Primordial follicles, the main source of oocytes in the ovary, are essential for
the maintenance of fertility throughout the reproductive lifespan. To the best
of our knowledge, there are no reports describing the effect of anethole on this
important ovarian follicle population. The aim of the study was to investigate
the effect of different anethole concentrations on the in vitro
culture of caprine preantral follicles enclosed in ovarian tissue. Randomized
ovarian fragments were fixed immediately (non-cultured treatment) or distributed
into five treatments: α-MEM+ (cultured control), α-MEM+
supplemented with ascorbic acid at 50 μg/mL (AA), and anethole at 30 (AN30), 300
(AN300), or 2000 µg/mL (AN2000), for 1 or 7 days. After 7 days of culture, a
significantly higher percentage of morphologically normal follicles was observed
when anethole at 2000 μg/mL was used. For both culture times, a greater
percentage of growing follicles was observed with the AN30 treatment compared to
AA and AN2000 treatments. Anethole at 30 and 2000 µg/mL concentrations at days 1
and 7 of culture resulted in significantly larger follicular diameter than in
the cultured control treatment. Anethole at 30 µg/mL concentration at day 7
showed significantly greater oocyte diameter than the other treatments, except
when compared to the AN2000 treatment. At day 7 of culture, levels of reactive
oxygen species (ROS) were significantly lower in the AN30 treatment than the
other treatments. In conclusion, supplementation of culture medium with anethole
improves survival and early follicle development at different concentrations in
the caprine species.
The aim of the present study was to evaluate the development of fresh and vitrified agouti ovarian tissue after xenografting to C57Bl/6 severe combined immunodeficiency (SCID) female mice. Ovaries were obtained from five female agoutis and divided into 16 fragments. Five fragments were transplanted immediately to ovariectomised SCID mice and the others were vitrified, stored for 2 weeks and transplanted only after rewarming. Tissue fragments were transplanted under the kidney capsule in recipients. The return of ovarian activity in recipients was monitored by the observation of external signs of oestrus and vaginal cytology over a period of 40 days after transplantation, after which the grafts were removed and evaluated for morphology, cell proliferation and the occurrence of DNA fragmentation. Ovarian activity returned in four of five mice that received fresh ovarian tissue from agoutis and in one of six mice that had received vitrified tissue a mean (±s.e.m.) 20.6±8.6 days after xenotransplantation. After graft removal, a predominance of primordial and primary follicles was observed in all grafts. Vitrification reduced cell proliferation and increased the occurrence of DNA fragmentation in grafted agouti ovarian tissue. In conclusion, the present study demonstrates that xenografted agouti ovarian tissue, fresh or vitrified, is able to promote the return of ovarian activity in ovariectomised SCID C57B1/6 mice. However, improvements to vitrification protocols for agouti ovarian tissue are necessary.
The present study evaluated the effect of supplementing in vitro culture medium with J. insularis compared to FSH on isolated secondary follicles and in vitro maturation of oocytes from those follicles. Secondary follicles were isolated from sheep ovaries and individually cultured for 18 days in α-MEM+ (Control), α-MEM+ supplemented with 100 ng/mL recombinant bovine follicle stimulating hormone (FSH) or with 0.3, 1.25, or 2.5 mg/mL of J. insularis extract (JI0.3, JI1.25, and JI2.5, respectively). Culture medium collected every 2 days was used to measure ROS levels. At the end of the culture period, cumulus oocytes complex (COCs) were collected and matured in vitro. Follicular walls were used for mRNA quantitation. JI0.3 led to a higher (P < 0.05) percentages of intact follicles than other groups after 18 days of culture. While follicular diameter remained unchanged from Day 6 onwards with JI0.3 and FSH, percentages of antral cavity formation were higher (P < 0.05) with JI0.3 at Day 6 than in all other treatments. No differences were observed between controls and treatment groups regarding ROS levels and mRNA expression of genes. Viability of resulting oocytes was higher (P < 0.05) in JI0.3 compared to FSH. Interestingly, in control experiment, supplementation of maturation medium with JI0.3 led to higher (P < 0.05) percentages of metaphase II compared to controls. Although more validations will be needed, it seems that this natural extract could be used as a cheap and easily available alternative to commercial FSH.
Although 100 μM/mL of ALA did not alter intracellular ROS, this concentration reduced the levels of ROS in the culture medium, preserved both the follicular morphology, as well as the mitochondrial activity, promoted follicle activation, and protected the follicles from DNA damage.
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