For many decades, the dogma prevailed that female mammals had a finite pool of oocytes at birth and this was gradually exhausted during a lifetime of reproductive function. However, in 2004, a new era began in the field of female oogenesis. A study was published that appeared to detect oocyte-stem cells capable of generating new eggs within mouse ovaries. This study was highly controversial and the years since this initial finding have produced extensive research and even more extensive debate into their possibility. Unequivocal evidence testifying to the existence of oocyte-stem cells (OSCs) has yet to be produced, meanwhile the spectrum of views from both sides of the debate are wide-ranging and surprisingly passionate. Although recent studies have presented some convincing results that germ cells exist and are capable of creating new oocytes, many questions remain. Are these cells present in humans? Do they exist in physiological conditions in a dormant state? This comprehensive review first examines where and how the dogma of a finite pool was established, how this has been challenged over the years and addresses the most pertinent questions as to the current status of their existence, their role in female fertility, and perhaps most importantly, if they do exist, how can we harness these cells to improve a woman’s oocyte reserve and treat conditions such as premature ovarian insufficiency (POI: also known as premature ovarian failure, POF).
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Corrina J Horan and Suzannah A Williams
Suzannah A Williams and Pamela Stanley
Complex and hybrid N-glycans generated by N-acetylglucosaminyltransferase I (GlcNAcT-I), encoded by Mgat1, affect the functions of glycoproteins. We have previously shown that females with oocyte-specific deletion of a floxed Mgat1 gene using a zona pellucida protein 3 (ZP3)Cre transgene produce fewer pups primarily due to a reduction in ovulation rate. Here, we show that the ovulation rate of mutant females is decreased due to aberrant development of preovulatory follicles. After a superovulatory regime of 48 h pregnant mare's serum (PMSG) and 9 h human chorionic gonadotropin (hCG), mutant ovaries weighed less and contained ∼60% fewer preovulatory follicles and more atretic and abnormal follicles than controls. Unlike controls, a proportion of mutant follicles underwent premature luteinization. In addition, mutant preovulatory oocytes exhibited gross abnormalities with ∼36% being blebbed or zona-free. While 97% of wild-type oocytes had a perivitelline space at the preovulatory stage, ∼54% of mutant oocytes did not. The cumulus mass surrounding mutant oocytes was also smaller with a decreased number of proliferating cells compared with controls, although hyaluronan around mutant oocytes was similar to controls. In addition, cumulus cells surrounding mutant eggs were resistant to removal by either hyaluronidase or incubation with capacitated sperm. Therefore, the absence of complex and hybrid N-glycans on oocyte glycoproteins leads to abnormal folliculogenesis resulting in a decreased ovulation rate.
Patricia Grasa, Heidy Kaune, and Suzannah A Williams
Female mice generating oocytes lacking complex N- and O-glycans (double mutants (DM)) produce only one small litter before undergoing premature ovarian failure (POF) by 3 months. Here we investigate the basis of the small litter by evaluating ovulation rate and embryo development in DM (Mgat1 F/F C1galt1 F/F:ZP3Cre) and Control (Mgat1 F/F C1galt1 F/F) females. Surprisingly, DM ovulation rate was normal at 6 weeks, but declined dramatically by 9 weeks. In vitro development of zygotes to blastocysts was equivalent to Controls although all embryos from DM females lacked a normal zona pellucida (ZP) and ∼30% lacked a ZP entirely. In contrast, in vivo preimplantation development resulted in less embryos recovered from DM females compared with Controls at 3.5 days post coitum (dpc) (3.2±1.3 vs 7.0±0.6). Furthermore, only 45% of mated DM females contained embryos at 3.5 dpc. Of the preimplantation embryos collected from DM females, approximately half were morulae unlike Controls where the majority were blastocysts, indicating delayed embryo development in DM females. Post-implantation development in DM females was analysed to determine whether delayed preimplantation development affected subsequent development. In DM females at 5.5 dpc, only ∼40% of embryos found at 3.5 dpc had implanted. However, at 6.5 dpc, implantation sites in DM females corresponded to embryo numbers at 3.5 dpc indicating delayed implantation. At 9.5 dpc, the number of decidua corresponded to embryo numbers 6 days earlier indicating that all implanted embryos progress to midgestation. Therefore, a lack of complex N- and O-glycans in oocytes during development impairs early embryo development and viability in vivo leading to delayed implantation and a small litter.
Belinda K M Lo, Sairah Sheikh, and Suzannah A Williams
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.
Panayiota Ploutarchou, Pedro Melo, Anthony J Day, Caroline M Milner, and Suzannah A Williams
During follicle development, oocytes secrete factors that influence the development of granulosa and cumulus cells (CCs). In response to oocyte and somatic cell signals, CCs produce extracellular matrix (ECM) molecules resulting in cumulus expansion, which is essential for ovulation, fertilisation, and is predictive of oocyte quality. The cumulus ECM is largely made up of hyaluronan (HA), TNF-stimulated gene-6 (TSG-6, also known as TNFAIP6), pentraxin-3 (PTX3), and the heavy chains (HCs) of serum-derived inter-α-inhibitor proteins. In contrast to other in vivo models where modified expansion impairs fertility, the cumulus mass of C1galt1 Mutants, which have oocyte-specific deletion of core 1-derived O-glycans, is modified without impairing fertility. In this report, we used C1galt1 Mutant (C1galt1 FF :ZP3Cre) and Control (C1galt1 FF ) mice to investigate how cumulus expansion is affected by oocyte-specific deletion of core 1-derived O-glycans without adversely affecting oocyte quality. Mutant cumulus–oocyte complexes (COCs) are smaller than Controls, with fewer CCs. Interestingly, the CCs in Mutant mice are functionally normal as each cell produced normal levels of the ECM molecules HA, TSG-6, and PTX3. However, HC levels were elevated in Mutant COCs. These data reveal that oocyte glycoproteins carrying core 1-derived O-glycans have a regulatory role in COC development. In addition, our study of Controls indicates that a functional COC can form provided all essential components are present above a minimum threshold level, and thus some variation in ECM composition does not adversely affect oocyte development, ovulation or fertilisation. These data have important implications for IVF and the use of cumulus expansion as a criterion for oocyte assessment.