Ooplasm Of Previtellogenic Oocytes Research Articles

Cyclin B protein undergoes increased expression and nuclear relocation during oocyte meiotic maturation of the freshwater prawn Macrobrachium rosenbergii and the Chinese mitten crab Eriocheir sinensis.

Cyclin B functions as a regulatory protein through association with its catalytic partner Cdc2 kinase forming M-phase promoting factor (MPF), which plays a central role in the meiotic maturation of oocyte. To gain insight into the molecular events, we here cloned a cyclin B cDNA from the ovary of the prawn Macrobrachium rosenbergii and compared its spatial-temporal expression patterns during oocyte maturation with those of crab Eriocheir sinensis. The prawn cyclin B cDNA encodes a 398 amino acid protein with predicted molecular weight of 45.16kDa. Immunodetection of cyclin B protein by Western blot showed that a target band of approximately 53kDa protein in the prawn ovaries at both late vitellogenesis (lVt) and germinal vesicle breakdown (GVBD) stages, whereas a 41kDa band was present in the crab ovaries. Cyclin B protein expression changes indicating that the newly synthesis of cyclin B proteins could be required for GVBD in both prawn and crab. Immunohistochemical analysis revealed that both the prawn and crab cyclin B proteins, were localized in the ooplasm of previtellogenic oocytes, then relocated into germinal vesicle at vitellogenesis stage and localized on meiotic spindle at M phase. These similar behaviors suggested that the prawn and the crab cyclin B proteins associated with Cdc2 kinase have conserved roles in inducing GVBD and regulating the formation of meiotic spindle. The similar expression patterns of the cyclin B proteins during oocyte maturation implicated that the molecular mechanisms for MPF activation could be identical between the prawn and the crab.

Isolation and expression analysis of Bystin 1 transcript and protein during ovarian development of the giant tiger shrimp Penaeus monodon.

The full-length cDNA of bystin isoform 1 (PmBys1) of the giant tiger shrimp Penaeus monodon was characterized. It was 1553 bp in length containing an ORF of 1365 bp corresponding to a polypeptide of 454 amino acids. The level of PmBys1 mRNA in ovaries was greater than that in other tissues of females and in testes of males in both juveniles and wild broodstock (P < .05). In non-ablated wild female broodstock, PmBys1 mRNA significantly and progressively increased in ovaries from stage I of development, peaking at stage IV (P < .05). Its level in stages I-IV of eyestalk-ablated broodstock was greater than that in non-ablated broodstock (P < .05). Injection of exogenous serotonin (50 μg/g body weight) into 18-month-old shrimp resulted in a significantly increase of ovarian PmBys1 mRNA at 6-48 h post injection (hpi) (P < .05). PmBys1 protein (52 kDa) was found in ovarian stages I-V of non-ablated wild broodstock and II-IV of ablated wild broodstock, respectively. Along with the 52 kDa band, immunoreactive bands of 50 and 43 kDa were also observed in ovarian stages II-IV of both non-ablated and ablated broodstock and in ovaries of post-spawning broodstock. The 43 KDa band was not observed in ovarian stage I of wild female broodstock or in premature juveniles. PmBys1 protein was localized in the ooplasm of previtellogenic oocytes, nucleo-cytoplasmic compartments of vitellogenic oocytes and cortical rods of mature oocytes in wild broodstock. The results implied a possible role for PmBys1 during ovarian development in P. monodon.

Expression of catechol O-methyltransferase during ovarian development and association between its SNP and reproduction-related parameters of the giant tiger shrimp Penaeus monodon

Catechol O-methyltransferase cDNA and gene of the giant tiger shrimp Penaeus monodon (PmCOMT) were characterized. Genomic sequence of PmCOMT spanned 1466bp in length containing 3 exons (194, 111 and 361bp) and 2 introns (143 and 147bp). The full-length cDNA of PmCOMT was 1176bp in length with an open reading frame (ORF) of 666bp corresponding to a polypeptide of 221 amino acids. PmCOMT mRNA was not differentially expressed during ovarian development in wild intact P. monodon adults (P>0.05). Eyestalk ablation resulted in up-regulation of PmCOMT in stages II (vitellogenic) and IV (mature) relative to stage I (previtellogenic) ovaries (P<0.05). In domesticated shrimp, its expression was reduced in 18-month-old adults compared with 6-month-old juveniles and 14-month-old adults (P<0.05). In situ hybridization indicated that PmCOMT mRNA was clearly localized in ooplasm of previtellogenic oocytes but not in oogonia and other oocytes stages. Recombinant PmCOMT protein and anti-rPmCOMT polyclonal antibody were successfully produced. The PmCOMT protein was localized in ooplasm of previtellogenic oocytes. It was temporally found in both ooplasm and nucleus of vitellogenic oocytes and subsequently in cortical rods (Crs) and the Cr membrane of mature oocytes. In addition, single nucleotide polymorphism (SNP) in PmCOMT of domesticated P. monodon (14-month-old shrimp, N=64) was identified by single-strand conformational polymorphism (SSCP) and PCR-direct sequencing. Two SSCP patterns (A and B) corresponding to G/Cc.305+g.84 and C/Cc.305+g.84 SNP genotypes were found. Shrimp carrying the former genotype (OW=0.92±0.37g and GSI=1.06±0.39%, N=31) had a greater average ovarian weight and GSI value than those carrying the latter genotype (OW=0.75±0.30g and GSI=0.86±0.32%, N=33) (P<0.05). Taken the information together, PmCOMT should play the functional role during ovarian development of P. monodon. Statement of relevanceReduced reproductive performance of the giant tiger shrimp Penaeus monodon in captivity prevents the potential application of domestication and selective breeding program for genetic improvement of this economically important species. Understanding molecular mechanisms controlling the development and maturation of ovaries/oocytes is essential for finding an approach to promote the reproductive maturation of P. monodon without the use of eyestalk ablation. Two kinds of O-methyltransferase (OMT) transcripts; farnesoic acid O-methyltransferase (FAMeT) and catechol O-methyltransferase (COMT) have been characterized in penaeid species. P. monodon FAMeT was differentially expressed during ovarian development and its SNPs showed association with ovarian weight of domesticated adults. Similarly, the expression profile of P. monodon COMT was examined and eyestalk ablation affected its expression level during vitellogenic and mature stages. Relationships between a SNP of P. monodon COMT and reproduction-related parameters (ovarian weight and GSI) were found.

Characterization and expression analysis of Cyclin-dependent kinase 7 gene and protein in ovaries of the giant tiger shrimp Penaeus monodon

The characterization of genes differentially expressed in ovaries is necessary for understanding the mechanisms involving ovarian developmental processes of the giant tiger shrimp ( Penaeus monodon ). Here, the full-length cDNA of P. monodon Cyclin-dependent kinase 7 ( PmCdk7 ) was characterized. It was 1407 bp in length (ORF of 1062 bp corresponding to 353 amino acids). The expression of PmCdk7 in the ovaries was greater than that in the testes ( P < 0.05). PmCdk7 was significantly up-regulated in stage IV (mature) ovaries in wild intact broodstock ( P < 0.05). In wild eyestalk-ablated broodstock, its expression was earlier increased in stage III (late vitellogenic) ovaries ( P < 0.05). The expression level of PmCdk7 during ovarian development (stages I–IV ovaries) in eyestalk-ablated broodstock was greater than that of the same ovarian stages in intact broodstock ( P < 0.05). 5-HT (50 μg/g body weight) injection promoted its expression level in ovaries of domesticated 18-month-old shrimp at 6–48 h post injection (hpi). The rPmCdk7 protein and its polyclonal antibody were successfully produced. The positive immunoreactive band (67 kDa) was not observed in premature ovaries of juveniles. Western blot analysis revealed differential expression of PmCdc7 in stages II–IV ovaries compared with stage I ovaries in intact broodstock but it was comparably expressed among different ovarian developmental stages in eyestalk-ablated broodstock. Immunofluorescence revealed the positive signals of PmCdk7 in the ooplasm of previtellogenic oocytes and its subsequent nucleo-cytoplasmic translocation during oocyte development. Results indicated that PmCdk7 seems to play functional roles in the development and maturation of oocytes/ovaries in P. monodon. • The full-length cDNA of Penaeus monodon Cdk7 (1407 bp) was characterized. • PmCdk7 mRNA and protein were differentially expressed during ovarian development. • The level of PmCdk7 mRNA in stages I–IV ovaries was induced by eyestalk ablation. • 5-HT injection promoted its expression level in domesticated shrimp at 6–48 hpi. • Nucleo-cytoplasmic translocation of PmCdk7 was observed during oocyte development.

Comparative proteomic profiling during ovarian development of the shrimp Metapenaeus ensis

Two-dimensional electrophoresis and mass spectrometry were used to identify proteins that are differentially expressed during ovarian maturation in Metapenaeus ensis. 87 spots with consistently significant quantitative differences (≥ 1.5-fold for vol%) among stage I, III and V ovaries were chosen for MS/MS analysis. 45 spots were significantly matched to known proteins in the database (Mascot score >40). Half of them were down-regulated, in contrast to 9 out of 45 proteins that were up-regulated as ovarian maturation proceeded. Functionally, these identified proteins could be classified into five major groups, including cytoskeleton (11 %), metabolism (18 %), signal transduction (32 %), gene expression (14 %) and immune response (7 %). Among the differentially expressed reproduction-related proteins, the mRNA expression level of cellular retinoic acid/retinol binding protein in M. ensis (MeCRABP) during ovarian maturation was further characterized by quantitative real-time PCR. It was down-regulated during ovarian maturation. In situ hybridization further revealed that MeCRABP transcript was localized in ooplasm of previtellogenic oocytes but not in vitellogenic oocytes. These results demonstrate the application of proteomic analysis for identification of proteins involved in shrimp ovarian maturation and they provide new insights into ovarian development.

Characterization, expression and localization of valosin-containing protein in ovaries of the giant tiger shrimp Penaeus monodon

Valosin-containing protein (VCP), a member of the ATPase-associated with diverse cellular activity (AAA) family, was identified from the giant tiger shrimp (Penaeus monodon). The full-length cDNA of the PmVCP mRNA consisted of 2724bp containing an ORF of 2367bp corresponding to a deduced polypeptide of 788 amino acids. The deduced PmVCP protein contained two putative Cdc48 domains (positions 17–103, E-value=2.00e−36 and 120–186, E-value=3.60e−11) and two putative AAA domains (positions 232–368, E-value=3.67e−24 and 505–644, E-value=3.73e−25). PmVCP mRNA expression in ovaries was greater than that in testes in both juveniles and broodstock. PmVCP was significantly up-regulated in stages II and IV ovaries in intact wild broodstock (P<0.05) but it was not differentially expressed during ovarian development in eyestalk-ablated broodstock (P>0.05). The expression level of PmVCP mRNA in ovaries of 14-month-old shrimp was not affected by progesterone injection (0.1μg/g body weight, P>0.05). In contrast, exogenous 5-HT administration (50μg/g body weight) resulted in an increase of PmVCP mRNA in ovaries of 18-month-old shrimp at 6 and 24h post-injection (hpi) (P<0.05). The rPmCdc48-VCP protein and its polyclonal antibody were successfully produced. Cellular localization revealed that PmVCP was localized in the ooplasm of previtellogenic oocytes. Subsequently, it was translocated into the germinal vesicle of vitellogenic oocytes. Interestingly, PmVCP was found in nucleo-cytoplasmic compartments, in the cytoskeletal architecture and in the plasma membrane of mature oocytes in both intact and eyestalk-ablated broodstock.

Isolation of cDNA, genomic organization and expression of small androgen receptor-interacting protein 1 (PmSARIP1) in the giant tiger shrimp Penaeus monodon

The full-length cDNA and genomic organization of small androgen receptor-interacting protein 1 (SARIP1; also called RWD-containing protein 1, Rwdd1) in the giant tiger shrimp (Penaeus monodon) was isolated and characterized. PmSARIP1 was 1366bp in length containing an open reading frame (ORF) of 738bp corresponding to a polypeptide of 245 amino acids. Its genomic sequence contained 5 exons, 4 introns and untranslated regions (UTRs) spanning 5937bp in length. Tissue distribution analysis indicated that PmSARIP1 was specifically expressed in gonads (ovaries>testes) but not in other tissues of wild P. monodon adults. The expression level of PmSARIP1 was not differentially expressed during ovarian maturation in intact wild adults (P>0.05). Eyestalk ablation resulted in up-regulation of PmSARIP1 throughout the ovarian maturation of wild adults where the peak level was observed at stage I (previtellogenic) ovaries (P<0.05). PmSARIP1 mRNA was clearly localized in ooplasm of previtellogenic oocytes. Serotonin injection (5-HT, 50μg/g body weight; 18-month-old shrimp) promoted the expression level of ovarian PmSARIP1 at 6–72h post injection (hpi) with the peak level at 12hpi (P<0.05). Exogenous progesterone administration (0.1μg/g BW; 14-month-old shrimp) did not significantly affect the expression level of ovarian PmSARIP1. In contrast, 17β-estradiol treatment (0.01μg/g BW) resulted in an increase of PmSARIP1 in 14-month-old shrimp at 7days post injection (dpi). Results from this study suggested that PmSARIP1 should play an important role during ovarian maturation of P. monodon.

Molecular cloning and partial characterization of an ovarian receptor with seven ligand binding repeats, an orthologue of low-density lipoprotein receptor, in the cutthroat trout (Oncorhynchus clarki)

Teleost fish eggs contain a substantial yolk mass consisting of lipids and proteins that provides essential nutrients for embryonic and larval development. The polar lipid and protein components of the yolk are delivered to oocytes by circulating vitellogenins, however the source(s) of the neutral lipid remains unknown. We cloned a cDNA encoding an orthologue of low-density-lipoprotein receptor (LDLR) from the ovary of cutthroat trout, Oncorhynchus clarki (ct-Ldlr). Predominant expression of ct-ldlr mRNA was observed in the ovary and moderate expression was detected in intestine, gill and brain. The relative abundance of ct-ldlr transcripts was highest in early pre-vitellogenic ovaries and significantly decreased during vitellogenesis, followed by a slight increase during final maturation and in post-ovulatory follicles. In situ hybridization revealed an intense and evenly distributed localization of ct-ldlr transcripts in the ooplasm of pre-vitellogenic oocytes and these signals disappeared in vitellogenic follicles. Collectively, these results suggest that the Ldlr is involved in deposition of yolk lipids in cutthroat trout oocytes. The ct-ldlr transcripts also were detected in theca and granulosa cells, suggesting that this receptor may be involved in cholesterol uptake for ovarian steroidogenesis. This is the first report on partial characterization of an ldlr orthologue in any fish species.

Characterization and expression analysis of the Broad-complex (Br-c) gene of the giant tiger shrimp Penaeus monodon

Broad-complex (Br-c) is the early ecdysone responsive gene encoding a family of zinc-finger transcription factors. In this study, the full-length cDNA of the Br-c gene of the giant tiger shrimp (Penaeus monodon) was identified. PmBr-c was 1897 bp in length containing an ORF of 1329 bp deducing to a polypeptide of 442 amino acids. PmBr-c was more abundantly expressed in ovaries than testes of P. monodon broodstock. The expression levels of PmBr-c mRNA in ovaries of juveniles was significantly greater than that in stages II (vitellogenic), IV (mature) and V (post-spawning) ovaries of intact broodstock. The expression level of PmBr-c was significantly increased in stage III (nearly mature) ovaries of intact wild broodstock and in stage IV ovaries of eyestalk-ablated broodstock (P<0.05). In domesticated broodstock, ovarian PmBr-c was expressed lower in 18-month-old shrimp compared to 6-month-old shrimp (P<0.05). In situ hybridization revealed that PmBr-c mRNA was localized in ooplasm of previtellogenic oocytes in various ovarian stages of P. monodon broodstock. Serotonin (5-HT, 50 μg/g body mass; 18-month-old shrimp) and progesterone (0.1 μg/g body mass; 14-month-old shrimp) injection significantly promoted the expression level of PmBr-c in ovaries of domesticated broodstock at 24 and 48-72 h post injection (hpi, P<0.05). The expression levels of PmBr-c in ovaries of juvenile P. monodon was significantly increased following 20-hydroxyecdysone treatment (1 μg/g body mass; 4-month-old shrimp) for 168 hpi (P<0.05). Taken together, PmBr-c seems to play an important role during ovarian development of P. monodon.

Molecular cloning and expression analysis of the Mitogen‐activating protein kinase 1 (MAPK1) gene and protein during ovarian development of the giant tiger shrimp Penaeus monodon

Isolation and characterization of genes and/or proteins differentially expressed in ovaries are necessary for understanding ovarian development in the giant tiger shrimp (Penaeus monodon). In this study, the full-length cDNA of P. monodon mitogen-activating protein kinase 1 (PmMAPK1) was characterized. PmMAPK1 was 1,398 bp in length containing an open reading frame of 1,098 bp that corresponded to a polypeptide of 365 amino acids. PmMAPK1 was more abundantly expressed in ovaries than in testes of P. monodon. Quantitative real-time PCR revealed differential expression levels of PmMAPK1 mRNA during ovarian development of intact broodstock, where it peaked in early cortical rod (stage III) ovaries (P < 0.05) and slightly decreased afterwards (P > 0.05). Likewise, the expression level of PmMAPK1 in early cortical rod and mature (IV) ovaries was significantly greater than that in previtellogenic (I) and vitellogenic (II) ovaries of eyestalk-ablated broodstock (P < 0.05). The PmMAPK1 transcript was localized in ooplasm of previtellogenic oocytes. In intact broodstock, the expression of the PmMAPK1 protein was clearly increased from previtellogenic ovaries in subsequent stages of ovarian development (P < 0.05). In contrast, the level of ovarian PmMAPK1 protein was comparable during oogenesis in eyestalk-ablated broodstock (P > 0.05). The PmMAPK1 protein was localized in ooplasm of previtellogenic and vitellogenic oocytes. It was also detected around the nuclear membrane of early cortical rod oocytes in both intact and eyestalk-ablated broodstock. Results indicated that PmMAPK1 gene products seem to play functional roles in the development and maturation of oocytes/ovaries in P. monodon.

Identification, characterization and expression of adipose differentiation-related protein ( ADRP) gene and protein in ovaries of the giant tiger shrimp Penaeus monodon

In this study, the full length cDNA of adipose differentiation-related protein, a gene involved in neutral lipid accumulation that is important in oocyte maturation, of the giant tiger shrimp, Penaeus monodon ( PmADRP) was identified and characterized. PmADRP was 1292 bp in length containing an ORF of 1107 bp corresponding to a polypeptide of 368 amino acids. PmADRP was abundantly expressed in ovaries but not in testes of P. monodon juveniles and broodstock or several tissues (e.g. intestine, eyestalk and gill) of P. monodon broodstock. The expression levels of PmADRP in ovaries of juvenile P. monodon were significantly induced following 5-HT administration at 48 h post treatment ( P < 0.05). Quantitative real-time PCR illustrated that the PmADRP mRNA in ovaries of juveniles was lower than that of broodstock ( P < 0.05). In intact broodstock, PmADRP was significantly up-regulated at early cortical rod (stage III) ovaries and after spawning ( P < 0.05). Likewise, the expression level of PmADRP in early cortical rod and mature (IV) ovaries was significantly greater than that in previtellogenic (I) and vitellogenic (II) ovaries of eyestalk-ablated broodstock ( P < 0.05). In situ hybridization indicated that PmADRP was localized in ooplasm of previtellogenic oocytes. The PmADRP protein was detected since the vitellogenic stage for intact broodstock and since the previtellogenic stage for eyestalk-ablated broodstock. Results suggested functionally important roles of PmADRP on oocyte and ovarian development of P. monodon.

Isolation and characterization of progesterone receptor-related protein p23 ( Pm-p23) differentially expressed during ovarian development of the giant tiger shrimp Penaeus monodon

A homologue of progesterone receptor-related protein p23 ( Pm-p23) was isolated from EST analysis of the cDNA library established from vitellogenic ovaries of the giant tiger shrimp ( Penaeus monodon). The full length cDNA of Pm-p23 was further characterized by RACE-PCR and it was 1943 bp, comprising an open reading frame (ORF) of 495 bp corresponding to 164 amino acid residues and the 5′ and 3′ UTRs of 7 and 1441 bp, respectively. Pm-p23 significantly matched p23-like protein of Nasonia vitripennis ( E-value = 7 × 10 − 46 ). The predicted molecular mass and p I of the deduced Pm-p23 protein was 19.07 kDa and 4.39, respectively. Quantitative real-time PCR analysis revealed that the expression levels of Pm-p23 in ovaries of both intact and eyestalk-ablated broodstock were significantly greater than that of juveniles (4-month-old shrimp) ( P < 0.05). Pm-p23 was up-regulated since stage II ovaries of intact and stage III ovaries of eyestalk-ablated P. monodon broodstock ( P < 0.05). The mRNA level of Pm-p23 after spawning was not significantly different from stages II–IV ovaries of intact broodstock ( P > 0.05). In situ hybridization indicated that Pm-p23 was localized in ooplasm of previtellogenic oocytes. Recombinant Pm-p23 protein was successfully expressed in vitro and its polyclonal antibody was successfully produced. Western blot analysis indicated that the level of ovarian Pm-p23 protein peaked at the vitellogenic stage and decreased as oogenesis progressed. Taken together, results from this study strongly suggested functionally important roles of Pm-p23 gene products during vitellogenesis of P. monodon oocytes.

The Maturation of Oocyte Follicular Epithelium of Podarcis s. sicula Is Promoted by d-Aspartic Acid

We investigated whether the maturation of oocyte follicular epithelium of lizard is affected by d-aspartic acid (d-Asp). Our results demonstrated that d-Asp is endogenously present in the oocytes, and its distribution varies during the reproductive cycle and following intraperitoneal administration. At previtellogenesis, it is observed in the cytoplasm and nucleus of pyriform cells, in intermediate cells, in some small cells of the granulosa, in the ooplasm, and in some thecal elements. At vitellogenesis, d-Asp is localized in the proximity of the zona pellucida, in the theca, and in the ooplasm. Injected d-Asp is mainly captured by pyriform cells and ooplasm of previtellogenic oocytes, but a moderate accumulation is evident in the cytoplasm of some small granulosa cells and in the theca. d-Asp also increases the ovarian and plasmatic levels of 17β-estradiol and decreases those of testosterone. As a direct and/or indirect consequence of d-Asp, previtellogenic oocytes grow up and mature, resulting in a higher accumulation of carbohydrates in the granulosa, zona pellucida, and ooplasm, but also a reduction in the thickness of the granulosa layer and an increase of the theca stratum. Taken together, our results show that d-Asp may be related to the synchrony of reproduction, either enhancing the growth and maturation of follicular epithelium or influencing its endocrine functions.