The Structure and Hormone Expression of Male Accessory Reproductive Glands of the Sunda Porcupine (Hystrix Javanica)

This research was to investigate the histological structure of male accessory reproductive glands of the Sunda porcupine and verify the presence of testosterone and Androgen Binding Protein (ABP) with immunolocalization. The research used three accessory reproductive gland samples of male Sunda porcupine. Tissues were processed using paraffin method and stained with Hematoxylin-Eosin and Immunohistochemistry methods. The histological structure and distribution of testosterone and ABP were analyzed descriptively. This research showed that male accessory reproductive glands of the Sunda porcupine are seminal vesicle, prostate, and bulbourethral glands. The seminal vesicle was lined by simple columnar epithelium, prostate was lined by simple columnar epithelium or simple cuboidal epithelium, and bulbourethral gland was lined by simple cuboidal epithelium. The presence of testosterone was found in adenomere cells of seminal vesicle and prostate glands; and in interstitial tissue of bulbourethral gland. The presence of ABP was detected in the adenomere cells of the bulbourethral gland. The male accessory reproductive glands of Sunda porcupine has been identified which consist of seminal vesicle, prostate, and bulbourethral glands. The immunolocalization of testosterone was found in adenomere cells of seminal vesicle and prostate glands, and ABP was detected in bulbourethral gland.


The Macroscopic Description of the Male Accessory Gland of Sunda Porcupine
Macroscopic representation of the genital of the Sunda porcupine gland indicates that the Sunda porcupine has three pairs of genital accessories glands, namely the seminal vesicle gland, the prostate gland, and the bulbourethral gland. The seminal vesicle gland is a pair of glands located on the dorsal surface ( Fig. 1) of the urinary vesicles or lateral cauda from the urinary vesicles. The prostate gland is a branched tubuloacinar gland located between the urethra and the seminal portion of the ventral section (Fig. 2), while the bulbourethral gland is a mixed tubuloacinar gland located outside the pelvic cavity next to the caudal the prostate-looting is covered by the musculus ischiocavernosus.

Histology Structure of Male Accessory Gland of Sunda Porcupine
Hematoxylin Eosin staining, results in an overview of the histological structures of the seminal vesicle gland, the prostate gland, and the bulbourethral gland. Figure 3 shows the seminal vesicle gland consisting of four lobes with each lobe surrounded by thick smooth muscle.  (Fig. 4A). The lamina epithelialis mucosae consist of epithelium columnar simplex with varying height, whereas lamina propia-submucosa is composed by loose collagenous connective tissue (Fig. 4C). The musculoskeletal tunic in this gland is divided into two laminas namely lamina muscularis circular internal and lamina longitudinal external (Fig. 4B). Lamina on the tunica muscularis is dominated by smooth muscles with difficult boundaries, while in tunica adventitia are composed by connective tissue and blood vessels (Fig. 4D). The seminal vesicle gland of the Sunda porcupine is included in the serous gland type as it has a rounded cell nucleus and is located in the center (Fig. 4C). shows that the seminal vesicle gland consists of several layers, including the lamina epithelialis mucosa (le), lamina propriasubmucosa (lp), tunica muscular (tm), and tunica adventitia (ta). (B) and (C) indicate the lamina epithelialis mucosa (le) is simple columnar epithelium, whereas the lamina propria-submucosa (lp) is composed of loose collagenous connective tissue. The tunica muscular (tm) in this gland is divided into two lamina which include the internal circulatory lamina muscular (tms) and external longitudinal lamina muscular (tml). (D) indicates that the lamina in the tunica muscular is dominated by smooth muscle with a boundary that is difficult to distinguish, while the tunica adventitia is composed of connective tissue and blood vessels. (C) also shows that the seminal vesicle gland is a type of serous gland (ks).
Advances in Life Science and Technology www.iiste.org ISSN 2224-7181 (Paper) ISSN 2225-062X (Online) Vol.78, 2020 Figure 5 shows that the prostate gland lobe has two parts, the corpus and the portions are disseminated. The dissemination portion is part of the prostate gland located around the urethra. Portions of the prostate gland are urethra, residual duct deference, and seminal vesicle glands. The lobe of the prostate gland is each compiled by several lobules of the glands. The lobules in the corpus are composed by the adenomere of the prostate gland and the part is composed by the excretory duct surrounded by the fibro myoelastic capsule, which composed of collagen connective tissues, elastic tissues and smooth muscles.  show the adenomere of the prostate gland is composed of the lamina epithelialis mucosa (le), lamina propriasubmucosa (lp), tunica muscular (tm), and tunica adventitia (ta). The lamina epithelialis mucosa (le) is in the form of simple columnar epithelium or simple cuboidal epithelium. The lamina propria-submucosa (lp) contains connective tissue, whereas the tunica muscular is composed of smooth muscle. The tunica adventitia is composed of connective tissue (ji) and blood vessels (pd). Excretory duct of the prostate gland is composed of several layers namely lamina epithelialis mucosae, Advances in Life Science and Technology www.iiste.org ISSN 2224-7181 (Paper) ISSN 2225-062X (Online) Vol.78, 2020 lamina propria-submucosa, tunica muscularis and tunica adventitia (Fig. 7A). Lamina epitelialis mucosa has the same epithelium as the adenomere, which is the epithelium columnar simplex or epithelium cuboid simplex (Fig.  7B). The Lamina propia-submucosa contains connective tissue and muscularis tunic composed of smooth muscles (Fig. 7B). The Tunica adventitia consists of connective tissues and blood vessels. The prostate gland in the Sunda porcupine is included in the serous gland type as it has a rounded cell nucleus and is located in the center. The last male gland is the bulbourethral gland. The gland is wrapped by fibroelastic capsule consisting of smooth muscles, connective tissues, and muscular skeletal (Fig 8). A single lobe of the bulbourethral gland ( Fig.  9A) consists of a lobule that is bordered by an interlobular septum that contains connective tissues and each lobule there are some adenomeres surrounded by fibrous connective tissues ( Fig. 9B and 9C). Image of 9D suggests that a bulbourethral gland adenomere is surrounded by a simplex cuboid epithelium. Bulbourethral glands of the Sunda porcupine belong to the mucous gland type because it has a flat-shaped cell nucleus and is located on the edge (Fig. 9D).

Distribution of Testosterone and ABP
The result of immunohistochemistry staining with testosterone antibodies suggests that there is immunized cytoplasm in the adenomere cells of the seminal vesicle gland (Fig. 10A), cytoplasm of the adenomere of the prostate gland (Fig. 10B), and the interstitials tissue of the bulbourethral gland (Fig. 10C). These immunoreactive cells are characterized by brown color, while the purple color comes from the counterstain using Harris Haematoxylin. The result of immunohistochemical staining with ABP/SHBG polyclonal antibodies suggests that only in the bulbourethral gland shows the immunoreactive cells of the adenomere cytoplasm (Fig.  10D).

Discussion
Macroscopic evaluation on Sunda porcupine gland indicates that there have three pairs of genital accessory glands, namely the seminal vesicle, the prostate gland, and the bulbourethral gland. Akbari et al. (2018) explained that the seminal vesicle gland located on the dorsal surface or lateral cauda of vesica urinaria. This indicates that there are differences in the gland type of genital accessories in the Sunda porcupine with other Rodentia, but it has a similarity to the location of the accessory's gland. The difference is the Sunda porcupine doesn't have an ampulla gland, while other Rhodesians have it.
HE staining results in an overview of the histological structures accessories glands. The seminal vesicle gland produces a yellowish, viscous liquid that serves as an active substance for sperm motility (Eroschenko 2008). The seminal vesicle gland consists of four lobes with each lobe surrounded by thick smooth muscle capsule. The seminal vesicle gland consists of several layers namely lamina epithelialis mucosae, lamina propria-submucosa, tunica muscular, and tunica adventitia. The lamina epitelialis mucosae consist of epithelium columnar simplex with varying height, whereas on the lamina propia-submucosa is composed by loose collagen connective tissue. The lamina on the tunica muscularis is dominated by smooth muscles with difficult boundaries, while in the tunica adventitia composed by connective tissue and blood vessels (Khojasteh et al. 2014). According to Treuting et al. (2018), the seminal vesicle gland of other Rodentia belongs to the serous gland type because it has a rounded cell nucleus and is located in the center of the cells, it shows similarities to the Sunda porcupines.
The prostate gland is a small branching gland that produces secretions and is slightly acidic. The results obtained from the observation that the prostate gland in Sunda porcupine has a similar structure to Rodentia, because it has a pair of lobes, namely the ventral lobe and dorsolateral lobe, but the porcupine does not have anterior lobes or coagulating glands (Neill 2006). The prostate gland lobe has two parts namely the corpus and the disseminated part. Disseminated is part of the prostate gland around the urethra. Disseminated part of the prostate gland is the urethra, the remaining deferens ducts, and the seminal vesicle gland. The lobes of the prostate gland are each composed by several glands' lobules (Ross & Pawlina 2011).
The histological structure of the prostate gland in the Sunda porcupine has a difference with other rodents. The difference is in the disseminated part of the lamina epithelial mucosa of the excretory duct of the Sunda porcupine prostate gland in the form of the epithelium columnar simplex or cuboid simplex epithelium, whereas in the other Rodentia is pseudo complex epithelium (Eroschenko 2008). According to Treuting et al. (2018) the prostate gland in other rodents is included in the type of serous gland because it has a rounded cell nucleus and is located in the middle of adenomere cells, this shows similarities in the Sunda porcupine so that it can be said that the type of prostate gland of Sunda porcupine gland is a type of serous gland.
The last male accessory genital gland is the bulbourethral gland. The bulbourethral gland is a mixed tubuloacinar gland capable of producing mixed secretions from mucous and seromucous material (Treuting et al. 2018). The bulbourethral gland adenomere is surrounded by a cuboid simplex epithelium (Scudamore 2014). This shows the similarity of the histological structure of the bulbourethral gland in Sunda porcupines with other rodents. According to Treuting et al. (2018) the bulbourethral glands in other rodents are included in the mucosal gland type because they have a flat cell nucleus and are located on the edge, this shows similarities in the Sunda porcupine so that it can be said that the type of bulbourethral gland is a type of mucosal gland.
The results of immunohistochemical staining with testosterone antibodies indicate that there are immunoreactive cells in the cytoplasm of the seminal vesicle gland adenomere cells, cytoplasm of the prostate gland adenomere cells, and interstitial tissues of the bulbourethral gland. Dihydrotestosterone (DHT) is an active form of testosterone that belongs to the strong androgenic metabolite (Chaves et al. 2011); and it's important for normal male genital, prostatic development, promote the growth and survival of prostate cells (Anawalt 2017;Tan et al. 2015;Zhou et al. 2015).
Androgen Binding Proteins is testicular glycoproteins that bind androgens with high affinity (del Mar Grasa et al. 2017;Laurent et al. 2016). ABP in the testis Rodentia is synthesized by Sertoli cells and is secreted into the seminiferous tubule lumen and into other vascular compartments. The results of immunohistochemical staining with polyclonal ABP/SHBG antibodies showed that only in the bulbourethral gland showed immunoreactive cells that were marked with a brownish color in the cytoplasm of adenomere cells.

Conclusion
The results of this study indicate that the male accessories glands of Sunda porcupine (Hystrix javanica) consists of seminal vesicles gland, prostate gland and bulbourethral gland. Seminal vesicle gland was lined by simplex columnar epithelium, prostate gland was lined by simplex columnar epithelium or simplex cuboid, and bulbourethral gland was lined by simplex cuboid epithelium. Immunohistochemical staining results show that testosterone is found in the adenomere cells of the seminal vesicle gland, the adenomere cells of the prostate gland, and the interstitial tissues of the bulbourethral gland. The ABP is detected in the cytoplasm of adenomere cells of the bulbourethral gland, but not seminal vesicle and prostate glands.