ZFIM 2019 | PISA
miRNAs as sperm
quality molecular markers in fish: effect of sperm quality on progeny
M.F. Riesco, D.G. Valcarce, J.M. Martínez-Vázquez, V. Robles
ABSTRACT
miRNAs are a conserved class
of small non-coding RNAs that control gene expression by inducing mRNA
degradation or by suppressing mRNA translation. These non coding RNAs have been
described as molecular markers of sperm quality and crucial factors during
early embryogenesis in mammals (Kotaja 2014). Sperm miRNA delivered in the
embryo are considered important regulatory elements for early embryo
transcriptional control and development. In mammals, some previous studies
suggest that specific miRNAs could be associated to sperm quality as (miR-200a,
miR-141 and 122-5p). Moreover, miR-let 7-d,
regulates the self-renewal of spermatogonial stem cells and spermatogenesis in
mouse. The aim of this work is to identify different miRNAs that could be used
as molecular markers of sperm quality in zebrafish and analyze their
contribution in the progeny. For this purpose, miR expression was analyzed in
two different zebrafish male groups selected by sperm motility (low and high
sperm motility). Moreover, sperm quality traditional analyses (concentration,
volume, kinetic parameters...), in vitro fertilization ability and methylation
status at different levels (global and dmrt1 promoter) were
analyzed in these groups. Our results confirmed significant differences in all
traditional parameters of sperm quality between groups as expected. However, no
differences were found when we analyzed global methylation status neither dmrt1 promoter
methylation. dmrt1 transcript was previously established as
molecular marker of good and bad breeders in zebrafish by our group. Different
miRNAs were significant upregulated in zebrafish bad breeders in respect to the
good ones (miR-122, miR-141-3p, miR-200a-5p). The modifications in these
miRNAs could contribute to transmission of certain phenotypes to the progeny
affecting fertilization success, early embryo development and malformation rate
according our data.
Acknowledgements
AGL2015-68330-C2-1-R project (MINECO-FEDER), Stolt Sea
Farm S.L. and staff from Planta de Cultivos El Bocal. PTA2016-11987-I contract (MINECO/FEDER) and AQUA-CIBUS
international net 318RT0549 (Cyted).
ZFIM 2019 | PISA
Correlation of the
effect of drug abuse with changes observed between reproductive perfomance in
zebrafish
M.F. Riesco, D.G. Valcarce, J.M. Martínez-Vázquez, A. Calderón-García, V.
González-Núñez, V. Robles
ABSTRACT
The use of different mRNAS,
potentially relevant in mature spermatozoa, as molecular markers of
reproductive performance was previously evaluated by our group in
testicular cells from good and bad zebrafish breeders. Our results allow us to
establish some molecular markers
in testes samples able to predict reproductive success and successful embryo
development. The alterations in the dopaminergic system after drug
addiction (morphine and cocaine) in zebrafish could have a direct
effect on reproduction and on male sperm quality, including molecular markers.
In this work we study the changes produced by drugs of abuse (cocaine and
morphine) on mRNAs described as markers for good reproductive performance by
qPCR. The set of mRNA previously described as molecular markers of breeding
performance in zebrafish are modified after drug treatments. Some of these
molecular markers of breeding performance, as fshb and myca, have
been affected in drug treatments correlating the effect of drug abuse with the
changes observed in those transcripts in bad breeders. bdnf could
be employed as molecular marker of drug effect in testes taking into account
the opposite effect observed between morphine and cocaine according to previous
studies in other species.
Acknowledgements
AGL2015-68330-C2-1-R and
AGL2015-68330-C2-2-R projects (MINECO-FEDER), Stolt Sea Farm S.L. and staff
from Planta de Cultivos El Bocal. PTA2016-11987-I contract (MINECO/FEDER) and AQUA-CIBUS
international net 318RT0549 (Cyted).
ZFIM 2019 | PISA
Development of a
treatment to improve male reproductive performance in teleost based on
probiotic administration
D.G. Valcarce, M.F.
Riesco, J.M. Martínez-Vázquez, V. Robles
ABSTRACT
A key issue for the
aquaculture sector is the improvement of reproductive performance of breeders
and the development of effective treatments to guarantee reproduction success.
The objective of this study was to analyse the effect of the ingestion of two selected
probiotics strains (Lactobacillus rhamnosus CECT8361 and Bifidobacterium
longum CECT7347) with antioxidant and anti-inflammatory properties on
zebrafish sperm samples in males showing initial intermediate-Iow spermatozoa
motility. The optimization of this approach on zebrafish is a model but results
may be transferred to other commercial teleost showing reproductive failure.
18 fish (six per experimental
group) were used in the study. All of them were tagged with Visible Implant
Elastomers (VIE) to track each animal in each keypoint in the experimental
design. Showing a total motility ≤ 60% was the inclusion criteria in
the population. Three groups were created, each one with a different feeding
regime: 1) Control, fed with a pelleted formulated diet; 2) MALTO; fed with the
formulated diet and maltodextrin (vehicle of the probiotics) and 3) PROBIO; fed
with the formulated diet and both probiotics mixture (10E9 CFU)
and maltodextrin. In each group, the quality of individual sperm samples was
evaluated at t=0 d and t=21 d after the beginning of the diet supplementation.
The temporal frame analysed was established in 21 days, corresponding to
zebrafish spermatogenesis.
Our results showed that the
ingestion of two probiotic lactic-acid bacteria (Lactobacillus rhamnosus CECT8361
and Bifidobacterium longum CECT7347) during a spermatogenesis
cycle in Danio rerio significantly improved sperm motility in
males. No statistical
differences were found regarding sperm kinetics or concentration, although a non-significant
tendency was observed in the number of cells per mL. The ingestion of the
microorganisms did not affect the animal weight. The results confirm the
suitable use of these strains as a treatment for sperm motility improvement in
teleost.
Acknowledments
Proyect AGL2015-68330-C2-1-R
(MINECO-FEDER), RED AQUA CIBUS CYTED 318RT0549,
PTA 201611987-I, Stolt Sea
Farm and staff from Planta de Cultivos El Bocal.
CRYOBIOLOGY 2018 | MADRID
Effect of sperm
quality and germ cell cryopreservation on DNA methylation pattern in teleosts
D.G. Valcarce, M.F.
Riesco, J.M. Martínez-Vázquez, V. Robles
ABSTRACT
Cryopreservation is a
technique commonly used for gene banking purposes in aquaculture. Molecular
modifications potentially produced by cryopreservation on key transcripts could
be relevant for fertilization success after cryopreservation. The reduction of
some transcripts or even the elimination of some of them as a consequence of
cryopreservation has also been reported by our group in different species. DNA
methylation is a major epigenetic modification that plays an important role in
regulating gene expression. Bisulphite sequencing analysis of CpG methylation
in the promoter of cxcr4b, pou 5f1, sox3 and vasa genes
in fresh and cryopreserved zebrafish genital ridges demonstrated that
cryopreservation produced an increase in methylation that could be correlated
with gene downregulation. When global methylation of teleost sperm samples
(from Danio rerio and Solea senegalensis) with
different qualities was analyzed using a commercial kit "epiJET DNA
Methylation analysis kit" that uses the MspI and HpaII restriction
enzymes, no significant differences were found, probably due to the high level
of methylation in sperm cells. To provide more precise information, we analyze
embryo methylation pattern after fertilization with different quality sperm
samples, in order to discard the possibility that molecular alterations that
could not be detected using this technique in the hypermethylated sperm cells,
could be altering embryo development after fertilization.
Funding: AGL2015-68330-C2-1-R
(MINECO-FEDER)
CRYOBIOLOGY 2018 | MADRID
Development of molecular markers for teleost breeder
selection previous to cryopreservation
M. F. Riesco, D.G. Valcarce, J.M.
Martínez-Vázquez, I. Martín, V. Robles
ABSTRACT
The availability of molecular
markers in sperm samples able to predict reproductive success and successful
embryo development it is highly valuable in reproductive management.
Cryopreservation could increase the presence of reactive oxygen species and
could decrease overall sperm quality after the process. Interestingly molecular
alterations have been also described in the spermatozoa after freezing-thawing
even when optimized cryopreservation protocols are used. For these reasons, the
use of optimal sperm samples for gene banking purposes it is relevant and
highly recommended. In this work we study the validity of different RNAm and
miRNA for their use as molecular markers for teleost sperm samples. We
used Danio rerio as a model species and Solea
senegalensis as an example of species with high commercial value. The
set of mRNA and miRNA derived from this study could be used as complementary
analysis to determined the molecular status of a specific sperm sample,
together with traditional analysis such as motility and viability.
Funding: AGL2015-68330-C2-1-R
(MINECO-FEDER)
ESDAR 2016 | LISBOA
Solea senegalensis
spermatozoa quality: are apoptotic cells and reactive oxygen species playing a
role in F1 reproductive failure?
D. G.Valcarce, O. Chereguini,
M. de la Hera, I. Martín, M. P. Herráez, I. Rasines,
C. Rodríguez and V. Robles
ABSTRACT
S.
senegalensis broodstock (F1
generation) presents a failure on spawning performance compared to
wild-captured counterparts. It is known that the problem relies on males in the
F1 generation. Courtship lack and low quality semen hinder the expansion of
sole aquaculture. The absence of courtship in F1 individuals leads to the use
of artificial fertilization protocols, which require sperm cryopreservation
prior to fertility trials. Cryopreservation can generate reactive oxygen
species (ROS) that could have a negative impact on spermatozoa function. The
aims of this study were: 1) to evaluate ROS in males born in captivity (F1) and
in wild individuals 2) to implement a selection method for optimal sperm
subpopulation recovery prior to cryopreservation. The percentage of positive
cells for dichlorofluorescein (DCF) and propidium iodide (PI) were determined
by flow cytometry in both groups males. The presence and distribution of H2O2
within the cells was analyzed by confocal microscopy. In order to select
non-apoptotic cell subpopulations magnetic activated cell sorting (MACS) was
used and YOPRO and caspases were determined in the recovered population.
Our results indicated a
decrease in viability (55%) and DCF+ cells (66%) in slow F1 spermatozoa.
Cryopreservation did not affect viability neither the presence of DCF+ cells in
samples from wild individuals but decreased viability in F1 samples. Confocal
studies demonstrated a colocalization of H2O2 with active mitochondria but also
with nuclear DNA. Finally, MACS significantly reduced the percentage of
apoptotic cells (54% and 75% removal in wild and F1 respectively) showing a
potential future application on aquaculture.
Funding: AGL2015-68330-C2-1-R
(MINECO-FEDER); AQUAGAMETE FA1205 COST Action; JCyL EDU1084/2012 and FSE