THE ANATOMICAL RECORD 236:635-640 (1993)

Localization of I nsulin-Li ke Growth Factor- I-Li ke I mmunoreactivity in the Reproductive Tract of the Vitellogenic Female American

Alligator, Alligator mississippiensis CATHY COX AND LOUIS J. GUILLETTE, JR.

Department of Zoology, University of Florida, Gainesville, Florida 3261 1

ABSTRACT Insulin-like growth factor I (IGF-I) is a 70 amino acid, mi- togenic polypeptide, which, in mammals, acts through an endocrine, para- crine, and/or autocrine pathway to regulate growth and development. The primary goal of this study was to determine whether or not IGF-I-like im- munoreactivity is present in the oviduct of the vitellogenic American alli- gator, Alligator mississippiensis, and if immunoreactivity patterns vary among the three functional oviducal regions: the albumen-secreting tube region, the anterior, fiber-secreting uterus, and the posterior, calcium-se- creting uterus. Immunolocalization of IGF-I-like immunoreactivity was ac- complished using a polyclonal antihuman rabbit antiserum with an immu- noperoxidase staining system. IGF-I-like immunoreactivity was detected in all three oviducal regions of the vitellogenic alligator. The presence of IGF- I-like immunoreactivity in the oviduct suggests this hormone could func- tion in the growth and proliferation of the alligator oviduct. Furthermore, the presence of IGF-I-like immunoreactivity in the tuba1 glands, which se- crete components of the egg white, suggests that growth factors such as IGF-I may be synthesized by these glands and incorporated into the albu- men during egg formation.

Key words: Insulin-like Growth Factor I, Immunocytochemistry, AlZiga-

o 1993 Wiley-Liss, Inc.

tor mississippiensis, Oviduct, Reproduction

The functional morphology of the oviduct of the American alligator, Alligator mississippiensis, differs from that described for other reptiles (Palmer and Guil- lette, 1992). The term “oviduct” is used here to refer to the entire structure derived from the embryonic Mul- lerian duct. Turtles, lizards, snakes, and tuatara have oviducts divided into two distinct regions: an anterior tube that secretes the egg albumen and a posterior uterus that secretes the proteinaceous fibers and cal- cium of the eggshell (Guillette et al., 1988; Palmer and Guillette, 1989; Cree and Guillette, unpub data). In contrast, although neonatal alligators exhibit no re- gional specialization of the oviduct (Austin, 1990), sex- ually mature females (those > 1.8 m in length) have an oviduct differentiated into three structurally and func- tionally distinct regions: a tube region that secretes albumen and a uterus divided into an anterior, fiber- secreting region and a posterior, calcium-secreting re- gion (Palmer and Guillette, 1992). The tube region of the alligator oviduct is similar in functional morphol- ogy to the avian magnum, whereas the fiber-secreting region of the uterus and the calcium-secreting region of the uterus are homologous to the avian isthmus and shell gland, respectively. Although the tube of the al- ligator is similar in morphology to that of a bird, the proteins secreted from this region are unique when compared to birds (Palmer, 1990; Palmer and Guillette, 1992). For example, ovalbumen, a major component of 0 1993 WILEY-LISS, INC.

avian albumen, is present in alligator egg white in only trace amounts. In addition to unique egg white pro- teins, alligator albumen also contains growth factors, specifically insulin-like growth factor-I (IGF-I) (Guil- lette and Williams, 1991).

Insulin-like growth factor I (IGF-I) is a 70 amino acid, mitogenic polypeptide that regulates develop- ment and somatic growth in mammals (Schoenle et al., 1982; D’Ercole et al., 1984; Froesch et al., 1985; Sara and Hall, 1990; Simmen and Simmen, 1991). It acts in an endocrine, paracrine, andlor autocrine fashion to promote cell division, cell differentiation, and tissue morphogenesis. In mammals, the liver is a major source of IGF-I, but this polypeptide also has been iso- lated from brain, skeletal muscle, heart, and uterine tissue (Kajimoto and Rotwein, 1989; Murphy and Frie- sen, 1989).

The amino acid sequence of IGF-I is highly conserved among mammals. Daughaday and Rotwein (1989) demonstrated that 66 of 70 amino acid residues are identical among human, bovine, rat, and mouse IGF-I. Limited information concerning IGF-I structure is available for nonmammalian vertebrates (see Bern et al., 1991). Kajimoto and Rotwein (1989) isolated and

Received December 10, 1992; accepted March 12, 1993.

636 C. COX AND L.J. GUILLETTE

characterized an IGF-I cDNA from the domestic chicken (Gallus domesticus) and reported a high corre- lation between the amino acid sequences of chicken and mammalian IGF-I. Similar observations have been reported for comparisons among the peptide sequences of mammalian IGF-I and those of an amphibian, Xe- nopus laevis (Shuldiner et al., 1990), and the coho salmon, Oncorhynchus kisutch (Cao et al., 1989). The hormonal regulation of IGF-I also appears to be evolu- tionarily conserved among vertebrates, as the expres- sion of IGF-I mRNA is stimulated by growth hormone, insulin, estrogens, thyroid hormone, and prolactin (Cao et al., 1989; Kajimoto and Rotwein, 1989; Simmen and Simmen, 1990). IGF-I has been implicated as a possible mediator of estradiol-induced proliferation of the fe- male reproductive tract (Murphy and Friesen, 1989).

The role of IGF-I in the egg white is unknown, al- though this hormone has been implicated in the control of mammalian embryonic differentiation and growth (Simmen and Simmen, 1990). Little is known about the structure and function of this hormone in reptiles. Wil- son and Hintz (1982) reported no IGF-I activity in tur- tle serum based on radioreceptor assays performed with human IGF-I receptor. However, Daughaday and Rotwein (1985) demonstrated the presence of IGF-I re- activity in the sera of the turtle, Pseudemys scripta elegans, and Bautista et al. (1990) demonstrated the presence of IGF-I in the skeletal tissue of a lizard (gecko). Given its presence in the egg white of the al- ligator, one major goal of this study was to determine if IGF-I was present in the oviduct, and specifically in the tuba1 glands of the vitellogenic alligator prior to ovu- lation. If present, we wanted to determine if the pat- terns of immunoreactivity varied among the three functional regions of the oviduct.

MATERIALS AND METHODS Specimens

Eight mature, vitellogenic female alligators (Alliga- tor mississippiensis) were captured from several lakes (Griffin, Orange, Okeechobee) in central Florida (per- mit #W88063). Within 24 hours of capture, the ani- mals were anesthetized with 20 mg/kg sodium pento- barbital, and their oviducts were surgically removed. Tissues from the three major functional regions of the reproductive tract (tube, fiber-secreting uterus, and calcium-secreting uterus) were dissected and immedi- ately fixed for basic histology and immunocytochemis- try in Bouin’s fixative (see Palmer and Guillette, 1992, for details of oviducal morphology). These animals were taken as part of a larger interdisciplinary re- search project (University of Florida, US . Fish and Wildlife Service, Florida Game and Freshwater Fish Commission) on the reproductive biology of the alliga- tor.

Basic Histology Tissues were washed, dehydrated in a series of

graded ethanols, cleared in xylene, embedded in paraf- fin, and serially sectioned at 8 pm on a rotary micro- tome (Humason, 1979). Sections from each oviducal re- gion of each alligator (tube, fiber uterus, calcium uterus) were gelatin-mounted on slides. Each slide had one section from each oviducal region of a single ani- mal. Fifteen slides were prepared for each animal.

Each set of 15 slides contained adjacent sections for each oviducal region so that comparisons of individual cells and glands could be made. Two slides from each set were stained with a connective tissue staining tech- nique using hematoxylin, eosin, Alcian blue (pH 2.5 for glycosaminoglycans), fast green (for connective tissue), orange G, and Biebrich scarlet (for proteins) dyes. The remainder of the slides were used for immunocy- tochemical validation and experimentation.

lrnmunocytochernical Staining Validation. Validation of the immunocytochemical

procedure was accomplished by (1) treating control slides without primary antibody or with nonimmune normal rabbit serum (similar results were obtained from these two control procedures) for each experimen- tal slide incubated with primary antibody, (2) saturat- ing the IGF-I antibody with insulin (1 kg IGF-I anti- body/1,000 pl TBS pH 7.4 obtained from Sigma) prior to incubation of the tissues with primary antibody (pos- itive control), (3) saturating the IGF-I antibody with human recombinant IGF-I (1 pg IGF-I antibody/1,000 k1 TBS pH 7.4 obtained from USB, Cleveland, OH) prior to incubation of the tissues with primary anti- body (negative control), and (4) exposing pig oviducal tissue known to be positive for IGF-I as a control with each set of gator slides. Additionally, the results ob- tained from the immunoperoxidase technique were compared with the results obtained from another stain- ing technique, immunogold silver staining (IntenSE M, Janssen Biotech).

Experimentation. The antihuman IGF-I antibody (polyclonal anti-IGF-I/somatomedin C rabbit antise- rum) was received as a gift from Drs. Louis Underwood and Judson J . Van Wyk (Division of Pediatric Endocri- nology, University of North Carolina at Chapel Hill; distributed for research use by the Hormone Distribu- tion Program of the National Institute of Diabetes and Digestive and Kidney Diseases through National Hor- mone and Pituitary Program). Immunocytochemical staining was performed using an immunoperoxidase system (Vectastain Elite ABC Kit, Vector Laborato- ries, Burlingame, CAI. Tissue sections were deparaf- finized in xylene and hydrated in graded alcohols. Slides were then treated with 3.0% H20? for 30 min- utes to block endogenous peroxidase activity, washed with tris-buffered saline (TBS, pH 7.4), and incubated with primary antibody diluted in 1.5% normal goat- blocking serum (experimental sections) or without pri- mary antibody (control sections). Tissues were then washed with TBS (pH 7.4) for 10 minutes, incubated with biotinylated secondary antibody, and washed with TBS (pH 7.4) for 10 minutes. Sections were then incu- bated with Vectastain Elite ABC reagent for 30 min- utes, washed with TBS (pH 7.4) for 10 minutes, and incubated with a peroxidase substrate solution (0.1% diaminobenzidine tetrachloride made in 0.1 M tris buffer, pH 7.2 and mixed with equal amounts of 0.02% H,02! for 5 minutes to localize the bound primary an- tibodies. Finally, slides were washed in tap water and coverslips were mounted. After staining, slides were viewed with differential interference contrast micros- copy and photographed. Patterns of immunoreactivity were determined by viewing slides directly and photo- graphs of all sections were then used to support these

ALLIGATOR OVIDUCAL IGF-I 637

determinations of distribution and relative staining in- tensity.

RESULTS Validation

Control sections that were incubated without anti- IGF-I or with nonimmune normal rabbit serum exhib- ited minimal background or no immunostaining. Sim- ilarly, control sections incubated in the presence of anti-IGF-I, which had been preabsorbed with human recombinant IGF-I, exhibited a significant reduction in immunostaining intensity, whereas sections incubated with anti-IGF-I preabsorbed with insulin showed no reduction in staining intensity. Similar staining distri- bution and intensity were obtained by both immuno- cytochemical techniques: the immunoperoxidase and immunogold silver staining procedures.

Experimental Results Whereas different histological characteristics were

observed in the three functionally distinct oviducal re- gions of the vitellogenic alligators, IGF-I immunoreac- tivity exhibited similar patterns in the tube, fiber uterus, and calcium uterus.

Tube. The luminal epithelium of the tube region, which stains intensely with Alcian blue for glycosami- noglycans (GAGS), contains two types of tall, simple columnar cells: ciliated cells with apical nuclei and nonciliated microvillous secretory cells with central, or sometimes basal, nuclei (see Palmer and Guillette, 1992). The glands within the tube region are branched acinar. Immunostaining for IGF-I was observed within the cells comprising the glands and in the myometrium (Fig. 1). Intense staining at the apical aspect of the epithelial cells also was noted in the tube (Fig. 1). Lit- tle or no staining was observed in the connective tissue surrounding the glands.

Fiber uterus. The luminal epithelium of the fiber-se- creting region of the uterus consists of simple columnar cells similar to those described for the tube region. However, these cells are lower in height and stain less intensely with Alcian blue. The glands are branched tubular. Immunoreactivity for IGF-I occurred in the epithelium, glands, and myometrium with little or no immunoreactivity in the stroma (Fig. 2). Immunostain- ing results from the fiber region of the oviduct were difficult to interpret due to high levels of background (nonspecific) staining. This high background staining may have resulted from either high levels of endoge- nous peroxidase activity or high levels of avidin within this oviducal region. Nonspecific staining was most prevalent in the glands.

Calcium uterus. The luminal epithelium of the cal- cium-secreting region of the uterus consists of low co- lumnar cells as described for the fiber uterus; however, very few cells stain positively with Alcian blue. The endometrial glands of this posterior uterine region are branched tubular. Immunostaining for IGF-I occurred in the epithelial cells, glands, and myometrium with little to no staining in the stroma (Fig. 3).

DISCUSSION Insulin-like growth factor I (IGF-I)-like immunore-

active material is present in the oviduct of the mature, female American alligator, Alligator mississippiensis,

Fig. 1 . Immunocytochemical localization of IGF-I-like material in the tube region of the oviduct from a vitellogenic alligator. A. Basic histology: epithelium (E), gland (G), stroma (S), and myometrium (M). B. Experimental tissue incubated with polyclonal antihuman IGF-I rabbit antiserum. Note immunoreactivity in apical tips of epithelial cells, arrow. C. Control tissue incubated with nonimmune normal rabbit serum.

during vitellogenesis. Positive immunostaining for IGF-I-like material in the alligator oviduct using a polyclonal mammalian IGF-I antibody suggests a high degree of homology between alligator IGF-I and mam- malian IGF-I, as reported for the chicken, frog, and

638 C. COX AND L.J. GUILLETTE

Fig. 2. Immunocytochemical localization of IGF-I-like material in the fiber uterus of the oviduct from a vitellogenic alligator. A. Basic histology: epithelium (E), gland (G), stroma (S). B. Experimental tis- sue incubated with polyclonal antihuman IGF-I rabbit antiserum. Note immunoreactivity in apical tips of epithelial cells, arrow. C. Control tissue incubated with nonimmune normal rabbit serum.

Fig. 3. Immunocytochemical localization of IGF-I-like material in the calcium uterus of the oviduct from a vitellogenic alligator. A. Basic histology: epithelium (E), gland (G), stroma (S). B. Experimen- tal tissue incubated with polyclonal antihuman IGF-I rabbit antise- rum. C. Control tissue incubated with nonimmune normal rabbit se- rum.

salmon (Cao et al., 1989; Kajimoto and Rotwein, 1989; Shuldiner et al., 1990). Furthermore, data that demon- strate growth-hormone regulated expression of IGF-I, in the coho salmon, Oncorhynchus kzsutch (Cao et al., 1989), domestic chicken, Gallus domesticus (Kajimoto

and Rotwein, 1989), and in several mammals (Murphy and Friesen, 1986) indicate that the conserved primary structure and growth hormone-regulated expression of IGF-I may occur throughout the vertebrates. Chan et al. (1990) cloned a hybrid insuliniinsulin-like growth

ALLIGATOR OVIDUCAL IGF-I 639

factor cDNA from amphioxus (Branchiostoma califor- niensis), a primitive cephalochordate that occupies a key position in chordate phylogeny, which may repre- sent a transitional form connecting insulin and IGF. IGF probably arose at an early stage in vertebrate ev- olution from an ancestral insulin gene. This high de- gree of conservation of structure and hormone-regu- lated expression further supports a fundamental role for IGF-I in general aspects of vertebrate growth, de- velopment, and differentiation.

Hormonal regulation of the development of the rep- tilian oviduct, as in other vertebrates, involves the ste- roid hormones estradiol-17P and progesterone. During follicular development of the alligator, ovarian follicles increase in size and secrete estradiol in response to pituitary gonadotropin secretion (Lance, 1989). Estra- diol secreted by the developing follicles serves two ba- sic functions in reptiles. First, estradiol stimulates the liver to synthesize the yolk precursor protein vitelloge- nin, which is then secreted into the blood, carried to the ovaries, taken up by the oocytes, and transformed into yolk (Ho et al., 1982). Second, estradiol stimulates growth and differentiation of the reproductive tract in preparation for oviposition (Mead et al., 1981; Jones and Guillete, 1982; Guillette et al., 1991). Estrogen stimulation of oviducal growth in alligators (Forbes, 1938), iguanid lizards (Callard et al., 1972), and snakes (Mead et al., 1981) indicates that estrogens serve a similar function in all reptiles.

Three types of glands are found in the reproductive tract of alligators. Glands found in the tube region of the oviduct are responsible for the secretion of albu- men, whereas glands found in the uterus are responsi- ble for either fiber or calcium secretion. These glands are present in greatly reduced numbers during repro- ductive quiescence. The numbers of glands increase dramatically as follicles begin to grow, suggesting that recruitment of glands is stimulated by estradiol, which is secreted in large quantities by growing follicles. These glands are derived from the luminal epithelium of the oviduct. The glands of the tube develop as the tubal epithelium invaginates to form deep pockets lined by secretory cells. The uterine glands form as the uterine epithelium invaginates, but these glands come to lie deep within the stroma in both the fiber uterus and the calcium uterus.

The response of the oviduct to estradiol includes cel- lular hypertrophy as well as hyperplasia as described above. Eliciting this response in vivo has not been dif- ficult; however, problems have arisen during attempts to elicit an estrogen response in cultured oviducal tis- sue. The observation that stromal tissue is necessary for estradiol responsiveness supports the idea that paracrine growth factors are involved in the mediation of estrogen-induced cellular growth and differentiation (Cooke et al., 1986; Cunha and Young, 1992). This was demonstrated clearly in recent studies showing that the growth hormone, epidermal growth factor (EGF), was capable of eliciting most of the effects attributed to estrogens in ovariectomized mice (Nelson et al., 1991). Further, treatment of mice with anti-EGF abolished most of the estrogenic response in the oviduct. Al- though EGF clearly mediates many estrogen-induced responses in the reproductive tract, other growth fac- tors have been isolated from reproductive tract tissue.

Simmen and Simmen (1991) reviewed the literature and reported that transforming growth factor, insulin- like growth factor I and 11, and epidermal growth factor were all implicated in endometrial cell proliferation and differentiation.

In addition to the possible role of IGF-I in the prolif- eration and differentiation of the alligator oviduct, the presence of IGF-I-like immunoreactivity at the apical aspects of the epithelial cells of the tube region and within the tubal glands suggests that the IGF-I incor- porated into the egg white (Guillette and Williams, 1991) is derived from the oviduct. Consequently it may play a critical role in embryonic development. The presence of growth factors such as IGF-I in the egg white provides an important model for studying the biological role of growth factors during early embry- onic development. Unlike mammals or other vivipa- rous vertebrates, the oviparous female must provide all of the growth factors, or their mRNAs, required for early development either in the yolk or albumen prior to oviposition.

ACKNOWLEDGMENTS We thank A. Woodward, H.F. Percival, G. Masson,

and K. Rice for assistance in collecting alligators; Dr. Fuller Bazer and his staff for the use of their surgical facilities and expertise; and Dr. Howard Bern for valu- able comments on this manuscript. This research was supported in part by a grant from the Florida Game and Freshwater Fish Commission to L.J.G.

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