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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 inactivation in primary follicles (Lhx8flx/flxZp3Cre) abolishes follicle growth. Histomorphological analysis was done on control (Lhx8flx/flx) and Lhx8 deficient ovaries (Lhx8flx/flxZp3Cre) at various stages of postnatal ovarian development ranging from newborn (PD0) to postnatal day 30 (PD30). a–f Periodic acid–Schiff (PAS) staining and counting of different follicle types in the newborn and PD7 ovaries showed no significant differences between control and Lhx8 deficient ovaries. g–o Anti-NOBOX antibodies were used to stain oocytes (brown immunoreactivity) in ovaries from PD14 (G and H), PD21 (J and K), and PD30 (M and N) mice. At PD14, the Lhx8flx/flxZp3Cre ovaries showed a significantly higher number of primary follicles (PrF) and significantly diminished number of secondary/preantral (SF/AF) follicles characterized by two or more layers of granulosa cells. At PD21 and PD30, the primary follicle pool did not differ significantly between Lhx8flx/flxZp3Cre and control ovaries; however, there was a marked decrease in the number of secondary and more advanced ovarian follicles in conditional knockouts including degenerating follicles without oocytes (marked by asterisks in insets in K and N). The primordial follicle (PF) pool remained relatively stable between PD14 and PD30, with no significant difference between Lhx8flx/flxZp3Cre and control ovaries. **P < 0.01. Scale bars: 100 μm (A, B, D, and E); 200 μm (G, H, J, K, M, and N); 50 μm (insets of K and N)
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Fig6: Lhx8 inactivation in primary follicles (Lhx8flx/flxZp3Cre) abolishes follicle growth. Histomorphological analysis was done on control (Lhx8flx/flx) and Lhx8 deficient ovaries (Lhx8flx/flxZp3Cre) at various stages of postnatal ovarian development ranging from newborn (PD0) to postnatal day 30 (PD30). a–f Periodic acid–Schiff (PAS) staining and counting of different follicle types in the newborn and PD7 ovaries showed no significant differences between control and Lhx8 deficient ovaries. g–o Anti-NOBOX antibodies were used to stain oocytes (brown immunoreactivity) in ovaries from PD14 (G and H), PD21 (J and K), and PD30 (M and N) mice. At PD14, the Lhx8flx/flxZp3Cre ovaries showed a significantly higher number of primary follicles (PrF) and significantly diminished number of secondary/preantral (SF/AF) follicles characterized by two or more layers of granulosa cells. At PD21 and PD30, the primary follicle pool did not differ significantly between Lhx8flx/flxZp3Cre and control ovaries; however, there was a marked decrease in the number of secondary and more advanced ovarian follicles in conditional knockouts including degenerating follicles without oocytes (marked by asterisks in insets in K and N). The primordial follicle (PF) pool remained relatively stable between PD14 and PD30, with no significant difference between Lhx8flx/flxZp3Cre and control ovaries. **P < 0.01. Scale bars: 100 μm (A, B, D, and E); 200 μm (G, H, J, K, M, and N); 50 μm (insets of K and N)

Mentions: Previous studies have shown that PTEN-regulated pathways are important in primordial oocyte activation, but not in primary oocytes [24]. We studied the role of Lhx8 in primary oocytes by generating Lhx8flx/flxZp3Cre mice. Zp3Cre is specifically expressed in primary oocytes, and Lhx8flx/flxZp3Cre ovaries continue to express LHX8 in primordial, but not primary, oocytes. Morphometric analyses revealed that the Lhx8flx/flxZp3Cre conditional knockout ovaries did not significantly differ from Lhx8flx/flx (control) mice at PD0 and PD7 (Fig. 6a–f). However, at PD14, we counted 159 ± 23 primary follicles and 29 ± 7 secondary/antral follicles per ovary in the Lhx8flx/flxZp3Cre mice, compared to 79 ± 6 primary follicles and 108 ± 7 secondary/antral follicles per ovary in the control mice (Fig. 6g–i). The relative increase of primary follicles at PD14 and the relative decrease of multilayer follicles in the Lhx8flx/flxZp3Cre ovary indicated that the transition from primary follicles to secondary follicles was blocked, which was consistent with the observation in Lhx8flx/flxGdf9Cre mice (Fig. 1f). At PD21 and PD30, we observed that many primary follicles were devoid of oocytes (Fig. 6k, n). We stained for LIN28A in PD21 ovaries and found that LIN28A was strongly expressed in Lhx8 deficient oocytes of the Lhx8flx/flxZp3Cre ovary, but no expression was detected in the empty follicles (see Additional file 6: Figure S5). However, excluding these empty primary follicles, the number of primary follicles between control and Lhx8flx/flxZp3Cre ovaries was not significantly different at PD21 or PD30 (Fig. 6l, o). For secondary/antral follicles, the number sharply dropped to 17 ± 3 at PD21 and to 2 ± 1 at PD30 in Lhx8flx/flxZp3Cre ovaries, compared to 170 ± 2 and 104 ± 4, respectively, in control mice. These findings imply that the growing follicle pool continued to be eliminated from the Lhx8flx/flxZp3Cre mice. Lhx8flx/flxZp3Cre mice were infertile (see Additional file 2: Figure S2A) and superovulation treatment of Lhx8flx/flxZp3Cre mice did not produce oocytes (see Additional file 2: Figure S2B). This result was in accord with the sharp fall of secondary/antral follicles in the Lhx8flx/flxZp3Cre ovary at PD21. Taken together, these data show that the folliculogenesis of Lhx8flx/flxZp3Cre mice is blocked in the transition from the primary to secondary follicle stage and results in primary oocyte death and infertility. The relative stability of the primordial follicle pool from PD14 to PD30 suggests that the PFA into primary follicles was not affected by the diminution in the number of secondary and more advanced ovarian follicles in Lhx8flx/flxZp3Cre ovaries.Fig. 6


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 inactivation in primary follicles (Lhx8flx/flxZp3Cre) abolishes follicle growth. Histomorphological analysis was done on control (Lhx8flx/flx) and Lhx8 deficient ovaries (Lhx8flx/flxZp3Cre) at various stages of postnatal ovarian development ranging from newborn (PD0) to postnatal day 30 (PD30). a–f Periodic acid–Schiff (PAS) staining and counting of different follicle types in the newborn and PD7 ovaries showed no significant differences between control and Lhx8 deficient ovaries. g–o Anti-NOBOX antibodies were used to stain oocytes (brown immunoreactivity) in ovaries from PD14 (G and H), PD21 (J and K), and PD30 (M and N) mice. At PD14, the Lhx8flx/flxZp3Cre ovaries showed a significantly higher number of primary follicles (PrF) and significantly diminished number of secondary/preantral (SF/AF) follicles characterized by two or more layers of granulosa cells. At PD21 and PD30, the primary follicle pool did not differ significantly between Lhx8flx/flxZp3Cre and control ovaries; however, there was a marked decrease in the number of secondary and more advanced ovarian follicles in conditional knockouts including degenerating follicles without oocytes (marked by asterisks in insets in K and N). The primordial follicle (PF) pool remained relatively stable between PD14 and PD30, with no significant difference between Lhx8flx/flxZp3Cre and control ovaries. **P < 0.01. Scale bars: 100 μm (A, B, D, and E); 200 μm (G, H, J, K, M, and N); 50 μm (insets of K and N)
© Copyright Policy - open-access
Related In: Results  -  Collection

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Fig6: Lhx8 inactivation in primary follicles (Lhx8flx/flxZp3Cre) abolishes follicle growth. Histomorphological analysis was done on control (Lhx8flx/flx) and Lhx8 deficient ovaries (Lhx8flx/flxZp3Cre) at various stages of postnatal ovarian development ranging from newborn (PD0) to postnatal day 30 (PD30). a–f Periodic acid–Schiff (PAS) staining and counting of different follicle types in the newborn and PD7 ovaries showed no significant differences between control and Lhx8 deficient ovaries. g–o Anti-NOBOX antibodies were used to stain oocytes (brown immunoreactivity) in ovaries from PD14 (G and H), PD21 (J and K), and PD30 (M and N) mice. At PD14, the Lhx8flx/flxZp3Cre ovaries showed a significantly higher number of primary follicles (PrF) and significantly diminished number of secondary/preantral (SF/AF) follicles characterized by two or more layers of granulosa cells. At PD21 and PD30, the primary follicle pool did not differ significantly between Lhx8flx/flxZp3Cre and control ovaries; however, there was a marked decrease in the number of secondary and more advanced ovarian follicles in conditional knockouts including degenerating follicles without oocytes (marked by asterisks in insets in K and N). The primordial follicle (PF) pool remained relatively stable between PD14 and PD30, with no significant difference between Lhx8flx/flxZp3Cre and control ovaries. **P < 0.01. Scale bars: 100 μm (A, B, D, and E); 200 μm (G, H, J, K, M, and N); 50 μm (insets of K and N)
Mentions: Previous studies have shown that PTEN-regulated pathways are important in primordial oocyte activation, but not in primary oocytes [24]. We studied the role of Lhx8 in primary oocytes by generating Lhx8flx/flxZp3Cre mice. Zp3Cre is specifically expressed in primary oocytes, and Lhx8flx/flxZp3Cre ovaries continue to express LHX8 in primordial, but not primary, oocytes. Morphometric analyses revealed that the Lhx8flx/flxZp3Cre conditional knockout ovaries did not significantly differ from Lhx8flx/flx (control) mice at PD0 and PD7 (Fig. 6a–f). However, at PD14, we counted 159 ± 23 primary follicles and 29 ± 7 secondary/antral follicles per ovary in the Lhx8flx/flxZp3Cre mice, compared to 79 ± 6 primary follicles and 108 ± 7 secondary/antral follicles per ovary in the control mice (Fig. 6g–i). The relative increase of primary follicles at PD14 and the relative decrease of multilayer follicles in the Lhx8flx/flxZp3Cre ovary indicated that the transition from primary follicles to secondary follicles was blocked, which was consistent with the observation in Lhx8flx/flxGdf9Cre mice (Fig. 1f). At PD21 and PD30, we observed that many primary follicles were devoid of oocytes (Fig. 6k, n). We stained for LIN28A in PD21 ovaries and found that LIN28A was strongly expressed in Lhx8 deficient oocytes of the Lhx8flx/flxZp3Cre ovary, but no expression was detected in the empty follicles (see Additional file 6: Figure S5). However, excluding these empty primary follicles, the number of primary follicles between control and Lhx8flx/flxZp3Cre ovaries was not significantly different at PD21 or PD30 (Fig. 6l, o). For secondary/antral follicles, the number sharply dropped to 17 ± 3 at PD21 and to 2 ± 1 at PD30 in Lhx8flx/flxZp3Cre ovaries, compared to 170 ± 2 and 104 ± 4, respectively, in control mice. These findings imply that the growing follicle pool continued to be eliminated from the Lhx8flx/flxZp3Cre mice. Lhx8flx/flxZp3Cre mice were infertile (see Additional file 2: Figure S2A) and superovulation treatment of Lhx8flx/flxZp3Cre mice did not produce oocytes (see Additional file 2: Figure S2B). This result was in accord with the sharp fall of secondary/antral follicles in the Lhx8flx/flxZp3Cre ovary at PD21. Taken together, these data show that the folliculogenesis of Lhx8flx/flxZp3Cre mice is blocked in the transition from the primary to secondary follicle stage and results in primary oocyte death and infertility. The relative stability of the primordial follicle pool from PD14 to PD30 suggests that the PFA into primary follicles was not affected by the diminution in the number of secondary and more advanced ovarian follicles in Lhx8flx/flxZp3Cre ovaries.Fig. 6

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