BIOLOGY OF REPRODUCTION (2012) 86(3):65, 111

Published online before print 16 November 2011.

DOI 10.1095/biolreprod.111.094243

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

ABSTRACT affecting the maturation phase.

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

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competence was examined in zebrafish (Danio rerio). L.
rhamnosus administration induced the responsiveness of incom-
petent 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. Molec-
ular analysis using PCR-DGGE (denaturing gradient gel electro-
phoresis) approach, providing information about only the most
abundant bacterial members of the microbial community,
revealed that the probiotic was able to populate the gastroin-
testinal 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 biochem-
ical composition of samples. Changes were found above all in
stage IIIa follicles from probiotic-exposed females; the modifi-
cations, 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,
1 In zebrafish, oocytes at the midvitellogenic stage, referred as
Supported by the Fondi di Ateneo 20082009 to O.C. and National
Institutes of Health grants DK063491, CA023100, and DK080506 to
G.H.
2
Correspondence: Oliana Carnevali, Dipartimento di Scienze del
Mare, Universita Politecnica delle Marche, Via Brecce Bianche,
60131 Ancona, Italy. E-mail: o.carnevali@univpm.it
Received: 22 June 2011.
First decision: 11 May 2011.
Accepted: 10 November 2011.
2012 by the Society for the Study of Reproduction, Inc.
eISSN: 1529-7268 http://www.biolreprod.org
ISSN: 0006-3363
1 Article 65
maturation, PCR-DGGE, real-time PCR, zebrafish
INTRODUCTION
Acquisition of maturational competence in oocytes repre-
sents a very important stage of oogenesis that signals the
transition from the growth phase to the maturation phase. This
process involves the ability of the follicle cells to produce
maturation-inducing hormone (MIH) and the responsiveness of
oocytes to MIH. The hormonal control of the acquisition of
oocyte maturational competence has been studied by several
authors, and the crucial role of luteinizing hormone (LH) on
this phase has been reported in several fish species, including
zebrafish [15].
In zebrafish, it has been more recently established that in
addition to the actions of LH, local factors directly produced by
the ovary play an important role in the acquisition of
maturational competence. Among these molecules are mem-
bers of the transforming growth factor-b (Tgfb) superfamily
and others molecules, such as the membrane receptors of
progesterone (Mprs) [38].
The zebrafish model provides several advantages for
studying the regulation of follicle development and maturation.
Zebrafish ovaries are asynchronous, containing follicles at all
stages of development [9] as well as mature eggs. Growth and
maturation of the oocyte occurs over a period of approximately
10 days [10, 11], and in laboratory conditions, eggs are
spawned throughout the year [12]. In addition, the mapped
genome of this model facilitates the use of molecular
techniques to identify the regulation of target genes.
stage IIIa, with a diameter of between 0.31 and 0.51 mm, are
known to be maturationally incompetent. In fact, they are
unable to undergo maturation in response to MIH. Only fully
grown, vitellogenic oocytes (stage IIIb), with a diameter of
between 0.52 and 0.70 mm, acquire the ability to respond to
MIH [1, 2], which by binding directly to membrane receptors
on the surface of fully grown oocytes leads to the final oocyte
maturation.
Using zebrafish as a model, our group recently found
evidence for the positive effect of a probiotic, Lactobacillus
rhamnosus, on reproduction; we demonstrated the induction of
 GIOACCHINI ET AL.
oocyte maturation and the increase of fecundity after probiotic
administration [1316]. These results led us to hypothesize that
probiotic could induce earlier oocyte maturation. Therefore, in
the present study, we investigated the role of L. rhamnosus
IMC 501 administration on the acquisition of oocyte
maturational competence in zebrafish.
The present study had several objectives. The first was to
determine whether L. rhamnosus administration could induce
the ability of the stage IIIa follicles to respond to MIH. The
second was to provide an overview of the transcriptome
follicles (stages IIIa and IIIb) from both probiotic-treated and
control females by DNA microarray analysis, highlighting
specifically the pathways that are affected, but also those that
are unaffected, by the probiotic treatment.
The third objective was to examine whether probiotic
treatment in stage IIIa and IIIb follicles modulates the
transcription of specific mRNAs involved in the acquisition
of competence. To this end, changes in gene expression of
transforming growth factor-b1 (tgfb1) and growth differentia-
tion factor 9 (gdf9), which are known to inhibit follicle
maturation [8, 17], were analyzed. In addition, expression of
activinbA1 (inhbaa), which stimulates oocyte maturation [10,
18]; LH receptor (lhr); b isoform of membrane progestin
receptor (mprb), which mediates the effects of MIH on oocyte
maturation; and pou5f1 were also examined.
The fourth objective was to assess the impact of the
probiotic on the gastrointestinal (GI) microbiome, an important
microbial population that is known to influence host develop-
ment, stress responses, nutrition, immunity, and disease
resistance [19, 20] and to identify any potential microbial
populations associated with the ovaries using PCR-DGGE
(denaturing gradient gel electrophoresis). Finally, the last
objective was to pinpoint, using FPA (focal plane array)
Fourier transform-infrared (FT-IR) imaging, the modifications
occurring in the biochemical composition of stage IIIa and IIIb
follicles as a result of probiotic administration [16, 21, 22].
MATERIALS AND METHODS
Animals and Probiotic Administration
Adult Danio rerio (zebrafish) females, purchased from a commercial dealer
(Acquario di Bologna), were kept in aquaria at 288C with oxygenated water.
Fish were fed four times daily, twice with commercial food (Vipagran) and
twice with Artemia salina. Eggs laid by parental fish were kept and grown to
approximately 6 mo of age to test the effects of the probiotic on reproductive
processes.
Two experimental groups were evaluated: a control group, which was fed a
commercial diet only, and a probiotic-treated group, which was fed the
commercial diet containing the lyophilized probiotic at a final concentration of
106 colony-forming units/g for 10 days. To exclude the possible effect of
feeding rate on reproduction and to focus only on possible probiotic effects on
reproduction, the fish were underfed (1.5%2% of body weight of commercial
diet per day). In addition, to avoid any possible external contaminations, both
groups (control and probiotic-treated) never received Artemia sp. during the 10
days of treatment. The experiment was conducted in triplicate, and for each
replicate tank, the final housing density was 15 females and 5 males. The
probiotic strain used was L. rhamnosus IMC 501, provided by Synbiotec s.r.l.,
and was mixed into the diet just before providing the food to the fish.
After 10 days of treatment, 10 females from each experimental group were
killed by a lethal overdose of anesthesia (500 mg/L of 3-aminobenzoic acid
ethyl ester [MS-222] buffered to pH 7.4; Sigma). Ovaries from each of the
experimental groups were teased into separate follicles using transfer pipettes
(Samco Scientific Corp.) without trypsinization. Therefore, follicles were
separated into different maturational stages according to their diameters as
determined with an ocular micrometer under a dissecting microscope. Stage IIIa
(diameter, 0.310.51 mm) and stage IIIb (diameter, 0.520.70 mm) follicles
were sampled for in vitro maturation, molecular, and FT-IR assays.
Procedures were performed in accordance with the guidelines on the
handling and training of laboratory animals by the Universities Federation for
2 Article 65
Animal Welfare (UFAW) and with the Italian animal welfare legislation (D.L.
116/92).
DNA Array
RNA extraction, fluorescent target labeling and microarray hybridiza-
tions. Total RNA was extracted from samples composed of 25 follicles (stage
IIIa or IIIb follicles, separately), were mechanically isolated from all
experimental groups using the Minikit RNAeasy (Qiagen) extraction kit
following the manufacturers protocol. Total RNA extracted was eluted in 25 ll
of RNase-free water. Total RNA was treated with DNase (10 IU at 378C for 10
min; MBI Fermentas), and final RNA concentrations were determined by
absorbance readings (optical density [OD]) at 260 nm using an ND-1000
(Nanodrop). RNA was further assessed for integrity with the 6000 Nano
LabChip assay (Agilent). Total RNA (100 ng) was converted into fluorescently
labeled Cy3 cRNA using the Low RNA Input Fluorescent Linear Amplification
Kit (Agilent). Fluorescent targets were purified to remove unincorporated
nucleotides using RNeasy (Qiagen). Absorbance (OD) at 260 nm was used to
quantify the cRNA concentrations, and absorbance at 550 and 650 nm was used
to measure the efficiency of Cy3 dye incorporation. An incorporation efficiency
of 9 pmol/lg or greater was deemed to be necessary before proceeding with
hybridization. The Agilent Zebrafish Oligo Microarray V1, containing 21 495
probes representing all existing predicted zebrafish genes from the most recent
public databases, such as RefSeq, Unigene, and TIGR Gene Index, was used.
Each array also contained quality-control probes for monitoring any
experimental variations. For each sample, 1 lg of fragmented cRNA was
hybridized to the zebrafish microarray in accordance with single-color Agilent
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hybridization protocols. Data were collected using an Agilent Microarray
Scanner and Feature Extraction Software V10.5.
Statistical and bioinformatic analysis of microarray data. Array data have
been deposited in the EBI ArrayExpress database (accession number pending).
Statistical analysis of our microarray experiment was carried out in three steps:
1) normalization of microarray data, 2) sorting of genes according to interest,
and 3) statistical analysis of pathways and gene ontology (GO) terms
represented by the sorted list of genes. For normalization of microarray data,
the Agilent Feature Extraction Software (v10.5) provides high-quality,
expression-level reports for Agilent microarrays. Nevertheless, these data must
be normalized to remove subtle biases due to variations in hybridization
conditions or, perhaps, manufacture. We normalized all samples simultaneous-
ly using a multiple-loess technique as described previously [23].
For sorting the genes according to interest, we borrowed ideas from Cole et
al. [24] and their software package Focus. The interest statistic reflects a
biologists understanding that a gene with a greater fold-change (in absolute
value) than other genes is potentially the more interesting one. Also, given two
genes with the same fold-changes, the gene with a higher expression level (and,
therefore, the higher absolute change) is the more interesting one.
For statistical analysis of GO terms, we note that the sorted list of genes
obtained in step 2 does not imply statistical significance for any individual
gene; however, it does contain information on which groups of genes are
responsible for the studied phenotype. We calculated P-values for groups of
genes by performing GO analysis (http://www.geneontology.org/). All GO
terms define groups of genes for which we calculate P-values. Because of the
hierarchical structure of the GO database (it is a directed acyclic graph in which
the nodes are GO terms), every term contains all the genes of its daughter
terms, and the daughter terms are not independent of the parent term(s). It is
therefore possible and, indeed, even likely that a term may appear to be
statistically significant because its parent or daughter term is statistically
significant. It is not clear, however, which term, or if both, should be reported.
The parent-daughter dependence poses a problem for a purists P-value
interpretation, but in practice, it does not matter whether the same biological
process gets reported multiple times. A heuristic method that eliminates the
problem of parent-daughter dependence on the GO graph was proposed
previously [25]. The parent-daughter dependence means the same biological
process may get reported multiple times, which makes it difficult to
meaningfully adjust the P-values for multiple testing. We find that a simple
Bonferroni adjustment is somewhat useful, even though it is very conservative.
Real-Time PCR
A total amount of 1 lg of RNA was used for cDNA synthesis, employing
iScript cDNA Synthesis Kit (Bio-Rad). PCRs were performed with SYBR
green method in an iQ5 iCycler thermal cycler (Bio-Rad). Triplicate PCR
reactions were carried out for each sample analyzed. The reactions were set on
a 96-well plate by mixing; each reaction mixture consisted of 2 ll of diluted
(1:10) cDNA and 10 ll of 23 concentrated iQ SYBR Green Supermix (Bio-
Rad) containing SYBR Green as a fluorescent intercalating agent, 0.3 lM of
forward primer, and 0.3 lM of reverse primer. The thermal profile for all
 L. RHAMNOSUS CONTROLS ZEBRAFISH FOLLICLE MATURATION
reactions was 3 min at 958C and then 45 cycles of 20 sec at 958C, 20 sec at
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,
showed a single peak.
Both actb and arp were used as housekeeping genes in each sample to
standardize the results by eliminating variation in mRNA and cDNA quantity and
quality. No amplification product was observed in negative controls, and no
primer-dimer formation was observed in the control templates. The data obtained
were analyzed using the iQ5 optical system software version 2.0 (Bio-Rad)
including GeneEx Macro iQ5 Conversion and genex Macro iQ5 files.
Modification of gene expression is represented with respect to the control
sampled at the same time as the treatment. The primer sequences, designed using
Primer3 (v. 0.4.0) software were as follows: lhr forward, 5 0 -ggcgaaggctagatgg
cacat-3 0 ; lhr reverse, 5 0 -tcgccatctggttcatcaata-3 0 ; inhb forward, 5 0 -caacttagatgga
cacgctg-3 0 ; inhb reverse, 5 0 -gtggatgtcgaggtcttgtc-3 0 ; mprb forward, 5 0 -
caacgagctgctgaatgtgt-30 ; mprb reverse,-5 0 gggccagttcagagtgagac-3 0 ; tgfb1 for-
ward, 5 0 -tcgctttgtctccaaggact-3 0 ; tgfb1 reverse, 5 0 -tgcaagagagttgccatttg-3 0 ; gdf9
forward, 5 0 -cgaccacaaccacctctctcc-3 0 ; gdf9 reverse, 5 0 -gggactgagtgctggatgcc-3 0 ;
pou5f1 forward, 5 0 -caaattacggcccaagctaa-30 ; pou5f1 reverse, 5 0 -cagaatcactg
catcctcca-3 0 ; actb forward, 5 0 -ggtacccatctcctgctccaa-3 0 ; actb reverse, 5 0 -
gagcgtggctactccttcacc-3 0 ; arp forward, 5 0 -ctgaacatctcgcccttctc; and arp reverse,
5 0 -tagccgatctgcagacacac-3. 0
Western Blot Analysis
For the Mprb assays, stage IIIa and IIIb follicles from each of the experimental
groups were electrophoresed and transferred into polyvinylidene fluoride as
described previously [26]. Briefly, 20 lg of each protein sample were separated
using a 4% stacking gel and a 10% separating SDS-PAGE and then electroblotted
onto a Bio-Rad filter using a Bio-Rad Mini Trans-Blot Electrophoretic Transfer
Cell. The transfer was carried out for 2 h at 250 mA at 48C using 25 mM Tris base,
192 mM glycine, and 20% methanol as electrode solution. The membrane was
soaked in 5% Nonidet-P40 for 1 h to remove SDS and then incubated overnight at
48C with 2% bovine serum albumin (Sigma) in PBS buffer. For the Mprb assay,
the primary antibody (anti-Mprb), diluted 1:1000, was kindly supplied by Dr.
Yong Zhu at the Department of Biology, East Carolina University, Greenville,
North Carolina, USA. Anti-b-tubulin antibody (l g/ml; Gene Tex, Inc.) was used
to normalize the sample loading. The antibody reaction was visualized with ECL-
PLUS (GE Healthcare) chemiluminescent reagent for Western blot. The
densitometric analysis was performed by ImageJ software (U.S. National
Institutes of Health, Bethesda, MD) for Windows.
In Vitro Follicle Maturation Assay
In vitro maturation assays were conducted as described previously [17, 27].
Briefly, stage IIIa and IIIb follicles derived from both control and probiotic-
treated groups were incubated in 1 ml of 60% Lebovitz L-15 Medium (Gibco,
Invitrogen) containing 200 lg/L of gentamicin (Sigma Aldrich) at 258C in the
dark in 24-well culture plates. Both stage IIIa and IIIb follicles isolated from
control and probiotic-treated ovaries were incubated separately in L15 and in
L15 plus MIH (1 lg/ml). The MIH used here was 17a,20b-dihydroxyproges-
terone (100 ng/ml; Sigma Aldrich). Maturation was scored after 18 h of
incubation. Follicles that underwent germinal vesicle breakdown (GVBD)
could be identified by their acquired translucency. Each experiment was
conducted in four wells, with approximately 20 follicles/well, and all
experiments were repeated three times.
PCR-DGGE Analysis of GI Tract and Ovarian Microbiota
At the end of the trial, six individual fish per tank were sampled aseptically
to isolate the ovaries and GI material. These samples were pooled by tank to
provide three samples of ovaries and three samples of the GI tract, each derived
from six individuals, for the assessment of probiotic populations and the impact
of such potential populations on indigenous microbial populations. DNA
extraction, PCR amplification of the 16S rRNA V3 region, and a 40%60%
DGGE were conducted as described previously [28]. A known sample of pure
L. rhamnosus culture was processed in the same manner to allow the
identification of the probiotic in the DGGE fingerprints. Selected bands of
interest, including the presumptive L. rhamnosus phylotype, were excised.
DNA was eluted from the excised bands in 50 ll of molecular-grade water at
48C overnight before a second PCR amplification. The PCR product was
cleaned (QIAquick PCR Purification Kit; Qiagen) and sequenced by GATC
Biotech. The nucleotide sequence (GenBank accession nos. JF431929 to
JF431934) were then submitted to a BLAST search in GenBank (http://blast.
ncbi.nlm.nih.gov/Blast.cgi) to retrieve the closest known alignment identities
for the partial 16S rRNA sequences.
3 Article 65
FT-IR Measurements
The FT-IR measurements were carried out at the infrared beamline SISSI@
ELETTRA synchrotron [29] using a Bruker VERTEX 70 interferometer
coupled with a Hyperion 3000 Vis-IR microscope and equipped with a liquid
nitrogen-cooled FPA detector (detector area, 64 3 64 pixels). Because of the
follicle dimensions, representative areas were chosen for IR imaging in
transmission mode at room temperature using a 153 condenser/objective (pixel
resolution, ;2.6 lm). Each IR image (OPUS 6.5 software package; Bruker),
acquiring simultaneously groups of 4096 spectra, was collected by averaging
128 scans for each detector pixel, with a spectral resolution of 4 cm1, rationing
the background single-channel image against the sample single-channel image.
All the samples were compared with independent trials. Total absorbance
cartograms, representing the total intensity of the infrared absorption, were
generated for each sample by integrating the total area between 1800 and 900
cm1 (not shown).
Average spectra were extracted from IR images by using OPUS 6.5, with
the number of selected spectra ranging from 4096 to 16 384. All the average
spectra were two-point baseline linear-fitted in the spectral range of 4000 to 900
cm1 and vector normalized [30].
To better evidence small changes observed in normal spectra, the second
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
subcomponents and to evaluate the corresponding absorbance intensity of a
single band. Using the GRAMS/AI 7.02 (Galactic Industries, Inc.) software
package, peak fitting was performed on average spectra (interpolated in the
range of 1775 to 1580 cm1 and two-point baseline linear-fitted); to identify the
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underlying component bands, the number of peaks together with their center
values were carefully individuated according to second derivative results and
fixed before running the iterative process to obtain the best reconstructed curve
(residual near zero). Mean values of areas and wavelength were calculated for
each component peak. Attribution of the bands was done as described in the
literature [3134].
For data handling, the following software packages were used: Opus 6.5,
Spectrum 6.3.1 (PerkinElmer), and GRAMS/AI 7.02.
Statistical Analysis
Gene expression, protein levels, and GVBD rate data are presented as the
mean 6 SD for all experiments. Significant differences between treatment
groups were determined with a factorial (one-way) ANOVA followed by
Bonferroni multiple-comparison test using the statistical software package
Prism5 (GraphPad Software, Inc.). Statistical significance was accepted at P ,
0.05.
The DGGE banding patterns were transformed into presence/absence
matrices for assessment between treatments using Quantity One software
(V4.6.3; Bio-Rad). Dice coefficients of similarity were calculated to
compare fingerprints, and band intensities were measured. Similarity
percentages (SIMPER), a one-way analysis of similarity (ANOSIM), and
a Bray-Curtis similarity matrix were used to represent the relative
similarities between treatments and replicates using Primer V6 [35]. The
species richness was assessed using the Margalef d measure of richness, and
species diversity was assessed using the Shannon-Weaver index. These
parameters were compared using an independent sample t-test (SPSS 17.0
software; IBM, Chicago, IL).
RESULTS
Stimulation of GVBD
In a series of four experiments, each replicated three times,
very few stage IIIa follicles isolated from control or probiotic-
treated females underwent spontaneous maturation (3% 6
1.11% and 5% 6 1.51%, respectively) after 18 h in the absence
of MIH. In contrast, a much higher proportion of control-
derived stage IIIa follicles underwent maturation (GVBD)
when exposed to MIH for 18 h (19.9% 6 1.7%), whereas a
significantly higher GVBD rate (41.8% 6 2.9%) was reached
by stage IIIa follicles isolated from probiotic-treated females
when exposed to MIH.
The stage IIIb follicles isolated from the ovaries of
probiotic-treated females incubated with MIH showed the
greatest rate (95.7% 6 4.1%) of GVBD (Fig. 1). In contrast,
 GIOACCHINI ET AL.

TABLE 2. List of Gene Ontology Terms from Biological Process and

Molecular Function that are altered significantly in stage IIIa follicles
treated with probiotic.
GO P Bonferroni
GO Terms ID value* P value

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

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control females incubated with MIH showed GVBD.
* The P values for groups of genes were calculated by performing Gene
Ontology (GO) analysis (htpp://geneontology.org).
GO Analysis
Numerous prominent and putatively regulated functions and samples, the GO analysis of Molecular Function also revealed
processes were revealed in the present study to be regulated by that gene families involved in transcription, heme binding,
L. rhamnosus during follicle development. In the control tetrapyrrole binding, and iron binding were modified (Table 1).
group, on going from stage IIIa to stage IIIb and, therefore, Comparing stage IIIa follicles from control versus probiotic-
during the normal progression of maturational competence, treated females, it was found that the probiotic administration
several genes involved in Biological Processes, such as modulated several genes belonging to Biological Processes
multicellular organismal development, organ and anatomical involved in the generation, transmission, reception, or
structure development, regulation of cell growth, and regula- interpretation of a signal (e.g., signal transmission, signal
tion of transcription, were modulated. In addition, in the same transduction, and response to chemical stimuli). Also interest-
ing is its effect in regulating transcripts associated with
TABLE 1. List of Gene Ontology Terms from Biological Process and development (regulation of anatomical structure size) and
Molecular Function that are altered significantly between untreated regulation of biological quality, which is involved in any
zebrafish stage IIIa and stage IIIb follicles. process that modulates the frequency, rate, or extent of a
GO P Bonferroni measurable attribute of an organism or part of an organism,
GO Terms ID value* P value such as size, mass, shape, and color (GO definition) (Table 2).
The GO analysis of Molecular Function in stage IIIa
Biological Process follicles revealed that L. rhamnosus regulated the expression of
Multicellular organismal development 7275 1.49E07 2.38E04 two main groups of genes: 1) those involved in the selective
Multicellular organismal process 32501 2.85E07 4.56E04
Developmental process 32502 3.27E07 5.23E04 interaction of a molecule with one or more specific sites on
Anatomical structure development 48856 3.54E07 5.66E04 another molecule, such as heme binding, tetrapyrrole binding,
System development 48731 4.65E06 7.44E03 iron ion binding, GTP binding, and guanyl ribonucleotide
Organ development 48513 7.70E06 1.23E02 binding; and 2) those involved in oxidoreductase activity and
Regulation of biosynthetic process 9889 1.19E05 1.91E02 mono-oxygenase activity (Table 2).
Regulation of cellular biosynthetic 31326 1.27E05 2.03E02 In stage IIIb follicles, both Biological Process and
process
Regulation of gene expression 10468 1.58E05 2.53E02 Molecular Function highlighted the regulation of many genes
Regulation of macromolecule 10556 2.02E05 3.23E02 involved in transcription, including regulation of transcription
biosynthetic process DNA-dependent (Biological Process) and transcription factor
Regulation of nitrogen compound 51171 2.40E05 3.83E02 activity, transcription regulator activity, and sequence-specific
metabolic process DNA binding (Molecular Function) (Table 3). Moreover, the
Regulation of cell growth 1558 2.83E05 4.52E02
Regulation of transcription 45449 2.84E05 4.55E02
Biological Process analysis also revealed a regulation of
Molecular Function transcripts involved in development: developmental process,
Transcription factor activity 3700 1.81E05 1.11E02 multicellular organismal development, anatomical structure
Heme binding 20037 2.10E05 1.29E02 development, and organ morphogenesis.
Transcription regulator activity 30528 2.49E05 1.53E02
Tetrapyrrole binding 46906 2.62E05 1.60E02
Iron ion binding 5506 4.71E05 2.89E02
Effects of L. rhamnosus Administration on lhr and inhbaa
Transition metal ion binding 46914 5.80E05 3.56E02 Gene Expression in Stage IIIa and IIIb Follicles
* The P values for groups of genes were calculated by performing Gene The quantitative measurements of lhr and inhbaa mRNA
Ontology (GO) analysis (http://www.geneontology.org/). are shown in Figure 2. Stage IIIa and IIIb follicles isolated from
4 Article 65
 L. RHAMNOSUS CONTROLS ZEBRAFISH FOLLICLE MATURATION

TABLE 3. List of Gene Ontology Terms from Biological Process and

Molecular Function that are altered significantly in stage IIIb follicles
treated with probiotic.
GO P Bonferroni
GO Terms ID value* P value

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).

Downloaded from www.biolreprod.org.

FIG. 3. mprb mRNA (top) normalized against actb and arp genes and
protein levels (bottom) normalized against b-tubulin 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).

probiotic-treated females showed significantly higher lhr and

inhbaa mRNA levels with respect to follicles isolated from the
control. In particular, the levels of these genes in stage IIIa
follicles from probiotic-treated females were similar to those
found in stage IIIb follicles isolated from control females.

Effects of L. rhamnosus Administration on mprb Gene

Expression and Protein Levels in Stage IIIa and IIIb Follicles
The stage IIIa and IIIb follicles isolated from probiotic-
treated females showed significantly higher mprb mRNA
levels compared to follicles isolated from control females. In
particular, stage IIIa follicles from probiotic-treated females
reached the same levels found in stage IIIb follicles isolated
from control females (Fig. 3).
Regarding the Mprb protein, as evidenced by Western blot
analysis, a similar trend of the gene expression was found.
However, the increase at protein levels was more modest in
respect to the gene expression (Fig. 3).

Effects of L. rhamnosus Administration on tgfb1 and gdf9

FIG. 2. lhr (top) and inhbaa (bottom) 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).
5 Article 65
Gene Expression in Stage IIIa and IIIb Follicles
The quantitative measurements of tgfb1 and gdf9 mRNA are
reported in Figure 4. Stage IIIb follicles isolated from
probiotic-treated females showed significantly lower tgfb1
mRNA levels with respect to follicles isolated from control
females. Regarding stage IIIa follicles, a significant decrease of
gdf9 gene expression was observed in follicles isolated from
probiotic-treated females. The levels were similar to those
found in stage IIIb follicles isolated from the controls.
 GIOACCHINI ET AL.

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).

Downloaded from www.biolreprod.org.

FIG. 4. tgfb1 (top) and gdf9 (bottom) 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 mean 6
SD (error bars). Different letters indicate statistically significant differences
(P , 0.05, ANOVA followed by Bonferroni multiple-comparison test).

Effects of L. rhamnosus Administration on pou5f1 Gene

Expression in Stage IIIa and IIIb Follicles
The quantitative measurements of pou5f1 mRNA are shown
in Figure 5. Stage IIIa and IIIb follicles isolated from probiotic-
treated females showed significantly higher pou5f1 mRNA
levels with respect to follicles isolated from control females. In
particular, the expression of this gene in stage IIIa follicles
from the probiotic-treated females was similar to that found in
stage IIIb follicles isolated from control females.

Effects of L. rhamnosus Administration on GI Tract and

Ovarian Microbiota
Lactobacillus rhamnosus was present in all replicates of the
probiotic-treated group and was absent in all replicates of the
control group. The presence of the probiotic had a clear and
distinctive effect on the microbial communities, causing a shift
in microbial fingerprints that is reflected by the dendrogram in
Figure 6; the replicates of the two treatments are clustered into
two distinct groups, characterized by low similarity of
approximately 50% between the groups. Despite the large
dissimilarity between the groups, no significant differences
were observed in terms of microbial community ecological
parameters (Table 4). However, trends toward differences were
observed in respect to microbial richness (P 0.105), evenness
(P 0.138), and diversity (P 0.112). Additionally, the
ANOSIM showed a trend toward significance (P 0.10), with
an R value of one (indicating that the replicates within the
FIG. 6. PCR-DGGE profiles and dendrogram of the GI microbial
groups are more similar to one another than they are to the populations of zebrafish. Each lane represents replicates of the control
replicates of the other group). (C1C3) or the probiotic-treated (P1P3) fish. Lane Lr represents the
The PCR-DGGE fingerprints revealed, as supported by the migration point of a known L. rhamnosus culture. The location of
high dissimilarity between the groups, that the impact of the sequenced phylotypes is also presented.

6 Article 65
 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
(bands, which represent presumed individual bacterial species/
strains) either by suppressing them (to nondetectable levels, in
some cases) or by stimulating the presence of certain
phylotypes that were absent or nondetectable in the other
group. A clear example was the low to undetectable levels of
the phylotype JF431931, identified as Streptococcus thermo-
in the range of 1775 to 1580 cm1, and the areas of the
component bands were calculated. In all specimens, it was
possible to distinguish intermolecular (1692 6 2 and 1615 6 2
cm1) from intramolecular (1679 6 1 and 1629 6 1 cm1) b
structures as well as three-turn helix (1667 6 1 cm1), a-helix
(1655 6 2 and 1646 6 2 cm1), and random coil (1639 6 1
cm1) components.

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philus (Table 5), from the control replicates, which was clearly
stimulated by the presence of the probiotic (Fig. 6). Other
phylotypes sequenced are displayed in Table 5. PCR-DGGE
analysis showed the ovaries to be devoid of L. rhamnosus;
indeed, these samples were devoid of any detectable bacterial
rRNA (data not shown).
Effects of L. rhamnosus Administration on Macromolecular
Building of Stage IIIa and IIIb Follicles
Average vibrational spectra of stage IIIa and IIIb follicles
(control and probiotic-treated groups) in the spectral range of
4000 to 900 cm1 and their corresponding second derivatives
(DII; five-point smoothing) in the range of 1800 to 900 cm1
are reported in Figure 7. The spectra corresponding to stage
IIIb follicles of both control and probiotic-treated groups were
similar, whereas changes were observed in spectra related to
stage IIIa follicles. In particular, modifications in amide I band
shape and in the bands associated with phosphate moieties
were found in the spectrum corresponding to stage IIIa oocytes
in the probiotic-treated group with respect to stage IIIa oocytes
in the control group.
The analysis of amide I, a convoluted band strictly related to
protein secondary structures, is considered crucial to approach
the proteic pattern of biological samples. In our case, because
the spectroscopic data were acquired on the whole follicles, this
band was affected by either cytoplasmic or membrane proteins.
To ascertain amide I component bands, the peak fitting
procedure (GRAMS/AI 7.02) was applied on average spectra
By analyzing the total areas relative to helical, b, and
random coil components, the following considerations were
drawn: First, in both groups, on maturation, helical and random
coil structures increased with respect to b structures. Second,
stage IIIa follicles derived from probiotic-treated females
showed helical and b structures with values intermediate
between those found in stage IIIa and IIIb follicles from the
control group. Third, stage IIIb follicles from both control and
probiotic-treated groups exhibited similar values for helical and
b structures. Fourth, the random coil moiety was always higher
in follicles of probiotic-treated females (Table 6).
On maturation, an increase in hydration was observed in
stage IIIa follicles of the probiotic-treated group, in which the
band at 1157 cm1 (mC-OH) was prevalent with respect to that at
1167 cm1 (mC-OP). In stage IIIa follicles of the control group,
an inverted trend was observed (Fig. 8).
The hydration increase observed in stage IIIa follicles of the
probiotic-treated group was also confirmed by the analysis of
the asymmetric and symmetric stretching modes of phosphates
at 1240 and 1085 cm1, respectively (DII spectra in the range
of 1275 to 1045 cm1) (Fig. 9). The asymmetric stretching
mode was composed by two bands at 1245 cm1 (non-
hydrogen-bonded) and 1236 cm1 (hydrogen-bonded): In stage
IIIa follicles isolated from control females, these two
components showed the same intensity, whereas in stage IIIa
follicles isolated from probiotic-treated females, the latter
component was prevalent. The same trend was also observed in
the symmetric stretching mode that, in the spectrum related to
stage IIIa follicles isolated from probiotic-treated females,

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)

OC1 JF431929 Uncultured bacterium 100% HQ420288

OC6 JF431930 Agrococcus sp. 97% EU372989
OC13 JF431931 Streptococcus thermophilus 100% HM059018
OC20 JF431932 Uncultured alpha proteobacterium 96% HM534257
Rhodobacter sp. 96% FJ906694
OC22 JF431933 Uncultured Brevundimonas sp. 100% HQ711915
Uncultured Caulobacter sp. 100% HQ711902
OC24 JF431934 Lactococcus plantarum 100% EF694029

7 Article 65
 GIOACCHINI ET AL.

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FIG. 7. Left) Average spectra of stage IIIa and IIIb follicles in the range of 4000 to 900 cm1. Right) Corresponding second derivatives (five-point
smoothing) in the range of 1800 to 900 cm1 (stage IIIa probiotic-treated [P], stage IIIb control [C], and stage IIIb P spectra were shifted along the y-axis; 
 control; , probiotic treated).

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

TABLE 6. Percentages of helical, b, and random coil structures in amide I

band (17751580 cm1) for IIIa and IIIb follicles from control and
probiotic-treated females.
Random
Follicles stages Helical (%) b (%) coil (%)

IIIa C 28.2 64.3 7.5

IIIb C 47.3 44.7 8.0
IIIa P 36.9 54.0 9.1 FIG. 8. Average spectra of stage IIIa and IIIb follicles (   , control; ,
IIIb P 45.3 47.8 9.6 probiotic treated) in the range of 1190 to 1130 cm1 (stage IIIb spectra were
shifted along the y-axis).

8 Article 65
 L. RHAMNOSUS CONTROLS ZEBRAFISH FOLLICLE MATURATION
FIG. 9. Second derivative spectra (five-point smoothing) of stage IIIa
control (C) and probiotic-treated (P) follicles in the range of 1275 to 1045
cm1 (   , control; , probiotic treated; stage IIIa P spectrum was
shifted along the y-axis).
processes and molecular functions modulated by L. rhamnosus
that may have been involved in the acquisition of maturational
competence.
The GO analysis revealed the modulation of genes
implicated in any biological process involved in the generation,
transmission, reception, or interpretation of a signal and in a
change in state or activity of a cell or an organism (e.g., in
terms of movement, secretion, enzyme production, or gene
expression) as a result of a stimulus that, in this case, was the
probiotic administration. After probiotic administration, we
found in stage IIIa follicles a modulation of several gene family
members involved in heme, tetrapyrrole, and ion binding,
which are modulated on going from stage IIIa to stage IIIb
during the normal progression of maturational competence. In
addition, probiotic treatment in stage IIIb follicles modulated
several genes belonging to Biological Processes and Molecular
Functions, such as multicellular organismal development and
process, anatomical structure development, and transcription
activity regulation, which also are modulated during normal
follicle development. These results can better explain the
mechanisms through which L. rhamnosus could stimulate still-
incompetent oocytes (stage IIIa) to become responsive to MIH,
inducing in the oocyte the prerequisite changes for the next
developmental stage and future embryonic stages.
The quantitative PCR results revealed that the probiotic
treatment induced lhr, inhbaa, and mprb levels and concom-
itantly reduced tgfb1 and gdf9 mRNA levels in stage IIIa
follicles in comparison with the control group. These
observations suggest that L. rhamnosus has the capability to
give advance acquisition of oocyte maturational competence by
modulating the gene and protein levels of some molecules that
have recently emerged as regulators of this delicate phase at
both endocrine and local/paracrine levels. The effect of LH on
the acquisition of oocyte maturational competence has been
9 Article 65
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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genes in the maturation pathway, including lhr and mprb [45].
The decrease in mRNA levels of these genes after probiotic
administration is in accord with previous results found in the
whole ovary [13].
In addition, pou5f1gene expression was evaluated in stage
IIIa and IIIb follicles, to our knowledge for the first time.
pou5f1 is a class V POU domain transcription factor expressed
in pluripotent embryonic and germ line cells. In mammals,
pou5f1 is critical to maintain pluripotency of the inner cell
mass and survival of germ cells [4648], and it is a key
regulator of embryonic development [49]. Recently, in mice, it
emerged that Pou5f1 was also involved on the molecular
events that govern the establishment of the developmental
competence of mouse oocytes [50, 51].
Patterns of pou5f1 mRNA and protein expression have been
well characterized during embryonic development in zebrafish
and medaka [52, 53] and in primordial germ cells and adult
gonads in medaka [53]; to our knowledge, however, the mRNA
levels of this gene have never been evaluated in follicles at
different stages of development. In control females of the
present study, the pou5f1 mRNA levels significantly increased
from stage IIIa to IIIb follicles, suggesting that this signal may
be involved in the acquisition of maturational competence. The
increase in pou5f1 mRNA levels in both stage IIIa and stage
IIIb follicles due to probiotic administration may be related to
enhanced fecundity as previously observed [1315] as well as
to advancing the acquisition of maturational competence.
For a deeper insight regarding the acquisition of matura-
tional competence due to probiotic administration, FPA FT-IR
imaging was applied to intact stage IIIa and IIIb follicles. FT-
IR imaging is a powerful technique to study the composition
and the macromolecular chemistry of cells and tissues,
providing a biochemical fingerprint of samples under investi-
gation, and it represents an objective and promising tool to
study oocyte building [16, 19, 22]. The use of the bidimen-
sional FPA detector was fundamental for the fast acquisition of
chemical images at high spatial resolution and good signal:-
noise ratio [54]. Using bidimensional IR detectors, a large
number of microspectra can be acquired, simultaneously
retrieving spatially resolved information on the biochemical
and structural features of the sample. In detail, 4096 micro-
spectra were collected in a sample area of 170 3 170 lm2 using
a 64- 3 64-pixel FPA detector, therefore achieving a pixel
 GIOACCHINI ET AL.
resolution of approximately 2.6 lm. The data obtained in the
current study showed significant differences between stage IIIa
follicles isolated from probiotic-treated females with respect to
controls.
In particular, the spectral data relative to the amide I band
(peak fitting procedure) pinpointed a modification of protein
secondary structures in follicles isolated from probiotic-treated
females with respect to those isolated from control females,
probably due to the de novo synthesis or uptake of proteic
components into the follicles [16, 22]. At the same time,
changes in the 1167/1157 cm1 band ratio were attributable to
an increase of the hydration process [16, 22], because under
normal conditions this process starts in late stage IIIb/IV
follicles, this indicates an early water uptake in stage IIIa
follicles from probiotic-treated females. This behavior was also
confirmed by the analysis of the asymmetric and symmetric
stretching modes of phosphates, at 1240 and 1085 cm1,
respectively, showing an increase in hydrogen bonding.
The microbiological results within the present investigation
indicate that the dietary application of the lactic acid bacteria
(LAB) L. rhamnosus was also able to elevate the abundance of
other indigenous LAB species, such as S. thermophilus.
Molecular analysis using PCR-DGGE revealed that the
probiotic was able to populate the GI tract and modulate the
microbial communities, causing a clear shift in those
communities. Analysis of the ovaries revealed that the
probiotic was not directly associated with the ovaries,
excluding a possible local, direct role of the probiotic in the
ovary and indicating that systemic effects were indirectly
mediated, most likely by host-microbe (e.g., probiotic and/or S.
thermophilus) interactions in the GI tract. These results are in
accordance with, and greatly support, data obtained in a
previous study [13], which offered evidenced that at both the
gut and the brain level, the probiotic treatment induced a
significant increase in leptin gene expression, a key hormone in
energy homeostasis and neuroendocrine functions. In that same
study, this increase was correlated with a significant rise of
kiss1, kiss2, and gnrh3 gene expression in the brain. Taken
together, these results could suggest that the probiotic may act
indirectly in the ovary by activating in the gut the synthesis of a
potent metabolic hormone, such as leptin, and that this
hormone (either alone or with other hormones) may represent
the link between the metabolic and reproductive systems in
which the probiotic may act. At the same time, the finding that
L. rhamnosus IMC 501 administration can modulate the
intraovarian signals, such as tgfb1 and gdf9, may suggest an
additional role of leptin directly on the ovary given that the
leptin receptor (lepr) is expressed on zebrafish ovary [55].
Future studies should elucidate if these changes are induced by
the presence of GI L. rhamnosus, elevated abundance of S.
thermophilus, or a combination of both.
In conclusion, the present results demonstrate the enhancing
role of L. rhamnosus on acquisition of maturational compe-
tence in zebrafish follicles, confirming the positive role of L.
rhamnosus in reproduction and representing an important step
in understanding of the mechanisms by which this functional
feed additive may act on follicle development.
ACKNOWLEDGMENT
Special thanks go to Francesca Maradonna, Chiara Piccinetti, Eugenia
Ricciardelli, and Silvia Falcinelli for their help at various stages of the
present study. Special thanks also go to Dr. Yong Zhu for kindly providing
the anti-Mprb, to James Sprague at the University of CaliforniaSan Diego
Biomedical Genomics Laboratory (BIOGEM) for Agilent microarray
processing, and Dr. Roman Sasik for help with the data analysis.
10
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