The Safe Use of a PTEN Inhibitor for the Activation of Dormant Mouse Primordial Follicles and Generation of Fertilizable Eggs

Primordial ovarian follicles, which are often present in the ovaries of premature ovarian failure (POF) patients or are cryopreserved from the ovaries of young cancer patients who are undergoing gonadotoxic anticancer therapies, cannot be used to generate mature oocytes for in vitro fertilization (IVF). There has been very little success in triggering growth of primordial follicles to obtain fertilizable oocytes due to the poor understanding of the biology of primordial follicle activation.We have recently reported that PTEN (phosphatase and tensin homolog deleted on chromosome ten) prevents primordial follicle activation in mice, and deletion of Pten from the oocytes of primordial follicles leads to follicular activation. Consequently, the PTEN inhibitor has been successfully used in vitro to activate primordial follicles in both mouse and human ovaries. These results suggest that PTEN inhibitors could be used in ovarian culture medium to trigger the activation of primordial follicle. To study the safety and efficacy of the use of such inhibitors, we activated primordial follicles from neonatal mouse ovaries by transient treatment with a PTEN inhibitor bpV(HOpic). These ovaries were then transplanted under the kidney capsules of recipient mice to generate mature oocytes. The mature oocytes were fertilized in vitro and progeny mice were obtained after embryo transfer.Long-term monitoring up to the second generation of progeny mice showed that the mice were reproductively active and were free from any overt signs or symptoms of chronic illnesses. Our results indicate that the use of PTEN inhibitors could be a safe and effective way of generating mature human oocytes for use in novel IVF techniques

Mastl is required for timely activation of APC/C in meiosis I and Cdk1 reactivation in meiosis II

In mitosis, the Greatwall kinase (called microtubule-associated serine/threonine kinase like [Mastl] in mammals) is essential for prometaphase entry or progression by suppressing protein phosphatase 2A (PP2A) activity. PP2A suppression in turn leads to high levels of Cdk1 substrate phosphorylation. We have used a mouse model with an oocyte-specific deletion of Mastl to show that Mastl-null oocytes resume meiosis I and reach metaphase I normally but that the onset and completion of anaphase I are delayed. Moreover, after the completion of meiosis I, Mastl-null oocytes failed to enter meiosis II (MII) because they reassembled a nuclear structure containing decondensed chromatin. Our results show that Mastl is required for the timely activation of anaphase-promoting complex/cyclosome to allow meiosis I exit and for the rapid rise of Cdk1 activity that is needed for the entry into MII in mouse oocytes

Pharmacological inhibition of mTORC1 prevents over-activation of the primordial follicle pool in response to elevated PI3K signaling.

The majority of ovarian primordial follicles must be preserved in a quiescent state to allow for the regular production of gametes over the female reproductive lifespan. However, the molecular mechanism that maintains the long quiescence of primordial follicles is poorly understood. Under certain pathological conditions, the entire pool of primordial follicles matures simultaneously leading to an accelerated loss of primordial follicles and to premature ovarian failure (POF). We have previously shown that loss of Pten (phosphatase and tensin homolog deleted on chromosome ten) in mouse oocytes leads to premature activation of the entire pool of primordial follicles, subsequent follicular depletion in early adulthood, and the onset of POF. Lack of PTEN leads to increased phosphatidylinositol 3-kinase (PI3K)-Akt and mammalian target of rapamycin complex 1 (mTORC1) signaling in the oocytes. To study the functional and pathological roles of elevated mTORC1 signaling in the oocytes, we treated the Pten-mutant mice with the specific mTORC1 inhibitor rapamycin. When administered to Pten-deficient mice prior to the activation of the primordial follicles, rapamycin effectively prevented global follicular activation and preserved the ovarian reserve. These results provide a rationale for exploring the possible use of rapamycin as a drug for the preservation of the primordial follicle pool, and the possible prevention of POF

Enhanced Akt-rpS6 activation and <i>in vitro</i> inhibition of rpS6 activation in Oo<i>Pten</i>^−/− oocytes by rapamycin.

<p>(<b>A</b>) Comparison of Akt-rpS6 signaling in Oo<i>Pten</i>^−/− and Oo<i>Pten</i>^+/+ oocytes. Oocytes were isolated from ovaries of mice at postnatal day 12–14 and immunoblotting was performed as described in <i>Materials and Methods</i>. Loss of PTEN led to enhanced PI3K signaling as indicated by an increase in phosphorylated Akt (p-Akt). The level of phosphorylated rpS6 (p-rpS6) was also increased in Oo<i>Pten</i>^−/− oocytes compared with Oo<i>Pten</i>^+/+ oocytes. Levels of total rpS6, Akt, and β-actin were used as internal controls. (<b>B</b>) Activation of rpS6 in Oo<i>Pten</i>^−/− oocytes is dependent on mTORC1 signaling. Oocytes were isolated from ovaries of Oo<i>Pten</i>^−/− mice at PD 12–14 as described in <i>Materials and Methods</i>. Treatment of oocytes with the mTORC1-specific inhibitor rapamycin (Rapa, 50 nM) for 2 h was found to largely suppress levels of phosphorylated rpS6 (p-rpS6), but did not affect the level of phosphorylated Akt (p-Akt). As a control, treatment of Oo<i>Pten</i>^−/− oocytes with the PI3K-specific inhibitor LY294002 (LY, 50 µM) for 2 h also largely suppressed levels of phosphorylated rpS6 (p-rpS6), but it also suppressed the level of phosphorylated Akt (p-Akt). This suggests that activation of rpS6 in Oo<i>Pten</i>^−/− oocytes is dependent on both PI3K and mTORC1 signaling. Levels of total Akt, rpS6, and β-actin were used as internal controls.</p

Transcriptomic survey of key reproductive and metabolic tissues in mouse models of polycystic ovary syndrome

Transcriptomic mapping of key reproductive and metabolic tissues and oocytes in mouse models of polycystic ovary syndrome reveals that the hypothalamus is susceptible to fetal programming while adipose and ovary are more affected upon postnatal androgen exposure

Fertility measurement of the female mice directly injected with bpV(HOpic).

<p>(<b>A</b>) Weekly comparison of the cumulative number of pups for the low dose bpV(HOpic)-injected mice (n = 2, blue bars), high dose-injected mice (n = 2, red bars) and control mice (n = 2, black bars). All mice had been bred with CD-1 strain males. (<b>B</b>) Weekly comparison of the cumulative number of pups produced by breeding the F1 mice. Breeding between F1 males and F1 females produced by low dose-injected females (n = 2, red bars), breeding between F1 males and F1 females produced by high dose-injected females (n = 2, green bars) and breeding between F1 males and F1 females produced by PBS-injected females (n = 2, black bars). n = number of breeding pairs used.</p

Prenatal androgen exposure causes a sexually dimorphic transgenerational increase in offspring susceptibility to anxiety disorders

If and how obesity and elevated androgens in women with polycystic ovary syndrome (PCOS) affect their offspring’s psychiatric health is unclear. Using data from Swedish population health registers, we showed that daughters of mothers with PCOS have a 78% increased risk of being diagnosed with anxiety disorders. We next generated a PCOS-like mouse (F0) model induced by androgen exposure during late gestation, with or without diet-induced maternal obesity, and showed that the first generation (F1) female offspring develop anxiety-like behavior, which is transgenerationally transmitted through the female germline into the third generation of female offspring (F3) in the androgenized lineage. In contrast, following the male germline, F3 male offspring (mF3) displayed anxiety-like behavior in the androgenized and the obese lineages. Using a targeted approach to search for molecular targets within the amygdala, we identified five differentially expressed genes involved in anxiety-like behavior in F3 females in the androgenized lineage and eight genes in the obese lineage. In mF3 male offspring, three genes were dysregulated in the obese lineage but none in the androgenized lineage. Finally, we performed in vitro fertilization (IVF) using a PCOS mouse model of continuous androgen exposure. We showed that the IVF generated F1 and F2 offspring in the female germline did not develop anxiety-like behavior, while the F2 male offspring (mF2) in the male germline did. Our findings provide evidence that elevated maternal androgens in PCOS and maternal obesity may underlie the risk of a transgenerational transmission of anxiety disorders in children of women with PCOS.CC BY 4.0Correspondence: Elisabet Stener-Victorin ([email protected])</p

Enhanced follicular development by transient treatment of neonatal mouse ovaries with the PTEN inhibitor bpV(HOpic).

<p>(<b>A</b>) Comparison between the sizes of treated and control ovaries transplanted under the kidney capsules. One ovary from a PD3 mouse was cultured for 24 h with 1 µM bpV(HOpic) and another ovary was cultured without bpV(HOpic) and then transplanted under the capsule of each kidney of the same ovariectomized recipient as described in <i>Materials and Methods</i>. Ovaries that were treated with bpV(HOpic) before transplantation grew bigger than the non-treated control ovaries. K represents kidney tissue from the recipient, O represents the transplanted ovary, and the ovarian border is outlined by dashed circles. Scale bar = 1 mm. (<b>B</b>) Morphological analysis of treated and control ovaries excised from the kidney capsules. Ovaries from PD3 mice were cultured for 24 h with or without 1 µM bpV(HOpic) before transplantation under each kidney capsule of the same ovariectomized recipient as described in <i>Materials and Methods</i>. One day after the transplantation, recipient mice were treated daily with 2 IU of pregnant mare serum gonadotropin for 18 days. Fourteen hours before being killed, the mice were treated with 5 IU of human chorionic gonadotropin. Ovaries were excised from the kidney capsules and embedded in paraffin, and serial sections of 8 µm thickness were prepared and stained with hematoxylin. A larger number of antral follicles were observed in the bpV(HOpic)-treated ovaries (arrows) than in the control ovaries. The experiments were repeated at least 4 times, and 5 mice were used each time. Scale bar = 250 µm.</p