Ultrastructural and metabolic changes associated with reproductive tract atrophy and adiposity in diabetic female mice.код для вставкиСкачать
THE ANATOMICAL RECORD 216:359-366 (1986) Ultrastructural and Metabolic Changes Associated With Reproductive Tract Atrophy and Adiposity in Diabetic Female Mice DAVID R. GARRIS, R. LEE WEST, AND PHILLIP H. PEKALA East Carolina University, School of Medicine, Greenville, NC 27834 ABSTRACT The effect of progressive, diabetes-associated adiposity on reproductive tract structure and function was examined in 4- to 16-week-old C57BL/KsJ, control (+/?) and diabetic (db/db) mice. Uterine and ovarian tissues were analyzed by transmission electron microscopy for ultrastructural changes associated with increased intracellular lipid accumulation. In addition, the same tissues were analyzed for changes in activity of tissue lipoprotein lipase, an enzyme that hydrolyzes lipoprotein-associatedtriacylglycerols and supports the cellular uptake and storage of free fatty acids. Between 8 and 16 weeks of age, intracellular lipid deposits increased dramatically in the ovarian granulosa, thecal and stromal cell populations, as well as in the uterine epithelium, of diabetic mice compared to controls. By 16 weeks of age, the lipid deposits essentially occupied the entire cytoplasmic area of both the ovarian and uterine cell types in diabetics. The basal lamina underlying the uterine epithelium was expanded in the diabetics relative to controls, and the hyperglycemic condition induced an observable increase in endometrial intercellular space that was occupied by a hyaline type of ground substance of unknown composition and origin. In association with these structural changes, both ovarian and uterine lipase activities were greatly increased in the dbldb mice compared with controls. These data suggest that the structural adiposity and functional decline in reproductive tract condition of the dbldb mutants are related to the enhanced cellular lipid deposition observed in this species. These changes in structural and metabolic parameters are related to the reproductive incompetence characteristic of this murine model. A decline in female reproductive performance is a recognized consequence of the diabetes syndrome in many mammalian species, including humans (Shipley and Danley, 1947; Lawrence and Cantopoulos, 1960; Chieri et al., 1969; Williams and Porte, 1974). In association with both type I (i.e., insulin-dependent) and type 11 (i.e., non-insulin-dependent) diabetic states, changes in the structure, metabolism, responsiveness, and function of the female reproductive tract tissues have been described (Garris et al., 1982, 1984a, 1985a; Garris and Smith, 1983; Johnson and Sidman, 1979; Vomachka and Johnson, 1982). Depending on the severity of the diabetic condition, anovulation (Shipley and Danley, 1947; Kirchick et al., 1978), altered cyclic patterns (Chieri et al., 1969),depressed follicular recruitment (Garris et al., 1985b), insensitivity to exogenous gonadotrophin therapy (Kirchick et al., 1978; Vomachka and Johnson, 1982), depressed ovarian steroidogenesis (Vomachka and Johnson, 1982; Garris et al., 1982, 198513))incomplete luteal development and function (Garris et al., 1984a; Garris, 1984), as well as ovarian atrophy (Davis et al., 1947; Lawrence and Cantopoulos, 1960; Garris, 1984)occur in association with the hyperglycemic state. In addition, changes in uterine sensitivity to gonadal steroids (Garris, 1985), structural organization of the epithelial and stromal layers (Garris, 1985; Garris et al., 1984b; Garris 0 1986 ALAN R. LISS, INC. and Smith, 1983),tissue carbohydrate metabolism (Garris et al., 1985a; Swigart et al., 196l), and responsivity to stimulation (Kirkland et al., 1981) characterize the condition of the reproductive tract in diabetics. These changes in utero-ovarian parameters have been associated with the recognized decline in fetal-placental maintenance during pregnancy in diabetic females (Hall and Tillman, 1951; Sinden and Longwell, 1949). In addition, these structural and functional changes in the reproductive tract become more pronounced with the increased duration of the diabetic condition (Garris, 1984; Garris et al., 1984b). The diabetes (db/db) mutation on chromosome 4 in the C57BLKsJ mouse induces a hyperglycemic, hyperinsulinemic and obese murine model that experiences reproductive tract atrophy (Coleman, 1978; Garris et al., 1985~). Recent studies indicate that the diabetes-induced changes in both utero-ovarian structure and function (Garris, 1985; Garris et al., 1985~)result in cellular involution and tissue atrophy associated with a pronounced intracellular adiposity (Garris et al., 198%). Between 8 and 16 weeks of age, the ovarian thecal, stromal, and granulosa cells, as well as uterine epithelium of diabetic mice exhibited a high concentration, Received January 8,1986; accepted April 21,1986. 360 D.R. GARRIS, R.L. WEST. AND P.H. PEKALA compared to controls, of intracellular lipid that was demonstrated by differential staining (Garris, 1985; Garris et aI., 1985~). The lipid accumulation was temporally associated with pronounced tissue atrophy and dysfunction and was suspected to be associated with the infertility problems common to this diabetes mutant. Similar changes in reproductive tract function observed in genetically obese rats have been linked to abnormal cellular lipid synthesis and activity of lipoprotein lipase (Gray and Greenwood, 1983, 1984), an enzyme that metabolizes triacylglycerols and permits the uptake and storage of free fatty acids in nonhepatic tissues. To date, an ultrastructural and metabolic examination of the cellular changes that occur in association with the progressive adiposity and atrophy of the reproductive tract of the db/db mutants has not been described. The present studies were undertaken to correlate the ultrastructural changes that occur in uterine and ovarian cell types with the related alterations in lipoprotein lipase activity and lipid accumulation in these peripheral tissues. MATERIALS AND METHODS Animals Adult, female C57BL/KsJ mice were obtained from the Jackson Laboratory (Bar Harbor, ME). Both control (+/?) and diabetic (db/db) genotypes were match-paired at 4,8, and 16 weeks of age. All mice were housed under controlled environmental conditions (23-25 "C) with an established photoperiod of 12 hr light per day (lights on: 0600 hr). Body weights and blood glucose concentrations (Technicon Autoanalyzer method) were determined at the time of sacrifice for all mice. All db/db mice that were obese andor hyperglycemic relative to age-matched controls by 4 weeks of age were considered diabetic (Table 1). Tissue Collection and Preparation The uteri and ovaries were collected from 8- and 16week-old control and diabetic mice for analysis by transmission electron microscopy (TEM). As previously described (Garris et al., 1982, 1984b), mice were anesthetized with sodium pentobarbitol and perfused with 50 ml of physiological saline (by intracardiac puncture), which was followed by 100 ml of Karnovsky's fixative solution. The tissues were cleaned, blotted, blocked, and embedded in plastic by conventional techniques. All tissues were subsequently sectioned and stained with osmium tetroxide prior to examination by TEM. All uterine specimens examined were from the midcornua zone. Ovarian cell changes were analyzed from specimens containing secondary follicles that exhibited the structural characteristics of viable follicles, as previously described (Garris et al., 1985~). Tissue Lipoprotein Lipase Assay Groups of mice corresponding to those used for TEM analysis were sacrificed by cervical dislocation and the ovaries, uterus, pancreas, and liver were removed and weighed. Each tissue was immediately rinsed in sterile Dulbecco's modified Eagles (DME) medium containing 2% bovine serum albumin, then blotted on sterile filter paper. The tissues were subsequently minced and placed into sterile polypropylene culture tubes containing 1mi of DME medium supplemented with 2% bovine serum albumin and 10 U of heparin. Tubes were then capped and incubated a t 37°C in a 90%02110%C02 atmosphere for 2 hr with gentle shaking. At the end of the incubation period, each tube was centrifuged (3,OOOg/ 30 min) and the supernatant was collected and used as the source of the tissue lipoprotein lipase in the assays. Assays for lipoprotein lipase were performed in triplicate pools within 30 min after the preparation of the tissue lipase samples according to the method of NilssonEhle and Schotz (1976). In brief, 75 p1 of the isolated enzyme preparation was mixed with 25 p1 of a substrate that consisted of 22.6 mM (3H)-triolein (1.4 pCdpmol), lecithin (2.5 mg/ml), bovine serum albumin (40mg/ml), 33% (vol/vol)human serum and 33%(vol/vol)glycerol in 0.27 M Tris-HC1(pH: 8.1) buffer and incubated at 37°C for 90 min. The reactions were subsequently terminated and the free fatty acids (FFA) were separated from the incubation mixture by liquid-liquid partition extraction as follows. To each tube, 3.25 ml of a methanol : chloroform : heptane (1.41 : 1.25 : 1vollvol) mixture was added, followed by 1.05 ml of a 0.1 M potassium carbonateborate buffer (pH 10.5). The combined solution was vigorously mixed and centrifuged at 3,OOOg for 15 min at 23°C. An aliquot (300 pl) was obtained from the methanol-water upper phase, which contained the free fatty acids. Radioactivity present in the free fatty acid fraction was determined by liquid scintillation analysis. One milliunit of enzyme activity was defined as the release of 1 nmol of fatty acid per 1 min, and all values were then expressed as nanomoles FFN1 m i d l gm tissue. The activity in all cases was inhibited by either the addition of 1 M NaCl or by the omission of serum from the assay. All values were expressed as group means (+SEMI with intergroup differences (P < 0.05) determined by the Student t test for each tissue group analyzed. RESULTS and blood glucose concentrations in C57BLMsJ mice The uterine epithelium of 8- to 16-week-old control Body weight Blood glucose mice appeared as a pseudostratified-to-simplecolumnar Age cell type with prominent, indented nuclei forming a (mgldl) (weeks) Group N (gm) well-organized nuclear zone (Fig. 1A).The apical borders 4 (+I?) 5 17.2 + 0.1 159.6 + 1.3 (db/db) 5 22.8 + 0.2* 148.3 + 3.1 6 18.7 + 0.4 90.6 + 4.7 8 (+I?) 4 38.0 + 0.4* 406.8 + 24.1* (dbldb) 4 24.0 + 0.7 90.3 + 9.6 Fig. 1. Representative photomicrographs ( X 2,100) of the uterine 16 (+I?) 502.2 + 13.2* epithelium in 8- to 16-week controls (A) and in 8-week-old(B) and 1650.8 + 0.7* (db/db) 5 TABLE 1. Age- and diabetes-related changes in body weight week-old (C) diabetic mice. Lipid deposits (L)were visible in all groups, All values are represented 88 group means (+SEMI for control (+/?) the most abundant concentrations being apparent in the diabetic mice. and diabetic (db/db) C57BL/KsJ mice. Significant differences between The basal lamina (BL) in the diabetic mice was thickened and the genotypes of each age group are denoted by an asterisk 8 < 0.05). basal pseudopodia emanating from the inferior epithelial surface were obvious. DIABETES AND REPRODUCTION 361 Fig. 2. Photomicrographs of the basal pole of control (A) ( X 7,000) diabetic 03) ( X 10,500) uterine epithelium demonstrating the high concentrations of lipid (L)deposited in this zone in 8-week-olddiabetic mice. The basal lamina (BL) underlying the epithelial layer surrounded the basal pole pseudopodia, which were common in the cells of diabetic mice. Periluminal stromal cells (S) remained in close apposition to the basal lamina. The nuclei (N) of the epithelial cells were prominent in all control and diabetic specimens. 363 DIABETES AND REPRODUCTION presented a thick, microvillous surface, whereas the basal membrane surface appeared smooth and adherent to the underlying basal lamina (Fig. 2A). A small amount of intracellular lipid was noted in the basal zone of the epithelial cells from typical 16-week-old control mice (Fig. 1A). In contrast, the uterine epithelium of both 8-week-old (Fig. 1B) and 16-week-old(Fig. 1C) diabetic mice exhibited very prominent, and expanded, lipid accumulations in all of the samples examined. In addition, lipid droplets were observed scattered throughout the cytoplasm of the epithelial cells from diabetic mice, the increased cellular lipid content being most prominent in the 16-week-oldmice. The basal lamina supporting the epithelial layer in diabetics was expanded (Fig. 2B) relative to controls. Basal membrane evaginations from the epithelial cells of the diabetic mice served to anchor the cellular layer to the endometrial basal lamina (Fig. 2B). In control mice, the ovarian granulosa layers were composed of ovoid cells with large, prominent nuclei and a mitochondria-rich cytoplasm (Fig. 3A). In contrast, the granulosa cells in the cumulus zone of 16-week-olddiabetic mice (Fig. 3B) contained an enormous amount of lipid, which occupied nearly the entire intracellular area of most cells. In cells where nuclei could be identified (Fig. 3B), the nuclear membranes appeared indented and shrunken relative to controls. In most specimens, little structural detail of the intracellular organization of the granulosa cells could be determined owing to the heavy lipid deposits present in the cells of diabetic mice. In contrast t o the ordered layers of thecal and stromal cells surrounding the granulosa layers of control ovaries (Fig. 4A), the stromal cells of diabetic mice appeared t o be undergoing degenerative changes between 8 and 16 weeks of age. The nuclei of the stromal cells were less prominent and smaller in the diabetics than in controls (Fig. 4B). In addition, there was an obvious lack of mitotic activity in the cells examined and a dispersed intranuclear chromatin pattern. By 16 weeks of age (Fig. 4C), the orderly array of periluminal stromal cells observed in control specimens was absent in the diabetic tissue samples. The cytoplasmic membranes of the dbl db stromal cells were indented and degenerative in appearance compared t o the smooth-surfaced features of controls (Fig. 4A). There was an apparent increase in the intercellular space between stromal cells in the diabetic endometrium that was occupied by a hyaline type of ground substance of unknown composition or origin Fig. 4C). No evidence of functional, intracellular organelles was observed in the periluminal stromal cells of 16-week-olddiabetic mice. Between 8 and 16 weeks of age, a dramatic increase in lipoprotein lipase activity was observed in the uteri and ovaries of diabetic mice relative to controls (Table 2). By 8 weeks of age, the lipase activity was approximately 1.5-5 times greater in the tissues collected from diabetic mice than in those from controls. By 16 weeks of age, these changes became even more pronounced as both the uteri and ovaries of the diabetics became infiltrated with lipid deposits. The continued increase in lipase activity in the ovarian and uterine tissues of the diabetic mice correlated temporally with the progressive increase in intracellular lipid deposits observed in the corresponding tissues examined by TEM. DISCUSSION The results of the present studies indicate that the diabetes-related decline in reproductive tract function in dbldb mice is associated with an increased intracellular lipid deposition in uterine epithelial cells and in ovarian granulosa, thecal, and stromal cells. The observed elevation in lipoid deposition was temporally related to the dramatic increase in tissue lipoprotein lipase activity in the diabetic mice compared with controls. These changes in cellular structure and metabolism were progressive with respect to the age of the dbldb mice, the 16-weekold groups exhibiting exaggerated cellular atrophy compared to the 8-week-oldmice. By 16 weeks of age, all of the ovarian cell types in the dbldb mice were completely infiltrated with lipid, a condition that we term the “fatty follicle syndrome” in this murine model. The condition apparently underlies the functional decline in ovarian activity that is characterized by an increased follicular atrophy, depressed luteal function, declining steroidogenic activity, and general structural degeneration of all cell types in this mutant (Garris et al., 1985~). Similar structural and metabolic changes in reproductive tract activity are common in other obese and diabetic rodent models (Saiduddin et al., 1973; Swerdloff et al., 1976; Chieri et al., 1969; Garris et al., 1982, 1984a; Garris, 19841, suggesting that the functional impairments in utero-ovarian activity in these species may have a common cellular deficit or incompetence for adjusting to the changes in systemic lipid and carbohydrate metabolism that characterize these models. The changes noted in tissue lipase activity in the present study support the reported presence of lipoprotein lipase activity in the uterus of obese Zucker rats (Gray and Greenwood, 1984) and the relationship between altered enzyme activity and reproductive problems in these animals. In both species, tissue lipase activities were altered in the reproductive tract tissues of these genetic mutants compared to controls. Estrogens have been demonstrated to modulate cell lipase activity (Gray and Greenwood, 1984)and in consequence possibly influence the rate of triglyceride uptake by gonadal steroidsensitive cell types in the ovary and uterus. The depressed circulating estradiol levels in diabetic mice (Garris et al., 1985~) may account for the elevated lipase activity noted in the present studies, since estradiol depresses lipase activity in the uterus of obese subjects (Gray and Greenwood, 1984). Recent evidence suggests that estrogens and related metabolites are also capable of modulating circulating glucose levels and related tissue parameters in the dbldb mouse and in the obese Zucker rat (Coleman et al., 1982; Gansler et al., 1985). Thus, the corrective effects induced by estradiol with respect to cellular metabolism may occur through the modulation of lipid catabolism, as well as carbohydrate utilization. Alterations in pancreatic (Coleman, 19781, renal (Like et al., 1972), CNS (Garris et al., 1985a,b), peripheral nerve (Hanker et al., 1980),and hepatic (Coleman, 1982) structure and function are recognized to occur in association with the diabetes syndrome in this murine model. The changes are influenced by the duration of the hyperglycemic-hyperinsulinemic condition that characterizes the expression of the genetic mutation in this murine 364 D.R. GARRIS, R.L. WEST, AND P.H. PEKALA Fig. 3.Photomicrographs ( x 3,812) of the granulosa cell layers of antral follicles from control (A) and diabetic (B) mice between 8 and 16 weeks of age. All cells from control mice exhibited distinct nuclei (N) and a mitochondria-rich cytoplasm. In contrast, the lipid deposits (L) present in the granulosa cells of diabetic mice essentially obliterated the cytoplasmic space. The nuclei present in the granulosa cells of diabetic mice appeared degenerative. 365 DIABETES AND REPRODUCTION model. Changes in the reproductive-neuroendocrineaxis, which modulates ovarian activity, have also been reported for this species (Johnson and Sidman, 1979; Garris et al., 1985b). Hence the abnormal metabolic and endocrine regulation of cellular function in the dbldb mice probably contribute to the reproductive tract involution observed in the present studies. However, preliminary studies (Garris, unpublished observations) suggest that, even after prolonged exposure to the adverse metabolic conditions that characterize this mutant, uterine and ovarian tissues will respond to endocrine replacement therapies, as indicated by normalized structural and metabolic parameters. These data suggest that the therapeutic normalization of the peripheral metabolic disturbances in this murine mutant may also correct or prevent the diabetes-associated depression of female reproductive tract function. The metabolic and structural changes that occur in uterine or ovarian cells of untreated human diabetics are not well understood. Since the availability of insulin for the treatment of diabetes in the mid-l920s, most humans are treated for the overt symptoms of the disease as soon as it is clinically detected. However, Krause (1936)reported that both cellular atrophy and adiposity characterized the ovarian cellular components collected from a small number of untreated, diabetic females. The structural changes described by Krause (1936)resemble those common to the db/db murine mutant, suggesting that this model may be useful in the testing of therapeutic approaches designed to correct the reproductive dysfunction that characterizes both rodent and human diabetics. In summary, the results of the present study indicate that the cellular accumulation of intracellular lipid by TABLE 2. Lipoprotein lipase activity in C57BLMs.J mice: Effects of age and diabetes (db/db) mutation Age (weeks) 8 Grour, (+I?) (db/db) (+/?) (db/db) 16 (+/?) (db/db) (+I?) (db/db) N Tissue Lipase activity (nmoles FFMmidmn) 5 5 5 5 4 5 4 5 Uterus Uterus Ovary Ovary Uterus Uterus Ovary Ovary 5.2 + 0.1 18.8 + 0.2* 14.4 0.1 69.8 4.1* 22.0 + 1.5 111.0 15.0* ND 217.0 + 25.0* + + + All values are expressed as group means (+SEM) for the indicated control (+R) and diabetic (dbidb) mice. All assays were run in triplicate from pooled samples of the indicated number of animals. Diabetesrelated differences in lipase activity are denoted by a n asterisk (p < 0.05) for each tissue relative to age-matched control values (ND: not detectable). Fig. 4. Photomicrographs of control (A) ( X 4,630) and diabetic (B,C) 3,050) ovarian stromal cells ( S )between 8 and 16 weeks of age. The orderly array of cells in the controls was in contrast with the rough- (x ened cytoplasmic membrane borders that characterized the diabetic cells by 8 weeks of age. In 16-week-old diabetics, the cytoplasmic contents and membranes appeared degenerative, with a n increased intercellular (IC)space apparent between cells that was occupied by a hyaline type of ground substance of unknown composition or origin. 366 D.R. GARRIS, R.L. WEST, AND P.H. PEKALA utero-ovarian tissues is temporally related to the increased lipase activity in these tissues of dbfdb mice. meextent of lipid infiltration appears to be cumulative, the aged mice demonstrating more intracellular lipid deposition than either younger diabetics or age-matched controls. mesedata suppofiprevious suggestionsthat utero-ovarian involution in these mutants may be relakd to lipid (Gamis et 1985c), and that the increased intracellular lipids cause the affected cell types to exhibit metabolic activities that are more characteristic Of adipocytes than their (Gmis et 1985a)*It is apparent that these structural and metabolic changes in utero-ovarian cell types contribute to the reproductive incompetence of this mutant murine model. ACKNOWLEDGMENTS The encouragement and support in terms of time, space, and donated by D ~Douglas . L. Coleman Of these studies are greatly appreciduring the ated. The excellent technical assistance provided by S.K. Williams, D. Whitehead, C. 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