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R A Wallace

Publications and source records attributed to R A Wallace.

At least 19 recordsLinked to original sources

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 heteroclitus ovarian follicles leading to the inhibition of oocyte meiosis reinitiation (oocyte maturation) in vitro induced by the naturally occurring maturation-inducing steroid 17 alpha, 20 beta-dihydroxy-4-pregnen-3-one (17,20 beta P) 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 17,20 beta P in IBMX-free culture media in the presence or absence of 10(-4) M 5-HT. The exposure of follicles to 17,20 beta P 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 17,20 beta P, 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 17,20 beta P-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 17,20 beta P-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.

Animals↗

Synthesis and preliminary evaluation of MP-2269: a novel, nonaromatic small-molecule blood-pool MR contrast agent.

A nonaromatic, small-molecule, gadolinium(3+)-chelate code named MP-2269 was synthesized and evaluated in animals as a potential MR contrast agent for blood pool. The ligand of MP-2269 was prepared by conjugating a lipophilic, albumin-binding moiety, 4-pentylbicyclo[2.2.2]octane-1-carboxylic acid, to an amino-functionalized DTPA derivative by means of a diaspartic acid linker. Proton relaxometry studies in vitro yielded spin-lattice relaxivities (R1) for MP-2269 of 6.2, 20.0 and 26.1 mM(-1)sec(-1) in water, rabbit blood, and human blood, respectively. The enhanced relaxivities in blood indicate significant binding of the agent to blood proteins. At a dose of 45 micromol/kg, MP-2269 showed a biphasic rabbit blood clearance profile with half-lives of 4.7 and 142 minutes, respectively, for the fast and slow components. In rats, the agent is cleared predominantly through the hepatobiliary pathway (approximately 70% in 24 h by this mode). The LD50 value of MP-2269 is approximately 3.0 mmol/kg in mice. Preliminary MR angiograms obtained in the rabbit showed excellent enhancement of blood vessels. Hence, MP-2269 has potential for future exploitation as a contrast agent for MR angiography.

Animals↗

Oocyte sensitivity to serotonergic regulation during the follicular cycle of the teleost Fundulus heteroclitus.

In the teleost Fundulus heteroclitus, serotonin (5-HT) reversibly inhibits oocyte maturation induced in vitro by the maturation-inducing steroid (MIS) 17,20beta-dihydroxy-4-pregnen-3-one (17,20betaP). The 5-HT inhibition of 17,20betaP-induced meiotic maturation was examined in ovarian follicles at different developmental stages or isolated at different times during the follicular cycle. Steroid treatment of late vitellogenic and early maturing follicles (1.2- to 1.7-mm diameter) promoted oocyte maturation in a size-dependent manner, and this maturation was inhibited by 5-HT in follicles of < 1.6- to 1.7-mm diameter. Thus, the 5-HT inhibition progressively decreased as follicles developed the ability to mature in the absence of 17,20betaP. The effectiveness of 5-HT to increase follicular cAMP remained similar within the same developmental stages, indicating that the reduction of 5-HT inhibitory action was not related to the competence of 5-HT to activate inhibitory signals in the oocyte. During the follicular cycle, fully grown follicles (1.3- to 1.4-mm diameter) showed a decreased maturational competence in response to gonadotropin or MIS stimulation after the follicular recruitment into maturation and spawning occurred, which coincided with an increase of the effectiveness of 5-HT at inhibiting 17,20betaP-induced maturation. In further experiments, preincubation of follicles with hCG was found to reduce 5-HT inhibitory action, but when follicles were incubated with either hCG in the presence of a steroidogenesis inhibitor or estradiol-17beta (E2), the 5-HT inhibition was unaffected. These findings suggest that 5-HT inhibition of the MIS-induced meiotic maturation is not under direct gonadotropin or E2 regulation but that it might be regulated in vivo by changes in the competence of the oocytes to undergo oocyte maturation after MIS stimulation.

Animals↗

Pharmacology of the serotonergic inhibition of steroid-induced reinitiation of oocyte meiosis in the teleost Fundulus heteroclitus.

Serotonin (5-HT) was found to inhibit steroid (17 alpha,20 beta-dihydroxy-4-pregnen-3-one; 17,20 beta P)-induced resumption of oocyte meiosis (oocyte maturation) in vitro in the teleost Fundulus heteroclitus. Serotonin inhibited both follicle-enclosed and denuded oocytes, which indicates the presence of oocyte-associated 5-HT sensitive sites. The response of oocytes to 5-HT was characterized pharmacologically, i.e., the capacity of serotonergic agonists and antagonists to mimic or block the 5-HT inhibition of the steroid-induced oocyte maturation was assessed by the changes in the percentage of oocyte germinal vesicle breakdown (GVBD). Dose-response curves for each compound were drawn and compared. The rank order of potency among the agonists was: 5-HT > 5-methoxytryptamine > tryptamine = 5,6-diHT = 5-carboxidotryptamine > 5,7-diHT = 5-methoxy-dimethyltryptamine > alpha-methyl-5HT > 2-methyl-5HT. Incubation of ovarian follicles with high doses of some antagonists (mianserin and metergoline) induced oocyte GVBD, although this effect was associated with high levels of oocyte atresia during GVBD or shortly after maturation. Consequently, doses of the antagonist too low to induce GVBD were tested for their ability to block the 5-HT inhibitory action; the rank order of potency was: MDL-72222 = metoclopramide > metergoline > propanolol > ketanserin. Dopamine, acetylcholine, epinephrine, and norepinephrine could also inhibit 17,20 beta P-induced GVBD, although at doses much higher than those of 5-HT; melatonin and histamine had no effect on oocyte maturation. These results suggest that specific receptors mediate the inhibitory action of 5-HT on the steroid-triggered meiosis resumption. The pharmacological profile of these 5-HT receptors is different from those of any known mammalian 5-HT receptor, although they showed some similarities to the 5-HT1A, 5-HT2, and 5-HT3 receptors, as well as to 5-HT receptors on oocytes of some bivalve molluscs.

17-alpha-Hydroxyprogesterone↗

The developing Xenopus oocyte specifies the type of gonadotropin-stimulated steroidogenesis performed by its associated follicle cells.

As a response to gonadotropin, amphibian ovarian follicles primarily synthesize and secrete estradiol-17 beta (E2) during vitellogenesis and progesterone (P) when fully grown. Stage IV (vitellogenic) and stage VI (full-grown) ovarian follicles from Xenopus laevis, as well as intermediate sizes, were used to explore this change in steroidogenesis. Optimum steroidogenesis occurred in both stage IV and stage VI follicle exposed for 6 h to 20 IU human chorionic gonadotropin/mL. Although the total amounts of steroid fund were about the same, the E2/P ratios ranged from 26 to 35 for intact stage IV follicles, but only 0.02-0.03 for intact stage VI follicles. Steroid-producing follicle cells were isolated from stage IV and stage VI follicles by non-enzymatic procedures, were washed and were tested for steroidogenic activity in the absence of oocytes. In both cases, P was the predominant steroid produced (E2/P = 0.004-0.04), so the presence of stage IV, but not stage VI, oocytes appears to be necessary for E2 production as a response to gonadotropin. Octanol had no significant effect on the E2/P ratio of intact stage IV follicle. Dissected oocyte/follicle cell preparations from stage IV follicles were also periodically challenged with gonadotropin over 72 h, during which time most follicle cells detached from the oocyte and formed a monolayer over the bottom of the culture dish. The relatively high E2/P ratios for such preparations showed no significant change when stimulated with gonadotropin at various times over the 72 h, as long as the medium was not replaced. We conclude that the estrogenic effect of stage IV oocytes is most likely mediated by a secretory product rather than by gap junctions or by cell contact. Because the X. laevis oocyte has been shown to be a self-differentiating cell, the steroidogenic shift that occurs in developing ovarian follicles appears to be fundamentally regulated by the growing oocyte as if undergoes a physiological change rather than by different gonadotropins.

Animals↗

Neuropeptide Y in the forebrain and retina of the killifish, Fundulus heteroclitus.

The organization of the neuropeptide Y (NPY)-immunoreactive system in the forebrain, pineal organ and retina of a biweekly spawning fish (Fundulus heteroclitus) was investigated. Immunoreactivity was encountered in neurons of the nucleus olfactoretinalis, in the large population of neurons in the floor of the telencephalon, and in the nucleus entopeduncularis. Isolated somata were encountered in the hypophysiotropic hypothalamic nuclei, viz., the nucleus preopticus periventricularis, nucleus preopticus, and nucleus lateralis tuberis. Immunoreactive somata were also seen in the nucleus dorsomedialis thalami. The olfactory bulb was abundantly innervated by NPY fibers. The telencephalon showed thick radiating processes basally and terminal fields with modest to high densities in the dorsal and lateral regions. NPY-immunoreactive fibers were also conspicuous in the preoptic area, suprachiasmatic nucleus, tuberal hypothalamus, pituitary gland, and paraventricular thalamic regions; discrete CSF-contacting sites were also encountered. Of special interest was the occurrence of NPY immunoreactivity in fibers of the pineal stalk and organ. In the retina, some amacrine cells displayed immunoreactivity, while the inner plexiform layer revealed a well-developed pattern of NPY fibers different from that previously described for the goldfish.

Animals↗

Semilunar follicular cycle of an intertidal fish: the Fundulus model.

In their habitat, Fundulus heteroclitus (Cyprinodontidae) spawn against a variety of specific substrates that become available only at spring tides; they thus exhibit a semilunar reproductive cyclicity. In the laboratory at 28 +/- 0.6 degrees C and 14L:10D, this cyclicity is free-running and can be observed by means of daily egg collection and frequent ovarian sampling. Daily egg collection cycles gathered from three different spawning groups were pooled chronologically; the cycles were determined by nonlinear regression sine-curve matching and so were dated from Days -6 to +6, with Day 0 as the peak egg collection day. Ovaries were sampled on Days -6, -4, -2, -1, +1, +2, and +4. The pooled data indicated that early-maturing follicles (1.3- to 1.6-mm diameter) are recruited from a constant reserve of vitellogenic follicles (0.7- to 1.2-mm diameter) and become abundant early in the cycle but are depleted during Days -6 to +4 by the formation of late-maturing follicles and ovulated eggs. A midcycle peak of ovulated eggs in the ovary corresponds to the egg collection peak. This progression of follicles followed by spawning decreases the total count of the follicles (> or = 0.7-mm diameter) and eggs in the ovary by 52% (from 454 to 219 per 10 g of female weight). The semilunar follicular cycle in this Fundulus model thus includes 1) an early-cycle follicle recruitment, 2) an early- and midcycle follicle maturation, and 3) a midcycle egg ovulation and spawning. This follicular cycle also proceeds in fish deprived of a spawning substrate. A female in the wild would thus execute the follicular cycling in a timely sequence, in anticipation of each recurring spring tide, whether or not a natural substrate becomes available. We conclude that F. heteroclitus provides a useful nonmammalian model for the study of cyclic reproductive activity in the laboratory.

Animals↗

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 bp were isolated through a combination of degenerate oligonucleotide probing of the library and PCR. The cDNA sequence contains a 5,112 bp 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.

Amino Acid Sequence↗

A precise structural analysis of a fertilization-associated carbohydrate-rich glycopeptide isolated from the fertilized eggs of euryhaline killi fish (Fundulus heteroclitus). Novel penta-antennary N-glycan chains with a bisecting N-acetylglucosaminyl residue.

A novel carbohydrate-rich sialoglycopeptide of apparent molecular mass approximately 6 kDa was isolated from the fertilized eggs of Fundulus heteroclitus (euryhaline killi fish). This glycopeptide is a member of the L-hyosophorin family, characterized by its high content of carbohydrate (80-90% by weight) and formed by depolymerization of the precursor glycopoly-protein (H-hyosophorin) upon fertilization. The structures of the N-glycan chains were unambiguously established by a combination of compositional analysis, methylation analysis, selective chemical degradation (periodate oxidation-Smith degradation and hydrazinolysis-nitrous acid deamination), enzymatic (peptide:N-glycosidase F, several beta-galactosidases, beta-hexosaminidase and alpha-galactosidase) digestions and instrumental analyses (1H-NMR and fast atom bombardment mass spectrometry) to have the novel and unique carbohydrate sequences, Gal alpha 1-->3(Gal beta 1-->4)Gal beta 1-->4GlcNAc beta 1--> and Gal alpha 1-->3(+/- GalNAc beta 1-->4GlcNAc beta 1-->3Gal beta 1-->4)Gal beta 1-->4GlcNAc beta 1-->. This study represents the first detailed investigation of the nature of bulky complex asparagine-linked penta-antennary glycans with a bisecting GlcNAc residue in glycoproteins. Expression of such bulky multiantennary glycan units on proteins may be essential during early embryogenesis.

Acetylglucosamine↗

Functional heterologous gap junctions in Fundulus ovarian follicles maintain meiotic arrest and permit hydration during oocyte maturation.

The physiological significance of heterologous gap junctions between granulosa cells and the oocyte was investigated in late vitellogenic ovarian follicles of the teleost Fundulus heteroclitus. Lucifer Yellow injected into the oocyte readily passed to the overlying granulosa cells, demonstrating effective dye-coupling. Passage of the fluorescent dye, and hence intercellular communication, was inhibited both by the tumor-promoting phorbol ester phorbol 12-myristate 13-acetate (PMA) and by 1-octanol, known uncouplers of gap junctions in a variety of invertebrate and vertebrate cell types. Octanol alone also initiated resumption of meiosis in follicle-enclosed oocytes, indicating that granulosa cells normally maintain meiotic arrest, as apparently occurs in mammalian and amphibian follicles. Both PMA and octanol also consistently inhibited the hydration process that normally accompanies meiotic maturation. These results support a previously suggested hypothesis that K+, which is the primary osmotic effector for oocyte hydration, is translocated via gap junction from granulosa cells to the maturing oocyte.

Analysis of Variance↗

Steroidogenesis in Fundulus heteroclitus V.: purification, characterization, and metabolism of 17 alpha,20 beta-dihydroxy-4-pregnen-3-one by intact follicles and its role in oocyte maturation.

In vitro steroidogenesis of ovarian follicles incubated with radioactive precursors or a Fundulus heteroclitus pituitary extract (FPE) was investigated. Steroids were extracted from both the medium and follicular tissue and fractionated by liquid or thin-layer chromatography. A similar pattern of steroid metabolites was obtained with either [14C]pregnenolone or [14C]progesterone as exogenous precursor. Several metabolites comigrated with known reference steroids and thus were tentatively identified. Some have been previously reported to induce germinal vesicle breakdown (GVBD) in this and other species, particularly 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (DHP). [3H]DHP added to intact follicles or denuded oocytes was also extensively metabolized. All the DHP metabolites produced by the intact follicle were tested for biological activity. Three of the metabolites were almost as effective inducers of GVBD as DHP itself, and two were tentatively identified as 5 alpha-pregnan-3 alpha,17 alpha,20 beta-triol and 5 alpha-pregnan-3 beta,17 alpha,20 beta-triol. However, DHP was the most potent and the quickest inducer of GVBD, indicating that its maturational action is not due to metabolic conversion to a more active form. In addition, we found two very active fractions after HPLC analysis of steroid extracts from FPE-stimulated follicles: one that corresponded to and was further identified (mass spectroscopy) as DHP and a second tentatively identified as the DHP metabolite 5 alpha-pregnan-3 beta,17 alpha,20 beta-triol. This study provides strong evidence that DHP plays the major role as a maturation inducing-steroid in F. heteroclitus, even though DHP is not the only active steroid produced by maturing follicles.

Animals↗

Steroidogenesis in Fundulus heteroclitus. IV. Dichotomous effects of a phorbol ester on ovarian steroid production and oocyte maturation.

The possible role of protein kinase C (PKC) activation in mediating the stimulatory actions of a Fundulus pituitary extract (FPE) on ovarian steroidogenesis and oocyte maturation was investigated. The phorbol ester, phorbol 12-myristate 13-acetate (PMA), alone slightly increased basal 17 alpha-hydroxy,20 beta-dihydroprogesterone (DHP) and 17 beta-estradiol (E2) synthesis and significantly stimulated germinal vesicle breakdown (GVBD). Addition of FPE promoted synthesis of DHP, testosterone (T), and E2, and initiated GVBD. Phorbol ester inhibited FPE-induced steroidogenesis but increased the number of oocytes that underwent GVBD. Phorbol ester also markedly impeded induction of steroidogenesis by dibutyryl cAMP and differentially affected the conversion of 25-hydroxycholesterol, pregnenolone, or progesterone to DHP, T, and E2: DHP production was not affected; T production diminished; and E2 synthesis increased (T aromatization also increased). These results suggest an inhibitory role for the PKC pathway on FPE-induced ovarian steroid production, with PMA appearing to affect various steroidogenic steps. The stimulatory action of PMA on oocyte maturation seems to be independent of follicular steroid production since aminoglutethimide, an inhibitor of steroidogenesis, did not block PMA-induced GVBD. Moreover, PMA had a marked stimulatory effect on GVBD in denuded oocytes. Thus, in contrast to the inhibitory role found for the PKC pathway on ovarian follicular steroidogenesis, activation of PKC in the oocyte may serve as a signal-transducing mechanism leading to GVBD.

Alkaloids↗

Analytical and experimental studies on the relationship between Na+, K+, and water uptake during volume increases associated with Fundulus oocyte maturation in vitro.

Oocytes of marine and estuarine teleosts often undergo pronounced volume increases during the maturation phase of development that precedes ovulation and fertilization. To examine the physiological correlates of these volume increases, prematuration follicles of the saltmarsh teleost, Fundulus heteroclitus, were cultured in vitro with a maturation-inducing steroid (17 alpha-hydroxy-20 beta-dihydroprogesterone). Mean follicle volume rose significantly (75%) during a 40-h incubation period. Similar to the situation previously found in vivo, uptake of water by the maturing follicle was responsible for this volume increase in vitro, with the water content increasing from 62% to 78% of the total follicle mass. The follicle contents of two probable osmotic effectors--Na+ and K(+)--also rose, the increase in K+ being twice that of Na+. The influx of K+ even exceeded water uptake, resulting in a net increase in the concentration of this cation. It thus appears that the influx of these cations, in particular K+, is a major cause of the uptake of osmotically obligated water and subsequent volume increase experienced by maturing F. heteroclitus follicles. In a search for operant mechanisms, it was found that follicle hydration, but not maturation, was strictly dependent on external K+ in a concentration-dependent manner. The mechanism by which K+ accumulates in the follicle was insensitive to ouabain, so that a typical Na+, K(+)-ATPase mechanism does not appear to be involved. The ability of external K+ to promote follicle hydration was gradually lost during the maturation process as the oocyte dissociated from the surrounding granulosa cells in preparation for ovulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Fundulus heteroclitus gonadotropins. 3. Cloning and sequencing of gonadotropic hormone (GTH) I and II beta-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.

Amino Acid Sequence↗

Preparation and rapid resolution of Xenopus phosvitins and phosvettes by high-performance liquid chromatography.

The presence of acidic phosivitins phosvettes in Xenopus laevis yolk platelets and their purification by (NH4)2SO4 precipitation of associated lipovitellin were documented by polyacrylamide gel electrophoresis followed by staining with Stains-all. Procedures were further developed to resolve the various entities present in the crude phosvitin/phosvette fraction by size-exclusion, anion-exchange, and hydrophobic interaction chromatography, using a Pharmacia FPLC system, and their resolution was documented by both electrophoresis and two-dimensional chromatography. Four major entities (phosvitins 1 and 2; phosvettes 1 and 2) were observed, but microheterogeneity was also apparent, particularly by hydrophobic interaction chromatography. The new separation procedures require min/h rather than h days.

Animals↗

Placement of small lipovitellin subunits within the vitellogenin precursor in Xenopus laevis.

N-terminal amino acid sequence data for the small lipovitellin subunits in Xenopus laevis crystalline yolk platelets indicate that LV2 beta is derived from vitellogenin A2 and that LV2 tau is most likely derived from vitellogenin A1. The small lipovitellin subunits apparently commence within the exon 24 region of the parental vitellogenins, flanking the C-terminal end of phosvitin. As a consequence, we conclude that most of the vitellogenin sequence encoded by exons 30 to 35 is not accounted for by the known yolk proteins.

Amino Acid Sequence↗