Harry J. Grier

Oogenesis of microlecithal oocytes in the viviparous teleost Heterandria formosa

Viviparous teleosts exhibit two patterns of embryonic nutrition: lecithotrophy (when nutrients are derived from yolk that is deposited in the oocyte during oogenesis) and matrotrophy (when nutrients are derived from the maternal blood stream during gestation). Nutrients contained in oocytes of matrotrophic species are not sufficient to support embryonic development until term. The smallest oocytes formed among the viviparous poeciliid fish occur in the least killifish, Heterandria formosa, these having diameters of only 400 μm. Accordingly, H. formosa presents the highest level of matrotrophy among poeciliids. This study provides histological details occurring during development of its microlecithal oocytes. Five stages occur during oogenesis: oogonial proliferation, chromatin nucleolus, primary growth (previtellogenesis), secondary growth (vitellogenesis), and oocyte maturation. H. formosa, as in all viviparous poeciliids, has intrafollicular fertilization and gestation. Therefore, there is no ovulation stage. The full‐grown oocyte of H. formosa contains a large oil globule, which occupies most of the cell volume. The oocyte periphery contains the germinal vesicle, and ooplasm that includes cortical alveoli, small oil droplets and only a few yolk globules. The follicular cell layer is initially composed of a single layer of squamous cells during early previtellogenesis, but these become columnar during early vitellogenesis. They are pseudostratified during late vitellogenesis and reduce their height becoming almost squamous in full‐grown oocytes. The microlecithal oocytes of H. formosa represent an extreme in fish oogenesis typified by scarce yolk deposition, a characteristic directly related to matrotrophy. J. Morphol., 2011.

Reproductive Histology of Tomeurus gracilis Eigenmann, 1909 (Teleostei: Atherinomorpha: Poeciliidae) With Comments on Evolution of Viviparity in Atherinomorph Fishes

Tomeurus gracilis is a species long considered pivotal in understanding the evolution of livebearing in atherinomorph fishes. Tomeurus gracilis is a zygoparous or embryoparous poeciliid: internal fertilization is followed by females laying fertilized eggs singly or retaining fertilized eggs until or near hatching. Tomeurus was hypothesized as the sister group of the viviparous poeciliids until it was proposed as a close relative of a derived viviparous poeciliid, Cnesterodon, hence nested among viviparous taxa rather than near the root of the tree. Here, we describe and compare reproductive morphological characters of the little‐known Tomeurus with those of representative atherinomorphs. In Tomeurus and Cnesterodon, sperm are packaged in naked sperm bundles, or spermatozeugmata, in a configuration considered here diagnostic of viviparous poeciliids. Testes are single and free sperm are stored in the ovary in both taxa in contrast to oviparous atherinomorphs in which testes are paired and sperm are not packaged and not stored in the ovary. Efferent ducts in Cnesterodon testes and other viviparous poeciliids have a PAS‐positive secretion demonstrating presence of a glycoprotein that inactivates sperm or prevents final sperm maturation. No PAS‐positive staining secretion was observed in Tomeurus or oviparous atherinomorphs. Tomeurus shares apomorphic reproductive characters, such as sperm bundle and testis morphology and a gonopodium, with viviparous poeciliids and plesiomorphic characters, such as a thick zona pellucida with filaments, with oviparous taxa. We do not postulate loss or reversal of viviparity in Tomeurus, and we corroborate its phylogenetic position as sister to the viviparous poeciliids. J. Morphol., 2010.

Ovarian structure and oogenesis of the oviparous goodeids Crenichthys baileyi (Gilbert, 1893) and Empetrichthys latos Miller, 1948 (teleostei, Cyprinodontiformes)

The cyprinodontiform family Goodeidae comprises two biogeographically disjunct subfamilies: the viviparous Goodeinae endemic to the Mexican Plateau, and the oviparous Empetrichthyinae, known only from relict taxa in Nevada and California. Ovarian characteristics of two oviparous species of goodeid, Crenichthys baileyi and Empetrichthys latos, studied using museum collections, are compared with those of viviparous species of goodeids. Both subfamilies have a single, cystovarian ovary. The ovary in the viviparous Goodeinae has an internal septum that divides the ovarian lumen into two compartments, and it may possess oogonia. There is no ovarian septum in the oviparous C. baileyi and E. latos. Oogenesis is similar in both subfamilies with regard to the proliferation of oogonia, initiation of meiosis, primary growth and development of an oocyte during secondary growth in which fluid yolk progressively fuses into a single globule. Notably, eggs of C. baileyi and E. latos are approximately double the size of those of the viviparous Goodeinae in which embryos develop inside the ovarian lumen and are nourished, in part, by nutrients transferred from the maternal tissues, a mode of embryo development called matrotrophy. Egg envelopes of the two subfamilies differ in that those of C. baileyi and E. latos have a relatively thick zona pellucida, attachment fibrils or filaments that develop between the follicle cells during oogenesis, and a micropyle observed only in E. latos. In contrast, viviparous goodeid eggs have a relatively thin zona pellucida, but lack adhesive fibrils, and a micropyle was not observed. These reproductive characters are compared with those of species of the eastern North American Fundulus, a representative oviparous cyprinodontiform. One newlyrecognized shared, derived character, a single, median ovoid ovary with no obvious external evidence of fusion, supports monophyly of the Goodeidae. Differences among the goodeid subfamilies and Fundulus are interpreted relative to the oviparous versus viviparous modes of reproduction. J. Morphol., 2012.

Conserved form and function of the germinal epithelium through 500 million years of vertebrate evolution.

The germinal epithelium, i.e., the site of germ cell production in males and females, has maintained a constant form and function throughout 500 million years of vertebrate evolution. The distinguishing characteristic of germinal epithelia among all vertebrates, males, and females, is the presence of germ cells among somatic epithelial cells. The somatic epithelial cells, Sertoli cells in males or follicle (granulosa) cells in females, encompass and isolate germ cells. Morphology of all vertebrate germinal epithelia conforms to the standard definition of an epithelium: epithelial cells are interconnected, border a body surface or lumen, are avascular and are supported by a basement membrane. Variation in morphology of gonads, which develop from the germinal epithelium, is correlated with the evolution of reproductive modes. In hagfishes, lampreys, and elasmobranchs, the germinal epithelia of males produce spermatocysts. A major rearrangement of testis morphology diagnoses osteichthyans: the spermatocysts are arranged in tubules or lobules. In protogynous (female to male) sex reversal in teleost fishes, female germinal epithelial cells (prefollicle cells) and oogonia transform into the first male somatic cells (Sertoli cells) and spermatogonia in the developing testis lobules. This common origin of cell types from the germinal epithelium in fishes with protogynous sex reversal supports the homology of Sertoli cells and follicle cells. Spermatogenesis in amphibians develops within spermatocysts in testis lobules. In amniotes vertebrates, the testis is composed of seminiferous tubules wherein spermatogenesis occurs radially. Emerging research indicates that some mammals do not have lifetime determinate fecundity. The fact emerged that germinal epithelia occur in the gonads of all vertebrates examined herein of both sexes and has the same form and function across all vertebrate taxa. Continued study of the form and function of the germinal epithelium in vertebrates will increasingly clarify our understanding of vertebrate reproduction. J. Morphol. 277:1014–1044, 2016.

Proliferation of Oogonia and folliculogenesis in the viviparous teleost Ilyodon whitei (Goodeidae)

Oogonial proliferation in fishes is an essential reproductive strategy to generate new ovarian follicles and is the basis for unlimited oogenesis. The reproductive cycle in viviparous teleosts, besides oogenesis, involves development of embryos inside the ovary, that is, intraovarian gestation. Oogonia are located in the germinal epithelium of the ovary. The germinal epithelium is the surface of ovarian lamellae and, therefore, borders the ovarian lumen. However, activity and seasonality of the germinal epithelium have not been described in any viviparous teleost species regarding oogonial proliferation and folliculogenesis. The goal of this study is to identify the histological features of oogonial proliferation and folliculogenesis during the reproductive cycle of the viviparous goodeid Ilyodon whitei. Ovaries during nongestation and early and late gestation were analyzed. Oogonial proliferation and folliculogenesis in I. whitei, where intraovarian gestation follows the maturation and fertilization of oocytes, do not correspond to the late oogenesis, as was observed in oviparous species, but correspond to late gestation. This observation offers an example of ovarian physiology correlated with viviparous reproduction and provides elements for understanding the regulation of the initiation of processes that ultimately result in the origin of the next generation. These processes include oogonia proliferation and development of the next batch of germ cells into the complex process of intraovarian gestation. J. Morphol. 275:1004–1015, 2014.

The occurrence of spermatozoa in the ovary of the gynogenetic viviparous teleost Poecilia formosa (POECILIIDAE)

The reproductive mode of the female viviparous teleost Poecilia formosa (Poeciliidae) represents the phenomenon known as gynogenesis; that is, parthenogenetic development is initiated by spermatozoa which are needed for physiological activation of the egg and the initiation of gestation, but spermatozoa are prevented from contributing to the genome of the embryo. For the reason that no previous histological analyses of the ovary of this species during the reproductive cycle has been published the present study has been conducted. This study examined the histology of the ovary of P. formosa during nongestation and gestation phases and identified the presence of spermatozoa inside the ovary. Spermatozoa were observed in folds of the ovarian epithelium of P. formosa during both the nongestation and gestation phases. Sperm storage as documented in this study is a very important trait for the gynogenetic viviparous fish P. formosa contributing to the understanding of this species reproduction. J. Morphol. 277:341–350, 2016.

Oogenesis: From Oogonia to Ovulation in the Flagfish, Jordanella floridae Goode and Bean, 1879 (Teleostei: Cyprinodontidae)

We provide histological details of the development of oocytes in the cyprinodontid flagfish, Jordanella floridae. There are six stages of oogenesis: Oogonial proliferation, chromatin nucleolus, primary growth (previtellogenesis [PG]), secondary growth (vitellogenesis), oocyte maturation and ovulation. The ovarian lamellae are lined by a germinal epithelium composed of epithelial cells and scattered oogonia. During primary growth, the development of cortical alveoli and oil droplets, are initiated simultaneously. During secondary growth, yolk globules coalesce into a fluid mass. The full‐grown oocyte contains a large globule of fluid yolk. The germinal vesicle is at the animal pole, and the cortical alveoli and oil droplets are located at the periphery. The disposition of oil droplets at the vegetal pole of the germinal vesicle during late secondary growth stage is a unique characteristic. The follicular cell layer is composed initially of a single layer of squamous cells during early PG which become columnar during early vitellogenesis. During primary and secondary growth stages, filaments develop among the follicular cells and also around the micropyle. The filaments are seen extending from the zona pellucida after ovulation. During ovulation, a space is evident between the oocyte and the zona pellucida. Asynchronous spawning activity is confirmed by the observation that, after ovulation, the ovarian lamellae contain follicles in both primary and secondary growth stages; in contrast, when the seasonal activity of oogenesis and spawning ends, after ovulation, the ovarian lamellae contain only follicles in the primary growth stage. J. Morphol. 277:1339–1354, 2016.

Insemination, intrafollicular fertilization and development of the fertilization plug during gestation in Heterandria formosa (Poeciliidae)

The viviparous teleost Heterandria formosa is a remarkable species for its reproductive characters including: (a) the smallest oocyte in viviparous fish species; (b) a high level of matrotrophy with a complex placenta; and (c) the highest level of superfetation. Superfetation involves (d) the continuous development of oocytes and fertilization at the same time with embryos in gestation. The sequential fertilization of oocytes requires (e) storage of spermatozoa in the ovary. Among these characteristics, fertilization is of fundamental interest, specifically the intrafollicular fertilization of poeciliids, species that do not present micropyle, and the consequent formation of the fertilization plug, a structure developed at the periphery of the follicle where the entrance of spermatozoa occurs. Both processes intrafollicular fertilization and formation of the fertilization plug have been rarely described. There is only one study illustrating, the fertilization plug of H. formosa with a drawing. In the context of reproductive aspects of H. formosa, the goal of this study is to describe the morphology of the ovary during insemination, intrafollicular fertilization and development of the fertilization plug. After insemination, spermatozoa enter the ovary and occupy folds of the lamella near follicles of all stages of oogenesis, the delle, where the germinal epithelium establishes contact with the follicular epithelium. The results of the present study provide evidence that both epithelia open at the distal end of the delle, this morphological change allow that the spermatozoa to make contact with the zona pellucida of the oocyte. After fertilization, the delle becomes blocked by proliferation of cells of the germinal epithelium, to form the fertilization plug that persists throughout gestation. Abundant reticular fibers and blood vessels are seen around the fertilization plug. Persistence of the fertilization plug suggests that it could be the site where the juvenile will gain entrance to the ovarian lumen during birth.

Change of lecithotrophic to matrotrophic nutrition during gestation in the viviparous teleost Xenotoca eiseni (Goodeidae)

Teleosts possess unique features of the female reproductive system compared with the rest of vertebrates, features that define the characteristics of their viviparity. Viviparity involves new maternal–embryonic relationships detailing the most diverse structures during gestation that include embryonic nutrition. In order to analyze the morphological features of the complex nutrition in viviparous teleosts during intraovarian gestation, this study utilizes the goodeid Xenotoca eiseni as a model. Ovarian gestation in X. eiseni, as in all goodeids, is intraluminal; the early embryo moves from the follicle to the ovarian lumen where gestation continues. The scarce yolk in the oocytes implies that the initial lecithotrophy is replaced by matrotrophy, with nutrients provided via maternal tissues. The nutrients are absorbed by the embryo mainly by trophotaenia, extensions of the embryonic intestine into the ovarian lumen. This histological study analyses the structures involved in these two types of nutrition and when they occur during gestation in X. eiseni. The morphology displayed in this study demonstrated the extended simultaneity of lecithotrophy and matrotrophy during gestation with the progressive reduction of lecithotrophy and increase of matrotrophy. Similarly, it describes the development of complex embryonic structures for metabolic exchange with the maternal tissues associated with matrotrophy; specifically the branchial placenta and mainly the trophotaenia.

Production of live young with cryopreserved sperm from the endangered livebearing fish Redtail Splitfin (Xenotoca eiseni, Rutter, 1896)

Previous studies of sperm cryopreservation of livebearing fish have been limited to two genera within the family Poeciliidae. The goal of the present study was to investigate the feasibility to produce live young of livebearing goodeids (family Goodeidae) with cryopreserved sperm, using aquarium-trade populations of the endangered species Redtail Splitfin (Xenotoca eiseni, Rutter, 1896). Reproductive condition of females was evaluated by histological categorization of ovarian development. A total of 117 females were inseminated with cryopreserved sperm, 81 were inseminated with fresh sperm, 27 were mixed with males for natural breeding, and 30 were maintained without males or insemination. Histological images of 34 mature females indicated 68% of ovaries had primary- or secondary-growth oocytes, and 32% had ovulated eggs. Ovarian development had no significant relationship (P =  0.508) with body wet weight, but had a relationship (P <  0.001) with ovary weight and gonadosomatic index. Sperm cells were observed within ovaries that were fixed at 12 h after insemination with fresh sperm. A total of 29 live young were produced from two females inseminated with thawed sperm (8% post-thaw motility with HBSS300 as extender, 20 min incubation in 15% DMSO, cooling rate at 10 °C/min, and thawing at 40 °C for 7 s), 12 were produced from two females with fresh sperm (1%-20% motility), 41 were produced from five naturally spawned females, and no live young were produced from the female-only group. This study provides a foundation for establishment of germplasm repositories for endangered goodeids to assist conservation programs.