Cat and dog primordial follicles enclosed in ovarian cortex sustain viability after in vitro culture on agarose gel in a protein-free medium.

Mammalian ovaries, including those of the cat and dog, contain thousands of follicles/oocytes that never mature, eventually degenerate or die with the host and, thus, are a valuable, but wasted resource. The ability to exploit this germplasm would offer endless opportunities for preserving the fertility and genetic diversity of valuable individuals, populations or entire species, including those that are endangered. More oocytes could be made available if there were consistently reliable means to provoke follicle development and ovulation, especially in the dog (Songsasen and Wildt 2007). However, an alternative would be to develop an approach for artificially growing in vitro a proportion of the thousands of primordial follicles available per ovary. It then would be possible to create a vast resource of oocytes that could be used for in vitro fertilization or stored until needed. Another benefit would be the applicability to animals of diverse ages, including pre-pubertal individuals that die before having the opportunity to reproduce. The biological feasibility of this approach has been demonstrated in the mouse (Eppig and O’Brien 1996). In that study, primordial follicles in newborn mouse ovarian cortical tissue were grown by organ culture for 8 days and then isolated follicles further matured to produce mature oocytes that were fertilizable in vitro. Of the 190 two-cell embryos produced, two resulted in pups. Although offspring have not been produced from in vitro-grown oocytes in larger-size animals, including in the cat (Jewgenow 1998) and dog (Songsasen and Wildt 2007), a wealth of information has emerged on the sensitivity of such immature follicles to the microenvironment. For example, it is now well known that constituents of the culture medium significantly influence growth and viability of ovarian follicles (Hovatta et al. 1997; Jewgenow 1998). More specifically, Earle’s balanced salt solution is superior to α-minimum essential medium (α-MEM) in maintaining in vitro viability of human primordial follicles (Hovatta et al. 1997). Historically, MEM has been one of the most widely used media for culturing cat ovarian follicles and oocytes (Johnston et al. 1993). Eagle’s α-MEM, which is a rich resource for non-essential amino acids, has been used with some success to advance pre-antral follicle culture in the dog (Serafim et al. 2010; Songsasen et al. 2011). Blood serum also has been commonly included as a protein supplement in follicle or oocyte culture systems (Eppig and O’Brien 1996; Lane and Gardner 2007). Serum as a source of albumin balances osmolarity and removes precursors of free-oxygen radicals (Freshney 2005). But, serum is comprised of many undefined factors, some of which can be detrimental to gamete and embryo development. For example, serum supplementation in culture has been shown to alter oocyte metabolism and gene expression profiles as well as cause excessive fetal size and pregnancy complications after embryo transfer in some species (Lane and Gardner 2007). One protein supplement substitute, a knock-out serum replacement (KSR, proprietary formulation), has been found to be superior for boosting the proportion of cattle blastocysts produced in vitro as well as increasing pregnancy success after embryo transfer (Moore et al. 2007). KSR was originally developed for culturing mouse embryonic stem cells and, more recently, has been determined to be superior to fetal calf serum in establishing embryonic germ cells from mouse primordial germ cells while effectively preventing differentiation (Horii et al. 2003). In terms of the actual culture system, ovarian tissue traditionally has been incubated in multi-well plates (Matos et al. 2007) or in a cell culture insert system (Eppig and O’Brien 1996). Compared to the former, the latter prevents tissues from attaching to the dish surface while increasing the surface area available for nutrient exchange in medium (Freshney 2005). Recently, an agarose gel system has been developed for culturing mouse testicular tissue, which maintains normal cellular structure while promoting spermatogenesis in vitro (Gohbara et al. 2010). The advantage of this approach is that tissues are placed on an agarose gel saturated with culture medium, thereby offering a liquid/gas interface that apparently facilitates oxygen and nutrient exchange. Interestingly, this same agarose gel tactic was demonstrated more than 50 years ago to maintain immature rat ovarian tissue structure for 9 days (Trowell 1959). Since that early experiment, this method has not been re-applied to culturing ovarian tissue from other mammals. The overall goal of the present study was to understand in vitro culture conditions that could sustain cat and dog primordial follicle survival in vitro. Although both are carnivores, these species differ markedly in fundamental reproductive physiology. Most of this variation has been demonstrated at the macro-level, especially in terms of oestrous cyclicity and stimulus, timing and endocrine control of ovulation (Wildt et al. 1981). There is little information available on the specifics of intraovarian follicular (and oocyte) sensitivity to ex situ culture, especially in pre-antral stages. Therefore, we were keen to conduct a comparative study in the cat vs the dog to determine similarities, and differences, in how primordial follicles within the ovarian cortex react to varied in vitro culture conditions. Our specific objectives were to determine the influences of (i) culture medium, (ii) protein supplementation and (iii) the microenvironment itself on subsequent in vitro viability of primordial follicles from freshly excised tissue slices.