Kelly Selman

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.

Dynamics of oocyte development and related changes in serum estradiol-17β, yolk precursor, and lipid levels in the teleostean fish, Leptocottus armatus

Abstract 1. 1. The dynamics of ovarian development in Leptocottus armatus were studied. Seasonal changes of serum estradiol-17β, serum vitellogenin, serum total lipids and visceral lipid reserves were monitored. 2. 2. Two to four distinct size classes (clutches) of oocytes occurred in the ovary preceding and during the spawning period. One of these clutches contained oocytes in the vitellogenic or post-vitellogenic phase, a second clutch contained oocytes “arrested” in the cortical alveoli stage and a third clutch contained non-yolky oocytes. Only one clutch was oviposited in the annual spawning season. 3. 3. Estradiol levels increased coincident with the appearance of yolky oocytes in the ovary. Serum estrogen levels were highest during the period of ovulation and spawning. During the spawning period estradiol titers were not significantly correlated with oocyte size or gonosomatic index. 4. 4. Serum vitellogenin levels increased in parallel with oocyte size and estradiol titers. Vitellogenin levels were relatively low during the early portion of the spawning period, becoming elevated again towards the end of the spawning period. In concordance with ovarian regression, serum vitellogenin levels declined and remained low during the ovarian quiescence phase. Neither GSI nor serum estradiol titers correlated significantly with vitellogenin levels. 5. 5. With initiation of ovarian recrudescence visceral lipid reserves began to decline and continued to decline through the vitellogenic and spawning phases. Visceral lipid reserves were inversely correlated with ovarian GSI. Serum lipid levels were high during ovarian recrudescence, remaining elevated through the spawning period. During the phase of ovarian quiescence serum lipid levels were low and visceral lipid reserves were high. Visceral lipid reserves showed an inverse correlation to serum estradiol titers.

Oocyte growth in the sheepshead minnow: Uptake of exogenous proteins by vitellogenic oocytes

The structure of the vitellogenic follicles of the sheepshead minnow, Cyprinodon variegatus, is described. Follicles enlarge primarily by protein yolk accumulation (vitellogenesis) and subsequently increase in size by hydration. This study uses the electron-dense tracer, horseradish peroxidase, and a larger heterologous protein, Xenopus laevis [3H]vitellogenin, to follow the fate of exogenous proteins from the maternal circulation to yolk spheres of the growing oocyte. Materials appear to leave the perifollicular capillaries via an interendothelial route, traverse the theca and the patent intercellular channels of the follicular epithelium and the pore canals of the vitelline envelope. At the oocyte surface they are incorporated via micropinocytosis and translocated to growing yolk spheres in the peripheral ooplasm. In contrast to other studies on oocyte growth in teleosts which suggest that yolk is an autosynthetic product, this study substantiates the importance of heterosynthetic processes during oocyte growth in C. variegatus.

Oogenesis in Fundulus heteroclitus. II. The transition from vitellogenesis into maturation.

Abstract Female Fundulus heteroclitus collected in the vicinity of Woods Hole have asynchronous ovaries which continuously ovulate eggs during the breeding season (early May until early July). At this time, vitellogenic oocytes become competent to respond to exogenous deoxycorticosterone (DOC) by undergoing maturation when follicles reach a diameter of 1.3 mm. Follicular oocytes with a diameter of 1.35 ± 0.05 mm can also give a 100% response to exogenous gonadotropins under appropriate conditions in vitro , indicating that endogenous gonadotropin(s) most likely acts directly on the ovary in situ by promoting the synthesis of maturation-inducing steroid. A response equivalent to one female pituitary is provided by 0.49 μg ovine luteinizing hormone, 6.7 μg human chorionic gonadotropin (hCG), and 31 μg ovine follicle-stimulating hormone. The response to gonadotropins is slower than that achieved by exogenous DOC alone and is potentiated (23–24 times) by the addition of physiological levels (100 ng/ml) of cortisol to the incubation medium. The available cytological evidence suggests that the granulosa cells rather than “special cells” in the theca are the progenitors of maturation-producing steroid. Once follicular oocytes reach a diameter greater than 1.4 mm diameter in situ , they become irreversibly committed to complete the process of maturation and ovulation. Cessation of feeding eliminates the competence of 1.3-mm-diameter follicles to respond to exogenous DOC and also the recruitment of 1.4-mm-diameter follicles into maturational stages, but hCG injection or refeeding restores both processes. Two hormonal mechanisms which can regulate recruitment of vitellogenic follicles into maturation have thus been identified (gonadotropin and/or cortisol production), but the sensitivity of these mechanisms to the nutritional state of the animal has not yet been elucidated.

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.

Steroidogenesis in Fundulus heteroclitus: II. Production of 17α-hydroxy-20β-dihydroprogesterone, testosterone, and 17β-estradiol by various components of the ovarian follicle

Fundulus heteroclitus prematurational follicles (1.3-1.4 mm) were dissected into various components and cultured in vitro to examine the type of cells involved in the synthesis of steroids upon F. heteroclitus pituitary extract (FPE) stimulation or addition of exogenous precursors (25-hydroxycholesterol or pregnenolone). Culture media and follicular tissue extracts were assayed for 17 alpha-hydroxy-20 beta-dihydroprogesterone (17 alpha-OH.20 beta-DHP), testosterone (T), and 17 beta-estradiol (E2) content using specific radioimmunoassays. Complete removal of the follicle wall (denuded oocytes) eliminated steroid accumulation induced by FPE treatment. Removal of the theca/epithelium layer (defolliculated oocytes) did not affect the steroidogenic response (17 alpha-OH,20 beta-DHP, T, E2 production) of the follicles to FPE or exogeneously added precursors (25-hydroxycholesterol or pregnenolone). Isolated theca/epithelium layers secreted only T. Isolated follicular preparations that did not contain the oocyte (theca/epithelium layers or follicle cells) secreted higher levels of steroids to the culture media than did intact follicles. We conclude from these results that (1) the follicle cells (granulosa cells) are the primary source of the various steroids produced by the F. heteroclitus ovarian follicle in response to FPE stimulation: (2) the synthesis of 17 alpha-OH,20 beta-DHP and E2 does not require the involvement of two cell types as shown in other teleosts; (3) the theca/epithelium layer is able to produce T but lacks the aromatase activity necessary for E2 synthesis; and (4) steroids synthesized in the follicle wall are both secreted to the medium and accumulated in the oocyte.

Preliminary observations on oocyte maturation and other aspects of reproductive biology in captive female snook, Centropomus undecimalis

Abstract Female snook collected from the field in mornings during the summer months appear to have asynchronous ovaries, with ovarian follicles generally present in all developmental stages up to prematurational stages. Methods were developed to maintain snook ovarian follicles successfully in culture for up to 48 h at 28°C. Oocytes within follicles larger than 0.5-mm diameter could be induced to undergo maturation (germinal vesicle breakdown, clarification, volume enlargement by 200%) in vitro with suitable concentrations of either human chorionic gonadotropin or homologous pituitary extract. Snook pituitaries were found to contain approximately 155 IU gonadotropic activity/mg. Steroids that have been found to be effective inducers of oocyte maturation in other teleosts were essentially ineffective on snook oocytes, so the endogenous maturation-inducing steroid produced by follicles under gonadotropic stimulation remains unknown. After capture, snook ovarian follicles could be used successfully for experimental purposes for up to 48 h, after which they became unresponsive to gonadotropin in vitro. Concomitant with the decline in follicle competence, rapid and massive degenerative changes occurred in the ovary, which was invaded by a large population of PAS-positive cells.

An autoradiographic study of vitellogenesis in the squid, Loligo pealei.

ABSTRACT The structure of the vitellogenic (stage IV) ovarian follicle in Loligo pealei is described. Ultrastructural observations indicate that the follicular syncytium is synthesizing yolk proteins. Growing follicles incubated in l -[ 3 H]leucine incorporate this amino acid into protein with linear kinetics for 3·5 hr, thus establishing the adequacy of our culture procedure for this time period. Autoradiographic studies show that immediately after a 30 min exposure to l -[ 3 H]leucine, the follicular syncytium is uniformly labeled, while the chorion and oocyte are essentially unlabeled. Subsequently, labeled protein accumulates in the apical region of the follicular syncytium, traverses the extracellular space between the follicular syncytium and the oocyte, and, by 3·5 hr is present in the peripheral yolk bodies of the oocyte The follicular syncytium thus appears to be the sole heterosynthetic source of yolk protein Yolk protein does not appear to be taken up into the oocyte by micropinocytosis, as occurs in other systems, but most likely is simply engulfed.

Serotonin inhibition of steroid-induced meiotic maturation in the teleost Fundulus heteroclitus: role of cyclic AMP and protein kinases.

The transduction of the serotonin (5‐HT) signal in Fundulus heteroclitusovarian follicles leading to the inhibition of oocyte meiosis reinitiation (oocyte maturation) in vitro induced by the naturally occurring maturation‐inducing steroid 17α,20β‐dihydroxy‐4‐pregnen‐3‐one (17,20βP) was investigated. Steroid‐induced oocyte maturation was inhibited by 5‐HT in a dose‐dependent manner; maximum inhibition (90%) was observed with 10−4 M 5‐HT. Groups of follicle‐enclosed oocytes were cultured in the presence of the phosphodiesterase inhibitor 3‐isobutyl‐1‐methylxanthine (IBMX) and treated with increasing doses of 5‐HT. Serotonin was found to slightly increase the levels of follicular 3′,5′‐cyclic adenosine monophosphate (cAMP) in a dose‐dependent manner; 10−4 M 5‐HT induced approximately a 3‐fold increase in cAMP with respect to the controls. The changes in cAMP were then evaluated in follicles treated with 17,20βP in IBMX‐free culture media in the presence or absence of 10−4 M 5‐HT. The exposure of follicles to 17,20βP alone produced a small and transient reduction in cAMP (40%) within 1–3 hr of steroid stimulation, and these early changes in cAMP appeared associated with a high incidence of germinal vesicle breakdown (80% GVBD) by 24 hr of incubation. Under these conditions, treatment of follicles with 5‐HT also increased significantly the production of cAMP, and when 5‐HT was combined with 17,20βP, the steroid‐mediated reduction in cAMP was prevented and the levels of GVBD inhibited by 95%. Meiosis also was reinitiated with either the protein kinase A (PKA) inhibitor H8 or the protein kinase C (PKC) activator PMA, and the 5‐HT inhibitory action on GVBD was found to be 100‐fold reduced or completely ineffective, respectively. Preincubation of follicles with the PKC inhibitor GF109203x abolished PMA‐induced GVBD in a dose‐dependent manner, whereas this inhibitor had no effect on 17,20βP‐triggered meiotic maturation, indicating that activation of PKC is apparently sufficient but not necessary to reinitiate meiosis. Taken together, these findings suggest that 5‐HT may inhibit 17,20βP‐induced meiotic reinitiation through the activation of a cAMP‐PKA transduction pathway and that PKC possibly induces oocyte maturation by a different pathway than the steroid and thus is not affected by 5‐HT. Mol. Reprod. Dev. 49:333–341, 1998.