Oviducal Anatomy and Sperm Storage Structures in Lizards

Oviducal Anatomy and Sperm Storage Structures in Lizards ORLANDO CUELLAR Texas Technological College, Lubbock, Texas ABSTRACT Gross and histological examination of lizard oviducts was made in 11 species of the family Iguanidae, and in one species of each of the families Gekkonidae and Eublepharidae. Lizard oviducts are bound dorsally by a mesentery which is continuous with the peritoneum, and ventrally by a smooth muscle band which extends from the segment of the vagina to the base of the infundibular ostium. The musculature of the vagina consists of an inner circular smooth muscle layer which is thickened ly, and an outer longitudinal layer which is arranged into longitudinal folds at about the utero-vaginal transition. In iguanid lizards the vaginal mucosa is arranged into longitudinal folds that extend the entire length of the vagina. Posteriorly, the folds are high and reduced in number. Anteriorly, they decrease gradually in height and become more numerous. In Phylloductylus homo- Eepidurus fold height and number remain essentially constant through the vagina. Seminal receptacles in the iguanids occur principally in the segment of the vagina. Receptacles in P. homolepidurus (Gekkonidae) and Coleonyx variegatus (Eublepharidae) appear to be confined to the tube between the uterus and the infundibulum. Most receptacles are located adjacent to the oviducal mesentery and to the smooth muscle band. The presence of seminal receptacles in the oviducts of reptiles was reported in colubrid (Fox, '6), viperid (Saint Girons, '7), and both typhlopid and leptotyphlopid (Fox and Dessauer, '62) snakes. As yet, no receptacles have been reported for turtles; however, observations on delayed fertilization indicate that this group may also possess structures capable of storing sperm. Receptacles have been described in lizards of the family Chameleonidae (Saint Girons, '62) and in the green anole, Anolis carolinensis (Fox, '63). The occurrence of receptacles in members of such divergent families suggests that these structures may occur widely among lizards. This study was undertaken with the following objectives: ( 1 ) to conduct an exploratory survey for the presence of seminal receptacles in a variety of lizard species; (2) to make a detailed examination of the vaginal portion of the oviduct. Recent studies have directed much attention to discussions of the seminal receptacles and have neglected detailed descriptions of the vagina in species in which the receptacles have been found (Saint Girons, '62; Fox, '63). Earlier studies of the anatomy and histology of oviducts have been J. MORPK., 119: 7-20. relatively superficial, particularly in descriptions of the vaginal segment (Brooks, '0; Giersberg, '22). MATERIALS AND METHODS Histological studies Histological examination of the oviducts was made in the following numbers of animals from the indicated localities : Crotaphytus collaris (3), Seeloporus undu- Zatus (4), Colorado National Monument, Colorado; Uta stansburiana ( ), Phrynosoma cornutum (l), Winkler County, Texas; Holbrookia propinqua ( 3 ), Padre Island, Texas; Coleonyx variegatus (2), Imperial County, California; Holbroohia elegans ( l ), San Carlos Bay, Sonora, Mexico. All others were collected from islands in the Gulf of California, Mexico: Callisaurus dracwnoides ( 3), Sator grandaevus (4), Cerralvo : U t u palmeri ( ), San Pedro Martir; Uta squamata (3), Santa Catalina; Urosaurus microscutatus (2), San Jose; Phyllodactylus homolepidurus (), San Pedro Nolasco. Lizard oviducts are delicate structures and easily damaged in the fresh state. In order to simplify dissection and reduce damage, the oviducts were fixed in situ by 7

8 ORLANDO CUELLAR preserving the animals in Bouin s solution. All viscera and large ovarian eggs were removed prior to fixation to avoid distorting the normal shape and position of the oviducts during the hardening process. The oviducts were removed by cutting the vagina at the level where it merges with the colon and enters the cloaca. Oviducts were dehydrated in Dioxane and imbedded in paraffin. Transverse as well as longitudinal sections were made at 10 IJ and stained with Delafields hemotoxylin and eosin. Measurements of fold height and tubule diameter were made with an ocular micrometer and measurements of tubule length by counting the number of sections included by the tubules. Fold height and number were measured in five alternate sections in each of the,, and regions of the vagina. Where more than 20 folds were present, each section was divided into quadrants and the height measured from five folds in each division. Measurements in tables 1-3 are based on a single specimen per species and should not be interpreted to denote interspecific or intergeneric differences. RESULTS Gross anatomy Lizard oviducts are paired structures and consist of an infundibulum with tube, a uterus, and a vagina. Dorsally, the oviduct is suspended by a mesentery which is continuous with the peritoneum and binds the vaginal and uterine segments to the middorsal wall and the infundibular segment to the doso-lateral wall. The membrane tapers ly and terminates at the level of the axillary region. At the infundibular region of the right oviduct, the membrane is attached to a narrow projection of the right liver lobe. Ventrally, the oviduct is bound by a smooth muscle band which extends from the vagina to the border of the infundibular ostium. In the segment of the vagina the band is imbedded in the thick musculature of that region. Anteriorly, the band projects from the vagina and is surrounded by the serosa and the outer longitudinal smooth muscle layer which serve to suspend it from the vaginal wall (figs. 2, 3, ). His tolog y The wall of the vagina consists of the mucosa, an inner circular and an outer longitudinal layer of smooth muscle, and the serosa which is attached to the outer muscle layer. Only the circular layer is present in the most segment of the vagina, where it forms an enlargement due to its greater thickness in this region (fig. 1 ). The muscle fibers of the circular layer are organized into bundles (fig. 6). The longitudinal layer forms a smooth sheet over the and regions of the vagina, but ly it is arranged into longitudinal folds (fig. 4) which are highest at the utero-vaginal transition and diminish in the uterus. The mucosa is arranged into longitudinal folds that extend the entire length of the vagina. All specimens studied except Phyllodactylus homolepidurus exhibit a consistent decrease in height of folds from to (table 2). All of the iguanids show a characteristic increase in number of folds from to (table 1). The three specimens of P. homolepidurus differ by possessing a relatively uniform number and height of folds throughout the vagina (tables 1, 2) and a thickened circular muscle layer at the utero-vaginal transition. Also, the number of folds in the segment are markedly reduced with respect to C. variegatus and the iguanids. Although C. variegatus shows only a slight increase in number of folds from to, the gradual decrease in fold height and the number of folds in the segment are more typical of the iguanid pattern. The increase in number results from the bifurcation of branched folds, which become most numerous in the, and gradually begin to converge, and diminish as fusion occurs in the utero-vaginal transition (fig. 3 ). Finally, the folds disappear in the uterus, where the epithelium flattens and forms an even layer. Posteriorly, the lumen of the vagina is reduced to a narrow opening by the higher folds in that region (fig. 1). Anteriorly, the lumen gradually widens by a decrease

LIZARD OVIDUCTS AND SEMINAL RECEPTACLES 9 TABLE 1 Comparisons of the number of folds i n different regions of the vagina in Iguanid, Gekkonid and Eublepharid lizards Species Iguanidae Callisaurus draconoides Crotaphytus collaris Holbrookia elegans Holbrookia propinqua Pkrynosoma cornutum Sator grandaevus Sceloporus undulatus Utrc palmeri Uta squamata Uta stansburiana Gekkonidae Pkyllodactylus homolepidurus Phyllodactylus homolepidurus Phyllodactylus homolepidurus Eublepharidae Coleonyx variegntus Coleonyx variegatus Dierent Mean reeons Sections number of of t,he counted folds per vaglna section 18 114 34 18 73 10 33 32 2 2 18 22 46 9 23 41 23 9 22 2 87 22 1 62 22 21 83 30 1 11 10 10 9 9 22 23 1 19 26 Range 18 107-7 2-41 18-19 69-7 9-11 33-34 2-60 30-33 24-2 46-6 1-20 20-2 42-0 6-1 1 23-24 40-42 9-14 20-2 88-106 11-14 24-27 77-91 21-2 13-1 60-63 19-2 7 21 7-93 22-37 11- -1 11 10 10 9 9 2 1-23 22-24 14-16 18-19 24-28 11-14

10 ORLANDO CUELLAR TABLE 2 Comparisons of the height of folds in different regions of the vagina in Iguanid, Gekkonid and Eublepharid lizards - Different Mean regions Sections height of S.E. Species of the counted folds per vagina section 1 Iguanidae Callisaurus draconoides Crotaphytus collaris Holbrookia elegans Holbrookia propinqua Phyrynosoma cornutum Sator grandaeuus Sceloporus undulatus Uta palmeri Uta squamata Uta stansburiana Eublepharidae Coleonyx variegatus Coleonyx variegatus Gekkonidae Phyllodactylus homolepidurus Phyllodactylus homodepidurus Phyllodactylus homolepidurus Fosterior 1 Height expressed in ocular units (3.6 fi per ocular unit). - 16.7. 3.7 11.1 9.9.9 14.8 10.3 10.2 11.9 7.3 6.2 22.7 6.0 4.2 14. 9.0. 16. 9.3 4.9 13.2 6.2 4.6 8.6.2 4.7 7.4 3.9 4.0 7.6.2 4.9 8.1.1 4. 8.6 7.4 10. 10.3 14.8 10.1 11.6 6.3 11.6 0.44 0.20 0.16 0.46 0.24 0.23 0.74 0.0 0.41 0.2 0.20 0.30 1.14 0.32 0.14 0.60 0.22 0.20 0.33 0.30 0.46 0.3 0.23 0.09 0.49 0.11 0.10 0.27 0. 0.11 0.30 0.22 0.17 0.33 0.1 0.16 0.30 0.38 0.32 0.48 0.46 0.4 0.41 0.33 0.30

LIZARD OVIDUCTS AND SEMINAL RECEPTACLES 11 in fold height and a general increase in vaginal diameter. The widest diameter is attained in the (fig. 2). Anteriorly, the vagina narrows but not as extensively as in the region (fig. 3 ). Posteriorly and medially, the vaginal epithelium is uniformly ciliated. At the segment of the vagina the ciliated epithelium becomes interrupted by narrow free surfaces. At the transitional zone the free surface increases greatly, and the cilia become restricted to a few cells around the duct. Single cell rows are thus formed which run through the entire uterus. These clumps of cilia generally assume a conical shape with the apex facing the lumen. Seminal receptacles All seminal receptacles examined consist of simple and branched tubules formed by a single layer of epithelial cells. Cilia line the cells (figs. 8, 11, 14) and occur throughout the entire tubule. Serial sections show that the tubules taper distally (table 3 ) due to a reduction in cell number, and terminate blindly in the wall of the oviduct. Most extend ly but some are occasionally found directed ly. They are not always straight and frequently twist and assume spiral configurations. In some instances where several tubules originate closely and extend more or less parallel to each other, their branches seem to interconnect. Tubule length varies considerably in all specimens (table 3). Seminal receptacles in the iguanids occur principally in the and parts of the vagina (table 4), but are most numerous at the utero-vaginal transition and frequently occur in the presence of TABLE 3 Size of seminal receptacles in Iguanid, Gelzkonid and Eublepharid lizards ::&;, Mean tubule diameter Mean Species tubule Range ~~~~~~s Proximal Medial Distal length Iguanidae Callisaurus draconoides Crotaphytus collaris Holbrookia elegans Holbrokia propinqua Phynosoma cornutum Sator grandaevus Sceloporus undulatus U t a palmeri Uta squamata Uta stansburiana Gekkonidae Phyllodactylus homolepidurus 1 od, outside diameter. 2 id, inside diameter. 6 6 4 7 7 4 4 7 8 7 od' 6 76 61 id2 40 47 36 od 1 83 72 id 6 32 32 od 94 79 61 id 8 47 29 od 82 6 43 id 40 29 14 od 100 83 61 id 8 40 18 od 90 79 8 id 8 4 29 od 86 86 83 id 8 8 40 od 80 76 8 id 0 47 28 od 76 76 47 id 36 40 14 od 14 14 i d 7 7 7 od 6 0 36 id 36 18 11 88 9 90 167 10 7 60 7 77 60-160 0-10 80-110 90-0 60-300 130-260 0-200 0-100 30-0 40-80 0-1 10

ORLANDO CUELLAR Species Iguanidae Callisaurus draconoides Crotaphytus collaris Holbrookia elegans Holbrookia propinqua Phynjnosoma cornutum Sator grandaevus Sceloporous undulatus Uta. pnlmeri Uta squamata L7ta stansburiana Urosaurus microscutatus Gekkonidae Phyllodactylus homolepidurus TABLE 4 Location of seminal receptacles i n the oviduct Oviduct regions Vaginal regions Vagina Infundibulum Posterior Middle Anterior.. x................ Eublepharidae Coleonyx variegatus...... x, presence of receptacles;.., absence of receptacles... shell glands at the margin of the uterus. They occur irregularly on the wall but are usually confined to a narrow strip conforming to the line of attachment of the mesentery on one side and the smooth muscle band on the other (figs., 7, 9). Tubules may arise in the following manners: ( 1) from invaginations at the base of the folds (figs. 10, 11); (2) from the fusion of two folds (figs. -14); (3) from the apex of the folds; and ( 4 ) from invaginations in the smooth, unfolded epithelium. The tubules may occur in the following locations: ( 1 ) within the lumen of the vagina (In this arrangement most of the wall of the tubule is surrounded by a layer of epithelial cells. The lamina propia lies beneath the epithelium and surrounds the entire tubule.) (fig. 14); (2) between the epithelium and the circular muscle layer (The epithelium does not surround the tubules. The lamina propria borders the wall facing the epithelium and does not appear to surround the entire tubule.) (fig. 11); (3) between the inner circular and outer longitudinal muscle layers; ( 4 ) within the muscle fibers of both layers; () on the outside wall of the longitudinal layer (fig. 1). In the last three conditions the tubules form small bulges which project to the outside of the vaginal wall. Heretofore, receptacles had been described only from the vagina; however, it is now apparent that they also occur in the infundibulum and the tube. Although in the iguanids none of the sections revealed sperm in the infundibular region, seminal receptacles do occur in that region in at least two species (table 4 and fig. 9). Receptacles in P. homolepidurus and Coleonyx variegatus do not appear to occur in the vagina and are strictly confined to the infundibular region. In both species, sperm were present in the receptacles. Concentration of receptacles in the site adjacent to the mesentery or muscle band is also apparent in the infundibular region (figs. 7-9). All specimens contained varying amounts of sperm in the receptacles. Several probably had a recent copulation as evidenced by large masses in the lumen and between the vaginal folds. Such spermatozoa generally assume an orderly distribution with the heads oriented toward the base of the folds (fig. 16). In the receptacles sperm are oriented distally and seem to occur most abundantly toward the end of the tubules. Many sperm appear to penetrate the cells by means of long, filamentous acrosomes. This condition is also manifest in the epithelial cells of the folds (fig. 16). In Sator

LIZARD OVIDUCTS AND SEMINAL RECEPTACLES 13 grundaevus (fig. 17), and to a lesser degree in Urosaurus microscutatus and Uta squamata, the entire heads of many spermatozoa occur intracellularly in the epithelial cells of the folds and are oriented toward the basement membrane, which appears to mark the level of penetration. In all cases where sperm bundles are in close proximity to the epithelial border or where sperm have penetrated the cells, the ciliary border is completely disintegrated and unrecognizable as such (fig. 17). This phenomenon is restricted to the vaginal folds. DISCUSSION Vaginal anatomy Anatomically, the vagina is the most complex division in lizard oviducts. It does not appear to be distinctive in all lizards as is shown by the departure of P. homolepidurus from the iguanid arrangement. The paucity of information on this segment of the oviduct makes it difficult to compare this study with others. Giersberg ('22) described briefly the vagina of 'hative lizards." His descriptions are vague and do not consider and changes in the musculature, vaginal diameter, or number of folds. Fox ('63) states that the folds caudal to the two-thirds of the vagina bifurcate ly in A. carolinensis. This does not seem likely in view of the fact that all the genera of iguanids treated in this report show that bifurcation occurs ly in the vaginal segment. Furthermore, a bifurcation would result in an increase in the number of folds in the region. The description of the vagina in chamaeleons (Saint Girons, '62) indicates that spatial changes in the musculature and number of folds are very similar to those described in this report and supports the contention that bifurcation occurs ly. Fox ('63) described the vagina in A. carolinensis as being divided into an vaginal tube and a vaginal pouch. I was unable to detect vaginal pouches in the specimens investigated in this report; however, the thickened circular muscle in the segment results in a slight enlargement which may have been interpreted as a pouch. Spatial differences in the arrangement of the folds, masculature, and vaginal diameter must reflect anatomical specializations in the different vaginal regions. Normally, the tall folds which fill most of the lumen in the segment may act as a valve to partially seal the vagina from the cloaca1 chamber. The thickened circular muscle layer surrounding the tall folds may act as a sphincter to regulate the diameter of that region. During the passage of eggs, the vagina distends greatly due to the pressure exerted on the wall by the passing eggs. This distention may result in a flattening of the folds due to a spreading of the troughs and consequent depression of the crests. The overall effect would be an increase in vaginal diameter to accomodate the eggs. After the passage of eggs, the vagina would return to its normal preovulatory condition by a compression of the folds. It seems reasonable to suppose that a narrow region, such as the segment of the vagina, would have to stretch to a greater degree than the wider segment. Thus, the tall folds in the segment would serve to facilitate the increased distention and, upon return to the pre-ovulatory stage, to fill the lumen as indicated above. Seminal receptacles The tubules described in chamaeleons (Saint Girons, '62) and in A. carolinensis (Fox, '63) do not differ histologically from those described in this report, except for the absence of cilia in the distal end. Fox ('63) noted that cilia were absent where the sperm were stored. Fox ('6) observed that in Thamnophis sirtalis the cytoplasm in the cells of the alveolar receptacles appears to diminish where sperm have invaded the vacuoles. Although vacuoles do not appear to be present in the tubular receptacles of lizards, a similar invasion of the cytoplasm was observed in many of the cells lining the tubules and especially in the epithelial cells of the mucosa. Saint Girons ('62) did not detect secretory activity in the tubules of chamaeleons

14 ORLANDO CUELLAR but found a mucous secretion which may come from the vaginal epithelium. He states that seminal receptacles only protect the sperm from mechanical damage and that their prolonged survival is assured by the mucous secretions of the vaginal epithelium. The observations of intracellular sperm in this report and the apparent penetration of cells in the tubules by unusually fine and pointed sperm heads leaves doubt to the role of receptacles as simple escapes for mechanical damage. In Crotalus viridis (Ludwig and Rahn, '43) sperm are stored in the vagina but in the absence of seminal receptacles. Although Rahn ('42) showed that C. viridis females retain motile sperm through the winter, it was not demonstrated that such sperm can survive beyond parturition. The fact that sperm can remain viable over a period of months in the lumen of the vagina, suggests that receptacles are not a necessity for prolonged storage and that the vaginal epithelium may be instrumental in the nourishment of sperm. Whether or not sperm are nourished from secretions of the vaginal epithelium, from secretions of the receptacles, by invading the cellular cytoplasm, or any combination of these remains a matter for speculation; however, recent evidence suggests that the role of seminal receptacles is undoubtedly instrumental for prolonged activity and function of stored sperm (Cuellar, in press). The question of how sperm are stimulated to activity at the time of ovulation has been treated by Fox ('6), who suggested that pressure exerted on the walls by the passing eggs would force out the sperm. This mechanism could easily operate in snakes with infundibular receptacles; but in the iguanids, where vaginal receptacles contain the stored sperm, the eggs are already shelled at the time of passage through the vagina and fertilization must occur before this stage. Therefore, the exertion of direct pressure on the vaginal wall is not a feasible mechanism in such forms. In iguanids the relative aggregation of receptacles adjacent to the muscle band and the oviducal mesentery suggests that these sites are more suited for the storage and delivery of sperm than are other areas along the oviducal wall. Presumably, at ovulation the muscle band and mesentery act as stress areas; and since both structures extend from the infundibulum through the vagina, the receptacles may be indirectly stimulated. The possibility of a chemical stimulator cannot be disregarded, but as yet no information is available in this regard. ACKNOWLEDGMENTS I am grateful to Dr. Kozo Imahori, Louis Irwin, Chester Rowell, and Dr. Russel W. Strandtmann for advice and assistance with the preparation of the plates. Many thanks are due to Royce Ballinger, Gary Ferguson, Charles McKinney, Jim Platz, and Mike Sabbath for help in the collection of many specimens. I am particularly grateful to my major professor, Dr. Donald W. Tinkle, for his patience, and valuable suggestions during the course of this study. This work was supported by a National Science Foundation grant GB-181 to Dr. Donald W. Tinkle. LITERATURE CITED Brooks, B. 190 Anatomy of the internal urogenital organs of certain North American lizards. Trans. Tex. Acad. Sci., 8: 23-38. Cuellar, 0. Delayed Fertilization in the lizard Uta stnnsbun'ana. Copeia, (in press). Fox, W. 196 Seminal receptacles of snakes. Anat. Rec., 4: 19-40. - 1963 Special tubules for sperm storage in female lizards. Nature, 198: 00-01. Fox, W., and H. C. Dessauer 1962 The single right oviduct and other urogenital structures of female Typhlops and Leptotyphlops. Copeia, 90-97. Giersberg, H. 1922 Untersuchungen uber Physiologie und Histologie des Eileiters der Reptilien und Vogel; nebst einem Beitrag zur Fasergenese. Ztschr. wiss,. Zool., 0: 1-97. Ludwig, M., and H. Rahn 1943 Sperm storage and copulatory adjustment in the prairie rattlesnake. Copeia, 1-18. Rahn, H. 1942 The reproductive cycle of the prairie rattler. Copeia, 223-240. Saint Girons, H. 197 Le cycle sexuel chez Viperu aspis (L.) d a m I'ouest de la France. Bull, Biol., 91: 294-30. - 1962 Presence de recptacles seminaux chex les camkleons. Beaufortia, 9: 16-172.

PLATES

PLATE 1 EPLANATION OF FIGURES 1 2 3 4 6 7 8 9 Holbrookiu propinqua. Cross section through vaginal region. Note thick circular muscle layer, tall folds, and reduced lumen. 48. Holbrookia propinqua. Cross section through vaginal region. Note reduction in fold height, increase in fold number, increase in diameter, and longitudinal muscle band (MB). x 48. Holbrookia propinqua. Cross section through utero-vaginal transition. Note reduction in fold height and number, flattening of the epithelium, and scattered uterine glands (UG). x 48. Utu palrneri. Cross section through vaginal region. Note convoluted outer longitudinal muscle layer (COM) and inner circular muscle layer (ICM). 208. Phrynosoma cornutnm. Cross section through vaginal region. Note seminal receptacles (SR) adjacent to longitudinal muscle band. 48. Holbroohia elegans. Longitudinal section through vaginal region. Note bundle arrangement of circular layer fibers (CLF). 48. Phyllodactylus homolepidurus. Cross section through infundibulum. Note seminal receptacle complex ( S R C ) adjacent to mesentery. 48. Enlarged view of seminal receptacle complex from figure 7. 208. Holbrookia propinqua. Cross section through the tube. Note seminal receptacle complex adjacent to the muscle band. x 48. 16

LIZARD OVIDUCTS A N D SEMINAL RECEPTACLES Orlando Cuellar PLATE 1 17

PLATE 2 EPLANATION OF FIGURES 10-1 1 Holbrookia elegnns. Cross section through vaginal region. Note sequence of seminal receptacle formation by invagination ( I ) of the epithelium. x 208. -14 Sator y r m d u e v u s. Cross section through vaginal region, Note sequence of seminal receptacle formation by the fusion of folds. Also note sperm bundles ( S B ) in the lumen. x 208. 1 Utcr squamata. Cross section through vaginal region. Note seminal receptacle outside of vaginal wall. x 208, 16 Phyllodactylus homolepidurus. Cross section through vaginal region. Note association of sperm heads with epithelial cells of the folds. 469. 17 Sator grandaevus. Cross section through vaginal region. Note absence of cilia on folds and complete penetration of sperm in epithelial cells. >; 469. 18

LIZARD OVIDUCTS AND SEMINAL RECEPTACLES Orlando Cuellar PLATE 2 19