Preservation Of Preantral Follicles Research Articles

Microbiological load and preantral follicle preservation using different systems for ovarian tissue vitrification in the red-rumped agouti

We evaluated the effect of open and closed systems used for ovarian tissue vitrification on the microbiological load and preservation of preantral follicles (PAFs) in the red-rumped agoutis. The ovaries from eight females were recovered and fragmented, with four cortexes fragments immediately fixed and evaluated (fresh group). The other fragments were processed for the solid-surface vitrification method (SSV) or an ovarian tissue cryosystem (OTC) using fetal calf serum, ethylene glycol, and sucrose as cryoprotectants, stored for two weeks, and rewarmed. Subsequently, fragments were subjected to a 24-h in vitro culture and assessed for microbiological load, PAF morphology, and DNA integrity. There was no fungal contamination; however, the vitrified samples from two individuals showed bacterial contamination of 79 200 colony forming units per milliliter (CFU)/mL for SSV and 3120 CFU/mL for OTC. From those samples, a total of eight different types of bacterial colonies were isolated and identified as coagulase-negative Staphylococci and Gram-positive bacilli. Regarding PAF morphology, both systems provided adequate preservation, with values higher than 70% normal follicles observed before and after culture. The TUNEL assay revealed that both SSV (52.39%) and OTC (41.67%) could preserve DNA integrity after vitrification and after 24 h of culture. In summary, both open and closed systems were equally efficient in preserving agouti ovarian tissues, especially concerning the preantral follicle morphology and DNA integrity; however, the OTC seems to provide a less adequate environment for bacterial proliferation.

Structure of preantral follicles, oxidative status and developmental competence of in vitro matured oocytes after ovary storage at 4\xa0\xb0C in the domestic cat model

BackgroundStorage conditions during transportation of explanted ovaries are a critical step in setting up fertility preservation protocols in both animal and human fields. Here, we evaluated the effects of ovary storage at 4 °C on the preservation of preantral follicles and oocytes retrieved from antral follicles using the domestic cat as model.MethodsOvaries were harvested from fifty-five healthy domestic queens during ovariectomy and stored at 4 °C for 0 (control), 24, 48, 72 and 96 h. In Experiment 1, the effects of the storage period at 4 °C on the morphology, cytoskeleton (α/β tubulin) and DNA integrity (phosphorylation of histone H2AX) of preantral follicles were investigated. In Experiment 2, oocytes recovered from antral follicles were matured and fertilized in vitro to evaluate their meiotic and developmental competence. Reactive oxygen species (ROS), glutathione (GSH) and lipid peroxidation were measured in matured oocytes.ResultsThe results showed that: a) storage up to 24 h did not affect the morphology and the DNA integrity of preantral follicles; b) extended storage times caused progressive morphological abnormalities, disassembling of microtubules and DNA damage; c) storage up to 48 h did not influence in vitro meiotic maturation of oocytes nor cleavage after in vitro fertilization. However, only oocytes stored within the ovary for 24 h produced blastocysts in a percentage similar to control oocytes; d) GSH levels of in vitro matured oocytes did not change at any time during ovary storage; a progressive increase in ROS levels was detected from 48 h associated with elevated lipid peroxidation at 72 and 96 h of storage.ConclusionsStorage of cat ovaries for up to 24 h caused minimal alteration of preantral follicles and oocytes. The extension of the storage period beyond 24 h progressively impaired the structure of follicles, and modified the oxidative status of in vitro matured oocytes and their developmental competence after in vitro fertilization. This information may help when setting up programs for fertility conservation, especially for wild feline species which die in geographic areas located far away from ARTs centers.

Vitrification and xenografting of human ovarian tissue

To assess the efficiency of two vitrification protocols to cryopreserve human preantral follicles with the use of a xenografting model. Pilot study. Gynecology research unit in a university hospital. Ovarian biopsies were obtained from seven women aged 30-41 years. Ovarian tissue fragments were subjected to one of three cryopreservation protocols (slow freezing, vitrification protocol 1, and vitrification protocol 2) and xenografted for 1 week to nude mice. The number of morphologically normal follicles after cryopreservation and grafting and fibrotic surface area were determined by histologic analysis. Apoptosis was assessed by the TUNEL method. Morphometric analysis of TUNEL-positive surface area also was performed. Follicle proliferation was evaluated by immunohistochemistry. After xenografting, a difference was observed between the cryopreservation procedures applied. According to TUNEL analysis, both vitrification protocols showed better preservation of preantral follicles than the conventional freezing method. Moreover, histologic evaluation showed a significantly higher proportion of primordial follicles in vitrified (protocol 2)-warmed ovarian tissue than in frozen-thawed tissue. The proportion of growing follicles and fibrotic surface area was similar in all groups. Vitrification procedures appeared to preserve not only the morphology and survival of preantral follicles after 1 week of xenografting, but also their ability to resume folliculogenesis. In addition, vitrification protocol 2 had a positive impact on the quiescent state of primordial follicles after xenografting.

Dissociation and preservation of preantral follicles and immature oocytes from female dasyurid marsupials

The mammalian ovary contains numerous immature preantral follicles that are not dependent on endocrine support, unlike the more mature hormone-dependent antral follicles. Preantral follicles can be enzymatically dissociated to yield immature oocytes that survive sub-zero preservation better as they lack a temperature-sensitive meiotic spindle. These techniques are highly applicable to gamete banking, which is an urgent requirement for Australian carnivorous marsupials as several species have rapidly declining populations and risk extinction. The present study developed protocols for the transport, dissociation, preservation and culture of granulosa cell-oocyte complexes (GOC) from the ovaries of dasyurid marsupials. High viability of GOC following enzymatic dissociation is reported and it was demonstrated that GOC are of significantly better quality following refrigerated storage for 24 h compared with storage at room temperature. Oocytes from primary follicles were not damaged by cold shock or the toxicity of vitrification media and following vitrification in liquid nitrogen 69.42+/-2.44% of oocytes were viable. However, the surrounding granulosa cells demonstrated significant damage post-thaw. These granulosa cells proliferated during a 48-h culture period resulting in significant improvements in GOC quality. The present study is a valuable step towards cryostorage of dasyurid gametes and represents fundamentally important methods by which we can contribute to the conservation of Australia's native predators.

Effects of lowered temperatures and media on short-term preservation of zebu ( Bos indicus) preantral ovarian follicles

The maintenance of follicle quality during the transportation of ovaries is essential for the successful cryopreservation and in vitro development of preantral follicles. The objective of this study was to evaluate the effect of cooling ovarian tissue on the conservation of zebu cow preantral follicles. Ovarian pieces were immersed in saline or coconut water (CW) solutions and maintained at 4 or 20 °C for 6, 12, or 18 h. Preantral follicles were evaluated by histology and transmission electron microscopy. Storage of ovarian pieces at 20 °C for 12 or 18 h significantly reduced the percentage of morphologically normal follicles compared to controls. In contrast, conservation at 4 °C for up to 18 h and at 20 °C for up to 6 h kept the percentage of normal follicles similar to controls. However, the type of solution that the ovaries were immersed in had little effect on the results. Decreased cellular metabolism probably accounted for better preservation of preantral follicles at 4 °C. In conclusion, zebu cow ovaries were successfully stored at 4 °C for up to 18 h with no morphological damage to preantral follicles. However, at 20 °C, ovaries could only be stored for 6 h.

Effects of storage time and temperature on atresia of goat ovarian preantral follicles held in M199 with or without indole-3-acetic acid supplementation

Maintenance of follicular quality after removal and during transport of ovaries is necessary for studies on development of preantral follicles in vitro. The present work investigated the effectiveness of M199 and M199IAA for preservation of goat preantral follicles in ovarian tissue. At the slaughterhouse, the ovarian pair of each animal was divided into 19 fragments. One ovarian fragment was immediately fixed (control — Time 0). The other 18 fragments were randomly distributed in M199 or M199IAA at 4, 20 or 39°C and stored for 4, 12 or 24 h. Histological analysis showed that storage of ovarian fragments in either solution at 20 or 39°C significantly reduced the percentage of normal preantral follicles when compared with the control, in all cases except after preservation in M199IAA at 20°C for 4 h. In contrast, preservation at 4°C, in either solution, kept the percentage of normal preantral follicles at control values. Reduced cellular metabolism may explain why the best preservation of preantral follicles was at 4°C. The addition of IAA to the TCM 199 was effective for goat preantral follicle preservation at 20°C for 4 h.

Effect of coconut water and Braun-Collins solutions at different temperatures and incubation times on the morphology of goat preantral follicles preserved in vitro

Preservation of preantral follicles becomes very important to ensure follicle quality at the onset of cryopreservation or in vitro culture. However, for domestic animals, the ovarian donor of preantral follicles for in vitro studies is commonly encountered far away from reproduction laboratories. We investigated the effectiveness of coconut water and Braun-Collins solutions on the preservation of goat preantral follicles. At the slaughterhouse, the ovarian pair of each animal was divided into 19 fragments. One ovarian fragment was immediately fixed (Control — Time 0). The other 18 fragments were randomly distributed into tubes containing 2 mL of coconut water or Braun-Collins solution at 4 °, 20 ° or 39 °C and then stored for 4, 12 or 24 h. Histological analysis showed that the storage of ovarian fragments in coconut water and Braun-Collins solutions at 20 ° or 39 °C for 12 or 24 h significantly reduced (P < 0.05) the percentage of morphologically normal preantral follicles when compared with the control. However, storage in coconut water at 20 °C for 4 h and in both solutions at 4 °C kept the percentage at control values. Ultrastructural analysis of follicles exposed to the stated conditions confirmed the integrity of preantral follicles stored at 4 °C in Braun-Collins and coconut water solutions for up to 12 and 24 h, respectively. Reduced cellular metabolism at 4 °C may explain why the best preservation of preantral follicles was at 4 °C, which may suggest a useful method for ovary transport in the future.