Reproduction

Sutton-McDowall, Melanie L; Gilchrist, Robert B; Thompson, Jeremy G

doi: 10.1530/rep-09-0345pmid: 20089664

The environment that the cumulus oocyte complex (COC) is exposed to during either in vivo or in vitro maturation (IVM) can have profound effects on the success of fertilisation and subsequent embryo development. Glucose is a pivotal metabolite for the COC and is metabolised by glycolysis, the pentose phosphate pathway (PPP), the hexosamine biosynthesis pathway (HBP) and the polyol pathway. Over the course of oocyte maturation, a large proportion of total glucose is metabolised via the glycolytic pathway to provide substrates such as pyruvate for energy production. Glucose is also the substrate for many cellular functions during oocyte maturation, including regulation of nuclear maturation and redox state via the PPP and for the synthesis of substrates of extracellular matrices (cumulus expansion) and O-linked glycosylation (cell signalling) via the HBP. However, the oocyte is susceptible to glucose concentration-dependent perturbations in nuclear and cytoplasmic maturation, leading to poor embryonic development post-fertilisation. For example, glucose concentrations either too high or too low result in precocious resumption of nuclear maturation. This review will discuss the relevant pathways of glucose metabolism by COCs during in vivo maturation and IVM, including the relative contribution of the somatic and gamete compartments of the COC to glucose metabolism. The consequences of exposing COCs to abnormal glucose concentrations will also be examined, either during IVM or by altered maternal environments, such as during hyperglycaemia induced by diabetes and obesity.

Sutton-McDowall, Melanie L; Gilchrist, Robert B; Thompson, Jeremy G

doi: 10.1530/REP-09-0345pmid: 20089664

The environment that the cumulus oocyte complex (COC) is exposed to during either in vivo or in vitro maturation (IVM) can have profound effects on the success of fertilisation and subsequent embryo development. Glucose isa pivotal metabolite for the COC and is metabolised by glycolysis, the pentose phosphate pathway (PPP), the hexosamine biosynthesispathway (HBP) and the polyol pathway. Over the course of oocyte maturation, a large proportion of total glucose is metabolisedvia the glycolytic pathway to provide substrates such as pyruvate for energy production. Glucose is also the substrate formany cellular functions during oocyte maturation, including regulation of nuclear maturation and redox state via the PPP andfor the synthesis of substrates of extracellular matrices (cumulus expansion) and O-linked glycosylation (cell signalling) via the HBP. However, the oocyte is susceptible to glucose concentration-dependentperturbations in nuclear and cytoplasmic maturation, leading to poor embryonic development post-fertilisation. For example,glucose concentrations either too high or too low result in precocious resumption of nuclear maturation. This review willdiscuss the relevant pathways of glucose metabolism by COCs during in vivo maturation and IVM, including the relative contribution of the somatic and gamete compartments of the COC to glucose metabolism.The consequences of exposing COCs to abnormal glucose concentrations will also be examined, either during IVM or by alteredmaternal environments, such as during hyperglycaemia induced by diabetes and obesity.

Dickinson, Rachel E; Duncan, W Colin

doi: 10.1530/rep-10-0017pmid: 20100881

The secreted SLIT glycoproteins and their Roundabout (ROBO) receptors were originally identified as important axon guidance molecules. They function as a repulsive cue with an evolutionarily conserved role in preventing axons from migrating to inappropriate locations during the assembly of the nervous system. In addition the SLIT-ROBO interaction is involved in the regulation of cell migration, cell death and angiogenesis and, as such, has a pivotal role during the development of other tissues such as the lung, kidney, liver and breast. The cellular functions that the SLIT/ROBO pathway controls during tissue morphogenesis are processes that are dysregulated during cancer development. Therefore inactivation of certain SLITs and ROBOs is associated with advanced tumour formation and progression in disparate tissues. Recent research has indicated that the SLIT/ROBO pathway could also have important functions in the reproductive system. The fetal ovary expresses most members of the SLIT and ROBO families. The SLITs and ROBOs also appear to be regulated by steroid hormones and regulate physiological cell functions in adult reproductive tissues such as the ovary and endometrium. Furthermore several SLITs and ROBOs are aberrantly expressed during the development of ovarian, endometrial, cervical and prostate cancer. This review will examine the roles this pathway could have in the development, physiology and pathology of the reproductive system and highlight areas for future research that could further dissect the influence of the SLIT/ROBO pathway in reproduction.

Dickinson, Rachel E; Duncan, W Colin

doi: 10.1530/REP-10-0017pmid: 20100881

The secreted SLIT glycoproteins and their Roundabout (ROBO) receptors were originally identified as important axon guidancemolecules. They function as a repulsive cue with an evolutionarily conserved role in preventing axons from migrating to inappropriatelocations during the assembly of the nervous system. In addition the SLIT-ROBO interaction is involved in the regulation ofcell migration, cell death and angiogenesis and, as such, has a pivotal role during the development of other tissues suchas the lung, kidney, liver and breast. The cellular functions that the SLIT/ROBO pathway controls during tissue morphogenesisare processes that are dysregulated during cancer development. Therefore inactivation of certain SLITs and ROBOs is associated with advanced tumour formation and progression in disparate tissues. Recent research has indicated that theSLIT/ROBO pathway could also have important functions in the reproductive system. The fetal ovary expresses most members ofthe SLIT and ROBO families. The SLITs and ROBOs also appear to be regulated by steroid hormones and regulate physiological cell functions in adult reproductive tissues suchas the ovary and endometrium. Furthermore several SLITs and ROBOs are aberrantly expressed during the development of ovarian, endometrial, cervical and prostate cancer. This review will examinethe roles this pathway could have in the development, physiology and pathology of the reproductive system and highlight areasfor future research that could further dissect the influence of the SLIT/ROBO pathway in reproduction.

Kwong, W Y; Adamiak, S J; Gwynn, A; Singh, R; Sinclair, K D

doi: 10.1530/REP-09-0517pmid: 20083604

Maternal B-vitamin status at conception can affect fertility and the health of offspring. This study details transcript expressionfor genes encoding key enzymes in the linked methionine/folate cycles in the bovine oocyte, somatic cells of the ovarian follicleand pre-implantation embryo. Transcripts for all 12 enzymes that were studied and for the two folate receptors (FOLR1 and FOLR2) and reduced folate carrier (SLC19A1) were expressed in liver cells, but transcripts for betaine-homocysteine methyltransferase and methionine adenosyl transferase1A were absent in all ovarian cells, and transcripts for FOLR2 were absent in embryonic cells. Transcripts for glycine methyltransferase were also absent/weak in cumulus and granulosacells. The absence of these enzymes could have a profound effect on single-carbon metabolism within the ovary and pre-implantationembryo. Immunocytochemical analysis revealed SLC19A1 protein expression on the plasma and basal-lateral membranes of the pre-implantationembryo. The folate antagonist methotrexate (MTX) enters the cell via SLC19A1, and in the current study, MTX inclusion in bovine/ovineculture media at either 1 or 10 μM from the 1-cell stage inhibited embryo development beyond the 8-cell stage. Hypoxanthineand thymidine (100 μM) increased the proportion of embryos that developed to blastocysts, but the cell number was reducedby 20%. The reduced uptake of [35S] methionine into intra-cellular S-adenosylmethionine and S-adenosylhomocysteine pools, together with reduced uptake of glutamate and tryptophan, was consistent with depleted intra-cellularpools of reduced folates. These data provide an insight into the importance of maternal dietary folate/B-vitamin status duringthe peri-conceptional period.

Kwong, W Y; Adamiak, S J; Gwynn, A; Singh, R; Sinclair, K D

doi: 10.1530/rep-09-0517pmid: 20083604

Maternal B-vitamin status at conception can affect fertility and the health of offspring. This study details transcript expression for genes encoding key enzymes in the linked methionine/folate cycles in the bovine oocyte, somatic cells of the ovarian follicle and pre-implantation embryo. Transcripts for all 12 enzymes that were studied and for the two folate receptors (FOLR1 and FOLR2) and reduced folate carrier (SLC19A1) were expressed in liver cells, but transcripts for betaine-homocysteine methyltransferase and methionine adenosyl transferase 1A were absent in all ovarian cells, and transcripts for FOLR2 were absent in embryonic cells. Transcripts for glycine methyltransferase were also absent/weak in cumulus and granulosa cells. The absence of these enzymes could have a profound effect on single-carbon metabolism within the ovary and pre-implantation embryo. Immunocytochemical analysis revealed SLC19A1 protein expression on the plasma and basal-lateral membranes of the pre-implantation embryo. The folate antagonist methotrexate (MTX) enters the cell via SLC19A1, and in the current study, MTX inclusion in bovine/ovine culture media at either 1 or 10 μM from the 1-cell stage inhibited embryo development beyond the 8-cell stage. Hypoxanthine and thymidine (100 μM) increased the proportion of embryos that developed to blastocysts, but the cell number was reduced by 20%. The reduced uptake of [35S] methionine into intra-cellular S-adenosylmethionine and S-adenosylhomocysteine pools, together with reduced uptake of glutamate and tryptophan, was consistent with depleted intra-cellular pools of reduced folates. These data provide an insight into the importance of maternal dietary folate/B-vitamin status during the peri-conceptional period.

Zhu, Li; Santos, Nadine C; Kim, Kwan Hee

doi: 10.1530/REP-09-0527pmid: 20130111

Retinoic acid receptor α (RARA) is critical for spermatogenesis, as shown by a sterility phenotype observed in Rara knockout mice. RARA is important in both Sertoli and germ cells of the testis. Here, we demonstrate that a disulfide isomeraseglucose-regulated protein 58 (GRp58) participates in the nuclear import and degradation of RARA in Sertoli cells. GRp58 interactedwith RARA in the presence of all-trans retinoic acid (ATRA) ligand and, as a complex, it was translocated from the cytoplasm to the nucleus and, then with time,GRp58 dissociated from RARA and was found in the cytoplasm. The GRp58 RNAi treatment disrupted ATRA-dependent RARA nuclearlocalization, indicating the requirement of GRp58 for RARA nuclear localization. Moreover, treatment with sulfhydryl-modifyingagents that oxidize SH-groups of cysteine residues to disulfide bonds abolished ATRA-mediated RARA nuclear localization, suggestingthat the thiol oxidoreductase activity of GRp58 may be required for RARA nuclear import. Additionally, the proteasome inhibitortreatment resulted in the co-localization of GRp58 and RARA at the endoplasmic reticulum (ER), suggesting that GRp58 may bringRARA to the ER for the ER-associated degradation (ERAD) of RARA before it is de-coupled from RARA for recycling. In this regard,proteasome inhibitor treatment also increased the interaction of RARA with UBE2J2, an ERAD-associated ubiquitin E2 enzyme.Collectively, the results indicate that GRp58 may act as a molecular chaperone that alters the protein conformation of RARAfor its delivery to the nucleus and, then with time, accompanies RARA to the ER for RARA ubiquitination and proteasome-mediatedERAD.

Zhu, Li; Santos, Nadine C; Kim, Kwan Hee

doi: 10.1530/rep-09-0527pmid: 20130111

Retinoic acid receptor α (RARA) is critical for spermatogenesis, as shown by a sterility phenotype observed in Rara knockout mice. RARA is important in both Sertoli and germ cells of the testis. Here, we demonstrate that a disulfide isomerase glucose-regulated protein 58 (GRp58) participates in the nuclear import and degradation of RARA in Sertoli cells. GRp58 interacted with RARA in the presence of all-trans retinoic acid (ATRA) ligand and, as a complex, it was translocated from the cytoplasm to the nucleus and, then with time, GRp58 dissociated from RARA and was found in the cytoplasm. The GRp58 RNAi treatment disrupted ATRA-dependent RARA nuclear localization, indicating the requirement of GRp58 for RARA nuclear localization. Moreover, treatment with sulfhydryl-modifying agents that oxidize SH-groups of cysteine residues to disulfide bonds abolished ATRA-mediated RARA nuclear localization, suggesting that the thiol oxidoreductase activity of GRp58 may be required for RARA nuclear import. Additionally, the proteasome inhibitor treatment resulted in the co-localization of GRp58 and RARA at the endoplasmic reticulum (ER), suggesting that GRp58 may bring RARA to the ER for the ER-associated degradation (ERAD) of RARA before it is de-coupled from RARA for recycling. In this regard, proteasome inhibitor treatment also increased the interaction of RARA with UBE2J2, an ERAD-associated ubiquitin E2 enzyme. Collectively, the results indicate that GRp58 may act as a molecular chaperone that alters the protein conformation of RARA for its delivery to the nucleus and, then with time, accompanies RARA to the ER for RARA ubiquitination and proteasome-mediated ERAD.

Higaki, Shogo; Eto, Yoshiki; Kawakami, Yutaka; Yamaha, Etsuro; Kagawa, Noriko; Kuwayama, Masashige; Nagano, Masashi; Katagiri, Seiji; Takahashi, Yoshiyuki

doi: 10.1530/REP-09-0549pmid: 20154175

This study aimed to produce fertile zebrafish (Danio rerio) possessing germ cells (gametes) that originated from cryopreserved primordial germ cells (PGCs). First, to improve the vitrificationprocedure of PGCs in segmentation stage embryos, dechorionated yolk-intact and yolk-removed embryos, the PGCs of which werelabeled with green fluorescent protein, were cooled rapidly after serial exposures to equilibration solution (ES) and vitrificationsolution (VS), which contained ethylene glycol, DMSO, and sucrose. Yolk removal well prevented ice formation in the embryosduring cooling and improved the viability of cryopreserved PGCs. The maximum recovery rate of live PGCs in the yolk-removedembryos vitrified after optimum exposure to ES and VS was estimated to be about 90%, and about 50% of the live PGCs showedpseudopodial movement. Next, to elucidate the ability of cryopreserved PGCs to differentiate into functional gametes, PGCsrecovered from the yolk-removed embryos (striped-type) that were vitrified under the optimum exposure to ES and VS were transplantedindividually into 218 sterilized recipient blastulae (golden-type). Two days after the transplantation, 7.5% (14/187) of morphologicallynormal embryos had PGC(s) in the genital ridges. Six (5 males and 1 female) of the 14 recipient embryos developed into maturefish and generated progeny with characteristics inherited from PGC donors. In conclusion, we demonstrated the successful cryopreservationof PGCs by vitrification of yolk-removed embryos and the production of fertile zebrafish possessing germ cells that originatedfrom the PGCs in vitrified embryos.

Higaki, Shogo; Eto, Yoshiki; Kawakami, Yutaka; Yamaha, Etsuro; Kagawa, Noriko; Kuwayama, Masashige; Nagano, Masashi; Katagiri, Seiji; Takahashi, Yoshiyuki

doi: 10.1530/rep-09-0549pmid: 20154175

This study aimed to produce fertile zebrafish (Danio rerio) possessing germ cells (gametes) that originated from cryopreserved primordial germ cells (PGCs). First, to improve the vitrification procedure of PGCs in segmentation stage embryos, dechorionated yolk-intact and yolk-removed embryos, the PGCs of which were labeled with green fluorescent protein, were cooled rapidly after serial exposures to equilibration solution (ES) and vitrification solution (VS), which contained ethylene glycol, DMSO, and sucrose. Yolk removal well prevented ice formation in the embryos during cooling and improved the viability of cryopreserved PGCs. The maximum recovery rate of live PGCs in the yolk-removed embryos vitrified after optimum exposure to ES and VS was estimated to be about 90%, and about 50% of the live PGCs showed pseudopodial movement. Next, to elucidate the ability of cryopreserved PGCs to differentiate into functional gametes, PGCs recovered from the yolk-removed embryos (striped-type) that were vitrified under the optimum exposure to ES and VS were transplanted individually into 218 sterilized recipient blastulae (golden-type). Two days after the transplantation, 7.5% (14/187) of morphologically normal embryos had PGC(s) in the genital ridges. Six (5 males and 1 female) of the 14 recipient embryos developed into mature fish and generated progeny with characteristics inherited from PGC donors. In conclusion, we demonstrated the successful cryopreservation of PGCs by vitrification of yolk-removed embryos and the production of fertile zebrafish possessing germ cells that originated from the PGCs in vitrified embryos.