Gross observations
The paired seminal vesicular glands of
Eidolon helvum appeared as large milky coloured and somewhat spiral shaped tubes located dorsolateral to the urinary bladder and prostate gland (Figs 1A and B). The seminal vesicular tubes appeared tapered at their openings into the urethra but enlarged as the tubes coiled centripetally outwards indicating availability of seminal vesicular glands unlike most bats that lacked this gland
(Christante et al., 2015; Martins et al., 2015; Beguelini et al., 2016; Santos et al., 2018). The spiral shape in
Eidolon helvum confirmed the variability in bats (
Krutzsch and Nellis, 2006;
Fard and Ghassemi, 2017) and other mammals
(Badia et al., 2006; Samuelson, 2007;
Adebayo et al., 2014; Nissar et al., 2014; Akbari et al., 2018) with seminal vesicular glands.
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Histology and histochemistry
The seminal vesicular gland was enclosed by a variably thick capsule of outer connective tissues and inner smooth muscle layers (Fig 2C). Originating from the capsular layer, thin trabeculae penetrated and divided the glandular parenchyma into numerous lobules, forming a delicate thin network of interlobular connective tissues. Each lobule was a tubular-alveolar glandular acinus and the mucosa lined by simple cuboidal epithelium made up of two cell types; cuboidal secretory cells and basal cells (Fig 2A). These observations were similar to other bats and Southern White-breasted Hedgehog (
Krutzsch and Nellis, 2006;
Fard and Ghassemi, 2017;
Akbari et al., 2018). This varied greatly from the numerous lobes and lobules occupied by longitudinally branching and sometimes anastomosing mucosal folds of most mammalian vesicular glands (
Risbridger and Taylor, 2006;
Wrobel and Bergmann, 2006;
Adebayo et al., 2014; Nissar et al., 2014). These differences probably confirmed the species-specific variations in the seminal vesicular glands of mammals. The cuboidal secretory cells which constituted the major epithelial cells possessed centrally located, euchromatic, oval nuclei with prominent nucleoli. However, binucleated cuboidal cells were frequently encountered in the epithelium. Similarly binucleation were observed in mammals and may represent different types or stages of the secretory cell as most mammalian vesicular glands possessed two types of columnar secretory (light and dense) cells (
Riva and Aumüller, 1994;
Badia et al., 2006; Adebayo et al., 2014).
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The apical cytoplasm of the secretory cuboidal cells stained lightly with eosinophilic granules and gelatinous eosinophilic apical blebs projected from the apical surface of the cuboidal cell into the lumen filled with numerous singly occurring gelatinous eosinophilic secretions. Similar observations had been reported in other bats and mammals (
Krutzsch and Nellis, 2006;
Wrobel and Bergmann 2006;
Adebayo et al., 2014; Fard and Ghassemi, 2017;
Akbari et al., 2018). PAS/Alcian blue stain showed that these secretions were PAS positive (Fig 2B) and similar PAS positive reactions had been described in the epithelial cells of mammalian seminal vesicular glands that secretes fructose, other sugars, prostaglandins, flavins, phosphorylcholine, vitamin C, proteins, neutral mucins and sialomucins (
Krutzsch, 2000;
Wrobel and Bergmann 2006). This may be related to the presence of glycogen moiety which occurred as glycoprotein or neutral mucins and sialomucins known to be nutritive and protective to the spermatozoa and the reproductive tracts. The Alcian blue negative reaction in this study suggests absence of acidic mucins which may be injurious to the spermatozoa and the organ, thus affecting reproductive health.
Electron microscopy
Ultrastructural analysis confirmed that the seminal vesicular glands of
Eidolon helvum were lined by simple cuboidal epithelium made up of cuboidal and basal cells. The epithelial cells rested on a thin basal lamina that separated the epithelium from the underlining interstitial connective tissues (Fig 3). The principal cuboidal cells contained centrally located mono or bi-nucleated oval euchromatic nuclei with nuclear membrane-associated heterochromatin condensations. The cytoplasm contained numerous Rough endoplasmic reticulum (RER), mitochondria and poly-ribosomes, Golgi complexes, electron lucid secretory vesicles, electron dense granules and lysosomes (Figs 4 and 5). These observations were similar to the columnar secretory cells of vesicular glands reported in man (
Riva and Aumüller, 1994), pig
(Badia et al., 2006) and rodents (
Oke and Aire, 1997;
Adebayo et al., 2014). The presence of abundant well developed RER, Golgi complex and mitochondria indicates that these cells are active with intense protein synthesis and secretion, with lots of energy expended in the process. The energy needed for protein synthesis had been quantified at about four high-energy phosphate bonds
(Albert et al., 2002) representing a great amount of ATP being expended
(Bardia et al., 2006). This energetic consumption probably explains the large number of mitochondria, sugars and lipids which can be converted into acetyl-CoA and oxidised to CO
2 via the citric acid cycle
(Badia et al., 2006). The presence of lysosomes in this study may aid in normal turnover, removal and digestion of old or worn-out organelles in the cell due to the active protein synthesis within these cells.
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The apical border showed numerous tall cilia that projected into the lumen. Numerous electron dense granules were attached to the cilia (Fig 5 and 6). The mono and bi-nucleated cells showed no difference in their structures and cytoplasmic contents while the basal cells are small and located between two secretory cells basally. The dark granules were observed secreted by exocytosis and upon extrusion were either observed singly in the lumen or lined the length of the apical cilia (6A) after which the granules were released into the lumen as a ball without the cilia (Fig 6B). Apocrine mode of secretion was also observed in which the apical cytoplasm was walled off by plasma membrane and its content budded off with the granules attached to the cilia (Fig 7). These methods of elaboration presents another unique mode of elaboration of seminal glands as most secretory methods reported in mammalian vesicular glands were mostly apocrine mode
(Badia et al., 2006; Adebayo et al., 2014). The content of the glandular lumen are balls of granules with some consisting of apical cytoplasm and cilia while others are balls of granules without apical cytoplasm and cilia (Figs 3, 5, 6 and 7). These secretions and the gelatinous gel-like nature of the secretions probably aids in vaginal plug formation in
Eidolon helvum as seen in numerous bat species (
Krutzsch, 2000) and rodents (
Banks, 1993).
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