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Lhx8 regulates primordial follicle activation and postnatal folliculogenesis.

Ren Y, Suzuki H, Jagarlamudi K, Golnoski K, McGuire M, Lopes R, Pachnis V, Rajkovic A - BMC Biol. (2015)

Bottom Line: The conditional deficiency of Lhx8 in the oocytes of primordial follicles leads to massive primordial oocyte activation, in part, by indirectly interacting with the PI3K-AKT pathway, as shown by synergistic effects on FOXO3 nucleocytoplasmic translocation and rpS6 activation.However, LHX8 does not directly regulate members of the PI3K-AKT pathway; instead, we show that LHX8 represses Lin28a expression, a known regulator of mammalian metabolism and of the AKT/mTOR pathway.Our results indicate that the LHX8-LIN28A pathway is essential in the earliest stages of primordial follicle activation, and LHX8 is an important oocyte-specific transcription factor in the ovary for regulating postnatal folliculogenesis.

View Article: PubMed Central - PubMed

Affiliation: Magee-Womens Research Institute, Department of Obstetrics, Gynecology and Reproductive Sciences, University of Pittsburgh, Pittsburgh, PA, 15213, USA. yur4@pitt.edu.

ABSTRACT

Background: The early stages of ovarian follicle formation-beginning with the breakdown of germ cell cysts and continuing with the formation of primordial follicles and transition to primary and secondary follicles-are critical in determining reproductive life span and fertility. Previously, we discovered that global knockouts of germ cell-specific transcriptional co-regulators Sohlh1, Sohlh2, Lhx8, and Nobox, cause rapid oocyte loss and ovarian failure. Also factors such as Nobox and Sohlh1 are associated with human premature ovarian failure. In this study, we developed a conditional knockout of Lhx8 to study oocyte-specific pathways in postnatal folliculogenesis.

Results: The conditional deficiency of Lhx8 in the oocytes of primordial follicles leads to massive primordial oocyte activation, in part, by indirectly interacting with the PI3K-AKT pathway, as shown by synergistic effects on FOXO3 nucleocytoplasmic translocation and rpS6 activation. However, LHX8 does not directly regulate members of the PI3K-AKT pathway; instead, we show that LHX8 represses Lin28a expression, a known regulator of mammalian metabolism and of the AKT/mTOR pathway. LHX8 can bind to the Lin28a promoter, and the depletion of Lin28a in Lhx8-deficient oocytes partially suppresses primordial oocyte activation. Moreover, unlike the PI3K-AKT pathway, LHX8 is critical beyond primordial follicle activation, and blocks the primary to secondary follicle transition.

Conclusions: Our results indicate that the LHX8-LIN28A pathway is essential in the earliest stages of primordial follicle activation, and LHX8 is an important oocyte-specific transcription factor in the ovary for regulating postnatal folliculogenesis.

No MeSH data available.


Related in: MedlinePlus

Lhx8 and Pten conditional knockout effects on FOXO3 localization. a–c In control mice (Lhx8flx/flx), FOXO3 is expressed in the nucleus and cytoplasm of primordial oocytes (PF, arrows in c'). d–f In Lhx8flx/flxGdf9Cre mice, the extensive nucleocytoplasmic translocation is not observed in activated primordial follicles between 20 and 30 μm (aPF, arrow in f') but is noted in activated primordial follicles larger than 30 μm (aPF, arrow in F'). d–i A similar expression pattern of FOXO3 localization exists in Pten conditional knockout (Ptenflx/flxGdf9Cre) mice. The arrows in i' represent primordial follicles (PF) with both nuclear and cytoplasm expression of FOXO3 and cytoplasm expression of FOXO3 in activated primordial follicles (aPF) below 30 μm. j–l However, in mice that are conditionally deficient in both Lhx8 and Pten (Lhx8flx/flxPtenflx/flxGdf9Cre), FOXO3 nucleocytoplasmic translocation is present in primordial, activated, and primary oocytes. The negative control is immunofluorescence in the presence of secondary antibodies and is shown in m–o. The boxed areas in C, F, I, L, and O are shown magnified in C', F', I', L', and O'. p Graphic representation of FOXO3 distribution (cytoplasm only or nucleus and cytoplasm). Oocytes were grouped by size (diameter) as less than 20 μm, between 20 and 30 μm, and greater than 30 μm. Only oocytes with clear DAPI nuclear staining were counted. Scale bars: 50 μm (A–C, D–F, G–I, J–L, and M–O); 20 μm (C', F', I', L', and O')
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Fig3: Lhx8 and Pten conditional knockout effects on FOXO3 localization. a–c In control mice (Lhx8flx/flx), FOXO3 is expressed in the nucleus and cytoplasm of primordial oocytes (PF, arrows in c'). d–f In Lhx8flx/flxGdf9Cre mice, the extensive nucleocytoplasmic translocation is not observed in activated primordial follicles between 20 and 30 μm (aPF, arrow in f') but is noted in activated primordial follicles larger than 30 μm (aPF, arrow in F'). d–i A similar expression pattern of FOXO3 localization exists in Pten conditional knockout (Ptenflx/flxGdf9Cre) mice. The arrows in i' represent primordial follicles (PF) with both nuclear and cytoplasm expression of FOXO3 and cytoplasm expression of FOXO3 in activated primordial follicles (aPF) below 30 μm. j–l However, in mice that are conditionally deficient in both Lhx8 and Pten (Lhx8flx/flxPtenflx/flxGdf9Cre), FOXO3 nucleocytoplasmic translocation is present in primordial, activated, and primary oocytes. The negative control is immunofluorescence in the presence of secondary antibodies and is shown in m–o. The boxed areas in C, F, I, L, and O are shown magnified in C', F', I', L', and O'. p Graphic representation of FOXO3 distribution (cytoplasm only or nucleus and cytoplasm). Oocytes were grouped by size (diameter) as less than 20 μm, between 20 and 30 μm, and greater than 30 μm. Only oocytes with clear DAPI nuclear staining were counted. Scale bars: 50 μm (A–C, D–F, G–I, J–L, and M–O); 20 μm (C', F', I', L', and O')

Mentions: Previous studies have shown FOXO3 nucleocytoplasmic translocation and rpS6 phosphorylation via the PI3K-AKT-mTOR pathways to be associated with PFA [5, 6, 8]. We examined FOXO3 nucleocytoplasmic translocation and rpS6 phosphorylation in PD7 Pten and Lhx8 conditional knockouts (Fig. 3 and Additional file 4: Figure S4). FOXO3 nucleocytoplasmic translocation was prominent in oocytes larger than 30 μm but did not show obvious translocation among oocytes between 20 and 30 μm in Lhx8flx/flxGdf9Cre mice (Fig. 3d–f, f', p). Oocytes of primordial follicles in Pten and Lhx8 conditional single knockouts as well as corresponding controls did not show FOXO3 nucleocytoplasmic translocation. However, FOXO3 nucleocytoplasmic translocation was significantly induced in the primordial (<20 μm) oocytes (Fig. 3j–l, l', p) of the PD7 double Lhx8/Pten conditional knockouts (Lhx8flx/flxPtenflx/flxGdf9Cre). These data indicate a synergistic action of the LHX8 and PTEN proteins on FOXO3 nucleocytoplasmic translocation.Fig. 3


Lhx8 regulates primordial follicle activation and postnatal folliculogenesis.

Ren Y, Suzuki H, Jagarlamudi K, Golnoski K, McGuire M, Lopes R, Pachnis V, Rajkovic A - BMC Biol. (2015)

Lhx8 and Pten conditional knockout effects on FOXO3 localization. a–c In control mice (Lhx8flx/flx), FOXO3 is expressed in the nucleus and cytoplasm of primordial oocytes (PF, arrows in c'). d–f In Lhx8flx/flxGdf9Cre mice, the extensive nucleocytoplasmic translocation is not observed in activated primordial follicles between 20 and 30 μm (aPF, arrow in f') but is noted in activated primordial follicles larger than 30 μm (aPF, arrow in F'). d–i A similar expression pattern of FOXO3 localization exists in Pten conditional knockout (Ptenflx/flxGdf9Cre) mice. The arrows in i' represent primordial follicles (PF) with both nuclear and cytoplasm expression of FOXO3 and cytoplasm expression of FOXO3 in activated primordial follicles (aPF) below 30 μm. j–l However, in mice that are conditionally deficient in both Lhx8 and Pten (Lhx8flx/flxPtenflx/flxGdf9Cre), FOXO3 nucleocytoplasmic translocation is present in primordial, activated, and primary oocytes. The negative control is immunofluorescence in the presence of secondary antibodies and is shown in m–o. The boxed areas in C, F, I, L, and O are shown magnified in C', F', I', L', and O'. p Graphic representation of FOXO3 distribution (cytoplasm only or nucleus and cytoplasm). Oocytes were grouped by size (diameter) as less than 20 μm, between 20 and 30 μm, and greater than 30 μm. Only oocytes with clear DAPI nuclear staining were counted. Scale bars: 50 μm (A–C, D–F, G–I, J–L, and M–O); 20 μm (C', F', I', L', and O')
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Related In: Results  -  Collection

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Fig3: Lhx8 and Pten conditional knockout effects on FOXO3 localization. a–c In control mice (Lhx8flx/flx), FOXO3 is expressed in the nucleus and cytoplasm of primordial oocytes (PF, arrows in c'). d–f In Lhx8flx/flxGdf9Cre mice, the extensive nucleocytoplasmic translocation is not observed in activated primordial follicles between 20 and 30 μm (aPF, arrow in f') but is noted in activated primordial follicles larger than 30 μm (aPF, arrow in F'). d–i A similar expression pattern of FOXO3 localization exists in Pten conditional knockout (Ptenflx/flxGdf9Cre) mice. The arrows in i' represent primordial follicles (PF) with both nuclear and cytoplasm expression of FOXO3 and cytoplasm expression of FOXO3 in activated primordial follicles (aPF) below 30 μm. j–l However, in mice that are conditionally deficient in both Lhx8 and Pten (Lhx8flx/flxPtenflx/flxGdf9Cre), FOXO3 nucleocytoplasmic translocation is present in primordial, activated, and primary oocytes. The negative control is immunofluorescence in the presence of secondary antibodies and is shown in m–o. The boxed areas in C, F, I, L, and O are shown magnified in C', F', I', L', and O'. p Graphic representation of FOXO3 distribution (cytoplasm only or nucleus and cytoplasm). Oocytes were grouped by size (diameter) as less than 20 μm, between 20 and 30 μm, and greater than 30 μm. Only oocytes with clear DAPI nuclear staining were counted. Scale bars: 50 μm (A–C, D–F, G–I, J–L, and M–O); 20 μm (C', F', I', L', and O')
Mentions: Previous studies have shown FOXO3 nucleocytoplasmic translocation and rpS6 phosphorylation via the PI3K-AKT-mTOR pathways to be associated with PFA [5, 6, 8]. We examined FOXO3 nucleocytoplasmic translocation and rpS6 phosphorylation in PD7 Pten and Lhx8 conditional knockouts (Fig. 3 and Additional file 4: Figure S4). FOXO3 nucleocytoplasmic translocation was prominent in oocytes larger than 30 μm but did not show obvious translocation among oocytes between 20 and 30 μm in Lhx8flx/flxGdf9Cre mice (Fig. 3d–f, f', p). Oocytes of primordial follicles in Pten and Lhx8 conditional single knockouts as well as corresponding controls did not show FOXO3 nucleocytoplasmic translocation. However, FOXO3 nucleocytoplasmic translocation was significantly induced in the primordial (<20 μm) oocytes (Fig. 3j–l, l', p) of the PD7 double Lhx8/Pten conditional knockouts (Lhx8flx/flxPtenflx/flxGdf9Cre). These data indicate a synergistic action of the LHX8 and PTEN proteins on FOXO3 nucleocytoplasmic translocation.Fig. 3

Bottom Line: The conditional deficiency of Lhx8 in the oocytes of primordial follicles leads to massive primordial oocyte activation, in part, by indirectly interacting with the PI3K-AKT pathway, as shown by synergistic effects on FOXO3 nucleocytoplasmic translocation and rpS6 activation.However, LHX8 does not directly regulate members of the PI3K-AKT pathway; instead, we show that LHX8 represses Lin28a expression, a known regulator of mammalian metabolism and of the AKT/mTOR pathway.Our results indicate that the LHX8-LIN28A pathway is essential in the earliest stages of primordial follicle activation, and LHX8 is an important oocyte-specific transcription factor in the ovary for regulating postnatal folliculogenesis.

View Article: PubMed Central - PubMed

Affiliation: Magee-Womens Research Institute, Department of Obstetrics, Gynecology and Reproductive Sciences, University of Pittsburgh, Pittsburgh, PA, 15213, USA. yur4@pitt.edu.

ABSTRACT

Background: The early stages of ovarian follicle formation-beginning with the breakdown of germ cell cysts and continuing with the formation of primordial follicles and transition to primary and secondary follicles-are critical in determining reproductive life span and fertility. Previously, we discovered that global knockouts of germ cell-specific transcriptional co-regulators Sohlh1, Sohlh2, Lhx8, and Nobox, cause rapid oocyte loss and ovarian failure. Also factors such as Nobox and Sohlh1 are associated with human premature ovarian failure. In this study, we developed a conditional knockout of Lhx8 to study oocyte-specific pathways in postnatal folliculogenesis.

Results: The conditional deficiency of Lhx8 in the oocytes of primordial follicles leads to massive primordial oocyte activation, in part, by indirectly interacting with the PI3K-AKT pathway, as shown by synergistic effects on FOXO3 nucleocytoplasmic translocation and rpS6 activation. However, LHX8 does not directly regulate members of the PI3K-AKT pathway; instead, we show that LHX8 represses Lin28a expression, a known regulator of mammalian metabolism and of the AKT/mTOR pathway. LHX8 can bind to the Lin28a promoter, and the depletion of Lin28a in Lhx8-deficient oocytes partially suppresses primordial oocyte activation. Moreover, unlike the PI3K-AKT pathway, LHX8 is critical beyond primordial follicle activation, and blocks the primary to secondary follicle transition.

Conclusions: Our results indicate that the LHX8-LIN28A pathway is essential in the earliest stages of primordial follicle activation, and LHX8 is an important oocyte-specific transcription factor in the ovary for regulating postnatal folliculogenesis.

No MeSH data available.


Related in: MedlinePlus