JOBNAME: 101#1 96 PAGE: 1 SESS: 4 OUTPUT: Wed Jun 12 20:05:05 1996 /xypage/worksmart/tsp000/66754g/17pu General and Comparative Endocrinology 101, 32–42 (1996) Article No. 0005 Reduction in Penis Size and Plasma Testosterone Concentrations in Juvenile Alligators Living in a Contaminated Environment Louis J. Guillette Jr.,* Daniel B. Pickford,* D. Andrew Crain,* Andrew A. Rooney,* and H. Franklin Percival† *Department of Zoology, 223 Bartram Hall, University of Florida, Gainesville, Florida 32611; and †National Biological Service, Florida Cooperative Fish and Wildlife Research Unit, Department of Wildlife Ecology and Conservation, University of Florida, Gainesville, Florida 32611 Accepted September 19, 1995 The development of the male reproductive ducts and drotestosterone. These sex steroids are responsible for external genitalia in vertebrates is dependent on el- the differentiation of the Wolffian duct into the male evated androgen concentrations during embryonic de- internal duct system as well as the formation of the velopment and the period of postnatal growth. We have external genitalia in reptiles (see Raynaud and Pieau, observed that a population of juvenile alligators living 1985). During embryonic development in alligators, on Lake Apopka exhibit significantly smaller penis size secretion of androgens and Müllerian-inhibiting hor- (24% average decrease) and lower plasma concentra- mone from the testis produces an internal anatomy tions of testosterone (70% lower concentrations) when similar to that commonly described in other reptiles compared to animals of similar size on Lake Woodruff. (Austin, 1989, 1990). In addition to embryologically In addition to smaller phalli, no relationship exists be- organizing the male reproductive system, androgens tween plasma testosterone concentrations and penile are also responsible for stimulating the ontogenetic size in males from Lake Apopka, whereas a positive re- development of this system during juvenile (especially lationship exists for males from Lake Woodruff. The during puberty) and adult periods (Raynaud and alligators on Lake Apopka are known to have elevated Pieau, 1985). Additionally, seasonal fluctuations in the concentrations of the antiandrogenic DDT breakdown secretory activity and size of the testis and glands of product p,p؅-DDE stored in their fat. We suggest a num- the reproductive system are androgen dependent ber of hypotheses that could explain the modification in (Licht, 1984; Raynaud and Pieau, 1985). the phenotype of the juvenile male living in Lake Reptiles exhibit three distinctly different patterns of Apopka. These modifications in phenotype include a copulatory organ development, as the squamates (liz- smaller penis size, lower plasma androgen concentra- ards and snakes) have paired hemipenes, the turtles tions, and lack of responsiveness of the penis to the and crocodiles (includes alligators and caiman) have a plasma androgens present. © 1996 Academic Press, Inc. single penis, and the tuatara have no copulatory organ (Raynaud and Pieau, 1985). There has been an exten- sive debate on the homologies of the various copula- tory organs in reptiles over the last century but a con- In vertebrates, the development of the male internal clusion is still forthcoming. The available data suggest reproductive ducts and external genitalia is largely de- that the embryological development of the hemipenes pendent on the androgens testosterone and dihy- in squamates is less complex than that seen in turtles 0016-6480/96 $18.00 Copyright © 1996 by Academic Press, Inc. 32 All rights of reproduction in any form reserved. JOBNAME: 101#1 96 PAGE: 2 SESS: 2 OUTPUT: Wed Jun 12 20:05:05 1996 /xypage/worksmart/tsp000/66754g/17pu Guillette et al. 33 (for a review see Raynaud and Pieau, 1985). Little is growth in juvenile female alligators (Pickford, 1995). It known of the embryological development of the copu- should be noted that, in alligators, incubation tem- latory organ in crocodilians. It is apparent that the perature also influences the size of the clitoris in fe- crocodilian males and females have a homologous males, with females derived from eggs incubated at structure generally similar in gross morphology. It has near male temperatures exhibiting larger clitorides been termed the cliteropenis by some workers (see than females from eggs incubated at lower tempera- Raynaud and Pieau, 1985; Allsteadt and Lang, 1995), tures (Allsteadt and Lang, 1995). However, the size, whereas others use the term penis or phallus for this shape, and coloration of the phallic structures in structure in males to denote its functional role (Forbes, 3-month-old juvenile males and females are distinc- 1939; Ramaswani and Jacob, 1965). Even in light of the tive, allowing the correct diagnosis of sex 96% of the possible lack of homology between squamate hemipe- time (Allsteadt and Lang, 1995). Thus, phallus size nes and the chelonian or crocodilian penis, all avail- could represent an obvious marker of abnormal an- able studies indicate that the steroid environment dur- drogen concentrations or functioning in reptiles as has ing embryonic development directly influences the been reported for mammals. development of the copulatory organ. That is, growth A number of environmental contaminants act as cel- and differentiation of the penile anlagen in some rep- lular signal transduction modifiers (Colborn et al., tilian species is stimulated by the presence of andro- 1993; McLachlan, 1993; McLachlan et al., 1992). These gens, as reported for eutherian mammals, whereas in contaminants interact with various cellular recep- other species phallic development is inhibited by es- tors—genetic transcription factors—so as to act as en- trogens, as observed in birds (Raynaud and Pieau, docrine system agonists or antagonists. The vast ma- 1985). jority of the compounds studied to date appear to bind Androgens are essential for normal maturation and to the vertebrate estrogen receptor, acting as xenoes- growth of the male reproductive system in reptiles. trogens or antiestrogens (McLachlan, 1981, 1993; Safe Seasonal growth of the testis produces elevated et al., 1991; Soto et al., 1992, 1994; Peterson et al., 1993). plasma concentrations of testosterone that are directly Exposure to these compounds during embryonic de- correlated with increased reproductive tract weight velopment can have either catastrophic (e.g., mortal- and synthesis of protein secretory products (Licht, ity, cancer) or subtle (e.g., changes in enzyme func- 1984; Raynaud and Pieau, 1985). Exogenous androgen tion) effects as they are capable of modifying the or- treatment of orchidectomized males reestablished re- ganization of differentiating cells and organs (for productive tract growth and secretory activity. Previ- discussion, see Guillette et al., 1995a). ous studies have demonstrated that phallus growth is A series of recent studies suggests that endocrine- androgen dependent in alligators (Forbes, 1939) and disrupting contaminants can interact with other cellu- crocodilians (Ramaswani and Jacob, 1965), as ob- lar receptor types, such as the androgen receptor served in other reptiles (Raynaud and Pieau, 1985) and (Gray et al., 1993; Kelce et al., 1994). For example, the in mammals (van Teinhoven, 1983). Interestingly, Ra- fungicide vinclozolin is not an active endocrine sys- maswani and Jacob (1965) observed that the penis of tem antagonist until it is metabolized to produce two juvenile Indian mugger crocodile (Crocodylus palustris) compounds that act as antiandrogens. Exposure of de- exhibited the greatest androgen responsiveness of any veloping rats to various doses of vinclozolin via ma- male reproductive organ they examined, which in- ternal exposure produced a series of developmental cluded the secondary sexual ducts, the Wolffian duct, abnormalities of the reproductive system in male off- and the renal sex segment of the kidney. Likewise, spring, including cryptorchidism, cleft phallus, hypo- recent studies have shown that significant penis spadias, and strophic seminal vesicles and prostate growth in juvenile alligators can be induced by exog- glands (Gray et al., 1993; Kelce et al., 1994). enous treatment with pharmacological levels of testos- Abnormalities of the male reproductive system have terone or dihydrotestosterone (DHT) (Jeff Lang, per- been reported in wildlife living in wetlands known to sonal communication), whereas treatment with DHT be contaminated with endocrine-disrupting chemicals at lower concentrations stimulated no significant (for a review see Colborn et al., 1993). For example, Copyright © 1996 by Academic Press, Inc. All rights of reproduction in any form reserved. JOBNAME: 101#1 96 PAGE: 3 SESS: 2 OUTPUT: Wed Jun 12 20:05:05 1996 /xypage/worksmart/tsp000/66754g/17pu 34 Sex Characteristics of Alligators in Polluted Water male alligators hatched from eggs collected from a and a control lake. As penis size is dependent on cir- contaminated lake in Florida, Lake Apopka, exhibit a culating androgen concentrations during fetal and ju- number of developmental abnormalities such as de- venile development, it represents an excellent marker pressed plasma testosterone concentrations, elevated of normal or abnormal steroid-induced development. testicular estradiol synthesis in vitro, and aberrant cel- lular structures in the seminiferous tubules (Guillette et al., 1994, 1995b). The underlying cause of these ab- MATERIALS AND METHODS normalities, as well as the reported developmental problems in females,