Acquisition Of Maturational Competence Research Articles

Expression of two insulin receptor subtypes, insra and insrb, in zebrafish (Danio rerio) ovary and involvement of insulin action in ovarian function

Present study reports differential expression of the two insulin receptor (IR) subtypes in zebrafish ovary at various stages of follicular growth and potential involvement of IR in insulin-induced oocyte maturation. The results showed that mRNA expression for IR subtypes, insra and insrb, exhibited higher levels in mid-vitellogenic (MV) and full-grown (FG) rather than pre-vitellogenic (PV) oocytes. Interestingly, compared to the levels in denuded oocytes, mRNAs for both insra and insrb were expressed at much higher level in the follicle layer harvested from FG oocytes. Immunoprecipitation using IRβ antibody could detect a protein band of desired size (∼95kDa) in FG oocyte lysates. Further, IRβ immunoreactivity was detected in ovarian tissue sections, especially at the follicle layer and oocyte membrane of MV and FG, but not PV stage oocytes. While hCG (10IU/ml) stimulation was without effect, priming with insulin (5μM) could promote oocyte maturation of MV oocytes in a manner sensitive to de novo protein and steroid biosynthesis. Compared to hCG, in insulin pre-incubated MV oocytes, stimulation with maturation inducing steroid (MIS), 17α,20β-dihydroxy-4-pregnen-3-one (DHP) elicited higher maturational response. Potential involvement of insulin-mediated action on acquisition of maturational competence and regulation of oocyte maturation was further manifested through up regulation of 20β-hydroxysteroid dehydrogenase (20β-hsd), MIS receptor (mPRα), insulin-like growth factor 3 (igf3) and IGF1 receptor (igf1rb), but not cyp19a expression in MV oocytes. Moreover, priming with anti-IRβ attenuated insulin action on meiotic G2-M1 transition indicating the specificity of insulin action and physiological relevance of IR in zebrafish ovary.

Probiotics Can Induce Follicle Maturational Competence: The Danio rerioCase1

In the present study, the effects of the probiotic Lactobacillus rhamnosus IMC 501 on the acquisition of oocyte maturational competence was examined in zebrafish (Danio rerio). L. rhamnosus administration induced the responsiveness of incompetent follicles (stage IIIa) to 17,20-dihydroxy-4-pregnen-3-one and their in vitro maturation. Acquisition of competence by the stage IIIa follicles was further validated by changes of lhr, mprb, inhbaa (activin betaA1), tgfb1, and gdf9 gene expression, which have recently emerged as key regulators of oocyte acquisition of maturational competence, and pou5f1 gene expression, which in other models has been shown to govern the establishment of developmental competence of oocytes. In addition, a DNA microarray experiment was conducted using the same follicles, and with relative gene ontology (GO) data analysis, the molecular effects of probiotic administration emerged. Molecular analysis using PCR-DGGE (denaturing gradient gel electrophoresis) approach, providing information about only the most abundant bacterial members of the microbial community, revealed that the probiotic was able to populate the gastrointestinal tract and modulate the microbial communities, causing a clear shift in them and specifically enhancing the presence of the lactic acid bacteria Streptococcus thermophilus. At the same time, PCR-DGGE analysis revealed that the probiotic was not directly associated with the ovaries. Finally, the effects of probiotic treatment on zebrafish follicle development were also analyzed by FPA (focal plane array) Fourier transform-infrared (FT-IR) imaging, a technique that provides the overall biochemical composition of samples. Changes were found above all in stage IIIa follicles from probiotic-exposed females; the modifications, observed in protein secondary structures as well as in hydration and in bands related to phosphate moieties, allowed us to hypothesize that probiotics act at this follicle stage, affecting the maturation phase.

Aromatase is expressed and active in the rainbow trout oocyte during final oocyte maturation

While it is generally well accepted that the ovarian follicular sites of estradiol-17β (E2) synthesis are restricted to somatic cells, the possible contribution of the germinal compartment has received little or no attention in teleosts. In order to demonstrate the expression of ovarian aromatase in the oocyte, cyp19a1a mRNA was studied in ovarian follicles by in situ hybridization. In addition, the expression of cyp19a1a was studied in both somatic and germinal compartments of the ovarian follicle in rainbow trout (Oncorhynchus mykiss) during final oocyte maturation (i.e., maturational competence acquisition and subsequent meiosis resumption) by real-time PCR. The enzymatic activity of ovarian aromatase was also studied in both somatic and germinal compartments of the ovarian follicle. Finally, E2 levels were monitored in follicle-enclosed oocytes throughout the pre-ovulatory period. We were able to demonstrate a significant ovarian aromatase expression and activity in the late vitellogenic oocyte. Furthermore, a dramatic decrease in aromatase expression and activity occurs in the oocyte during late oogenesis, concomitantly with the trend observed in surrounding follicular layers. We also report an unexpected increase of E2 levels in the oocyte during the pre-ovulatory period. To our knowledge, these observations are reported for the first time in any teleost species. Together, our data support the hypothesis of the participation of the germinal compartment in follicular estrogen synthesis and a biological role of E2 during oocyte and/or early embryo development.

The role of the insulin-like growth factor (IGF) system in zebrafish ( Danio rerio) ovarian development

The presence of an ovarian IGF system in teleosts suggests a distinct role in reproductive physiology. This study investigates the role of the ovarian IGF system in oocyte maturation, the acquisition of maturational competence and steroidogenesis in the zebrafish ( Danio rerio). Recombinant human IGF-I and IGF-II stimulated germinal vesicle breakdown (GVBD) in early vitellogenic (EV; 0.35–0.44 mm), midvitellogenic (MV; 0.45–0.56 mm) and full grown (FG; 0.57–0.65 mm) follicles incubated in vitro. By comparison, the maturation inducing steroid 17α,20β-dihydroxy-4-pregnen-3-one (17,20β-P) only induced GVBD in MV and FG follicles. Collectively these studies suggest that IGF is involved in oocyte maturation and that follicles become responsive to IGFs at an earlier stage compared to 17,20β-P. IGF-I also increased the responsiveness of the follicle to 17,20β-P, suggesting a role in promoting maturational competence. IGF-I alone and in combination with human chorionic gonadotropin (hCG) stimulated the production of 17,20β-P by ovarian follicles incubated in vitro. However, IGF-I had no effect on the production of 17β-estradiol (E 2) or the expression of genes involved in steroidogenesis (20β-hydroxysteroid dehydrogenase; 20β-HSD and P450c17-II). These results provide evidence that the IGF system plays an important role in the promotion of oocyte maturation and ovarian development in the zebrafish.

Comparative transcriptomic analysis of follicle-enclosed oocyte maturational and developmental competence acquisition in two non-mammalian vertebrates

BackgroundIn vertebrates, late oogenesis is a key period during which the oocyte acquires its ability to resume meiosis (i.e. maturational competence) and to develop, once fertilized, into a normal embryo (i.e. developmental competence). However, the molecular mechanisms involved in these key biological processes are far from being fully understood. In order to identify key mechanisms conserved among teleosts and amphibians, we performed a comparative analysis using ovarian tissue sampled at successive steps of the maturational competence acquisition process in the rainbow trout (Oncorhynchus mykiss) and in the clawed toad (Xenopus laevis). Our study aimed at identifying common differentially expressed genes during late oogenesis in both species. Using an existing transcriptomic analysis that had previously been carried out in rainbow trout, candidate genes were selected for subsequent quantitative PCR-based comparative analysis.ResultsAmong the 1200 differentially expressed clones in rainbow trout, twenty-six candidate genes were selected for further analysis by real-time PCR in both species during late oogenesis. Among these genes, eight had similar expression profiles in trout and Xenopus. Six genes were down-regulated during oocyte maturation (cyp19a1, cyp17a1, tescalcin, tfr1, cmah, hsd11b3) while two genes exhibited an opposite pattern (apoc1, star). In order to document possibly conserved molecular mechanisms, four genes (star, cyp19a1, cyp17a1 and hsd11b3) were further studied due to their known or suspected role in steroidogenesis after characterization of the orthology relationships between rainbow trout and Xenopus genes. Apoc1 was also selected for further analysis because of its reported function in cholesterol transport, which may modulate steroidogenesis by regulating cholesterol bioavailability in the steroidogenic cells.ConclusionsWe have successfully identified orthologous genes exhibiting conserved expression profiles in the ovarian follicle during late oogenesis in both trout and Xenopus. While some identified genes were previously uncharacterized during Xenopus late oogenesis, the nature of these genes has pointed out molecular mechanisms possibly conserved in amphibians and teleosts. It should also be stressed that in addition to the already suspected importance of steroidogenesis in maturational competence acquisition, our approach has shed light on other regulatory pathways which may be involved in maturational and developmental competence acquisitions that will require further studies.

Temporal mRNA expression of transforming growth factor-beta superfamily members and inhibitors in the developing rainbow trout ovary

During mammalian ovarian development transforming growth factor-beta (TGFβ) superfamily members and their inhibitors are critical paracrine regulators, yet the intraovarian functions of these proteins have received less attention in fish. Using quantitative real-time RT-PCR, changes in ovarian mRNA expression of six TGFβ members and two inhibitors were investigated in rainbow trout across a wide range of fish ovarian stages (i.e., early perinucleous stage through acquisition of maturational competence). Transcript changes for insulin-like growth factor 1 and 2, and five enzymes associated with steroidogenesis, as well as plasma levels of three sex steroids were also measured to provide a framework of established intraovarian regulators in trout. Expression of bone morphogenetic protein 4 ( bmp4), bone morphogenetic protein 7 ( bmp7), and growth differentiation factor 9 ( gdf9) peaked during pre-vitellogenic stages and steadily decreased through advancing stages implicating these genes in early ovarian development. A dramatic increase in inhibin beta A and decrease in follistatin expression occurred during early to mid-vitellogenic stages, which corresponded with increased 17β-estradiol plasma levels suggesting a vitellogenic role for ovarian activin A. Follicles that were competent to respond to the maturation-inducing hormone had decreased levels of inhibin beta B and increased expression of bambi ( bmp and activin membrane-bound inhibitor) suggesting their roles in maturation processes . Furthermore, bmp4, bmp7 and gdf9 are primarily expressed in the oocyte whereas the inhibin subunits, follistatin, and bambi are primarily expressed in the somatic follicle cells. These results support TGFβ superfamily members and their inhibitors have wide-ranging and disparate roles in regulating ovarian development in fish.

Comparative Genomic Analysis of Follicle-Enclosed Oocyte Maturational and Developmental Competence Acquisition in Two Lower Vertebrates, Oncorhynchus mykiss and Xenopus laevis.

Maturational competence is defined as the oocyte ability to resume meiosis while developmental competence is defined as the oocyte ability to be fertilized and to subsequently develop into a normal embryo. In vertebrates, late oogenesis is a key period for the acquisition of both maturational and developmental competence. Throughout late oogenesis, several genes exhibit a differential expression in both oocyte and follicular cells to ensure proper oocyte maturation or to store transcripts and proteins that will act as maternal factors during early embryogenesis. However, the molecular mechanisms involved in these processes are far from being fully understood. In order to identify key conserved mechanisms in lower vertebrates, we performed a comparative genomic analysis using a teleost, the rainbow trout (Oncorhynchus mykiss) and an amphibian, the South African toad (Xenopus laevis). Comparison of competence acquisition mechanisms between fish and amphibians is really valuable as they share common physiological mechanisms and similar follicular structure but are distant from nearly 100 millions years. Our study aimed at identifying differentially expressed genes during late oogenesis in both species. Ovarian rainbow trout transcriptome was studied during late vitellogenesis, post-vitellogenesis and oocyte maturation using a 9K cDNA microarray. Using Significance Analysis of Microarray of hybridization signals with a False Discovery Rate of 5%, we identified 1200 differentially expressed genes. A subset of candidate genes exhibiting the most differential expression was kept for validation by real-time PCR in both species. For Xenopus, ovarian follicles at stage IV (late vitellogenesis), immature stage VI (post-vitellogenesis) and after in vitro maturation were used. In addition, genes exhibiting a lower differential expression and possibly involved in maturational or developmental competence acquisition according to the literature were also selected. As a first step, we focused on 27 genes and thoroughly analyzed the orthology relationship between trout and Xenopus sequences in order to identify true Xenopus orthologs of the identified trout genes. These genes are involved in various functions such as steroidogenesis, post-translational modifications, and cell cycle. For instance, cmah, the cytidine monophosphate-N-acetylneuraminic acid hydroxylase, exhibited decrease of mRNA expression at the time of oocyte maturation, in both species. In addition, we identified 4 genes, cyp19a1, cyp17a1, star and hsd11b3 that are important for the steroidogenic control of follicular differentiation in both species. cyp19a1 expression decreased dramatically during post-vitellogenesis in both species. cyp17a1 and hsd11b3 expression decreased, to a lower extent, during the same period. In contrast, a sharp and concomitant increase of star expression was observed in both species. In summary, we have successfully identified orthologous genes exhibiting conserved expression profile in the late oogenetic ovarian follicle of trout and Xenopus. These genes have pointed out molecular mechanisms possibly conserved in lower vertebrates. It should also be stressed that in addition to the already documented participation of steroidogenesis in maturational competence acquisition, our approach has also shed light on new mechanisms of maturational and developmental competence acquisition that we are currently further investigating. Supported by ANR-08-GENM-033 OSCILE. (poster)

Variation in spawning responses, egg and larvae productions from induced rohu ( Labeo rohita) during pre-monsoon and monsoon seasons: Relationship with hormonal changes and oocyte responsiveness during final maturation

Early induced spawning in captive rohu ( Labeo rohita) often encounters with reduced spawning performances and devaluation of final product. The present study attempted to gain insight into the problems associated with poor performance of rohu during pre-monsoon spawning. A combination of sGnRHa and domperidone was used to induce final oocyte maturation (FOM) and ovulation in rohu during early (pre-monsoon, PM) and normal (monsoon, MN) spawning. The spawning performance parameters such as, spawning response, production and quality of egg and larvae showed significantly lower values ( p < 0.05) in PM, when compared with MN spawning. The egg and spawn productions were recorded as 2.6 ± 0.05 and 2.41 ± 0.05 during the MN season, which were reduced by almost 50% in the PM season. Moreover the quality of egg and hatchling was devaluated significantly ( p < 0.05) and exhibited higher percentage of mortality and abnormality in PM than those recorded in the MN season. The plasma concentration of carp gonadotropin (cGtH), 17β-estradiol (E) and 17α20β-dihydroxy-4-pregnen-3-one (DP) in relation to progress of FOM and ovulation at different seasons exhibited marked variation in hormonal profiles particularly in E and DP of PM fish. Higher initial plasma E (3.8 ± 0.3) and a distinct E peak clearly indicated the lack of transition from vitellogenic to post-vitellogenic stages that prevailed in PM rohu. Delayed DP and cGtH surge during FOM and ovulation resulted in longer latency period in spite of higher water temperature (31.5 °C) that prevailed during the PM period. In-vitro study on oocyte maturational competence (OMC) clearly depicted the lack of maturational competence in ovarian follicles during PM than MN in rohu. However priming the fish with purified carp gonadotropin (PCG) enhanced the acquisition of OMC in PM rohu in such an extent, that no marked seasonal differences ( p > 0.05) in OMC were remained further, when compared with MN follicles. The PCG mediated acquisition of maturational competence was found to be dependent fully on new mRNA and protein synthesis in PM fish. The present study clearly demonstrated that the oocytes' unresponsiveness to hormonal induction was mainly responsible for reduced spawning performance in PM rohu, which could be ameliorated through PCG priming to achieve better spawning response in rohu during the pre-monsoon period. Thereby, the rohu fry production could be initiated successfully as early as May, allowing public and private hatcheries to produce larger age-0 rohu fingerlings ensuring reliable steady source of stocking materials for grow-out system earlier in the season.

Ovarian function of the trout preovulatory ovary: New insights from recent gene expression studies

During the preovulatory period the follicle-enclosed oocyte progressively acquires maturational and developmental competence. In addition, the follicle is also preparing for the release of the oocyte from the follicle at ovulation. Using real-time PCR and cDNA microarrays we have investigated the molecular mechanisms of oocyte competence acquisition and ovulation in rainbow trout ( Oncorhynchus mykiss) by monitoring gene expression in the preovulatory ovary. These studies have demonstrated that many molecular events related to maturational competence and developmental competence acquisition, and ovulation occur concomitantly in the preovulatory ovarian follicle. Oocyte maturational competence acquisition is associated with a decrease of estrogen synthesis and signaling capacities. We also observed a differential expression of genes encoding for igfs and related binding protein, members of the TGF beta superfamily, proteins involved in ion and water transport, bone morphogenetic proteins, and cathepsins. In addition, our observation of a strong up-regulation, prior to ovulation, of genes encoding for proteins putatively involved in proteolysis, inflammation, coagulation, vasodilatation, and angiogenesis further supports the hypothesis comparing ovulation with an inflammatory-like reaction. Together, our results suggest that a finely tuned cross-talk exists between oocyte and follicular layers and between the ovulatory process and the oocyte maturational and developmental competence acquisition processes.

Targeted gene expression profiling in the rainbow trout (Oncorhynchus mykiss) ovary during maturational competence acquisition and oocyte maturation.

A real-time polymerase chain reaction-based gene expression survey was performed using 37 target genes and 22 female rainbow trout sampled during follicular maturational competence (FMC) acquisition or during oocyte maturation. In females sampled before meiosis resumption, FMC was estimated using an in vitro assay. Several growth factors, bone morphogenetic proteins, steroidogenic enzymes, cathepsins, genes known to play a role in the fish preovulatory ovary, as well as previously unstudied genes, were analyzed in this survey. Gene expression profiling was performed using a supervised clustering analysis in order to identify groups of genes exhibiting similar expression profiles in the ovary during FMC acquisition and follicular maturation. From the clustering analysis, three clusters exhibiting a specific expression during FMC acquisition or at the time of oocyte maturation were identified. Cluster 1 was characterized by a progressive increase in gene expression during FMC acquisition, whereas cluster 2 exhibited an increased expression at the time of oocyte maturation. In contrast, cluster 3 was characterized by a decreased mRNA expression at the time of oocyte maturation. Among the 37 target genes used in this survey, 18 were significantly regulated during maturational competence acquisition or at the time of oocyte maturation. Among these 18 genes, 16 belonged to one of the three clusters identified. Although the results allowed a global description of gene expression profiles, they also suggest an important role for several factors, including some previously unstudied bone morphogenetic proteins, in the paracrine control of FMC acquisition and meiosis resumption.

Rainbow trout follicular maturational competence acquisition is associated with an increased expression of follicle stimulating hormone receptor and insulin-like growth factor 2 messenger RNAs.

Post-vitellogenic female rainbow trout (Oncorhynchus mykiss) were assayed in vitro for follicular maturational competence (FMC). Ovarian follicles were stimulated with a range of concentrations of partially purified gonadotropin. The efficient concentration for 50% germinal vesicle breakdown (GVBD) was calculated and used as an indicator of FMC. Before in vitro assay, ovarian tissue was sampled in order to quantify mRNA abundance of specific genes in the ovarian follicle by real-time PCR. In addition, maturation-inducing steroid (MIS, 17, 20 beta-dihydroxy-4-pregnen-3-one) and estradiol (E2) plasma levels were measured by radioimmunoassay. The mRNA expression of several genes such as luteinizing hormone receptor (LH-r), follicular stimulating hormone receptor (FSH-r), insulin-like growth factor 1 (IGF1), insulin-like growth factor 2 (IGF2), insulin-like growth factor receptor 1a (IGF-r1a), and 20 beta-hydroxysteroid dehydrogenase (20 beta-HSD) that are putatively expressed in the preovulatory ovary, was studied in females of varying FMC using real-time PCR. FMC acquisition is characterized by an increase of MIS circulating levels and a concomitant drop of E2 levels. At the ovarian level, no significant variation of LH-r, 20 beta-HSD, IGF1, and IGF-r1a mRNA abundance was observed among females of varying FMC. In contrast, FSH-r and IGF2 mRNA levels were significantly higher in females exhibiting high FMC. In addition, correlation analyses showed that IGF2 and FSH-r, mRNA levels were positively correlated with FMC. These results indicate that FMC acquisition is associated with an increased expression of these gene products that may be useful markers of FMC.

Proteome analysis: a new approach to identify key proteins involved in acquisition of maturational competence and oocyte maturation of medaka oocytes

We have applied analytical methods of proteomics to identify key proteins that are involved in physiologically important processes on fish reproduction. We have improved methods of proteome analysis for fish oocytes and revealed changes of expression patterns of oocyte proteins and protein phosphorylation during acquisition of maturational competence and signal transduction of oocyte maturation of medaka oocytes.

Induced maturation and spawning: opportunities and applications for research on oogenesis

Broodstock management requires the ability to detect and regulate oocyte growth, acquisition of maturational competence, maturation of oocytes, and onset of ovarian atresia. Our research on temperate basses (genus Morone) has supported development of these capabilities. These investigations have revealed that accumulation of neutral lipid droplets and deposition of vitellogenin-derived yolk proteins in growing oocytes are independent processes with different sensitivities to changing day length and water temperature. In these fishes, completion of oocyte growth is marked by disappearance of vitellogenin from ovarian biopsy samples. Competence of females for induced spawning is predicted by the ability of biopsied follicles to initiate oocyte meiosis in vitro in response to insulin-like growth factor I. Cytoplasmic maturation of the oocytes is triggered by the maturation-inducing steroid hormone and can be monitored by evaluating degradation of the yolk proteins. Onset of ovarian atresia is indicated by the appearance of edema in the granulosa cell layer of biopsied follicles, and can be delayed for months by holding gravid females at abnormally low temperature (`cold banking'). These novel findings hold strong promise for application to other farmed fishes.

Specific gene expression profiles are associated with follicular maturational competence acquisition in rainbow trout (Oncorhynchus mykiss)

The expression profiles of several mRNAs were characterized during follicular maturational competence (FMC) acquisition. Post-vitellogenic female rainbow trout (Oncorhynchus mykiss) were assayed in vitro for follicular maturational competence (FMC). Ovarian follicles were stimulated in vitro for 60 h at 12 °C with a range of concentrations of partially purified gonadotropin and the efficient concentration for 50% germinal vesicle breakdown was calculated and used as an indicator of follicular maturational competence. Before in vitro assay, ovarian tissue was sampled in order to quantify mRNA abundance in the ovarian follicle by real-time PCR. The mRNA expression of Luteinizing Hormone receptor (LH-r), Follicular Stimulating Hormone receptor (FSH-r), Insulin-like Growth Factor 1 (IGF1), Insulin-like Growth Factor 2 (IGF2), Insulin-like Growth Factor receptor 1a (IGF-r1a) and 20β-hydroxysteroid dehydrogenase (20β-HSD), that are putatively expressed in the preovulatory ovary, was thus studied in females of varying FMC.

Connexin Messenger Ribonucleic Acids in the Ovary of Atlantic Croaker: Molecular Cloning and Characterization, Hormonal Control, and Correlation With Appearance Of Oocyte Maturational Competence1

Intercellular gap junction channels are composed of connexin (Cx) protein. In the Atlantic croaker ovary, gonadotropin (GtH)-induced oocyte maturational competence is accompanied by an obligatory synthesis of RNA and protein and by the formation of granulosa cell-oocyte gap junctions. However, the GtH control of Cx gene expression in relation to follicular differentiation (including maturational competence) has not been examined in Atlantic croaker or in any other vertebrate species. Moreover, Cx genes and their products have not been characterized in fishes. Therefore, the objectives of this study were to characterize ovarian Cx mRNAs in Atlantic croaker and to determine their regulation by GtH during the acquisition of maturational competence. Polymerase chain reaction was used to amplify Cx cDNA prepared from maturationally incompetent or competent ovaries. Mixed primers for this reaction were based on the high sequence homology of selected regions of known connexins. One major fragment (Cx1) was amplified from incompetent ovary cDNA, and two major fragments (Cx1' and Cx2) from competent ovary cDNA. Sequence analysis showed that Cx1 and Cx1' are identical and that Cx1 and Cx2 have high sequence homology to other Cx cDNAs. A cDNA library made from competent ovaries was screened with Cx1 or Cx2 used as probes. Two cDNAs were isolated: one (Cx1) encoded 282 amino acids (32,693 daltons; Cx32.7), and the other (Cx2) encoded 285 amino acids (32,169 daltons; Cx32.2). Both amino acid sequences showed all key features of Cx. Northern blot analysis showed a negligible level of Cx32.2 mRNA in incompetent ovaries, but a substantial rise in its levels accompanied the GtH induction of maturational competence. However, Cx32.7 mRNA levels in incompetent and competent ovaries were similar. In conclusion, selective activation of the ovarian Cx32.2 gene by GtH seems to occur during the acquisition of oocyte maturational competence.

Ultrastructural Changes in Follicle Cell-Oocyte Associations during Development and Maturation of the Ovarian Follicle in Atlantic Croaker

Full-grown ovarian follicles of the marine teleost Atlantic croaker ( Micropogonias undulatus) are unresponsive to steroidal maturation-inducing substance (MIS). However, responsiveness to MIS (maturational competence) can be induced in vivo or in vitro by brief stimulation with gonadotropin (e.g., human chorionic gonadotropin, hCG). The objective of this study was to examine ultrastructural changes in follicle cell-oocyte associations related to the acquisition of maturational competence and maturation in croaker ovarian follicles. Ultrathin sections of lanthanum-infiltrated samples were observed by transmission electron microscopy at various points during follicular development and hormone-induced maturation: vitellogenic oocytes (<350 μm), full-grown oocytes (350-400 μm), hCG-stimulated (maturationally competent) oocytes, hCC/MIS-stimulated (early-maturation, hydrating) oocytes, and ovulated oocytes. In general, prior to ovulation, oocyte microvilli made contact with granulosa cells, and gap junction-like structures were seen at these points of contact. Midvitellogenic follicles (<350 μm) had relatively high levels of follicle cell-oocyte associations and gapjunctional contacts, but by late vitellogenesis (350-400 μm) these levels had declined markedly. However, high levels of intercellular associations and gap junctional contacts were reestablished upon induction of maturational competence with hCG, and these levels remained high in early-maturation oocytes. These changes seemed to be mainly due to alterations in oocyte microvilli length and surface contact with granulosa cells rather than to alterations in the numbers of microvilli. However, retraction and disappearance of microvilli occurred during ovulation. Therefore, the appearance of maturational competence coincided with the reestablishment of heterocellular gap junctional contacts in fullgrown follicles. One function of these contacts may be to allow transfer of maturational signals between the granulosa cells and the oocyte during the early stages of follicular maturation.