Prenatal development of submandibular salivary gland of New-Zealand rabbits

ARTICLE INFO The present study aimed to ellucidate the prenatal developmental stages of the submandibular salivary gland of the New-Zealand rabbits. To conduct that, twenty New-Zealand rabbit fetuses ranged from 11 to 30 days-old were used. The head region of fetuses and gland specimens were fixed, processed and stained with histological stains to be examined by light microscope. The submandibular primordia was firstly seen at the 12 th day of the prenatal life as bilateral invaginated epithelial buds from the linguo-gingival groove. At 15 days-old , such buds continued deep down growth forming cord-like structure ended by compact bulges that forming the future primitive acini. At 17 days, such cords were branched off forming the future primary ducts. Canalization of the ducts appeared at 18 days. At 22 days, the primitive capsule initiated around the gland and the lobulation was recognized. At 25 days, the capsule became well developed, the duct system was completed and the parenchyma occupied by serous adenomeres surrounded by myoepithelial cells. At the full term, the submandibular gland became fully developed and became typically compound tubulo-acinar nature, the parenchyma showed seromucoidadenomeres. Strong positive PAS reaction was noticed in the striated ducts, while the cytoplasm of the acinar cells reacted weakly. Article history:


Introduction
The maintenance of normal oral biology is achieved by the saliva lubricating and antimicrobial actions (Hsu and Yamda, 2010). Additionally, food digestion by substantial saliva produced is an essential function of the major salivary glands. The paired submandibular gland is one of these major salivary glands (Adnyane et al., 2010). Studies reported that the submandibular and sublingual are endodermal in origin (Emami et al., 1991). However, the major salivary gland, including the submandibular gland, is primarily ectodermal due to its interaction with neural crest-derived mesenchyme (Jaskoll et al.,2002; Rothova et al.,2012). Prenatally, the development of major salivary glands starts with the submandibular gland, followed by sublingual and parotid glands (Tucker, 2007). The prenatal developmental stages of the submandibular gland were investigated in different animals, including the rats (Culter and Moordian, 1987), mice (Jasckoll and Melnick, 1999), pigs (Pospienzy et al., 2010), cats (Knospe and Bohme, 1995), buffalos (Amanand Opinder, 2017) and human (Velasco et al., 1993). However, little literature reported their development in rabbits. Therefore, the present study aimed at highlighting the submandibular salivary gland prenatal development stages in New Zealand rabbits.

Materials and methods
Total number of 20 normal and healthy New-Zealand rabbit fetuses ranging from 11-30 days of gestation were used in this study. The head regions of fetuses (11-20 days of gestation), and the dissected glands of the fetuses (21-30 days old fetuses) were obtained, fixed in 10% neutral formalin, Suza, and Helley's fluid, processed and embedded in paraffin blocks. Cross and/or sagittal step serial sections of 4-6 um thick were obtained and stained with Harris's Haematoxylin and Eosin, Masson's trichrome stain, Periodic acid Schiff technique (PAS), and Alcian blue method ( PH 2.5) as outlined by (Bancroft and Gamble, 2008).

Results
The submandibular salivary gland's primordia were first recognized at 12 days-old New Zealand rabbit embryos as bilateral solid epithelial buds invaginated from the linguogingival groove at the base of the developing tongue (Fig.1). Such buds were formed of clusters of irregularly arranged undifferentiated cells which were rounded or ovoid in shape with rounded nuclei surrounded by faintly basophilic cytoplasm. Some primordial cells showed mitotic activity (Fig.2). On the13 th day of the rabbit embryo, the submandibular buds grew deeply throughout underlying mesenchymal tissue forming solid epithelial cords with closely packed cellular masses of ill-distinct cell boundaries (Fig.3&4). On reaching the 15 th day, the developing cords continued their deep down growth and showed compact terminal bulges forming the primitive acini, which was surrounded by a large amount of primitive stroma with many fibroblasts and mesenchymal cells (Fig.5). The primitive acini at this stage were lined by multilayered polyhedral cells of basophilic cytoplasm and darkly stained nuclei with loose cellular central mass ( Fig.6). At 17 days-old rabbit embryo, progressive branching of the developing cords connected with primitive acini which surrounded by loose mesenchymal tissue (Fig.7). The primitive acini were lined by closely packed cells with numerous mitotic divisions. The developing cords at this stage showed more differentiation and designated to form the future primitive ducts. Their cellular clusters formed of outer regularly arranged closely packed layer of columnar cells with oval nuclei while the inner cells were loosely arranged (Fig.8). The primitive ducts began to be canalized on the 18 th day of prenatal life. They were lined by one to two layers of cuboidal to columnar cells housing rounded or oval nuclei surrounded by pale basophilic cytoplasm. The acini were still illuminized ( Fig.9). On reaching 22 days, the embryonic mesenchymal tissue became differentiated into primitive capsules and trabeculae that divided the gland into different lobes and lobules. The developing adenomeres continued to increase in number but loosely arranged within the lobules. The interlobular ducts were lined by double layers of epithelial cells with central lumina (Fig.10). At 25 days-old rabbit embryo, the submandibular gland became larger in size and highly organized into well-developed stroma and a distinct increase in lobulation and vascularity. The capsule became well-developed formed dense collagenous connective tissue with many fibroblasts (Fig.11). The gland adenomeres were progressively increased and closely packed with each other. Most of them became luminized and lined by truncated pyramidal cells with basal basophilic cytoplasm and slightly acidophilic apically. An elongated curved myo-epithelial cells with flattened nuclei and scanty cytoplasm appeared to surround the adenomeres (Fig.12&13). Extensive branching of ducts were observed. All types of ducts including intercalated, striated and interlobular ducts were noticed at this stage. The lining epithelium of some striated ducts changed into single layer of columnar cells with oval nuclei (Fig.14). Both fibrous elements either in capsule or in the trabeculae and basal lamina of adenomeres and ducts showed positive PAS reaction, while the cytoplasm of acinar cells showed weak reaction (Fig.15). From 27 till the end of the prenatal life, submandibular gland became highly developed and became typical compound tubulo-acinar gland. The glandular lobules were increased on the expense of the interstitial tissue (Fig.16).The stroma became fully developed formed mainly of collagen fibers and fibroblasts. The trabeculae carried interlobular ducts (Fig.17). The glandular acini became differentiated into mucous and serous. The mucous adenomeres were more prominent and larger in size than the serous ones. They lined byhigh cuboidal cells with basally ovoid or flattened nuclei surrounded by vacuolated or foamy cytoplasm. While the serous adenomeres appeared smaller in size and lined by truncated pyramidal cells with basal spherical nuclei and basophilic cytoplasm. Some serous demilunes showed to be capped the mucous adenomeres (Fig.18). Both types of gland adenomeres were surrounded by myoepithelial cells. During this stage, the fibrous stroma and the basement membrane of adenomeres and ducts showed positive PAS reaction. Also, the cellular cytoplasm of some striated ducts showed strong positive PAS reaction, while the cytoplasm of acinar cells still showed weak reaction (Fig.19).

Discussion
In the current study, the submandibular salivary gland primordia arise as an epithelial bud formed of epithelial cell clusters. These findings further supporting the previously known morphological development of the submandibular salivary gland from the epithelial lining of the linguo-gingival groove (William et al., 1989;Klein, 2002;Sivakumar et al., 2003;Patel and Hoffman, 2014;and Kwon &larsen, 2015). The submandibular salivary gland's primordia development began as early as 12 days in the New Zealand rabbit embryos. The development on the 12 th day is similar to that observed in rats (Kleinman, 2003). However, Soliman (2006) first recognized the submandibular primordia development in rabbits by the 13 th day of prenatal life. Compared to mice, the submandibular primordia development could be seen at an earlier age, "11.5 th days" (Tucker, 2007).
The epithelial buds progressively developed with underlying mesenchyme and branch, as previously reported by Melnick et al. (2001). The epithelial buds initially grow deeply down through the underlying mesenchymal tissue to form cord-like structures that branched off as the glandular primitive ducts and acini (BathBalogh & Fehrenbach, 2011;Abuzaid et al., 1990). The present study results are in accordance with the previous reports indicating the formation of compact bulges of stratified epithelium that develop to the primitive acini of the submandibular salivary gland (Soliman, 2006;Patel, 2014). additionally, Klein (2002) augments that a continuous basal lamina primarily surrounds the developing ducts or acini of the submandibular salivary gland. We observed a highly mitotic activity in the developed gland's cellular elements that indicate a proceeding development process (Noden and Lahunta, 1985;Soliman, 2006) and (Teshima et al., 2015).
In this study, the canalization of the glandular duct system began on the 18 th day of prenatal life (Kwon and Larsen, 2015). In contrast, Soliman (2006) reported the canalization of the ducts at earlier stagy by the 17 th day. The developing salivary gland parenchymal duct canalization occurred due to the centrally located cell apoptosis (Teshima et al., 2015).
The rabbits developing gland parenchyma surrounded by mesenchymal tissue from which the glandular stroma was differentiated (Soliman, 2006). Upon development proceeding, the mesenchymal tissue underwent condensing and gradually shifted to a fibrous tissue containing many fibroblasts (Melnick et al.,2001). The developing gland became then surrounded by a prominent, continuous fibrous capsule by the 22 nd day of the rabbit embryo's prenatal life (Soliman, 2006). The sizes and the numbers of the secretory alveoli increase. Subsequently, the developing gland size increased gradually due to the cord's progressive branching and new alveoli formation (Noden and Lahunta, 1985;Soliman, 2006).
By day 25 of prenatal life, the developed alveoli are canalized and lined by pyramidal cells as previously recorded (Soliman, 2006). The myoepithelial cells were first observed as flattened curved cells with elongated nuclei on the 25 th day of pregnancy. These cells surrounded the secretory acini, and their numbers increased gradually toward the fullterm as previously reported by Sivakumar et al., (2003) and Patel and Hoffman (2014).
Conversely, late development of the myoepithelial cells was recorded on the 29 th day of the embryonic rabbit life (Soliman, 2006). Most histological studies showed that the myoepithelial cells have a contractile function that evacuates the saliva in the alveolar lumen into the alveolar ducts (Sivakumar et al., 2003;Soliman et al., 2006 andTeshima et al., 2015).