Methodology and characterization of an in vitro perfusion model for the mouse ovary

Perfusion of intact ovaries in vitro has proved to be a suitable model for the study of ovarian events, such as ovulation and corpus luteum function, with advantages over cell culture systems of preserved three-dimensional structure with intact intercellular communication. This methodology has been described for several larger experimental animals. Development of genetically manipulated mouse strains and the larger availability of recombinant proteins and monoclonal antibodies in this species prompted the development of a mouse ovary perfusion system. In the present study, we describe the methodology for perfusion of mouse ovaries in vitro and characterize the model with respect to steroidogenesis, ovulatory efficiency and morphology. Swiss mice (30-45 g) were synchronized with a single injection of a GnRH agonist and laparotomy was performed on the morning of pro-oestrus. The right ovary and its vasculature, after cannulation on the arterial side, were surgically isolated and transferred to a recirculating perfusion system, and were perfused for 20 h. The ultrastructure of the ovaries after 20 h of perfusion was examined by transmission electron microscopy, which showed well-preserved cellular organelles and nuclei and distinct tight junctions between endothelial cells. Unstimulated ovaries did not ovulate and secreted small quantities of progesterone. After addition of hCG (50 iu) or ovine LH (0.1 microgram ml-1), ovulations were observed in all perfused ovaries (2.6 +/- 0.5 and 2.0 +/- 0.3 ovulations per treated ovary, respectively). There was a distinct rise in progesterone output after hormone addition. When a phosphodiesterase inhibitor, isobutylmethylxanthine (0.2 mumol l-1), was added in combination with LH, the ovulation rate (7.4 +/- 1.2) was similar to that in vivo and progesterone secretion was further increased compared with other treatments. Since the mouse has become a very important experimental animal in medical research and we have the ability to genetically manipulate this species, this methodology for perfusing mouse ovaries in vitro may be a useful tool in future studies of ovarian physiology.