Although the development, morphology, and physiology of external male genitalia are relatively well studied in mammals (Yamada et al.,2003; Klonisch et al.,2004; Ramm,2007; Simmons and Jones,2007), these structures have received less examination in reptiles. Aspects of the gross anatomy and physiology of chelonian and crocodilian phalli have been described for over 150 years (Todd,1852). More recently, the genital morphology and the general erectile physiology have been investigated in turtles (Zug,1966) and the anatomy and basic physiology of male external genitalia, the hemipenes, have been examined in snakes and lizards (Savage,1997; Rhen et al.,1999; Zaher,1999). Crocodilian genital morphology (Ziegler and Olbort,2007) and functional histology of spectacled caiman (Caiman crocodilus crocodilus) phalli (Cabrera et al.,2007; Cabrera and Garcia,2007) have received more in-depth investigation.
Here we present a histological evaluation of the overall general tissue morphologies, erectile structures, and secretory morphology of the American alligator (Alligator mississippiensis) juvenile male phallus. We focus on the cross-sectional histological architecture of phallic components and the epithelial morphologies of the sulcus (alternatively penile groove or seminal groove) and phallus body. To better capture the complexity of this dynamic organ, we also employ histochemical staining and three-dimensional reconstruction techniques. From these observations we have begun to better define the roles and functions of the various structures of the alligator phallus.
Intromittent organs are primarily male structures designed to transfer gametes to mates for internal fertilization. Phalli have independently evolved multiple times among vertebrates and the frequency and chronology of the evolution of intromittent organs in amniotes is debated (Kelly,2002). However, it is hypothesized that a phallus of monophyletic origin is present in Squamata (lizards and snakes), Testudines (turtles and tortoises), Crocodylia (alligators and crocodiles), and within extant birds phalli are found in all species of Paleognathae and some families of Galloanserae (Montgomerie and Briskie,2007; Brennan et al.,2008). Common to all, these phalli satisfy the basic functional requirements of sufficient stiffness for copulation and gamete transfer mechanisms (Kelly,2002).
Phallus stiffness can be achieved in various ways, including the use of stiff materials like cartilage or through changes in hydrostatic pressure in the organ as fluid fills a central vascular space surrounded by an inelastic tensile membrane reinforced with collagen fibers (Kelly,2007). Some testudines, crocodilians, and birds use both cartilage and hydrostatic pressure to achieve a rigid intromittent organ. These intracloacal phalli are composed of bilateral, rigid, and fused fibrous bodies (alternatively called fibroelastic, fibrovascular, corpora fibrolymphatica, or corpus fibrosum) which develop as a thickening of the ventral wall of the proctodeum and terminate in glans-like structures containing vascular erectile tissues (Montgomerie and Briskie,2007; Isles,2009). Here, we identify both fibrous bodies and vascular erectile spaces in the juvenile alligator phallus using histochemical techniques to visualize surrounding structural connective tissue fibers associated with erectile tissues.
In mammals, secretions from accessory glands, such as the prostate, determine many biochemical characteristics and influence fertility-related endpoints of semen (Poiani,2006). Extratesticular factors often contribute to sperm maturation and fertilization efficiency (Amann et al.,1993; Jones et al.,2007). Mucin glycoproteins in mammalian reproductive tracts perform many functions including facilitation of gamete transport, protection against infection by acting as a physical barrier and a matrix of antimicrobial molecules, lubrication of epithelial surfaces, and prevention of tissue dehydration (Lagow et al.,1999; Linden et al.,2008). However, the distribution and functions of male reproductive tract mucins have received far less study than those of female reproductive structures (Lagow et al.,1999; Russo et al.,2006). Further, studies of seminal fluid production in nonmammalian vertebrates are relatively sparse (Holmes and Gist,2004) with the exception of accessory reproductive fluids produced by some birds (Fujihara,1992). These avian studies show that variation in seminal fluids can influence reproductive endpoints. For example, dominant male chickens can differentially allocate ejaculate size of seminal fluids and thus, in turn, affect sperm swimming velocity (Cornwallis and O'Connor,2009).
Crocodilians are not reported to possess accessory reproductive glands that contribute to semen production. However, in Caiman crocodilus, portions of the sexual duct system (epididymis and ductus deferens) show morphology and histochemical properties indicative of seminal fluid production and/or modification (Guerrero et al.,2004). Further, seminal fluids containing sperm are found in the phallic sulcus of male American alligator, Nile crocodile (Crocodylus niloticus), and dwarf crocodile (Osteolaemus tetraspis) phalli during the breeding season (Joanen and McNease,1980; Kofron,1990; Kofron and Steiner,1994). More recently, direct contributions by the phallic sulcus to seminal fluid secretions have been demonstrated in Caiman crocodilus (Cabrera et al.,2007; Cabrera and Garcia,2007). Here we expand on these observations through an investigation of sulcus and penile body glandular morphologies and associated mucin histochemical reactivity.
MATERIALS AND METHODS
Four male juvenile American alligators (Alligator mississippiensis, snout-vent lengths: 49.7–52.5 cm, body masses 2.5–4.7 kg) were collected by hand at Lake Woodruff Wildlife Refuge, Florida (Permit# WX01310) on September 10th, 2007 and transported to the University of Florida (Gainesville, Florida) for necropsy (IACUC #D525). Cloaca temperatures immediately upon collection ranged from 25.9 to 28.4°. Female alligators of a similar size were also collected and necropsied, results other than comparisons of gross phallic morphology between sexes are not reported within. Male phalluses were fixed in Bouin's solution for 24 hr, dehydrated, paraffin embedded, and serial sectioned transversely at 10 μm. Three phalluses were stained using Masson's trichrome (TC) and used for computer-aided, three-dimensional reconstructions. Masson's trichrome was used to highlight nuclei (dark purple), connective tissue (blue-green), cytoplasm, and muscle (red-orange). Every fifth serial section was imaged using an Olympus BH-2 light microscope and photographed at a fixed magnification using a Pixelink PL-B623CU 3.0 megapixel digital camera. Adjacent sections were photographed and noted for the reconstruction parameters for absent or torn sections. All images were sequentially imported into Reconstruct (Fiala,2005) and manually oriented using the proximal terminus of the sulcus as reference. Reconstructed thicknesses of program generated Boissonnat surfaces were 50 μm each. Tissue sections from the remaining phallus were alternately stained with TC, McMannus' periodic acid Schiff's (PAS), alcian blue (AB) at pH 2.5 or 1.0, or periodic acid methionine silver with an omission of gold toning (PAMS). Both PAS and AB stain mucopolysaccharides with PAS staining a variety of mucin carbohydrates (magenta) while AB selectively staining acidic mucins (blue). Further, AB (pH 2.5) will detect acidic carboxylated and/or sulfated glycoconjugates while AB (pH 1.0) is selective for sulphated acidic glycoconjugates. Alcian blue reactive tissues stained equally at either pH, therefore in the results section tissues will be reported solely as AB-reactive. PAMS staining detects collagen, reticular fibers, and some mucins (brown) (Herrera and Lott,1996).
We characterize the gross anatomy of the alligator phallus as three distinct subunits: the base, cuff, and tip (Fig. 1A,B). The base and cuff form the proximal portion of the phallus and the tip composes the distal portion. On the ventral side of the phallus, the sulcus extends from the base to the tip and its width decreases from proximal to distal (Fig. 1C). Cross-sectional analyses along the length of the phallus (Fig. 2A, oblique view of reconstruction image with transverse planes corresponding to the histology presented in Fig. 2B–D, respectively) reveal multiple stromal and epithelial tissue morphologies in the base (Plane B), cuff (Plane C), and tip (Plane D). First, we present a panoramic overview of the tissue morphologies in these regions (Fig. 2B–D) and later present individual morphologies, as highlighted by figure insets with roman numerals, in greater resolution (as shown in Figs. 3, 4, 5, 6, 7, 3–7).
At the base (Fig. 2B), the phallus presents outer base epithelium (OBE) frequently convoluted by underlying lymphatic aggregates and overlying stromal tissues, including symmetrically paired fibrous bodies (FB) of dense connective tissues with intercalated muscle fibers. Ventrally, a broad sulcus lumen (SL) is defined by a thinner sulcus epithelium (SE), when compared with the OBE. While the sulcus continues along the ventral shaft of the phallus to the distal tip of the phallus, the FB terminates within the base, proximal to the start of the cuff. The cuff region (Fig. 2C) is bounded by the outer cuff epithelium (OCE) and the SE, but also by the inner cuff epithelium (ICE) which forms through an involution of the OCE around the distal aspect of the cuff (note cuff shape in reconstruction image in Fig. 2A). This involution results in the formation of the cuff lumen (CL). Simple branched alveolar glands are subjacent to both the OCE and ICE. The SE defines a smaller SL, when compared to the SL of the base. The tip region (Fig. 2D) is bounded by an outer tip epithelium (OTE) and the SE defining the SL.
Within the cuff, tissues show distinct morphological stratification, as defined by PAS- (not shown) and PAMS-reactive connective tissue fibers of varying architecture (Fig. 3A, an elaboration of Fig. 2C inset I morphology). Glandular tissue layers (G) are intercalated in dense connective tissue fibers directly subjacent to the stratified squamous, non-keratinized OCE and ICE (Fig. 3A). Underlying the OCE and adjacent to the glandular tissues are alternating, orthogonally arranged lateral and radial fiber bundles (LFB & RFB) of PAMS-reactive connective tissues (Fig. 3A,B). Longitudinally oriented fiber bundles (LFB) are medially located within the cuff (Fig. 3A,C). Radially oriented fibers bundles (RFB) located adjacent to the ICE and associated glandular tissues and traverse a large, blood filled sinus (BS) (Fig. 3A,D).
Using digital 3D image reconstruction, we determined the shape of the BS (as defined by the zone of RFB surrounding the BS) as a single, contiguous sinus that originates in the base, expands in the cuff, and continues into the proximal half of the tip (Fig. 3E). During dissection we observed the inflatable region of the male phalli, as defined by the location BS, flushed red in comparison to other, pale phallic tissues including proximal base, distal tip, and cloaca. This flushing continued after the tissue was excised from the body (Supporting Information Fig. 1) and was not observed in female phalli collected during the same dissection.
The tissues adjacent to the sulcus in the distal base, cuff, and proximal tip regions display morphological stratification similar to that observed in the cuff (Fig. 4A, an elaboration of Fig. 2C inset II morphology). Between the stratified squamous ICE and the stratified columnar SE, connective tissues fiber bundles define a blood filled sinus. This blood sinus is contiguous with the one observed in the dorsal cuff body (Fig. 3E). PAS- (not shown) and PAMS-reactive connective tissue fibers of varying orientation and organization span the tissue (Fig. 4B). Subjacent to the glandular tissues of the ICE, fiber bundles are orthogonally arranged in radial and longitudinal orientations to the shaft of the phallus. Radial-oriented fiber bundles transverse the medially placed BS. Subjacent to the SE, smooth muscle fiber bundles are intercalated between PAMS-reactive fibrous tissues (Figs. 4B, 5D, and Supporting Information Fig. 2A).
The phallic sulcus presents varying epithelial morphologies (Fig. 5A, an elaboration of Fig. 2C inset III morphology). At the distal aspect of the sulcus, the phallus body epithelia (OBE, OCE, or OTE) invaginates and this stratified squamous epithelia transitions to a ciliated, stratified columnar epithelium (Fig. 5A–D; morphological transitions at arrowheads). The sulcus epithelium becomes increasingly convoluted deeper within the sulcus. The deepest aspect of the sulcus displays pronounced invaginations of a pseudostratified ciliated epithelium (Fig. 4A, 5B–D inset ii). Goblet cells within the SE show regional variations of PAS-, AB-, and PAMS-reactivity. The morphological transition of phallus body epithelia to SE displays PAS- and AB-reactivity. In the stratified body epithelia adjacent to the transition, PAS- and AB-reactivity is a gradient with apical epithelium staining greater than basal (Fig. 5B,C: note epithelia to the left of the arrowheads). The localization of this histochemical reactivity shifts from the cytoplasms of squamous and cuboidal cells of body epithelia to the cytoplasms of the columnar SE cells (Fig. 5 B,C respectively: note epithelia to the right of the arrowhead). Columnar cell nuclei are round to oval and basally located. At the epithelial transition to the columnar SE, PAMS-reactivity is not observed (Fig. 5D, arrowhead). Within the medial sulcus region (Fig. 5B-D, defined by inset i) the ciliated columnar epithelia cells are heterogeneously reactive to PAS, AB, and PAMS staining (greater magnification images are presented in Supporting Information Fig. 2). PAS- or AB-reactive columnar cells are more frequent than PAMS-reactive cells. Columnar cell PAS-, AB-, and PAMS-reactivity decreases deeper within the sulcus and is greatly diminished or absent from the pseudostratified ciliated epithelium of the deepest, most convoluted portion of the sulcus (Fig. 5B–D, inset ii; greater magnification images presented in Supporting Information Fig. 3).
Numerous multicellular exocrine glands are associated with the epithelia of the base, outer cuff (Figs. 2C inset IV, 3A, and 6A), inner cuff (Figs. 3A, 4A and Supporting Information Fig. 4A), and tip. These glands are more numerous on the dorsal aspect of the phallus. Histological observation and digital 3D reconstruction (Fig. 6B–D) shows mostly simple branched alveolar morphologies. Glandular cells show tall columnar cytoplasms and basally placed, round to flattened nuclei. Gland cell cytoplasms, secreted materials in the glandular lumen, and associated ducts exhibit PAS- and AB-reactivity (Fig. 6E,F, Supporting Information Fig. 4B–D). Further, these PAS- and AB-reactive glandular materials are contiguous with histochemically reactive materials located on and within adjacent epithelia (OCE: Fig. 6E,F; ICE: Supporting Information Fig. 4B–C). In these stratified epithelia cells, mucin histochemical detection is greater on the apical aspect of cells cytoplasm and this reactivity is greater in cells nearer to the apical epithelium. Thus, cells present an apically facing crescent of histochemical reactive materials. In contrast, PAMS-reactivity is localized to the gland cell cytoplasm and associated ducting and is minimally observed in the secreted materials within the glandular lumen or the apical epithelium surrounding the gland (OCE: Fig. 6I; ICE: Supporting Information Fig. 4D).
Lymphatic structures are observed subjacent to the phallic epithelia. Lymphatic aggregates (LA), identified by clustered, intensely stained basophilic round nuclei are frequently observed throughout the entire phallus subjacent to the epithelia (Fig. 7, elaboration of Fig. 2B,D insets Va,b). Lymphatic aggregates were observed in association with intracloacal and phallic petechiae- small (1–2 mm) red spots caused by minor hemorrhage, and blisters in both male and female animals (Fig. 8A,B, respectively).
We have demonstrated that phalli of immature male American alligators have a complex functional anatomy, show active exocrine gland activity, and present evidence of active immune functions. The age of wild caught alligators is difficult to ascertain due to variable growth rates between individuals (Wilkinson and Rhodes,1997). However, we can use the relative body sizes of these animals to place their phalli in a developmental context. Phalli of hatchling alligators display only a moderate sexual dimorphism in length, but this differential markedly increases during post-hatching growth (Allsteadt and Lang,1995). Juvenile alligators with snout-vent lengths ˜45 cm (slightly shorter than the snout-vent lengths of animals used in this study: 49.7–52.5 cm) possess sexually dimorphic phallus lengths (male phalli being three to four times longer than female phalli). This sexually dimorphic growth continues throughout juvenile growth and results in adult male phalli which are up to two orders of magnitude longer than female counterparts (Ziegler and Olbort,2007).
It has been hypothesized that this sexually dimorphic growth pattern is a function of differing circulating sex hormone concentrations, namely levels of 5α-dihydrotestosterone (DHT, an androgenic sex steroid hormone). In addition to ontogenetic phallus development, androgen mediated regulation of phallus size may be seasonally dynamic. In Rhea americana, phallus size positively correlated with circulating testosterone levels between breeding and non-breeding seasons (Goes et al.,2010). Therefore, the phallic morphologies presented in this manuscript are of a developing juvenile anatomy which will greatly enlarge and elaborate with increased exposure to sex hormones and sexual maturity. Little is known of the histological anatomy and cellular morphologies of adult alligator phalli. Therefore, to understand if the transition from juvenile to adult phallus morphology is just an enlargement and elaboration of the observed juvenile morphology or if fundamental structural and histochemical changes occur requires further empirical investigation.
In the functional anatomy of the alligator phallus, muscle associated with the cloaca work via the fibrous bodies to cause protrusion of the phallus from the cloaca while blood engorges the distal tissues (Ziegler and Olbort,2007). We have shown that the base of the juvenile alligator phalli contains paired fibrous bodies which will facilitate erection upon adult sexual activity. This mode of crocodilian erection is similar to those described in turtles and ostrich (Struthio camelus). Turtle phalli are composed proximally of rigid connective tissues (corpus fibrosa) and distally of erectile tissues composed of a sinus defined by vascularized connective tissues (corpora spongiosa) (Zug,1966). Similarly, the ostrich phallus presents muscles articulated to a pair of rigid fibrous bodies and a vascular body surrounded by a thick outer layer of elastic tissues that begins at the middle of the phallus, expands in volume, and extend into the tip (Montgomerie and Briskie,2007).
In turtles and mammals, engorgement of vascular spaces within multiple layers of collagen fibers arrayed in alternating, orthogonal geometries results in an erect structure that is resistant to bending during copulation (Kelly,2002,2004). Here we presented high-resolution histological images that demonstrate similar connective tissue morphology in the distal base, cuff, and tip of the alligator phallus and propose they are erectile in function. Further functional analysis of these vascular spaces will better characterize the mechanism of alligator erection and intromission.
Along with vascular spaces, smooth muscle fibers are located in tissues adjacent to each wall of the sulcus. The sulcus of Caiman crocodilus is similarly lined with putative erectile tissues and a muscularis mucosae, leading to a hypothesis that the sulcus may close into a tube during erection by way of inflation of the blood sulcus (Cabrera and Garcia,2007). Further, muscular contractions may facilitate the movement of semen along this closed sulcus. Our observations of juvenile alligator phallus morphology support this hypothesis. The highly convoluted, deepest aspect of the sulcus may act a semen conduit while the more laminar aspects of the medial to distal sulcus juxtapose with the engorgement of the adjacent blood sinuses during erection. This proposed functional difference is in line with the differing histochemical reactivity observed between the reactive distal sulcus regions and the nonreactive proximal/deep sulcus (Fig. 5, insets i and ii, respectively). We hypothesize that these two regions of the sulcus play different roles during erection, intromission, and ejaculation.
While components of the male reproductive duct system of Caiman crocodilus show epithelial secretory granules, it presents limited histochemical reactivity for mucins; alcian blue reactivity is restricted to the epididymis (Guerrero et al.,2004). In contrast, the alligator phallus shows broadly distributed exocrine glands with robust mucin histochemistry reactivity. Intraepithelial glands in the sulcus produce both neutral and acidic mucins that putatively contribute to seminal fluids and sperm quality in adult phalli. As observed in other species, the role of these mucins may include acting as a suspending media or capacitator of sperm.
The body (nonsulcus) epithelia of the base, cuff, and tip present mucin-producing simple branched alveolar glands and stratified epithelia. While better known for their roles in female reproductive tracts, mucins also play roles in male urogenital tracts (Lagow et al.,1999) and are produced by phalli of other species. For example, mucins are produced in human foreskin (Russo et al.,2006) and the apical stratified squamous epithelia of the Guinea fowl (Numida meleagris) phallus (Sasaki et al.,1983). The role that phallus-produced mucins play in the male reproductive tracts need elaboration; we hypothesize that these roles extend beyond facilitating intromission and germ cell transfer.
While many birds have lost an intromittent phallus, they retain a reduced intracloacal phallic structure which often presents paired fibrous bodies, inflatable sinuses, and a sulcus (Montgomerie and Briskie,2007; Brennan et al.,2008). The vascular bodies of the chicken (Gallus gallus) copulatory structure form a ring around the base of the ejaculatory ducts in the urodeum and present a pseudo-stratified columnar epithelium in which glands extend into the submucosa and produce acidic mucopolysccharides that contributes to fertility (Lake,1957; Tingari and Lake,1972). These mucosal secretions could be homologous with the glandular activity observed in alligator phalli as some birds with a non-intromittent phallus rely on these functional secretions.
The alligator phallus is located in the cloacal proctodeum, between the urodeum and coprodeum which sequesters urine and feces, respectively, and the external aquatic environment beyond the vent. This placement constantly exposes the phallus to both endogenous gut flora and environmental microbes. American alligators (Johnston et al.,2010) and Chinese alligators (Alligator sinenesis) (Ma et al.,2008) possess a cloacal bacteria flora that differs from, and is greater than, those isolated from the animal's aquatic habitat. The phallus is found within this bacteria-rich environment.
Alligators possess both an innate and active immune system. Alligator serum has an innate complement system that is effective against West Nile and Herpes simplex virus (Merchant et al.,2005). In this study, we observed multiple morphologies that illustrate multiple lines of immune defense in the juvenile alligator phallus. Phalli present a secreted mucus coat. Mucin glycoproteins form a physical barrier against pathogens and often have antimicrobial activities. In addition to secreted surface mucins from simple branched alveolar glands, stratified epithelial cells displayed PAS- and AB-reactivity at the apical aspects of their cytoplasms. Vaginal stratified squamous epithelia can produce PAS- and AB-reactive mucins in non-glandular, subapical cells (Gipson et al.,1995). Two mucins detected in vagina epithelium, MUC1 and MUC4, have barrier forming, antimicrobial activity and can be membrane tethered (Hjelm et al.,2010). Similarly, we hypothesize that the alligator phallus presents a mucosal barrier of adherent, membrane-anchored cell surface mucin glycoproteins of an origin different from mucins secreted from the epithelia glands.
Alligators do not possess lymph nodes, but have cell-mediated immunity mediated, in part, through lymphocytes (Zimmerman et al.,2010) and lymphoid tissues such as thymus and spleen (Tanaka and Elsey,1997; Rooney et al.,2003). Lymphoid aggregates have been documented in post-hatching alligator ovaries (Moore et al.,2008). In alligator phalli, we frequently observed interspersed intraepithelial lymphocytes in the basal aspect of epithelia and subepithelial lymphoid aggregates. Lymphoid aggregates have also been documented in male juvenile alligator phalli from zoological and farm rearing facilities (Govett et al.,2005). These aggregates were associated with petechiae and PCR detection of crocodylid herpes virus HV1 in phallic tissues, though herpes has not been confirmed as a causative agent of the aggregate or lesions. In one observation, 4.5 year-old alligators were culled from the North Carolina farm rearing facility due to West Nile virus outbreak. The animals were originally obtained from a Florida facility one week after hatching. Upon gross examination at dissection, 38% of these animals exhibited cloaca lesions. Histological analysis revealed lymphoid aggregates and PCR analysis of one female cloaca amplified a fragment of herpes virus. The intensity of lymphoid aggregates was hypothesized to positively correlate with environmental temperature, with greater clinical signs from 21 to 30°C. Cloacal temperatures of alligators within this study were within this range upon capture.
Tissues samples employed for this study were collected at Lake Woodruff, FL, a site with minimal anthropogenic environmental contamination and consistently high reproductive success (Milnes and Guillette,2008; Garrison et al.,2010). Field assessment of alligator snapping turtle (Macrochelys temminckii) health in Florida found 7 of 11 animals sampled at one site to be expressing herpes viral antibodies (Chaffin et al.,2008). We propose that the observed cloacal lesions and phallic lymphoid aggregates may be pathological, but not abnormal for wildlife populations.
This study was made possible by the continuing logistical support of our alligator research by the Florida Fish and Wildlife Conservation Commission; specifically we thank Allan Woodward for his continued assistance with fieldwork and permitting. Thank you to Dr. María del Carmen Uribe Aranzábal with expert assistance in histological analysis.