JOURNAL OF MORPHOLOGY 000:000–000 (2015)

Impact of the Processes of Testicular Regression and Recrudescence in the Prostatic Complex of the Bat Myotis nigricans (Chiroptera: Vespertilionidae)  es,1 Paula Rahal,1 Eliana Morielle-Versute,2 and Mateus R. Beguelini,1* Rejane M. Go 1 ~ o R. Taboga Sebastia 1

Department of Biology, UNESP—Univ. Estadual Paulista, S~ ao Jose do Rio Preto, S~ ao Paulo, Brazil, 15054-000 Department of Zoology and Botany, UNESP—Univ. Estadual Paulista, S~ ao Jose do Rio Preto, S~ ao Paulo, Brazil, 15054-000 2

ABSTRACT Myotis nigricans is a species of vespertilionid bat, whose males show two periods of total testicular regression during the annual reproductive cycle in the northwest S~ ao Paulo State, Brazil. Thus, the aim of this study was to investigate the impact of total testicular regression on the prostatic morphophisyology and its regulation. The prostatic complex (PC) of animals from the four periods of the reproductive cycle (active, regressing, regressed, and recrudescence) was analyzed by different histological, morphometric, and immunohistochemical procedures to characterize its variations, analyze its hormonal regulation and evaluate whether the prostate is affected by the processes of testicular regression and recrudescence. The results indicated a decrease in the prostatic parameters from the active to regressed periods, which are related to decreases in the testicular production of testosterone and in the prostatic expression of androgen receptor (AR), estrogen receptor a (ERa) and aromatase. However, in regressed-recrudescence periods, the prostatic expression of AR, ERa and aromatase increased, indicating the reactivation of the PC. Despite this, the PC appears to have a slower reactivation and seems not to follow the testicular recrudescence in morphological and morphometric terms. With these data, we demonstrate that the prostatic physiology is directly affected by total testicular regression and conclude that it is regulated by testosterone and estrogen, via the production of testosterone by the testes, its conversion to dihydrotestosterone by 5aredutase and to estrogen by aromatase, and the activation/deactivation of AR and ERa in epithelial cells, which regulate cell expression and proliferation. J. Morphol. 000:000–000, 2015. VC 2015 Wiley Periodicals, Inc.

Panama (Wilson and Findley, 1970), and a short period of gestation of about 60 days. The first peak occurs in February and is followed by postpartum estrus that results in peaks of births in April–May and August. The third peak is followed by a period of decline in reproductive activity until late December, when a new cycle begins (Wilson and Laval, 1974). Data relating to this annual reproductive cycle are scarce, with only few previous studies available (Wilson and Findley, 1970, 1971; Wilson, 1971; Wilson and Laval, 1974). Wilson and Findley (1971) and Krutzsch (1979), suggested that the reproductive cycle of M. nigricans is geographically variable, with specimens from Paraguay showing an active pattern throughout the year, those from Panama showing an active pattern for most of the year, but becoming reproductively quiescent for about 3 months (September to November), and animals from Mexico resembling temperate zone bats in their reproductive patterns. Beguelini et al. (2013a) showed that animals from northwest S~ ao Paulo State, Brazil, displayed two peaks of spermatogenic activity followed by two periods of total testicular regression, in the

Contract grant sponsor: Brazilian National Research and Development Council (CNPq) Processes; Contract grant numbers: 300163/2008-8 and 301596/2011-5 (S.R.T.), 302008/2010-1 (E.M.V.).

KEY WORDS: hormones; prostate; regulation; reproduction; seasonality

*Correspondence to: Mateus R. Beguelini; Center of Biological and Health Sciences, UFOB–Univ. Federal do Oeste da Bahia-Rua assaros, 47808Prof. Jos e Seabra de Lemos n 316, Recanto dos P 021, Barreiras, Bahia, Brazil. E-mail: [email protected]

INTRODUCTION

Mateus R. Beguelini is currently at Center of Biological and Health Sciences, UFOB—Univ. Federal do Oeste da Bahia, Barreiras, Bahia, Brazil47808-021

Myotis nigricans is a bat species of the Vespertilionidae family, which is endemic to the Neotropical region, with records from the southern edge of the Mexican plateau to just below the Tropic of Capricorn (Wilson and Laval, 1974). Data indicate that females have a distinctive reproductive cycle, with three peaks of births in C 2015 WILEY PERIODICALS, INC. V

Received 6 December 2014; Revised 15 January 2015; Accepted 17 January 2015. Published online 00 Month 2015 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jmor.20373

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same annual reproductive cycle. The authors predicted that this pattern appears to be only indirectly influenced by abiotic factors; it is not directly linked to apoptosis and is controlled by the expression of the androgen receptor (AR), because, throughout the year, AR expression is high in periods of testicular recrudescence and low in periods of regression. Similarly, Beguelini et al. (2014) proposed that the processes of total testicular regression and recrudescence are directly regulated by testosterone and estrogen, via variations in the production of testosterone by 17b-HSD, its conversion to estrogen by aromatase, and the activation/deactivation of the AR in Sertoli cells and the estrogen receptor a (ERa) in spermatogonia. Likewise, Negrin et al. (2014) demonstrated that the reproductive accessory glands (RAGs) of M. nigricans are comprised of a multilobed complex, three bilobed prostatic regions (ventral, dorsolateral and dorsal), associated with the urethra and a pair of inguinal bulbourethral glands, with no ampullary gland or seminal vesicles. The authors also predicted that each prostatic region had unique and distinctive characteristics, which varied seasonally, but were synchronized for the main reproductive peak of the species in summer. Another notable characteristic was an asynchrony in the activity of primary (testes) and secondary reproductive organs (RAGs) for this species (Negrin et al. 2014). Based on these data, the aim of this study was to analyze animals from the four periods of the reproductive cycle (active, regressing, regressed and recrudescence) by different histological, morphometric, and immunohistochemical procedures to characterize their variations, analyze their hormonal regulation and evaluate whether the prostate is affected by the processes of testicular regression and recrudescence.

darkness at 25–30 C, from captures until the following morning, when they were sacrificed by cervical dislocation and the tissues (testes and prostate) were histologically processed.

Species, Ageing and Experiment The species analyzed was M. nigricans, (Miller, 1897), which is an exclusively Neotropical species of vespertilionid bat that is not listed as endangered on the International Union for Conservation of Nature Red List of Threatened Species; however, it is a species that is scarce and difficult to collect, thus, we took care not to disturb the females and only a few adult males were used in this study. The bats were aged as adults based on body weight, complete ossification of the metacarpal-phalangeal epiphyses, wear of the teeth (De Knegt et al., 2005), the positioning of the testes and the presence of sperm inside the testes, cauda epididymis and/ or urethra (Beguelini et al., 2013a, 2013b, 2013c; Puga et al., 2013, 2014). Twenty-three sexually mature specimens were used in this study, including at least five specimens collected for each period of the testicular cycle (four sample groups): 1) Active (September, five specimens); 2) Regressing (October, five specimens); 3) Regressed (November, five specimens); and 4) Recrudescence (December, six specimens; January, two specimens). These periods of collection were determined based on the reproductive cycle of the species, as described by Beguelini et al. (2013a, 2013b).

Processing of Animals Following sacrifice, the weights of the body, gonad and prostatic complex (PC) were measured and the PC was removed and fixed by a methanol:chloroform:acetic acid (6:3:1) fixative solution for 3 h at 4 C, then dehydrated in ethanol, clarified in xylene, embedded in paraffin, sectioned (4 lm thick) and submitted to histological, morphometric and immunohistochemical analyses. All specimens are housed in the Chiroptera collection at the S~ ao Paulo State University (DZSJRP—UNESP).

Histology The PC was serially sectioned (4 mm thick), stained with hematoxylin-eosin (Ribeiro and Lima, 2000) and analyzed using an Olympus BX60 microscope (Olympus Optical Co, Tokyo, Japan) coupled with an image analyzer (Image Pro Plus version R 1993–2006 Media Cybernetics). 6.1 for Windows—Copyright V

MATERIAL AND METHODS Study Area, Capture, and Licenses

Morphometric Analysis

The animals were collected in the city of S~ ao Jos e do Rio Preto, in northwest S~ ao Paulo State, Brazil, (49W220 4500 20S490 1100 ). The capture was performed between September 2009 and January 2010 at night, using five mist-nets (3 3 6 m) set to intercept bats flying 1–3 m above ground. The nets were precisely set on possible flight paths or at exits from shelters. The study was carried out on private lands, locations in which specific permission was not required. The capture and captivity of bats were authorized by the Brazilian institution responsible for wild animal care (Instituto Brasileiro do Meio Ambiente, —Process: 21707-1), and the ethics committee at the Institute of Biosciences, Letters and Exact Sciences at S~ ao Paulo State University (IBILCE-UNESP) authorized all experimental procedures (Process: 013/09— CEEA). The animals were treated according to the recommendations of the Committee on the Care and Use of Laboratory Animals from the Institute of Laboratory Animal Resources, National Research Council, “Guide for the Care and Use of Laboratory Animals.” Following capture, the animals were kept in individual cages (40 3 20 3 20 cm) with water ad libitum, in a specific room in

The relative percentage of glandular compartments (epithelial, luminal, and interstitial tissues) and the epithelium height were measured for the PC (ventral, dorsolateral, and dorsal regions) cross-sections using Image Pro-Plus-Media CybernetR computer software for image ics, version 6.1 for WindowsV analysis. Stereology. The relative percentage of the epithelial, luminal, and interstitial tissues was estimated according to the procedure of Weibel et al. (1966) using a 168-point grid-test system. Data were obtained from 30 random microscopic fields selected from each region (ventral, dorsolateral, and dorsal regions) for each sample group (active, regressing, regressed, and recrudescence) at 2003 magnification. The relative percentage (%) was calculated after counting the number of points that coincided with each of the tissue compartments (epithelium, lumen, and interstitial tissue). Morphometry. To measure the epithelium height, 20 random fields from each region (dorsolateral and dorsal regions) were imaged from each animal at 4003 magnification and three measurements were performed in each field, totaling 300 measurements for each group (active, regressing, regressed, and recrudescence). The epithelium height was measured only

Journal of Morphology

PROSTATIC REGULATION IN Myotis nigricans in sections where the urethra was sectioned transversely; oblique cuts were discarded. Similarly, only acini with a single layer of cells, with no overlapping nuclei or apical membranes were measured. The epithelium height was taken as the linear length from the base of the epithelium (basal lamina) to the apical edge. Analyses were conducted only in the dorsal and dorsolateral regions, since the ventral region had an undefined epithelium, which hinders this type of analysis in this region.

Immunohistochemistry After microwaving for antigen retrieval, nonspecific antibody binding was blocked using 3% of bovine serum albumin prior to incubation with primary antibodies against: the androgen receptor (AR—rabbit polyclonal IgG, sc-816, Santa Cruz Biotechnology, Santa Cruz, CA, 1:100); proliferating cell nuclear antigen (PCNA—mouse monoclonal IgG, sc-56, Santa Cruz Biotechnology, Santa Cruz, CA, 1:100); estrogen receptor a (ERa— rabbit polyclonal IgG, sc-542, Santa Cruz Biotechnology, Santa Cruz, CA, 1:100) and aromatase (CYP19—rabbit polyclonal IgG, sc-30086, Santa Cruz Biotechnology, EUA, 1:100). The sections were submitted to diaminobenzidine and counterstained with Harris’s hematoxylin. For negative controls, the sections received phosphate buffered saline (PBS) instead of the primary antibody. To confirm the results, immunoreactions were performed in triplicate sets. The relative percentage of positive cells in the acinar epithelium was determined using measurement fields consisting of the entire length of the slides. The incidence was estimated by calculating the number of positive cells in the total cell population.

Statistical Analysis Means and standard deviations were calculated for all data sets. Differences between groups were evaluated using one-way analysis of variance, followed by pairwise comparisons with Tukey’s test, using the program Statistica 7.0 (Statsoft, Tulsa, OK). A value of P  0.05 was accepted as statistically significant.

RESULTS Histology All specimens were easily classified into one of the four periods of the testicular cycle, predicted by Beguelini et al. (2013a, 2013b): 1) Active period: normal spermatogenesis, with the presence of all germinative cell types (Fig. 1A); 2) Regressing period: initiation of the process of regression, with the absence of type B spermatogonia and other cell types (Fig. 1B); 3) Regressed period: presence of only Sertoli cells and spermatogonia, with no spermatogenesis (Fig. 1C); and 4) Recrudescence period: reactivation of spermatogenesis (Fig. 1D). Histologically, the PC appeared to undergo an accentuated decrease in activity during the process of testicular regression (Fig. 1E–P). In the Active period, the three regions had a highly secretory aspect, with a large lumen, occupied by a large amount of secretions, and large, welldeveloped secretory cells (Fig. 1E,I,M). However, from the Active to the Regressed periods, the luminal proportion gradually decreased in the three regions, the amount of epithelial and interstitial tissues increased, and showed an approximation/ compression of the secretory cells (Fig. 1E–G,I–

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K,M–O), indicating a continuous decrease in secretory activity, which appeared to follow the testicular regression pattern (Fig. 1A–C). However, the PC did not appear to follow testicular recrudescence (Fig. 1D), since the three prostatic regions showed no apparent changes in morphology from the Regressed to the Recrudescence periods (Fig. 1G–H,K–L,O–P). Body, Gonad, and Prostatic Weights Body weight did not differ significantly between the analyzed periods (Active: 4.1 g 6 0.14; Regressing: 3.94 g 6 0.15; Regressed: 3.89 g 6 0.18; Recrudescence: 3.91 g 6 0.33), with a mean of 3.9 g (Fig. 2A). However, the gonad and prostatic weights were significantly different. The maximum gonad weight occurred in the Active period (14 mg 6 1.1), and then gradually decreased in the Regressing (7 mg 6 0.5) and subsequently, in the Regressed periods, to reach a minimum value (3 mg 6 0.2). A later significant increase in the Recrudescence period (8 mg 6 4.3) was also observed (Fig. 2B). The maximum prostatic weight was also observed in the Active period (20 mg 6 3.5) and decreased gradually in the Regressing (9 mg 6 1.6) and Regressed periods (3 mg 6 0.7); however, the prostatic weight in the Recrudescence period (3 mg 6 0.5) was not significantly different to that in the Regressed period (Fig. 2C). Stereology and Morphometry Despite the inherent characteristics of each prostatic region, the three regions varied similarly during the four periods analyzed, with a significant gradual decrease in the lumen and a significant gradual increase in the epithelium and interstitial tissue from the Active to Regressed periods. Similarly, the three regions showed almost no significant differences between the Regressed and Recrudescence periods (Fig. 3). The ventral region had a higher proportion of lumen in all periods, except in the recrudescence period, when the epithelium was higher or equivalent in amount; followed by the epithelium and interstitial tissue (Fig. 3A–E). The lumen showed a peak in the Active period (70.77% 6 4.66, Fig. 3B), which was significantly different from that in subsequent periods (Regressing: 57.03% 6 4.02; Fig. 3C; Regressed: 44.77% 6 4.32, Fig. 3D; and Recrudescence: 38.64% 6 4.72, Fig. 3E). In contrast, the epithelium and interstitial tissue showed an opposite behavior, with a maximum proportion occurring in the Recrudescence period (Epithelium: 39.51% 6 4.65; Interstitial tissue: 21.85% 6 4.5, Fig. 3E), and a minimum in the Active period (Epithelium: 22.32% 6 4.08; Interstitial tissue: 6.89% 6 2.66, Fig. 3B). This region showed a less drastic reduction in lumen, showing a value of 38.64% 6 4.72 in the Recrudescence period. Journal of Morphology

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Fig. 1. Myotis nigricans, pattern of the testes and the prostate during the four periods of the annual reproductive cycle. Histological sections stained with hematoxylin-eosin. (A–D) Testes: [(A) Active period. Note the continuous spermatogenesis, with the presence of all germinative cell types; (B) Regressing period. Note the initiation of the process of regression, with the absence of type B spermatogonia; (C) Regressed period. Note the presence of only Sertoli cells and spermatogonia, with no spermatogenesis; and (D) Recrudescence period. Note the reactivation of spermatogenesis]. (E–P) Prostatic regions [ventral: (E–H); dorsolateral: (I–L); and dorsal: (M– P)]. Note the large lumen and the large secretory cells (S) in the three prostatic regions during the Active period (E, I, and M); the gradual decrease in the luminal proportion, the increase in the amount of epithelial and interstitial tissues and the approximation/ compression of the secretory cells in the three regions from the Active to the Regressed periods; and the absence of morphological changes in the three prostatic regions from the Regressed to the Recrudescence periods. (B, basal cell; ES, elongated spermatids; I, spermatocytes; Rd, round spermatids; S, secretory cell; Se, Sertoli cells; and Sg, spermatogonia). Scale bars 5 10 mm.

In contrast to the general predominance of the lumen in the ventral region, the dorsolateral (Fig. 3F–J) and dorsal regions (Fig. 3K–O) showed an inversion in the values during the periods. The lumen predominated in both regions in the Active (Dorsolateral: 63.36% 6 6.73, Fig. 3G; Dorsal: 76.82% 6 6.23, Fig. 3L) and Regressing periods (Dorsolateral: 35.64% 6 5.11, Fig. 3H; Dorsal: 43.49% 6 5.15, Fig. 3M); however, the interstitial tissue predominated in the Regressed (Dorsolateral: 52.72% 6 4.05, Fig. 3I; Dorsal: 47.57% 6 6.34, Fig. 3N) and Recrudescence periods (Dorsolateral: 52.34% 6 6.18, Fig. 3J; Dorsal: 54.12% 6 4.88, Fig. 3O). This occurred because the luminal proportion decreased considerably from Active to Regressing period, displaying minimum values below 10% (Fig. 3F,K). Journal of Morphology

The dorsolateral region did not show significant differences in the height of the epithelium during the periods (Fig. 4A–C), with a mean of 4.7 mm. However, the dorsal region had a significantly thicker epithelium in the Active period (7.75 mm 6 1.63, Fig. 4A,D), which significantly decreased in the Regressing (5.99 mm 6 1.31, Fig. 4A) and Regressed periods (5.38 mm 6 0.96, Fig. 4A,E), but remained constant in the Recrudescence period (5.33 mm 6 0.95). Immunohistochemistry Cell proliferation. Similar to that observed for stereology, the percentage of cells that were immunoreactive to the PCNA varied in the three regions during the four periods analyzed (Fig. 5A–J), with a significant decrease from the Active

PROSTATIC REGULATION IN Myotis nigricans

Fig. 2. Myotis nigricans, body, gonad and prostatic weights variations during the four periods of the annual reproductive cycle. (A) Variations in mean body weight. (B) Variations in mean gonad weight. (C) Variations in the prostatic weight. Different letters indicate statistically significant differences (ANOVA at P < 0.05).

(Ventral: 11.63% 6 6.67, Fig. 5B; Dorsolateral: 52.1% 6 8.29, Fig. 5D; Dorsal: 19.38% 6 8.19; Fig. 5F) to the Regressing period (Ventral: 5.69% 6 2.62; Dorsolateral: 39.54% 6 8.14; Dorsal: 12.38% 6 5.75); a significant increase in the Regressed period (Ventral: 30.02% 6 7.9, Fig. 5C; Dorsolateral: 76.55% 6 3.11, Fig. 5E; Dorsal: 42.1% 6 12.48; Fig. 5G); and a further significant decrease in the Recrudescence period (Ventral: 16.48% 6

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9.65; Dorsolateral: 65.96% 6 10.16; Dorsal: 34.58% 6 9.48). Figure 5H–J represents the negative controls of the immunoreactions for the ventral, dorsolateral and dorsal regions, respectively. The percentage of cell proliferation was highest in the dorsolateral region in all periods of the reproductive cycle, followed by in the dorsal region and the lowest value was observed in the ventral region (Fig. 5A). Androgen receptor. The AR expression also varied synchronously in the three regions during the four periods analyzed (Fig. 5K–T), with a decrease from the Active period (Ventral: 24.24% 6 9.35, Fig. 5L; Dorsolateral: 50.48% 6 8.62, Fig. 5N; Dorsal: 54.72% 6 10; Fig. 5P) to the Regressing period (Ventral: 18.56% 6 6.08; Dorsolateral: 46.87% 6 7.26; Dorsal: 35.87% 6 9.35), but which was only significant in the dorsal region; a significant increase in the Regressed period (Ventral: 47.79% 6 7.14, Fig. 5M; Dorsolateral: 76.34% 6 6.75, Fig. 5O; Dorsal: 87.76% 6 7.64; Fig. 5Q); and a further significant increase in the Recrudescence period (Ventral: 54.96% 6 9.4; Dorsolateral: 92.44% 6 6.93; Dorsal: 95.78% 6 3.77), which was only nonsignificant in the dorsal region. Figure 5R–T represents the negative controls of the immunoreactions for the ventral, dorsolateral and dorsal regions, respectively. The amount of AR expression was highest in dorsal region in all periods of the reproductive cycle, except in the Regressing period, followed by the dorsolateral region and the lowest value was observed in the ventral region (Fig. 5K). Estrogen receptor alpha. The pattern of ERa expression was different to that observed for other markers (Fig. 6A–J). The dorsal region showed a constant and higher ERa expression in all studied periods (Active: 99.31% 6 1.7, Fig. 6F; Regressing: 98.21% 6 2.17; Regressed: 93.01% 6 7.72, Fig. 6G; Recrudescence: 98.69% 6 1.72), and only showed a significant decrease in the Regressed period. The ventral and dorsolateral regions varied synchronously in all studied periods, with a significant decrease from the Active (Ventral: 40.66% 6 9.31, Fig. 6B; Dorsolateral: 58.08% 6 8.98, Fig. 6D) to the Regressing period (Ventral: 27.41% 6 7.7; Dorsolateral: 45.15% 6 8.86); a significant increase in the Regressed period (Ventral: 84.64% 6 7.92, Fig. 6C; Dorsolateral: 95.78% 6 5.39, Fig. 6E); and maintained a high expression in the Recrudescence period (Ventral: 80.41% 6 8.79; Dorsolateral: 93.52% 6 5.91). Figure 6H–J represents the negative controls of the immunoreactions for the ventral, dorsolateral and dorsal regions, respectively. The percentage of ERa expression was highest in the dorsal region in all periods of the reproductive cycle, except in the Regressed period, followed by the dorsolateral region and the lowest value was observed in the ventral region (Fig. 6A). Journal of Morphology

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Fig. 3. Myotis nigricans, variation of the glandular components in the PC during the four periods of the annual reproductive cycle. (A) Graph showing the variation in the ventral region. (B–E) Photomicrographs showing the general pattern of the ventral region in Active (B), Regressing (C), Regressed (D), and Recrudescence periods (E). (F) Graph showing the variation in the dorsolateral region. (G–J) Photomicrographs showing the general pattern of the dorsolateral region in Active (G), Regressing (H), Regressed (I), and Recrudescence periods (J). (K) Graph showing the seasonal variation in the dorsal region. (L–O) Photomicrographs showing the general pattern of the dorsal region in Active (L), Regressing (M), Regressed (N), and Recrudescence periods (O). Data in the graphs are given as the mean 6 SD. Different letters indicate statistically significant differences (ANOVA at P < 0.05). Scale bars 5 50 mm.

Aromatase. Aromatase expression also varied synchronously in the three regions during the four periods analyzed (Fig. 6K–Y), with expression restricted to the interstitial tissue. Although it is a cytoplasmic marker, which is difficult to quantify, there was a large variation in aromatase expression during the four periods. The highest expression was observed in the Active period Journal of Morphology

(Fig. 6K,P,U); there was almost no expression in the Regressing period (Fig. 6L,Q,V); expression was reactivated in the Regressed period (Fig. 6M,R,X); and gradually increased in the Recrudescence period (Fig. 6N-N’,S,W). Figure 6O,T,Y represent the negative controls of the immunoreactions for the ventral, dorsolateral and dorsal regions, respectively.

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Fig. 4. Myotis nigricans, variation in epithelium height in the dorsolateral and dorsal prostate during the four periods of its annual reproductive cycle. (A) Graph showing the variation in epithelium height in the dorsolateral and dorsal regions. (B,C) Photomicrographs showing the general pattern of the dorsolateral region in Active (B) and Regressed periods (C). (D,E) Photomicrographs showing the general pattern of the dorsolateral region in Active (D) and Regressed periods (E). Data in the graphs are given as the mean 6 SD. Different letters indicate statistically significant differences (ANOVA at P < 0.05). Scale bars 5 10 mm.

DISCUSSION Impact of the Processes of Testicular Regression and Recrudescence in the Prostatic Complex of M. nigricans Most male mammals display a relatively constant annual reproductive activity of the testes, epididymis, and prostate, with little seasonal variation. For example, male humans present continuous spermatogenesis throughout the year, with the prostate, after epithelial differentiation, showing a steady-state equilibrium between programmed cell death (apoptosis) and cellular proliferation (Isaacs, 1985; van Leenders and Schalken, 2001). Similarly, some rodents also demonstrate continuous spermatogenesis; however, in the prostate, the incidence of apoptosis and proliferation varies regionally, according to the lobes and location (Lee et al., 1990). Despite this, seasonal reproduction is also common among mammals and occurs at all latitudes and in different taxa (Bronson, 2009). Most species of bats reproduce seasonally (Racey, 1982; Crichton and Krutzsch, 2000;); reproduction is timed so that lactation, the most energetically expensive part of reproduction (Kurta et al., 1989), coincides with the peak of food availability (Racey, 1982). Bats from temperate zones generally breed seasonally, with the annual reproductive cycle being directly affected by low temperatures (Gustafson, 1979). However, tropical bats show different patterns (Krutzsch, 1979), which vary from species that, due to their habits, might not be subject to the constraints of seasonal reproduction, such as Desmodus rotundus (Wilson, 1979); to species that showed continuous spermatogenesis during the whole year, but with periods of increased activity, such as Artibeus

planirostris (Beguelini et al., 2013c); to species that had periods of total testicular regression in their annual reproductive cycle, such as Myotis. levis (Ara ujo et al., 2013), Eptesicus furinalis (Bueno et al., 2014), Histiotus velatus, Lasiurus blossevillii, Myotis. albescens (Beguelini et al., 2013d) and M. nigricans (Beguelini et al., 2013a, 2013b, 2013d, 2014). Despite this wide variation, the impact of these different cycles on the PC has largely remained unexplored. Christante et al. (2014) postulated that the PC of Molossus molossus (Molossidae) is synchronized with testicular physiology and is active throughout all the seasons in S~ ao Paulo State (Brazil). Puga et al. (2014) also predicted that the PC of A. planirostris (Phyllostomidae) is active throughout the seasons, in the same region, however, they observed two secretory peaks, which were related to a gradual increase in the rate of circulating testosterone and testicular increase. In contrast, Negrin et al. (2014) demonstrated an asynchrony in the activity of primary (testis and epididymis) and secondary (RAGs) reproductive organs in the annual reproductive cycle of M. nigricans (Vespertilionidae), where prostatic physiology did not follow the process of total testicular regression, and remained active throughout all seasons. A similar asynchrony was also observed in Taphozous longimanus (Singh and Krishna, 2000) and Taphozous. georgianus (Jolly and Blackshaw, 1989), two species of the Emballonuridae family. Unlike the asynchronicity between the primary and secondary reproductive patterns observed by Negrin et al. (2014) for M. nigricans, our data indicate that the process of total testicular regression directly impacts on prostatic morphophysiology, and also causes a regression in its epithelium. Journal of Morphology

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Fig. 5. Myotis nigricans, variation in PCNA (A–J) and AR (K–T) expression in the three prostatic regions during the four periods of its annual reproductive cycle. A. Graph showing the variation of PCNA expression in the three prostatic regions during the four periods of the annual reproductive cycle. (B,C) Photomicrographs showing the general pattern of the ventral region in Active (B) and Regressed periods (C). (D,E) Photomicrographs showing the general pattern of the dorsolateral region in Active (D) and Regressed periods (E). (F,G) Photomicrographs showing the general pattern of the dorsal region in Active (F) and Regressed periods (G). (H–J) Negative controls of the PCNA immunoreaction of the ventral (H), dorsolateral (I), and dorsal regions (J). (K) Graph showing the variation of AR expression in the three prostatic regions during the four periods of the annual reproductive cycle. (L,M) Photomicrographs showing the general pattern of the ventral region in Active (L) and Regressed periods (M). (N,O) Photomicrographs showing the general pattern of the dorsolateral region in Active (N) and Regressed periods (O). (P,Q) Photomicrographs showing the general pattern of the dorsal region in Active (P) and Regressed periods (Q). (R–T) Negative controls of the AR immunoreaction of the ventral (R), dorsolateral (S), and dorsal regions (T). Data in the graphs are given as the mean 6 SD. Different letters indicate statistically significant differences (ANOVA at P < 0.05). Scale bars 5 10 mm.

Journal of Morphology

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Fig. 6. Myotis nigricans, variation in ERa (A–J) and aromatase (K–Y) expression in the three prostatic regions during the four periods of its annual reproductive cycle. (A) Graph showing the variation of ERa expression in the three prostatic regions during the four periods of the annual reproductive cycle. (B–C) Photomicrographs showing the general pattern of the ventral region in Active (B) and Regressed periods (C). (D,E) Photomicrographs showing the general pattern of the dorsolateral region in Active (D) and Regressed periods (E). (F,G) Photomicrographs showing the general pattern of the dorsal region in Active (F) and Regressed periods (G). (H,J) Negative controls of the ERa immunoreaction of the ventral (H), dorsolateral (I), and dorsal regions (J). (K–O) Photomicrographs showing the general pattern of aromatase expression in the ventral region in Active (K), Regressing (L), Regressed (M), and Recrudescence periods (N-N’). (O) Negative control of the aromatase immunoreaction of the ventral region. (P–S) Photomicrographs showing the general pattern of aromatase expression in the dorsolateral region in Active (P), Regressing (Q), Regressed (R), and Recrudescence periods (S). (T) Negative control of the aromatase immunoreaction of the dorsolateral region. (U–X) Photomicrographs showing the general pattern of aromatase expression in the dorsal region in Active (U), Regressing (V), Regressed (W), and Recrudescence periods (X). (Y) Negative control of the aromatase immunoreaction of the dorsal region. Data in the graph is given as the mean 6 SD. Different letters indicate statistically significant differences (ANOVA at P < 0.05). Scale bars 5 10 mm.

Journal of Morphology

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Data showed that reductions in prostatic parameters such as weight, luminal proportion and epithelium height, directly correlated with reductions in testicular parameters, from the Active to Regressing, and subsequently, to the Regressed period (process of total testicular regression). This difference in results might relate mainly to differences in sampling and data analysis, because Negrin et al. (2014) performed a seasonal analysis, whereas this study examined different months. Conversely, the testicular recrudescence process was not followed by the reactivation of the prostatic epithelium, which did not differ significantly between Regressed and Recrudescence periods. Thus, in this study, we observed a continuous decrease in prostatic parameters, such as weight, luminal proportion, and epithelium height, from the Active to Regressed periods, which appears to be related to an accentuated decrease in the testicular production of testosterone (Beguelini et al., 2014) and to a reduction in the prostatic expression of AR, ERa and aromatase. These changes lead to a large decrease in proliferation and production-secretion of metabolites by the glandular epithelium, which causes the morphological regression of the PC. In contrast, in the regressedrecrudescence periods, the prostatic expression of AR, ERa and aromatase greatly increased, indicating the onset of the reactivation of the PC. However, despite the increase in the concentration of circulating testosterone (Beguelini et al., 2014) and in the expression of receptors and enzymes in this period, the PC appears to have a slower reactivation, which does not follow testicular recrudescence, in morphological and morphometric terms. It is important to note that although each of the three prostatic regions are inherently different, they are all similarly affected by total testicular regression and demonstrated a concomitant regression from Active to Regressed periods and a slow reactivation in the Recrudescence period, which does not follow testicular recovery. Hormonal Regulation of the Prostatic Complex of M. nigricans During the Four Phases of the Reproductive Cycle Studies have demonstrated that the mature prostate is mainly regulated by circulating testosterone, which stimulates/regulates epithelial and stromal cells, and triggers different paracrine signaling pathways, to maintain their fully differentiated growth-quiescent epithelium (Prins et al., 1991, 1992). Stimulation via epithelial cells is given by the transformation of testosterone into dihydrotestosterone (DHT), which is catalyzed by 5a-redutase enzyme, which is mainly present in nchez et al., the cytoplasm of epithelial cells (Sa 2013). This conversion is important, because DHT has an increased excitatory activity by having a Journal of Morphology

greater affinity to AR (Cunha et al., 1987). The binding of DHT to the AR of epithelial cells triggers different cascades of reactions that primarily regulate their production and secretion. Similarly, the stimulation via stromal cells is provided by its conversion into estradiol (E2), which is catalyzed by aromatase, mainly present in the cytoplasm of stromal cells (Ellem et al., 2004; Ellem and Risbridger, 2010). Estradiol is essential for normal tissue homeostasis within the prostate, and has opposing dual roles in prostatic tissue, with the activation of ERa leading to proliferation and the activation of estrogen receptor-beta (ER-b) mediating antiproliferative effects (apoptosis), which balance the proliferative action of androgens on the epithelia (Heldring et al., 2007; Ellem and Risbridger, 2009). Based in these patterns and on our results, we propose that in the Active period, the regular concentration of circulating testosterone, which is mainly produced by the active testes (Beguelini et al., 2014), continuously stimulates/regulates the functionality of the PC, and integrates at least two different pathways: First, circulating testosterone can directly reach the secretory epithelial cells, where it is converted into DHT by the action of 5a-reductase enzyme, which regulates the normal expression of the cell (production and secretion) by associating to epithelial AR. Second, circulating testosterone can reach the stromal cells, where they are converted into E2 by the action of aromatase enzyme, which can be transferred to epithelial cells and associate with epithelial ERs, to regulate the normal rate of cell proliferation (proliferation 3 apoptosis—ERa 3 ERb) (Fig. 7A). In the Regressing period, with the onset of total testicular regression (Beguelini et al., 2013a), there is a clear decrease in the testicular production of testosterone—low expression of 17b-HSD (Beguelini et al., 2014), which directly impacts on the amount of circulating testosterone available to stimulate the PC. Similarly, there is an accentuated decrease in the expression of aromatase in the stromal cells of the prostate. As a result, there is not enough circulating testosterone in this period, to stimulate prostatic expression and secretion, via the activation of AR, and proliferation, via its conversion to E2 and the activation of ERa (Fig. 7B). Thus, the process of total testicular regression induces a process of prostatic regression. During the Regressed period, the amount of circulating testosterone remained low (low expression of 17b-HSD [Beguelini et al., 2014]); however, the expression of AR and ERa clearly increased. This increase in receptor expression causes a concomitant increase in the uptake of the few signals (DHT and E2) that are available in this period, thereby increasing prostatic expression and proliferation in this period (Fig. 7C). However, this

PROSTATIC REGULATION IN Myotis nigricans

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Fig. 7. Myotis nigricans, regulation of the PC of during the four phases of its reproductive cycle. Schematic figure showing the pattern of the animals from the northwest of the S~ ao Paulo State (Brazil). Note that the process of total testicular regression directly impact in the prostatic complex, causing a concomitant prostatic regression, which is regulated by the amount of the circulating testosterone, via its conversion into DHT and binding/activation of AR, and via its conversion into estradiol (E2) and binding/activation of ER.

increase is not sufficient to cause morphological changes in the PC, which still shows a pattern of inactivation. In the Recrudescence period, the increase in the production of testosterone—high expression of 17b-HSD (Beguelini et al., 2014), increases the amount of circulating testosterone and combined with the high expression of prostatic AR and ER, triggers the reactivation of the prostate (Fig. 7D). Despite the apparent physiological reactivation, prostatic morphology alters little during this period, showing that the reactivation of the PC is possibly delayed compared to that of the testes.

ogy of the prostate. Prostatic physiology is regulated by testosterone and estrogen, via the production of testosterone by the testes, its conversion to DHT by 5a-redutase in epithelial cells, and to estrogen by aromatase in stromal cells, and by the activation/deactivation of AR and ERa in epithelial cells, which regulates the cell expression and proliferation. ACKNOWLEDGMENT The scholarship awarded to Mateus Rodrigues Beguelini by the S~ ao Paulo State Research Foundation (FAPESP—Process: 2012/09194-0) is gratefully acknowledged.

CONCLUSION With these data, we demonstrate that the processes of total testicular regression and posterior recrudescence suffered by M. nigricans from Sep~o tember to January in the northwest of the Sa Paulo State, Brazil, directly impacts the physiol-

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