Reproduction

Garcia-Segura, Luis Miguel; Lorenz, Betty; DonCarlos, Lydia L

doi: 10.1530/REP-07-0540pmid: 18367504

Neuron-to-glia, glia-to-neuron, and glia-to-glia communication are implicated in the modulation of neuronal activity and synaptictransmission relevant to reproduction. Glial cells play an important role in neuroendocrine regulation and participate inthe sexual differentiation of neuronal connectivity of brain regions involved in the control of reproductive neuroendocrineoutput. During puberty, modifications in the morphology and chemistry of astrocytes and tanycytes in the hypothalamus andmedian eminence influence the maturation of the neuronal circuits controlling the secretion of GnRH. During adult reproductivelife, the glial cells participate in the transient remodeling of neuronal connectivity in the preoptic area, the arcuate nucleus,the median eminence, and other brain regions involved in the control of reproduction. Gonadal hormones regulate glial plasticityby direct and indirect effects and regulate various other endocrine signals, local soluble factors and adhesion moleculesthat also affect glial function and glia-to-neuron communication. The glial cells, therefore, are central to the coordinationof endocrine and local inputs that bring about neural plasticity and adapt reproductive capacity to homeostatic signals.

Garcia-Segura, Luis Miguel; Lorenz, Betty; DonCarlos, Lydia L

doi: 10.1530/rep-07-0540pmid: 18367504

Neuron-to-glia, glia-to-neuron, and glia-to-glia communication are implicated in the modulation of neuronal activity and synaptic transmission relevant to reproduction. Glial cells play an important role in neuroendocrine regulation and participate in the sexual differentiation of neuronal connectivity of brain regions involved in the control of reproductive neuroendocrine output. During puberty, modifications in the morphology and chemistry of astrocytes and tanycytes in the hypothalamus and median eminence influence the maturation of the neuronal circuits controlling the secretion of GnRH. During adult reproductive life, the glial cells participate in the transient remodeling of neuronal connectivity in the preoptic area, the arcuate nucleus, the median eminence, and other brain regions involved in the control of reproduction. Gonadal hormones regulate glial plasticity by direct and indirect effects and regulate various other endocrine signals, local soluble factors and adhesion molecules that also affect glial function and glia-to-neuron communication. The glial cells, therefore, are central to the coordination of endocrine and local inputs that bring about neural plasticity and adapt reproductive capacity to homeostatic signals.

Gall, Laurence; Bourhis, Daniel Le; Ruffini, Sylvie; Boulesteix, Claire; Vignon, Xavier

doi: 10.1530/REP-07-0284pmid: 18296513

It is clear from a wide range of studies that the nuclear/cytoplasmic distribution of Cdc25C has important functional consequencesfor cell cycle control. It is now admitted that in somatic cells, the localization of Cdc25C in the cytoplasm is requiredto maintain the cell in an interphasic state and that Cdc25C has to translocate to the nucleus just before M-phase to inducemitotic events. We characterized the expression and localization of Cdc25C during oocyte maturation, the first embryo mitosis,and the first steps of somatic cell nuclear transfer (SCNT) in cattle. We demonstrated that Cdc25C was expressed throughoutthe maturation process and the early development. We clearly showed that Cdc25C was localized in the nucleus at the germinalvesicle stage and during the early development until the blastocyst stage. However, the signal change in blastocyst and Cdc25Cbecame cytoplasmic as is the case in somatic cells. Thus, oocytes and early embryonic cells presented a specific nuclear Cdc25Clocalization different from the one observed in somatic cells, suggesting that Cdc25C could have a particular localization/regulationin undifferentiated cells. Following SCNT, Cdc25C became nuclear as soon as the nucleus swelled, and this localization persisteduntil the blastocyst stage, as is the case in in vitro fertilized embryos. The Cdc25C nuclear localization appeared to constitute a major change, which could be associated withthe reorganization of the somatic nucleus upon nuclear transfer.

Gall, Laurence; Le Bourhis, Daniel; Ruffini, Sylvie; Boulesteix, Claire; Vignon, Xavier

doi: 10.1530/rep-07-0284pmid: 18296513

It is clear from a wide range of studies that the nuclear/cytoplasmic distribution of Cdc25C has important functional consequences for cell cycle control. It is now admitted that in somatic cells, the localization of Cdc25C in the cytoplasm is required to maintain the cell in an interphasic state and that Cdc25C has to translocate to the nucleus just before M-phase to induce mitotic events. We characterized the expression and localization of Cdc25C during oocyte maturation, the first embryo mitosis, and the first steps of somatic cell nuclear transfer (SCNT) in cattle. We demonstrated that Cdc25C was expressed throughout the maturation process and the early development. We clearly showed that Cdc25C was localized in the nucleus at the germinal vesicle stage and during the early development until the blastocyst stage. However, the signal change in blastocyst and Cdc25C became cytoplasmic as is the case in somatic cells. Thus, oocytes and early embryonic cells presented a specific nuclear Cdc25C localization different from the one observed in somatic cells, suggesting that Cdc25C could have a particular localization/regulation in undifferentiated cells. Following SCNT, Cdc25C became nuclear as soon as the nucleus swelled, and this localization persisted until the blastocyst stage, as is the case in in vitro fertilized embryos. The Cdc25C nuclear localization appeared to constitute a major change, which could be associated with the reorganization of the somatic nucleus upon nuclear transfer.

Vallée, Maud; Aiba, Kazuhiro; Piao, Yulan; Palin, Marie-France; Ko, Minoru S H; Sirard, Marc-André

doi: 10.1530/REP-07-0342pmid: 18367505

Cross-species comparison of gene expression is a powerful approach for discovering genes that have been conserved throughoutevolution. Conserved genes are presumably very important in the mechanisms related to the unique molecular functions in oocytes.The objective of this study was to identify genes expressed in the oocyte and conserved across three diverse vertebrate species.We report the global gene expression profiles of Bos taurus and Xenopus laevis oocytes on an NIA mouse development microarray that consists of 60-mer oligonucleotide probes representing more than 20 000mouse transcripts derived from stem cell, oocyte, and early embryo cDNA libraries. Analysis based on intensity values revealedthat 9853 and 10 046 genes are expressed in bovine and Xenopus oocytes respectively. Furthermore, previously published microarray data on preimplantation development in the mouse wereused for a comparative analysis of global oocyte gene expression profiles. Interestingly, a substantial proportion of thegenes expressed in mouse oocytes is conserved between the three species (74%, 7275 genes). Moreover, functional annotationof these conserved oocyte-expressed genes confirmed that certain functions are conserved among the three species. RNA metabolismand cell cycle were among the over-represented Gene Ontology terms in the biological process category. Finally, a pattern-matchinganalysis identified 208 conserved maternally expressed genes. Results from these cross-species hybridizations allowed numerousgenes expressed in oocytes and conserved between Mus musculus, B. taurus, and X. laevis to be identified. This comparative analysis of oocyte transcript profiles revealed a high degree of conservation among species.

Vallée, Maud; Aiba, Kazuhiro; Piao, Yulan; Palin, Marie-France; Ko, Minoru S H; Sirard, Marc-André

doi: 10.1530/rep-07-0342pmid: 18367505

Cross-species comparison of gene expression is a powerful approach for discovering genes that have been conserved throughout evolution. Conserved genes are presumably very important in the mechanisms related to the unique molecular functions in oocytes. The objective of this study was to identify genes expressed in the oocyte and conserved across three diverse vertebrate species. We report the global gene expression profiles of Bos taurus and Xenopus laevis oocytes on an NIA mouse development microarray that consists of 60-mer oligonucleotide probes representing more than 20 000 mouse transcripts derived from stem cell, oocyte, and early embryo cDNA libraries. Analysis based on intensity values revealed that 9853 and 10 046 genes are expressed in bovine and Xenopus oocytes respectively. Furthermore, previously published microarray data on preimplantation development in the mouse were used for a comparative analysis of global oocyte gene expression profiles. Interestingly, a substantial proportion of the genes expressed in mouse oocytes is conserved between the three species (74%, 7275 genes). Moreover, functional annotation of these conserved oocyte-expressed genes confirmed that certain functions are conserved among the three species. RNA metabolism and cell cycle were among the over-represented Gene Ontology terms in the biological process category. Finally, a pattern-matching analysis identified 208 conserved maternally expressed genes. Results from these cross-species hybridizations allowed numerous genes expressed in oocytes and conserved between Mus musculus, B. taurus, and X. laevis to be identified. This comparative analysis of oocyte transcript profiles revealed a high degree of conservation among species.

Martínez-Pastor, Felipe; Cabrita, Elsa; Soares, Florbela; Anel, Luis; Dinis, Maria Teresa

doi: 10.1530/REP-07-0376pmid: 18367506

Computer-assisted sperm analysis (CASA) and clustering analysis have enabled to study sperm subpopulations in mammals, buttheir use in fish sperm has been limited. We have used spermatozoa from Senegalese sole (Solea senegalensis) as a model for subpopulation analysis in teleostei using two different activating solutions. Semen from six males was activatedusing 1100 mOsm/kg solutions: artificial seawater (ASW) or sucrose solution (SUC). Motility was acquired at 15, 30, 45, and60 s post-activation. CASA parameters were combined into two principal components, which were used in a non-hierarchical clusteringanalysis, obtaining four subpopulations (CL): CL1 (slow/non-linear), CL2 (slow/linear), CL3 (fast/non-linear), and CL4 (fast/linear).We detected spermatozoa lysis, especially in ASW. Sperm motility was higher for SUC and decreased with time. The subpopulationproportions varied with time and activating treatment, showing both an increase in CL1 and CL2 and a decrease in CL3 and CL4with time. Both CL3 and CL4 were higher in samples activated with SUC, at least in early post-activation. Proportions of CL3and CL4 at 15 s were associated with higher quality at 60 s and with lower lysis. A second clustering analysis was conducted,classifying the males accordingly to their motility subpopulations. This analysis showed a high heterogeneity between samples.Subpopulation analysis of CASA data can be applied to Solea spermatozoa, allowing identification of potentially interesting sperm subpopulations. Future studies might benefit from thesetechniques to establish the relationship of these subpopulations with fish sperm quality and fertility, helping to characterizemales according to their reproductive potential.

Martínez-Pastor, Felipe; Cabrita, Elsa; Soares, Florbela; Anel, Luis; Dinis, Maria Teresa

doi: 10.1530/rep-07-0376pmid: 18367506

Computer-assisted sperm analysis (CASA) and clustering analysis have enabled to study sperm subpopulations in mammals, but their use in fish sperm has been limited. We have used spermatozoa from Senegalese sole (Solea senegalensis) as a model for subpopulation analysis in teleostei using two different activating solutions. Semen from six males was activated using 1100 mOsm/kg solutions: artificial seawater (ASW) or sucrose solution (SUC). Motility was acquired at 15, 30, 45, and 60 s post-activation. CASA parameters were combined into two principal components, which were used in a non-hierarchical clustering analysis, obtaining four subpopulations (CL): CL1 (slow/non-linear), CL2 (slow/linear), CL3 (fast/non-linear), and CL4 (fast/linear). We detected spermatozoa lysis, especially in ASW. Sperm motility was higher for SUC and decreased with time. The subpopulation proportions varied with time and activating treatment, showing both an increase in CL1 and CL2 and a decrease in CL3 and CL4 with time. Both CL3 and CL4 were higher in samples activated with SUC, at least in early post-activation. Proportions of CL3 and CL4 at 15 s were associated with higher quality at 60 s and with lower lysis. A second clustering analysis was conducted, classifying the males accordingly to their motility subpopulations. This analysis showed a high heterogeneity between samples. Subpopulation analysis of CASA data can be applied to Solea spermatozoa, allowing identification of potentially interesting sperm subpopulations. Future studies might benefit from these techniques to establish the relationship of these subpopulations with fish sperm quality and fertility, helping to characterize males according to their reproductive potential.

Ardón, Florencia; Helms, Dietmar; Sahin, Evrim; Bollwein, Heinrich; Töpfer-Petersen, Edda; Waberski, Dagmar

doi: 10.1530/rep-07-0333pmid: 18367507

In the present study, the prevalence of chromatin instability in the fertilizing-competent sperm population in the porcine oviduct in vivo was examined through qualitative analysis of the chromatin structure status of accessory boar sperm found in in vivo-derived embryos. The binding of chromatin-unstable sperm to oviductal epithelium in vitro was also studied. To examine the sperm chromatin state, a modified fluorescence microscopic sperm chromatin structure assay was used. Among a population of 173 fertile boars, individuals were selected for according to their chromatin status: 25 animals showed more than 5% of chromatin-unstable sperm in their ejaculates, and 7 showed consistently elevated percentages of chromatin-unstable sperm in three successively collected semen samples. A positive correlation was found between incidence of chromatin instability and attached cytoplasmic droplets (r=0.44, P<0.01). Analyses of accessory spermatozoa from in vivo-derived embryos demonstrated that the proportion of chromatin-unstable sperm was significantly (P<0.05) reduced in the population of fertilizing-competent sperm in the oviduct compared with the inseminated sperm. Populations of sperm bound to the oviduct in vitro had significantly (P<0.05) lower percentages of chromatin instability than in the original diluted semen sample. In conclusion, numbers of sperm with unstable chromatin are reduced in the oviductal sperm reservoir, possibly because of associated changes in the plasma membrane that prevent sperm from binding to the oviductal epithelium. We conclude that in vivo the likelihood that sperm with unstable chromatin will reach the egg and fertilize it is low.

Ardón, Florencia; Helms, Dietmar; Sahin, Evrim; Bollwein, Heinrich; Töpfer-Petersen, Edda; Waberski, Dagmar

doi: 10.1530/REP-07-0333pmid: 18367507

In the present study, the prevalence of chromatin instability in the fertilizing-competent sperm population in the porcineoviduct in vivo was examined through qualitative analysis of the chromatin structure status of accessory boar sperm found in in vivo-derived embryos. The binding of chromatin-unstable sperm to oviductal epithelium in vitro was also studied. To examine the sperm chromatin state, a modified fluorescence microscopic sperm chromatin structure assaywas used. Among a population of 173 fertile boars, individuals were selected for according to their chromatin status: 25 animalsshowed more than 5% of chromatin-unstable sperm in their ejaculates, and 7 showed consistently elevated percentages of chromatin-unstablesperm in three successively collected semen samples. A positive correlation was found between incidence of chromatin instabilityand attached cytoplasmic droplets (r=0.44, P<0.01). Analyses of accessory spermatozoa from in vivo-derived embryos demonstrated that the proportion of chromatin-unstable sperm was significantly (P<0.05) reduced in the population of fertilizing-competent sperm in the oviduct compared with the inseminated sperm. Populationsof sperm bound to the oviduct in vitro had significantly (P<0.05) lower percentages of chromatin instability than in the original diluted semen sample. In conclusion, numbers of spermwith unstable chromatin are reduced in the oviductal sperm reservoir, possibly because of associated changes in the plasmamembrane that prevent sperm from binding to the oviductal epithelium. We conclude that in vivo the likelihood that sperm with unstable chromatin will reach the egg and fertilize it is low.