Hatchlings and Neonate Turtle Gonads Have Spatially Restricted Neural Processes

Abstract

Morphological and molecular evidence explains the lack of nociception ("pain") associated with very small, laparoscopic gonadal biopsy in neonate turtles. This safe procedure serves to verify neonate sex of late-maturing species, such as sea turtles. Ethical concerns about the potential for biopsy pain, inferred from mammals, limited access to sex verification biopsy for decades. Yet, standard behavioral evidence of pain during biopsy (e.g., escape attempts, biting, guarding behavior after biopsy, inappetence) were negative. Morphological and molecular evidence early in ontogeny, shows that, unlike mice, young turtles have limited neural processes to the gonadal medulla and none reach the cortical layer.

Figure 1. Turtle gonad study panels and controls

(A) Red eared slider turtle ( T. scripta ) embryo (St.12) labeled with HuC/D (green) shows that HuC/D reliably labels developing neural tissue in the CNS and early PNS; note the neural tube, sensory vesicles, prominent peripheral nerve roots, and the linear mesonephros are bright green. (B) Representative images of St.26 T. scripta ovary and testis whole mounts labeled with HuC/D (green) and show elliptical germ cells (GCs) labeled green; many are in the ovary and very few are in the testis. The Sox9 (red-labeled) supportive (Sertoli) cells dominate the testis. Dashed lines outline the gonad (Cort: cortex, Med: medulla; Mes: Mesonephros). (C) Cross sections of St.26 T. scripta ovary and testis that identify the elliptical germ cells are isolated in the cortex of both males and females. Laminin (cyan), is a key part of the basement membrane. Based on the location and morphology of the cells labeled with HuC/D, we determined that all HuC/D positive cells observed in these samples were GCs and not neural bodies. Note that HuC/D labeling in the germ cells is distinct and remote from the labeling observed in the neural bodies. (D) Neonate C. caretta ovary (left) and testis (right) sections stained with H&E provide clear structural comparisons between the multicellular cortex of the ovary overlying a “disorganized” medulla. In contrast, the testis has a single-cell layered cortex (= “tunica”) overlying organized medullary cords, photographed at 400X. (E) Representative sagittal series image of optical Z-sections of neonate C. caretta ovary wholemount labeled with TuJ1 (red) for neural processes and Laminin (cyan) showing the distinct neural processes and a few neural bodies concentrated in the mesonephros. Note that few processes extend into the medulla, and none in the cortex. Dashed lines outline the ovary. No C. caretta testis was available due to high nest temperatures. (F) Representative cross-sections of a neonate C. caretta ovary, mesovarium, and mesonephros are labeled with HuC/D, Cyan, and TuJ1 (red). Mesonephros (Mes) and mesovarium (under the *), have positive neural processes. The roughly triangular gonad, toward the right in each panel, has sparse positive neural projections that are restricted to the ovarian medulla and do not reach the outer (cortical) domain. Neural processes are filamentous whereas germ cells are roughly elliptical. The HuC/D (green) labeled GCs in the ovarian cortex are similar to the T. scripta samples. Note, there are no signs of HuC/D positive neural bodies in the gonad. The presence of Tuj1 projections does not identify the type of neuron (sensory, motor, or interneuron) that might form later in ontogeny. (E-F) Gonads are outlined in white dashed lines. (G) Positive control and antibody labeling controls (primary and secondary) for fluorescent antibody labeling of T. scripta ovaries (upper row) and testis (lower row). Middle panel, testis 647, 488, and Cy3 identify the wavelengths excited by the fluorescent labels. FPT: female promoting incubation temperatures; MPT: male promoting incubation temperatures; St: developmental stage.