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Probiotics Can Induce Follicle Maturational Competence: The Danio rerio Case1
Giorgia Gioacchini,3 Elisabetta Giorgini,4 Daniel L. Merrifield,5 Gary Hardiman,6,7 Andrea Borini,8 Lisa
Vaccari,9 and Oliana Carnevali2,3
3
Dipartimento di Scienze del Mare, Universita Politecnica delle Marche, Ancona, Italy
4
Dipartimento di Idraulica, Strade, Ambiente e Chimica ISAC, Sezione Chimica, Universita Politecnica delle Marche,
Ancona, Italy
5
Aquaculture and Fish Nutrition Research Group, School of Biomedical and Biological Sciences, University of
Plymouth, Plymouth, Devon, United Kingdom
6
Department of Medicine, University of CaliforniaSan Diego, La Jolla, California
7
BIOGEM, School of Medicine, University of CaliforniaSan Diego, La Jolla, California
8
Tecnobios Procreazione, Centre for Reproductive Health, Bologna, Italy
9
SISSI Beamline, ELETTRA Synchrotron Light Laboratory, Basovizza, Trieste, Italy
In the present study, the effects of the probiotic Lactobacillus DNA array, fish reproduction, FPA FT-IR imaging, follicle
rhamnosus IMC 501 on the acquisition of oocyte maturational maturation, gamete biology, gene expression, nutrition, oocyte
oocyte maturation and the increase of fecundity after probiotic Animal Welfare (UFAW) and with the Italian animal welfare legislation (D.L.
administration [1316]. These results led us to hypothesize that 116/92).
probiotic could induce earlier oocyte maturation. Therefore, in
the present study, we investigated the role of L. rhamnosus DNA Array
IMC 501 administration on the acquisition of oocyte RNA extraction, fluorescent target labeling and microarray hybridiza-
maturational competence in zebrafish. tions. Total RNA was extracted from samples composed of 25 follicles (stage
The present study had several objectives. The first was to IIIa or IIIb follicles, separately), were mechanically isolated from all
determine whether L. rhamnosus administration could induce experimental groups using the Minikit RNAeasy (Qiagen) extraction kit
following the manufacturers protocol. Total RNA extracted was eluted in 25 ll
the ability of the stage IIIa follicles to respond to MIH. The of RNase-free water. Total RNA was treated with DNase (10 IU at 378C for 10
second was to provide an overview of the transcriptome min; MBI Fermentas), and final RNA concentrations were determined by
follicles (stages IIIa and IIIb) from both probiotic-treated and absorbance readings (optical density [OD]) at 260 nm using an ND-1000
control females by DNA microarray analysis, highlighting (Nanodrop). RNA was further assessed for integrity with the 6000 Nano
specifically the pathways that are affected, but also those that LabChip assay (Agilent). Total RNA (100 ng) was converted into fluorescently
are unaffected, by the probiotic treatment. labeled Cy3 cRNA using the Low RNA Input Fluorescent Linear Amplification
Kit (Agilent). Fluorescent targets were purified to remove unincorporated
The third objective was to examine whether probiotic nucleotides using RNeasy (Qiagen). Absorbance (OD) at 260 nm was used to
treatment in stage IIIa and IIIb follicles modulates the quantify the cRNA concentrations, and absorbance at 550 and 650 nm was used
transcription of specific mRNAs involved in the acquisition to measure the efficiency of Cy3 dye incorporation. An incorporation efficiency
of competence. To this end, changes in gene expression of of 9 pmol/lg or greater was deemed to be necessary before proceeding with
transforming growth factor-b1 (tgfb1) and growth differentia- hybridization. The Agilent Zebrafish Oligo Microarray V1, containing 21 495
probes representing all existing predicted zebrafish genes from the most recent
tion factor 9 (gdf9), which are known to inhibit follicle public databases, such as RefSeq, Unigene, and TIGR Gene Index, was used.
maturation [8, 17], were analyzed. In addition, expression of Each array also contained quality-control probes for monitoring any
activinbA1 (inhbaa), which stimulates oocyte maturation [10, experimental variations. For each sample, 1 lg of fragmented cRNA was
18]; LH receptor (lhr); b isoform of membrane progestin hybridized to the zebrafish microarray in accordance with single-color Agilent
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L. RHAMNOSUS CONTROLS ZEBRAFISH FOLLICLE MATURATION
reactions was 3 min at 958C and then 45 cycles of 20 sec at 958C, 20 sec at FT-IR Measurements
608C, and 20 sec at 728C. Fluorescence monitoring occurred at the end of each
cycle. Additional dissociation-curve analysis was performed and, in all cases, The FT-IR measurements were carried out at the infrared beamline SISSI@
showed a single peak. ELETTRA synchrotron [29] using a Bruker VERTEX 70 interferometer
Both actb and arp were used as housekeeping genes in each sample to coupled with a Hyperion 3000 Vis-IR microscope and equipped with a liquid
standardize the results by eliminating variation in mRNA and cDNA quantity and nitrogen-cooled FPA detector (detector area, 64 3 64 pixels). Because of the
quality. No amplification product was observed in negative controls, and no follicle dimensions, representative areas were chosen for IR imaging in
primer-dimer formation was observed in the control templates. The data obtained transmission mode at room temperature using a 153 condenser/objective (pixel
were analyzed using the iQ5 optical system software version 2.0 (Bio-Rad) resolution, ;2.6 lm). Each IR image (OPUS 6.5 software package; Bruker),
including GeneEx Macro iQ5 Conversion and genex Macro iQ5 files. acquiring simultaneously groups of 4096 spectra, was collected by averaging
Modification of gene expression is represented with respect to the control 128 scans for each detector pixel, with a spectral resolution of 4 cm1, rationing
sampled at the same time as the treatment. The primer sequences, designed using the background single-channel image against the sample single-channel image.
Primer3 (v. 0.4.0) software were as follows: lhr forward, 5 0 -ggcgaaggctagatgg All the samples were compared with independent trials. Total absorbance
cacat-3 0 ; lhr reverse, 5 0 -tcgccatctggttcatcaata-3 0 ; inhb forward, 5 0 -caacttagatgga cartograms, representing the total intensity of the infrared absorption, were
cacgctg-3 0 ; inhb reverse, 5 0 -gtggatgtcgaggtcttgtc-3 0 ; mprb forward, 5 0 - generated for each sample by integrating the total area between 1800 and 900
caacgagctgctgaatgtgt-30 ; mprb reverse,-5 0 gggccagttcagagtgagac-3 0 ; tgfb1 for- cm1 (not shown).
ward, 5 0 -tcgctttgtctccaaggact-3 0 ; tgfb1 reverse, 5 0 -tgcaagagagttgccatttg-3 0 ; gdf9 Average spectra were extracted from IR images by using OPUS 6.5, with
forward, 5 0 -cgaccacaaccacctctctcc-3 0 ; gdf9 reverse, 5 0 -gggactgagtgctggatgcc-3 0 ; the number of selected spectra ranging from 4096 to 16 384. All the average
pou5f1 forward, 5 0 -caaattacggcccaagctaa-30 ; pou5f1 reverse, 5 0 -cagaatcactg spectra were two-point baseline linear-fitted in the spectral range of 4000 to 900
catcctcca-3 0 ; actb forward, 5 0 -ggtacccatctcctgctccaa-3 0 ; actb reverse, 5 0 - cm1 and vector normalized [30].
gagcgtggctactccttcacc-3 0 ; arp forward, 5 0 -ctgaacatctcgcccttctc; and arp reverse, To better evidence small changes observed in normal spectra, the second
5 0 -tagccgatctgcagacacac-3. 0 derivative procedure was applied. At the same time, peak fitting procedure was
useful to discriminate in a convoluted band (like amide I and II) the various
Western Blot Analysis subcomponents and to evaluate the corresponding absorbance intensity of a
single band. Using the GRAMS/AI 7.02 (Galactic Industries, Inc.) software
For the Mprb assays, stage IIIa and IIIb follicles from each of the experimental package, peak fitting was performed on average spectra (interpolated in the
groups were electrophoresed and transferred into polyvinylidene fluoride as range of 1775 to 1580 cm1 and two-point baseline linear-fitted); to identify the
Biological Process
Regulation of anatomical structure size 90066 1.16E05 1.86E02
Oxidation reduction 55114 1.36E05 2.17E02
Signal transduction 7165 1.48E05 2.37E02
Regulation of biological quality 65008 2.23E05 3.57E02
Signal transmission 23060 2.51E05 4.01E02
Signaling process 23046 2.51E05 4.01E02
Response to chemical stimulus 42221 2.88E05 4.61E02
Molecular Function
Heme binding 20037 4.34E07 2.66E04
FIG. 1. Rate (%) of GVBD in D. rerio stage IIIa and IIIb follicles isolated
Tetrapyrrole binding 46906 5.60E07 3.44E04
from control group ovaries incubated in L15 (CTRL) and in L15 plus MIH
Oxidoreductase activity, acting on 16705 2.93E06 1.80E03
(1 lg/ml; MIH) and in D. rerio stage IIIa and IIIb follicles isolated from
paired donors, with incorporation or
treated group ovaries incubated in L15 (PROBIO) and in L15 plus MIH
reduction of molecular oxygen
(PROBIO MIH; n 20). Raw data are presented as the mean 6 SD (error
Oxidoreductase activity 16491 3.23E06 1.98E03
bars). Different letters indicate statistically significant differences (P ,
Electron carrier activity 9055 6.18E06 3.79E03
0.05, ANOVA followed by Bonferroni multiple-comparison test) after
Iron ion binding 5506 7.79E06 4.77E03
arcosin transformation of raw data.
Guanyl nucleotide binding 19001 2.35E05 1.44E02
Guanyl ribonucleotide binding 32561 2.35E05 1.44E02
only 69.8% 6 3.8% of stage IIIb follicles isolated from the GTP binding 5525 3.20E05 1.96E02
Monooxygenase activity 4497 5.10E05 3.13E02
Biological Process
Regulation of transcription, 6355 1.17E06 1.88E03
DNA-dependent
Regulation of RNA metabolic process 51252 1.63E06 2.60E03
Transcription, DNA-dependent 6351 4.18E06 6.69E03
RNA biosynthetic process 32774 5.11E06 8.17E03
Developmental process 32502 7.02E06 1.12E02
Multicellular organismal development 7275 7.22E06 1.15E02
Striated muscle cell development 55002 1.74E05 2.79E02
Multicellular organismal process 32501 1.85E05 2.95E02
Anatomical structure development 48856 2.45E05 3.92E02
Organ morphogenesis 9887 2.86E05 4.57E02
Molecular Function
Transcription factor activity 3700 9.72E06 5.96E03
sequence-specific DNA binding 43565 1.24E05 7.57E03
Transcription regulator activity 30528 2.82E05 1.73E02
Oxygen binding 19825 6.75E05 4.14E02
* The P values for groups of genes were calculated by performing Gene
Ontology (GO) analysis (htpp://geneontology.org).
FIG. 5. pou5f1 mRNA levels normalized against actb and arp genes in
stage IIIa and IIIb follicles of control (CTRL) and probiotic-treated
(PROBIO) fish (n 10). Data are presented as the mean 6 SD (error
bars). Different letters indicate statistically significant differences (P ,
0.05, ANOVA followed by Bonferroni multiple-comparison test).
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L. RHAMNOSUS CONTROLS ZEBRAFISH FOLLICLE MATURATION
TABLE 4. Microbial community analysis from PCR-DGGE fingerprints of the GI microbiota zebrafish from each group (n 3 per group).a
ANOSIMg
Experimental groups Nb Richnessc Evenessd Diversitye SIMPERf R value P value Dissimilarity (%)
Control 21.33 6 1.53 2.01 6 0.12 0.988 6 0.01 3.02 6 0.06 74.1 6 6.2
Probiotic 19.33 6 1.53 1.77 6 0.15 0.924 6 0.06 2.73 6 0.23 80.6 6 4.4
Control vs. Probiotic 1.00 0.10 50.46 6 5.04
a
Values expressed as means 6 SD.
b
Average number of bands=presumed species.
c
Margalef species richness: d (S 1) = log (N).
d
Pielou evenness: J 0 H 0 /log(S). P
e
Shannon diversity index: H 0 (pi(ln pi)).
f
SIMPER, similarity percentage within group replicates.
g
ANOSIM, analysis of similarities between groups.
probiotic on the GI populations affected individual phylotypes in the range of 1775 to 1580 cm1, and the areas of the
(bands, which represent presumed individual bacterial species/ component bands were calculated. In all specimens, it was
strains) either by suppressing them (to nondetectable levels, in possible to distinguish intermolecular (1692 6 2 and 1615 6 2
some cases) or by stimulating the presence of certain cm1) from intramolecular (1679 6 1 and 1629 6 1 cm1) b
phylotypes that were absent or nondetectable in the other structures as well as three-turn helix (1667 6 1 cm1), a-helix
group. A clear example was the low to undetectable levels of (1655 6 2 and 1646 6 2 cm1), and random coil (1639 6 1
the phylotype JF431931, identified as Streptococcus thermo- cm1) components.
TABLE 5. Isolated bacterial bands and their closest relatives (BLAST) from PCR-DGGE of the GI communities of zebrafish.
Similarity to Accession no. of
Band Accession no. Nearest neighbor(s) nearest neighbor(s) nearest neighbor(s)
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GIOACCHINI ET AL.
resulted in a split into two bands at 1089 and 1082 cm1 (non- that 10-day administration of L. rhamnosus significantly
hydrogen- and hydrogen-bonded, respectively). enhanced responsiveness of stage IIIa follicles to MIH. In
addition, and to our knowledge for the first time, we performed
DISCUSSION a DNA microarray experiment to elucidate the biological
Recent research on the molecular biology and genomics of
probiotics has focused on the interaction of gut microbiota with
the immune system [36], brain development [37], potential as
an anticancer agent [38], and potential as a biotherapeutic agent
in many diseases [39]. Very recently, our group observed a
positive role of a probiotic, L. rhamnosus IMC 501, on
fecundity and on endocrine and paracrine control of follicle
development in zebrafish females [1315]. Ten-day adminis-
tration of this bacterial species affected the number of ovulated
eggs in vivo and the GVBD rate in vitro [13]. In addition, using
FT-IR microspectroscopy analysis to investigate ovarian
sections, we clearly observed molecular changes induced by
probiotic administration on follicle molecular building, such as
the increase of water uptake and phosphorylation processes,
together with the modification of protein secondary structures.
However, no direct evidence for the effect of this probiotic on
the acquisition of maturational competence in the oocyte was
found.
In the present investigation, we studied the possible role of
L. rhamnosus on zebrafish oocyte competence. We showed
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L. RHAMNOSUS CONTROLS ZEBRAFISH FOLLICLE MATURATION
reported in several fish species [2, 40, 41], and the increase of
the mRNA of its receptor indicates a major responsiveness of
the follicles to the LH produced and released by the pituitary.
At the same time, activin is an important member of the
transforming growth factor superfamily that has been exten-
sively studied in mammals [42] and zebrafish [8], and its role
in regulation of the development of oocyte maturational
competence and in stimulation of the final maturation of full-
grown oocytes in the maturational stage has been demonstrated
[1, 4, 8, 43].
In accordance with the levels previously reported by other
authors [4, 8], the mprb mRNA and protein levels significantly
increase in stage IIIb control follicles compared to stage IIIa.
The stage IIIa follicles isolated from probiotic-treated females
showed a major capability to respond to MIH by significantly
enhancing the mRNA and protein levels of its membrane
receptor. At the same time, these increases were associated
with the down-regulation of local factors, such as tgfb1 and
gdf9 genes. These two intraovarian molecules inhibit follicle
maturation by blocking the resumption of meiosis [8, 17],
acting at multiple sites. In particular, tgfb1 suppresses both
human chorionic gonadotropin- and MIH-induced oocyte
maturation [44] by regulating the expression of several key
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L. RHAMNOSUS CONTROLS ZEBRAFISH FOLLICLE MATURATION
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