Robin A. Wallace

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, 20β‐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.

Major protein changes during vitellogenesis and maturation of Fundulus oocytes

The protein content of various size follicles was measured in Fundulus heteroclitus and indicated four phases of increase relative to follicle volume: Phases I (previtellogenic; estimated to be less than 0.01 mg/mm3), II (vitellogenic; 0.20 mg/mm3), III (early maturation; 0.03 mg/mm3), and IV (late maturation; 0 mg/mm3). A pronounced and rapid size increase occurs during maturation due to hydration, but protein uptake, which was also documented cytologically, contributes to about 16% of the volume increment during early maturation. Protein incorporation appears to stop abruptly at the time of germinal vesicle breakdown, most likely reflecting an altered physiological state of the oocyte. SDS-polacrylamide gel electrophoretic patterns of various size follicles indicated that five major protein bands (molecular weights = 122, 103, 45, 26, and 20 k) accumulate during vitellogenesis and presumably are proteolytically derived from a 200-kDa vitellogenin precursor. During maturation, the 122- and 45-kDa proteins disappear and several new, lower molecular weight bands appear. Proteolysis of specific yolk proteins may thus help generate part of the osmotic pressure gradient required for water uptake during oocyte maturation.

Fundulus heteroclitus vitellogenin: The deduced primary structure of a piscine precursor to noncrystalline, liquid-phase yolk protein

We have cloned and sequenced a cDNA encoding a vitellogenin (Vtg) from the mummichog, Fundulus heteroclitus, an estuarine teleost. We constructed a liver cDNA library against RNA from estrogen-treated male mummichogs. Five overlapping cDNA clones totalling 5,197 by were isolated through a combination of degenerate oligonucleotide probing of the library and PCR. The cDNA sequence contains a 5,112 by open reading frame. The predicted primary structure of the deduced 1,704-amino-acid protein is 30–40% identical to other documented chordate Vtgs, establishing this Vtg as a member of the ancient Vtg gene family. Of the previously reported chordate Vtg sequences (Xenopus laevis, Gallus domesticus, Ichthyomyzon unicuspis, and Acipenser transmontanus), all four act as precursor proteins to a yolk which is eventually rendered insoluble under physiological conditions, either as crystalline platelets or as noncrystalline granules. The yolk of F. heteroclitus, on the other hand, remains in a soluble state throughout oocyte growth. The putative F. heteroclitus Vtg contains a polyserine region with a relative serine composition that is 10–20% higher than that observed for the other Vtgs. The trinucleotide repeats encoding the characteristic polyserine tracts of the phosvitin region follow a previously reported trend: TCX codons on the 5′ end and AGY codons toward the 3′ end. Whether the difference in Vtg primary structure between F. heteroclitus and that of other chordates is responsible for the differences in yolk structure remains to be elucidated. As the first complete teleost Vtg to be reported, these data will aid in designing nucleotide and immunological probes for detecting Vtg as a reproductive status indicator in F. heteroclitus and other piscine species.

Changes in teleost yolk proteins during oocyte maturation: correlation of yolk proteolysis with oocyte hydration

Yolk proteins of prematuration occytes and postmaturation eggs were compared by SDS gel electrophoresis in several teleosts, including freshwater species that produce demersal eggs, estuarine and marine species with demersal eggs, and marine species with pelagic eggs. In certain teleosts distinct changes in yolk protein banding patterns during oocyte maturation are suggestive of extensive secondary proteolysis of yolk proteins at this time; proteolysis is most pronounced in marine fishes with pelagic eggs. In many teleosts the oocyte swells by hydration during maturation; this hydration is also most pronounced in marine fishes with pelagic eggs. The extent of yolk proteolysis is well correlated with the extent of oocyte hydration during maturation.

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

Abstract 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.

Oocyte maturation in the mummichog (Fundulus heteroclitus): effects of steroids on germinal vesicle breakdown of intact follicles in vitro.

The effects of several steroids on the maturation of follicle-enclosed oocytes of the mummichog Fundulus heteroclitus in vitro were examined. At a relatively high concentration (1.0 microgram/ml), a number of different steroids, including pregnenolone, 17 alpha-hydroxypregnenolone, corticosterone, cortisol, 11-deoxycorticosterone, 11-deoxycortisol, androstenedione, testosterone, progesterone, 17 alpha-hydroxyprogesterone, 20 beta-dihydroprogesterone, and 17 alpha-hydroxy-20 beta-dihydroprogesterone, were able to induce germinal vesicle breakdown (GVBD) in prematuration oocytes. Cholesterol, 17 beta-estradiol, 11-ketotestosterone, and 11 beta-hydroxytestosterone were totally ineffective. In general, 11-oxysteroids tended to be less effective than their 11-deoxysteroid counterparts. Two 20 beta-dihydroprogestins--17 alpha-hydroxy-20 beta-dihydroprogesterone and 20 beta-dihydroprogesterone--were the most potent maturation-inducing steroids, initiating 50% GVBD at 1 ng/ml in follicles obtained from ovaries containing mature or maturing follicles in vivo, or at 2.5-4.0 ng/ml in follicles from ovaries lacking mature or maturing oocytes in vivo. These results are consistent with several previous studies involving salmonids and various other teleosts, and suggest a possible physiological role for a 20 beta-dihydroprogestin in the resumption of meiotic maturation in F. heteroclitus.

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 [3H]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‐20β‐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.

Fundulus heteroclitus gonadotropins. 3. Cloning and sequencing of gonadotropic hormone (GTH) I and II β-subunits using the polymerase chain reaction ☆

mRNA was isolated from Fundulus heteroclitus pituitaries and used to construct a cDNA library in lambda gt22A. A series of synthetic oligonucleotides, based on conserved regions of teleost gonadotropic hormone (GTH) beta-subunits, were constructed and used as primers in the polymerase chain reaction (PCR) to amplify GTH cDNAs. Appropriate length PCR products were subcloned and sequenced. Eight clones were eventually identified as cDNAs encoding two distinct beta-subunits of F. heteroclitus, GTH I and GTH II. By comparison with known GTH sequences, putative signal sequences of 19 end 21 amino acids and mature beta-subunits of 95 and 115 amino acids were found for GTH I and GTH II, respectively. Both beta-subunits had well conserved cysteine positions when aligned with other members of the glycoprotein family. The elucidation of the complete nucleotide sequences of two types of F. heteroclitus GTH provides definitive proof that in this species there are at least two distinct forms of pituitary GTH analogous to the classical luteinizing hormone-follicle stimulating hormone family.

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.

Effects of insulin-like growth factor-I on final oocyte maturation and steroid production in Fundulus heteroclitus

Recent evidence has indicated the presence of IGF-I and IGF-I receptors in mammalian and teleost ovarian follicles. Since growth hormone (GH), which can be secreted from the pituitary concomitant with a gonadotropin as a response to gonadotropin-releasing hormone, generally acts to release IGF-I from tissues including the ovary, the effect of IGF-I itself on ovarian steroidogenesis and oocyte maturation was investigated in the model teleost, Fundulus heteroclitus. IGF-I was found to be without effect on ovarian follicle steroidogenesis, but initiated oocyte maturation in a dose-dependent manner even more rapidly and effectively than 17α,20β-dihydroxy-4-pregnene-3-one (DHP), the naturally occurring maturation-inducing steroid. IGF-II also induced oocyte maturation in a dose-dependent manner. IGF-I induction of oocyte maturation occurred in the absence of DHP production by the granulosa cells (which is normally stimulated by gonadotropin), and could be inhibited by cycloheximide but not actinomycin D, thus implicating the role of protein synthesis. These results suggest that GH-stimulated release of ovarian IGF-I may have an even more direct role than DHP on the reinitiation of oocyte maturation.