Lobe Identity in the Mongolian Gerbil Prostatic Complex: A New Rodent Model for Prostate Study

Authors

  • Sabrina Santos Rochel,

    1. São Paulo State University, Institute of Biosciences, Humanities and Exact Sciences, IBILCE/UNESP, Department of Biology and Animal Biology Graduate Program, São José do Rio Preto, SP, Brazil
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  • Alexandre Bruni-Cardoso,

    1. São Paulo State University, Institute of Biosciences, Humanities and Exact Sciences, IBILCE/UNESP, Department of Biology and Animal Biology Graduate Program, São José do Rio Preto, SP, Brazil
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  • Sebastião Roberto Taboga,

    1. São Paulo State University, Institute of Biosciences, Humanities and Exact Sciences, IBILCE/UNESP, Department of Biology and Animal Biology Graduate Program, São José do Rio Preto, SP, Brazil
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  • Patricia Simone Leite Vilamaior,

    1. São Paulo State University, Institute of Biosciences, Humanities and Exact Sciences, IBILCE/UNESP, Department of Biology and Animal Biology Graduate Program, São José do Rio Preto, SP, Brazil
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  • Rejane Maira Góes

    Corresponding author
    1. São Paulo State University, Institute of Biosciences, Humanities and Exact Sciences, IBILCE/UNESP, Department of Biology and Animal Biology Graduate Program, São José do Rio Preto, SP, Brazil
    • Rua Cristóvão Colombo n° 2265, CEP 15054-000, São José do Rio Preto, SP, Brazil
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    • Fax: 55-17-32212390


Abstract

Knowledge of structural and physiological differences among the prostatic lobes (PL) is the basis for development of experimental studies in traditional laboratory rodents. Although Mongolian gerbil reproductive organs have been increasingly investigated, its prostate structure is far from being properly known, and investigations of this organ focused on the ventral lobe (VL). Thus, the present study provides a thorough morphological description of prostatic complex in the male adult gerbil on the basis of topographic, histological, and ultrastructural analysis and ductal branching. Like other rodents, four pairs of PL were observed. However, in contrast to the rat and mouse, the VL is the least voluminous component and the dorsolateral lobe (DLL) is the most prominent and spatially isolated from remaining PL. The occurrence of a dorsal lobe (DL), hidden between bladder and insertion of seminal vesicles, has not been mentioned in previous reports with Mongolian gerbil. Collagenase digestion followed by microdissection revealed that, except for DL, which has a tubular-acinar organization, all PL exhibit tubular organization and variable ductal branching. Distinct histological and ultrastructural features such as secretory epithelium, aspect of luminal secretion and stromal organization are reported for each PL and are confirmed by morphometric and stereological methods. Histological sections showed at least three intralobar segments in VL and DL. Ultrastructural analysis evidenced that, although luminal epithelial cells of PL share typical features of exocrine secretory cells, there are striking lobe phenotypical variations. Both merocrine and apocrine pathways are observed in variable rates in all PL, with the predominance of the former in the DLL and the latter in the CG. The morphological observations presented herein point to distinct structural identities for each PL, which probably reflects specific functional compromise of seminal fluid secretion. These data also point to the gerbil as a good model for investigations concerning the regulation of prostate development and homeostasis, mainly with regard to the dorsal and dorsolateral PL. Anat Rec, 290:1233-1247, 2007. © 2007 Wiley-Liss, Inc.

The prostate is a fundamental accessory gland of the male reproductive tract, as it secretes products that provide survival, viability, and motility to spermatozoa. This gland is usually found in most mammals, but its morphology varies significantly among classes. In rodents, for instance, the prostate is formed by four distinct bilaterally symmetrical lobes that encircle the urethra at the base of the bladder, designated as the ventral lobe (VL), lateral lobe (LL), and dorsal lobe (DL), and, attached to the seminal vesicles, the anterior lobe or coagulating gland (CG; Price and Williams-Ashman, 1961; Price, 1963; Jesik et al., 1982). On the other hand, in other mammals, such as the dog and human, the multilobar organization is not observed in the adult stage, and the prostate contains closely associated distinct zones, to constitute a compact organ (Price, 1963; McNeal, 1983).

Although the presence of isolated lobes is a common feature of most rodents investigated to date, some variations have been reported. In rat and mouse prostate, for example, the LL and the DL are often dissected and analyzed together as the dorsolateral lobe, because these components exhibit a ductal origin from the same region, a topographical relationship and a delayed physiological response to androgen withdrawal (Price and Williams-Ashman, 1961; Jesik et al., 1982; Sugimura et al., 1986a, b). On the contrary, the VL of these species is the most prominent component of the complex, exhibits a more rapid epithelial response to androgen ablation, and is the main experimental model used to understand the biology of the prostate (Jesik et al., 1982; Sugimura et al., 1986b; Shappell et al., 2004). In the hopping mouse, the VL is the only component of the prostatic complex (Tsonis et al., 1981). Thus several studies have reported that there are considerable differences in the ductal array, histology, and hormonal sensitivity among the prostatic complex lobes in laboratory rodents (Jesik et al., 1982; Sugimura et al., 1986a; Lee et al., 1990; Hayashi et al., 1991; Banerjee et al., 1994; Kinbara and Cunha, 1996). It is known that these differences are, at least in part, a consequence of morphogenesis (Sugimura et al., 1986a), and recently the Hox genes have been indicated as distinct expression of genetic patternsinvolved in lobe identity (Huang et al., 2004). Each prostate lobe contributes with specific secretory products to the semen (Lee et al., 1985; Doncajour et al., 1990; Hayashi et al., 1991; Chow et al., 1992). However, the implications of these morphological variations for semen composition, as well as for the regulatory mechanisms involved in the development of prostatic lobe, is far from being totally understood.

Comparisons of the prostate development in humans and rodents have shown that the morphogenesis occurs in an analogous manner (Price, 1963; Timms et al., 1994). However, much controversy still exists concerning the homology between rodent prostatic lobes and human prostatic zones. It is a consensus opinion that there is no existing supporting evidence for a direct relationship between the specific mouse prostate lobe and the specific zones in the human prostate and, until these relationships can be more precisely defined, it is not possible to conclude which lobe is the most relevant to address clinicopathologic alterations in human prostate carcinoma (Shappell et al., 2004). Thus knowledge of the prostatic complex morphophysiology in other rodents will be useful in permitting better comparisons among them and with human prostatic zones.

Although there is no clear homology between prostatic lobes and human prostatic zones, most of the experimental studies concerning the action of carcinogens or hormonal manipulations have focused on the ventral lobe, probably due to its larger size, androgen sensitivity, and incidence of hyperplasia and neoplasia in this region of the prostate (Reznik et al., 1981; Banerjee et al., 1998; Vilamaior et al., 2000; Shappell et al., 2004). This emphasis on the ventral lobe has also been observed on the Mongolian gerbil Meriones unguiculatus (Corradi et al., 2004; Pegorin de Campos et al., 2006; Oliveira et al., 2007), a small rodent that has been widely used in studies of the prostate, in the males and females comparatively (Custódio et al., 2004; Santos et al., 2003, 2006).

The Mongolian gerbil is a rodent that has been known for its suitability for laboratory use since the 1960s (Williams, 1974). By nature, it is a curious and friendly rodent, almost without odor, with monogamous behavior and physiological mechanisms of corporal-water conservation, presenting relative absence of natural illnesses (Pinheiro et al., 2003). The usefulness of this animal in biomedical research has been recognized in immunology (Nawa et al., 1994), physiology (Nolan et al., 1990), and morphology (Pinheiro et al., 2003; Santos et al., 2003, 2006; Custódio et al., 2004; Corradi et al., 2004; Santos and Taboga, 2006). More recently, the gerbil has also been suggested as a suitable model for studies on mammalian aging (Spangler et al., 1997; Pegorin de Campos et al., 2006). The gerbil prostate, although multilobulate, has compact lobes, with relative similarity to those of the human prostate, but unlike those of rats and mice (Price, 1963; Pinheiro et al., 2003; Góes et al., 2007). Previous data from our laboratory have demonstrated that histological, histochemical, and ultrastructural features of the adult gerbil prostate are comparable with those of the human prostate, such as the smooth muscle cell structure and ultrastructure (Corradi et al., 2004) and epithelium cell types (Santos et al., 2003; Pegorin de Campos et al., 2006).

Considering that a descriptive study of the gerbil prostatic lobes has not been reported, and that the use of this animal in experimental research has increased in the past few years, in the present study, aiming to have a wider view about the morphophysiology of prostatic complex of this rodent, we present a descriptive analysis of the anatomy, histology, ductal branching, and ultrastructure of the Mongolian gerbil prostate.

MATERIALS AND METHODS

Animals

Twenty-seven adult (18-week-old) male gerbils (M. unguiculatus, Gerbilinae: Muridae), supplied from the State University of Sao Paulo, UNESP (Botucatu, SP, Brazil), were housed under adequate conditions of luminosity (12-hr light: 12-hr dark) and temperature (24°C) and were fed rodent ration (Labina®, Purina, Agribrands do Brasil Ltda) and water ad libitum. The treatment of the animals followed the instructions established by the Brazilian College of Animal Experimentation.

Dissection of Prostatic Complex

The accessory glands of the male genital system were exposed by abdominal incision, immediately after the killing of gerbils by CO2 inhalation. Careful dissections of these organs under a stereoscopic microscope (Leica MZ6) provided exploratory observations of the prostatic complex's topographical relationships with other organs of male genital system. Therefore, this complex, associated with seminal vesicles, urinary bladder, and urethra, was dissected from ureters and ductus deferens for posterior analysis. Three animals were used for direct anatomical observations of fresh prostate; the organs from three other animals were immediately immersed in Karnosvsky solution (2.5% glutaraldehyde, 2% formaldehyde in phosphate buffer, pH 7.2) after the excision. This brief fixation (5–15 min) was adopted aiming to harden the fragile prostatic tissue and preserve it during the dissection and removal of associated adipose tissue, providing a better identification of each lobe, especially the dorsal one.

Microdissection of Duct Arrays

Three animals were used in the microdissection experiments performed according to the procedures of Sugimura et al. (1986a). After the excision of bladder and the removal of associated fat, the set containing the seminal vesicle, the prostatic complex, and the periurethral muscles was incubated in Hank's solution containing 1% collagenase (Sigma, St. Louis, MO), at 25°C, for 10–20 min. First, the attachment of CG to seminal vesicle was sectioned. Then, the ductal secretory units of VL and dorsolateral lobes (DLL) were carefully dissected using fine forceps, and the set of ducts of each lobe, with a part of periurethral muscle attached, was sequentially removed. DL in the gerbil prostate is found hidden in a triangular area between the dorsal base of the bladder and the insertions of seminal vesicle. Additional collagenase incubations were performed to separate individual ducts. After incubations, the duct arrays were rinsed in saline, stained with drops of methylene blue and whole-mounted. The analyses were performed using a Leica MZ6 Stereoscopic Microscope attached to a digital camera (Cannon Powershot S40).

Light Microscopy

For the analysis under light microscopy, the complex containing the prostatic lobes of five animals was sectioned in the median sagittal or transverse direction and fixed by immersion in formaldehyde 4% just prepared in phosphate buffer, pH 7.2, for 24 hr, being, sequentially dehydrated and processed for inclusion in Paraplast (Histosec-Merck) and Historesin (Leica historesin embedding kit). Additionally, histological inclusions of the separate lobes were realized after the dissection under stereoscopic microscopy (three animals). Histological sections in Paraplast (4 μm thick) or Historesin (3 μm thick) were stained with hematoxylin–eosin (HE) and with Gömöri's Trichrome. The microscopy analyses were performed with an Olympus BX-60 photomicroscope. The Image-Pro Plus computer software, version 4.5 (© Media Cybernetics) for Windows®, was used to digitize the histological images.

Quantitative Analyses

Morphometrical and stereological parameters were only evaluated in the intermediate region of each lobe. The epithelial height, thickness of subepithelial stroma, and thickness of smooth muscle cells were measured in nonoblique historesin sections of the acinus stained with HE and the above-mentioned computer software. All data were obtained from 30 random microscopic fields per animal at ×100 objective, totaling 90 measurements per parameter. The volumetric fractions of the prostate components were determined in histological sections stained with HE using the Weibel's system and a 134-point grid was applied to all prostatic lobes following Huttunen and coworkers (1981) used previously by Vilamaior and collaborators (2006). For this procedure, isotropic and random orthogonal triplet probe sections were made, and the intermediate region of the each ductal system was selected for the counting. Mean values for each group were determined using three different prostate fragments per animal and 20 sequential contiguous visual fields (×200) per histological section. Thus, the relative volume of each prostate component was determined in terms of percentage of coincident total points.

Transmission Electron Microscopy

Tissue fragments of five animals were immersed in 2.5% glutaraldehyde, plus 0.25% tannic acid solution in Millonig's buffer for 2 hr, washed and post-fixed in 1% osmium tetroxide in the same buffer for 1 hr, washed again, dehydrated in graded acetone, and embedded in Araldite. Semithin sections were stained with Toluidin Blue and analyzed under light microscopy. Ultrathin silver sections were obtained with a diamond knife and contrasted with 2% alcoholic uranyl acetate and then with 2% lead citrate in 1 N sodium hydroxide solution for 10 min. Grids were examined under a Leo-Zeiss 906 transmission electron microscope, operating at 80 kV.

Scanning Electron Microscopy

The prostatic complexes of five other animals were processed for scanning electron microscopy (SEM). The dorsal and ventral lobes were fixed in 2.5% glutaraldehyde in phosphate buffer (0.1 M, pH 7.2), for 2 hr, washed, and post-fixed in 1% osmium tetroxide in the same buffer for another 2 hr. The fragments were dehydrated in graded ethanol series, dried at critical point with liquid CO2 (Balzers, CPD-010), coated with gold (Ion Sputter - Balzers SCD-040), and analyzed with a Jeol 5200 scanning electron microscope.

RESULTS

Architecture of Prostatic Complex

The prostatic complex of the gerbil is formed by four pairs of lobes closed associate with the urethra (Fig. 1). Except for the set of anterior lobes, or CG, which appears attached to the smaller curvature of the seminal vesicle, the prostatic lobes are located around the proximal portion of pelvic urethra, in association with urethral muscle (Fig. 1). The DLL are the most voluminous components of the prostatic complex in this rodent, and they fuse partially in the medial region, assuming a butterfly shape (Fig. 1B,D). This region of the prostate is easily differentiated from the others, not only because of its typical shape, but also because of the larger dimensions and parallel arrangement of the tubular secretory units. The VL are also connected in the medial region, forming a compact oval structure (Fig. 1C). The fourth pair of lobes is situated craniolaterally, hidden between the bladder and the ducts of the seminal vesicle and CG (Fig. 1B,D). As shown schematically in Figure 1, these DL exhibit an intermediate position in comparison with the DLL and VL and its glandular units can show a more diffuse arrangement. In this region, the deferent ducts are inserted.

Figure 1.

A–C: Lateral (A), dorsal (B,D), and ventral (C) view of the adult gerbil prostatic complex, fresh and schematically. BL, Bladder; CG, coagulating gland; DL, dorsal lobe; DLL, dorsolateral lobe; SV, seminal vesicle; VL, ventral lobe; UR, pelvic urethra and urethral muscle.

The collagenase digestion experiments followed by microdissection indicated that the ramification pattern is very distinct among the lobes (Fig. 2). Thus, as shown in Figure 2A, the CG has a large main duct, which emits 8–12 wide not-ramified branches. The remaining lobes are organized as ramified and composed of tubulo-acinar glands, but the degree of ramification is highly variable among them. So, the analysis of the serial histological sections in association with the microdissection experiments revealed that each ventral lobe exhibits two to three main ducts, each of which ramifies four to six times (Fig. 2B). The ductal ramification in the DLL is similar to VL, but the tubules are longer because the ramification occurs at greater distances in this lobe (Fig. 2D). On the other hand, the DL exhibited very short branches that give the gland an acinar aspect after collagenase digestion (Fig. 2C). Furthermore, the identification of ductal arrays after enzymatic digestion revealed that, while the CG, DLL, and DL show tubules with the same diameters along the entire gland, the VL has a set of tubules with ample lumen and a much narrower set of tubules.

Figure 2.

Structure of the prostatic lobes after microdissection followed by collagenase digestion. A: In coagulating gland note the main duct (d) and large lateral tubules (t) not branched. B: In the ventral lobe, note the wider (arrows) and thinner (arrowheads) tubules. C: The dorsal lobe shows a tubule–acinar organization, and the density of stroma made it difficult to isolate tubular arrays. D: Note the dichotomous ramification pattern of the dorsolateral lobe. Scale bars = 100 μm.

Lobe Histology and Ultrastructure

Under light microscopic analysis, all the prostate lobes showed a similar pattern of histological organization—they are composed of a secretory epithelial tissue, which forms the tubular units, and a stroma (Figs. 3, 4). The stromal compartment has a subepithelial region and a layer of smooth muscle cells that surround the tubules, in addition to the adjacent region of interstitial stroma situated between adjacent tubules (Figs. 3, 4). However, as shown in Figure 3, important histological differences are observed in these lobes related to the dimensions of the secretion units, epithelial height, secretion aspect and stromal organization. These differential features are shown quantitatively in Table 1 and will be described below. Although the frequency and distribution of stromal cells are variable among the lobes, their ultrastructural features are very similar (Fig. 7).

Figure 3.

A–L: Histological sections stained with hematoxylin–eosin (A,B,D,E,G,H,J,K) and thin sections stained with Toluidine Blue (C,F,I,L) of adult male gerbil prostate lobes. Coagulating gland (A–C) and ventral (D–F), dorsolateral (G–I), and dorsal (J–L) lobes. ap, aposomes; dd, ductus deferens; ep, epithelium; fb, fibroblast; lm, lumen; smc, smooth muscle cell; sbs, subepitelial stroma; st, stroma. In D, the arrowheads point to the main ducts and the dotted line marks the transition region next to the urethra. Scale bars = 500 μm in A,D,G,J, 100 μm in B,E,H,K, 25 μm in C,F,I,L.

Figure 4.

A–F: Histological sections stained with hematoxylin–eosin showing distal (A,D), intermediate (B,E), and transitional proximal segments (C,F) of ventral (A–C) and dorsal (D–F) lobes. The insets indicate the corresponding epithelium. Note that, in all segments, the stroma is denser in the dorsal lobe (two-headed arrow). lm, lumen; ep, epithelium. Magnification = ×200 in A,C,D,F, B,E, ×100 in B,E, ×400 in insets.

Table 1. Morphometrical and Stereological Data of Prostate Lobes From Adult Mongolian Gerbila
 CGVLDLDLL
  • a

    CG, coagulating gland; VL, ventral lobe; DL, dorsal lobe; DLL, dorsolateral lobe.

Morphometrical data (μm)
 Height of secretory epithelium15.4 ± 2.914.2 ± 321.7 ± 3.218.5 ± 4
 Thickness of subepithelial conjunctive tissue1 ± 0.43.6 ± 1.62.4 ± 0.71 ± 0.5
 Thickness of smooth muscle cell layer25.4 ± 5.215.7 ± 4.19.1 ± 217.3 ± 2.6
Stereological data (relative volume %)
 Secretory epithelium40.4 ± 6.615.7 ± 6.339.8 ± 6.119.9 ± 6
 Tubule lumen34.6 ± 5.860.8 ± 10.131.1 ± 5.460 ± 8.1
 Stroma24.9 ± 2.623.4 ± 6.629 ± 5.220 ± 6

Coagulating gland

The CG contains very large tubules with highly folded mucosae (Fig. 3) and a very thick layer of smooth muscle cells, with more than eight cellular strata. (Table 1; Fig. 3). The epithelium is cubic or low columnar, with elliptical nuclei, while the cytoplasm is rich in rough endoplasmic reticulum (RER) and the cisternae are very dilated (Figs. 3, 5). The various stacks of the Golgi complex occupy a great part of the apical cytoplasm, where also the secretion granules are concentrated. The interior of the granules presents a slightly electron-lucid aspect with sufficiently electron-dense spherical condensations. The apical face of the plasma membrane contains various short microvilli, and the cytoplasm in this region is very electron-dense (Fig. 5A), with apocrine secretion vesicles. In histological sections stained with HE, the secretion appears very acidophilic, exhibiting a high electron density and a filamentous aspect under transmission electron microscopy.

Figure 5.

Electron micrographs of gerbil coagulating gland. A: Simple epithelium with columnar cells. The apical cytoplasm appears very electron-dense, with short dispersed microvilli (mv) and apical blebs (arrowheads) of apocrine secretion. Note the short invaginations of the basal membrane (bm) and subepithelial stroma among adjacent epithelial cells (arrows). The apical portion of the cell is dark due to the secretion. B,C: The apical portion of the epithelial cell is filled with rough endoplasmic reticulum (RER) cisternae and secretion vesicles (sg) and grumes of electron-dense secretion (s) are observed next to this region. D,E: Fibromuscular stroma adjacent to the epithelium shows smooth muscle cells (smc) rich in contractile elements and interspersed by collagen fibrils (cf). cf, collagen fibrils; G, Golgi complex stacks; N- nucleus. Scale bars = 2.3 μm in A, 1.2 μm in B,C, 2 μm in D, 1.56 μm in E.

The epithelial–stromal interface shows short infoldings of the subepithelial connective tissue, rich in collagen fibers, toward the epithelium (Fig. 5A). Although most of the stromal compartment is formed by the smooth muscle cells, the intercellular regions are occupied by a high quantity of highly packed collagen fibrils.

Ventral lobe

The VL histological sections show marked variations along the glandular units. In addition to the main ducts, located close to the urethra and immersed in the urethral muscle, the VL contains a proximal, a secretory intermediate, and a distal tip segment (Figs. 3D, 4A–C). The proximal segment is a transition region, with tubular organization and intermediate characteristics between the main ducts and the secretory portion (Fig. 4C). In this region, the glandular epithelium shows a high degree of folding and the lumen is reduced. The intermediate segment, the more voluminous region of the lobe, shows a glandular epithelium lower than the other segments and usually does not exhibit folds (Figs. 3D–F, 4B). The distal tips of tubules have tall columnar simple epithelium, with discrete folds supported by a thick layer of subepithelial and fibromuscular stroma (Fig. 4A).

With regard to the relative volume of all main gland compartments, the VL is very similar to the DLL (Table 1). However, the VL distinguishes itself from the others by presenting a looser organization of the stromal tissue and thinner smooth muscle cells (Fig. 3D,E). The subepithelial compartment is thicker and contains two to four fibroblast layers surrounded by collagen fibrils and amorphous material (Figs. 3F, 6).

Figure 6.

Transmission electron micrographs of gerbil ventral prostate lobe. A: Secretory epithelium with luminal cells. Luminal cells are tall and columnar, contain long rough endoplasmic reticulum (RER) cisternae, granules of merocrine secretion (msg), with a granular aspect, and many lipid droplets (ld) in basal regions. Note that the luminal secretion (s) also exhibits a granular aspect. B: Shown is part of the secretory tubule through the scanning electron microscope evidencing the epithelium (ep) and secretion (s). C: Basal region of secretory cell rich in lipid droplets. D: Supranuclear region showing close associations of granules of merocrine secretion with Golgi complex stacks (G). E: Fibromuscular stroma containing fibroblast prolongation (fb) in subepithelial compartment, together with smooth muscle cells (smc) with a parallel arrangement. bm, basal membrane; N, nucleus. Scale bars = 1.3 μm in A, 20 μm in B, 0 75 μm in C, 1.2 μm in D,E.

Large amounts of lipid droplets form accumulations in the basal cytoplasm of the luminal epithelial cell under transmission electron microscopy (Fig. 6A). The cytoplasm exhibits fewer RER cisternae than the other lobes, and they are narrower, but presenting, in compensation, larger quantities of free ribosomes. The apical portion of the cell is filled with secretion granules, whose content is similar to the secretion found in the lumen (Fig. 6A,C). The secretion also exhibits a flaked effect as the DLL, but the granulation is more delicate (Fig. 6). On the histological sections stained with HE, the secretion shows itself more stained than that of the VL and the apocrine secretion vesicles are less frequent (Fig. 3). The secretory vesicles are also visualized in scanning electron microscopy, inside the acinar lumen (Fig. 6B).

Dorsolateral lobe

The tubulo-acinar units of this lobe have a smaller diameter when compared with the other lobes, and they are surrounded by a dense stroma, which presents many thick smooth muscle cells (Figs. 3G–I, 7). The epithelium is formed by a single layer of cubic or columnar secretion cells, which present basal nuclei, from round to oval (Fig. 7). The epithelial compartment corresponds to around 20% of total lobe volume (Table 1). The epithelial secretory cells of the DLL are differentiated by the large amount of RER cisternae, generally very dilated and rich in flaked material in the lumen, found distributed in all regions of the cytoplasm and also in the basal compartment (Fig. 7).

Figure 7.

A–C: Aspects of gerbil dorsolateral prostate lobe showing the epithelium (A) and periluminal cytoplasm (B,C) under transmission electron microscope. A: The epithelium shows secretory luminal cells with elliptical nuclei (N) parallel or perpendicular to the cell surface and large amounts of dilated rough endoplasmic reticulum (RER) cisternae. Fibroblasts (fb) are seen in the subepithelial stroma (sbs). The granules of merocrine secretion (msg) are seen in all cytoplasmic regions, including the cell base, where lipid droplets (ld) are concentrated. They exhibit an electron-lucid matrix with dispersed granular secretory material, which is also observed in tubule lumen. B: Shown is the apical portion of the secretory cell with secretory vesicles filled with secretion also seen in the lumen (dotted arrow). C: Detail of the apocrine and merocrine secretion granules. A,C: Aposomes (ap) are shown through the transmission electron microscope and present variable aspects. Some of them contain RER profiles (arrowheads), and others have a homogenous granular content (arrows). Scale bars: 3.4 μm in A; 1.3 μm in B,C.

The nucleus, generally elliptical, can be located parallel to the epithelium in regions of high secretory activity. Eventual lipid droplets are observed usually in the cellular base. The Golgi complex is also sufficiently developed and the secretion granules are dispersed throughout the cell, including the cellular base (Fig. 7A).

The secretion of DLL has a slightly acidophilic and flaked aspect after HE staining (Fig. 3G–I). The examination under transmission electron microscopy revealed that this secretion is formed mainly by the content of secretion granules (Fig. 7) but also by the loss of apical cytoplasm vesicles (Fig. 7A,C). In the vicinity of the apical surface cytoplasm, secretory vesicles are found, probably at several phases of maturation—the newly formed that contain cytoplasmic organelles, those formed by a mass of degenerated cytoplasm, and vesicles filled with an amorphous material (Fig. 7).

Dorsal lobe

The DL, like the VL, also presents histological intralobular variations and proximal, intermediate, and distal tips that can be easily recognized (Fig. 4D–F). However, it differs histologically from the VL because the tubular units are wider and its secretion exhibits a basophilic and vesiculous aspect after HE staining (Fig. 3J,K). Furthermore, the stroma is more dense, with predominance of fibromuscular component and thicker smooth muscle cells (Fig. 3L).

The epithelial cells are cubic or low columnar, with basal nuclei and convex apices (Figs. 3L, 8A). Voluminous lysosomes and some lipid droplets are observed on basal cytoplasm. The RER cisternae are placed in concentric arrangements, preferentially in the peripheral cytoplasm, while the cisternae of the Golgi apparatus form a round arrangement in the apical cytoplasm with the secretion granules located in the center (Fig. 8A,B). Each vesicle has a slightly granular interior and a highly condensed spherical region. Although the apical cytoplasmic portions can also contribute to the secretion of the DL, the major part is formed by the content of secretion granules, that concentrates, forming a substantially electron-dense homogeneous substance in the interior of the tubules (Figs. 3, 8C). This homogeneous aspect of DL secretion can be visualized by SEM (Fig. 8D).

Figure 8.

A–D: Aspects of the gerbil dorsal prostate lobe showing the epithelium (A), apical cytoplasm (B), and luminal secretion (C) under the transmission electron microscope and part of the secretory tubule in scanning electron microscope (D). A: Epithelial luminal cells with basal nuclei (N) and apical cytoplasm, exhibiting a concentric disposal of peripheral rough endoplasmic reticulum (RER) cisternae, several Golgi complex stacks (G), and granules of merocrine secretion (mgs) with electron-dense spherical condensation. Multivesicular body (mvb). B: Apical cytoplasm of epithelial cells shows granules of merocrine secretion (mgs) adjacent to a cytoplasmic bleb containing dilated RER cisternae. C: Detail of the luminal secretion (s) showing its homogeneous and electron-dense aspect next to an aposome (ap). D: Note the dome-shaped epithelial cells and the homogeneous aspect of luminal secretion. Scale bars = 2.6 μm in A, 1.56 μm in B, 20 μm in C,D.

DISCUSSION

The Mongolian gerbil is a small rodent of the Muridae family, subfamily Gerbillinae, that has been increasingly used as an experimental model in studies concerning male histophysiology (Corradi et al., 2004; Góes et al., 2007; Oliveira et al., 2007) and female prostate (Santos et al., 2003; Custódio et al.. 2004; Santos et al., 2006). As mentioned previously, the investigations on the male prostate in gerbil are focused on the ventral lobe (Corradi et al., 2004; Oliveira et al., 2007). A report on the relationship between prostatic ducts and the pelvic urethra is also available (Pinheiro et al., 2003). Despite this finding, the structure of male prostatic complex in this species is not well known and the lack of information about the lobular identity makes it difficult to compare with other murine models and, also, with the development of experimental studies.

In general, the prostatic complex of the gerbil is similar to that described in other rodents, but some relevant differences seem to occur in gross morphology and histology. The macroscopic analyses of fresh or recently fixed accessory sex glands showed that the prostatic complex of Mongolian gerbil, like that of other rodents (Price, 1963; Jesik et al., 1982), is composed of four isolated pairs of lobes named according to the position around the urethra (ventral, lateral, dorsal, and anterior) or coagulating gland. Thus, these anatomical observations, reinforced by collagenase digestion followed by microdissection experiments, allowed the identification of a fourth pair of lobes—herein designated as dorsolateral lobes (DLL)—which have not been mentioned in previous studies in this species (Williams, 1974; Pinheiro et al., 2003). As observed in the microsdissection experiments, the DLL, in comparison with the other lobes, presents a more compact arrangement of the tubulo- acinarunits due to its stromal density, which makes it harder to separate. However, either at the histological or ultrastructural level, a marked similarity with the VL is verified. Such similarity to the gerbil, indicated in the present work, was previously described for the mouse and rat (Sugimura et al., 1986a; Hayashi et al., 1991). According to Sugimura et al. (1986a), the ducts of the lateral portion of the prostate originate next to the dorsal branches and, during gland morphogenesis, extend ventrolaterally, surrounding the urethra toward the VL, facts which, at least in part, explain the similarities such as the ramification pattern and histological characteristics of DLL with VL. According to Kinbara and Cunha (1996), in the rat, the separation plan of the dorsolateral lobe in dorsal and lateral is hard to discern, being frequently considered as a single structure; but, when analyzed carefully, lateral and dorsal lobes can be identified and individualized. The close association between the DL and DLL does not occur in the Mongolian gerbil, because the ducts of the former are easily differentiable macroscopically. Thus it is important to emphasize that each prostate lobe of the gerbil is enclosed by a delicate capsule of mesothelial cells and, although in older animals part of the DLL can be next to the VL and the cranial part of the DL can be next to the DLL, a complete fusion of lobes was not observed in this animal as well as for the rodents mentioned above. Then, our previous observations of an apparent higher degree of similarity between the human and gerbil prostate on the basis of a compactness of lobes (Corradi et al., 2004; Pegorin de Campos et al., 2006; Oliveira et al., 2007) is no longer supported.

The data shown in Table 1 indicate that the prostate DLL and VL of the Mongolian gerbil present similar relative volumes between epithelial and stromal compartments, together occupying approximately 40% of the total organ. This result indicates that these lobes are the ones that present a closer similarity to the human prostatic peripheral zone according the zonal model of ductal architecture described by McNeal (1983), whose stromal and epithelial compartments occupy approximately half of the gland. Rodents and human comparative observations of prostate gland in adult phase does not seem to be appropriate, although the comparison between these species may be used during the prostatic development. In this initial phase, Thomson and Marker (2006) have demonstrated that branching morphogenesis begins in an analogous manner in both rodents and humans with several distinct sets of epithelial buds growing out of the urethra into a surrounding mass of urogenital sinus mesenchyme (Timms et al., 1994).

In rodents, each prostatic lobe is formed by a complex duct system that originates from the urethra and finishes in many distal branches near the capsule (Price and Williams-Ashman, 1961; Sugimura et al., 1986a; Hayashi et al., 1991; Kinbara and Cunha, 1996). In the present study, the application of the microdissection method pioneered by Sugimura et al. (1986a) revealed that the CG, VL, and DLL of gerbil are composed of ductal glands without true acinar components, and the DL is the only one of these to show tubule–acinar organization.

The DLL is the most voluminous component of the Mongolian gerbil, which differs from other rodents such as the rat and mouse, where the DL is the least developed and the VL corresponds to 50% of the total volume of the whole prostatic complex (Hayashi et al., 1991). As the microdissection experiments revealed that the ductal branching pattern in the gerbil DLL does not differ significantly from those of the rat and mouse (Sugimura et al., 1986a; Hayashi et al., 1991), it is possible that the increased volume is probably related to the dilation of the tubular units. On the other hand, the microdissection experiments also showed that the smaller size of the VL in the gerbil is related to a lesser degree of ramification of its ductal units.

The Mongolian gerbil CG, in comparison to that of the rat, also presents a lesser degree of branching, because it has a main duct that ramifies one time, originating ample ducts, while in the rat, the main duct branches into 40–50 main terminal ducts (Hayashi et al., 1991). This prostatic region of rodents is responsible for the secretion of proteins involved in the semen coagulation and copulatory-plug formation in the female tract after copulation (Aumüller and Seitz, 1990). As verified here and in previous reports (Jesik et al., 1982; Hayashi et al., 1991), the CG is the component that differs most from the other lobes due to a lesser degree of ramification, greater thickness of the muscular layer, and the highly folded mucosa. These histological characteristics resemble those of the seminal vesicle.

Although the DLL, VL, and DL present a common pattern of histological organization in the pluristratified epithelium and fibromuscular stroma, important characteristics such as the secretion aspect, epithelial infolding, and stromal organization give distinct identities to each lobe. Moreover, VL and DL show clear intralobular differences. Many authors have demonstrated the existence of intralobular regional differences in other rodents, either in the dorsal or ventral prostate lobes. DeKlerk and Coffey (1978) and Sugimura et al., (1986a, b) reported that the epithelial and stromal cells of different regions of the prostatic lobes respond differently to androgenic privation and stimulation, suggesting a functional heterogeneity inside the lobe. Therefore, in addition to the main ducts in prostate lobes of some rodents already studied, three morphologically and functionally distinct regions called distal, intermediate, and proximal, according to the position occupied in relation to the urethra, have been recognized (Lee et al., 1990; Banerjee et al., 1994; Nemeth and Lee, 1996). Nemeth and Lee (1996) have described how the regional differences along the duct system of the VL in rats, even in the acinar epithelium as in the stromal compartment, reflect differences in secretion levels, proliferation, and cellular death.

The ultrastructural analysis shows that, although luminal epithelial cells rich in organelles involved in the synthesis and processing of secretory products constitute the main cell type in the secretor epithelium, they exhibit phenotypical variations in each lobe. Analyzed as a set, these characteristics reflect a distinct functional commitment of each lobe in the production of the seminal fluid. In this matter, the DL of this rodent is sufficiently different from the DLL and VL and also from the DL of other traditional rodent models for the study of the prostate, and therefore shows a highly basophilic secretion of colloidal aspect. When observed on the ultrastructural level, it is perceived that the secretion produced in the DL is very similar to that of the CG concerning the organelles involved in its production, release mechanism, and luminal aspect. These findings agree with biochemical data that show a common secretory protein profile for these lobes (Hayashi et al., 1991).

The ultrastructural observations lead to confirmation that all prostatic lobes exhibit the two pathways of secretion export—the merocrine and the apocrine. In the CG, as verified even with conventional light microscopy (Fig. 3A), the apocrine is probably the main pathway involved. This finding is so far known in rats, whose CG has been used as a model to study this secretion mode (Wiche et al., 2003). The apical cytoplasmic blebs, called aposomes, are also found frequently in the VL. In this case, the early secreted aposomes contain RER cisternae that later appear to degenerate. It is possible that these organelles can contribute to Ca2+ and Zn2+ concentrations of VL secretion.

As has been suggested for other rodents, the gross aspect and other morphological variations among the gerbil prostate lobes are a consequence of differential interactions during organ morphogenesis (Sugimura et al., 1986a). Although further studies are necessary to elucidate how this process occurs in this species, it is probable that the variations in the size of the detected lobes reflect distinct needs in terms of the seminal fluid components.

In summary, our analyses show that, in addition to the coagulating gland and the VL and DL, the prostatic complex of the Mongolian gerbil also has a DLL. In contrast to other rodent models widely used in studies of the prostate, such as the mouse and rat, the gerbil presents DLL that are more developed than the ventral ones, differing from these by virtue of larger size, parallel organization of the duct–acinar units, which are little ramified. This context indicates the Mongolian gerbil as an interesting model for studies focusing on this lobe. Taken together, the analysis of ductal branching, histology, and ultrastructure, indicate that each lobe presents a distinct identity and, probably, a specific contribution to prostate physiology. Similarly to other murine species, the VL and DL of this small rodent present clear intralobar histological differences, and distal, intermediate, and proximal segments can be recognized in relation to the urethra. Thus, the information about the prostate organization of the Mongolian gerbil extends the knowledge of the murine prostate structure and supplies the morphological basis for the development of further studies on the regulation of the development and maturation of the prostate lobes and their relationships to prostate disorders.

Acknowledgements

This study is part of the undergraduate final essay of A.B.C. orientated by S.R.T. and Master's Thesis of S.S.R. orientated by R.M.G. and P.S.L.V., and was presented at the International Symposium of Animal Biology of Reproduction, Belo Horizonte, Brazil. Comments provided by the anonymous referees have helped to improve our original manuscript. Gratitude is also expressed to Prof. Dr. Denise de C. Rossa Feres for the use of the stereoscopic microscope and Luiz Roberto Falleiros Júnior and Maria D.S. Ferreira for their technical assistance.

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