Rescue of follicle development after oocyte-induced ovary dysfunction and infertility in a model of POI.

The mechanisms and aetiology underlying the development of premature ovarian insufficiency (POI) are poorly understood. However, the oocyte clearly has a role as demonstrated by the Double Mutant (DM) mouse model where ovarian dysfunction (6 weeks) is followed by POI (3 months) due to oocyte-specific deletion of complex and hybrid N- and O-glycans. The ovaries of DM mice contain more primary follicles (3a stage) accompanied by fewer developing follicles, indicating a block in follicle development. To investigate this block, we first analysed early follicle development in postnatal (8-day), pre-pubertal (3-week) and post-pubertal (6-week and 3-month) DM (C1galt1 F/F Mgat1 F/F:ZP3Cre) and Control (C1galt1 F/F Mgat1 F/F) mice. Second, we investigated if transplantation of DM ovaries into a "normal" endocrine environment would restore follicle development. Third, we determined if replacing DM ovarian somatic cells would rescue development of DM oocytes. At 3-week, DM primary 3a follicles contain large oocytes accompanied by early development of a second GC layer and increased GC proliferation. At 6-week, DM primary 3a follicles contain abnormally large oocytes, accompanied with decreased GC proliferation. Transplantation of DM ovaries into a 'normal' endocrine environment did not restore normal follicle development. However, replacing somatic cells by generating reaggregated ovaries (ROs) did enable follicle development to progress and thus highlighted intra-ovarian factors were responsible for the onset of POI in DM females. Thus, these studies demonstrate oocyte-initiated altered communication between GCs and oocytes results in abnormal primary follicles which fail to progress and leads to POI.

In vitro and in vivo mouse follicle development in ovaries and reaggregated ovaries.

Follicle development requires complex and coordinated interactions between both the oocyte and its associated somatic cells. In ovarian dysfunction, follicle development may be abnormal due to defective somatic cell function; for example, premature ovarian insufficiency or malignancies. Replacing defective somatic cells, using the reaggregated ovary (RO) technique, may 'rescue' follicle development. ROs containing mature follicles have been generated when transplanted to a host mouse to develop. We have developed a RO culture technique and the aims were to determine how follicle development differed between transplanted and cultured ROs, and the influence of ovarian age (P2 vs P6). Mouse ROs were cultured for 14 days; P2 and P6 ovaries cultured as Controls. Follicle development was compared to ROs transplanted for 14 days and ovaries from P16 and P20 mice. ROs generated from either P2 or P6 exhibited similar follicle development in culture whereas in vivo follicle development was more advanced in P6 ROs. Follicles were more developed in cultured ROs than transplanted ROs. However, follicles in cultured ROs and ovaries had smaller oocytes with fewer theca and granulosa cells than in vivo counterparts. Our results demonstrate the fluidity of follicle development despite ovary dissociation and that environment is more important to basal lamina formation and theca cell development. Furthermore, follicle development within cultured ROs appears to be independent of oocyte nest breakdown and primordial follicle formation in source ovaries. Our results highlight the need for understanding follicle development in vitro, particularly in the development of the RO technique as a potential fertility treatment.

Oocyte-specific ablation of N- and O-glycans alters cumulus cell signalling and extracellular matrix composition.

Cumulus-oocyte complex (COC) expansion is essential for ovulation and fertilisation and is linked to oocyte quality. Hyaluronan (HA), the major matrix constituent, is cross-linked via inter-α-inhibitor heavy chains (HCs), pentraxin 3 (PTX3) and tumour necrosis factor-stimulated gene 6 (TSG-6). All except HCs are secreted by cumulus cells in response to oocyte-secreted factors, which signal via SMAD pathways. The double mutant (DM) mouse generates oocytes lacking complex N- and O-glycans due to oocyte-specific deletion of core 1 ß1,3-galactosyltransferase (C1galt1) and N-acetylglucosaminyltransferase I (Mgat1) and has modified cumulus expansion. We compared COCs before expansion (48 h-post-pregnant mare serum gonadotrophin (PMSG)) and at late-stage expansion (9 h-post-human chorionic gonadotrophin (hCG); control n=3 mice, DM n=3 per group). Using histochemistry the levels of HA, HCs, PTX3, TSG-6 and phosphorylated-SMAD1/5/8 and -SMAD2 (12-25 COCs per group) were assessed. DM COCs did not differ from Controls in cumulus size or cell density at 9 h-post-hCG; however, HA and HC levels and phosphorylated-SMAD1/5/8 were reduced. Furthermore, no correlations were found between the levels of matrix molecules and cumulus area in DM or Control samples. These data suggest that HA and HCs can support cumulus expansion provided that they are present above minimum threshold levels. We propose that oocyte-specific ablation of C1galt1 and Mgat1 may affect bone morphogenetic protein 15 synthesis or bioactivity, thereby reducing SMAD1/5/8 phosphorylation and HA production.