Formation of multiple-oocyte follicles in culture.

Basement membranes are found in every organ of the body. They provide structure and a selective filter for molecules. The ovary is no different with the follicular basal lamina (FBL) separating the granulosa and theca cells, facilitating regulation of the changing follicular environment providing appropriate conditions for the developing oocyte. The FBL is modified in C1galt1 Mutant mice (C1galt1 FF:ZP3Cre) resulting from oocyte-specific deletion of C1galt1. Changes in the FBL lead to follicles joining to generate multiple-oocyte follicles (MOFs); where two or more oocytes are contained within a single follicle. This study aimed to determine if single-oocyte follicles could join in culture to become MOFs by co-culturing preantral follicles from Control or Mutant mice. Co-cultured follicles from both Control and Mutant follicles could superficially fuse (73% of Control follicle pairs; 84% of Mutant). Confocal microscopy revealed alterations in the organization of the space between follicles but was unable to discern MOFs. When co-cultured follicle pairs were embedded, sectioned and stained with haematoxylin, it was revealed that MOFs had formed from 50% of Mutant follicle pairs but none from Control follicle pairs. In conclusion, MOFs can form from C1galt1 Mutant follicles in culture and this model is a useful tool to elucidate the role of the oocyte in follicle development and the generation and function of the FBL. Furthermore, understanding the relationship between oocyte function and FBL generation will likely provide insight into optimizing conditions for follicle culture, which is important for fertility treatments and ART.

Oocyte-derived Smad4 is not required for development of the oocyte or the preimplantation embryo.

UNLABELLED: The generation of a competent egg requires complex molecular interactions between the oocyte and the ovary, and transforming growth factor ß (TGF-ß) is a major signaling pathway. Smad4 is a central regulator of the TGF-ß signaling pathway as it mediates gene expression triggered by activation of TGF-ß receptors. Deletion of Smad4 in granulosa cells disrupts follicle development; however, the role of Smad4 in the oocyte has not been confirmed. Furthermore, the role of Smad4 in embryo development has not been confirmed because previous studies of Smad4(del/del) embryos were generated from heterozygous parents, and thus it is possible that maternal transcripts rescue development before embryonic day 6.5 (E6.5) when Smad4(del/del) embryos die. To determine the role of TGF-ß signaling in oocyte and embryo development, mice with oocyte-specific deletion of Smad4 were studied. Fertility was evaluated in Mutant (Smad4(F/F):ZP3Cre) and CONTROL (Smad4(F/F)) females mated continuously with control males during a 6-month period. Surprisingly, Mutant females were fertile with the same litter size (Mutants, 9.23 ± 0.4; CONTROLs, 9.42 ± 0.4) and interlitter period as CONTROLs. Ovulation rate induced using a superovulation regime did not differ between CONTROLs and Mutants at both 6 weeks and 6 months. Embryo development was assessed at E6.5 using CONTROL and Mutant females mated with heterozygous males. Development of Smad4(del/del) embryos at E6.5 was retarded consistent with previous studies of embryos generated from heterozygous parents indicating that there is no rescue of preimplantation development by maternal transcripts. The numbers of implanted embryos at 6.5 dpc also did not differ ( CONTROL: 9.1 ± 0.4; Mutant: 7.0 ± 0.9). However, only 26.3% of E6.5 embryos carried by Mutant females were Smad4(del/del) compared with the expected ratio of 50%. Since litter size was not decreased, this indicates that either the number of Smad4(del) sperm fertilizing the oocytes is reduced or implantation of Smad4(del/del) embryos is suboptimal. In summary, we have shown that Smad4 in the oocyte, and thus TGF-ß signaling, is not required for oocyte or follicle development, ovulation, fertilization, preimplantation development, or implantation.