Immunolocalization of aromatase and estrogen and androgen receptors in the gonads and mesonephros of Podocnemis expansa during the first year of life Maria Fabiele Silva Oliveira, Layla Ianca Queiroz Rocha, Lucas Castanhola Dias, André de Macedo Medeiros, Moacir Franco de Oliveira, et al. Animal Reproduction, 2026 The endocrine regulation of testicular and ovarian development during early life in chelonians remains poorly understood, despite major morphophysiological changes occurring in this period. This study investigated the immunolocalization of estrogen (ER1 and ER2), androgen (AR), and aromatase (AROM) in the gonads and mesonephros of Podocnemis expansa during the first year after hatching (n = 5; males: 1, 3, 12 months; females: 2, 4 months). After euthanasia, gonads and mesonephros were collected, fixed in 10% buffered formaldehyde, and processed for immunohistochemistry. The intensity of immunoreactions varied according to age and sex, but not with tissue distribution. AROM, AR, ER1, and ER2 were detected in both gonads and mesonephros, confirming the persistence of steroidogenic and steroid-responsive activity after hatching. In males, AROM showed weak to moderate labeling in germ and interstitial cells of seminiferous tubules, whereas ER2 expression predominated in spermatogonia and interstitial endocrine cells. In females, AROM and AR were moderately expressed in the germinal epithelium and oogonia at two months, decreasing at four months, while ER2 persisted in follicles and oogonia. These findings suggest a dynamic endocrine environment influencing posthatch gonadal maturation, with the mesonephros acting as a transient extragonadal source of steroids. Despite the small, sex-unbalanced sample (due to lack of dimorphism), the results provide valuable baseline information and support future quantitative and functional studies on the reproductive endocrinology of P. expansa and other chelonians with temperature-dependent sex determination.
Gonadal Morphology of Stillborn Hawksbill Sea Turtle (Eretmochelys imbricata) Hatchlings Emanuel Lucas Bezerra Rocha, Lucas Gabriel Verissimo Pinheiro da Silva, Isadora Natália Rocha Barreto, Marcela dos Santos Magalhães, Lucas Castanhola Dias, et al. Microscopy and Microanalysis, 2024 This study aimed to investigate sexual dimorphism in stillborn hawksbill sea turtles (Eretmochelys imbricata) through gonadal morphological characterizations. Macroscopic, light microscopy, and transmission electron analyses were performed for 30 gonad-mesonephros complexes. Female gonads were spindle-shaped and present a translucent whitish appearance with a grainy texture. Male gonads were approximately ovoid with a smooth opaque white surface. A primary sexual difference concerns different marrow structures, with females presenting organized cellularity featuring oocytes, lacunae, and blood vessels, while males presented a distinct organizational medulla pattern marked by testicular cords extending throughout the gonad length. Ultrastructurally, female’s stroma presented interstitial cells and an abundant cytoplasm rich in electrodense droplets and large oval germline cells, with a conspicuous and noncentral nucleus. Males, on the other hand, presented testicular cord cells containing small amounts of heterochromatin and approximately triangular apical and basal cytoplasms with an evident nucleolus characteristic of support cells. Additionally, there were cells with a large spherical nucleus compared with the cell size and a relatively scarce cytoplasm, identified as gonocytes. These findings indicate that macroscopic, microscopic, and ultrastructural evaluations are effective and reliable techniques for the sexual identification of stillborn E. imbricata hatchlings.
Pronephros and mesonephros characterization during the embryonic development of the giant South American river turtle, Podocnemis expansa (Podocnemididae: Testudines) Marcela dos Santos Magalhães, Lucas Castanhola Dias, Moacir Franco de Oliveira, Antônio Sebben, Carlos Eduardo B. de Moura Anatomical Record, 2023 This study aimed to describe pronephros and mesonephros morphology during the embryonic development of Podocnemis expansa. Eggs were collected on an artificial beach at Balbina, Amazonas, Brazil, during the entire incubation period (mean of 59 days). The kidney‐gonad complex was processed using light microscopy and the mesonephros using transmission electron microscopy. The pronephros was present for the first time on stage 4, composed of external glomeruli devoid of a capsule, protruding into the coelomic cavity, and internally composed of a capillary network. The pronephros degenerated after development stage 15. The first sign of the appearance of the mesonephros occurred around stage 8, indicated by the early formation of renal corpuscles. The mesonephros comprised an renal corpuscles, neck segment, proximal tubule, intermediate segment, distal tubule, collector tubule, and collector duct. Ultrastructural analysis of the mesonephros brush border was done in the proximal tubule, and the presence of cells with structural characters indicative of secretory activity was detected in the juxtatubular region. Renal corpuscles and proximal tubules were the main components that underwent morphological alterations during mesonephros degeneration. The pronephros is a transient kidney, and the mesonephros became the functional embryonic kidney in P. expansa. Mesonephros degeneration occurs in the cranial‐caudal direction, and histologically, the degeneration is identified by changes in the morphology of the renal corpuscle and proximal tubule. However, the mesonephros is still present after hatching.
Heart morphology during the embryonic development of Podocnemis unifilis Trosquel 1948 (Testudines: Podocnemididae) Layla Ianca Queiroz Rocha, Maria Fabiele da Silva Oliveira, Lucas Castanhola Dias, Moacir Franco de Oliveira, Carlos Eduardo Bezerra de Moura, et al. Anatomical Record, 2023 Cardiogenesis is similar in all vertebrates, but differences in the valvuloseptal morphogenesis among non‐crocodilian reptiles, birds, and mammals are noted. The origin of mesenchymal structures such as valves that regulate the passage of blood and the formation of partial septa that prevent the complete mixing of oxygen‐rich and low‐oxygen blood present in adult chelonians are essential in the evolutionary understanding of complete septation, endothermy and malformations, even in mammals. In this context, this study analyzed the heart morphogenesis of Podocnemis unifilis (Testudines: Podocnemididae) from the 4th to the 60th day of incubation. We identified the tubular heart stage, folding of the cardiac tube and expansion of the atrial and ventricular compartments followed by atrial septation by the septum primum, ventricle septation by partial septa, outflow tract septation and the formation of bicuspid valves with cartilage differentiation at the base. The formation of the first atrial septum with the mesenchymal cap is noted during the development of the atrial septum, joining the atrioventricular cushion on the 17th day and completely dividing the atria. Small secondary perforations appeared in the mid‐cranial part, observed up to the 45th day. Partial ventricle septation into the pulmonary, venous, and arterial subcompartments takes place by trabeculae carneae thickening and grouping on the 15th day. The outflow tract forms the aorticopulmonary and interaortic septa on the 16th day and the bicuspid valves, on the 20th day. Therefore, after the first 20 days, the heart exhibits a general anatomical conformation similar to that of adult turtles.
Embryonic development of eggs and stereological analysis of body of Neoechinorhynchus buttnerae (Golvan, 1956) (Eoacanthocephala: Neoechinorhynchidae) Mayra da Silva Gonçalves, Oscar Tadeu Ferreira da Costa, Germán Augusto Murrieta Morey, Lucas Castanhola Dias, Edsandra Campos Chagas, et al. Parasitology, 2022 The egg is one of the fundamental parts of the life cycle of Neoechinorhynchus buttnerae, and this stage involves the acanthor larva. It is also the infection phase for the intermediate host. Under normal conditions, the larva inside the egg can survive for months in the environment; however, information regarding this phase of life of the parasite is scarce. In addition, there is no quantitative information about the structural composition of the parasite's body from a histological point of view. Such information is essential in order to support decisions aimed at controlling infestations by these parasites in fish farming. This study aimed to present a detailed description of the stages of embryonic development of N. buttnerae eggs, as well as a stereological evaluation of the body of adult females of the parasite. Three phases of development characterized the eggs: cell division (with four stages), formation of the internal nuclear mass (with four stages) and formation of the acanthor larva (with five stages). The ovary comprised 26.61% of the volume of the animal and most of it contained eggs (21.28%), ovarian balls (3.88%) and empty spaces (1.45%). These results are of great importance and will support future studies that seek to interrupt the life cycle of this parasite.
Natural nests incubated in two different soil types lead to an overall balanced sex ratio in Podocnemis unifilis hatchlings on the lower Purus river, Brazil Salamandra, 2020
