Derivation of major yolk proteins from parental vitellogenins and alternative processing during oocyte maturation in Fundulus heteroclitus.

Various Coomassie blue-staining yolk proteins (YPs) present in oocytes and eggs of Fundulus heteroclitus, a teleost that produces low hydrated, demersal eggs (benthophil species), were subjected to N-terminal microsequencing. Four YPs were N-terminally blocked, while five yielded sequence information. Of the latter, four corresponded to internal sequences of vitellogenin 1 (Vg1), whereas a fifth band corresponded to the N-terminal sequence of Vg2. Phosphorylated YPs (phosvitins and phosvettes) derived from the polyserine domain of Vg were not successfully sequenced. The major N-terminally blocked 122-and 103-kDa YPs both represented the lipovitellin heavy chain of Vg1 (LvH1), and thus most of the oocyte YPs were derived from Vg1. During oocyte maturation in vivo and in vitro, the LvH1 122 is degraded, concomitant with an increased enzymatic activity of cathepsin B, while the 45-kDa YP is converted to a 42-kDa YP. The LvH1 122 was found to contain a consensus site for proteolytic degradation (PEST) near its C-terminus, which is missing from its stable, but truncated twin sequence, LvH1 103. We suggest that this site becomes exposed to cathepsin B during the hydration process that accompanies oocyte maturation and renders the LvH1 122 susceptible to proteolysis. PEST sites are found in Vg sequences from other benthophil fish, whereas, interestingly, they are missing in marine teleosts that spawn highly hydrated, pelagic eggs (pelagophil species), displaying a different pattern of Vg incorporation into YPs and LvH1 and LvH2 processing to that found in F. heteroclitus. Thus, different models of Vg/YP precursor/product relationship and further processing during oocyte maturation and hydration are proposed for pelagophil and benthophil teleosts.

Fundulus heteroclitus gonadotropins.5: Small scale chromatographic fractionation of pituitary extracts into components with different steroidogenic activities using homologous bioassays.

Fractionation and characterization of gonadotropins (GtH) from Fundulus heteroclitus pituitary extracts were carried out using a biocompatible liquid chromatographic procedure (Pharmacia FPLC system). Chromatographic fractions were monitored for gonadotropic activities (induction of oocyte maturation and steroid production) using homologous follicle bioassays in vitro. Size-exclusion chromatography eluted gonadotropic activity in one major protein peak (Mr approximately 30,000). Anion-exchange and hydrophobic-interaction chromatography (HIC) yielded two distinct peaks of 17beta-estradiol (E2)- and 17alpha-hydroxy,20beta-dihydroprogesterone (DHP)-promoting activity with associated oocyte maturation. Two-dimensional chromatography (chromatofocusing followed by HIC) resolved pituitary extracts into two active fractions; both induced E2 synthesis, but one was relatively poor in eliciting DHP and testosterone production. Thus, using homologous bioassays, at least two quantitatively different gonadotropic (steroidogenic) activities: an E2-promoting gonadotropin (GtH I-like) and a DHP-promoting gonadotropin (GtH II-like), which has a lower isoelectric point but greater hydrophobicity than the former, can be distinguished from F. heteroclitus pituitaries by a variety of chromatographic procedures. This study complements previous biochemical and molecular data in F. heteroclitus and substantiates the duality of GtH function in a multiple-spawning teleost.

Stages of oocyte development in the zebrafish, Brachydanio rerio.

Oocyte development has been divided into five stages in the zebrafish Brachydanio rerio, based on morphological criteria and on physiological and biochemical events. In stage I (primary growth stage), oocytes reside in nests with other oocytes (Stage IA) and then within a definitive follicle (Stage IB), where they greatly increase in size. In stage II (cortical alveolus stage), oocytes are distinguished by the appearance of variably sized cortical alveoli and the vitelline envelope becomes prominent. In stage III (vitellogenesis), yolk proteins appear in oocytes and yolk bodies with crystalline yolk accrue during this major growth stage. Ooctes develop the capacity to respond in vitro to the steroid 17α, 20ß-dihydroxy-4-pregnen-3-one (DHP) by undergoing oocyte maturation. In stage IV (oocyte maturation), oocytes increase slightly in size, become translucent, and their yolk becomes non-crystalline as they undergo final meiotic maturation in vivo (and in response to DHP in vitro). In stage V (mature egg), eggs (approx. 0.75 mm) are ovulated into the ovarian lumen and are capable of fertilization. This staging series lays the foundation for future studies on the cellular processes occurring during oocyte development in zebrafish and should be useful for experimentation that requires an understanding of stage-specific events. © 1993 Wiley-Liss, Inc.

Ovary of the seahorse, Hippocampus erectus.

The ovary of the seahorse, Hippocampus erectus, is a cylindrical tube bounded by an outer layer consisting of a mesothelium and muscular wall and by an inner luminal epithelium, with a single row of developing follicles sandwiched between the two layers. Follicles are produced by a germinal ridge, which contains oogonia, early oocytes, and prefollicle cells, and which runs along the length of the ovary. The germinal ridge is an outpocketing of the luminal epithelium, as indicated by a continuous underlying basal lamina. Prefollicle cells invest diplotene oocytes and the complex eventually pinches off the germinal ridge as a primordial follicle surrounded by a basal lamina derived from the germinal ridge. Subsequent investment of the primordial follicle by elements of the theca complete the process of folliculogenesis. H. erectus has two ovaries and each ovary has two dorsally located germinal ridges. Thus, in each ovary the derived follicular lamina is bilaterally symmetrical: two temporally and spatially arranged sequences of developing follicles are produced, with the largest follicles found along the ventral midline of the ovary. The advantages of developmental, kinetic, and systemic analyses of these unusual ovaries are indicated.

The Influence of Yolk Protein Proteolysis on Hydration in the Oocytes of Fundulus heteroclitus: (oocyte hydration/meiotic maturation/proteolysis/yolk proteins/teleost).

Growth in oocytes of many marine teleosts can be attributed to a combination of yolk accumulation during the vitellogenic phase of development and water uptake during meiotic maturation. In the salt marsh fish, Fundulus heteroclitus, hydration associated with maturation gives rise to a greater than two-fold increase in oocyte volume. It has been proposed that a concurrent proteolysis of specific yolk proteins may be the mechanism driving this water uptake. To test this hypothesis, we used various in vitro culture techniques to block or significantly reduce oocyte hydration while allowing meiotic maturation to continue, then examined yolk proteins by SDS-polyacrylamide gel electrophoresis. We were able to dissociate yolk proteolysis from both hydration and nuclear maturation stimulated by a maturation-inducing steroid, 17α-hydroxy- 20ß-dihydroprogesterone. It therefore appears that the proteolysis of specific yolk proteins observed in maturing oocytes of marine teleosts is an independent developmental event, and is not directly involved in the hydration mechanism.

Stages of oocyte development in the pipefish, Syngnathus scovelli.

Oocyte development occurs in a temporal and spatial pattern in the ovary of the pipefish, Syngnathus scovelli. Cytological observations combined with [3 H]thymidine uptake and cell culture were used as criteria to identify six stages of oocyte development in this species. Stage I-Oogonia, found within the germinal ridge. Stage II-Oocytes in primary growth, subdivided into (1) chromatin-nucleolus oocytes in early prophase I of meiosis, also found within the germinal ridge, and (2) perinucleolar oocytes, which possess multiple nucleoli and are within definitive follicles (the Balbiani vitelline body is the main cytoplasmic component in these latter oocytes). Stage III-Oocytes distinguished by the appearance of cortical alveoli, the vitelline envelope, and lipid. Stage IV-Vitellogenic oocytes, which accumulate yolk proteins and possess primary, transitional, and mature yolk spheres. Stage V-Maturational oocytes, which are competent to undergo final meiotic maturation in vivo and in response to the steroid 17 α-hydroxy-20ß-dihydroprogesterone in vitro. Stage VI-Ovulated mature eggs, which are present in the ovary lumen and are capable of being fertilized. These studies provide a staging series for oogenesis and oocyte development in the pipefish and relate these stages to the unusual ovary in this animal.

Ovary of the pipefish, Syngnathus scovelli.

Gross dissection, light microscopy, and transmission electron microscopy were used to generate a detailed understanding of the ovarian anatomy of the pipefish, Syngnathus scovelli. The ovary is a cylindrical tube bounded by an outer layer consisting of a smooth muscle wall and an inner layer of luminal epithelium, with follicles sandwiched between the two layers. A remarkable feature of this ovary is a sequential pattern of follicle development. This pattern begins at the germinal ridge with a gradient of follicles of increasing developmental age extending to the mature edge. The germinal ridge is an outpocketed region of the luminal epithelium containing early germinal cells and somatic prefollicular cells. Therefore, the germinal ridge and luminal epithelium share the same ovarian compartment and follicle formation occurs within this compartment. The mature edge is defined as the site of oocyte maturation and ovulation. The outer ovarian wall contains unmyelinated nerve fibers throughout. Longitudinally oriented unmyelinated nerves are also observed near the smooth muscle bundles associated with the mature edge. Oocytes near the mature edge are polarized such that the germinal vesicle (nucleus) is generally oriented toward the luminal epithelium. The sandwichlike organization of the ovary results in follicles that have a shared theca. An extensive lymphatic network is also interspersed among the follicles. Thus, the exceptional features of the pipefish ovary make it particularly well suited for the examination of early events in oogenesis. Specifically, we characterize pipefish folliculogenesis in detail.