Ultrastructural study of the ventral lobe ofthe prostate of mice with streptozotocininduced diabetes (C57BL/6J)

V. H. A. Cagnon,1 A. M. Camargo,1 R. M. Rosa,1 R. Fabiani,1

C. R. Padovani,2 F. E. Martinez2

Keywords : diabetes, ultrastructural, prostate

Abstract Morphological and functional changes caused by diabetes in the accessory sex organs and especiallythe prostate have been reported by several investigators. The aim of the present study was to examine thepossible deleterious effects of experimentally induced diabetes on the secretory epithelium of the ventral prostateof mice. Sixteen adult male C57BL/6J mice were divided into two groups. The diabetic group received a strepto-zotocin injection of 75 mg/kg, while the control group received only 0.1 ml citrate buffer, i.p. After 30 days, thediabetic state was ascertained, the animals were sacrificed and the ventral lobe of the prostate was collected forhistological and ultrastructural examination. The results showed reduction in glandular epithelium cell height,increased numbers of cytoplasmic vacuoles and thickening of the extracellular matrix. In conclusion, experimentaldiabetes has harmful effects on the secretory epithelial cells of the ventral lobe of the prostate of mice. © 2000Harcourt Publishers Ltd

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Introduction

Diabetes mellitus is a universal disease, with approxima35 000 new cases diagnosed per year in the USA (Robet al., 1989), corresponding to 1–2% of the adult populaDiabetes mellitus is a chronic disorder that affects metabolism of proteins, carbohydrates and lipids. The mcharacteristic of diabetes is hyperglycemia, a reflex ofdeficient utilization of carbohydrates as a result of abnormal secretion of insulin or of its abnormal action

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1Department of Anatomy, Institute of Biology, State University of Campinas,Campinas-SP, Brazil. (E-mail-quitete@uol.com.br)2Department of Anatomy, Institute of Bioscience, São Paulo State University,Botucatu-SP, Brazil

Received 14 October, 1999Accepted 30 May, 2000

Correspondence to: V. H. Cagnon, Department of Anatomy/Institute ofBiology, State University of Campinas, Campinas-SP, Brazil, 13083-970, P.O Box 6109, Brazil

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tissues (Robbins et al., 1989). The viability of experimenmodels for the induction of diabetes has permitted the invtigation of various dysfunctions caused by the diabetic sin different organs composing the genitourinary tra(Latifpour et al., 1991). In addition to classical pancreattomy, several diabetogenic agents are being used forinduction of diabetes, such as streptozotocin and allo(Robbins et al., 1989). These diabetogenic agents lead todestruction of pancreatic beta cells which produce insuAdequate doses of these agents produce animals insulin deficiency similar to that of human diabetes type(Carson, 1979).

Several investigators have reported changes in the mreproductive system caused by the diabetic state bothmen and in laboratory animals submitted to experimeninduction of diabetes (Faerman, et al., 1972; Kolodny, et 1974; Ficher, et al., 1984; Ho, 1991). The decreasefertility is accompanied by changes in the testicles aaccessory sex glands, as determined by different meth

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276 CAGNON ET AL.

Table 1 Mean, standard deviation and result of the statistical analysis ofthe height (µm) of the secretory epithelium of the ventral lobe of theprostate for comparison of the groups studied.

GROUPS TEST RESULTS “P” VALUE

CONTROL DIABETIC10.52 ± 1.28 5.57 ± 0.52 10.13 P<0.0001

(Hunt & Bailey, 1961; Hellamn et al., 1963). According Paz et al., 1978, experiments on male rats using strepttocin as a diabetogenic agent demonstrated morphologand functional changes in the testicles and in the accessex glands. These investigators observed a reductiotestosterone levels in blood, as well as atrophy of the setory epithelium of the ventral lobe of the prostate, of coagulation gland and of the seminal vesicle. Gonods Bevier, 1995 observed moderate to severe alteration inspermatogenesis of non-obese diabetic mouse after inadministration during initial appearance of diabechanges. The animals with diabetes that did not recinsulin demonstrated extensive spermatogenic disruptio

The prostate is androgen dependent and plays a fumental role in the reproductive process (Price & WilliamAshman, 1961; Cavazos, 1975; Mann & Lutwak-Man1981). It secretes a complex mixture of nutrients foundseminal fluid, which are essential for sperm motility anutrition (Costello & Franklin, 1994). In rodents it is complex gland formed by three pairs of lobes (ventlateral and dorsal) distributed around the urethra (VaalasHervonen, 1979; Jesik et al., 1982). According to Bouand Danielli, 1966, the secretory epithelium of the venlobe of the prostate of mice consists of a single layecuboidal cells with nuclei located in the basal region oclearly visible basement membrane. Ultrastructurallydeveloped Golgi complex and mitochondria dispersthrough the cytoplasm can be seen. The cisterns of the gular endoplasmic reticulum are parallel and flattened occupy the perinuclear region of the cytoplasm. The aimthe present study was to determine the possible changhistology, ultrastructure and morphometry caused by eximentally induced diabetes in the secretory epithelium ofventral lobe of the prostate of mice.

Materials and methods

A total of 16 adult male mice (C57BL/6J) aged 3 monwere divided into two groups of 8 animals each, control experimental.

The experimental group received streptozotocin as diabetogenic agent (Sigma Chemical Company), andvehicle for administration was 0.1 M citrate buffer, pH 4Each animal received five intraperitoneal injections at 7 intervals. The first three doses were 75 mg/kg body weand the two remaining doses were 150 mg/kg, for a tota525 mg/kg per animal. The control group simultaneoureceived only 0.1 ml 0.1 M citrate buffer, pH 4.4, by tintraperitoneal route. All animals received balanced gralated solid Purina chow ad libitum. The diabetic state of theanimals was characterized using Multistix 10-SG reagstrips (Bayer) to determine the approximate variationglucose (mg/dl) in urine. Two Multistix 10-SG tests weperformed on all animals before the first streptozotoinjection for quantitative analysis of glucose as a con

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standard. Thirty days after the characterization of diabetic status the animals of both groups were anestheand sacrificed with excess ether inhalation. The venlobes of the prostate were then removed from four miceach group and fixed in Bouin’s fluid. Paraffin-embeddtissue sections were processed for routine light microscand stained with hematoxylin and eosin. The four remainmice were perfused with 2% paraformaldehyde in phphate buffer, pH 7.4. Samples of the ventral lobe of prostate were immediately removed and fixed in 3glutaraldehyde and 1% paraformaldehyde in 0.1 M phphate buffer, pH 7.4, for a period of 3 h, and postfixed in osmium tetroxide in the same buffer for 2 h. The matewas then dehydrated in a growing alcohol series embedded in plastic resin (Araldite). Sections of 0.5µmwere stained with toluidine blue and prepared for ligmicroscopy for the selection of specific areas to be exined by transmission electron microscopy. Ultrathsections were obtained with an LKB ultramicrotome acontrasted with uranil acetate (Watson, 1958) and lcitrate (Reynolds, 1963). Electron micrographs wobtained with a LEO 906 electron microscope.

Material obtained from four control group animals and foexperimental group was submitted to morphometric analyTen light microscope sections were selected per animal. acini in each section and three distinct areas in the acini submitted to measurements of the height of the secreepithelium of the ventral lobe of the prostate. The measments were made using a graded eyepiece (10 mm/coupled to a Carl Zeiss microscope with a 40X objective. eyepiece was calibrated using a slide with a calibrationfrom Carl Zeiss. The celular limits used for the epitheheight measurements was the basement membrane ancell apex. Data were analyzed statistically by Student’s t-testfor independent samples (Norman & Streiner, 1994)

Results

Urine analysisThe control group animals had an average glucose levelmg/dl in the urine, on 100% of the samples. The diabgroup animals had an average glucose level of 950 mg/the urine, during the four-week experiment.

Light microscopyThe glandular portion of the ventral prostate of control mwas of the tubuloalveolar type, presenting acini of differ

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ULTRASTRUCTURAL STUDY OF THE VENTRAL LOBE OF THE PROSTRATE OF MICE 277

Fig. 1 Photomicrographs of the secretory epithelium of the ventral lobe of the prostate of mice 30 days after characterization of the diabetic state. AControl group. Simple epithelium with high cuboidal cells and basally located nucleus (→). A light area is observed above the nucleus. The stroma showsa thin smooth muscle layer and sparse remaining components of the extracellular matrix. H.E. staining. X480 B Diabetic group. Atrophied simple epithe-lium with cuboidal cells and the nucleus occupying large part of the cytoplasm. The stroma presents marked thickening of extracellular matrix compo-nents. H.E. staining, X480

sizes with a folded mucosa. A thin smooth muscle layer wvisible around the acini with respect to the lumen of t

aseorgan, as well as connective tissue (Fig. 1A). The secreepithelium was simple, with high cuboidal cells resting

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278 CAGNON ET AL.

the basement membrane (Fig. 1A). The oval-shaped nucwas located in the basal region, with a clearly visible nuolus (Fig. 1A). In contrast, the animals of the diabetic groshowed marked atrophy of secretory epithelium cells wthe nucleus occupying most of the cytoplasmic region (F1B and Table 1). In addition, the glandular mucosa shoa significant decrease in folding. An increase in the regoccupied by its components was observed in the glandstroma (Fig. 1B). The lumen of the acini presented epithecells dispersed in the secretion.

Electron microscopyThe epithelial cells of the control group were high cuboiwith basally located oval-shaped nuclei, with clearly visibnuclear envelope and nucleolus. Homogeneous nucchromatin with sites of higher condensation near the nucperiphery was observed (Fig. 2A). In the cytoplasm, Golgi complex was developed and the granular endoplasreticulum was formed by numerous flattened and paracisterns located in the perinuclear and basal region ofcell (Fig. 2A, B). Rounded secretory vacuoles containsecretion granules of floccular appearance were observthe apical cytoplasm (Fig. 2D). Microvilli covered thsurface of the cell facing the lumen (Fig. 2D). The glandustroma was thin and consisted of smooth muscle fibcollagen fibers, fibroblasts. (Fig. 2C).

The secretory epithelium of the experimental groshowed morphological alterations 30 days after characzation of the diabetic state. There was atrophy of the setory epithelium characterized by a general reduction of cytoplasm (Fig. 3A). The basally located nucleus occupielarge part of the cytoplasm (Fig. 3A). The clearly visibnuclear membrane and nucleolus and the chromatin wdistributed in a homogeneous manner throughout nucleus and were condensed at the periphery of the nuenvelope (Fig. 3A, B). The cisterns of the Golgi compwere dilated (Fig. 3B, D) and those of the granular enplasmic reticulum were distributed in a concentric manthroughout the cytoplasm (Fig. 3B, D). A concentric formtion probably corresponding to membrane portions wobserved in the apical cytoplasm (Fig. 4B). Accumuladense bodies were detected in the basal region of the(Figs. 3 C and 4D). The microvilli on the cell surface wediscontinuous (Fig. 4B, C) and the apical region of the cyplasm was vacuolized (Fig. 3A, D and 4B, C). Thickeniof the elements of the extracellular matrix and of collagfibers in particular was observed in the stroma, in interclular spaces similar to the trabeculae of connective tis(Figs. 3B, C and 4D).

Fig. 2 Electronmicrographs of the ventral lobe of the prostate from the well-developed Golgi complex (GC), and secretory vaculoes containing Lumen (L), secretory vacuoles (V), flattened cisterns of the Golgi complthe lumen (MV). X15,500 C: Basal region. Nucleus (N) with a clearly visgranular endoplasmic reticulum (GER) can be seen. The basal lamina ismuscle fibers (S). X15, 500 D: Apical region. Lumen (L). Microvilli (MV) (M) can be seen. X27,125.

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Discussion and conclusionLight and transmission electron microscopy demonstratmarked atrophy of the glandular secretory epithelium ofprostate, with reduction of the cytoplasm in diabetic animwhen compared to the controls. These results agree those reported for diabetic rats (Paz et al., 1978). Inpresent study, in addition to cytoplasm vacuolizatwithout the presence of the secretory product, we observed ultrastructural changes of the various organinvolved in the secretory process of the prostate. On basis, we suggest that diabetes may have a negative on the morphology of the prostate.

No morphological studies of the changes in the differcell organelles of the secretory epithelium of the venlobe of the prostate due to the diabetic conditions wfound in the literature. However, we observed that thstructural changes, both of a quantitative and qualitanature, are similar to those detected in castrated and sanimals in the different accessory sex glands of rodents

Castration is known to induce rapid regression of prostate, a process that can be reversed by androgen aistration (Brandes, 1966; Tuohimaa, 1980). Castration be performed by removal of the gonads or by treatment hormonal antagonists such as estrogen or antiestro(Lung & Cunha, 1981). Brandes et al., 1961 studcastrated rats and observed the occurrence of electronbodies and atrophy of the cisterns of the granular enplasmic reticulum of cells of the secretory epithelium of coagulation gland. Later, Harkin, 1963 observed collapsthe cisterns of the granular endoplasmic reticulum, reduction in the number of mitochondria and free ribsomes, and of electrondense bodies in the supranuregion of the cells of the prostatic lobes (ventral, dorsal lateral) of castrated rats. Cavazos, 1975 also observedatrophy, loss of secretory activity, reduction of the cisteof the Golgi complex, disorganization of the cisterns of endoplasmic reticulum and accumulation of lipid dropsthe ventral lobe of the rat prostate. This author propothat, with the reduced levels of circulating testostercaused by castration, there is alteration of the catabmechanism of epithelial cells, which is mobilized aaccounts for the accumulation of dense bodies in the cplasm. Other investigators such as Thompson et al., 1and Tam and Wong, 1991 studied the seminal vesicle ofand the lateral lobe of the prostate of guinea-pigs acastration and observed results similar to those mentioabove. With respect to the stroma of castrated animalsCarvalho et al., 1997 observed an accumulation of elafibers and of type VI collagen in the fibrillar components

control group. A Simple epithelium. high cuboidal cell with a basal nucleus (N),material of floccular appearance in the apical region. X5,390 B: Apical region.ex (GC), and elongated mitochondria (M). Microvilli line the cell surface facingible nucleous. Mitochondria (M) and flattened and parallel cisterns of the clearly visible (→). Glandular stroma observes collagen fibers (C) and smooth

cover the cell surface and secretory vacuoles (V) and elongated mitochondria

ULTRASTRUCTURAL STUDY OF THE VENTRAL LOBE OF THE PROSTRATE OF MICE 279

280 CAGNON ET AL.

Fig. 3 Electronmicrograph of the ventral lobe of the prostate from the experimental group. A Atrophied epithelium with cuboidal cells. Basal nucleus(N) with a clearly visible nucleolus. Vacuolization of the apical region of the cytoplasm. Lumen (L). X5,395 B Basal region. Nucleus (N). Dilation of theGolgi complex (GC). Cisterns of the granular endoplasmic reticulum (GER) organized in a concentric manner, collagen fibers (C), smooth muscle (S).X12,000 C Basal region. Collagen fibers (C) infiltrating the intracellular spaces. Clearly visible basal lamina (→), smooth muscle fibers (S), Nucleus (N).X15,500 D Apical region. Cytoplasm vacuolization (→). Dilation of the Golgi complex (GC). Cisterns of the granular endoplasmic reticulum organized ina concentric manner (GER) X15,500.

ULTRASTRUCTURAL STUDY OF THE VENTRAL LOBE OF THE PROSTRATE OF MICE 281

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Fig. 4 Electronmicrograph of the ventral lobe of the prostate from the experimental group. A Atrophied simple epithelium with cuboidal cells. Nucleus(N) occupying large part of the cytoplasm. Chromatin distributed in a homogeneous form, which becomes condensed at the periphery of the nuclearmembrane. Lumen (L). Clearly visible basal lamina (→). X9,292 B Apical region. Lumen (L). Microvilli presenting discontinuity (MV). Concentricformation (F). Mitochondria (M). Cytoplasm vacuolization (V). Disorganization of the Golgi complex (GC). X20,000 C Apical region. Lumen (L).Cytoplasm vacuolization (V). Microvilli presenting discontinuity (MV). X25,860 D: Basal region. Nucleus (N). Accumulation of dense bodies (→), mito-chondria (M). Collagen fibers (C) infiltrating the intercellular spaces. Clearly visible basal lamina (→). X15,500

the extracellular matrix. These investigators suggested this morphological characteristic is of fundamental imptance for the structural maintenance of the integrity of organ.

With respect to senility, it has been confirmed that age of an individual determines his hormonal equilibriuMoore, 1936 and Brandes, 1963 observed morphologchanges in the secretory epithelium of the different proslobes. In general, clearly visible ultrastructural changwere the large amount of electrondense bodies and the amulation of lipid drops.

In the present study, on the basis of the similaritymorphological changes, we infer that a fall in androglevels may have occurred in the diabetic animals as a coquence of the alteration in the hypothalamus-pituitagonadal axis, which was similar to that of animals submitto castration and to that of senile animals.

However, there are doubts about the etiology of prosalteration in diabetes, especially with respect to insulin dciency interfering with hormonal equilibrium. According Seethalakshmi et al., 1987 insulin and testosterone hasynergistic effect on spermatogenesis when administratestreptozotocin-induced diabetic rats. In a study on rSufrin & Scott, 1972 observed that the insulin/testostercombination is of fundamental importance for the restoraof the histological and biochemical functions of the ventprostate. However, the reduction in the organ size is content with the decreased level of circulating testosterpreviously reported by several investigators (Paz et 1978). Seethalakshmi et al., 1987 considered that thresponsible for reproductive dysfunction in the diabeanimal, are both pituitary and testicular abnormalities.

We may conclude that experimental diabetes leadsevere changes in the cell organelles of the secretory eplium of the mouse prostate, contributing to a decreasfertility. Functional weakening clearly follows, leading impaired fertility.

ACKNOWLEDGEMENTS

The authors thank Dr Iara Maria Silva De Luca for valuacollaboration with the morphometric analysis and tElectron Microscopy Center of IB, UNICAMP.

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