Extrafollicular mediation of oocyte maturation by radial nerve factor in starfish Pisaster ochraceus

In starfish ovaries follicle cells that envelop each oocyte are thought to mediate the production of a maturation inducing substance (MIS), identified as 1-methyladenine, that induces maturation and spawning of oocytes after exposure to a gonadotropic substance secreted by the radial nerve (RNF). Studies were carried out to assess the possible role of extrafollicular cells within the ovarian wall in mediating this signal transduction process in the ovary of Pisaster ochraceus. Oocyte maturation and spawning occurred following the addition of RNF to intact ovarian tissue in vitro whereas no maturation occurred following the addition of RNF to germinal vesicle (GV) oocytes or GV oocytes surrounded by follicle cells. In contrast, oocyte maturation occurred when small ovarian wall fragments, lacking mature follicles, were incubated with GV oocytes and RNF. Neither actinomycin D nor cycloheximide altered RNF induction of oocyte maturation in the presence of the ovarian wall tissue whereas preheating (boiling water for 5 min) the tissue obliterated its response to RNF. Non-ovarian tissues failed to produce MIS in response to RNF. Results suggest that ovarian components other than the follicle cells that envelop fully grown immature oocyte are responsive to RNF and represent a significant and previously unrecognised intra-ovarian source of MIS.     

In vitro inhibition of oocyte and follicular maturation and spawning in starfish (Asterias forbesi) by 2-4-dinitrophenol

The in vitro effects of 2-4-dinitrophenol (DNP) on spawning and follicular and oocyte maturation in starfish ovaries and its various cellular components were investigated. Spawning and oocyte and follicular maturation induced by starfish gonadotropin radial nerve factor (RNF) in isolated ovarian fragments were all inhibited by appropriate doses of DNP. DNP inhibits processes which occur shortly after addition of the gonadotropin; in ovarian fragments insensitivity to DNP inhibition occurred shortly after addition of RNF but prior to initiation of spawning. Spontaneous follicular and oocyte maturation which occurred following release of ovarian follicles into sea water was prevented by DNP. In non-spontaneously maturing follicles released from the ovary, DNP inhibited both follicle and oocyte maturation induced by the secondary stimulator of spawning and maturation, 1-methyladenine (1-MA). DNP also inhibited 1-MA induced meiotic maturation in isolated immature oocytes incubated in the absence of follicle cells. Inhibition of oocyte maturation was not associated with inhibition of 3H-1-MA incorporation by isolated oocytes. Immature oocytes incubated in the presence of DNP underwent maturation following washing and subsequent exposure to 1-MA. Immature oocytes initially exposed to both 1-MA and DNP, however, showed decreased maturation responsiveness following washing and re-exposure to 1-MA. The results suggest that the inhibitory effects of DNP on spawning and oocyte maturation are the result of direct effects on the oocytes and possibly other cells and tissues within the ovary.     

In vitro Inhibition of Oocyte and Follicular Maturation and Spawning in Starfish (Asterias forbesi) by 2-4-Dinitrophenol

The in vitro effects of 2-4-dinitrophenol (DNP) on spawning and follicular and oocyte maturation in starfish ovaries and its various cellular components were investigated. Spawning and oocyte and follicular maturation induced by starfish gonadotropin radial nerve factor (RNF) in isolated ovarian fragments were all inhibited by appropriate doses of DNP. DNP inhibits processes which occur shortly after addition of the gonadotropin; in ovarian fragments insensitivity to DNP inhibition occurred shortly after addition of RNF but prior to initiation of spawning. Spontaneous follicular and oocyte maturation which occurred following release of ovarian follicles into sea water was prevented by DNP. In non-spontaneously maturing follicles released from the ovary, DNP inhibited both follicle and oocyte maturation induced by the secondary stimulator of spawning and maturation, 1-methyladenine (1-MA). DNP also inhibited 1-MA induced meiotic maturation in isolated immature oocytes incubated in the absence of follicle cells. Inhibition of oocyte maturation was not associated with inhibition of ³H-1-MA incorporation by isolated oocytes. Immature oocytes incubated in the presence of DNP underwent maturation following washing and subsequent exposure to 1-MA. Immature oocytes initially exposed to both 1-MA and DNP, however, showed decreased maturation responsiveness following washing and re-exposure to 1-MA. The results suggest that the inhibitory effects of DNP on spawning and oocyte maturation are the result of direct effects on the oocytes and possibly other cells and tissues within the ovary.     

Ultrastructure of the ovary and oogenesis in the polychaete Capitella jonesi (Hartman, 1959)

Ultrastructural features of the ovary and oogenesis in the polychaete Capitella jonesi (Hartman, '59) have been described. The ovaries are paired, sac-like follicles suspended by mesenteries in the ventral coelom throughout the midbody region of the mature worm. Oogenesis is unsynchronized and occurs entirely within the ovary, where developing gametogenic stages are segregated spatially within a germinal and a growth zone. Multiplication of oogonia and differentiation of oocytes into the late stages of vitellogenesis occur in the germinal region of the ovary, whereas late-stage vitellogenic oocytes and mature eggs are located in a growth zone. Follicle cells envelop the oocytes in the germinal zone of the ovary and undergo hypertrophy and ultrastructural changes that correlate with the onset of vitellogenesis. These changes include the development of extensive arrays of rough ER and numerous Golgi complexes, formation of microvilli along the surface of the ovary, and the initiation of extensive endocytotic activity. Oocytes undergo similar, concomitant changes such as the differentiation of surface microvilli, the formation of abundant endocytotic pits and vesicles along the oolemma, and the appearance of numerous Golgi complexes, cisternae of rough ER, and yolk bodies. Yolk synthesis appears to occur by both autosynthetic and heterosynthetic processes involving the conjoined efforts of the Golgi complex and rough ER of the oocyte and the probable addition of extraovarian (heterosynthetic) yolk precursors. Evidence is presented that implicates the follicle cells in the synthesis of yolk precursors for transport to the oocytes. At ovulation, mature oocytes are released from the overy after the overlying follicle cells apparently withdraw. Bundles of microfilaments within the follicle cells may play a role in this withdrawal process.     

Germinal epithelium, folliculogenesis, and postovulatory follicles in ovaries of rainbow trout, Oncorhynchus mykiss (Walbaum, 1792) (Teleostei, protacanthopterygii, salmoniformes)

The rainbow trout, Oncorhynchus mykiss (Walbaum, 1792), is a salmoniform fish that spawns once per year. Ripe females that had ovulated naturally, and those induced to ovulate using salmon gonadotropin-releasing hormone, were studied to determine whether follicles were forming at the time of spawning and to describe the process of folliculogenesis. After ovulation, the ovaries of postspawned rainbow trout were examined histologically, using the periodic acid-Schiff procedure, to stain basement membranes that subtend the germinal epithelium and to interpret and define the activity of the germinal epithelium. After spawning, the ovary contained a few ripe oocytes that did not ovulate, numerous primary growth oocytes including oocytes with cortical alveoli, and postovulatory follicles. The germinal epithelium was active in postspawned rainbow trout, as determined by the presence of numerous cell nests, composed of oogonia, mitotic oogonia, early diplotene oocytes, and prefollicle cells. Cell nests were separated from the stroma by a basement membrane continuous with that subtending the germinal epithelium. Furthermore, follicles containing primary growth oocytes were connected to the germinal epithelium; the basement membrane surrounding the follicle joined that of the germinal epithelium. After ovulation, the basement membrane of the postovulatory follicle was continuous with that of the germinal epithelium. We observed consistent separation of the follicle, composed of an oocyte and surrounding follicle cells, from the ovarian stroma by a basement membrane. The follicle is derived from the germinal epithelium. As with the germinal epithelium, follicle cells derived from it never contact those of the connective tissue stroma. As with epithelia, they are always separated from connective tissue by a basement membrane.     

Breakdown of starfish ovarian follicle induced by maturation-promoting factor

Immature starfish oocytes are surrounded by envelopes consisting of follicular cells. These cells adhere to each other and to the oocyte, immobilizing the latter within the ovary. When isolated oocytes in their follicles are treated with 1-methyladenine (1-MeAde), germinal vesicle breakdown (GVBD) and follicular envelope breakdown (FEBD) occur simultaneously. The 1-MeAde acts on the oocyte surface to produce a maturation-promoting factor (MPF) in the cytoplasm, which brings about GVBD. In the present study, MPF was found to induce FEBD as well as GVBD when injected into immature oocytes with their follicles in Asterina pectinifera. Although GVBD was induced by MPF in the presence of cytochalasin D, this drug prevented MPF-induced FEBD, and each follicular cell remained in situ on the surface of the oocyte. However, desmosomes connecting the processes of the follicle cell with the oocyte surface were disrupted following MPF injection even in the presence of cytochalasin D, and the processes became detached from the oocyte. FEBD occurred in these oocytes when cytochalasin D was removed, resulting in the formation of a small follicular clump by microfilament-mediated contraction of the follicle cells. These results show that FEBD is not brought about by the direct action of 1-MeAde but by the action of MPF. Therefore, in starfish, spawning as well as oocyte maturation is directly triggered by MPF produced under the influence of 1-MeAde.     

Pigmented Follicle Cells and the Maturation of Oocytes in the Sand Dollar, Dendraster excentricus

Early in the spawning season female D. excentricus can be induced to spawn oocytes in the late stages of oogenesis. Observations with light microscopy indicate that pigment cells migrate in the jelly coat away from the surface of oocytes coincident with the final growth of the oocyte and its maturation. The pigment cells undergo a series of changes in shape and the oocyte elaborates arrays of long microvilli as the cells elevate from the surface of the oocyte. Transmission electron microscopy (TEM) observations of oocytes fixed within the ovary indicate that features of the sequence observed in the spawned oocytes normally take place within the ovary, prior to spawning. Extracts of the pigment cells induce a low level of germinal vesicle breakdown in asteroid oocytes. It is proposed that these cells are homologous to the follicle cells of other echinoderms and are involved in stimulating the maturation of oocytes.     

Inhibitory effect of a SALMFamide neuropeptide on secretion of gonad-stimulating substance from radial nerves in the starfish Asterina pectinifera

In starfish, the peptide hormone gonad-stimulating substance (GSS) secreted from nervous tissue stimulates oocyte maturation to induce 1-methyladenine (1-MeAde) production by ovarian follicle cells. The SALMFamide family is also known to an echinoderm neuropeptide. The present study examined effect of SALMFamide 1 (S1) on oocyte maturation of starfish Asterina pectinifera. Unlike GSS, S1 did not induce spawning in starfish ovary. In contrast, S1 was found to inhibit GSS secretion from radial nerves by treatment with high K+ concentration. Fifty percent inhibition was obtained by 0.1 mM S1. S1 did not have any effect on GSS- and 1-MeAde-induced oocyte maturation. Following incubation with a S1 antibody and subsequently with rhodamine-conjugated second antibody, neural networks were observed in ovaries. The networks were restricted mainly to their surface with little evidence of immunoreactivity inside the basement membranes. This indicates that neural networks are distributed in the ovarian wall. The result further suggests that S1 plays a role in oocyte maturation to regulate GSS secretion from the nervous system.     

Incorporation of tritiated adenosine into mouse ovum RNA

The total RNA of ovulated mouse ova has been examined by polyacrylamide gel electrophoresis. The amount of RNA present in the two main peaks observed, 28 S and 18 S ribosomal RNA, has been estimated as 0.20 ng.The RNA of ovulated mouse ova was labeled by exposure of growing mouse oocytes to adenosine-8-³H in vivo. For this purpose a small volume of a concentrated solution of the precursor was injected into the ovarian bursa, and ova were collected by superovulation at various subsequent times. The major growth phase of the oocyte is known to lie between 20 and 6 days before ovulation. Significant incorporation into egg RNA was observed when bursal injection was performed between 19 and 7 days, but not between 5 days and 1 day before ovulation.The types of labeled RNA in ova ovulated at five intervals between 19 and 7 days after bursal injection of adenosine-8-³H or uridine-5,6-³H were analyzed by polyacrylamide gel electrophoresis. The distribution of label on the gels demonstrated that the bulk of the label appeared in ribosomal RNA and transfer RNA. In addition labeled heterogeneous RNA was estimated to represent 10–15% of the total incorporation.     

The ovarian maturation cycle of the Norway lobster Nephrops norvegicus (Linnaeus, 1758) (Crustacea,Decapoda) from the western Mediterranean Sea

Summary

The Norway lobster Nephrops norvegicus lives in the continental margins of the western Mediterranean Sea at depths between 100 and 600 m. It constitutes an important fisheries resource and presents a seasonal reproductive pattern. Female Norway lobsters were obtained each month from a vessel fishing off Barcelona. One hundred females caught in June 2002 were kept in the laboratory. After spawning, ovarian samples were taken every 30 days with the objective of monitoring the first steps in ovarian maturation. The gonado-somatic index (GSI) remained low over the 6 months during which the females carried their eggs, plus for a further 2–3 months. However, this study suggests that ovarian maturation is a continuous process with two different phases, taking at least 6–8 months during which the female carries its eggs. There is an increase in oocyte numbers; the germinal zone produces oogonia; and the oocytes that develop migrate to the periphery, pushing the post-ovulatory follicles to the wall of the ovary and reinforcing it for subsequent spawning. Besides this increase in the number of oocytes, vitellogenesis begins 2–3 months after the eggs hatch. Oocytes then grow and the ovaries gain weight and change from a cream color to a blackish-green. When the GSI reached 10, spawning occurred and, from then on, the ovary is mainly composed of post-ovulatory follicles.     

A reversible, hydrogen ion blockade of spontaneous oocyte maturation in the starfish: locus of action.

Starfish (Asterias forbesi) oocytes encased within their follicle cells mature spontaneously during a portion of the normal reproductive period when released from the ovary into seawater. A previous report has shown that oocytes isolated in acidic seawater do not mature spontaneously but retain the capacity to do so when returned to normal seawater. The object of this study was to determine the mechanism by which acidic pH reversibly blocks spontaneous oocyte maturation in isolated follicles. Incidence of spontaneous oocyte maturation in follicles isolated in acidic seawater decreased as pH decreased from 7 to 4. Oocytes in which spontaneous maturation was inhibited (ASW at pH 4.7 TO 5.4) underwent germinal vesicle breakdown with the addition of 1-methyladenine. Oocytes isolated in acidic seawater (pH 4 or 5) with intact follicle cells matured spontaneously when transferred immediately to normal seawater pH 8); after four hours, 60-65% of the follicles incubated in seawater at pH 5 matured spontaneously when returned to normal seawater as compared to less than 10% of the follicles maintained at pH 4. Inhibition of spontaneous maturation was not reversible in the absence of the follicle cells. Oocytes isolated in acidic seawater with their follicle cells did not spontaneously mature when transferred to calcium-free seawater at pH 8. The results obtained support the hypothesis that acidic seawater reversibly inhibits spontaneous oocyte maturation by interfering with the release of meiosis-inducing substance from the follicle cells.     

Degeneration of germ line cells in amphibian ovary

Ogielska, M., Rozenblut, B., Augustyńska, R., Kotusz, A. 2010. Degeneration of germ line cells in amphibian ovary. —Acta Zoologica (Stockholm) 91 : 319–327 We studied the morphology of degenerating ovarian follicles in juvenile and adult frogs Rana temporaria, Rana lessonae and Rana ridibunda. Degeneration of primordial germ cells was never observed and was extremely rare in oogonia and early oocytes in a cyst phase in juveniles. Previtellogenic oocytes were rarely affected. Three main types of atresia were identified. In type I (subdivided into stages A–D), vitellogenic oocytes are digested by proliferating follicle cells that hypertrophy and become phagocytic. A – germinal vesicle shrinks, nucleoli fuse, oocyte envelope interrupts, and follicular cells hypertrophy; B – follicular cells multiply and invade the oocyte; C – entire vesicle is filled by phagocytic cells; D – degenerating phagocytes accumulate black pigment. Type II is rare and resembles breakdown of follicles and release of ooplasm. In type III, observed in previtellogenic and early vitellogenic oocytes, ooplasm and germinal vesicle shrink, follicle cells do not invade the vesicle, and condensed ooplasm becomes fragmented. The residual oogonia in adult ovaries (germ patches) multiply, but soon degenerate.     

Synthesis and characterization of amplified DNA in oocytes of the house cricket,Acheta domesticus (Orthoptera: Gryllidae)

At a time in the life cycle when a large proportion of the oocytes of Acheta incorporate ³H-thymidine into an extrachromosomal DNA body, synthesis of a satellite or minor band DNA, the density of which is greater than main band DNA, is readily detected. Synthesis of the satellite DNA is not detectable in tissues, the cells of which do not have a DNA body, or in ovaries in which synthesis of extrachromosomal DNA by the oocytes is completed. The DNA body contains the amplified genes which code for ribosomal RNA. However, less than 1 percent of the satellite DNA, all of which appears to be amplified in the oocyte, is complementary to ribosomal 18S and 28S RNA. In situ hybridization demonstrates that non-ribosomal elements, like the ribosomal elements of the satellite DNA, are localized in the DNA body.Abbreviations used rRNA ribosomal RNA, includes 18S and 28S RNA - rDNA gene sequences complementary to rRNA - cRNA complementary RNA synthesized in vitro     

Comparative biology of oogenesis in nemertean worms

Stricker, S. A., Smythe, T. L., Miller, L. and Norenburg, J. L. 2001. Comparative biology of oogenesis in nemertean worms. — Acta Zoologica (Stockholm) 82 : 213–230
In order to supplement previous analyses of oogenesis in nemertean worms, this study uses light and electron microscopy to compare the ovaries and oocytes in 16 species of nemerteans that represent various taxa within the phylum. Nemertean ovaries comprise serially repeated sacs with an ovarian wall that characteristically includes myofilament-containing cells interspersed among the germinal epithelium. Each oocyte can attach to the germinal epithelium by a vegetally situated stalk and resides in the ovarian lumen without being surrounded by follicle cells. In the ovary, oocytes arrest at prophase I of meiosis and contain a hypertrophied nucleus ('germinal vesicle') that often possesses multiple nucleoli. Intraovarian growth apparently involves an autosynthetic mode of yolk formation in most nemerteans and generates oocytes that measure ~60 µm to 1 mm. When fully developed, oocytes can be discharged through a short gonoduct and are either spawned freely or deposited within egg cases. In most species, oocytes released from the ovary possess extracellular coats and resume maturation by undergoing germinal vesicle breakdown (GVBD). Such post-GVBD specimens also form a punctate endoplasmic reticulum that may facilitate fertilization and development.     

Characterization and action of meiotic maturation inhibitors in starfish ovary

Mechanical release of oocytes from the ovary of the starfish Asterias amurensis into sea water results in “spontaneous” meiotic maturation of the oocytes. The substances blocking the maturation of Asterias oocytes have been purified from the ovary and shown to be steroid glycosides named asterosaponins A and B. The extract prepared from isolated oocytes was incapable of inhibiting oocyte maturation. The ovarian extract inhibited the production of 1-methyladenine (1-MA) in follicle cells surrounding the oocyte. The ovarian extract failed to influence 1-MA-induced maturation of the oocyte with or without follicle cells. It can be concluded from the present results that the role of the ovarian extract containing steroid glycosides is to arrest “spontaneous” production of 1-MA in follicle cells. The suppression can be overcome by the action of a gonadotropic peptide hormone released from the nerve tissue.     

Metaphase I arrest of starfish oocytes induced via the MAP kinase pathway is released by an increase of intracellular pH

Reinitiation of meiosis in oocytes usually occurs as a two-step process during which release from the prophase block is followed by an arrest in metaphase of the first or second meiotic division [metaphase I (MI) or metaphase II (MII)]. The mechanism of MI arrest in meiosis is poorly understood, although it is a widely observed phenomenon in invertebrates. The blockage of fully grown starfish oocytes in prophase of meiosis I is released by the hormone 1-methyladenine. It has been believed that meiosis of starfish oocytes proceeds completely without MI or MII arrest, even when fertilization does not occur. Here we show that MI arrest of starfish oocytes occurs in the ovary after germinal vesicle breakdown. This arrest is maintained both by the Mos/MEK/MAP kinase pathway and the blockage of an increase of intracellular pH in the ovary before spawning. Immediately after spawning into seawater, activation of Na+/H+ antiporters via a heterotrimeric G protein coupling to a 1-methyladenine receptor in the oocyte leads to an intracellular pH increase that can overcome the MI arrest even in the presence of active MAP kinase.     

Ovary of the golden crab,Chaceon fenneri: I. Post-spawning and oosorption

Oosorption has been considered an important strategy in many invertebrate species which occurs in response to behavioral, ecological, or physiological factors. In crustaceans, the early light microscopic studies of the ovary attributed a role in oosorption to follicle cells, hemocytes, or phagocytes. In this study, ovaries were collected from female golden crabs following spawning and processed for examination by electron microscopy. Following spawning, several unspawned oocytes which had become dissociated from their follicle cells were found in the ovaries. They appeared to be lodged within the lumen. Such oocytes were observed undergoing various stages of autolysis. At no time were hemocytes or recognizable phagocytes found in the lumen of the ovaries or in contact with the degenerating oocytes. Follicle cells which had surrounded the oocytes prior to the time of spawning exhibited disrupted membranes. Resorption of unspawned eggs appears to occur by autolysis of the individual oocytes. Several of the females who had recently spawned had numerous sperm in their ovaries. Such sperm may have been pressed into the lumen at the time of spawning or during the fixation process.     

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.     

Ultrastructure and melatonin 1a receptor distribution in the ovaries of African ostrich chicks

Healthy 90-day-old ostrich chicks were used in the present study. The ultrastructure and melatonin 1a receptor (MT1) distribution in the ovaries of ostrich chicks was observed by transmission electron microscope and light microscope. The results showed that the ostrich chick ovary contained primordial follicles, primary follicles and secondary follicles, but no mature follicles. There are some unique ultrastructural characteristics observed in the secondary follicle, such as the cortical granule, which was located in cytoplasm beside the nucleus and appeared first in the oocyte. The zona radiata appeared in the secondary follicle, and there was an obvious vitelline membrane. There were intraovarian rete, connecting rete, and extraovarian rete in the ovaries of ostrich chicks. This is the first study that provides immunohistochemical evidence for the localization of the melatonin MT1 in the ostrich chick ovary. The germinal epithelium, follicular cell layer of every grade of follicle, cytoplasm of the oocyte and interstitial cells all expressed MT1. The expression of positive immunoreactivity materials was the strongest in the follicular cell layer of the primordial follicle and germinal epithelium, was weaker in the follicular cell layer of the primary follicle and secondary follicle, and was weakest in the oocytes of all grades of follicle. In addition, the extraovarian rete displayed strong positive expression of MT1, while there was no positive expression in the intraovarian rete or connecting rete. The positive expression of MT1 immunoreactivity in the ovary was very strong, implying that the ovary is an important organ for synthesizing MT1.