Aggressive behaviors in adult SF-1 knockout mice that are not exposed to gonadal steroids during development

Sex hormones are a major factor responsible for the development of sex differences. Steroidogenic factor 1 (SF-1) is a key regulator of gonadal and adrenal development, and SF-1 knockout mice (SF-1 KO) are born without gonads and adrenal glands. Consequently, these mice are not exposed to gonadal sex steroids. SF-1 KO pups die shortly after birth due to adrenal deficiency. In the present study, SF-1 KO mice were rescued by neonatal corticosteroid injections followed by adrenal transplantations on day 7-8 postnatally. Control mice received corticosteroid injections and were gonadectomized prior to puberty. Mice were observed interacting with ovariectomized hormone primed females and gonad-intact males. In the absence of sex steroid replacement, adult SF-1 KO mice were significantly more aggressive than control mice in tests with stimulus females. After testosterone treatment, control males displayed significantly more aggression towards male intruders than control female mice, or male and female SF-1 KO mice, suggesting a developmental role of gonadal hormones in the expression of aggressive behavior and affirming SF-1 KO mice as a behavioral model to investigate affects of fetal gonad deficiency.     

Age-related alterations in the lacrimal gland of adult albino rat: A light and electron microscopic study

BackgroundAge related changes in the lacrimal gland are associated with alterations in the structural organization and functional response in the gland of diverse mammalian species. Dry eye syndrome is one of the most common ocular problems in the world especially in old age. It results when the lacrimal gland fails to secrete proteins and fluid in sufficient quantity or appropriate composition.Aim of the workThe present study is designed to demonstrate the influence of aging on the structure of the lacrimal gland of albino rat and to provide a morphological basis to explain the pathogenesis of the dry eye syndrome with ageing. It also aims to carry out a comparative analysis of age-dependent changes in male and female rats and to address how the lacrimal gland ages in each sex.Material and MethodsEighty albino rats were used in this study. The animals were divided into two age groups, young adult and senile. Tear secretion was measured using a modified Schirmer test. Corneal impression cytology of the anesthetized rats was done. The glands were subjected to gross morphologic examination, microscopic examination using H&E, PAS, Masson's trichrome and Giemsa stains. Electron microscopic examination was done in addition to quantitative histomorphometric estimations included acinar density, ductal count and mast cell count.ResultsLight microscopic examination of the lacimal glands of the senile rats revealed different pathological changes. These included acinar, ductal as well as stromal changes. Electron microscope examination of the lacrimal gland of the senile group showed a decrease in the electron dense secretory vesicles, mitochondrial swelling and lipofuscin-like inclusions were frequently seen in the cytoplasm of acinar cells in senile rats.ConclusionThe structural changes in the lacrimal glands of senile rats were associated with reduction in tear secretion as well as alterations in corneal epithelium. Gender difference in lacrimal gland structure was recorded.     

Immunohistopathology of Sjögren's syndrome

Sj?gren's syndrome (SS) is characterized by keratoconjunctivitis sicca and xerostomia, which occur in an autoimmune lacrimal and salivary gland disease characterized by lymphocyte infiltrates of exocrine glands and/or Sj?gren's syndrome autoantibody production. It has been reported that aquaporin-5 distribution is abnormal in SS, perhaps as a result of paracrine effect of TNF-alpha. Also the neurogenic regulation of the salivary gland is impaired in SS. Apart from functional changes, the syndrome is also characterized by structural abnormalities of the secretory acinar apparatus. The acinar basement membrane is abnormal as it lacks laminin alpha1 chain, which may impair its capability to induce the progenitor cells to differentiate to acinar cells. CRISP-3 and TMPRSS-2 can be used as androgen markers and LIV-1 and Cyr61 as estrogen markers to study the sexual dimorphism of the salivary glands. Patients with SS seem to have low concentrations of dehydroepiandrosterone, which may predispose women and the exocrine glands to this syndrome.     

Encepalomyocarditis virus-induced apoptosis and ultrastructural changes in the lacrimal and parotid glands of mice

Development of acinar cell apoptosis and ultrastructural changes in the exorbital lacrimal and parotid glands was examined in DBA/2 mice infected with 10(2) PFU/mouse of EMC-D virus. Pyknotic acinar cells, most of which were positive for TUNEL and cleaved caspase-3 and had ultrastructural characteristics of apoptotic cells, developed earlier and were more frequently observed in the parotid gland than in the exorbital lacrimal gland, while the total damage of acinar cells and interstitial infiltration of macrophages were more prominent in the latter than in the former. These findings indicate that EMC-D virus induces acinar cell apoptosis in these glands. In addition, corresponding to the results of the detection of viral RNA signals by in situ hybridization, small aggregates of virus-like particles having typical size and structure of EMC virus were frequently observed in both the cytoplasm and the nucleus of acinar cells in the exorbital lacrimal gland, while they were found only in the cytoplasm of a few acinar cells in the parotid gland. In conclusion, between the exorbital lacrimal and parotid glands, there was a reverse relationship observed between the development of acinar cell apoptosis and that of total damage of acinar cells.     

Radiation-induced damage to lacrimal glands: an ultrastructural study in Sprague Dawley rats

Injury to lacrimal glands represents a major health problem after radiation therapy of the head and neck malignancies. Accordingly, this study aimed to investigate significant ultrastructural changes of lacrimal glands and some of their underlying mechanisms following the exposure to different fractionated doses of irradiation. In this study, 28 Sprague Dawley (SD) rats were assigned to four groups (seven rats each): Group I acted as control and received no irradiation. Groups II–IV received fractionated irradiation of 5 Gy (100 cGy/fraction daily for 5 days), 9 Gy (300 cGy/fraction daily for 3 days), and 20 Gy (one fraction), respectively. One month after the experiment, examination of lacrimal glands with transmission electron microscopy (TEM) demonstrated dose-dependent ultrastructural changes in the lacrimal acinar and intralobular ductal epithelial cells. In the acinar cells, there were swollen rough endoplasmic reticulum, irregularly shaped nuclei with chromatin condensation, mitochondrial damage, and retention of secretory granules. Intaralobular ductal epithelial cells showed loss of surface microvilli and damage to mitochondria. In addition to the potential direct effects of irradiation on lacrimal acinar and intralobular ductal epithelial cells, damage to blood vessels and nerve endings seemed to mediate some of the underlying mechanisms of these irradiation-induced ultrastructural changes. In conclusion, using TEM reveals that lacrimal gland is highly sensitive to even small doses of irradiation therapy; in addition, swelling of rough endoplasmic reticulum and aberrant nuclei are the most encountered structural changes. Damage to blood vessels and nerve endings might mediate some of the underlying mechanisms of irradiation-induced secondary injury in lacrimal glands.     

Influence of hyperprolactinemia on collagen fibers in the lacrimal gland of female mice

OBJECTIVE:

To quantify the collagen fibers in the lacrimal gland of female mice with hyperprolactinemia.

METHODS:

Forty adult female mice were randomly divided into two groups with 20 animals each: nonpregnant control (CTR1, control group, 0.2 mL of saline solution) and nonpregnant experimental (HPRL1, experimental group, 200 µg/day metoclopramide). Treatments lasted for 50 consecutive days. On day 50, 10 females from each group (control and experimental) were euthanized in the proestrus phase; then, the blood was collected and the lacrimal glands were removed. Thereafter, the remaining females were placed with the mates and continued to receive treatment with saline solution or metoclopramide. On the 6th post-coital day, 10 pregnant females from the control group (CTR2) and 10 pregnant females from the experimental group (HPRL2) were euthanized, after which blood was collected and the lacrimal glands removed. The lacrimal glands were processed for morphological analyses and collagen quantification, and prolactin and sex steroid levels were measured in the blood samples. Data were statistically analyzed using an unpaired Student t test (p<0.05).

RESULTS:

Morphological analysis revealed greater structural tissue disorganization of the lacrimal glands in the metoclopramide-treated groups. The total collagen content was significantly higher in the HPRL1 group than in the CTR1 group (p<0.05), whereas the difference between the CTR2 and HPRL2 groups was not significant.

CONCLUSION:

Our data suggest an impairment in the functioning of the lacrimal gland as a consequence of increased prolactin levels and decreased serum levels of estrogen and progesterone.     

Age-related dysfunction of the lacrimal gland and oxidative stress: evidence from the Cu,Zn-superoxide dismutase-1 (Sod1) knockout mice

An imbalance between free radical generation and radical scavenging antioxidant systems results in oxidative stress, which has been associated with cell injury observed in many age-related diseases. The superoxide dismutase (SOD) family is a major antioxidant system, and deficiency of Cu,Zn-superoxide dismutase-1 (Sod1) in mice leads to many different phenotypes that resemble accelerated aging. In this study we examined the morphologic features and the secretory functions of the lacrimal glands in Sod1(-/-) mice. Lacrimal glands showed atrophy of acinar units; fibrosis; infiltration with CD4(+) T cells, monocytes, and neutrophils; increased staining with both 4-hydroxy-2-nonenal and 8-hydroxy-2'-deoxyguanosine; increases in apoptotic cells; and the presence of the epithelial-mesenchymal transition in senescent Sod1(-/-) mice. Electron microscopy findings revealed evidence of epithelial-mesenchymal transition, presence of swollen and degenerated mitochondria, and the presence of apoptotic cell death in the lacrimal glands of senescent Sod1(-/-) mice. These alterations were also associated with the accumulation of secretory vesicles in acinar epithelial cells, decreased production of both stimulated and nonstimulated tears, and a decline in total protein secretion from the lacrimal glands. Our results suggest that Sod1(-/-) mice may be a good model system in which to study the mechanism of reactive oxygen species-mediated lacrimal gland alterations.     

Chronic changes in male rats' hormone-sensitive systems after suprathreshold pulses of testosterone

Adult male rats were castrated and maintained on daily SC injections of a threshold amount (200 micrograms) of testosterone propionate (TP). To mimic naturally occurring pulses of suprathreshold testicular hormones in intact males, animals in the experimental groups also received either one (single TP) or five (multiple TP) injections of 800 micrograms TP over 12 days. The rats were examined on the following day (acute) or 15 days later (chronic) for changes in hormone-sensitive behavior, physiology, and morphology. The hypothesis tested was that the hormonal pulses function to provoke chronic changes in substrates underlying the reproductive system. The results were that multiple doses of suprathreshold TP provoked acute modifications in aggressive behavior, sex accessory glands, and glans penis integrity. Chronic changes were observed in sex accessory gland functioning and penile morphology, particularly in the size of penile papillae. A single exposure to suprathreshold TP was considerably less effective, though there was some evidence of acute changes in sex accessory glands and chronic changes in penile papillae. There was substantial variation in the responses of individual animals, particularly the chronic responses. The data were interpreted as supporting the hypothesis.     

Immunolocalization of carbonic anhydrase isozymes in rat and mouse salivary and exorbital lacrimal glands

Carbonic anhydrase (CA) isozyme I and isozyme II have been localized with the immunoperoxidase bridge method in cells of mouse and rat salivary glands and exorbital lacrimal glands. Immunostaining proved optimal in Carnoy fixed specimens for some sites and in Bouin fixed glands for other sites. Staining in mouse largely resembled that in rat glands, but minor species differences were observed. Serous acinar cells in the submandibular gland stained uniformly and exclusively for CA I. From 50 to 100% of the serous acinar cells in the parotid glands evidenced content of both CA I and CA II. A minor population of serous acinar cells in the mouse exorbital lacrimal gland stained for CA I and CA II, but these glands in the rat failed to stain. Immunostaining was observed in ducts in Bouin fixed glands. Some cells in striated ducts of submandibular and sublingual glands stained for CA I and CA II and other cells in these ducts were negative. Such cellular heterogeneity was also observed in excretory ducts of submandibular and sublingual glands. These findings thus demonstrate the presence of CA in two morphologically and functionally diverse cell populations in rodent salivary glands. Immunolocalization of the CA isozymes in serous acinar cells and intercalated duct cells, presumably packaged in secretory granules, implies a role for this enzyme in salivary secretions whereas localization of CA in striated and excretory ducts suggests their traditional function in fluid and electrolyte transport.     

Androgen control of autoimmune expression in lacrimal glands of MRL/Mp-lpr/lpr mice

The purpose of the present study was to determine, by utilizing an animal model of Sj?gren's syndrome, whether androgen therapy might ameliorate autoimmune sequelae in the lacrimal gland. Age-matched female MRL/Mp-lpr/lpr mice were administered subcutaneous implants of placebo- or testosterone-containing pellets after the onset of disease. Lacrimal glands and, for comparison, submandibular glands were collected from sacrificed mice immediately prior to androgen administration and following 17 and 34 days of maintained hormone exposure. Tissues were processed for light microscopy and examined with a computer-assisted image analysis system. Results demonstrated that testosterone exposure dramatically reduced lymphocyte infiltration in lacrimal tissue: following 34 days of treatment, the percentage infiltrate had undergone a 12-fold decrease. This hormone action, which was time dependent, involved significant abrogations in both infiltrate size and number. Testosterone administration also induced a significant 2- to 3-fold rise in lacrimal gland weight and acinar area and a 2-fold reduction in acinar density/field, compared to values in placebo-treated controls. In addition, androgen administration significantly decreased the magnitude of lymphocyte infiltration in submandibular glands. Overall, our findings demonstrate that androgen therapy may reverse autoimmune sequelae in lacrimal, as well as submandibular, glands in a mouse model of Sj?gren's syndrome.     

人泪腺上皮细胞的体外培养及鉴定

目的　探讨人泪腺上皮细胞原代体外培养、鉴定、冻存和复苏的方法。方法　应用组织块培养法和混合消化液培养法对健康猝死成年人泪腺上皮细胞进行体外原代培养 ,应用免疫组织化学染色方法对培养有第 2代上皮细胞进行Pan keratin蛋白染色 ,以进行鉴定 ,取第 2、4代细胞进行冻存 ,1个月后取出复苏。结果　组织块培养法和混合消化液培养法均可获得较纯的泪腺上皮细胞 ,以组织块培养法接种的细胞早期生长较快 ,但第一代以后两种方法所获得的细胞生长无明显差别。两种方法所获取的泪腺上皮均为贴壁生长 ,未能形成生理状态下泪腺的囊腔结构 ,亦未见分泌小泡的形成。培养的泪腺上皮免疫组化染色Pan keratin蛋白染色阳性。经冻存和复苏 ,细胞保持良好活性。结论　组织块培养法和混合消化液培养法均可获得较纯的泪腺上皮细胞 ,但不能保持其泪腺束腔结构     

Gender and Androgen Treatment Influence the Expression of Proto-oncogenes and Apoptotic Factors in Lacrimal and Salivary Tissues of MRL/lprMice

The objectives of this study were to (1) determine whether Fas antigen, Fas ligand, p53, and proto-oncogene mRNAs may be detected in lacrimal and submandibular glands of the MRL/lprmouse model of Sjögren's syndrome, and (2) examine whether gender and androgen or cyclophosphamide therapy influence the mRNA expression of these apoptotic factors. Tissues were obtained from treated or untreated MRL/lprmice after the onset of disease and processed for the analysis of mRNAs by RT-PCR and Southern blot hybridization. Our results demonstrated that (1) Fas antigen (exons 1 → 2 or 3 → 7+), Fas ligand, c-myb, c-myc, bcl-2, Bax, p53, and androgen receptor (AR) mRNAs are present in exocrine tissues of MRL/lprmice; (2) the amounts of c-myb, c-myc, bcl-2, p53, and AR mRNA are higher (P< 0.05) and the level of Fas antigen (exons 1 → 2) mRNA is lower (P< 0.05) in lacrimal glands of female compared to male mice. In contrast, the content of c-myb and p53 mRNA is greater (P< 0.05) in submandibular tissues of female relative to those of male mice; and (3) testosterone or cyclophosphamide treatment led to a significant (P< 0.05) decline in the mRNA levels of c-myb, bcl-2, and/or AR, but an increase (P< 0.05) in the mRNA amount of Bax, in lacrimal, but not in salivary, glands of female mice. These findings demonstrate that gender-associated differences exist in the expression of apoptotic factor mRNAs in exocrine tissues of autoimmune mice and that some of these differences appear to be due to the influence of androgens.     

Selective down-regulation of aquaporin-1 in salivary glands in primary Sjögren's syndrome

Salivary and lacrimal gland secretions are reduced in primary Sj?gren's syndrome (pSS). Aquaporins (AQPs) are involved in transmembrane water transport, and different isoforms show specific cellular and subcellular distributions in salivary and lacrimal glands. Changes in expression of AQP molecules have therefore been suggested to contribute to the glandular dysfunction in pSS. AQP-5 is present in the apical membrane of acinar cells, where it mediates fluid outflow; however, we have recently shown that its expression is not altered in pSS. We therefore studied whether expression of other isoforms of AQP would be altered in pSS. Using high-resolution confocal microscopy, we determined the distribution of AQP-1 and AQP-3 in labial salivary gland biopsies from 11 patients with pSS and 9 healthy controls. AQP-1 is present in myoepithelial cells surrounding acini, and its expression in these cells was decreased by 38% in pSS glands. By contrast, expression of AQP-1 in endothelial cells of nonfenestrated capillaries was not altered in pSS. AQP-3 was expressed in the basolateral membrane of acinar epithelial cells, and its expression was not altered in disease. We therefore conclude that AQP-1 expression in myoepithelial cells is selectively down-regulated in pSS and that myoepithelial cell dysfunction may play a crucial role in the pathology of this disease.     

Harderian gland dependency of immunoglobulin A production in the lacrimal fluid of chicken

Involvement of the Harderian gland (HG) in the production of lacrimal immunoglobulin (especially IgA) was investigated. The lacrimal concentration of each immunoglobulin class was not affected by surgical bursectomy but was reduced by cyclophosphamide (CY) and testosterone (TP) treatments. Surgical removal of the Harderian gland caused a remarkable reduction of both the lacrimal concentration of each immunoglobulin class and the specific antibody titre, and and IgA was almost undetectable. The lacrimal concentration of each immunoglobulin class, as well as the specific antibody titre, was not affected by surgical removal of the Lacrimal gland (LG). The route of antigen administration produced no difference in the class of lacrimal immunoglobulin produced. The results indicate that the production of immunoglobulin in chicken tears may be dependent on the HG and that lacrimal immunoglobulin may be synthesized and secreted locally in the HG. Lymphocytes of the HG are of bursa of Fabricius origin and are seeded into the HG prior to hatching and its lymphocytes do not appear to be involved in systemic immunity.     

Proliferative and structural differences between male and female mouse submandibular glands

Sexual dimorphism has been observed in salivary glands of many species. In this study, evidence for sexual differences in adult mouse submandibular gland is extended beyond parenchymal cell composition, size, and volumes to include patterns of DNA synthesis and complexity of ductal branching. Computer-assisted three-dimensional reconstructions also revealed differences in overall organization of secretory complexes. Consistent with observations by others, granular intercalated duct cells were absent, while striated granular duct cells were low in proportion in the male glands relative to female glands. When the mean of average cell volumes were compared, acinar (AC) cells were smaller than granular duct (GD) cells in the male, but in the female the reverse was true. Furthermore, in addition to differences in average volumes of GD cells, the average volume of AC cells was significantly greater in females than males. The most dramatic evidence for sexual dimorphism was observed following a 90-min labeling with ³H-thymidine. Though all cell types showed DNA replication activity, the intercalated duct (ID) cells were substantially more active than AC and GD cells in the female, while in the maléeA the GD cells, ID cells, and AC cells all showed approximately equal activity. Three-dimensional reconstructions indicated that the female possessed a more highly branched intercalated duct system and that the GD usually terminated within a secretory complex, whereas in males the GD typically passes through a secretory complex and forms a prominent cap-like structure on the opposite side. © 1993 Wiley-Liss, Inc.     

Comparative analysis of senescent exorbital lacrimal glands in male and female albino rats

In our previous study we described a bilateral-macroscopic and structural dimorphism of young rat exorbital lacrimal gland (Loewenthal's gland), which was the probable cause of the bibliographic discrepancies in the entity and the onset of its sexual dimorphism. Relevant literature also reported sex-dependent alterations in gland structure during senescence. The present study aims to carry out a comparative analysis on age-dependent changes in glands of both sides from male and female rats, using histological, histochemical and transmission electron microscopy, to evaluate whether the gland bilateral-macroscopic and structural dimorphism might influence the kind of alterations which occur in senescence. Our findings indicate that the macroscopic and structural side-specific dimorphism is not so evident in comparison with young rats. The side-specific dimorphism is evident only in male rats, in which the roundish gland appears to be more Sudan-positive in comparison with the ellipsoidal gland. The gland bilateral-macroscopic and structural dimorphism, although more evident in comparison with young animals, does not seem to influence these kinds of alterations due to senescence, a time-window in which we still observed some sexual differences also in more aged rats.     

An alternative perspective to the immune response in autoimmune exocrinopathy: induction of functional quiescence rather than destructive autoaggression

Sjögren's syndrome is characterized by dryness of the eyes and the mouth due to mononuclear cell infiltration of the lacrimal and salivary glands. The aetiology is unknown but autoimmunity is considered to play a significant role in the pathogenesis. Recent studies have focused on the fact that tear and salivary flow involves an entire functional system that includes the mucosal surfaces with adnexes (the site of inflammation), efferent nerve signals sent to the midbrain (lacrimal and salivary response region), and afferent neural signals from the brain to the acinar/ductal epithelial structures in the gland. Mononuclear cell infiltration in exocrine glands can lead to glandular destruction, suggested to be mediated through apoptosis. However, the functional impairment of exocrine glands could be regulated by cytokines and/or antibodies against the muscarinic M3 receptor by inhibiting the neural stimulation of the residual glands. This review discusses the possibility that the pathogenesis of Sjögren's syndrome comprises aberrant immune-mediated neuro-hormonal events.     

Novel animal models for Sjögren's syndrome: expression and transfer of salivary gland dysfunction from regulatory T cell-deficient mice

IL-2 knockout (KO), IL-2Ralpha KO and scurfy mice lack the CD4+CD25+ regulatory T (Treg) cells and develop severe inflammation in multiple organs, although organs affected vary among these strains. We asked if salivary and lacrimal glands, the main organs affected in Sj?gren's syndrome, are targeted in these strains. Severe lymphocyte and neutrophil infiltration in the salivary and lacrimal glands and a decrease in salivary secretory function were observed in IL-2 KO and IL-2Ralpha KO mice, but not in scurfy mice. Interestingly, transfer of lymph node cells from scurfy mice to RAG-1 KO recipients rapidly and effectively induced inflammation and loss of function in the salivary glands. Furthermore, we observed that daily LPS feeding in scurfy mice also induced inflammation in the salivary glands. Our study demonstrates several novel models for Sj?gren's syndrome, including an adoptive transfer model that shows that scurfy mice have dormant salivary gland-specific autoreactive lymphocytes that can be activated by certain environmental factors, such as those present in RAG-1 KO mice.