Cells Of Female Rats Research Articles

Effects of Centrally Applied Somatostatin on Pituitary Adrenocorticotropes in Female Rats

Effects of intracerebroventricular (i.c.v.) application of somatostatin (SRIH-14 or SRIH-28) on growth and function of pituitary adrenocorticotropes (ACTH cells) were examined in adult female Wistar rats. Animals were subjected to i.c.v. administration of three 1-μg doses of SRIH-14 or SRIH-28 dissolved in 5 μl saline every second day. Controls were treated in the same way with the same volume of saline only. ACTH-producing cells were studied using the peroxidase-antiperoxidase (PAP) immunohistochemical procedure; blood samples were collected for hormone analyses 5 days after the last injection. SRIH-28 treatment decreased (p < 0.05) all morphometric parameters compared to control rats. Volume of ACTH cells decreased by 10%, nuclei by 36% and volume density by 13%. No significant changes (p > 0.05) in these parameters occurred after SRIH-14 treatment. Plasma concentration of ACTH in SRIH-28-treated rats was significantly lower (p < 0.05) than in control rats by 35%. In SRIH-14-treated rats, plasma concentration of ACTH was slightly, but not significantly (p > 0.05) increased by 13% compared to saline treatment. These observations suggest that centrally administered somatostatin-28, but not somatostatin-14, is specifically involved in the control of growth and secretory activity of ACTH cells in female rats. Thus, selective pharmacological manipulation of SRIH-28 receptors reached from CSF may affect ACTH activity without altering actions usually attributed to receptors sensitive to SRIF-14.

Effects of somatostatins on gonadotrophic cells in female rats

The effects of intracerebroventricular application of SRIF-14 and SRIF-28 on pituitary gonadotrophic cells (FSH and LH) were examined using immunocytochemical and morphometrical methods in adult female Wistar rats. FSH- and LH-producing cells were studied using the peroxidase-antiperoxidase (PAP) immunohistochemical procedure. Morphometry and stereology were used to evaluate changes in the number, volume densities and relative volume densities of LH- and FSH-immunopositive cells. In females treated with SRIF-14 or SRIF-28, the gonadotrophs were smaller, often pycnotic and more intensely stained. The number of LH-positive cells per unit area (mm2) was significantly decreased in both somatostatin-treated groups, while the number of FSH-positive cells was similar to that in the controls. Volume densities of perykarya of LH- and FSH-positive cells were decreased in all treated females, but extremely different in LH-positive cells after SRIF-14 administration. The relative volume density of LH cells was significantly decreased in both somatostatin-treated groups, while immunopositive FSH cells were not significantly decreased compared with the controls. It can be concluded that centrally applied somatostatins lead to changes in the immunocytochemical and morphometric properties of LH cells, while there is no significant effect on FSH cells.

Ovarian, uterine and brain mast cells in female rats: Cyclic changes and contribution to tissue histamine

Using histochemical techniques, we determined mast cell content in ovarian, uterine and brain tissues throughout the estrus cycle of the rat. In one series of experiments, 26 cycling female rats were used for the measurement of follicle stimulating hormone (FSH) in plasma and evaluation of mast cells in the tissues. In a second series, cycling female rats were used for the determination of tissue histamine. The number, degranulation pattern and staining characteristics of mast cells changed synchronously in rat ovarian, uterine and brain tissues during the estrus cycle. A great majority of mast cells in tissues were stained by Alcian blue at proestrus and metestrus. Safranin-stained mast cells were abundant in all tissues during estrus and diestrus. Alcian blue-stained mast cells contribute to the change of tissues histamine level. In ovarian tissue, histamine level increased significantly at proestrus and metestrus. The lowest ovarian histamine level was determined at estrus, in which virtually all mast cells were stained by safranin only. Mast cells in ovarian, uterine and brain tissues seem to change their histamine content throughout the estrus cycle. Mast cells are absent from the thalamus during proestrus and are present in the hypothalamus only during the estrus phase. Plasma FSH concentrations (mlU ml −1) did not significantly change throughout the estrus cycle (proestrus: 0.81±0.11, estrus: 0.69±0.07, metestrus: 0.82±0.13, diestrus: 0.67±0.19).

Influence of androgenicity on adipocytes and precursor cells in female rats.

We examined the effects of overexposure of testosterone (T) on fat cell morphology and adipocyte precursor pools in inguinal and retroperitoneal fat depots of ovariectomized rats. In both tissues peripubertal T decreased weights without affecting adipocyte mean cell size or the size distribution profiles, but adipocyte number was decreased by 65% in the inguinal and by 38% in the retroperitoneal depots. Immunofluorescent flow cytometry utilizing a specific antibody to rat adipose tissue lipoprotein lipase was used to quantify regional precursor cell populations. T sharply reduced the percentages of differentiated and undifferentiated preadipocytes in the inguinal depot, from 43.2 +/- 5.3 to 23.5 +/- 2.1% and from 57.7 +/- 4.0 to 43.6 +/- 5.3%, respectively, with a concomitant increase in fibroblasts from 1.6 to 32.9%. On the other hand, T had no effect on retroperitoneal preadipocyte pools. Perinatal androgenization exacerbated the decline in the inguinal weight (1.4 +/- 0.1 vs. 2.2 +/- 0.1g) but otherwise did not influence the actions of peripubertal T. Androgens may thus act in a tissue-specific manner to regulate fat cell growth potential in the femoral region in the female.

Estrogen increases the release of epidermal growth factor from individual pituitary cells in female rats.

Rat mixed anterior pituitary cells from cycling females were microscopically demonstrated to produce epidermal growth factor (EGF), in culture, using a reverse hemolytic plaque assay. Anterior pituitary cells of proestrous female rats secrete more EGF than those of metestrous rats, as evaluated by the mean area and the percentage of EGF plaque-forming cells. Estradiol-17 beta (10 nM) treatment of metestrous culture for 24 h increased significantly the size of EGF plaque-forming cells (from 1290 +/- 58 to 2566 +/- 57 microns 2, P < 0.01) and the percentage of EGF plaque-forming cells (from 20.57 to 28.19%; P < 0.01) to a level as high as observed in proestrous cultures (basal mean area 2171 +/- 157 microns 2, and basal percentage of EGF plaque-forming cells 22.71%). These results suggest that the hormonal status of female rats influences EGF secretion in the anterior pituitary gland and that estradiol is implicated in this phenomenon.

Changes in the ultrastructural distribution of prolactin and growth hormone mRNAs in pituitary cells of female rats after estrogen and bromocriptine treatment, studied using in situ hybridization with biotinylated oligonucleotide probes

The expression and distribution of prolactin (PRL) mRNA and their alterations induced by estrogen and bromocriptine were investigated using non-radioisotopic in situ hybridization (ISH) at the electron microscopic (EM) level. Our EM-ISH studies using biotinylated oligonucleotide probes showed that estrogen induced whirling changes of the rough endoplasmic reticulum (RER) of female rat PRL cells and increased transcription of PRL genes located on the polysomes of the whirling RER. The presence of mammosomatotroph cells in the rat pituitary gland was also verified in our EM-ISH studies. After bromocriptine administration, PRL cells contained many secretory granules due to the inhibition of secretion. Pre- and post-embedding EM-ISH and northern hybridization studies revealed that bromocriptine induced the distorted, vesiculated, and dilated RER, and also the suppressed PRL mRNA expression. The activity of protein kinase C (PKC), which mediates PRL gene expression, tended to be elevated by estrogen and suppressed by bromocriptine. Therefore, it is considered that the ultrastructural and quantitative changes in PRL mRNA expression evoked by estrogen and bromocriptine may be mediated by the intracellular signal transduction system, including PKC.

Estradiol-induced changes in TSH-like immunoreactivity of pituitary cells in female rats.

Beta-thyrotropin (TSH)-producing cells in the pituitary pars distalis of female rats were studied using rabbit anti-rat beta-thyrotropin (TSH) serum and a peroxidase-antiperoxidase (PAP) immunohistochemical procedure. Animals were neonatally treated with 1 mg estradiol-dipropionate (EDP) and sacrificed at different stages of development up to adulthood. Intact females of the corresponding age served as the controls. Morphometry and stereology were used to evaluate the changes in TSH-cell number and volume densities of the cells and nuclei. All morphometric parameters examined in estradiol-treated animals showed a significant decrease in comparison with immunoreactive TSH cells of age-matched controls. The most prominent EDP-induced changes were evident in peripubertal 38-day-old rats, the number and volumetric densities of both TSH cells and their nuclei being reduced by about 90% compared to intact pituitary. This decrease in the number and volume densities of TSH cells in EDP-treated rats explicitly demonstrated that this hormone, applied neonatally, has an inhibitory effect on TSH-immunoreactive cells up to adulthood, in accordance with our earlier data obtained by light and electron microscopy.

Role of estrogens in the regulation of prolactin receptors in liver cells of female rats

Strong expression of prolactin receptors in sinusoidal domains and cytoplasmic granules of hepatocytes, which is independent of cell location in the hepatic lobule and is positively regulated by estrogens, is revealed in pubertal female rats. In estrogen-treated animals, Prolactin receptors are also exposed in the perinuclear space of some hepatocytes surrounding the central veins. Estrogens regulate the intensity of prolactin receptors expression in hepatocytes, but not the number of cells containing these receptors.

Effects of ipriflavone on calcitonin synthesis in C cells of the rat thyroid.

Ipriflavone is known to stimulate calcitonin (CT) secretion from the thyroid glands of female animals, but the exact mechanism of this action remains unknown. In the present study, an increase of CT production in thyroid C cells of female rats, but not of male rats, was proven immunohistochemically. Furthermore, the parallel increase of CT mRNA in those thyroid glands was confirmed by Northern blot analysis. These results proved that ipriflavone stimulates not only CT secretion but also CT synthesis in thyroid C cells and that the changes were gender dependent (greater in females). In order to investigate the effect of estrogen on the ipriflavone-induced increase of CT in female rat thyroid gland, CT and CT mRNA in the thyroid glands of untreated, ovariectomized, and estrone-treated (postovariectomy) rats were examined by both immunohistochemistry and Northern blot technique. Serum levels of CT and calcium were also examined. Against expectation, estrone failed to produce any significant effect on the ability of ipriflavone to induce CT synthesis and secretion.

Prodynorphin and proenkephalin gene expression in the anteroventral periventricular nucleus of the rat: Sexual differentiation and hormonal regulation

Opioid peptides are generally thought to exert hormone-dependent regulatory influences on gonadotropin secretion and the anteroventral periventricular nucleus (AVPv) has been shown to play a critical role in the neural control of this sexually dimorphic function. In the present study we used in situ hybridization to compare the numbers of proenkephalin (PENK) and prodynorphin (PDYN) mRNA-containing neurons in the AVPv of male and female rats and to evaluate the influence of circulating sex steroid hormones on the development and mature regulation of PENK and PDYN gene expression in these neurons. In agreement with earlier immunohistochemical observations, the number of PENK mRNA-containing neurons in the AVPv of male rats was found to be twice that of female animals. In contrast, the AVPv contains over four times the number of PDYN mRNA-containing cells in female rats, relative to intact males. Treatment of newborn female rats with testosterone increases the number of PENK mRNA-containing neurons in the AVPv, but decreases the number of PDYN mRNA-containing neurons in the AVPv compared with untreated females. Treatment of adult ovariectomized female rats with estradiol significantly increased PDYN mRNA levels in the AVPv; however, comparable changes in levels of PENK mRNA were not detected. In adult male rats, neither PDYN, nor PENK mRNA were significantly altered by orchidectomy or testosterone treatment. Thus, the maintenance of enkephalinergic neurons and the loss of hormone-sensitive dynorphin-containing neurons in the AVPv may represent important developmental influences of neonatal androgens on the sexually differentiated neural circuitry controlling gonadotropin secretion.

Alterations in Splenic Lymphoid Cell Subsets and Activation Antigens in Copper-Deficient Rats

Rats were nursed by dams fed a diet containing adequate (6 micrograms/g) or deficient (0.6 micrograms/g) Cu during the lactation period and weaned to the same diet. Splenic mononuclear cells were isolated and the phenotypic profile determined by flow cytometry after immunolabelling with monoclonal antibodies to cell surface markers. Total splenic mononuclear cell yield and the relative percentage and absolute number of T-cells and the CD4+ (helper) and CD8+ (cytotoxic) T-subsets were decreased in Cu-deficient male rats. The relative percentage, but not the absolute numbers, of splenic B-cells and macrophages was increased by Cu deficiency. The percentage of splenic mononuclear cells from male rats that expressed interleukin-2 receptors and transferrin receptors in vivo was increased by Cu deficiency. In contrast, dietary Cu deficiency did not affect the yield and phenotypic profile of splenic mononuclear cells in female rats. Reactivity of splenic mononuclear cells to T-cell mitogens was decreased in Cu-depleted male and female rats. However, mitogen-induced increases in levels of interleukin-2 receptor and transferrin receptor were similar in cultures of splenic mononuclear cells obtained from control rats and rats subjected to restricted dietary intake of Cu only during the postlactation period. Thus, decreased mitogenic blastogenesis on exposure of cells from Cu-deficient rats does not reflect a nonspecific impairment of cellular activation.

Hormonal control of the development and regulation of tyrosine hydroxylase expression within a sexually dimorphic population of dopaminergic cells in the hypothalamus

In situ hybridization histochemistry was used to examine the development and regulation of tyrosine hydroxylase (TH) mRNA within the sexually dimorphic population of dopaminergic cells in the anteroventral periventricular nucleus (AVPv) of the hypothalamus. The AVPv contains over 3 times as many TH mRNA-containing cells in female rats, compared with males. This sexual dimorphism appears to be dependent on perinatal levels of gonadal steroids since orchidectomy of newborn males increased, and treatment of newborn females with testosterone decreased, the number of TH mRNA-containing cells detected within the AVPv. In addition, circulating gonadal steroids appear to downregulate TH expression within these cells in both adult male and female rats. In adult male animals, gonadectomy increased the number of TH mRNA cells in the AVPv within 7 days. Similarly, estradiol treatment prevented the increase in the number of TH mRNA-containing cells within the AVPv seen in ovariectomized female rats. No sexual differences were detected in the number of TH mRNA-containing cells within the suprachiasmatic preoptic nucleus, located just ventral to the AVPv. These findings indicate that perinatal gonadal steroids influence the number of cells within the AVPv that express TH in detectable amounts by determining the number of cells that are capable of producing sufficient quantities of TH message, as opposed to sex-specific alterations in the post-translational mechanisms. In the adult, circulating gonadal steroids appear to downregulate TH expression within these cells suggesting that testosterone and/or estrogen may exert a sustained influence on the biosynthetic activity of this sexually dimorphic population of dopaminergic cells.

Morphofunctional study of mammotropic cells following intraventricular administration of met-enkephalin

An ultrastructural and morphometric study was carried out on the adenohypophyseal mammotropic cells of rats treated intraventricularly with an acute dose (150 micrograms) of Met-enkephalin. In the female rats, clear features of cellular hyperactivity appeared after opioid administration. The changes affected the Golgi complex, the rough endoplasmic reticulum, the mature and immature secretory granules and the images of exocytosis. Such changes did not appear when naloxone was administered before the opioid, and naloxone induced an increase in the numerical density of lysosomal dense bodies with lipoid inclusions. In the male animals, administration of an identical dose of Met-enkephalin caused only a few significant changes, similar to those observed in the controls. It is concluded that Met-enkephalin administered intraventricularly causes evident modifications in the mammotropic cells of female rats whereas such changes in the male animals are not significant.

Distribution of cholecystokinin-immunoreactive cell bodies in the male and female rat: II. Bed nucleus of the stria terminalis and amygdala.

The distribution of cholecystokinin-immunoreactive (CCK-I) cell bodies was studied in the bed nucleus of the stria terminalis (BST) and amygdaloid complex of colchicine-treated male and female rats. Immunoreactive cells were visualized in the BST medial amygdaloid (MeA), central lateral, basolateral, basolateral ventral, medial, intercalated, anterior cortical, and posterior cortical nuclei and the amygdalohippocampal zone. Several significant sex differences were observed. In the male, a dense aggregation of CCK-I cell bodies was visualized in the MeA, especially in the dorsocaudal part and in the encapsulated part of the BST. In comparison, female rats had relatively fewer immunoreactive cells in both of these regions. In the lateral and basolateral amygdaloid nuclei, however, more CCK-I cells were visualized in the female than in the male, but the difference was not statistically significant. These data provide characterization of a sexually differentiated CCK system. In addition, we observed that the number of CCK-I cells in the BST and posterodorsal part of the MeA was substantially reduced after castration. The number of CCK-I cells in female rats, however, was not significantly reduced after ovariectomy in any of the regions studied. These findings imply that the steroid regulation of CCK is sexually differentiated. The sexually dimorphic distribution of CCK-I cells in areas that are targets of steroid hormones and regulate reproductive processes is consistent with the possibility that CCK participates in central integration of sensory and steroidal input that modulates reproductive behavior.

Prolactin cells of female rats treated with the carcinogen 7,12-dimethylbenz(A) anthracene (DMBA) in vivo. An ultrastructural study.

Pituitary prolactin (PRL) cell microadenomata developed in females of a strain of Sprague-Dawley rats (SD1) following intragastric treatment with the carcinogenic hydrocarbon 7,12-dimethylbenz(a) anthracene (DMBA). Similar treatment of another strain of Sprague-Dawley rats (SD2) resulted in mammary tumour development but no PRL cell microadenomata. In SD1 strain rats morphologically distinct populations of PRL cells appeared after DMBA treatment, one composed of cells characterised by abundant, organised but very dilated RER and with large hormone storage granules, 500-600 nm in diameter (P1). The other cell type had electron-dense cytoplasm, narrow organised arrays of RER and moderately large, pleomorphic granules (P2). Both cell types appeared active with large Golgi and prominent nucleoli. P2 cells were most numerous 2 months after DMBA treatment but had virtually disappeared at 6 months and microadenomata were common at 8 months. PRL cells of SD2 rats were uniform in morphology, characterised by only moderate accumulations of RER, pleomorphic hormone storage granules, large Golgi and prominent nucleoli, and showed no close resemblance to either P1 or P2 cells of SD1 strain rats. It is possible that the morphological variations which developed in SD1 PRL cells may represent changes in responsiveness to factors controlling PRL cell secretion and proliferation and which may be pertinent to microadenoma development.

Measurement of lipogenesis in isolated preputial gland cells of the rat and the effect of oestrogen.

Lipogenesis was measured in isolated preputial gland cells of female rats after ovariectomy and after the administration of oestradiol benzoate. Ovariectomy decreased preputial gland cell lipogenesis and also altered the pattern of lipid synthesis, producing a relative decrease in the proportion of polar lipids and an increase in the proportion of 'triglycerides'. Although daily administration of 2 or 10 micrograms oestradiol benzoate for 7 days produced slight increases in preputial gland cell lipogenesis in ovariectomized rats, the effects were not significant. A single injection of 10 micrograms oestradiol benzoate, however, produced significant increases in preputial gland cell lipogenesis of ovariectomized rats at both 2 and 24 h and, moreover, at 24 h the pattern of polar lipid and triglyceride labelling was restored to normal. Prior administration of actinomycin D reduced the lipogenic effect of oestradiol benzoate. Oestradiol benzoate had little or no effect on preputial gland cell lipogenesis in male rats. These results confirm that oestrogen is able to stimulate preputial lipogenesis in female rats. Whether this action of oestrogen is related to its pheromone-producing effect on the preputial glands is not yet known.

Mitotic activity of prolactin cells in the pituitary glands of male and female rats of different ages.

The anterior pituitary of colchicine-pretreated male and female rats from 20 days to 12 months of age was stained immunohistochemically with anti-rat prolactin serum. Immunoreactive mitotic cells were identified in all groups of rats. In adult female rats the mitotic index of prolactin cells was higher at oestrus than at other stages of the oestrus cycle and than that in male rats of comparable ages. If adult female rats were ovariectomized on the second day of dioestrus or on the day of proestrus, the mitotic indices at presumptive oestrus were less than those in sham-operated controls at oestrus. Estrogen administration to ovariectomized rats significantly elevated the mitotic index of prolactin cells at 48 h after the treatment. The mitotic indices of prolactin cells in female rats reached a peak at 60 days of age, and then decreased with age. In male rats the mitotic indices showed a steady decrease from the value at 20 days of age. A sex difference in the mitotic indices of prolactin cells was noted from 60 days to 12 months of age. The present results clearly demonstrate that differentiated prolactin cells can undergo mitosis and that a sex difference in the mitotic activity of prolactin cells is present during adult life.

Effects of stopping training on size and response to insulin of fat cells in female rats.

The parametrial adipocytes of exercise-trained female rats were smaller and had greater rates of 2-deoxyglucose uptake and glucose oxidation over a wide range of insulin concentrations than the fat cells of sedentary animals of the same age. The fat cells of the sedentary animals had a 180% greater volume than those of the trained rats, despite only a 9% difference in body weight. Following cessation of training adipocyte size increased rapidly; approximately two-thirds of the initial difference in fat cell volume between the trained and sedentary rats had disappeared after 9 days without exercise. Concomitant with the increase in fat cell size, the response of 2-deoxyglucose uptake and glucose oxidation to insulin diminished. There was a close correlation between fat cell size and the magnitude of the response to insulin of both glucose oxidation and 2-deoxyglucose uptake. It appears from these results that the effect of exercise training on the response of adipocytes to insulin is rapidly lost when exercise is stopped.

Interaction of Human Growth Hormone With Isolated Rat Liver Cells

The interaction of 125I-labeled human growth hormone (hGH) with isolated rat liver cells is a specific, time dependent and saturable process. In male rats, one cell binds a maximum of 2000 hormone molecules; the dissociation constant of the cell-hGH interaction is about 3 X 10(-10)M. Liver cells of female rats bind 5 to 10 times more hGH than do those of male rats at equivalent hormone concentrations. Binding of 125I-labeled hGH to liver cells is readily inhibited by native hGH; 50% inhibition occurs at about 2 X 10(-9)M hGH irrespective of sex. In male rats, bovine growth hormone (bGH) is almost as potent as hGH in inhibiting 125I-labeled hGH binding; no displacement occurs with ovine prolactin (oPRL) except at very high (greater than 10(-6)M) concentrations. In female rats, bGH competes less effectively, and oPRL, more effectively, than they do in males; in addition, oPRL demonstrates a higher apparent affinity for the hGH binding sites (4 X 10(-9)M) than does bGH (1 X 10(-8M). These findings suggest that in female rats hGH, unlike bGH, interacts with additional, "lactogenic" binding sites that are distinct from the "growth hormone" binding sites. The 125I-labeled hGH eluted from liver cells as well as that which remains in the incubation medium retains full biological activity, as judged on its ability to bind specifically to liver membranes. Treatment of liver cells by phospholipase A causes a 5-fold increase in cell binding capacity. Liver cells bind about the same amount of hGH as do crude particulate fractions from these cells; this suggests that in the intact cell, binding occurs at relatively accessible sites, presumably localized in the plasma membrane.

Ultrastructural immunocytochemical characterization of the thyrotroph in rat and human pituitaries.

The beta chain of thyroid stimulating hormone (TSH) was localized in normal rat and human pituitaries with the unlabeled antibody--peroxidase-antiperoxidase complex technique and antisera to rat or bovine TSH beta. Araldite embedded tissues, immunoreactivity of TSH was best preserved after fixation with 1% glutaraldehyde or picric acid formaldehyde. In the TSH cells, the immunocytochemical stain was located on granules. However, there was a variation in staining of individual granules, and among the population of granules. Rat TSH cells were ovoid or angular to stellate, and contained granules ranging in size from 60-175 nm. The human TSH cell was polyhedral, and contained scattered granules of 150-300 nm diameter. In both species, granule distribution was either in 1-2 rows at the periphery, or scattered throughout the cytoplasm with some concentration in cytoplasmic processes. TSH cells in female rats in estrous contained more granules than those of other stages. TSH cells were distinguished from adrenocorticotropic hormone cells and luteinizing hormone cells on the basis of granule size and distribution, and cell shape.