Enhanced hyperplasia in muscles of transgenic zebrafish expressing <Emphasis Type="Italic">Follistatin1</Emphasis>

Myostatin is a member of the transforming growth factor-β (TGF-β) super-family and functions as a negative regulator of muscle growth. Binding of the specific receptor, Activin receptor IIB (Act RIIB), with myostatin or other related TGF-β members, could be inhibited by the activin-binding protein follistatin (Fst) in mammals. Overexpressing Fst in mouse skeletal muscle leads to muscle hypertrophy and hyperplasia. To determine if Fst has similar roles in fish, we generated transgenic zebrafish expressing high levels of zebrafish Fst1 using the promoter of the zebrafish skeletal muscle-specific gene, myosin, light polypeptide 2, skeletal muscle (Mylz2). Independent transgenic zebrafish lines exhibited elevated expression levels of myogenic regulatory genes MyoD and Pax7 in muscle cells. Adult Fst1 overexpressing transgenic zebrafish exhibited a slight body weight increase. The high level of Fst1 expression dramatically increased myofiber numbers in skeletal muscle, without significantly changing the fiber size. Our findings suggest that Fst1 overexpression can promote zebrafish muscle growth by enhancing myofiber hyperplasia.     

Nitroreductase-mediated Gonadal Dysgenesis for Infertility Control of Genetically Modified Zebrafish

Genetically modified (GM) fish with desirable features such as rapid growth, disease resistance, and cold tolerance, among other traits, have been established in aquaculture. However, commercially available GM fish are restricted because of global concerns over the incomplete assessments of food safety and ecological impact. The ecological impact concerns include gene flow and escape of the GM fish, which may cause extinction of wild natural fish stocks. Infertility control is a core technology for overcoming this obstacle. Although polyploidy technology, GnRH-specific antisense RNA, and RNAi against GnRH gene expression have been used to cause infertility in fish, these approaches are not 100% reliable and are not heritable. In the present study, zebrafish was used as a model to establish an inducible platform of infertility control in GM fish. Nitroreductase, which converts metronidazole substrate into cytotoxin, was fused with EGFP and expressed specifically by oocytes in the Tg(ZP:NTR-EGFP) by a zona pellucida promoter. Through consecutive immersion of metronidazole from 28 to 42 days posthatching, oocyte-specific EGFP expression was eliminated, and atrophy of the gonads was detected by anatomical analysis. These findings reveal that oocyte-specific nitroreductase-mediated catalysis of metronidazole blocks oogenesis and leads to an undeveloped oocyte. Furthermore, oocyte cell death via apoptosis was detected by a TUNEL assay. We found that the gonadal dysgenesis induced by metronidazole resulted in activation of the ovarian killer gene bok, which is a proapoptotic gene member of the Bcl-2 family and led to infertility. These results show that oocyte-specific nitroreductase-mediated catalysis of metronidazole can cause reliable infertility in zebrafish and could potentially be used as a model for other aquaculture fish species.     

Roles of the activin regulatory system in fish reproduction

Activin (betaAbetaA, betaAbetaB, and betaBbetaB) is a dimeric growth factor with diverse biological activities in vertebrate reproduction. Activin exerts its actions by binding to its specific type II and type I receptors. The activity of activin is regulated by follistatin, its binding protein, and the antagonists inhibin and antivin. All major components of the activin-inhibin-follistatin system have been identified in fish except the alpha subunit of inhibin. Using goldfish as a model, we have demonstrated that activin is expressed in the pituitary and the recombinant goldfish activin B has novel inverse effects on the expression of GTH beta subunits. Activin increases the mRNA level of GTH-Ibeta while significantly suppressing the expression of GTH-IIbeta. We have also demonstrated the expression of activin and its receptors in the goldfish and zebrafish ovary. Using an in vitro ovarian follicle incubation as the system, we have investigated the involvement of the activin system in the process of final oocyte maturation. Our evidence clearly indicates that activin has potent effect of promoting final oocyte maturation, and that it may play a role in mediating the stimulatory effect of pituitary gonadotropin in the event of oocyte maturation.     

Betacellulin overexpression in the mouse ovary leads to MAPK3/MAPK1 hyperactivation and reduces litter size by impairing fertilization

The epidermal growth factor receptor (EGFR) and its ligands are emerging as key molecules in regulating female reproduction. Here, we used a transgenic mouse model to evaluate whether and at which level of the reproduction cascade higher-than-normal levels of the EGFR ligand betacellulin (BTC) in the reproductive organs affect fertility. Western blots and immunohistochemistry revealed increased BTC levels in uterus and ovaries from transgenic females, particularly evident in granulosa cells of antral follicles. Onset of puberty, estrous cyclicity, and the anatomy and histology of reproductive organs at puberty were not altered as compared to control females. Fertility tests revealed a reduction (~50%) in litter size as the major reproductive deficit of transgenic females. Embryo implantation was delayed in transgenic females, but this was not the reason for the reduced litter size. Transgenic females produced a normal number of oocytes after natural ovulation. The in vivo fertilization rate was significantly reduced in untreated transgenic females but returned to normal levels after superovulation. Impaired oocyte fertilization in the absence of superovulation treatment was associated with MAPK3/MAPK1 hyperactivation in BTC transgenic ovaries, whereas similar levels of MAPK3/MAPK1 activation were detected in transgenic and control ovaries after superovulation treatment. Thus, tight regulation of MAPK3/MAPK1 activity appears to be essential for appropriate granulosa cell function during oocyte maturation. Our study identified hitherto unknown effects of BTC overabundance in reproduction and suggests BTC as a novel candidate protein for the modulation of fertility.     

Overexpression of follistatin-like 3 in gonads causes defects in gonadal development and function in transgenic mice

Activin has numerous biological activities including regulation of follicular development, spermatogenesis, and steroidogenesis within the gonads. Activities of activin are regulated by follistatin (FST), an activin binding protein, and perhaps follistatin-like 3 (FSTL3; also known as FLRG and FSRP). FSTL3 is a recently described member of the FST family having an overall structure and activity profile similar to that of FST, including binding and neutralization of activin. FSTL3 is most highly expressed in the placenta and testis, whereas FST is highest in the ovary and kidney, suggesting that FSTL3 has biological actions that do not entirely overlap those of FST. To investigate the role of local FSTL3 as a potential regulator of activin action in gonad development and function, we examined FSTL3 expression in the mouse testis. FSTL3 protein was localized to Leydig cells, spermatagonia, and mature spermatids in normal male mice. We then created transgenic mice using a human FSTL3 cDNA driven by the mouse alpha-inhibin promoter. Three of five transgenic founders were fertile and were bred to establish lines. In the highest expressing line 3, transgene expression was largely restricted to gonads, with pituitary, adrenal, brain, and uterine expression being substantially lower. Gonad weights, sperm counts, and fertility were significantly reduced in transgenic males, and reduced litter size was evident in line 3 females. Within the testis, highest transgene expression was observed in Sertoli cells, and although most tubules showed evidence of normal spermatogenic development, degenerating tubules devoid of germ cells and Leydig cell hyperplasia were also evident in every line 3 animal examined. Ovaries from line 3 females contained fewer antral follicles and more apparent follicular atresia. Although circulating human FSTL3 levels were undetectable, FSH and LH levels in adult transgenic mice were not significantly different from wild-type animals. However, testosterone levels were significantly increased at d 21 and significantly reduced at d 60 compared with wild-type males. These results suggest that FSTL3 is likely to be a local regulator of activin action in gonadal development and gametogenesis and, further, that activin appears to have important actions in gonadal development and function that are critical for normal reproduction.     

Role of activin, transforming growth factor-beta and bone morphogenetic protein 15 in regulating zebrafish oocyte maturation

The transforming growth factor-beta (TGF-beta) superfamily is a large group of peptide growth and differentiation factors that have important functions in many physiological processes, including reproduction. We previously reported that several members of the TGF-beta superfamily, including activin-A, bone morphogenetic protein-15 (BMP-15) and TGF-beta1, regulate oocyte maturation in zebrafish. The aim of this study was to further examine the functions and mechanisms of these growth factors in regulating zebrafish oocyte maturation. First, the interaction among three regulators was examined. Overexpression of BMP-15 reduced the effect of activin-A on oocyte maturation. Inhibition of BMP-15 function or expression increased oocyte maturation but had no additive effect with activin-A. TGF-beta1 suppressed activin-A-, as well as BMP-15 antiserum-induced oocyte maturation. Second, the role of Smad 2, an intracellular mediator of activin and TGF-beta, in oocyte maturation was investigated. Western blot analysis revealed that both activin-A and TGF-beta1 activate Smad2 in zebrafish follicles. Injection of morpholino antisense olignucleotides against Smad2 into oocytes reduced Smad2 expression and completely blocked activin-A-induced oocyte maturation. Knockdown of Smad 2 also significantly decreased basal and hCG-induced oocyte maturation. These findings suggest that activin-A, TGF-beta1, and BMP-15 may target common gene(s) to regulate oocyte maturation and demonstrate that Smad2 plays an important role in oocyte maturation.     

Spatial expression patterns of activin and its signaling system in the zebrafish ovarian follicle: evidence for paracrine action of activin on the oocytes

We have previously demonstrated that activin is likely an ovarian mediator of pituitary gonadotropin(s) and local epidermal growth factor in their stimulating oocyte maturation and maturational competence in the zebrafish. However, the downstream events controlled by activin remain unknown. One possible mechanism is that activin may directly work on the oocytes to promote the development of oocyte maturational competence. To substantiate this hypothesis, we performed the present study to demonstrate the expression of the activin system in different compartments of zebrafish follicles, namely, the follicle cells and oocytes. The proteins examined include activin subunits (betaA and betaB), activin-binding protein (follistatin), activin type II receptors (type IIA and IIB), the type I activin receptor-like kinases (ALK1-like, ALK2-like, and ALK4-like), and the intracellular activin signaling molecules (Smad2, Smad3, Smad4, and Smad7). The results showed that the entire activin signaling system is expressed by the full-grown immature zebrafish oocytes ( approximately 0.65 mm in diameter), including ALK4-like (ActRIB), ALK2-like (ActRIA), ActRIIA, ActRIIB, Smad2, Smad3, Smad4, and Smad7, therefore supporting our hypothesis that the oocytes are one of the direct targets of activin actions in the zebrafish ovary. In contrast, activin itself (betaA and betaB) and ALK1-like type I receptor are predominantly expressed in the follicle cells surrounding the oocytes. Interestingly, although follistatin is expressed in both the follicle cells and oocytes, its level of expression is significantly higher in the oocytes than the follicle cells, implying that follistatin may serve as a signal from the oocytes to modulate the activity of activin produced by the follicle cells. Taken together, the present study provides convincing evidence that although all members of the activin system are expressed in the whole follicle, they exhibit distinct spatial patterns of expression among different compartments of the follicle. It is likely that activin works directly on the oocytes in a paracrine manner to promote oocyte maturation and maturational competence. On the other hand, instead of being controlled passively by the follicle cells, the oocytes may actively participate in the regulation of follicle development by releasing various modulating molecules such as follistatin.     

Evolution of follistatin in teleosts revealed through phylogenetic, genomic and expression analyses

Follistatin (Fst) inhibits transforming growth factor-β (TGF-B) proteins and is a known regulator of amniote myogenesis. Here, we used phylogenetic, genomic and experimental approaches to study its evolution in teleosts. Phylogenetic analyses suggested that one fst gene (fst1) is common to euteleosts, but a second gene (fst2) is conserved specifically within the Ostariophysi. Zebrafish fst1/2 respectively appear on chromosomes 5 and 10 in two genomic regions, each with conserved synteny to a single region in tetrapods. Interestingly, other teleosts have two corresponding chromosomal regions with a similar repertoire of paralogues. Phylogenetic reconstruction clustered these gene duplicates into two sister clades branching from tetrapod sequences. We suggest that an ancestral fst-containing chromosome was duplicated during the teleost whole genome duplication, but that fst2 was lost in lineages external to the Ostariophysi. We show that Fst1 of teleosts/mammals has evolved under strong purifying selection, but the N-terminal of Fst2 may have evolved under positive selection. Furthermore, the tissue-specific expression of zebrafish fst2 was restricted to fewer tissues compared to its paralogue and the single fst1 orthologue of Atlantic salmon (Salmo salar). Zebrafish fst1/2 may have subfunctionalized relative to non-duplicated vertebrate lineages, as several regions in the fst promoter of tetrapods were conserved with one paralogue, but not both. Finally, we examined the embryonic expression of fst1 in a teleost outside the Ostariophysi (Atlantic salmon). During segmentation, fst1 was expressed in the anterior somite compartment but was excluded from muscle progenitors that strongly expressed myogenic regulatory factors (MRFs). Later, fst1 was expressed in myogenic progenitors of the pectoral fin buds and also within the pax7 ⁺ cell layer external to the myotome. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Multiple rapid progestin actions and progestin membrane receptor subtypes in fish

Progestin hormones exert rapid, nongenomic actions on a variety of target tissues in fish. The induction of oocyte maturation and the progestin membrane receptor (mPR) that mediates this action of progestins have been well characterized in fishes. Progestins also act on Atlantic croaker spermatozoa via an mPR to rapidly increase sperm motility. Preliminary results indicate that progestins can also exert rapid actions in the preoptic anterior hypothalamus (POAH) in this species to down-regulate gonadotropin-releasing hormone (GnRH) secretion. Recently, we reported the cloning, sequencing and characterization of a novel cDNA in a closely related species, spotted seatrout, that has the characteristics of the mPR involved in the progestin induction of oocyte maturation. Three distinct mPR subtypes, named alpha, beta, and gamma, have been identified in both fishes and mammals. The tissue distribution of the mPRalpha protein in seatrout suggests the alpha-subtype mediates progestin actions on GnRH secretion, sperm motility and oocyte maturation. However, mPRbeta antisense experiments in zebrafish oocytes suggest the beta-subtype also participates in the control of oocyte maturation in zebrafish.     

Transgene expression in zebrafish: A comparison of retroviral-vector and DNA-injection approaches.

To assess alternative methods for introducing expressing transgenes into the germ line of zebrafish, transgenic fish that express a nuclear-targeted, enhanced, green fluorescent protein (eGFP) gene were produced using both pseudotyped retroviral vector infection and DNA microinjection of embryos. Germ-line transgenic founders were identified and the embryonic progeny of these founders were evaluated for the extent and pattern of eGFP expression. To compare the two modes of transgenesis, both vectors used the Xenopus translational elongation factor 1-alpha enhancer/promoter regulatory cassette. Several transgenic founder fish which transferred eGFP expression to their progeny were identified. The gene expression patterns are described and compared for the two modes of gene transfer. Transient expression of eGFP was detected 1 day after introducing the transgenes via either DNA microinjection or retroviral vector infection. In both cases of gene transfer, transgenic females produced eGFP-positive progeny even before the zygotic genome was turned on. Therefore, GFP was being provided by the oocyte before fertilization. A transgenic female revealed eGFP expression in her ovarian follicles. The qualitative patterns of gene expression in the transgenic progeny embryos after zygotic induction of gene expression were similar and independent of the mode of transgenesis. The appearance of newly synthesized GFP is detectable within 5-7 h after fertilization. The variability of the extent of eGFP expression from transgenic founder to transgenic founder was wider for the DNA-injection transgenics than for the retroviral vector-produced transgenics. The ability to provide expressing germ-line transgenic progeny via retroviral vector infection provides both an alternative mode of transgenesis for zebrafish work and a possible means of easily assessing the insertional mutagenesis frequency of retroviral vector infection of zebrafish embryos. However, because of the transfer of GFP from oocyte to embryo, the stability of GFP may create problems of analysis in embryos which develop as quickly as those of zebrafish.     

Lactobacillus rhamnosus Accelerates Zebrafish Backbone Calcification and Gonadal Differentiation through Effects on the GnRH and IGF Systems

Endogenous microbiota play essential roles in the host’s immune system, physiology, reproduction and nutrient metabolism. We hypothesized that a continuous administration of an exogenous probiotic might also influence the host’s development. Thus, we treated zebrafish from birth to sexual maturation (2-months treatment) with Lactobacillus rhamnosus, a probiotic species intended for human use. We monitored for the presence of L. rhamnosus during the entire treatment. Zebrafish at 6 days post fertilization (dpf) exhibited elevated gene expression levels for Insulin-like growth factors -I and -II, Peroxisome proliferator activated receptors -α and -β, VDR-α and RAR-γ when compared to untreated-10 days old zebrafish. Using a gonadotropin-releasing hormone 3 GFP transgenic zebrafish (GnRH3-GFP), higher GnRH3 expression was found at 6, 8 and 10 dpf upon L. rhamnosus treatment. The same larvae exhibited earlier backbone calcification and gonad maturation. Noteworthy in the gonad development was the presence of first testes differentiation at 3 weeks post fertilization in the treated zebrafish population -which normally occurs at 8 weeks- and a dramatic sex ratio modulation (93% females, 7% males in control vs. 55% females, 45% males in the treated group). We infer that administration of L. rhamnosus stimulated the IGF system, leading to a faster backbone calcification. Moreover we hypothesize a role for administration of L. rhamnosus on GnRH3 modulation during early larval development, which in turn affects gonadal development and sex differentiation. These findings suggest a significant role of the microbiota composition on the host organism development profile and open new perspectives in the study of probiotics usage and application.     

Expression Patterns of CREBs in Oocyte Growth and Maturation of Fish

In fish, oocyte meiotic maturation is regulated by 17α, 20β-dihydroxy-progesterone through cAMP. To study the role of cAMP response element binding protein (CREB) in meiotic maturation, we cloned and characterized the expression pattern of CREBs from two fish models, the Nile tilapia and catfish. In the Nile tilapia three different CREBs were identified where in CREB1 was found in many tissues including gonads with abundant expression in testis. CREB2, few amino acids shorter than CREB1, was expressed in several tissues with abundant expression in ovary. In addition, a 3’UTR variant form, CREB3 was exclusively found in ovary. During natural 14-day ovarian cycle of the Nile tilapia, CREB1 expression was stable throughout vitellogenesis with a sharp decrease on the day of spawning. In contrast, CREB2 remain unchanged throughout the ovarian cycle, however elevated in 11-day full-grown immature ovarian follicle and after hCG-induction. Interestingly, CREB3 expression was induced three folds on the day of spawning as well as during hCG-induced oocyte maturation. Based on the synergistic expression pattern, CREB1 is likely to control oocyte growth, whereas CREB 2 and 3 contribute to oocyte maturation in tilapia and the latter seems to be critical. In catfish, a single form of CREB showed a maximum expression during spawning phase and hCG-induced maturation both in vivo and in vitro augmented CREB expression. These results suggest that spatial and temporal expression of CREBs seems to be important for final oocyte maturation and may also regulate oocyte growth in fish.     

野生圆口铜鱼卵巢成熟内分泌信号表达分析

圆口铜鱼(Coreius guichenoti)是一种呈群同步发育繁殖的鱼类, 对其卵巢成熟各时期特征分析, 可进一步了解鱼类卵巢成熟的分子调控机制, 并为人工催产实践提供理论支持。研究以野生雌性圆口铜鱼不同发育时期的卵巢为研究对象, 通过现场卵巢解剖观察以及实验室内组织学分析, 收集处于Ⅲ、Ⅳ和Ⅴ期的卵巢组织样本进行转录组分析。研究显示野生圆口铜鱼卵巢组织在其成熟发育各阶段具有明确的组织学和转录表达特征。研究采用全序列转录组测序分析, 以鲤科模式鱼类斑马鱼的基因序列作为主要参考序列, 共检测得到11 495 bp基因片段。聚焦内分泌/自分泌信号分子的转录表达, 发现促乳素(Prolactin, prl)、卵泡刺激素(Follicle-stimulating hormone, fsh)、雌激素(Estrogen)、前列腺素E2(Prostaglandin E2, PGE2)合成酶类、生长/分化因子9(Growth/differentiation factor 9, gdf9)、激活素(Activin)、和抗穆氏管激素(Anti-Mllerian hormone,amh)等转录表达和卵巢发育成熟呈现出显著的关联动态变化。通过对样品中卵巢成熟诱导激素(Maturationinducinghormone, MIH)合成酶分子的表达水平的观察, 发现涉及MIH的17, 20-dihydroxy-4-pregnen-3-one的合成酶分子表达水平较高, 而另一种MIH分子11-deoxycorticosteron的合成酶的表达水平处于检测水平之下,因此, 提示17, 20-dihydroxy-4-pregnen-3-one可能是圆口铜鱼卵巢成熟后期的主要促进因子。同时, 发现了完整的胆酸合成酶系列分子在卵巢转录组中的存在, 这是鱼类卵巢组织中具有合成胆酸能力的首次报道, 其生理作用有待进一步研究。总之, 作为一种群同步发育的鱼类, 此项研究为鱼类卵巢成熟发育过程中的内分泌信号调控网络提供了大量的数据参考。     

Identification of a putative oocyte-specific small nuclear ribonucleoprotein polypeptide C in gibel carp

Previous studies have demonstrated that germinal vesicle of amphibian oocyte contains small nuclear ribonucleoprotein polypeptide C (SNRPC). In this study, a putative member of SNRPC was identified from Carassius auratus gibelio oocyte cDNA library. Its full-length cDNA has an open reading frame of 201 nt for encoding a peptide of 66 aa, a short 5'-UTR of 19 nt and a long 3'-UTR of 347 nt including a polyadenylation signal and poly- (A) tail, and the deduced amino acid sequence has 47% identity with the C-terminal of the zebrafish small nuclear ribonucleoprotein polypeptide C. Western blot analysis revealed its oocyte-specific expression. Immunofluorescence localization indicated that its gene product localized to numerous nucleoli within the oocytes and showed dynamic changes with the nucleoli during oocyte maturation. RT-PCR and Western blot analysis further revealed its constant presence in the oocytes and in the embryos until hatching. The data suggested that the newly identified CagOSNRPC might be a nucleolar protein.     

Transgenic RNAi-mediated reduction of MSY2 in mouse oocytes results in reduced fertility

MSY2 is implicated in regulating the stability and translation of maternal mRNAs during mouse oogenesis. We report here that by driving the expression of a transgene encoding an Msy2 hairpin dsRNA in growing oocytes using the oocyte-specific Zp3 promoter, the amount of MSY2 protein was reduced by at least 60% in fully grown oocytes. The decrease appeared specific because no decrease was observed in either non-targeted mRNAs or proteins. Fertility of transgenic females was severely reduced. Although transgenic eggs could be inseminated, the eggs did not exhibit the normal series of oscillations in intracellular Ca2+, resume meiosis, undergo cortical granule exocytosis, or ZP2 cleavage to ZP2f. Transgenic oocytes also displayed a higher incidence of both the non-surrounded nucleolus chromatin morphology, and abnormal meiotic spindle formation was observed following oocyte maturation. Transgenic oocytes contained less total mRNA (approximately 75-80% that of non-transgenic oocytes) and displayed a reduced level of protein synthesis. Moreover, several of the maturation-associated changes in protein synthesis failed to occur in the transgenic oocytes. These results support a role for MSY2 in stabilizing maternal mRNAs in growing oocytes, a process essential to generate meiotically and developmentally competent oocytes.     

GH overexpression causes muscle hypertrophy independent from local IGF-I in a zebrafish transgenic model

The aim of the present study was to analyse the morphology of white skeletal muscle in males and females from the GH-transgenic zebrafish (Danio rerio) lineage F0104, comparing the expression of genes related to the somatotrophic axis and myogenesis. Histological analysis demonstrated that transgenic fish presented enhanced muscle hypertrophy when compared to non-transgenic fish, with transgenic females being more hypertrophic than transgenic males. The expression of genes related to muscle growth revealed that transgenic hypertrophy is independent from local induction of insulin-like growth factor 1 gene (igf1). In addition, transgenic males exhibited significant induction of myogenin gene (myog) expression, indicating that myog may mediate hypertrophic growth in zebrafish males overexpressing GH. Induction of the α-actin gene (acta1) in males, independently from transgenesis, also was observed. There were no significant differences in total protein content from the muscle. Our results show that muscle hypertrophy is independent from muscle igf1, and is likely to be a direct effect of excess circulating GH and/or IGF1 in this transgenic zebrafish lineage.