Glycolytic enzymes localize to ribonucleoprotein granules in Drosophila germ cells, bind Tudor and protect from transposable elements.

Germ cells give rise to all cell lineages in the next-generation and are responsible for the continuity of life. In a variety of organisms, germ cells and stem cells contain large ribonucleoprotein granules. Although these particles were discovered more than 100 years ago, their assembly and functions are not well understood. Here we report that glycolytic enzymes are components of these granules in Drosophila germ cells and both their mRNAs and the enzymes themselves are enriched in germ cells. We show that these enzymes are specifically required for germ cell development and that they protect their genomes from transposable elements, providing the first link between metabolism and transposon silencing. We further demonstrate that in the granules, glycolytic enzymes associate with the evolutionarily conserved Tudor protein. Our biochemical and single-particle EM structural analyses of purified Tudor show a flexible molecule and suggest a mechanism for the recruitment of glycolytic enzymes to the granules. Our data indicate that germ cells, similarly to stem cells and tumor cells, might prefer to produce energy through the glycolytic pathway, thus linking a particular metabolism to pluripotency.

Cross talk between estradiol and mTOR kinase in the regulation of ovarian granulosa proliferation.

Treatment of ovarian granulosa cells and follicles with the mammalian target of rapamycin (mTOR) kinase inhibitor results in biphasic effects where nanomolar rapamycin (RAP) results in reduced proliferation, mitotic anomalies, and attenuated follicle growth, while the picomolar RAP results in accelerated follicle growth. Here, we tested whether such effects are specific to RAP or could be mimicked by 2 alternative mTOR inhibitors, everolimus (EV) and temsirolimus (TEM), and whether these effects were dependent on the presence of estradiol (E2). Spontaneously immortalized rat granulosa cells (SIGCs) were cultured in dose curves of RAP, EV, TEM, or vehicle with or without E2. Proliferation and phosphorylation of mTOR targets p70S6 kinase and 4E-binding protein (BP) were determined. Cell cycle gene array analysis and confirmatory quantitative reverse transcriptase polymerase chain reaction were performed upon cells treated with picomolar RAP versus controls. Nanomolar RAP, EV, and TEM reduced SIGC proliferation and decreased phospho-p70 and 4E-BP. Picomolar concentrations accelerated proliferation without affecting mTOR substrate phosphorylation. Acceleration of growth by picomolar inhibitor required E2. Picomolar drug treatment altered the transcription of cell cycle regulators, increasing Integrin beta 1 and calcineurin expression, and decreasing inhibin alpha, Chek1, p16ARF, p27/Kip1, and Sestrin2 expression. At nanomolar concentrations, mTOR inhibitors attenuated granulosa proliferation. Accelerated growth and alterations in cell cycle gene transcription found with picomolar concentrations required E2 within the intrafollicular concentration range. The low concentrations of inhibitors required to increase granulosa proliferation suggest a novel use to support the growth of ovarian follicles.

Oocyte destruction is activated during viral infection.

Viral infection has been associated with a starvation-like state in Drosophila melanogaster. Because starvation and inhibiting TOR kinase activity in vivo result in blocked oocyte production, we hypothesized that viral infection would also result in compromised oogenesis. Wild-type flies were injected with flock house virus (FHV) and survival and embryo production were monitored. Infected flies had a dose-responsive loss of fecundity that corresponded to a global reduction in Akt/TOR signaling. Highly penetrant egg chamber destruction mid-way through oogenesis was noted and FHV coat protein was detected within developing egg chambers. As seen with in vivo TOR inhibition, oogenesis was partially rescued in loss of function discs large and merlin mutants. As expected, mutants in genes known to be involved in virus internalization and trafficking [Clathrin heavy chain (chc) and synaptotagmin] survive longer during infection. However, oogenesis was rescued only in chc mutants. This suggests that viral response mechanisms that control fly survival and egg chamber survival are separable. The genetic and signaling requirements for oocyte destruction delineated here represent a novel host-virus interaction with implications for the control of both fly and virus populations.

Adaptation to P element transposon invasion in Drosophila melanogaster.

Transposons evolve rapidly and can mobilize and trigger genetic instability. Piwi-interacting RNAs (piRNAs) silence these genome pathogens, but it is unclear how the piRNA pathway adapts to invasion of new transposons. In Drosophila, piRNAs are encoded by heterochromatic clusters and maternally deposited in the embryo. Paternally inherited P element transposons thus escape silencing and trigger a hybrid sterility syndrome termed P-M hybrid dysgenesis. We show that P-M hybrid dysgenesis activates both P elements and resident transposons and disrupts the piRNA biogenesis machinery. As dysgenic hybrids age, however, fertility is restored, P elements are silenced, and P element piRNAs are produced de novo. In addition, the piRNA biogenesis machinery assembles, and resident elements are silenced. Significantly, resident transposons insert into piRNA clusters, and these new insertions are transmitted to progeny, produce novel piRNAs, and are associated with reduced transposition. P element invasion thus triggers heritable changes in genome structure that appear to enhance transposon silencing.

mTOR kinase inhibition results in oocyte loss characterized by empty follicles in human ovarian cortical strips cultured in vitro.

OBJECTIVE: To determine whether oocyte loss is induced by mTOR kinase inhibition in human cortical strips as seen in model organisms in vivo and in vitro. DESIGN: Ovarian cortex was collected at two centers and cut into small strips. Strips were cultured for 6 days with or without the mTOR inhibitor rapamycin (RAP; 100 nM). Strips were then embedded in paraffin, and serial sections were prepared. SETTING: Samples were collected in general obstetric (Edinburgh), gynecologic surgery (New Haven), and fertility preservation assisted reproductive technology (ART) (New Haven) practices. PATIENT(S): Ovarian cortex collected from patients (15-34 years of age) during cesarean section (donated tissue) was removed for the purposes of fertility preservation or was prepared after oophorectomy. INTERVENTION(S): Tissue was used for research purposes only, with no subsequent patient intervention. MAIN OUTCOME MEASURE(S): Follicles were counted and assessed in each serial section. Caspase activity was monitored to determine whether mTOR inhibition activated apoptosis. RESULT(S): The RAP inclusion in cultures results in significantly fewer follicles compared with ethanol vehicle-treated controls. Furthermore, RAP treatment resulted in the induction of follicles that lacked an oocyte in any serial section (30/161 follicles vs. 1/347 ethanol vehicle-treated follicles). Caspase activity was not elevated by RAP treatment. CONCLUSION(S): mTOR inhibition results in a conserved destruction of the oocyte by adjacent granulosa cells (GC) in the absence of increased caspase activity. This model of oocyte loss is not consistent with classic apoptosis/atresia.

Intrinsic and extrinsic mechanisms of oocyte loss.

A great deal of evolutionary conservation has been found in the control of oocyte development, from invertebrates to women. However, little is known of mechanisms that control oocyte loss over time. Oocyte loss is often assumed to be a result of oocyte-intrinsic deficiencies or damage. In fruit flies, starvation results in halted oocyte production by germline stem cells and induces oocyte loss midway through development. When we fed wild-type flies the bacterial compound Rapamycin (RAP) to mimic starvation, production of new oocytes continued, but mid-stage loss sterilized the animals. Surprisingly, follicle cell invasion and phagocytosis of the oocyte preceded any signs of germ cell death. RAP-induced egg chamber loss was prevented when RAP receptor FKBP12 was knocked down specifically in follicle cells. Oogenesis continued past the mid-stages, and these mutants continued to lay embryos that could develop into normal adults. Hence, intact healthy oocytes can be destroyed by somatic cells responding to extrinsic stimuli. We termed this process inducible somatic oocyte destruction. RAP treatment of mouse follicles in vitro resulted in phagocytic uptake of the oocyte by granulosa cells as seen in flies. We hypothesize that extrinsic modes of oocyte loss occur in mammals.