Gonadal mRNA expression levels of TGFbeta superfamily signaling factors correspond with post-hatching morphological development in American alligators

Abstract

Paracrine factor signaling regulates many aspects of vertebrate gonadal development. We investigated key ovarian and testicular morphological markers of the American alligator (Alligator mississippiensis) during the first 5 months post-hatching and correlated gonadal development with mRNA expression levels of a suite of regulatory factors. In both sexes, we observed significant morphology changes, including ovarian follicle assembly and meiotic progression of testicular germ cells. Concomitant with these changes were sexually dimorphic and ontogenetically variable mRNA expressions. In ovaries, FOXL2, aromatase, and follistatin mRNA expression was greater than in testes at all ages. At one week after hatching, we observed ovarian medullary remodeling in association with elevated activin/inhibin beta A subunit, follistatin, and aromatase mRNA expressions. Three and 5 months following hatching and concomitant with follicle assembly, ovaries showed increased mRNA expression levels of GDF9 and the mitotic factor PCNA. In testes, the activin/inhibin alpha and beta B subunit transcript levels were greater than in ovaries at all ages. Elevated testicular expression of GDF9 mRNA levels at 5 months after hatching aligned with increased spermatogenic activity. We propose that the mRNA expression levels and concomitant morphological changes observed here affect the establishment of alligator reproductive health and later fertility.

Fig. 1

Ovary of Alligator mississippiensis at 1 week (A, E), 1 month (B, F), 3 months (C, G), and 5 months (D, H) after hatching in parasaggital view (A–D) and enlarged detail (E–H). At 1 week after hatching the ovarian cortex contains nests of germ cells (N) along the coelomic (*) border. These nests contain Stage-1 oocytes (S1) in close interaction with pre-follicular cells (pF). Underlying the cortex, lacunae (L) form within degenerating medullary cords. The gonad overlies mesonephric (M) and adrenal tissues (A). At 1 month after hatching, the medullary lacunae (L) have expanded. At 3 and 5 months after hatching, the cortex contains Stage-2 (S2) and −3 oocytes (S3), displaying Balbiani bodies (B). S3 oocytes are contained in follicles with a complete complement of squamous to cuboidal follicular cells (F). Germ cell nests continue to present oogonia with mitotic chromosomes (Gch) at 5 months after hatching. Scale bars = 50 μm.

Fig. 2

Testis of Alligator mississippiensis at 1 week (A, E), 1 month (B, F), 3 months (C, G), and 5 months (D, H) after hatching in parasagittal view (A–D) and enlarged detail (E–H). The coelomic (*) border is defined by a tunica (T) that thickens with age. The gonads contain well-defined seminiferous tubules (S) lined with spermatogonia A (SA) and overlie mesonephros tissue (M) containing glomeruli (Gl) and mesonephric ducts (Md) and adrenal tissues (A). At 3 and 5 months after hatching, interstitial tissues (I) between tubules expand and present Leydig cells (Le). At 3 months after hatching, seminiferous tubules present spermatogonia A and B (SA, SB) and primary spermatocytes (PS). At 5 months after hatching, tubules contain germ cells including round spermatids (Rst). Scale bars = 50 μm.

Fig. 3

Mean (± SEM) gonadal mRNA expression of INHBA (A, B), INHBB (C, D), INHA (E, F), FST (G, H), GDF9 (I, J), PCNA (K, L), FOXL2 (M, N), CYP19A1 (O, P) in female (A, C, E, G, I, K, M, and O) and male (B, D, F, H, J, L, N, and P) alligators. Days after hatching: black bars = 7, diagonal lined bars = 30, white bars = 90, crosshatched bars = 150. All sample means were normalized using ribosomal protein L8 (RPL8) expression. Different letters above the bars of each graph of mRNA transcript levels indicate statistical significance at p < 0.05 across both ages and sexes.