Kevin M. Gribbins

Comparative analysis of glucagonergic cells, glia, and the circumferential marginal zone in the reptilian retina

Retinal progenitors in the circumferential marginal zone (CMZ) and Müller glia‐derived progenitors have been well described for the eyes of fish, amphibians, and birds. However, there is no information regarding a CMZ and the nature of retinal glia in species phylogenetically bridging amphibians and birds. The purpose of this study was to examine the retinal glia and investigate whether a CMZ is present in the eyes of reptilian species. We used immunohistochemical analyses to study retinal glia, neurons that could influence CMZ progenitors, the retinal margin, and the nonpigmented epithelium of ciliary body of garter snakes, queen snakes, anole lizards, snapping turtles, and painted turtles. We compare our observations on reptile eyes to the CMZ and glia of fish, amphibians, and birds. In all species, Sox9, Pax6, and the glucocorticoid receptor are expressed by Müller glia and cells at the retinal margin. However, proliferating cells were found only in the CMZ of turtles and not in the eyes of anoles and snakes. Similar to eyes of chickens, the retinal margin in turtles contains accumulations of GLP1/glucagonergic neurites. We find that filamentous proteins, vimentin and GFAP, are expressed by Müller glia, but have different patterns of subcellular localization in the different species of reptiles. We provide evidence that the reptile retina may contain nonastrocytic inner retinal glial cells, similar to those described in the avian retina. We conclude that the retinal glia, glucagonergic neurons, and CMZ of turtles appear to be most similar to those of fish, amphibians, and birds. J. Comp. Neurol. 524:74–89, 2016.

Ultrastructural analysis of spermiogenesis in the Eastern Fence Lizard, Sceloporus undulatus (Squamata: Phrynosomatidae)

Studies on reptilian sperm morphology have shown that variation exists at various taxonomic levels but studies on the ontogeny of variation are rare. Sperm development follows a generalized bauplan that includes acrosome development, nuclear condensation and elongation, and flagellar development. However, minute differences can be observed such as the presence/absence of manchette microtubules, structural organization during nuclear condensation, and presence/absence of a nuclear lacuna. The purpose of this investigation was to examine sperm development within the Sceloporus genus. The process begins with the development of an acrosomal complex from Golgi vesicles followed by nuclear condensation and elongation, which results in the presence of a nuclear lacuna. As the acrosomal complex differentiates, flagellar development commences with elongation of the distal centriole. Spermatid development culminates in a mature spermatid with a highly differentiated acrosomal complex, a condensed nucleus with a nuclear lacuna, and a differentiated flagellum. Although the overall developmental pattern is consistent with other squamate species, minute differences are observed, even within the same genus. For example there is variation in the presence/absence of an endoplasmic reticulum complex during acrosome development, presence/absence of a nuclear lacuna, and presence/absence of manchette microtubules within the three species of Sceloporus studied to date. Future studies concerning sperm morphology in closely related species will aid in our understanding of variation in sperm development and may prove to be useful in testing phylogenetic and evolutionary hypotheses.

Testicular histology and germ cell cytology during spermatogenesis in the Mississippi map turtle, Graptemys pseudogeographica kohnii, from Northeast Arkansas

The testicular histology and cytology of spermatogenesis in Graptemys pseudogeographica kohnii were examined using specimens collected between July 1996 and May 2004 from counties in northeastern Arkansas. A histological examination of the testes and germ cell cytology indicates a postnuptial testicular cycle of spermatogenesis and a major fall spermiation event. The majority of the germ cell populations in May and June specimens are represented by resting spermatogonia, type A spermatogonia, type B spermatogonia, pre-leptotene spermatocytes, and numerous Sertoli cell nuclei near the basement membrane. The start of proliferation is evident as spermatogonia in metaphase are present near the basal lamina and many of these germ cells have entered meiosis in June seminiferous tubules. Major spermatogenic events occur in the June and July specimens and result in an increased height of the seminiferous epithelium and increased diameter of the seminiferous tubules. The germ cell population during this time is represented by spermatogonia (type A, B, and resting), hypertrophic cells, large populations of early primary spermatocytes, and early round spermatids. By September, the major germ cell population has progressed past meiosis with abundant round and early elongating spermatids dominating the seminiferous epithelium. October seminiferous epithelia are marked by a decreas in height and mature spermatozoa fill the luminal space. Round and elongating spermatids constitute the largest portion of the germ cell population. Following the spermiation event, the testes enter a period of quiescence that lasts till the next spermatogenic cycle, which begins in the subsequent spring. Based on the cytological development of the seminiferous tubules revealed by our study, Graptemys pseudogeographica kohnii demonstrates a temporal germ cell development strategy similar to other temperate reptiles. A single major generation of germ cells progresses through spermatogenesis each year resulting in a single spermiation event with sperm stored within the epididymis until the next spring mating season.

Spermatogenic cycle of a plethodontid salamander, Eurycea longicauda (Amphibia, Urodela).

Previous investigators have described the spermatogenic cycles of numerous species of plethodontid salamanders. Most studies describe a fairly stereotypical cycle with meiotic divisions of spermatogenesis commencing in the spring/summer. However, many studies lack details obtainable from histological examination and/or testicular squashes and, instead, provide only mensural data from the testes. Studies that lacked microscopic evaluation often revealed spermatogenic cycles that varied greatly from that of the stereotypical cycle with meiotic divisions commencing in the fall/winter. Those studies hamper comparisons between the spermatogenic cycles of different species and their environments, as they do not provide a correlation between testicular size and any aspect of the spermatogenic cycle. In the following manuscript, we elucidate the spermatogenic cycle of Eurycea longicauda longicauda in an effort to outline an appropriate protocol for analyzing spermatogenesis in salamanders that will facilitate future comparative studies. Like many Nearctic plethodontids, E. l. longicauda exhibits a meiotic wave that travels through the testes during the summer; this process is followed by spermiogenesis, spermiation, and recrudescence in the fall, winter, and spring.

Ultrastructural analysis of the mature spermatozoon in the copperhead, Agkistrodon contortrix (Linnaeus, 1766)

Reptilian sperm morphology studies are continually increasing, and sperm biology is presently being utilized in other areas of study such as sexual selection, evolution, and phylogenetic analysis. The numbers of studies concerning sperm morphology in Squamata, however, are still limited compared to the diversity and number of lizard and snake species and the taxonomic distribution of these studies is widespread and random. Comparisons between closely related taxa are not feasible until further spermatozoal studies investigating designated comparative taxa are completed; therefore, this study aims to add a description of the mature spermatozoa of Agkistrodon contortrix (Linnaeus, 1766) and compare these data to the mature spermatid morphology of its related taxon, Agkistrodon piscivorus (Lacepede, 1789). Although the general architecture of the mature spermatozoa in A. contortrix is consistent with previous reports of mature sperm morphology in snakes, minor differences are noted including the location of the fibrous sheath starting at mitochondrial tier 4, a location that has not yet been reported for snakes. These data further suggest that minute differences in sperm ultrastructure can be observed in closely related taxa and may be beneficial in future phylogenetic and evolutionary analyses.

Ultrastructure of spermatid development within the testis of the Yellow-Bellied Sea Snake, Pelamis platurus (Squamata: Elapidae)

ABSTRACT Little is known about spermatid development during spermiogenesis in snakes, as there is only one complete study in ophidians, which details the spermatid ultrastructure within the viperid, Agkistrodon piscivorus. Thus, the following study will add to our understanding of the ontogenic steps of spermiogenesis in snakes by examining spermatid maturation in the elapid, Pelamis platurus, which were collected in Costa Rica in 2009. The spermatids of P. platurus share many similar ultrastructural characteristics to that described for other squamates during spermiogenesis. Three notable differences between the spermatids of P. platurus and those of other snakes is a round and shorter epinuclear lucent zone, enlarged caudal nuclear shoulders, and more prominent 3 and 8 peripheral fibers in the principal and endpieces. Also, the midpiece is much longer in P. platurus and is similar to that reported for all snakes studied to date. Other features of chromatin condensation and morphology of the acrosome complex are similar to what has been observed in A. piscivorus and other squamates. Though the spermatids in P. platurus appear to be quite similar to other snakes and lizards studied to date, some differences in subcellular details are still observed. Analysis of developing spermatids in P. platurus and other snakes could reveals morphologically conserved traits between different species along with subtle changes that could help determine phylogenetic relationships once a suitable number of species have been examined for ophidians and other squamates.