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Keywords:

  • penis;
  • alligator;
  • erectile tissues;
  • cloaca

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGEMENTS
  7. LITERATURE CITED

The intromittent organs of most amniotes contain variable-volume hydrostatic skeletons that are stored in a flexible state and inflate with fluid before or during copulation. However, the penis in male crocodilians is notable because its shaft does not seem to change either its shape or bending stiffness as blood enters its vascular spaces before copulation. Here I report that crocodilians may have evolved a mechanism for penile shaft erection that does not require inflation and detumescence. Dissections of the cloaca in sexually mature male American alligators (Alligator mississippiensis) show that the cross section of the proximal shaft of the alligator penis contains dense collagenous tissues that do not significantly change shape when fluid is added to the central vascular space. The large amount of collagen in the wall and central space of the alligator penis stiffen the structure so it can be simply everted for copulation and rapidly retracted at its completion. Because no muscles insert directly onto the penis, eversion and retraction must be produced indirectly. My results suggest that the contraction of paired levator cloacae muscles around the anterior end of the cloaca rotates the penis out of the cloacal opening and strains the ligamentum rami that connect the base of the penis to the ischia. When the cloacal muscles relax, the elastic recoil of the ligamentum rami can return the penis to its original position inside the cloaca. Anat Rec, 296:488–494, 2013. © 2013 Wiley Periodicals, Inc.

Male crocodilians use a specialized intromittent organ for copulation which has been called either a phallus (King, 1981; Moore et al., 2012) or a penis (Powell, 2000; Cabrera and Garcia, 2004; Cabrera et al., 2007) in the published literature. It is an unpaired organ found on the ventral wall of the cloaca, with a shape approximating a laterally compressed cylinder (Ziegler and Olbort, 2007). A medial groove on its surface called the sulcus spermaticus (King, 1981) conveys sperm to the female's cloaca: a pair of spermatic ducts open at the proximal end of the sulcus (Kuchel and Franklin, 2000), and sperm travels along its length to the distal end of the glans (Ziegler and Olbort, 2007; Moore et al., 2012). It is not clear whether these tissues are homologous with the tissues that make up the mammalian penis and glans: both alligator and mammalian tissues develop from a genital tubercle in the cloacal region, suggesting homology (Seifert et al., 2009), but recent amniote phylogenies place the lineage containing archosaurs as the sister group to the Rhynchocephalia (which either lack a penis or have lateral hemipenes) (Lyson et al., 2012; Crawford et al., 2012) suggesting a penis could have evolved independently in mammals and archosaurs.

In transverse section, the crocodilian penile shaft contains a layer of dense tissue arranged around a central, comparatively open vascular space (Cabrera and Garcia, 2004; Cabrera et al., 2007; Ziegler and Olbort, 2007). Blood is thought to enter the vascular spaces of the crocodilian penis before copulation because both the glans and the tissue around the sulcus spermaticus inflate when males prepare to mate (Gadow, 1887; Moore et al., 2012). But unlike other amniote penises, the crocodilian penile shaft does not appear to significantly change either its shape or bending stiffness (Gadow, 1887; Ziegler and Olbort, 2007) during this process.

In most amniotes inflation is a key element of penile function, producing changes in the shape, size, or bending stiffness of the structure. Before erection, inflatable penile tissue may be protruding or protrusible, as in mammals and turtles (Eckstein and Zuckerman, 1956; Zug, 1966; Miller and Dinkelacker, 2007), or fully involuted as in squamates and waterfowl (Dowling and Savage, 1960; King, 1981; Ruiz and Wade, 2002; Brennan et al., 2010). In each of these taxa the penile tissue changes shape as fluid enters its central space: an associated increase in bending stiffness occurs in mammal (Kelly, 1999), turtle (Miller and Dinkelacker, 2007), and squamate (Pope, 1941; Conner and Crews, 1980) penises, but not in Anseriformes, whose penises inflate directly into the female reproductive tract and remain flexible as they increase in length (Brennan et al., 2010).

It is not clear why blood entering the penile vascular spaces would not change the shape of the crocodilian penile shaft as it does in other amniotes. Tissue in the shaft region has been described as fibrous in the spectacled caiman (Caiman crocodilus crocodilus) (Cabrera and Garcia, 2004; Cabrera et al., 2007) and cartilaginous in the Nile crocodile (Crocodylus niloticus) (Lankester and Hernandez-Divers, 2005), suggesting that a large proportion of the structure is collagen. It is possible that the arrangement and density of collagen fibers in the penile shaft prevents it from changing shape as blood enters the vascular spaces: densely collagenous tissues in the penises of turtles (Zug, 1966) and mammalian artiodactyls (Frandson et al., 2009) and cetaceans (Pabst et al., 1998) are known to restrict shape changes during erection.

Powell (2000) and Whitaker et al. (1980) suggest that penile protrusion in crocodilians is primarily caused by contractions of the cloacal musculature. But while it is true that muscular actions are involved in protrusion and retraction in the erectile system of mammals (Gadow, 1887; Nickel et al., 1973), turtles (Gadow, 1887), squamates (Ruiz and Wade, 2002), and birds (King, 1981; Brennan and Prum, 2011), all of these taxa have one or more muscular insertions on the penile crurae or shaft. It is not known how crocodilians would produce similar behaviors as none of their cloacal muscles have insertion sites on the penis (Reese, 1915).

Here I examine the penis and surrounding cloacal musculature of four male American alligators (Alligator mississippiensis) and propose a functional model for crocodilian penile eversion and retraction.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGEMENTS
  7. LITERATURE CITED

Three adult male American alligators (Alligator mississippiensis) and one subadult male were donated to this study after euthanasia by staff biologists at the Rockefeller Wildlife Refuge (Grand Chenier, LA). Adult animals were more than 2 m in length (Snout-to-tail lengths of the animals were 1.64, 2.15, 2.40, and 4.64 m), suggesting that they were mature and in breeding condition (Lance, 1989).

Gross dissections of the pelvic and cloacal region of each animal were used to assess the anatomical relationships of the penis, cloaca, and surrounding musculature. Two animals were dissected with a ventral approach, one from a lateral approach, and one with a dorsal approach. In each case, the penis, cloaca, and associated muscles and connective tissues were removed and placed in 10% buffered neutral formalin; detailed dissections of these tissues were completed after they spent a minimum of 48 h in fixative. To assess the dimensional changes of the tissue during erection, the proximal ends of two penises were tied shut with cotton thread before fixation and the vascular space of the penis was injected with physiological saline to simulate erection. Injection continued until fluid leaked out its proximal opening, indicating that the space was at or near maximum volume.

Transverse sections that included both the penile wall and medial spongy tissue were cut from the mid-shaft region of one penis on the side opposite the sulcus spermaticus. Sections were dehydrated in ethanol, cleared in Hemo-De (Fisher Scientific, Fair Lawn, NJ) and embedded in paraffin. Prepared samples were cut at 10 µm with a rotary microtome in both the transverse and sagittal planes and stained for collagen and muscle with Milligan's Trichrome (Kiernan, 1990).

The proportion of dense tissues inside the vascular space was quantified using automated thresholding. A series of grayscale images spanning the diameter of a transverse section of a penis were photographed using the 10× objective and opened in Image J 1.43u (National Institutes of Health). The collagenous area of each image was measured by thresholding the white spaces in each image and subtracting them from the total area of the section; the percentage of image area made up of dense materials was calculated from these measures.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGEMENTS
  7. LITERATURE CITED

The Alligator Penis is Highly Collagenous and does not Inflate to Erection

The Alligator mississippiensis penis can be divided into three regions: a proximal pair of crurae located dorsal to the ischia which fuse to form the free penile shaft and the distal glans (Fig. 1). The shaft of the penis is nearly circular in cross section (Table 1). The sulcus spermaticus is a V-shaped groove that extends the full length of the penile shaft and glans. The penile shaft is not straight: the crurae and shaft form a sigmoid curve proximal to the origin of the free part of the penis. Other than differences in size, there was no obvious variation in penile anatomy between adult and subadult males.

image

Figure 1. Lateral view of an isolated American alligator (Alligator mississippiensis) penis showing the locations of the crurae, shaft, and glans. The ischium is sketched in to show the relative position of the penis relative to the pelvis when retracted; left is rostral, right is caudal. Scale bar = 2 cm.

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Table 1. American alligator body length and penis dimensions
  Penile diameters (mm)
Body length (m)Shaft length (mm)DorsoventralLateral
Adult   
2.1564.2417.8614.69
2.4077.3816.9417.23
4.64112.5721.8628.01
Subadult   
1.6426.956.626.77
mean (±SE)70.29 ± 17.6815.82 ± 3.2516.68 ± 4.39

In cross section the penile shaft contains a central region that has been interpreted as a vascular space (King, 1981) surrounded by a thin wall containing smooth muscle and fibers with the staining characteristics of collagen (Fig. 2A,B). Collagen fibers in the wall are slightly crimped and arranged in regular parallel bundles around the outside edge of the vascular space (Fig. 2B). These fibers run parallel to the long axis of the penis; there is no evidence of a complimentary layer of circumferential fibers in the wall, suggesting that the axial orthogonal array that reinforces mammalian and turtle penises during erection (Kelly, 2004) is not present in alligators.

image

Figure 2. Microanatomy of the American alligator penis. A. Transverse section through the shaft region of the penis showing dense collagen inside the vascular space (V) and the dorsal groove of the sulcus spermaticus (SS). Inset boxes on the section refer to histological sections in B and C. Scale bar = 0.5 cm. B. Photomicrograph of a sagittal section of wall tissue from the penile shaft stained with Milligan's trichrome. The wall is made up primarily of collagen (blue) with some smooth muscle fibers (red) arranged along the length of the penis. Collagen fibers on the inside of the wall bend medially into the vascular space. C. Photomicrograph of a transverse section of tissue from the vascular space in the penile shaft. The vascular space is filled with collagen fibers. Scale bar for B and C = 0.25 mm. D. Percentage of the area inside the vascular space made up of dense tissues from samples between the sulcus spermaticus and lateral penile wall.

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A large proportion of the central vascular space of the penile shaft is filled with collagen (Fig. 2C): sampling regions across the diameter of the vascular space show that up to 93% of the cross sectional area is comprised of collagen fibers and other dense materials (Fig. 2D). In cross-section, collagen fibers in this region appear to originate in the wall tissue around the dorsal and lateral sides of the penis and insert at or near the wall of the sulcus spermaticus. Extensive anastomoses connect the vascular spaces of the penile shaft and the sulcus spermaticus making them functionally one space.

Attempts to artificially inflate the proximal penile shaft with physiological saline produced no significant change in either its length or diameter. This behavior is quite different from the tissues in the glans and those associated with the sulcus spermaticus, both of which are known to inflate before copulation (Gadow, 1887; Moore et al., 2012). Interestingly, the glans and sulcus tissues expanded only slightly during artificial inflation. Because artificial inflation was induced directly through the vascular space of the penile shaft, it is possible that the fluids that inflate the sulcus spermaticus and glans may enter their vascular spaces by a different route.

The Alligator Penis is Everted for Copulation by Muscular Contractions

When retracted, the alligator penis rests on the ventral surface of the cloaca, where it fills much of the internal volume of the proctodeum. Its distal end is located immediately dorsal to the cloacal opening; its proximal end splits into a pair of crurae at the cranial end of the cloaca, immediately dorsal to the ischia. The sulcus spermaticus originates at the base of the penile shaft and extends along the dorsal surface of the retracted penis to its distal tip. The outer surface of the penis is covered with the same epithelial tissue as the inner surface of the cloaca.

The penis is anchored to the ischia by connective tissue structures attached to the proximal end of the penile shaft. One pair, called the ligamentum rami by Powell (2000), originate on the dorsal midline of the caudal end of the ischia (Fig. 3A), then pass between the penile crurae to insert on the dorsal surface of each crus (Fig. 3B). In addition, a short tendon originates on the ventral surface of the caudal end of each ischium and inserts on the ventral surface of the penile shaft immediately distal to the crurae (Fig. 3C).

image

Figure 3. Penile crurae anatomy of the American alligator. In each photo, penile tissues are shown in their retracted position; left is rostral, right is caudal. The free part of the penis has been removed. A. Lateral view with crurae partly reflected to show the origin of the ligamentum rami on the dorsocaudal portion of the ischia. B. Dorsal view of the crurae showing the insertion of the ligamentum rami on the dorsal surface of the crurae. C. Lateral view of the crurae showing the origin and insertion of the ventral penile tendon and the location of the levator cloacae muscles (cut). Key: penile crurae (Cr), ischium (I), sulcus spermaticus (SS), ligamentum rami (Lr); levator cloacae muscle (LC); ventral penile tendon (Vpt). Scale bar = 1 cm.

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A paired muscle, the levator cloacae (Powell, 2000) originates on the ventrocaudal surface of the ischium and flanks the cloacal wall laterally before attaching to the dorsal midline of the cloaca, dorsal and slightly distal to the convergence of the penile crurae (Fig. 3A,C). Some fibers of the levator cloacae attach to the lateral surfaces of the ventral penile tendon. Caudal to the levator cloacae, a group of intrinsic cloacal muscles cover the cloacal wall (Fig. 4). These muscles are in turn surrounded by a layer of connective tissue that separates them from the more superficial cloacal sphincter muscles as well as the abdominal, leg, and tail musculature. Pulling the m. levator cloacae along its line of action everts the penis from the cloacal opening; releasing them produces rapid penile retraction. Pulling other cloacal muscles along their lines of action has no obvious effect on penile position.

image

Figure 4. Deep cloacal muscles of the American alligator in lateral view. The penis is retracted, left is rostral, right is caudal. The superficial cloacal sphincter muscles and tail muscles have been removed, exposing the light colored intrinsic cloacal muscles and darker levator cloacae muscle. The position of the ischium, penile crurae and cloacal vent are indicated. Key: ischium (I), penile crurae (Cr); levator cloacae muscle (LC); intrinsic cloacal muscles (IntC); vent (V) Scale bar = 2 cm.

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Upon eversion, the base of the penis rotates dorsocaudally ∼90° and the ligamentum rami are put into tension. The sigmoid curve in the penile shaft means this rotation shifts the distal tip of the penis to point cranially (Fig. 5). The sulcus spermaticus is located on the ventral midline of the everted penis. The cloaca is compressed cranially and dorsoventrally, and its wall and associated sphincter muscles are displaced laterally and ventrally by the rotated penile crurae.

image

Figure 5. Schematic showing the change in position of the American alligator penis during eversion. Left is rostral, right is caudal. When the penis rotates ∼90°, the distal tip of the penis points cranially and the ligamentum rami are placed in tension. Key: penile crurae (Cr), ischium (I), ligamentum rami (Lr); ventral penile tendon (Vpt). Scale bar = 2 cm.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGEMENTS
  7. LITERATURE CITED

Although the A. mississippiensis penis contains a blood-vascular system that can inflate the tissues of the sulcus spermaticus and glans (Moore et al., 2012), it also contains a dense three-dimensional network of collagen fibers inside its vascular space and wall that prevents significant changes in the overall length or diameter of the penile shaft. Thus, unlike other amniotes, alligators cannot inflate their penile shaft to erection with either blood or lymphatic fluid. Instead, their penis has a set shape that resists bending, and is simply everted for copulation and retracted at its completion. It is likely that penile eversion is produced by the indirect action of cloacal muscles and penile retraction by elastic recoil.

Effects of Collagen Fiber Arrangement in the Alligator Penis

The alligator penile shaft does not significantly change length or diameter before copulation, even though the expansion of the contiguous sulcus and glans tissues indicate that blood enters its vascular system. Its restricted tissue expansion can be attributed to the small degree of collagen fiber crimping inside the penile wall and vascular space. Crimped fibers let collagen-reinforced tissues extend under load (Orton and Brodie, 1987; Brainerd, 1994; Kelly, 1999): folded fibers are initially compliant to extension, but resist it when they straighten and are put into tension.

Thus, relatively small amounts of expansion are sufficient to put the collagen fibers in alligator penile tissue into tension. Collagen fibers in the outer wall of the shaft region are arranged parallel to the long axis of the penis: increases in tissue length will therefore put these fibers into tension and resist further lengthwise expansion of the tissue. Collagen fibers also span the shaft's vascular space and connect its lateral and dorsal walls. Radially oriented fibers have been shown to restrict the expansion of penile diameter in mammals (Kelly, 1999); similarly, relatively small increases in alligator penis diameter can put the collagen fibers inside its vascular space into tension and resist expansion. The net effect is a penile shaft that does not straighten when blood enters its vascular space, but instead maintains its size and sigmoidal shape as the glans tissue inflates.

Restricted radial expansion during erection means that there is little change in the second moment of area of the alligator penis. Second moment of area is one factor contributing to a beam's resistance to bending or flexural stiffness; it describes the distribution of structural material around a bending plane (Wainwright et al., 1976; Ennos, 2012). A larger second moment of area increases a structure's resistance to bending. If second moment of area does not significantly change during erection, it suggests that tissue stiffness, the second variable contributing to flexural stiffness (Ennos, 2012), has a larger effect on the mechanical behavior of the alligator penis in bending.

Increasing collagen fiber density in a tissue raises the intrinsic stiffness of that tissue (Billiar, 2011). Therefore, the high concentration of collagen fibers inside the vascular space of the alligator penile shaft makes those tissues stiffer and increases the overall flexural stiffness of the penile shaft. Similar tissues are found in the corpus fibrosum of turtles (Zug, 1966; McDowell, 1983) and the fibroelastic penises of artiodactyls and cetaceans (Slijper, 1966; Frandson et al., 2009): these structures are notably less extensible and stiffer in bending than the inflatable tissues of either the turtle corpus spongiosum or the vascular type penises found in most mammals. However, neither of these structures are completely rigid before erection. In both turtles and mammals maximum penile rigidity is the result of an interaction between increased fluid pressure in the vascular space and wall tissue that is reinforced with collagen fibers arranged orthogonally to the long axis of the penis (Kelly, 1999). It is possible that changes in vascular pressure play a role in increasing the flexural stiffness of the alligator penile shaft, but the absence of orthogonally arranged collagen fibers to reinforce the wall tissue against inflationary stresses suggests this effect is minor. Instead, the alligator penis seems to be stiff in the absence of inflation due to the density and arrangement of collagen fibers in its walls and vascular space. Bending tests of alligator penile tissue would help determine the relative importance of wall stiffness and internal pressure during erection and copulation. A high and stable bending stiffness may be an important factor in crocodilian breeding behavior, as intromission typically occurs underwater and may be repeated several times in succession (Joanen and McNease, 1972) and copulation is prolonged, lasting ∼2–4 min in the American alligator (Alligator mississippiensis) (Joanen and McNease, 1972), American crocodile (Garrick and Lang, 1977), spectacled caiman (Caiman crocodilus) (Staton and Dixon, 1977) and an average of 58 seconds in the Nile crocodile (Crocodylus niloticus) (Modha, 1967).

Eversion, not Inflation: The Mechanism of Penis Protrusion in Alligators

Because the alligator penile shaft does not inflate, muscular actions are required to evert it from the cloaca. Moore (pers. com.) reports that spontaneous penile eversion can occur during the dissection of fresh alligator carcasses when a metal instrument depolarizes pelvic nerves and triggers muscle contraction. Muscular involvement in penile erection is common in amniotes: mammals (Bassett, 1961; Nickel et al., 1973), turtles (Gadow, 1887), squamates (Ruiz and Wade, 2002) and ratites (King, 1981) have retractor muscles that insert on the dorsal or lateral surfaces of the erectile bodies and act to pull the exposed penis into the body. Squamates and ratites also have muscles associated with penile eversion. In squamates, the transversus penis muscles wrap around the ventral surface of each hemipene and act to evert the reproductive organs (Gadow, 1887; Ruiz and Wade, 2002). In ratites the penis is protruded by the levator phalli, a pair of muscles attached to the lateral surfaces of the penile crurae (King, 1981; Brennan and Prum, 2011). However, there are no muscle insertions on the surface of the alligator penis, suggesting that its eversion is effected indirectly by part of the surrounding cloacal musculature.

I propose that actions of the m. levator cloacae evert and rotate the penis. Although these muscles are lateral to the cloacal wall, they form an arch around the portion of the penis that is firmly attached to the inner wall of the proctodeum. Contraction of the m. levator cloacae should therefore pull the walls of the proctodeum cranially and compress it dorsoventrally, putting compressive forces on the dorsal side of the penis and pushing its distal end out of the vent. In addition, each levator cloaca muscle sends fibers to insert on a lateral surface of the ventral penile tendon. This suggests that their contraction can pull the tendon cranially and rotate the penis relative to the ischia. Because the penile shaft is curved, a small rotation can significantly displace the tip of the penis. As suggested by Powell (2000), penile rotation would also stretch the ligamentum rami and store energy in tension; upon relaxation of the levator cloacae these structures could rapidly return to their original length and pull the penis back to its original position inside the cloaca.

Powell (2000) suggested that the alligator penis is everted by elements of the intrinsic cloacal musculature, but I found that pulling these muscles along their lines of action did not produce penile eversion. Moreover, these muscles are pale in color compared to the dark red levator cloacae (Fig. 4), suggesting that the intrinsic cloacal muscles contain a large number of fast glycolytic muscle fibers while the levator cloacae contain many myoglobin-rich slow oxidative fibers (Liem et al., 2001). If true, this difference implies that the levator cloacae are optimized for the isometric contractions that would hold the penis in its everted state for long periods of time while the intrinsic cloacal muscles are better adapted for rapidly stabilizing the penis during copulation. It is also possible that intrinsic cloacal muscles contract in concert with the levator cloacae during penile eversion. Histochemical studies of the cloacal musculature, materials testing of their tendons and ligaments, and electromyography of individual muscles during natural or artificially stimulated penile eversion and retraction would help resolve these questions.

ACKNOWLEDGEMENTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGEMENTS
  7. LITERATURE CITED

This study would not have been possible without the generous donation of alligator tissue from Ruth Elsey and the staff of the Rockefeller Wildlife Refuge (Grand Chenier, Louisiana). Thanks also go to Lynn Bengston for her assistance with the preparation of histological specimens. Discussions with Valentine Lance, Brandon Moore, and Patricia Brennan were invaluable in the development of this manuscript, and the author is grateful for the comments from two anonymous reviewers that helped improve it.

LITERATURE CITED

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGEMENTS
  7. LITERATURE CITED
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