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Oocyte-Specific Deletion of Complex and Hybrid N-Glycans Leads To

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Oocyte-Specific Deletion of Complex and Hybrid N-Glycans Leads To REPRODUCTIONRESEARCH Oocyte-specific deletion of complex and hybrid N-glycans leads to defects in preovulatory follicle and cumulus mass development Suzannah A Williams and Pamela Stanley Department of Cell Biology, Albert Einstein College of Medicine, New York, New York 10461, USA Correspondence should be addressed to P Stanley; Email: [email protected] S A Williams is now at Department of Physiology, Anatomy and Genetics, Le Gros Clark Building, University of Oxford, South Parks Road, Oxford OX1 3QX, UK Abstract 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 w60% 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 w36% being blebbed or zona-free. While 97% of wild-type oocytes had a perivitelline space at the preovulatory stage, w54% 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. Reproduction (2009) 137 321–331 Introduction synthesis of hybrid and complex N-glycans solely in oocytes, have decreased fertility due to a decreased The development of a functional oocyte from the w ovulation rate, and compromised preimplantation primordial to ovulatory stage, takes 3 weeks in female embryonic development (Shi et al. 2004). Hybrid and mice (Pedersen & Peters 1968, Peters 1969) or potentially complex N-glycans are generated following the addition longer as observed in rats (Hirshfield 1989). Each oocyte of N-acetylglucosamine (GlcNAc) by N-acetylglucosa- develops within a follicle which provides the appropriate minyltransferase I (GlcNAcT-I) to Man5GlcNAc2Asn at environment for oogenesis. Each follicle develops certain N-X-S/T sites in glycoproteins (Fig. 1). Mutant independently of gonadotropins, from recruitment as a oocytes were generated by females carrying a floxed meiotically quiescent, primordial follicle through folli- Mgat1 gene and a zona pellucida protein 3 (ZP3) Cre culogenesis to the late preantral stage when FSH is transgene (Shi et al. 2004). ZP3 is expressed from the required. LH stimulates preovulatory follicle develop- primary stage of folliculogenesis (Philpott et al. 1987), ment, oocyte maturation, and ovulation. The oocyte, 2–3 weeks prior to ovulation. Females with no complex despite being meiotically quiescent throughout follicu- or hybrid N-glycans on oocyte glycoproteins produce logenesis until ovulation, has an active role in the litters w50% smaller than controls. The decline in litter development of the follicle. It is now clear that there is size is primarily due to a decrease in the number of eggs considerable oocyte–follicle communication that is ovulated (Shi et al. 2004). The remaining reduction in essential for successful oogenesis, and that the oocyte fertility is due to aberrant preimplantation development carefully regulates the surrounding environment (Matzuk in embryos generated from Mgat1F/F:ZP3Cre females. et al. 2002, Gilchrist et al. 2004, Hutt & Albertini 2007). While about half the embryos generated by fertilization We have previously shown that female mice with a of mutant eggs develop aberrantly, w50% of these conditional Mgat1 gene deletion, which precludes the resume development upon implantation (Shi et al. 2004). q 2009 Society for Reproduction and Fertility DOI: 10.1530/REP-07-0469 ISSN 1470–1626 (paper) 1741–7899 (online) Online version via www.reproduction-online.org Downloaded from Bioscientifica.com at 10/01/2021 10:43:12AM via free access 322 S A Williams and P Stanley Table 1 Ovary weights from unstimulated and hormone-stimulated Mgat1F/F:ZP3Cre and control female mice. Control Mutant Unstimulateda nZ4 nZ4 Body (g) 18.2G1.5 17.2G1.1 Ovary (mg) 26.4G7.5 27.9G3.8 PMSGChCGb nZ3 nZ5 Body (g) 22.6G1.7 22.2G2.8 Ovary (mg) 78.2G16.0 56.9G4.1* Values are meanGS.D.*P!0.05. a6.5-week littermates from two sets of parents. b7-week females from four sets of parents analyzed in Table 2. females after the superovulatory regime were less than control ovaries (Table 1). To identify all preovulatory follicles, every unstained section from one Bouin’s-fixed ovary from each mouse was examined by light microscopy. The mutant ovaries were found to contain significantly fewer preovulatory follicles than control ovaries (20.0G2.6 per control ovary (nZ3) vs 8.2G2.7 Figure 1 (A) The oligomannosyl substrate of GlcNAcT-I, which is Z ! encoded by the Mgat1 gene, and a complex N-glycan whose synthesis per mutant ovary (n 5); P 0.001). is initiated by the action of GlcNAcT-I. (B) Genotype of female mice To determine the stage at which folliculogenesis was F/F and their oocytes. F, floxed. modified in Mgat1 :ZP3Cre females, follicles were counted and staged in every 15th section of the same The resumption of normal development demonstrates Bouin’s-fixed ovaries after staining with hematoxylin and that the aberrant embryonic development during blas- eosin (H&E). The follicles were counted if the oocyte togenesis is not due to parthenogenic activation, or nucleus was visible, and preovulatory follicles were penetration of the modified mutant zona and fertilization counted if the oocyte was clearly visible. The follicles by multiple sperm, because both of these events are lethal were defined as normal, atretic, or otherwise abnormal. (Sun 2003, Kono et al. 2004, Findlay et al. 2007). The decrease in ovary weight in Mgat1F/F:ZP3Cre females Therefore, although complex and hybrid N-glycans are was reflected in a 58% decrease in the number of normal not essential for oogenesis, fertilization, or preimplanta- preovulatory follicles (controls: 15.7G2.5 (nZ3), mutant: tion embryonic development, they play functional role(s) 6.6G2.9 (nZ5)). The mutant ovaries contained fewer in oogenesis and ovulation. follicles in all categories compared with controls In this paper, we show that the follicles with oocytes (Table 2). Interestingly, approximately two luteinizing lacking complex and hybrid N-glycans have aberrant follicles were present in each mutant ovary (nZ5), preovulatory follicular development, reflected in whereas none were observed in control ovaries (nZ3; reduced numbers of preovulatory follicles, morpho- Fig. 2B and C). The luteinizing granulosa cells of these logical abnormalities, and premature luteinization. follicles were larger and disorganized with the most Mutant oocytes are also surrounded by a reduced luteinized cells being furthest from the oocyte (Fig. 2B0 number of proliferating cumulus cells and generate and C0). The percent of normal preovulatory follicles eggs with a small cumulus mass. versus atretic/abnormal preovulatory follicles in Mgat1 mutants was significantly different from controls (Table 2; 2 Results P!0.0001, c -test). Overall, 27% of mutant follicles were lost from the growing pool by atresia or premature Reduced ovulatory response in oocytes lacking complex luteinization compared with 8% of control follicles. and hybrid N-glycans To determine whether the decreased ovulation rate of F/F Morphological analysis of preovulatory follicles with an Mgat1 :ZP3Cre females reported previously (Shi et al. oocyte lacking complex and hybrid N-glycans 2004) is due to aberrant development of preovulatory follicles or impaired ovulation, the number of ovarian Abnormalities of preovulatory follicles in Mgat1 mutant follicles was determined in ovaries collected from ovaries that were clearly not luteinizing or undergoing females treated with pregnant mare’s serum (PMSG) for atresia were common. In thin sections of glutaraldehyde- 48 h followed by human chorionic gonadotropin (hCG) fixed mutant ovaries, the second ovary obtained from the for 9 h. Mutant ovaries from unstimulated females were same mice as the Bouin’s-fixed ovaries analyzed in of the same weight as control ovaries (Table 1). However, Table 2, oocytes had misshapen zona pellucidae, the weights of ovaries collected from Mgat1F/F:ZP3Cre cumulus cells were observed beneath the zona, and Reproduction (2009) 137 321–331 www.reproduction-online.org Downloaded from Bioscientifica.com at 10/01/2021 10:43:12AM via free access N-glycans in preovulatory follicle development 323 Table 2 Follicle counts of Mgat1F/F:ZP3Cre ovaries after hormone stimulation. Preantral Antral Preovulatory Primary (% atretic) (% atretic/abnormal) (% atretic/abnormal) Total Control (nZ3) 72.0G13.5 43.7G15.1 (8G8) 6.7G3.8 (32G16) 17.0G1.0 (8G14) 139.3G31.8 Mutant (nZ5) 49.4G25.6 14.8G5.8 (9G6) 2.8G1.9 (37G41) 9.2G3.3 (27G21)a 76.2G24.5 Follicles were counted in one ovary per mouse collected at 57 h after administration of PMSG at 0 h and hCG at 48 h. Values are mean GS.D. aAbnormal preovulatory follicles included luteinizing follicles and corpora lutea containing an oocyte. K K there were oocytes separated from their zona (Fig. 3A–D). cumulus mass size. Mgat1 / preovulatory oocytes To quantify the abnormalities, sections through the lacking hybrid and complex N-linked glycans contained center of oocytes of preovulatory follicles from the large vesicles that reacted with the PAS reagent Bouin’s-fixed ovaries were stained with hematoxylin and compared with the much smaller vesicles present in periodic acid Schiff reagent (H&PAS) and examined.
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