Signalling pathways and mechanistic cues highlighted by transcriptomic analysis of primordial, primary, and secondary ovarian follicles in domestic cat

In vitro growth (IVG) of dormant primordial ovarian follicles aims to produce mature competent oocytes for assisted reproduction. Success is dependent on optimal in vitro conditions complemented with an understanding of oocyte and ovarian follicle development in vivo. Complete IVG has not been achieved in any other mammalian species besides mice. Furthermore, ovarian folliculogenesis remains sparsely understood overall. Here, gene expression patterns were characterised by RNA-sequencing in primordial (PrF), primary (PF), and secondary (SF) ovarian follicles from Felis catus (domestic cat) ovaries. Two major transitions were investigated: PrF-PF and PF-SF. Transcriptional analysis revealed a higher proportion in gene expression changes during the PrF-PF transition. Key influencing factors during this transition included the interaction between the extracellular matrix (ECM) and matrix metalloproteinase (MMPs) along with nuclear components such as, histone HIST1H1T (H1.6). Conserved signalling factors and expression patterns previously described during mammalian ovarian folliculogenesis were observed. Species-specific features during domestic cat ovarian folliculogenesis were also found. The signalling pathway terms "PI3K-Akt", "transforming growth factor-beta receptor", "ErbB", and "HIF-1" from the functional annotation analysis were studied. Some results highlighted mechanistic cues potentially involved in PrF development in the domestic cat. Overall, this study provides an insight into regulatory factors and pathways during preantral ovarian folliculogenesis in domestic cat

Short-term preservation of canine preantral follicles : effects of temperature, medium and time

The use of the large pool of preantral follicles is a promising alternative to provide high numbers of fertilizable oocytes to reproductive biotechnology. This issue is particularly important to canids, since current rates of success of in vitro techniques using oocytes are very limited, and many species within this family are threatened by extinction. The aim of this study was to evaluate effects of temperature, medium and time on morphology and viability of canine preantral follicles during short-term preservation. Canine ovaries were cut into fragments which were incubated in 0.9% NaCl solution or in minimum essential medium (MEM) at 4, 20 or 38 °C for 2, 6, 12 or 24 h. Afterwards, preantral follicles were analyzed by histology, transmission electron microscopy and viability testing using trypan blue, calcein-AM and ethidium homodimer-1. Percentages of morphological normal and viable follicles were maintained similar to control (time 0 h) after incubation in 0.9% NaCl at 4 or 20 °C for up to 6 h and at 38 °C for 2 h. Using MEM, such preservation was possible for 12 h at 4 or 20 °C, and for 6 h at 38 °C. These results indicate that preservation of canine preantral follicles might be better accomplished through hypothermic (4 or 20 °C) storage in MEM, which ensures maintenance of morphology and viability for up to 12 h

Osmotic tolerance and freezability of isolated caprine early-staged follicles

Isolated caprine early-staged follicles were submitted to osmotic tolerance tests in the presence of sucrose, ethylene glycol (EG), or NaCl solutions and were exposed to and cryopreserved (by slow or rapid cooling) in MEM alone or MEM supplemented with sucrose, EG (1.0 or 4.0 M), or both. When follicles were exposed to 1.5 M NaCl, only 2% of the follicles were viable, whereas 87% of the follicles were viable after exposure to 4.0 M EG. Regarding exposure time, the highest percentage of viable follicles was obtained when follicles were exposed for 10 min to 1.0 M EG + 0.5 M sucrose; exposure for 60 s to 4.0 M EG + 0.5 M sucrose also maintained high percentage viability in follicles. Slow cooling in the presence of 1.0 M EG + 0.5 M sucrose (75%) or rapid cooling in the presence of 4.0 M EG + 0.5 M sucrose (71%) resulted in a significantly higher proportion of viable follicles than all other treatments (P < 0.05). A 24-h culture of frozen-thawed follicles was used to assess survival; only slow-frozen follicles showed viability rates similar to control follicles (64% vs. 69% respectively; P > 0.05). Interestingly, the percentage of viable rapid-cooled follicles (59%) was similar to that obtained after in vitro culture of conventional slow-cooled follicles but was significantly lower than that in controls. Thus, in addition to determining improved procedures for the exposure of follicles to EG and sucrose before and after freezing of caprine early-staged follicles, we report the development of rapid- and slow-cooling protocols

Current achievements and future research directions in ovarian tissue culture, in vitro follicle development and transplantation: implications for fertility preservation

Female cancer patients are offered 'banking' of gametes before starting fertility-threatening cancer therapy. Transplants of fresh and frozen ovarian tissue between healthy fertile and infertile women have demonstrated the utility of the tissue banked for restoration of endocrine and fertility function. Additional methods, like follicle culture and isolated follicle transplantation, are in development. Specialist reproductive medicine scientists and clinicians with complementary expertise in ovarian tissue culture and transplantation presented relevant published literature in their field of expertise and also unpublished promising data for discussion. As the major aims were to identify the current gaps prohibiting advancement, to share technical experience and to orient new research, contributors were allowed to provide their opinioned expert views on future research. Normal healthy children have been born in cancer survivors after orthotopic transplantation of their cryopreserved ovarian tissue. Longevity of the graft might be optimized by using new vitrification techniques and by promoting rapid revascularization of the graft. For the in vitro culture of follicles, a successive battery of culture methods including the use of defined media, growth factors and three-dimensional extracellular matrix support might overcome growth arrest of the follicles. Molecular methods and immunoassay can evaluate stage of maturation and guide adequate differentiation. Large animals, including non-human primates, are essential working models. Experiments on ovarian tissue from non-human primate models and from consenting fertile and infertile patients benefit from a multidisciplinary approach. The new discipline of oncofertility requires professionalization, multidisciplinarity and mobilization of funding for basic and translational research