Leydig cell involvement in the paracrine regulation of mast cells in the testicular interstitium of the rat

The accumulation of mast cells in the rat testicular interstitium was studied under different experimental conditions in order to correlate this accumulation with the alterations of specific testicular tissue compartments or cell types. Estrogen treatment was effective in inducing mast cell proliferation when administered on Day 1 or at higher doses at 10 days of age. Estrogens were ineffective beyond 20 days of age. Postnatal treatment of neonatal-estrogen-treated rats with FSH and LH prevented the appearance of mast cells. In contrast, treatment with the Leydig cell cytotoxic ethylene dimethane sulphonate (EDS) was effective in inducing mast cell accumulation only when administered to adult rats, inducing small numbers of mast cells at 45 days of age; it was ineffective on 30-day-old rats. Hypophysectomy alone did not determine the appearance of mast cells. However, when atrophic Leydig cells were destroyed with EDS, high numbers of mast cells accumulated in the testis. These results support the existence of Leydig cell-related inhibitory factors for mast cells in the rat testicular interstitium.     

Suppression of testicular maturation and fertility following androgen administration to neonatal male rats

Administration of supraphysiologic doses of androgen to male rats within the first few neonatal days markedly suppresses subsequent testicular maturation; this effect diminishes as androgen is injected on succeeding postnatal days. Testosterone propionate (TP) administered neonatally at dosages up to 3.5 mg appreciably diminished postnatal testicular growth; postpubertal androgen secretion, as assessed by accessory sex organ weights and serum testosterone concentrations and as reflected by a castrationlike developmental pattern of the hepatic enzyme, histidase; spermatogenesis; and fertility. Beyond three mo of age testicular growth rates and androgen secretion--but not fertility--tended to be restored. These effects of neonatal androgen do not require aromatization to estrogen; indeed 5 alpha-dihydrotestosterone elicited more profound testicular suppression than TP, which was sustained until at least 100 days of age. Testes of neonatally androgenized rats were capable of responding to gonadotropins administered at three wk of age with increases in weight and androgen secretion. These findings suggest that a developmental event, suppressible by pharmacologic doses of androgen, occurs at a nontesticular site during the first few post partum days in the male rat; this event programs subsequent testicular maturation.     

Postnatal development of growth hormone and prolactin cells in male and female rat pituitary. An immunocytochemical light and electron microscopic study

Localization and ultrastructural maturation of prolactin (PRL) and growth hormone (GH) cells were studied in pituitaries from neonatal, immature (4-6 weeks old), and adult rats (2-3 months old) by light and electron microscopic immunocytochemistry. The distribution pattern of these cells did not change with age. Both cell types were concentrated laterodorsally, with PRL cells adjacent to the intermediate lobe and GH cells nearer the center of the pars distalis. Labeling density of the immunogold reaction was highest for both hormones in immature rats. In neonatal and immature rats, one PRL cell type with granules 200 nm in diameter was present. In adult rats, two types of PRL cells were present: one containing polymorphous granules measuring about 500 nm (prevalent in female rats), the other with spherical granules about 200 nm (prevalent in male rats). No changes were detected in GH cells during maturation.     

Maturation of adult rat peritoneal and mesenteric mast cells

Summary Repopulation and maturation of rat mesenteric and peritoneal mast cells were studied after mast cell depletion by intraperitoneal injection of distilled water. Immature mast cells were first identified in the mesentery and peritoneal fluid 5 and 6 days, respectively, after water injection. The most immature mast cells that could be identified contained a few orthochromatic granules. Upon maturation, the granules became metachromatic and increased in size and number. Heparin, revealed by toluidine blue staining and berberine sulfate fluorescence, appeared simultaneously with orthophthaldialdehyde (OPT)-induced histamine fluorescence. Paraformaldehyde-induced serotonin fluorescence appeared somewhat later. Repopulation of mesentery and peritoneal fluid by mast cells seemed to be independent of each other and to occur from undifferentiated precursor cells.     

Secretory granules of prolactin cells of neonatally thyroidectomized female rats

In the anterior pituitary glands of neonatally thyroidectomized female rats sacrificed at 30 days of age, the prolactin granules were small and spherical in shape. The administration of thyroxine to neonatally thyroidectomized rats produced an obvious increase in the number and size of secretory granules in prolactin cells; comparatively large, pleomorphic secretory granules were frequently observed in these cells. These enlarged and pleomorphic granules closely resembled those observed in the prolactin cells of sham-operated control rats. These results may indicate that thyroxine stimulates the basic metabolism or cellular function of prolactin cells of neonatally thyroidectomized rats and leads to the formation of prolactin granules that are similar to those of sham-operated control rats.     

The density of myocardial and pericardial rat mast cells in isoproterenol-induced heart failure

Myocardial mast cells (MC) respond to cardiovascular pathology. The behavior of MC population in myocardium and pericardium of rats has been studied 24 h, 14, 28 and 60 days after two isoproterenol injections (at 24 h intervals). The extent of heart failure has been estimated by supersonic inspection 28 and 60 days after isoproterenol injections. The density of MCs of different degrees of maturity was estimated on paraffin sections stained with Alcian blue--Safranin. The MC density in myocardium of intact and experimental rats was relatively low: from 4 to 6 cells/mm2. The MC density in pericardium of intact rats was several times higher than in myocardium: 48.6 +/- 13.0 cells/mm2. In 24 h and 14 days after isoproterenol injections the pericardial MC density was 1.5 times higher than in control rats (P < 0.05) at the expense of increase in the number of mature MCs with Safranine-positive granules without the increase in the number of immature cells with Alcian blue-positive granules. In 28 days the pericardial MC density was 2 times higher than in intact rats (P < 0.05) at the expense of increase in number of immature and mature cells. In 60 days after isoproterenol injections the pericardial MC density and the ratio of immature and mature cells compared with control did not reach statistical significance. The changes in pericardial MC population corresponded to severity of heart failure according to functional indices. The findings show active reaction of pericardial MCs on myocardium dysfunction that stimulates the maturation of resident immature MCs in pericardium and migration of immature cells to pericardium of damage heart.     

Changes in the testis interstitium of Sprague Dawley rats from birth to sexual maturity

Changes in the rat testis interstitium from birth to adulthood were studied using Sprague Dawley rats of 1, 7, 14, 21, 28, 40, 60, and 90 days of age. Our objectives were 1) to understand the fate of the fetal Leydig cells (FLC) in the postnatal rat testis, 2) to determine the volume changes in testicular interstitial components and testicular steroidogenic capacity in vitro with age, 3) to differentially quantify FLC, adult Leydig cells (ALC), and different connective tissue cell types by number and average volume, and 4) to investigate the relationship between mesenchymal and ALC numbers during testicular development. FLC were present in rat testes from birth to 90 days, and they were the only steroidogenic cells in the testis interstitium at Days 1 and 7. Except for FLC, all other interstitial cell numbers and volumes increased from birth to 90 days. The average volume of an FLC and the absolute volume of FLC per testis were similar at all ages except at Day 21, when lower values were observed for both parameters. FLC number per testis remained constant from birth through 90 days. The observations suggested that the significance of FLC in the neonatal-prepubertal rat testis is to produce testosterone to activate the hypothalamo-hypophyseal-testicular axis for the continued development of the male reproductive system. ALC were the abundant Leydig cell type by number and absolute volume per testis from Day 14 onwards. The absolute numbers of ALC and mesenchymal cells per testis increased linearly from birth to 90 days, with a slope ratio of 2:1, respectively, indicating that the rate of production of Leydig cells is 2-fold greater than that of mesenchymal cells in the postnatal rat testis through 90 days. In addition, this study showed that the mesenchymal cells are an active cell population during testis development and that their numbers do not decrease but increase with Leydig cell differentiation and testicular growth up to sexual maturity (90 days).     

Morphologic mast cell cycles

During ultrastructural studies of the early kinetics of anaphylactic degranulation and early and late recovery intervals from these release reactions induced in isolated, partially purified, cultured human lung mast cells stimulated to release histamine and granules by exposure to anti-IgE, we noted the ability of mast cells to undergo morphologic cycles. Following release of most cytoplasmic granules and shedding of large amounts of membranes and cellular processes, small, immature mast cells, nearly devoid of granules, were seen. These lymphocyte-like cells had condensed nuclear chromatin and large nucleoli. Blast transformation of these small cells and expansion of synthetic machinery-laden cytoplasm resulted in large, immature mast cells with small numbers of small, immature progranules and large numbers of lipid bodies in their cytoplasm. This cycle of mast cell morphologic change has considerable similarities to, as well as some differences from, lymphocyte morphologic cycles.     

Cutaneous mast cell depletion and recovery in murine graft-vs-host disease

Mast cells as studied by light microscopy with metachromatic staining, have been noted to "disappear" from the skin of mice with chronic graft-vs-host disease (GVHD) produced across minor histocompatibility barriers. This mast cell disappearance is accompanied by ultrastructural evidence of loss of granule contents. In this study, we followed cutaneous mast cells in chronic GVHD over 9 mo by three methods: Light microscopy of toluidine blue-stained sections showed that mast cells not seen at day 42 reappeared between days 94 and 125, were supramaximal at days 146 and 164, and returned to normal levels at days 195 and 280. Double immunofluorescent staining of mast cells for the presence of surface IgE receptors and cytoplasmic granules (avidin) revealed IgE receptor-bearing cells that lacked avidin-binding granules at the time when mast cells were not apparent on light microscopy. By electron microscopy, reappearing mast cells have the morphology of immature dermal mast cells. Ultrastructural abnormalities of mast cells persist some 150 days after GVHD induction. The possible relationship of these mast cell changes to the development of dermal fibrosis in chronic GVHD is discussed.     

Loss of histochemical identity in mast cells lacking carboxypeptidase A

Mast cell carboxypeptidase A (Mc-cpa) is a highly conserved secretory granule protease. The onset of expression in mast cell progenitors and lineage specificity suggest an important role for Mc-cpa in mast cells. To address the function of Mc-cpa, we generated Mc-cpa-null mice. Mc-cpa-/- mast cells lacked carboxypeptidase activity, revealing that Mc-cpa is a nonredundant enzyme. While Mc-cpa-/- peritoneal mast cells were ultrastructurally normal and synthesized normal amounts of heparin, they displayed striking histochemical and biochemical hallmarks of immature mast cells. Wild-type peritoneal mast cells had a mature phenotype characterized by differential histochemical staining with proteoglycan-reactive dyes (cells do not stain with alcian blue but stain with safranin and with berberine) and a high side scatter to forward scatter ratio by flow cytometry and were detergent resistant. In contrast, Mc-cpa-/- peritoneal mast cells, like immature bone marrow-derived cultured mast cells, stained with alcian blue normally or weakly and either did not stain with safranin and berberine or stained weakly, had a low side scatter to forward scatter ratio, and were detergent sensitive. This phenotype was partially ameliorated with age. Thus, histochemistry and flow cytometry, commonly used to measure mast cell maturation, deviated from morphology in Mc-cpa-/- mice. The Mc-cpa-/- mast cell phenotype was not associated with defects in degranulation in vitro or passive cutaneous anaphylaxis in vivo. Collectively, Mc-cpa plays a crucial role for the generation of phenotypically mature mast cells.     

DIFFERENTIATION AND PROLIFERATION OF EMBRYONIC MAST CELLS OF THE RAT

Histochemical reactions and radioautography were used to investigate the sequence of mast cell development in rat embryos. Mast cells arise ubiquitously in and are confined to the loose connective tissue in the embryo. The alcian blue-safranin reaction distinguishes between weakly sulfated and strongly sulfated mucopolysaccharides by a shift from alcian blue to safranin staining. Based on this reaction and morphologic characteristics, four stages were identified. Stage I mast cells are lymphocyte-like cells with cytoplasmic granules which invariably stain blue with the alcian blue-safranin reaction. In Stage II cells the majority of granules are alcian blue-positive, but some safranin-positive granules have appeared. Stage III mast cells are distinguished by a majority of safranin-positive cytoplasmic granules; some alcian blue-positive granules still remain. Stage IV cells contain only safranin-positive granules. Thymidine-H³ uptake and identification of mitotic figures indicates that mast cells in Stages I and II comprise a mitotic pool while those in Stages III and IV are mitotically inactive. The pattern of S³⁵O₄ incorporation and the sequence of appearance of histochemically identifiable mast cell constituents corroborates division of the proliferation and differentiation of embryonic mast cells into the four stages described above. The process of formation of mast cell granules is interpreted as reflecting the synthesis and accumulation of a heparin precursor in alcian blue positive granules followed by the synthesis and accumulation of highly N-sulfated heparin along with mast cell chymase and finally histamine in safranin-positive granules.     

Ultrastructural visualization of sulphated complex carbohydrates in blood and epithelial cells with the high iron diamine procedure

Synopsis The high iron diamine (HID) method has been found to impart density at the ultrastructural level selectively to sites known to contain sulphated complex carbohydrates. Thus, immature primary granules in rabbit heterophils, immature précrystalloid granules in rabbit eosinophils, all granules of rabbit basophils, mouse and rat mast cells and the nucleoids of -granules of rabbit platelets were stained by HID. Granules of mast cells in rat cervical lymph node varied in the distribution pattern of the HID-reactive component. Mucous droplets within goblets of mouse colonic epithelial cells varied in HID reactivity. Sites known to contain sialomucin but no sulphates, such as mucous cells and apical plasmalemmae in mouse rectosigmoid colon, failed to stain with HID in contrast to their reactivity for dialysed iron at the ultrastructural level. The surface of mast cells and blood cells lacked affinity for HID, indicating that the dialysed iron binding at the surfaces can be attributed to neuraminic acid. HID proved more effective than dialysed iron in visualizing acid mucosubstance in precursor forms of the crystalloid granules in the eosinophil and in mast cell granules. Inclusion of 0.5% glycerol in the HID solution enhanced staining in mouse colon.     

Ultrastructural and cytochemical studies of acid phosphatase and trimetaphosphatase in rat peritoneal mast cells developing in vivo

Summary The ultrastructural and cytochemical features of peritoneal mast cells of the rat were studied. Immature mast cells show specific cytoplasmic granules of different sizes, the smaller ones localized in the Golgi region. The rough endoplasmic reticulum and Golgi apparatus are well developed, and mitochondria are numerous. Nuclei show deep indentations. Acid phosphatase is present in the Golgi saccules, in GERL (Golgi apparatus-endoplasmic reticulumlysosome) and in some small granules. It is not present in mature granules. Trimetaphosphatase is present in the Golgi saccules, in GERL, in most immature granules and in some mature granules. These enzymes appear to be transported and packaged into granules by the Golgi apparatus, suggesting that the specific mast cell granules may be a form of lysosome. The results of this study are consistent with the hypothesis that peritoneal mast cells may be derived from macrophage-like precursors.     

Cystogenesis of antral follicles induced by dehydroepiandrosterone (DHEA) stimulates mast cell proliferation and maturation in the house rat (Rattus rattus) ovary

Mast cell dynamics has been studied in relation to cystogenesis of ovarian follicles in the house rat. Immature rats were injected (s.c.) daily with DHEA (6.0 mg/100 g body weight) and were sacrificed on the day 8, 16 and 24 of the start of treatment. Ovarian sections of the treated rats had majority of the antral follicles undergoing atresia or in early stages of cystogenesis. Completely developed cysts were evident from the ovarian surface after 24 days of daily treatment. Treatment for 8 days resulted in significant increase in the number of alcian blue-positive ovarian mast cells. Ovaries after 16 days of DHEA treatment showed no marked change with regard to the number of total mast cells per unit area and staining characteristics. However, a significant rise in ovarian mast cell counts was recorded after 24 days of treatment and most of the cells contained safranin-positive red granules. This increase was attributed due to the increase in their number in medulla and stroma around the cystic follicles.     

Translation and granule localization of mouse mast cell protease-5. Immunodetection with specific antipeptide Ig.

An antibody to mouse mast cell protease-5 (MMCP-5) was obtained by immunizing a rabbit with a 17-residue synthetic peptide corresponding to the unique amino acid sequence at residues 146 to 162 in this serine protease. After affinity purification, anti-MMCP-5(146-162) Ig reacted in SDS-PAGE immunoblots to recombinant MMCP-5 and to the native MMCP-5 protein present in the lysates of mouse serosal mast cells and the MC5 line of Kirsten sarcoma virus-immortalized mouse mast cells. Immunocytochemical staining localized MMCP-5 to the cytoplasmic granules of serosal mast cells and Kirsten sarcoma virus-immortalized mouse mast cells. Because mouse bone marrow-derived mast cells express abundant amounts of MMCP-5 mRNA, anti-MMCP-5(146-162) Ig was used to study the translation and granule accumulation of this protease when progenitor cells differentiate into these immature mouse mast cells. Maximal expression of MMCP-5 mRNA occurred after bone marrow cells had been cultured for 2 wk in IL-3-rich WEHI-3 cell conditioned medium, and MMCP-5 protein was detected in these cells. However, electron-microscopic analysis with gold-labeled antibody revealed that the amount of MMCP-5 in the individual granules of bone marrow-derived mast cells varied. The highest concentration of MMCP-5 was found in the most electron-dense secretory granules of the cells. These studies demonstrate the ultrastructural localization of the earliest transcribed mouse mast cell chymase, MMCP-5, and its granule accumulation during the differentiation of mouse bone marrow progenitor cells into immature mouse mast cells.     

Dynamics of the restoration of a population of peritoneal mast cells following their destruction

Removal of the mast cells from the mouse abdominal cavity by repeated washings as well as their osmotic destruction with distilled water are suitable methods to study regeneration of mast cells in postnatal ontogenesis. Simultaneous application of their differential staining with alcian blue-safranin makes it possible to reveal successive stages of the newly formed mast cells maturation and determine duration of every stage. The time of complete renovation of the peritoneal mast cells has been stated to be, according to the mode of the stimulation, 60--80 days. Total duration of the first two stages of maturation is 1--2 days. Mast cell differentiation is fully completed by the 11th day.     

Changes in germ cell adenylate cyclase and protein carboxyl methylase activities in rat testicular tissue during bilateral cryptorchidism and after orchidopexy

Soluble Mn2+-dependent adenylate cyclase and protein carboxyl methylase are two enzymes that are primarily localized in haploid germ cels of rat testicular tissue, and both enzymes exhibit an increase in activity in association with sexual maturation. Experimental cryptorchidism (surgery at 17 days of age) in immature rats prevented the age-dependent increase in the activity of these two testicular enzymes. After orchidopexy at 34 days of age the activities of these two enzymes increased to normal control values in association with testicular growth. These observations show that biochemical markers such as soluble Mn2+-dependent adenylate cyclase and protein carboxyl methylase can be used to follow germ cell differentiation.     

Effect of neonatal head X-irradiation on growth of costal and tibial cartilage in rats: a histochemical and electron microscopic study

Long-Evans rats were exposed to a single dose of head X-irradiation (600 rads) at 2 days of age. Experimental and sham irradiated rats were sacrificed at 14, 20-21, 23, 41-45, and 70-71 days. Tibial epiphyseal width and the number of cells in the epiphyseal plate were determined. Histochemical and electron microscopic studies were carried out on both costal and epiphyseal cartilage. Histochemical techniques revealed a reduction in chondroitin sulfate at 14 days in both costal and epiphyseal cartilage of X-irradiated rats. Epiphyseal cartilage demonstrated recovery subsequently, and this was followed by a normal decrease of chondroitin sulfate with increasing age, but costal cartilage did not recover. Collagen synthesis was also reduced in both costal and epiphyseal cartilage, but not as dramatically as chondroitin sulfate. Except for some electron dense cells and reduced scalloping of the cell membrane, costal chondrocytes from irradiated rats did not show major ultrastructural alterations. In contrast, epiphyseal chondrocytes demonstrated radiation induced alterations in organelles, in enhanced glycogen deposition, and in retardation of chondrocyte maturation. Extracellularly in both costal and epiphyseal cartilage of irradiated rats, collagen density and matrix granules were reduced, while calcification of the matrix was enhanced. Beyond 45 days, the effects of irradiation were markedly reduced. Comparisons of the histochemical results with metabolic studies carried out previously in cartilage from the same animals indicated a more direct concordance of the histochemical results with the pattern of physical growth and supported the usefulness of morphologic and histochemical techniques in the analysis of the growth disorder in the head-irradiated rat.     

新生大鼠雌激素注射后睾丸肥大细胞的变化

新生大鼠注射雌二醇后,睾丸肥大细胞于第30天可见到,细胞数量随年龄增长而增多,生后4－6个月,睾丸网附近仍可见大量肥大细胞。睾丸内的肥大细胞比皮肤内的结缔组织肥大细胞（CTMC）小而与小肠粘膜的粘膜肥大细胞（MMC）相近,AB－S染色后基本着蓝色,硫酸小檗碱荧光染色后呈现中等强度黄色荧光,结果提示,新生大鼠雌激素注射后睾丸内肥大细胞的增多可能与免疫过程有关,睾丸内肥大细胞与CTMC和MMC皆有所不同。     

Changes in the concentration and size of testicular macrophages during development

Structural and functional interactions exist between Leydig cells and testicular macrophages of adult rats. Since the function of Leydig cells changes during critical periods of development and postnatal maturation, it is possible that macrophages are in part involved in regulating this process. As a first step towards gaining an understanding of the development of this paracrine phenomenon, I have undertaken a series of studies designed to determine when macrophages first become identifiable in the fetal tests and to determine whether the concentration or size of macrophages changes during important stages of testicular maturation. Macrophages were identified immunohistochemically in frozen sections of testis from rats at various prenatal and postnatal ages using commercially available monoclonal antibodies to proteins specific to rat macrophages. It was found that macrophages positive for these antigens were found only within the interstitial compartment and were commonly associated with clusters of presumptive Leydig cells that were negative for these antigens. Macrophages were first identifiable in the testis at Day 19 of fetal development. The number of macrophages/unit area of interstitium increased 15-fold between Day 20 of gestation and Day 47 postpartum. The cross-sectional area of the macrophages increased 1.7-fold between Days 13 and 47 postpartum. These results demonstrate that the number and size of testicular macrophages changes with age, suggesting a role for these cells during important times of testicular development and maturation.