Embryotoxicity of silica nanoparticles in the drug delivery of domperidone in zebrafish.

Domperidone is a dopamine D2 receptor inhibitor that stimulates pituitary gonadotropins. It is usually associated with synthetic GnRHa to promote spawning in fish. However, the route of administration used, intramuscular injection, can be quite stressful. Little is known about the effects of domperidone, as well as other routes. This study aims to evaluate the toxicity of domperidone encapsulated by silica nanoparticles in zebrafish embryos. The study involved four groups with three concentrations: 1. domperidone (DP) 0.0001, 0.0002 and 0.0004 mg/mL; 2. DP associated with silica nanoparticles (SiNPs) 0.0001 + 1.1, 0.0002 + 2.2 and 0.0004 + 4.4 mg/mL; 3. SiNPs 1.1, 2.2 and 4.4 mg/mL and 4. Control (E3), with four repetitions per group. Survival, teratogen and heart rate (HR) were evaluated over a period of 168 hpf. Survival was higher in DP + SiNPs treatment, HR was lower in treatment with 4.4 mg/mL of SiNPs, while treatment with 0.004 mg/mL of DP increased HR. This study demonstrated that the association of DP and SiNPs decreased the toxicity of both DP and SiNPs, demonstrating that this may be a viable alternative to reduce the possible cardiotoxic effects of DP.

How origin of ovaries influences the vitrification outcome of bovine ovarian tissue: effects of side of ovaries and corpus luteum.

Although cryopreservation of ovarian tissue has advanced greatly, it remains a challenge, and protocols should be optimized to handle the heterogeneous nature of ovarian samples. In an effort to address this factor, the present study evaluated the effects of corpus luteum (CL) and side of ovaries (right versus left) on cellular morphology and viability of vitrified bovine ovarian fragments in a closed system. The ovaries were categorized according to whether they had a CL and which side they were on, and then divided into six groups: 1) CL+ (with CL) group; 2) CL- (without CL) group; 3) right ovaries group; 4) left ovaries group; 5) fresh control group (ovaries without vitrification or culture that were not selected for CL or ovarian side) and 6) In vitro culture medium control group (non-vitrified ovaries that were not selected for the presence or absence of CL or side of the ovaries). The current study shows that the CL- and right groups had the greatest percentage of follicles with normal morphology compared to other vitrified-warmed groups. Furthermore, the levels of necrosis and tissue damage of the right cultured group were the lowest compared to other groups. It was shown that bovine ovarian tissues derived from right ovaries and ovaries without a corpus luteum can be functionally and morphologically preserved after vitrification. For the first time, the present study suggests that bovine ovarian tissue vitrification can be improved by considering the origin of the ovaries.

Addition of synthetic polymer in the freezing solution of mesenchymal stem cells from equine adipose tissue as a future perspective for reducing of DMSO concentration.

The regenerative therapies with stem cells (SC) has been increased by the cryopreservation, permitting cell storage for extended periods. However, the permeating cryoprotectant agents (CPAs) such as dimethylsulfoxide (DMSO) can cause severe adverse effects. Therefore, this study evaluated equine mesenchymal stem cells derived from adipose tissue (eAT-MSCs) in fresh (Control) or after slow freezing (SF) in different freezing solutions (FS). The FS comprise DMSO and non-permeating CPAs [Trehalose (T) and the SuperCool X-1000 (X)] in association or not, totalizing seven different FS: (DMSO; T; X; DMSO+T; DMSO+X; T+X, and DMSO+T+X). Before and after cryopreservation were evaluated, viability, colony forming unit (CFU), and cellular differentiation capacity. After freezing-thawing, the viability of the eAT-MSCs reduced (P< 0.05) in all treatments compared to the control. However, the viability of frozen eAT-MSCs in DMSO (80.3 ± 0.6) was superior (P<0.05) to the other FS. Regarding CFU, no difference (P>0.05) was observed between fresh and frozen cells. After freezing-thawing, the eAT-MSCs showed osteogenic, chondrogenic, and adipogenic lineages differentiation potential. Nonetheless, despite the significative reduction in the osteogenic differentiation capacity between fresh and frozen cells, no differences (P > 0.05) were observed among FS. Furthermore, the number of chondrogenic differentiation cells frozen in DMSO+X solution reduced (P<0.05) comparing to the control, without differ (P>0.05) to the other FS. The adipogenic differentiation did not differ (P>0.05) among treatments. In conclusion, although these findings confirm the success of DMSO to cryopreserve eAT-MSCs, the Super Cool X-1000 could be a promise to reduce the DMSO concentration in a FS.

As terapias regenerativas com células-tronco (CT) têm sido incrementadas pela criopreservação, permitindo o armazenamento celular. No entanto, os agentes crioprotetores (ACPs) penetrantes, como DMSO, podem causar efeitos adversos graves. Portanto, este estudo avaliou células-tronco mesenquimais equinas derivadas de tecido adiposo (CTM-TAe) in natura (Controle) ou após congelamento lento (CL) em diferentes soluções de congelamento (SC). As SCs compreendem DMSO e ACPs não permeáveis [Trealose (T) e o SuperCool X-1000 (X)] associados ou não: (DMSO; T; X; DMSO+T; DMSO+X; T +X e DMSO+T+X). Antes e após a criopreservação foram avaliados, viabilidade, unidade formadora de colônia (UFC) e capacidade de diferenciação celular. Após o congelamento-descongelamento, a viabilidade das CTM-TAe reduziu (P< 0,05) em todos os tratamentos em relação ao controle. Entretanto, a viabilidade das CTM-TAe congeladas em DMSO (80,3 ± 0,6) foi superior (P<0,05) às demais SC. Em relação às UFC, não houve diferença (P>0,05) entre células frescas e congeladas. Após congelamento-descongelamento, as CTM-TAe apresentaram potencial de diferenciação de linhagens osteogênicas, condrogênicas e adipogênicas. No entanto, apesar da redução significativa na capacidade de diferenciação osteogênica entre células frescas e congeladas, não foram observadas diferenças (P > 0,05) entre SCs. Além disso, o número de células de diferenciação condrogênica congeladas em solução de DMSO+X reduziu (P<0,05) em relação ao controle, sem diferir (P>0,05) das demais SCs. A diferenciação adipogênica não diferiu (P>0,05) entre os tratamentos. Em conclusão, embora esses achados confirmem o sucesso do DMSO para criopreservar CTM-TAe, o Super Cool X-1000 pode ser uma promessa para reduzir a concentração de DMSO.

Development of sheep secondary follicles and preservation of aromatase and metalloproteinases 2 and 9 after vitrification and in vitro culture.

The cryopreservation of secondary follicles (SF) is a promising alternative to preserve the reproductive potential both in humans and animals in situations in which the transplantation of ovarian tissue is not possible. The objective of the present study was cryopreserved SF isolated sheep. Beyond follicular morphology, viability and development, we investigated proteins related to steroidogenic function and basement membrane remodeling [metalloproteinases 2 (MMP-2) and 9 (MMP-9)] in fresh SF (FSF) and vitrified SF (VSF) followed by in vitro culture for 6 (D6) or 12 days (D12). The percentage of intact follicles, follicular and oocyte diameter of the VSF were lower than FSF on both days of culture (P < 0.05). The VSF viability was statistically reduced from D6 (95.5%) to D12 (77.3%) but did not differ from the FSF on both days (D6:96.2% to D12:86.5%). Antrum formation in the VSF (D6: 59.13%; D12: 79.56%) was significantly lower than the FSF (D6: 79.61%; D12: 92.23%). However, an increase in this percentage was observed from D6 to D12 in both groups. Aromatase showed stronger labeling on FSF D6 and VSF D12 compared to other treatments (P < 0.05). MMP-2 showed a similar pattern of labeling in FSF D6 and VSF D12, similarly to that observed in FSF D12 and VSF D6. MMP-9 was similar in FSF and VSF cultivated for 6 and 12 days. In conclusion, VSF are able to grow and develop during 12 days of in vitro culture and showed evidence of preservation of steroidogenic function and remodeling of the basement membrane.

Ovarian tissue features assessed in bovine fetuses after vitrification and xenotransplantation procedures.

Cryopreservation and transplantation of ovarian tissue are proposed methods for the restoration of endocrine function and reproductive potential. Therefore, this study aimed to evaluate the effects of vitrification and xenotransplantation on follicle viability, activation, stromal cell integrity, vascularization, and micronuclei formation. Bovine fetal ovaries were fragmented and assigned to the following groups: Fresh control (FC), ovarian fragments immediately fixed; Vitrified control (VC), ovarian fragments vitrified; Vitrified xenotransplanted (VX), ovarian fragments vitrified and xenotransplanted; and Fresh xenotransplanted (FX), ovarian fragments xenotransplanted. Ovarian fragments were grafted in female BALB/c mice and recovered after 14 days. Follicular viability was preserved (P > 0.05) in VC group. The rate of developing follicles was greater (P < 0.05) in the FX group compared to other groups. Follicular density was higher (P < 0.05) in the VC group than the FC, VX, and FX groups. A decrease (P < 0.05) of stromal cell density was recorded after vitrification (VC vs. FX). Blood vessel density decreased in VC, VX, and FX groups compared with the FC group, and blood vessel density was correlated with follicular viability (positively; P = 0.07) and developing follicles (negatively; P < 0.001). Both vitrification and xenotransplantation groups (VC, VX, and FX) had a greater (P < 0.05) number of cells with one MN compared to the FC group. In summary, our findings showed that both vitrification and xenotransplantation modified blood vessel, follicular and stromal cell densities, follicular viability and activation, and micronuclei formation in ovarian tissue.

Induced-damages on preantral follicles by withanolide D, a potent chemotherapy candidate are not attenuated by melatonin.

Withanolide D (WD) has been investigated as an antineoplastic drug. This study aimed to evaluate whether melatonin (MT) could attenuate toxic effects on preantral follicles enclosed in the ovarian cortex (experiment 1 - E1) or on isolated secondary follicles (experiment 2 - E2) exposed to WD. For E1, ovarian cortex was incubated for 48 h to: (1) α-MEM+; (2) α-MEM+ plus 6 µM WD; (3) α-MEM+ plus 3 mmol/L MT or (4) α-MEM+ plus WD and MT. For E2, secondary follicles were exposed for until 96 h in. (1) only to basic medium (α-MEM++/α-MEM++); (2) α-MEM++ plus 3 mmol/L MT (MT/MT); (3) α-MEM++ until 48 h, followed by more 48 h in 6 µM WD (α-MEM++/WD) or (4) a pre-exposure to MT for until 48 h, followed by more 48 h of exposure to WD plus MT (MT/MT + WD). The main results obtained showed that exposure to drugs caused damage to follicular morphology (WD or WD + MT) and diameter (WD) in the ovarian cortex or in isolated follicles. In pre-antral follicles in situ, ATM expression increased in the presence of WD, MT or association. As for the secondary follicles, ATM and γH2AX were immunostained in the granulosa and theca cells and oocytes in all treatments. TAp63α was immunostained in follicles included in the ovarian cortex and in isolated follicles. We conclude that melatonin did not provide protection and could have enhanced the toxic effect of WD to follicles surrounded or not by the ovarian cortex.

Vitrification of canine ovarian tissue using the Ovarian Tissue Cryosystem (OTC) device.

The present study evaluated the effect of Ovarian Tissue Cryosystem (OTC) on follicular morphology and density, as well as on stromal cell density of vitrified canine ovarian tissue. Canine ovarian fragments collected from adult female dogs in stages of the random oestrous cycle were fixed (FC, fresh control) or vitrified (VIT) with an OTC device. After vitrification and warming, the fragments were fixed for histological analysis. Overall, the mean percentage of normal pre-antral follicles decreased after vitrification procedure (FC: 74.5% ± 1.6% vs. VIT: 52.05% ± 1.5%). Although the rates of normal primordial (71.1% ± 1.8%) and secondary (0.7% ± 0.4%) follicles vitrified showed a reduction (p < .05), vitrification using OTC showed considerable preservation of follicles, when compared to the fresh control (81.1% ± 1.5% and 2.3% ± 0.6%, respectively). The mean follicular density was maintained after vitrification (FC: 199.65 ± 12.8 vs. VIT: 199.68 ± 10.8), whereas the stromal cell density decreased in the VIT group. Based on the results, we recommend the use of OTC for vitrification of canine ovarian tissue.

Alpha Lipoic Acid Supplementation Improves Ovarian Tissue Vitrification Outcome: An Alternative to Preserve the Ovarian Function of Morada Nova Ewe.

This study evaluated the effect of adding alpha lipoic acid (ALA) to the vitrification solution of sheep ovarian tissue on 7 days of in vitro culture or 15 days of xenotransplantion. ALA was used at two different concentrations (100 µM: ALA100 and 150 µM: ALA150). Ovarian tissue was evaluated by classical histology (follicular morphology, development, and stromal cell density); immunohistochemistry for forkhead box O3a (FOXO3a); Ki67 (cell proliferation); cluster of differentiation 31 (CD31); and alpha smooth muscle actin (α-SMA). Reactive oxygen species (ROS) levels in ovarian tissue, as well as malondialdehyde (MDA) and nitrite levels in the culture medium, were assessed. Similar percentage of morphologically normal follicles was found in the vitrified ovarian tissue in the presence of ALA100 or ALA150 after in vitro culture or xenotransplantation. Follicular development from all treatments was higher (P < 0.05) than the control group. Moreover, an activation of primordial follicles was observed by FOXO3a. Stromal cell density and immunostaining for Ki67 and CD31 were significantly higher (P < 0.05) in ALA150 vitrified tissue. No difference (P > 0.05) was found in α-SMA between ALA concentrations after in vitro culture or xenograft. ROS levels in the ovarian tissue were similar (P > 0.05) in all treatments, as well as MDA and nitrite levels after 7 days of culture. We concluded that the addition of ALA 150 is able to better preserve the stromal cell density favoring granulosa cell proliferation and neovascularization.

Equine ovarian tissue xenografting: impacts of cooling, vitrification, and VEGF.

Ovarian tissue transplantation methods using cooled and cryopreserved samples have been attractive options for fertility preservation in animal models and humans. The aim of this study was to evaluate the impact of previous exposure to cooling, cryopreservation, and VEGF on the overall efficiency of equine ovarian tissue after heterotopic xenotransplantation in mice. The end points evaluated were follicular morphology and development, follicular and stromal cell densities, angiogenesis (i.e. the density of new and mature blood vessels), collagen types I and III fiber densities, and total fibrosis. Ovaries of adult mares were harvested after ovariectomy, and ovarian fragments were xenografted in the i.p. wall of BALB nude mice. Ten types of treatments involving different combinations of cooling, cryopreservation, xenografting procedures, and VEGF exposure were compared. The novel aspect of this study was the use of equine ovarian tissue xenotransplantation in mice, challenging the fragments with different combinations of treatments. The main findings were (i) cooling but not cryopreservation was effective in preserving the follicular morphology, (ii) a greater percentage of developing follicles but lower follicular and stromal cell densities were observed after ovarian tissue engraftment, (iii) exposure to VEGF increased new and mature vessels in cryopreserved-transplanted tissue, and (iv) an appropriate balance in the collagen types I and III fiber ratio in cooling-transplanted tissue was observed after exposure to VEGF. This study contributes to advancing knowledge in the preservation of ovarian tissue after cooling-cryopreservation and transplantation aiming to be applied to genetically superior/valuable horses, livestock, endangered animals, and, possibly, humans. LAY SUMMARY: Due to ethical limitations involving humans, the female horse (mare) has recently emerged as an alternative model for reproductive comparisons with women to optimize fertility restoration using ovarian tissue transplantation techniques. This study determined if ovarian tissue from donor mares (n = 3), exposed or not to vascular endothelial growth factor (VEGF) before transplantation, better survives for 7 days after transplantation into mouse hosts (n = 12). Tissues submitted to different combinations of cooling, freezing, and transplanting treatments, along with control groups, were evaluated using the parameters morphology, development, the density of immature eggs (follicles), the density of supportive (stromal) cells, collagen protein proportions, and density of blood vessels. Frozen-thawed treatments had lower percentages of normal follicles. Exposure to VEGF increased blood vessel densities in frozen tissue and favored adequate collagen levels in cooled-transplanted treatments. In conclusion, VEGF exposure seems to be beneficial for mare ovarian tissue transplantation and warrants further investigation.

Sperm head morphometry and chromatin condensation are in constant change at seminiferous tubules, epididymis, and ductus deferens in bulls.

The aim of this study was to evaluate the sperm head morphometry and chromatin condensation at different regions of the reproductive tract in bulls. Sperm smears of seminiferous tubules (ST), epididymis head (EH), body (EB), and tail (ET), and ductus deferens (DD) were stained with toluidine blue. Afterwards, the sperm head morphometry and chromatin alteration types were evaluated by a computational image analysis. Overall, spermatozoa of ST had lower (P < 0.05) area (A), perimeter (P), width (W), length (L), ellipticity (E), and Fourier harmonics (F0, F1, and F2). The chromatin decondensation (CD) and heterogeneity (CH) were higher (P < 0.05) in the ST region and decreased (P < 0.0001) during the migration along the reproductive tract (ST - DD direction). Considering the factors extracted (Factors 1 and 2) by the principal component analysis, the parameters A, P, W, L, and F0 were responsible for ∼36% of the Factor 1, while the E, F0, F1, and anterior-posterior symmetry (APS) contributed ∼27% to Factor 2. Both, CD and CH were associated with Factor 1 in the EH and ET regions and Factor 2 in the ST. Also, a well-defined difference between sperm heads collected from the ST and DD regions was observed by canonical analysis. The distribution of each chromatin alteration type was recorded. The proportion of normal sperm was lower (P < 0.05) in ST compared to other regions. Moreover, the chromatin influenced the morphometry and sperm heads with whole chromatin alteration type showed a smaller (P < 0.05) A, P, W, L, and E. In summary, the epididymal maturation is important for chromatin compaction and final morphometry of the sperm head. Also, the identification and quantification of the sperm chromatin condensation in different regions of reproductive tract can be used as potential biomarkers to predict the fertility in bulls.

Blastocoel fluid removal and melatonin supplementation in the culture medium improve the viability of vitrified bovine embryos.

In this study, we investigated the effects of melatonin supplementation in the culture medium and blastocoel fluid removal (BFR) before vitrification on the quality and viability of in vitro-derived bovine embryos. After fertilization, presumptive zygotes were assigned to one of the following treatments: control, in vitro standard culture (IVC) medium; IVC + M10-9, IVC medium supplemented 10-9 M melatonin; or IVC + M10-9 BFR, IVC medium supplemented with 10-9 M melatonin plus BFR on day 7 (D7) of culture. D7 blastocysts were vitrified by the Cryotop method and, after 5 mo of storage, were warmed and incubated for an additional 72 h. The re-expansion rate was evaluated after 2 and 24 h, and the hatching rate was evaluated after 24, 48, and 72 h. At 72 h, the total number of cells (TNC); number of apoptotic cells (NAC); and expression of genes related to oxidative stress (HSPA5), cell metabolism (SLC2A3), cell repair (MSH6), placentation (KRT8 and PLAC8), and implantation (FOSL1) were assessed in the blastocysts. Less than 30% of the control blastocysts re-expanded until 2 h, whereas more than 85% of the IVC + M10-9 and IVC + M10-9 BFR blastocysts re-expanded (P < 0.05). The hatching rate of IVC + M10-9 BFR blastocysts increased at all time points (P < 0.05), reaching 66.8% at 72 h of incubation. The TNC was similar among treatments (P > 0.05), regardless of vitrification/warming and re-cultivation. The NAC:TNC was smaller for melatonin-treated blastocysts (P < 0.05). BFR increased HSPA5 (P = 0.0118) expression and did not affect SLC2A3, MSH6, KRT8, and FOSL1 expression (P > 0.05). In conclusion, melatonin (10-9 M) supplementation in the culture medium and BFR on D7 of culture increased the hatching rate 24, 48, and 72 h after warming of the vitrified embryos, indicating an improvement in cryotolerance.

Ultra-diluted Folliculinum 6 cH impairs ovine oocyte viability and maturation after in vitro culture.

This study investigated the effect of Folliculinum 6 cH on the oocyte meiosis resumption and viability rates, progesterone production and mitochondrial activity after in vitro maturation of cumulus-oocyte complexes (COCs) in sheep. Sheep ovaries were collected at a local slaughterhouse and COCs were recovered by slicing technique. The selected COCs were maturated in TCM199 (Control treatment), or control medium supplemented with 0.05% ethanol (v/v) (the vehicle of the homeopathic preparation - Ethanol treatment) or with Folliculinum 6 cH. After 24 h of in vitro maturation (IVM), oocytes were mechanically denuded and incubated with Hoechst 33342 and MitoTracker (0.5 µM) Orange CMTMRos for analysis of viability and chromatin configuration, and mitochondrial activity, respectively. The results showed that Folliculinum 6 cH addition increased oocyte degeneration and reduced meiotic resumption compared to the control (P < 0.05). Interestingly, the percentages meiotic resumption and oocyte maturation were lower in the Folliculinum 6 cH treatment compared to its vehicle (Ethanol treatment) (P < 0.05). On the other hand, when the treatments were compared, higher mitochondrial activity was observed in the Ethanol treatment (P < 0.05). In conclusion, contrary to its vehicle, the addition of Folliculinum 6 cH to the IVM medium promoted oocyte degeneration and affected negatively the mitochondrial distribution, impairing meiosis resumption.

Vitrification of caprine secondary and early antral follicles as a perspective to preserve fertility function.

The present study aimed to evaluate the structure, survival and development of isolated caprine (secondary-SEC and early antral-EANT) follicles, after vitrification in the presence of synthetic polymers and in vitro culture. Additionally, transzonal projections (TZPs) and p450 aromatase enzyme were evaluated. After isolation, SEC and EANT follicles were in vitro cultured for six days or vitrified. After one week, SEC and EANT follicles were warmed and also in vitro cultured for six days. Data revealed that the percentage of morphologically normal follicles was similar between fresh and vitrified follicles in both follicular categories and antrum formation rate was similar between fresh and vitrified SEC follicles. Fluorescence by calcein-AM did not show difference between fresh and vitrified (SEC and EANT) follicles, however, the trypan blue test showed low viability for vitrified follicles. The integrity of TZPs was not affected between fresh and vitrified SEC follicles, however, in vitrified EANT follicles, there were signs of TZPs loss. Regarding steroidogenic function, it was observed a positive staining for p450 aromatase enzyme in fresh and vitrified SEC and EANT follicles. It was concluded that SEC follicles seem to be more resistant to vitrification than EANT follicles, as shown by the trypan blue test and TZPs assay. Future studies may confirm this hypothesis, in order to consolidate the use of SEC and EANT follicles as an alternative to ovary cryopreservation.

Impacts of different synthetic polymers on vitrification of ovarian tissue.

Type and concentration of cryoprotective agents (CPAs) are important factors which influence the likelihood of a successful ovarian tissue vitrification outcome. In an attempt to address this factor, the present study was conducted to evaluate the impacts of different synthetic polymers (Supercool X-1000, Supercool Z-1000 and PVP K-12) on vitrification of bovine ovarian tissue. From each ovarian pair, fragments were recovered and immediately fixed for analysis (fresh control) or submitted to vitrification, either or not followed by in vitro culture for one or five days. Vitrification was performed using the ovarian tissue cryosystem (OTC) system. The ovarian tissues were intended for histological and viability analysis [Reactive oxygen species (ROS) production and degenerate cells assay (Ethidium homodimer-1)], as well as immunolocalization of AQP3 and AQP9 were measured. The results showed that during almost all the periods after warming, in treatment groups which contain polymer (X-1000, Z-1000 and PVP), the percentage of morphologically normal follicles was the highest in the X-1000 samples. Furthermore, post-thawed X-1000 group revealed stronger labeling for AQP9 in primordial and transitional follicles, when compared with others. However, morphology after cryopreservation did not correlate with follicle viability and function where the levels of degeneration and tissue damage of PVP K-12 group were lower in comparison with X-1000 group and only in PVP K-12 group, ROS level was similar to that of the fresh control group. We believe that in addition to permeating CPAs, the addition of one (Supercool X-1000) or maybe a combination (Supercool X-1000 and PVP K-12) of non-permeating polymers could be useful to improve the outcome for vitrified bovine ovarian tissue.

Use of synthetic polymers improves the quality of vitrified caprine preantral follicles in the ovarian tissue.

The aim of this study was to evaluate whether the addition of synthetic polymers to the vitrification solution affected follicular morphology and development and the expression of Ki-67, Aquaporin 3 (AQP3) and cleaved Caspase-3 proteins in ovarian tissue of the caprine species. Caprine ovaries were fragmented and two fragments were immediately fixed (Fresh Control) for morphological evaluation, while other two were in vitro cultured for 7 days (Cultured Control) and fixed as well. The remaining fragments were distributed in two different vitrification groups: Vitrified and Vitrified/Cultured. Each group was composed of 4 different treatments: 1) Sucrose (SUC); 2) SuperCool X-1000 0.2 % (X-1000); 3) SuperCool Z-1000 0.4 % (Z-1000) or 4) with polyvinylpyrrolidone K-12 0.2 % (PVP). Also, Fresh Control, Cultured Control, SUC and X-1000 were destined to immunohistochemical detection of Ki-67, AQP3 and cleaved Caspase-3 proteins. Morphologically, the treatment with X-1000 showed no significant difference with the Fresh Control group and was superior to the other treatments. After the cleaved caspase-3 analysis, X-1000 showed the lowest percentages of strong immunostaining while Cultured Control showed the highest. Also, a positive correlation was found between the percentages of degenerated follicles and the percentages of strong staining intensity follicles. Regarding the AQP3 analysis, the highest percentages of strong AQP3 staining intensity were found in X-1000. In conclusion, we have demonstrated that the addition of the synthetic polymer SuperCool X-1000 to the vitrification solution improved the current vitrification protocol of caprine ovarian tissue.

Response of preantral follicles exposed to quinoxaline: A new compound with anticancer potential.

The culture of preantral follicles as an in vitro model to evaluate the toxicity of new anticancer drug has being established. Therefore, the aim of this study was to evaluate the effect of quinoxaline derivative the 2 2- (XYZC 6H 3 -CH=N-NH)-quinoxaline, 1 (QX) on caprine preantral follicles. We evaluate the follicular morphology and activation, proliferation and apoptosis of granulosa cells and finally the protein (ABCB1) and genes expression (cyclin/Cdks), respectively involved in multidrug resistance and cell cycle progression. Ovarian fragments containing primordial and developing follicles were exposed (in vitro culture) to different concentrations of QX (QX1.5, QX3.0 or QX6.0 µM/mL) during 6 days. To evaluate the effect of QX, the ovarian tissue was exposed to Paclitaxel 0.1 µg/mL (PTX - negative control) or in culture media without QX (MEM). At the end of exposure time, we realized that the QX (all concentrations) increased (P < .05) the normal morphology of preantral follicles compared to control (not treated ovarian tissue) or MEM. However, QX6.0 showed a enhanced (P < .05) on follicular activation (burnout) and apoptosis than QX1.5 and QX3.0. Expression of ABCB1 was similar between QX1.5 and QX6.0 and both were lower than control, MEM and PTX. Interestingly, the apoptosis rate in QX3.0 was similar to control and MEM and lower then QX1.5; QX6.0 and PTX. We conclude that quinoxaline may be a promising chemotherapeutic agent, however, other concentrations within a defined range (2-5.5 µM) could be widely investigated.

Early ovine preantral follicles have a potential to grow until antral stage in two-step culture system in the presence of aqueous extract of Justicia insularis.

The objective of this study was to determine whether preantral follicles cultured in vitro for 7 days within ovine ovarian cortical strips could be isolated at the secondary follicles (SF) and grown until antral stage during an additional 6 days period of in vitro culture in the presence of aqueous extract of Justicia insularis. Fresh ovarian fragments from 16 adult sheep were fixed for histological analysis (Control 1) or in vitro cultured individually in α-MEM+ supplemented with 0.3 mg/ml J. insularis (Step 1) for 7 days. Part of the fragments then were fixed for histological analysis (in vitro culture group). Remaining fragments were exposed stepwise to increasing trehalose concentrations before immediate isolation of SF and viability assessment (Control 2) or after 6 days of culture in α-MEM++ supplemented with 0.3 mg/ml J. insularis (Step 2). In Step 1, percentage of follicular activation was 80%. In Step 2, a significant increase (p < 0.05) in follicular diameter and antrum formation within 6 days in vitro culture of isolated follicles was achieved. The total antioxidant capacity from both steps significantly increase (p < 0.05) from day 2 to day 6. Confocal analysis of oocytes showed 57.14% oocytes with homogeneous distribution and 42.86% with peri-cortical distribution. In conclusion, SF can be successfully isolated from sheep ovarian cortex after 7 days of culture and are capable of surviving and forming an antral cavity if cultured in vitro for an additional 6 days in the presence of 0.3 mg/ml J. insularis.

Three-dimensional levitation culture improves in-vitro growth of secondary follicles in bovine model.

RESEARCH QUESTION: Does a three-dimensional culture system based on magnetic levitation with nanoparticles assembly maintain the follicular structure and viability with adequate growth rates leading to oocyte maturation after long-term culture? DESIGN: Randomized-controlled trial of treatments in a bovine model. Secondary follicles (n = 213) isolated from bovine ovaries were cultured in a two-dimensional system (two-dimensional control) or three-dimensional levitation system with different concentrations (three-dimensional 50 µl/ml, 100 µl/ml and 200 µl/ml) of magnetic nanoparticles. Follicular growth (diameter, daily growth and growth patterns), morphology (normal, degenerated and extruded follicles), antrum formation, oocyte viability and chromatin configuration were assessed. RESULTS: Secondary follicles of three-dimensional 200-µl/ml treatment showed higher viability, antrum formation and lower degeneration rates than two-dimensional control. Also, follicles cultured in the three-dimensional 200-µl/ml treatment presented a most homogenous daily growth rate as shown by the lowest variance and standard deviation. Compared with the two-dimensional control, the proportion of non-growing and slow-growing follicles were 3.8-fold lower and 1.6-fold higher, respectively, in the three-dimensional 200-µl/ml treatment. After in-vitro maturation, the three-dimensional 200-µl/ml had a greater proportion of viable oocytes (1.7-fold) and meiotic resumption rates (2.4-fold) than the two-dimensional control treatment. CONCLUSION: The three-dimensional levitation culture system improves the viability of in-vitro development of bovine secondary follicles, antrum formation and lower extrusion and degeneration rates and adequate growth rate leading to relevant oocyte viability and meiotic resumption after in-vitro maturation. This approach does not require a specific medium, and has the potential as an alternative method to in-vitro follicle culture in several species, including humans.

Xenotransplantation of goat ovary as an alternative to analyse follicles after vitrification.

The aim of this study was to evaluate the caprine preantral follicles enclosed on vitrified/warmed ovarian cortex grafted to nude BALB/mice during 1 month. The ovarian cortex from goats was fragmented (3 × 3 × 0.5 mm) and divided into four groups: fresh control, vitrified control, fresh transplant and vitrified transplant. Follicular morphology, development and density, fibrosis as well as apoptosis, and tissue revascularization were evaluated. It was also observed a significant decrease in morphologically normal preantral (primordial, transition, primary and secondary) follicles in both vitrified control and vitrified transplant treatments when compared with both fresh control and fresh transplant. However, fresh control and fresh transplant exhibited a similar percentage of developing follicles. Additionally, Vitrified control showed a significant increase in developing follicles in comparison with both fresh control and fresh transplant. Follicular density significantly decreased in all treatments in comparison with fresh control. We observed high fibrosis in both fresh transplant and vitrified transplant. The mRNA expression of caspase 3 was lower in both fresh transplant and vitrified transplant in comparison with vitrified control. In conclusion, xenotransplantation is an excellent strategy to maintain normal preantral follicle morphology after vitrification/warming of goat ovarian tissue. Yet, in order to ensure the survival and development of these follicles, it is essential to improve the revascularization of the graft.

In vitro study of Withanolide D toxicity on goat preantral follicles and its effects on the cell cycle.

The Withanolide D is a chemotherapeutic potential against the human tumor cell. However, there is no report on the effect of this compound on ovarian function, especially on preantral folliculogenesis. The aim of this study was to evaluate the toxicity of a new candidate to anticancer drug, Withanolide D (WD) on morphologic integrity, development (activation and granulosa cell proliferation) and gene expression of ABCB1 protein of caprine preantral follicles. Ovarian fragments were cultured in vitro for 2 or 6 days in α-MEM or α-MEM added with paclitaxel (PTX -0.1 µg/mL; negative control) and different concentrations of WD (WD1.5, WD3.0 or WD6.0). The higher dose of WD showed a toxic effect similar to PTX and higher (P < 0.05) than other treatments after 2 and 6 days. In addition, WD6.0 reduced the cell proliferating compared to PTX or mild dose. The expression of ABCB1 remained unchanged in the presence of the chemotherapeutic agents (PTX and WD) throughout the culture period. In conclusion, WD exerted a toxic effect observed by decreasing follicular survival and cell proliferation, on the preantral caprine follicles similar to PTX, whose negative effect on folliculogenesis is already widely known.