Jose Ricardo Figueiredo

In vitro reproductive biotechnologies show promise for fertility preservation but still face challenges, including oxidative stress from high oxygen tension, which impairs cell viability and development. Antioxidants have been widely explored to attenuate oxidative stress during culture. Among them, anethole, a plant‐derived phenylpropanoid, stands out for its promising properties. This review explores the mechanisms and applications of anethole in reproductive physiology and its potential to enhance in vitro reproductive systems. Findings indicate that anethole modulates key pathways and may improve outcomes in in vitro follicle culture, oocyte in vitro maturation and in vitro embryo culture. These insights support future research and the strategic inclusion of anethole in reproductive biotechnology protocols.

This study This study aimed to evaluate two conservation times (2h vs 6h) at 4 ºC of prepubertal goat testicles for subsequent slow freezing and 3D in vitro culture. Testicular fragments from six animals were preserved at 4 ºC for 2 or 6 h. After 2 or 6 h of conservation, the fragments were randomly fixed, constituting the control (CTR2; CTR6), cultured in vitro (IVC2; IVC6), frozen (SFZ2; SFZ6), or frozen and cultured in vitro (SFZIVC2; SFZIVC6). All fragments were analyzed by classical histology (morphology), immunohistochemistry (PCNA), immunofluorescence (β-catenin), PCRq-RT (Oct4, C- kit, Bax, Bcl-2), transmission (TEM), and scanning (SEM) electron microscopy, respectively. The histomorphological data showed that regardless of the conservation time, whether followed by cryopreservation or not, epithelial (Ealt) and nuclear (Nalt) alterations increased after 48 h of IVC (p<0.05). Immunostaining for β-catenin was lower (p < 0.05) in SFZIVC2 and SFZIVC6 fragments compared with IVC2 and IVC6, respectively. Oct4 expression in SFZ6 was lower than that in CTR6 (p < 0.05). Conversely, C-kit expression was similar among CTR2, CTR6, SFZ2, and SFZ6 fragments. Bax and Bcl-2 expression were also similar among IVC2, IVC6, SFZIVC2, and SFZIVC6 fragments. The Bax/Bcl-2 ratio in IVC2 and SFZIVC6 was higher than that observed in CTR2 and SFZ6, respectively. In conclusion, our results demonstrate that controlling the duration of testicular storage at 4 °C before slow freezing and/or in vitro culture directly affects the viability and quality of testicular tissue, thereby influencing the success of reproductive function preservation.

Graphical Understanding how to effectively isolate a large number of healthy follicles is essential for increasing success rates in ARTs, such as in vitro culture and cryopreservation. Late secondary (preantral) and early tertiary (antral) follicles isolated from the intermediary portion of equine ovarian tissue sliced at two different thicknesses (0.25 vs 0.50 mm) were characterized according to regional distribution (dorsal, medial, and ventral), morphofunctional features, and epigenetic profiles. After isolation via microdissection, follicles were evaluated for membrane integrity (trypan blue), viability (propidium iodide and Hoechst 33342), mitochondrial membrane potential (JC-1), reactive oxygen species (ROS; H2DCFDA), and histone trimethylation patterns (H3K4me3 and H3K9me3). Regardless of the ovarian tissue slice thickness or region, all isolated follicles had intact membranes, confirming the effectiveness of mechanical microdissection. The key findings from this study reveal that i) recovery of preantral and antral follicles within different ovarian regions is influenced by tissue slicing thickness; ii) the abundance of recoverable follicles varies among the ovarian regions; iii) preantral and antral follicle viability and ROS levels remain consistent between developmental stages, irrespective of tissue slicing thickness, although mitochondrial membrane potential is influenced by slicing thickness; iv) in antral follicles, viability and ROS levels vary depending on location within the ovarian regions; and v) epigenetic markers (H3K4me3 and H3K9me3) exhibit different patterns, depending upon follicular developmental stage. Altogether, these results demonstrate that optimal follicle isolation depends on tissue thickness and ovarian region and that viable, good-quality preantral and antral follicles can be successfully isolated from equine ovarian tissue. Lay summary: Follicles are the functional units of the ovary that contain and support the egg. Depending on the presence of a fluid-filled cavity called an antrum, follicles are developmentally classified as preantral (no antrum) or antral (antrum present). In this study, small preantral and antral horse follicles were removed with needles from ovarian tissue sliced at different thicknesses and classified by their location and other characteristics. The health of the harvested follicles was then examined in several ways (cell survival, toxic by-product levels, and certain types of gene control) to evaluate how slice thickness during follicle removal affects these structures. This study reports, for the first time, that tissue slice thickness influences the removal of late preantral and early antral follicles in horses and that the location of these follicles depends on their developmental stage and quality, with these follicles showing distinct levels of gene control.

This work investigated the effect of zinc oxide nanoparticles functionalized with curcumin (ZnO _(np) +CUR) supplementation during the in vitro maturation (IVM) of bovine oocytes on the in vitro embryo production and the cellular antioxidant response. A total of 1,625 cumulus-oocyte complexes (COCs) were cultured in the maturation medium in the absence (0 µM - control) or presence of different concentrations of ZnO _(np) +CUR (3 µM, 6 µM or 12 µM). After IVM, COCs were destined either to 1) in vitro embryo production or 2) analysis of reactive oxygen species production, superoxide dismutase (SOD) activity, catalase (CAT) activity and total antioxidant capacity (FRAP). The results demonstrated that the addition of 6 and 12 µM ZnO _(np) +CUR during in vitro maturation showed a higher rate of blastocyst production when compared to the control ( p < 0.05). However, only 12 µM ZnO _(np) +CUR treatment showed higher rates of embryo production when compared to 3µM ZnO _(np) +CUR treatment. Supplementation of IVM medium with 6 µM ZnO _(np) +CUR reduced ROS production ( p < 0.05) compared to control and 12 µM ZnO _(np) +CUR treatments. Also, the treatment containing ZnO _(np) +CUR at 12 µM had lower SOD activity after IVM than control treatment. In conclusion, the best outcome for in vitro embryo production was obtained when 6 and 12 µM ZnO _(np) +CUR was added during IVM of bovine oocytes. However, this improvement in in vitro embryo production was not associated with either the reduction of ROS production or SOD and CAT activities.

This study assessed the potential protective effects of Mangiferin (MANGI) at various concentrations (10, 50 and 100 μM) on follicular survival and activation, enzyme activity of catalase (CAT) and superoxide dismutase (SOD), as well as the relative mRNA expression for BAX, BCL2 and SOD in porcine ovarian tissue cultured in vitro for 48 h with and without chemical stress induced by 0.3 μg/mL doxorubicin (DOX). The results indicated that when MANGI was associated with DOX, regardless of the tested concentration, MANGI increased (p < 0.05) follicle survival when compared to DOX alone, with only MANGI 10 + DOX and MANGI 50 + DOX treatments reducing follicular degeneration to levels comparable with the culture control. Regarding antioxidant enzymes, all treated groups decreased (p < 0.05) SOD activity compared to cultured control, while MANGI 10 treatment showed lower (p < 0.05) CAT activity compared to DOX treatment. Compared to cultured control, the mRNA levels of SOD were reduced (p < 0.05) in the MANGI 100, MANGI 10 + DOX and MANGI 100 + DOX treatments. In conclusion, supplementing the culture medium with MANGI attenuated the toxicity induced by DOX during the in vitro culture of pig preantral follicles enclosed in ovarian tissue.

Ovarian tissue transplantation (OTT) has been suggested as an alternative to preserving female fertility in livestock species, as currently performed in women. The OTT technique has been tested as xenografting or autografting in different body sites and animal species. Currently, there are no reports available regarding the autotransplantation of ovarian tissue using the bovine model and also testing the effect of vascular endothelial growth factor (VEGF) on graft survival. This study evaluated the effects of ovarian tissue short‐term exposure to VEGF before heterotopic autotransplantation into a subcutaneous site (flank region) for 7 days in cattle. The initial finding was that after OTT and without pre‐exposure to VEGF, the ovarian grafts had 42% of normal preantral follicles, which indicates a substantial step forward for this technique in cattle. Furthermore, VEGF exposure actively facilitated the process of neoangiogenesis, the proliferation capability of the stromal cells for activation of the cell cycle, maintained the balance between types I and III collagen fibers, and reduced the total collagen of the grafted tissue. Moreover, ovarian fragments previously exposed to VEGF tended to have greater follicular density; however, a detrimental effect of VEGF on follicular morphology was noticed. In conclusion, this study marks a significant step forward in bovine OTT and provides a foundation for further investigations into the specific pathways, stages, and durations of VEGF exposure to unveil strategies for refining ovarian transplantation techniques in cattle and other species.

: In brief: Conditioned medium from Wharton’s jelly mesenchymal stem cells improved tissue and preantral follicle outcomes, preventing adverse effects of oxidative stress, apoptosis, and epigenetic changes. This study investigated the methylation patterns of H3K4me3 and H3K9me3, as well as the mRNA expression of genes encoding the epigenetic regulators KDM1AX1, KDM1AX2, and KDM3A in goat preantral follicles developed in vivo (Uncultured control) or after in vitro culture for 7 days in either the absence (α-MEM+) or presence of conditioned medium (α-MEM+ + CM) from Wharton’s jelly mesenchymal stem cells (WJ-MSCs). In the invivo setting, all follicular categories exhibited similar H3K4me3 and H3K9me3 patterns, and transcripts of KDM1AX1, KDM1AX2, and KDM3A were detected in all samples. During in vitro culture, α-MEM+ + CM enhanced several important aspects. It increased the percentage of normal growing follicles, oocyte diameters across all categories, stromal cell density, and the H3K4me3 methylation pattern in preantral follicles. Simultaneously, it decreased the levels of reduced thiols and reactive oxygen species in the spent media, diminished the presence of lipofuscin aggresomes, lowered granulosa cell apoptotic rates, and reduced the H3K9me3 methylation pattern in preantral follicles. In conclusion, the findings from this study provide compelling evidence that supplementing the in vitro culture medium (α-MEM+) with CM from WJ-MSCs has a protective effect on goat preantral follicles. Notably, CM supplementation preserved follicular survival, as evidenced by enhanced follicular and oocyte growth and increased stromal cell density when compared to the standard culture conditions in the α-MEM+ medium. Furthermore, CM reduced oxidative stress and apoptosis and promoted alterations in H3K4me3 and H3K9me3 patterns.

Oviduct fluid extracellular vesicles (oEV) are essential for periconceptional events. The presence of EV has already been identified in the oviduct fluid (OF) from mammalian species, except in caprine. Therefore, this study aimed to isolate and characterize the caprine oEV (coEV). Initially, in Experiment 1, coEV were isolated from the OF of either each animal individually or from a pool of three animals. In experiment 2, coEV were isolated during the follicular or luteal phases of the estrous cycle. The coEV were characterized by size distribution, polydispersity index (PDI), and zeta potential using dynamic light scattering (DLS) analysis, as well as, by transmission electron microscopy (TEM) and dot blotting (DB). Our results indicated that the physicochemical characteristics of the coEV were similar (P > 0.05), regardless of the isolation method (individual or pool). However, coEV collected during the luteal phase were larger (P < 0.05) than those during the follicular phase. The TEM showed spherical and cup-shaped particles, characteristic of exosomes. The DB revealed the presence of exosomal proteins involved in the biogenesis of coEV. In conclusion, it is possible to isolate and characterize coEV from a single caprine female and the estrous cycle phase influences the vesicles average size and PDI.