular and paraventricular nuclei as well as in the pituitary gland under physiological and stress conditions were determined, while in rats the metabolism of brain serotonin was investigated. Footshocking in rats induced significant decrease in 5-HT concentrations in the fronto-parietal brain cortex, hippocampus, striatum, medial basal hypothalamus and the preoptic-anterior hypothalamic area. These results allow to suggest that the decline in brain 5-HT under stress conditions has some associations with the impairments in the course of oestrous cycle. Measurements of the beta-END in perfusates of medial basal hypothalamus (nucl. infundibularis) in sheep evidenced significant increase of this opioid under stress conditions and it was postulated that this increase might be the main cause of the stress-induced impairments in the course of oestrous cycle and inhibition of LH-release. In addition, it was found that beta-END suppressed the secretion of cortisol and attenuated some noxious consequences of general nature for organism.     

冬眠周期长短不同的蒙古黄鼠（Citellus dauricus）...

Adult Mongolian ground squirrels (Citellus dauricus) were kept at 5 degrees C in winter and divided into four experimental groups according to the bout length. The first group was not hibernating until decapitation. The bout length of the second group was between 4-10 days, the third group 11-17 days and the fourth group longer than 20 days. All pineals were sampled at the end of January. Morphometric analytical procedures were used to study the ultrastructure of the distal part of the pineal gland. The statistical results demonstrated that 1) the euthermic animals have larger cross areas of pinealocyte, longer and narrower Golgi apparatus and more number of saccules of each Golgi apparatus (P less than 0.01). But they also have smaller volume density of vaculoes, less lipid droplets and associated vesicles around Golgi apparatus (P less than 0.01). 2) the hibernating animals with variety of bout length had no significant differences in the number of mitochondria, lipid droplets, lysosomes, the size of Golgi apparatus and the cross areas of nucleus and cytoplasm (P greater than 0.05). However, the number and the cross areas of vacuoles were significantly increased with the bout length (P less than 0.01). This might suggest that the bout length was not related to the metabolic activity of pinealocytes in Citellus dauricus and vacuoles might play some important roles in maintenance of individual bout of hibernation in this species.     

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.     

Microglia play a role in mediating the effects of cytokines on the structure and function of the rat pineal gland

The role of the pineal gland in regulating immune function has been extensively investigated. However, there is little information about possible feedback mechanisms of immunological factors on pineal gland neuroendocrine functions. Therefore, experiments were designed to test the effects of cytokines (interferon-gamma, IFN-gamma, interleukin-1 beta, IL-1 beta; tumor necrosis factor-alpha, TNF-alpha; transforming growth factor-beta 1, TGF-beta 1) on pinealocytes and the role of pineal microglia in mediating these cytokine effects in the pineal gland of the rat. Our studies showed that IFN-gamma enhanced 5-hydroxytryptamine (5-HT) content (measured by high-performance liquid chromatography, HPLC) and increased pinealocyte process length in pineal cultures. IL-1 beta treatment decreased 5-HT content in both cell and organ culture, but exhibited no effect on pinealocyte process length. 5-HT content and process length were decreased by TNF-alpha treatment. IFN-gamma and IL-1 beta exhibited no significant effect in the absence of microglia in cell cultures. In contrast, TNF-alpha caused a further decline in 5-HT content even in the absence of microglia in the cultures. The effects of TNF-alpha were probably due to toxic effects, since an increased number of pyknotic nuclei were observed in treated cultured explants. TGF-beta 1 treatment caused aggregation of pinealocytes in cultures and suppressed process length and 5-HT content. In conclusion, cytokine effects on pinealocytes may be mediated by microglia (IFN-gamma and IL-1 beta) or act directly on pinealocytes (TNF-alpha). The presence of IL-1 beta and TGF-beta 1 protein in the pineal gland and the suppressive effect of TGF-beta 1 on pinealocytes in cultures further suggest that endogenous cytokines play regulatory roles in response to peripheral homeostatic changes.     

Changes in female attractivity and proceptivity throughout the oestrous cycle of the Syrian hamster (Mesocricetus auratus)

Interactions between male and female hamsters were recorded on each day of the oestrous cycle in terms of proximity (approaching, leaving and following by each member of the pair). Time spent sniffing the partner was also recorded. Measures were extracted which related to the females' stimulus value to the male (attractivity) and to the females' initiation and maintenance of proximity to the male (proceptivity). Both were found to change not only at oestrus but throughout the three dioestrous days of the cycle. Although the concepts of attractivity and proceptivity were originally introduced in relation to the oestrous female (Beach 1976) their extension to the dioestrous period may lead to a better understanding of the social and sexual factors underlying the interaction.