Oocyte recruitment and maturation is a critical process requiring spatial and temporal expression of specific factors that contribute to the unique cell cycle regulation dynamics of female germ cells. Phosphatase and tension homolog (PTEN) antagonizes the action of oocyte phosphoinositide 3-kinase (PI3K), known to regulate primordial follicle recruitment, and consequently PTEN knockout mice demonstrate impaired primordial follicle activation. Oocyte PTEN expression can be influenced through interactions via miRNA, specifically miR-21. We have previously demonstrated an increase in oocyte and granulosa cell miR-21 expression during in vitro oocyte maturation, thus the objective of this study was to identify PTEN and miR-21 expression changes during recruitment (in vivo) and PTEN expression changes during maturation (in vitro) of oocytes in the porcine ovary. A time point (age and weight) at which approximately 50% of prepubertal pigs have begun tertiary follicle development was identified (98 ± 4d of age; 35 ± 2 kg BW). These two stages were designated as naive (no tertiary follicle; N) or advanced (with tertiary follicle, A). In order to determine changes in PTEN and miR-21 during recruitment, ovaries were collected from three gilts at each stage (N or A), preserved in 4% paraformaldehyde and utilized for in situ hybridization (miR-21) and immunohistochemistry (PTEN). Additionally, to determine PTEN expression during follicle maturation, sow ovaries were obtained from a local abattoir and aspirated to collect cumulus oocyte complexes (COC) from follicles of 3-5 mm in diameter. Germinal vesicle stage oocytes (GV) were collected immediately from COC’s. Remaining COCs containing several compact layers of cumulus cells were washed and placed in maturation media for 42-44 hr, stripped of expanded cumulus cells, and metaphase II (MII) arrested oocytes, as indicated by the presence of an extruded polar body, were collected. PTEN mRNA and protein levels were measured by quantitative RT-PCR and immunostaining with quantification using ImageJ software, respectively, in GV and MII oocytes. Immunolocalization demonstrated that PTEN expression in the oocyte increases in an oocyte-specific manner during transition from primary to secondary to tertiary follicle. In situ hybridization demonstrated increased miR-21 in the oocyte that followed the same pattern as that of PTEN. MiR-21 was expressed in granulosa cells at all follicle stages. Following in vitro maturation of oocytes, there was no difference in PTEN protein level between GV and MII-arrested oocytes (P = 0.65). Furthermore, oocyte PTEN mRNA did not differ (P = 0.97) between GV and MII oocytes matured in vitro. Surprisingly, there was no PTEN protein suppression temporally associated with increased miR21 expression as previously demonstrated with another miR-21 target gene, PDCD4. These data indicate that PTEN protein expression in the oocyte increases significantly in proliferating secondary follicle oocytes, with highest PTEN expression occurring in tertiary follicle oocytes. Additionally, these results support a potential role for and interaction between miR-21 and PTEN during recruitment from the primary to tertiary follicle stage in the porcine ovary, rather than in the maturation steps that take place thereafter. Project was supported by National Research Initiative Competitive Grant no. 2008-35205-05309 and 2008-35205-18712 from the USDA National Institute of Food and Agriculture. (poster)