These studies demonstrate differential activation and role of caspases in cisplatin injury, and provide the first evidence of cisplatin-induced induction of the Akt/PKB phosphorylation pathway, inhibition of which enhances activation of caspase-3 and caspase-9.

MicroRNAs (miRNAs) mediate posttranscriptional gene regulation by binding to the 3' untranslated region of messenger RNAs to either inhibit or enhance translation. The extent and hormonal regulation of miRNA expression by ovarian granulosa cells and their role in ovulation and luteinization is unknown. In the present study, miRNA array analysis was used to identify 212 mature miRNAs as expressed and 13 as differentially expressed in periovulatory granulosa cells collected before and after an ovulatory dose of hCG. Two miRNAs, Mirn132 and Mirn212 (also known as miR-132 and miR-212), were found to be highly upregulated following LH/hCG induction and were further analyzed. In vivo and in vitro temporal expression analysis by quantitative RT-PCR confirmed that LH/hCG and cAMP, respectively, increased transcription of the precursor transcript as well as the mature miRNAs. Locked nucleic acid oligonucleotides complementary to Mirn132 and Mirn212 were shown to block cAMP-mediated mature miRNA expression and function. Computational analyses indicated that 77 putative mRNA targets of Mirn132 and Mirn212 were expressed in ovarian granulosa cells. Furthermore, upon knockdown of Mirn132 and Mirn212, a known target of Mirn132, C-terminal binding protein 1, showed decreased protein levels but no change in mRNA levels. The following studies are the first to describe the extent of miRNA expression within ovarian granulosa cells and the first to demonstrate that LH/hCG regulates the expression of select miRNAs, which affect posttranscriptional gene regulation within these cells.

The ribonuclease III endonuclease, Dicer1 (also known as Dicer), is essential for the synthesis of the 19-25 nucleotide noncoding RNAs known as micro-RNAs (miRNAs). These miRNAs associate with the RNA-induced silencing complex to regulate gene expression posttranscriptionally by base pairing with 3'untranslated regions of complementary mRNA targets. Although it is established that miRNAs are expressed in the reproductive tract, their functional role and effect on reproductive disease remain unknown. The studies herein establish for the first time the reproductive phenotype of mice with loxP insertions in the Dicer1 gene (Dicer1fl/fl) when crossed with mice expressing Cre-recombinase driven by the anti-müllerian hormone receptor 2 promoter (Amhr2Cre/+). Adult female Dicer1fl/fl;Amhr2Cre/+ mice displayed normal mating behavior but failed to produce offspring when exposed to fertile males during a 5-month breeding trial. Morphological and histological assessments of the reproductive tracts of immature and adult mice indicated that the uterus and oviduct were hypotrophic, and the oviduct was highly disorganized. Natural mating of Dicer1fl/fl;Amhr2Cre/+ females resulted in successful fertilization as evidenced by the recovery of fertilized oocytes on d 1 pregnancy, which developed normally to blastocysts in culture. Developmentally delayed embryos were collected from Dicer1fl/fl; Amhr2Cre/+ mice on d 3 pregnancy when compared with controls. Oviductal transport was disrupted in the Dicer1fl/fl;Amhr2Cre/+ mouse as evidenced by the failure of embryos to enter the uterus on d 4 pregnancy. These studies implicate Dicer1/miRNA mediated posttranscriptional gene regulation in reproductive somatic tissues as critical for the normal development and function of these tissues and for female fertility.

Expansion of the cumulus complex surrounding the oocyte is critical for ovulation of a fertilizable egg. The ovulation-inducing surge of luteinizing hormone leads to an increased expression of genes such as prostaglandin-endoperoxide synthase 2 (Ptgs2), pentraxin-related protein 3 (Ptx3), and tumor necrosis factor alpha-induced protein 6 (Tnfaip6) that support cumulus expansion. Factors released by mural granulosa and cumulus granulosa cells into the follicular fluid induce paracrine signaling within the follicular compartment. The follicular fluid that separates these distinct granulosa cell types is an enriched fluid containing numerous proteins, nucleic acids, and other macromolecules. Extracellular vesicles (EVs) are also present; however, no physiologically relevant functions of follicular EVs have yet been demonstrated. In our study, the effect of follicular EVs on cumulus-oocyte complex (COC) expansion and relevant gene expression was assayed. Follicular EVs were isolated using ultracentrifugation from follicular fluid of small (3-5 mm) and large (>9 mm) antral bovine follicles, then characterized by nanoparticle tracking analysis, electron microscopy, and Western blot analysis. To test for bioactivity, mouse and bovine COCs were cultured with follicular EVs. Cumulus expansion and Ptgs2, Ptx3, and Tnfaip6 gene expression were measured following COC maturation culture. The results demonstrated that follicular EVs can support both measurable cumulus expansion and increased gene expression.

The molecular mechanisms that regulate the pivotal transformation processes observed in the follicular wall following the preovulatory LH surge, are still not established, particularly for cells of the thecal layer. To elucidate thecal cell (TC) and granulosa cell (GC) type-specific biologic functions and signaling pathways, large dominant bovine follicles were collected before and 21 hours after an exogenous GnRH-induced LH surge. Antral GCs (aGCs; aspirated by follicular puncture) and membrane-associated GCs (mGCs; scraped from the follicular wall) were compared with TC expression profiles determined by mRNA microarrays. Of the approximately 11 000 total genes expressed in the periovulatory follicle, only 2% of thecal vs 25% of the granulosa genes changed in response to the LH surge. The majority of the 203 LH-regulated thecal genes were also LH regulated in GCs, leaving a total of 57 genes as LH-regulated TC-specific genes. Of the 57 thecal-specific LH-regulated genes, 74% were down-regulated including CYP17A1 and NR5A1, whereas most other genes are being identified for the first time within theca. Many of the newly identified up-regulated thecal genes (eg, PTX3, RND3, PPP4R4) were also up-regulated in granulosa. Minimal expression differences were observed between aGCs and mGCs; however, transcripts encoding extracellular proteins (NID2) and matrix modulators (ADAMTS1, SASH1) dominated these differences. We also identified large numbers of unknown LH-regulated GC genes and discuss their putative roles in ovarian function. This Research Resource provides an easy-to-access global evaluation of LH regulation in TCs and GCs that implicates numerous molecular pathways heretofore unknown within the follicle.

Autophagy is an important cellular process that serves as a companion pathway to the ubiquitin-proteasome system to degrade long-lived proteins and organelles to maintain cell homeostasis. Although initially characterized in yeast, autophagy is being realized as an important regulator of development and disease in mammals. Beclin1 (Becn1) is a putative tumor suppressor gene that has been shown to undergo a loss of heterozygosity in 40-75% of human breast, ovarian, and prostate cancers. Because Becn1 is a key regulator of autophagy, we sought to investigate its role in female reproduction by using a conditional knockout approach in mice. We find that pregnant females lacking Becn1 in the ovarian granulosa cell population have a defect in progesterone production and a subsequent preterm labor phenotype. Luteal cells in this model exhibit defective autophagy and a failure to accumulate lipid droplets needed for steroidogenesis. Collectively, we show that Becn1 provides essential functions in the ovary that are essential for mammalian reproduction.A utophagy is an evolutionarily conserved cellular process from yeast to mammals that recycles long-lived proteins and organelles to maintain cell energy homeostasis, and is classically activated through the nutrient sensor, mammalian target of rapamycin complex (1-3). Cellular material is encapsulated within a double phospholipid bilayer organelle called the "autophagosome," which fuses with a lysosome to degrade the internalized debris. Using genetic screens in yeast, more than 34 autophagyrelated (Atg) genes have been identified, with most having clear homologs in higher eukaryotes through protein-sequence identity (4-6). BECLIN1 (BECN1), a haploinsufficient tumor suppressor, is an essential autophagy protein that directs the initiation phase and maturation-fusion phase through its participation in two distinct phosphoinositide 3-kinase class III complexes (7-9). Autophagy is not only important for normal development, but it is linked to an array of diseases including neurodegenerative disorders, liver disease, heart disease, inflammatory diseases, and cancer (10, 11). We previously showed a role for BECN1 and autophagy in the establishment of the murine primordial follicle pools in a germ-line knockout model (12). In this study, we follow up on these results and investigate the role of BECN1 in ovarian granulosa cells.In mammals, the success of early pregnancy from embryo implantation to development of the conceptus depends on the function of an ephemeral endocrine gland, the "corpus luteum" (CL), which rapidly forms in the ovary from the remains of the ovulated follicle and migrating somatic cells through a process called "luteinization." Progesterone is an essential hormone that acts at several different levels during pregnancy, including the development of the endometrium for implantation of the embryo and the maintenance of pregnancy through quiescence of the myometrium (13,14). Luteal cells within the CL use cholesterol for steroidogenesis to produce progesterone ...

Our data indicate that ceramide is a key modulator for DNA damage and cell death in chemical hypoxia to renal tubular epithelial cells.

MicroRNAs (miRNAs) are small, non-coding RNA molecules which post-transcriptionally regulate gene expression. We have previously demonstrated that within the uterus, miRNA expression is under steroidal control and that disruption of Dicer1, the enzyme which generates mature miRNAs, leads to abnormalities in the development and function of the female reproductive tract. Despite the apparent importance of miRNAs and the enzymes which lead to their generation, little to no information exists on the mechanisms which regulate the expression of this system in the female reproductive tract. The objective of the current study was to examine steroidal regulation of the miRNAs biogenesis enzymes, Drosha, Dgcr8, Exportin-5 and Dicer1 in the mouse uterus. The results of this study indicate that estrogen and progesterone significantly increased Exportin-5 mRNA expression while only progesterone increased Dicer1 expression. We conclude from these studies that the miRNA biogenesis components Drosha, Dgcr8, Exportin-5 and Dicer1 are expressed in the mouse uterus and that Exportin-5 and Dicer1 appear to be the major steroid regulated components in the miRNA biogenesis pathway. These observations suggest that in addition to steroids modulating miRNA expression at the level of transcription, they may also influence miRNA expression by regulating the expression of the miRNA biogenesis components necessary for their processing to the mature cytoplasmic form.