Acquisition Of Oocyte Maturational Competence Research Articles

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.

Expression and gonadotropin regulation of membrane progestin receptor alpha in Atlantic croaker ( Micropogonias undulatus) gonads: Role in gamete maturation

Recent results suggest that membrane progestin receptor alpha (mPRα) mediates nongenomic actions of progestin hormones to induce oocyte maturation and sperm hypermotility in several teleost species. The role of mPRα in gamete and gonadal physiology was further evaluated in the present study by examining gonadal expression of mPRα during gamete maturation in Atlantic croaker ( Micropogonias undulatus), a well-characterized teleost model of oocyte maturation and sperm motility. Sequencing of the croaker mPRα gene isolated from croaker ovaries showed it is 98% homologous at the nucleotide level to spotted seatrout mPRα. The mPRα mRNA and protein were detected in both somatic and gonadal tissues. In croaker ovaries, the mPRα protein was present throughout the gonadal cycle and was upregulated by gonadotropin in vitro, coincident with the acquisition of oocyte maturational competence (i.e., ability to respond to progestin hormones and complete oocyte maturation). Both mPRα mRNA and protein were also expressed in croaker testes throughout the gonadal cycle. Expression of mPRα protein was weakly upregulated in testes after 18 h of in vitro gonadotropin treatment. Immunocytochemical staining showed mPRα was localized to both germ and interstitial cells. Finally, elevated levels of mPRα protein in croaker sperm were associated with high sperm motility. Taken together, these data strongly support the hypothesis that mPRα mediates progestin induction of oocyte maturation and upregulation of sperm motility in teleosts.

Heterologous Gap Junctions between Granulosa Cells and Oocytes in Ayu (Plecoglossus altivelis): Formation and Role during Luteinizing Hormone-dependent Acquisition of Oocyte Maturational Competence

Gap junctions (GJs) are intercellular channels. Molecules with a molecular weight of 1 kDa or less can pass back and forth between adjacent cells through GJs. Communication between oocytes and the somatic cells that surround them via GJs is known to play key roles to initiate oocyte maturation in many vertebrates. However, little is known of the detailed functions of ovarian GJs during oocyte maturation in fish. The oocyte maturation of fish is induced by a maturation-inducing steroid (MIS). The sensitivity of oocytes to the MIS is known as oocyte maturational competence (OMC) and is induced by luteinizing hormone (LH). However, LH receptors are found on the surface of granulosa cells rather than oocytes. We therefore proposed that the LH signals received by granulosa cells were passed to oocytes via GJs. This review describes current knowledge of the role of GJs between granulosa cells and oocytes during the LH-induced acquisition of OMC in fish.

Differential expression and localization of four connexins in the ovary of the ayu (Plecoglossus Altivelis)

The post-vitellogenic oocytes of teleost fish are generally unresponsive to maturation-inducing hormone (MIH) until a luteinizing hormone (LH) surge stimulates sensitivity via the acquisition of oocyte-maturational competence (OMC). Heterologous gap junctions (GJs) between granulosa cells and the oocyte have been previously implicated in the regulation of oocyte maturation in various vertebrate species. Although heterologous GJ are present in ovarian follicles of ayu (Plecoglossus altivelis), their role in maturation remains unclear. In the present study, we cloned and characterized complementary DNAs for GJ protein connexin (Cx), and examined the expression pattern of Cx messenger RNAs in the ayu ovary. Four Cx cDNAs with predicted molecular masses of 32.1 (Cx32.1), 34.9 (Cx34.9), 44.1 (Cx44.1), and 44.2 (Cx44.2) kDa, respectively, were cloned. Northern blot analysis revealed that the levels of Cx44.1 and Cx44.2 transcripts were similar during the vitellogenic and ovulatory stages. Cx32.1 transcripts were more abundant during the vitellogenic stage, but their levels declined thereafter. Cx34.9 transcript levels increased during the vitellogenic stage and remained high during the acquisition of OMC. In situ hybridization revealed that Cx44.1 and Cx44.2 signals were restricted to the oocyte, whereas the Cx32.1 and Cx34.9 signals were detected in both cellular fractions. Furthermore, a dye-transfer assay revealed the presence of functional GJs between the oocytes and follicle cells. These results suggest that Cx34.9 contributes to the formation of heterologous GJs between oocytes and granulosa cells. Moreover, GJs formed by Cx34.9 may function during the LH-dependent acquisition of OMC and the MIH-dependent resumption of meiosis in ayu.

卵母細胞が黄体形成ホルモンの刺激により、卵成熟誘起物質に対する感受性を獲得する際、どの様な細胞内情報伝達系が活性化されているのか？

卵母細胞が黄体形成ホルモンの刺激により、卵成熟誘起物質に対する感受性を獲得する際、どの様な細胞内情報伝達系が活性化されているのか？

Seasonal changes in plasma steroid levels in Indian major carp Labeo rohita: influence of homologous pituitary extract on steroid production and development of oocyte maturational competence

Circannual variations in plasma levels of testosterone (T), 17β-estradiol (E 2), and 17α,20β-dihydroxy-4-pregnen-3-one (DHP) as well as seasonal fluctuations in ovarian steroid synthetic potential were observed in Indian major carp, Labeo rohita. A study was also conducted to examine the mechanism of the development of gonadotropin-induced maturational competence in oocytes of this fish. The present study recorded the lowest values of plasma E 2 and T in L. rohita during the period from October to January. A mild increase in the levels of these steroids observed in February was followed by their rapid rise reaching peak values in April, when the ovary contained mostly the vitellogenic follicles. In the month of May, as the postvitellogenic follicles predominated in the ovary, there was a decline in plasma concentrations of both T and E 2. Low levels of these steroids in plasma remained until January, except a small elevation detectable during June and July (spawning stage). DHP was not detected in the plasma of this fish collected during the period from August to March. Existence of DHP was first recorded in blood in the month of April (vitellogenic stage) and it quickly reached the peak value in May (postvitellogenic stage), followed by a sudden decline in the month of June. Under stimulation of fish pituitary extract (FPE), as a source of gonadotropin, in vitro production of E 2 and T by the vitellogenic follicles was shown to be highest compared to their production rate in other stages, while the postvitellogenic follicles recorded the highest rate of DHP synthesis. Acquisition of oocyte maturational competence (OMC) was shown to develop either by priming the vitellogenic stage fish with a single dose of FPE or by in vitro addition of FPE in culture. In vitro treatment of trilostane, an inhibitor of 3β-HSD, blocked the FPE-stimulated steroid production but not the development of OMC. Presence of cycloheximide and actinomycin D in the incubation was shown to inhibit FPE-induced development of OMC, indicating the requirement of de novo protein synthesis for this process.

Gonadotropin and activin enhance maturational competence of oocytes in the zebrafish (Danio rerio).

In most teleosts, 17alpha,20beta-dihydroxy-4-pregnen-3-one (DHP) serves as the most potent maturation-inducing steroid (MIS) to initiate final oocyte maturation. The maturational competence or the responsiveness of oocytes to DHP increases when the ovarian follicles approach the final stage of growth. In the zebrafish, we demonstrated in the present study that full-grown oocytes (approximately 0.7 mm) exhibited the highest maturational competence, which diminished progressively with decreasing size of the follicles. Using midvitellogenic follicles (0.49-0.56 mm), which had little response to DHP, as the material, the present study aimed at investigating the endocrine and paracrine mechanisms that regulate maturational competence of the oocytes. In agreement with the results of studies in other teleost fish, pretreatment of follicles with gonadotropin (hCG) significantly enhanced the responsiveness of midvitellogenic oocytes to DHP in a clear time- and dose-dependent manner. Interestingly, activin, an ovarian growth factor, also had a potent stimulatory effect on the acquisition of oocyte maturational competence. Pretreatment with either recombinant human activin A or goldfish activin B significantly increased the rate of DHP-induced oocyte maturation from approximately 3% to approximately 70%, also in a clear dose-dependent manner. Similar to the results with hCG, pretreatment with activin alone had no effect in inducing maturation of midvitellogenic oocytes without subsequent DHP treatment, although both exhibited a strong effect in promoting maturation of full-grown oocytes. The effect of activin on maturational competence of oocytes could be reduced by cotreatment with follistatin, a potent activin-binding protein. Interestingly, follistatin treatment also significantly reduced the effect of hCG on maturational competence of oocytes, suggesting a mediating role for endogenous activin or activin-related molecules in the action of gonadotropin. The effects of hCG and activin on maturational competence of oocytes could be significantly inhibited by actinomycin D (1 microg/ml) and completely blocked by cycloheximide (1 microg/ml), suggesting that the hCG and activin-induced acquisition of oocyte maturational competence involves de novo protein synthesis at both the transcriptional and translational levels.

Ovarian follicle growth, maturation, and ovulation in teleost fish

Knowledge of ovarian follicle growth, maturation, and ovulation in teleosts is important to many aspects of basic and biomedical research, fisheries management, aquaculture, and environmental science. Follicle growth includes previtellogenic and vitellogenic stages. Although little progress has been made in our understanding of previtellogenic development, information on the regulation and mechanisms of vitellogenic growth has increased substantially in recent years. This new information pertains to the multiplicity of vitellogenin (Vg) genes, the oocyte Vg receptor, Vg processing enzymes (cathepsins) and their specific yolk products, and synthesis of various vitelline envelope precursor proteins in liver and ovary. Oocyte acquisition of lipids from sources other than Vg dominates follicle growth in many teleosts, especially perciformes, yet our understanding of the basic processes involved remains rudimentary. Considerable advances also have been made in our understanding of the endocrine regulation and mechanisms of ovarian follicle maturation. We have learned that ovarian follicle maturation involves a number of events including luteinizing hormone (LH)-dependent acquisition of oocyte maturational competence, LH induction of maturation-inducing hormone (MIH) synthesis, and MIH-dependent meiotic resumption (nuclear maturation) and cytoplasmic maturation. While much has been learned about mechanisms of maturational competence, MIH synthesis and meiotic resumption, our knowledge of cytoplasmic maturation is limited to descriptions of MIH-dependent yolk protein hydrolysis and associated oocyte hydration in a few species. It has become apparent that ovulation requires genomic activation regulated by nuclear MIH receptors, but the transduction pathways for the ovulatory MIH signal appear to be complex and are poorly understood.

Molecular cloning and hormonal control in the ovary of connexin 31.5 mRNA and correlation with the appearance of oocyte maturational competence in red seabream.

Gap junctions are aggregates of intercellular channels, composed of the protein connexin (Cx), between adjacent cells. This study examined whether, in the ovary of the red seabream Pagrus major, the connexin gene essential for the production of RNA and protein during the acquisition of oocyte maturational competence is active. Mixed primers for this reaction were designed on the basis of the high sequence homology of selected regions of known connexin genes. Polymerase-chain-reaction-amplified cDNA fragments generated by 3' and 5' rapid amplication of cDNA ends were combined to generate full-length cDNA sequences. The resulting 2400 base pair cDNA had an open reading frame encoding a polypeptide containing 275 amino acid residues (31493 Da; Cx31.5). Hydropathicity analysis of the predicted amino acid sequence indicated that red seabream Cx31.5 has four major hydrophobic regions and four major hydrophilic regions indicative of a topology similar to that of known connexins. Typical connexin consensus sequences were also observed in the first and second extracellular loops. During the acquisition of oocyte maturational competence, red seabream Cx31.5 mRNA transcription levels increased after treatment with gonadotropin-II. It is therefore proposed that expression of Cx31.5 contributes to the acquisition of oocyte maturational competence in this species.

Cellular apoptosis susceptibility gene messenger ribonucleic acid in the ovary of red seabream: molecular cloning and correlation with acquisition of oocyte maturational competence

This study has used differential display-PCR, to amplify genes transcribed during oocyte maturation induced by human chorionic gonadotropin (hCG). A cDNA expressed in time of acquisition of oocyte maturation of red seabream (Pagrus major) by treatment of hCG was amplified and cloned. A full-length cDNA of the cellular apoptosis susceptibility gene for red seabream was isolated using differential display-PCR and 5′ RACE. This cDNA clone contained 3560 nucleotides including the open reading frame (ORF) that encoded a protein of 971 amino acids. The deduced amino acid sequence shows 87% identity and 97% similarity to human cellular apoptosis susceptibility gene. Consensus sequences consistent with sites for phosphorylation by protein kinase C was identified. Red seabream cellular apoptosis susceptibility gene (CAS) transcript level increased between 9 and 12 h after the incubation with hCG. Red seabream CAS mRNA transcript level was increased by treatment of GtH-II (300 ng ml−1). Moreover, in vivo, red seabream CAS mRNA was strongly expressed after, but not before the acquisition of oocyte maturational competence. These results suggest that an obligatory element of the hCG induction of maturational competence is the de novo synthesis of red seabream, Pagrus major CAS (PmCAS).

Regulation of Gap Junctions and Oocyte Maturational Competence by Gonadotropin and Insulin-like Growth Factor-I in Ovarian Follicles of Red Seabream

The in vitro effects of human chorionic gonadotropin (hCG) and human insulin-like growth factor-I (IGF-I) on the formation of oocyte–granulosa cell (heterologous) and granulosa cell–granulosa cell (homologous) gap junctions (GJ) were determined during the course of development of oocyte maturational competence (OMC) in ovarian follicles of red seabream. Incompetent, full-grown follicles were incubated in hCG (10 IU/ml), IGF-I (10 nM), or hormone-free medium for 0, 6, and 12 h and examined by electron microscopy. Qualitative analysis indicated that homologous and heterologous GJ were relatively rare in incompetent follicles and remained rare if they were maintained in control medium. However, both types of GJ were readily observed in follicles treated with hCG or IGF-I. For quantitative analysis, we measured changes in the combined numbers of both GJ types, since discrimination between the two was not always possible. The numbers of GJ along 125 μm of granulosa cell–zona radiata border were counted in three follicles/treatment. GJ markedly increased with time in the presence of hCG or IGF-I but not in control medium (Duncan's multiple range test; P<0.05). Functional assays confirmed that the hormonal treatments also induced OMC. Naturally competent follicles sampled at the appropriate stage of the reproductive cycle contained high levels of both types of GJ suggesting that the OMC-associated increase in GJ is physiologically relevant. In view of similar, earlier results with the Atlantic croaker, the present results support the concept that an increase in homologous and heterologous GJ is a general developmental event associated with the acquisition of OMC in teleost ovarian follicles. Also, a novel finding of this study is that IGF-I can regulate heterologous and homologous GJ in intact ovarian follicles.

Developmental and Protein Kinase-Dependent Regulation of Ovarian Connexin mRNA and Oocyte Maturational Competence in Atlantic Croaker

The acquisition of oocyte maturational competence (OMC) in ovarian follicles of Atlantic croaker is associated with increased gap junction (GJ) contacts and increased levels of ovarian connexin (Cx) 32.2 mRNA. However, the developmental control of ovarian Cx gene expression and the mechanisms of OMC acquisition are unknown. Ovarian Cx32.2 and Cx32.7 mRNA levels were determined in fish with gonadosomatic indices (GSI; gonad weight-to-body weight ratio) ranging from 0.1 to 13%. The mRNA level for both Cx increased from a low level in previtellogenic ovaries (GSI, <1%) to a peak level during the midstage of ovarian growth (GSI, 6–7%). Levels of Cx32.2 mRNA, but not Cx32.7 mRNA, declined markedly during late ovarian vitellogenic growth (GSI, 7–13%), and increased again upon stimulation of OMC by human chorionic gonadotropin (hCG). These changes in ovarian Cx32.2 mRNA seem to parallel previously reported changes in the incidence of oocyte–granulosa cell GJ during follicular growth and early maturation.In vitrotreatment with hCG and protein kinase A (PKA) activators (dbcAMP and forskolin) induced ovarian Cx32.2 mRNA levels and OMC. The effects of hCG were blocked by PKA inhibitors (H89, H7). Protein kinase C (PKC) inhibitors (GF 109207X) had little effect on hCG-induced Cx32.2 mRNA or OMC, whereas PKC activators (PMA) blocked both events. There was no association between changes in Cx32.7 mRNA levels and OMC status in these experiments. In conclusion, changes in Cx32.2 gene expression seem to be involved in the regulation of oocyte–granulosa cell GJ during growth and differentiation of the croaker ovarian follicle. Also, the stimulation of OMC and Cx32.2 mRNA levels by hCG is mediated by PKA-dependent pathways and antagonized by PKC-dependent mechanisms.

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.