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Research Project
The imprinted characteristics of spermatogonia antioxidant capacity and their role in oxidative stress tolerance
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Post-thaw quality assessment of testicular fragments as a source of spermatogonial cells for surrogate production in the flatfish Solea senegalensis
Publication . Cabrita, Elsa; Pacchiarini, Tiziana; Fatsini, Elvira; Sarasquete, Carmen; Herráez, María Paz
Cryopreservation of germ cells would
facilitate the availability of cells at any time allow ing the selection of donors and maintaining quality
control for further applications such as transplan tation and germline recovery. In the present study,
we analyzed the efciency of four cryopreservation
protocols applied either to isolated cell suspensions
or to testes fragments from Senegalese sole. In tes tes fragments, the quality of cryopreserved germ
cells was analyzed in vitro in terms of cell recovery,
integrity and viability, DNA integrity (fragmentation
and apoptosis), and lipid peroxidation (malondialde hyde levels). Transplantation of cryopreserved germ
cells was performed to check the capacity of cells to
in vivo incorporate into the gonadal primordium of
Senegalese sole early larval stages (6 days after hatch ing (dah), pelagic live), during metamorphosis (10
dah) and at post-metamorphic stages (16 dah and 20
dah, benthonic life). Protocols incorporating dimethyl
sulfoxide (DMSO) as a cryoprotectant showed higher
number of recovered spermatogonia, especially in
samples cryopreserved with L-15 + DMSO (0.39 ±
0.18 × 106
cells). Lipid peroxidation and DNA frag mentation were also signifcantly lower in this treat ment compared with other treatments. An important
increase in oxidation (MDA levels) was detected
in samples containing glycerol as a cryoprotectant,
refected also in terms of DNA damage. Transplan tation of L-15 + DMSO cryopreserved germ cells
into larvae during early metamorphosis (10 dah, 5.2
mm) showed higher incorporation of cells (27.30 ±
5.27%) than other larval stages (lower than 11%).
Cryopreservation of germ cells using testes fragments
frozen with L-15 + DMSO was demonstrated to be
a useful technique to store Senegalese sole germline.
Methodological approaches for spermatogonia population enrichment and effect of cryopreservation on Senegalese sole spermatogonia quality
Publication . Almeida, Maria Mafalda Amaral Fonseca de; Cabrita, Elsa; Fatsini, Elvira
Senegalese sole (Solea senegalensis) is an emerging species for aquaculture that shows reproductive issues in aquaculture conditions. The transplantation of cryopreserved spermatogonia into closely related species allows the production of surrogated broodstocks. However, the cryopreservation of spermatogonia might cause damage on the quality of spermatogonia, meaning quality assays must be performed. For these assays, it is important to perform cell enrichment protocols to achieve only pure spermatogonia population. Therefore, the aim of this project was to develop an efficient protocol for Senegalese sole spermatogonia enrichment and observe how the cryopreservation protocol affects these germ cells. For this purpose, two experiments were performed using fresh and cryopreserved using dimethyl sulphoxide (DMSO) as cryoprotectant solution. In experiment 1, cells were cultivated in several extracellular matrixes (uncoated, collagen, gelatine and laminin) to perform differential plating for cell enrichment. The results showed that gelatine and collagen plates showed higher spermatogonia presence, however due to the low percentage of spermatogonia recovery obtained this technique was considered unfit for the target species. Afterwards, serial strainers were used to observe that the appropriate size was the 5 μm strainer, which contained higher percentage of spermatogonia recovery. Then, two 5 μm strainers were used and showed slightly better and promising results. This method was validated by RT- PCR where three relevant spermatogonia markers (gfra1, pou5f1/oct4 and nanos2) were up regulated in the samples from the strainers, yet no significance difference was observed. In experiment 2, effect of cryopreservation on the quality of spermatogonia was evaluated where the results showed that viability, DNA integrity and epigenetic were affected by cryopreservation. No difference in lipid peroxidation was observed. Future studies should focus on the limitations of the enrichment techniques to improve this method and improving the cryopreservation protocol to decrease the damaged suffered by cells during this technique.
Cryopreservation did not affect spermatogonia global methylation profile in Senegalese sole (Solea senegalensis)
Publication . Almeida, Maria Mafalda; Cabrita, Elsa; Laizé, Vincent; Brionne, Aurélien; Labbé, Catherine; Fatsini Fernández, Elvira
Spermatogonia cryopreservation is a method to preserve valuable genomes from both maternal and paternal origin. The damage associated with the application of this technology on post-thaw cell quality is important to assess, including at the epigenetic level. This study aimed to assess post-thawed spermatogonia quality by evaluating alterations in plasma membrane integrity, DNA integrity (fragmentation and apoptosis), lipid peroxidation (malondialdehyde levels) and epigenetic modifications (DNA methylation profile). We observed that plasma membrane integrity (fresh 78.98 % f 5.66; cryopreserved 62.81 % f 3.25; P = 0.003) and DNA integrity (fresh 32.95% f 2.28; cryopreserved 37.28% f 1.87; P = 0.0026) were affected by cryopreservation, while no difference in lipid peroxidation was observed (fresh 1.13 % f 0.45; cryopreserved 0.91 % f 0.96; P = 0.701). While global levels of DNA methylation were unaffected by cryopreservation (fresh 82.80 % f 0.47; cryopreserved 83.32 % f 0.81; P = 0.745), some differentially methylated cytosines (DMC) were observed in cryopreserved versus fresh spermatogonia (156 DMC). This study showed that spermatogonia cryopreserved according to our protocol provides a good supply of undamaged cells for several applications. The significance of the few detected DMCs deserves further attention since it may affect gamete differentiation and epigenetic profile.
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Funding agency
Fundação para a Ciência e a Tecnologia
Funding programme
3599-PPCDT
Funding Award Number
EXPL/CVT-CVT/0305/2021