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.

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.

Effect of aquaporin 3 knockdown by RNA interference on antrum formation in sheep secondary follicles cultured in vitro.

SummaryThe objectives were to develop an effective protocol for transfection of ovine secondary follicles and to assess the effect of attenuating aquaporin 3 (AQP3) using a small interfering RNA (siRNA-AQP3) on antrum formation and follicular growth in vitro. Various combinations of Lipofectamine® volumes (0.5, 0.75 or 1.0 µl), fluorescent oligonucleotide (BLOCK-iT ™) concentrations (3.18, 27.12 or 36.16 nM) and exposure times (12, 14, 16, 18 or 20 h) were tested. The BLOCK-iT™ was replaced by siRNA-AQP3 in the transfection complex. Ovine secondary follicles were isolated and cultured in vitro for 6 days using standard protocols. Follicles were transfected on day 0 or 3 or on both days (0 and 3) and then cultured for an additional 3 or 6 days. As revealed by the fluorescence signal, the Lipofectamine®/BLOCK-iT™ complex (0.75 µl + 27.12 nM by 12 h of incubation) crossed the basement membrane and granulosa cell and reached the oocytes. In general, the rate of intact follicles was higher and the rate of antrum formation was lower in transfected follicles compared with control follicles. In conclusion, ovine secondary follicles can be successfully transfected during in vitro culture, and siRNA-mediated attenuation of AQP3 gene reduced antrum formation of secondary follicles.

Supraphysiological doses of nandrolone decanoate disrupts spermatogenesis but did not interfere on embryo rate.

The aim of this study was to investigate the effects of 10 mg/kg/week of nandrolone decanoate (DECA - Deca Durabolin®) on body composition, hormonal levels, spermatic parameters, redox status, and morphometric parameters of testicle and epididymis; furthermore, the fertility capacity of Wistar rats was measured thought in vitro fertilization (IVF). The animals (n = 16) were divided into two groups: control group (CTRL, n = 8), which received only vehicle composed by peanut oil and 10% of the benzoic alcohol and nandrolone decanoate group (DECA, n = 8), which received intramuscular injections of DECA for 8 weeks, both groups were treated for 8 weeks. The results demonstrate significative decrease in visceral fat, testosterone levels, and thiol content on epididymis, reduction on normal sperm parameters, and deleterious effect on testicles and epididymis tissue morphology showing reduction of germ height and luminal diameter on the DECA group. Thus, it can be concluded that high doses of nandrolone decanoate impairs male reproductive parameters.

Use of green microalgae Chlorella as a nutritional supplement to support oocyte and embryo production in goats.

This study aimed to evaluate the use of green microalgae as a nutritional supplement for oocyte and embryo production in goats. Two experiments were performed on adult goats to obtain oocytes (EVO; n = 14) and in vivo embryos (IVD; n = 14). In both, the donors were divided into control (n = 7) and Chlorella (n = 7) groups. All goats received a base diet, and donors were orally supplemented with Chlorella pyrenoidosa (CH) in the Chlorella groups. For EVO, donors received 10 g CH for 14 days, and for IVD, 20 g CH was given for six days before embryo recovery. In EVO and IVD, food intake in the CH group was comparatively low, and it showed relatively high subcutaneous adipose deposition. In addition, the CH group exhibited an increase in triglyceride, cholesterol, and plasma glucose levels. In IVD, a significant increase in peripheral glutathione peroxidase levels was noticed. In EVO, the CH group showed relatively large follicular size and an increase in intrafollicular levels of triglycerides, glucose, and glutathione peroxidase. No differences were observed in the oocyte collected, and CH oocytes showed a low intensity of MitoTracker fluorescence (MT). In IVD, the CH group had a high proportion of transferable embryos, and these structures exhibited high fluorescence intensities for MT and H2DCFDA probes. We concluded that under these conditions, CH did not enhance the quality of the recovered oocytes. However, a daily dose of 20 g CH improved the quality of embryos and stimulated their mitochondrial functionality.

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.

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.

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.

Powdered coconut water (ACP 406®) as an alternative base culture medium for in vitro culture of goat preantral follicles enclosed in ovarian tissue.

This study evaluated a powdered coconut water solution (ACP 406®) as a base culture medium on the in vitro survival and development of in situ goat preantral follicles. The ovarian fragments were either immediately fixed in Carnoy solution (non-cultured control) or individually cultured for 2 or 6 days. The following culture media (all containing 100 µg/mL penicillin and 100 µg/mL streptomycin) were evaluated: α-MEM (α-MEM alone, without additional supplementation); α-MEM+ (supplemented α-MEM); ACP (ACP®406 alone); or ACP+ (supplemented ACP®406). Additional supplementation includes: 1.25 mg/mL bovine serum albumin, 10 µg/mL insulin, 5.5 µg/mL transferrin, 5 ng/mL selenium, 2 mM glutamine, and 2 mM hypoxanthine. The endpoints (i) follicular morphology; (ii) development; (iii) estradiol production; and (iv) reactive oxygen species (ROS) were recorded. Data were analyzed using chi-square, Turkey, t-test or One-Way ANOVA. Differences were considered significant when P < 0.05. At day 2 of culture, a greater (P < 0.05) percentage of morphologically normal follicles was observed between ACP+ and ACP treatments. Moreover, at day 2 of culture, no hormonal difference (P < 0.05) was observed between ACP+ and both α-MEM treatments. At day 6 of culture when ACP and α-MEM treatments were compared the percentage of healthy follicles were similar (P > 0.05) among treatments. Overall, all treatments had lower primordial follicles (P < 0.05) accompany by greater developing follicles (P < 0.05) percentages than non-cultured control treatment, indicating primordial follicle activation. However, at day 6 of culture, the percentage of primordial follicle development were similar (P > 0.05) among the treatments. Likewise, no differences (P > 0.05) were observed for ROS production and follicular and oocyte diameters among treatments. Therefore, ACP+ has the equivalent efficiency to MEM+ in maintaining the survival and development of goat preantral follicles, representing an alternative plant-based low-cost culture medium for in vitro culture.

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.

Sheep Isolated Secondary Follicles Are Able to Produce Metaphase II Oocytes After Vitrification and Long-Term In Vitro Growth.

The vitrification of preantral follicles followed by in vitro growth (IVG) could be valuable to produce fertilizable oocytes. However, the meiotic resumption rates of oocytes cultured from vitrified secondary follicles (SF) have been reported as suboptimal. This study aimed to verify two base media (alpha modification of minimum essential medium, α-MEM, and tissue culture medium 199, TCM199) on vitrified SF regarding different requirements during IVG. Sheep ovarian fragments were divided in six groups: (1) Fresh groups (Control α-MEM and TCM199): SF without vitrification; (2) Follicle-Vitrified (Follicle-Vit α-MEM and TCM199): SF vitrified after isolation; and (3) Tissue-Vitrified (Tissue-Vit α-MEM and TCM199): SF vitrified enclosed in ovarian fragments and, subsequently, isolated. The isolated SF were submitted to IVG for 18 days. Thereafter, the recovered cumulus-oocyte complexes (COCs) underwent in vitro maturation (IVM) and evaluation of chromatin configuration. Follicular granulosa cells were analyzed for their gene expression of Bax, Bcl2, and Connexins (CX) 37 and 43. COCs from in vivo antral follicles were used as in vivo control. Data were analyzed by analysis of variance, Tukey, and chi-square tests. Differences were considered significant if p-value is <0.05. Follicle-Vit groups had higher (p < 0.05) percentage of antrum formation compared with Tissue-Vit groups. Vitrification did not affect (p > 0.05) oocyte diameter postmaturation. Oocytes from Follicle-Vit in α-MEM reached metaphase II stage after IVM. Gene expression for CX37, CX43, and Bax was lower in Tissue-Vit groups. For Bcl2, the gene expression was the opposite. In conclusion, during IVG for 18 days, maximal oocyte meiotic resumption was not negatively impacted by vitrification and was greatest for isolated SF using α-MEM as a medium.