Patricia L. Morris

A novel class of small RNAs bind to MILI protein in mouse testes.

Small RNAs bound to Argonaute proteins recognize partially or fully complementary nucleic acid targets in diverse gene-silencing processes. A subgroup of the Argonaute proteins—known as the ‘Piwi family’—is required for germ- and stem-cell development in invertebrates, and two Piwi members—MILI and MIWI—are essential for spermatogenesis in mouse. Here we describe a new class of small RNAs that bind to MILI in mouse male germ cells, where they accumulate at the onset of meiosis. The sequences of the over 1,000 identified unique molecules share a strong preference for a 5′ uridine, but otherwise cannot be readily classified into sequence families. Genomic mapping of these small RNAs reveals a limited number of clusters, suggesting that these RNAs are processed from long primary transcripts. The small RNAs are 26–31 nucleotides (nt) in length—clearly distinct from the 21–23u2009nt of microRNAs (miRNAs) or short interfering RNAs (siRNAs)—and we refer to them as ‘Piwi-interacting RNAs’ or piRNAs. Orthologous human chromosomal regions also give rise to small RNAs with the characteristics of piRNAs, but the cloned sequences are distinct. The identification of this new class of small RNAs provides an important starting point to determine the molecular function of Piwi proteins in mammalian spermatogenesis.

Functional role for the c-Abl tyrosine kinase in meiosis I.

The c-Abl tyrosine kinase is activated by ionizing radiation and certain other DNA-damaging agents. The DNA-dependent protein kinase (DNA–PK) and the ataxia telangiectasia mutated (ATM) gene product, effectors in the DNA damage response, contribute to the induction of c-Abl activity. The present study demonstrates that c-Abl is expressed in mouse and rat testes, and predominantly in pachytene spermatocytes of meiosisu2009I. The results also demonstrate that c-Abl interacts directly with meiotic chromosomes. In concert with a requirement for c-Abl at the pachytene stage, we show that, in contrast to wild-type mice, testes from Abl−/− mice exhibit defects in spermatogenesis. These findings provide the first demonstration that c-Abl plays a functional role in meiosis.

Identification and Regulation of Testicular Interferon-γ (IFNγ) Receptor Subunits: IFNγ Enhances Interferon Regulatory Factor-1 and Interleukin-1β Converting Enzyme Expression1

Interferon-γ (IFNγ) transmits its signal through a specific cell surface receptor (IFNγR), which consists of a primary ligand bindingα -chain (IFNγRα) and a signaling β-chain (IFNγRβ). Recent studies identified the cytokines IFNγ, interleukin-6 (IL-6), IL-1α, and tumor necrosis factor-α in testicular cells. Therefore, we: 1) examined the expression of IFNγRα and IFNγRβ subunits in freshly isolated and purified rat testicular cells; 2) examined the differential regulation of receptor components by cytokines using primary cultures of Sertoli cells; 3) identified the cell signaling pathway components of testicular IFNγR; and 4) characterized the functional role of testicular IFNγ using primary Sertoli cells. We demonstrated the messenger RNAs for both chains of IFNγR in rat testicular cells using Northern hybridization analysis. Western blot analysis and immunocytochemistry showed that both specific IFNγR protein subunits were present in cultured primary Leydig and Sertoli cells prepared from the testes of i...

Transcriptional Regulation of Sertoli Cell Immediate Early Genes by Interleukin-6 and Interferon-γ Is Mediated through Phosphorylation of STAT-3 and STAT-1 Proteins1

The immediate early genes are regulated by a variety of extracellular signals, including pleiotropic cytokines. The effects of the testicular cytokines, interleukin-6 (IL-6) and interferon-γ (IFN-γ), on signal transducers and activators of transcription 3 and 1 (STAT-3 and STAT-1) and on c-fos gene expression in primary Sertoli cells are suggestive of their roles in differential function. Using the tyrosine phosphorylation inhibitor, genistein, and electrophoretic mobility shift assay, we show that IL-6 and IFN-γ induce nuclear factor STAT-3 and STAT-1 DNA-binding activity to the sis-inducible element of c-fos in a genistein-dependent pathway. Quantitative solution hybridization, Northern blot, and nuclear run-on analysis show that differential induction of c-fos, junB, and c-myc messenger RNA (mRNA) by these cytokines occur at transcriptional levels. IL-6 stimulates c-fos mRNA levels by 6-fold while increasing junB levels by 2-fold. IFN-γ increases c-fos message 2-fold, but has no effect on junB mRNA lev...

Recommendations concerning the new U.S. National Institutes of Health initiative to balance the sex of cells and animals in preclinical research

The U.S. National Institutes of Health (NIH) announced last May that steps will be taken to address the over‐reliance on male cells and animals in preclinical research. To further address this announcement, in September 2014, scientists with varying perspectives came together at Georgetown University to discuss the following questions. (1) What metrics should the NIH use to assess tangible progress on policy changes designed to address the over‐reliance on male cells and animals in preclinical research? (2) How effective can education be in reducing the over‐reliance on male cells and animals in preclinical research and what educational initiatives sponsored by the NIH would most likely effect change? (3) What criteria should the NIH use to determine rigorously defined exceptions to the future proposal requirement of a balance of male and female cells and animals in preclinical studies? (4) What additional strategies in addition to proposal requirements should NIH use to reduce the overreliance of male cells and animals in preclinical research? The resulting consensus presented herein includes input from researchers not only from diverse disciplines of basic and translational science including biology, cell and molecular biology, biochemistry, physiology, pharmacology, neuroscience, cardiology, endocrinology, nephrology, psychiatry, and obstetrics and gynecology, but also from recognized experts in publishing, industry, advocacy, science policy, clinical medicine, and population health. We offer our recommendations to aid the NIH as it selects, implements, monitors, and optimizes strategies to correct the over‐reliance on male cells and animals in preclinical research.—Sandberg, K., Umans, J. G., Georgetown Consensus Conference Work Group. Recommendations concerning the new U.S. National Institutes of Health initiative to balance the sex of cells and animals in preclinical research. FASEB J. 29, 1646‐1652 (2015). www.fasebj.org

Kzf1 - a novel KRAB zinc finger protein encoding gene expressed during rat spermatogenesis.

Two novel KRAB (Krüppel associated box) type zinc finger protein encoding cDNAs, named Kzf1 and Kzf2 (Kzf for KRAB zinc finger), were identified by screening of a rat embryonic brain cDNA library with a human ZNF91 KRAB probe. Kzf1 and Kzf2 encode proteins with an amino-terminal KRAB domain and a carboxy-terminal zinc finger cluster containing 9 and 13 zinc finger units, respectively. While Kzf2 appears to be ubiquitously expressed, Kzf1 is preferentially expressed in the testis. Within the testis, Kzf1 mRNA is restricted to germ cells. The Kzf1 protein exhibits DNA binding activity and its KRAB domain can function as a repressor module in transcription. Using somatic cell hybrid analysis, the Kzf1 gene was mapped to chromosome 6.

The effect of mono(2-ethylhexyl) phthalate on Sertoli cell transferrin secretion in vitro

We have examined the effect of mono(2-ethylhexyl) phthalate (MEHP) on the secretion of rat transferrin (rTf) by Sertoli cells (SC) in the presence and absence of FSH. Significant decreases in rTf secretion were observed in SC cultures exposed to 50 and 75 microM MEHP, in the absence of FSH. Treatment of SC with FSH (300 ng/ml) obviated this suppression of rTf secretion by MEHP. These findings indicate an effect of MEHP on rTf secretion by SC in vitro which could account for the testicular toxicity of MEHP in vivo.

Interleukin-6 regulation of kappa opioid receptor gene expression in primary sertoli cells

Three classes of opioid receptors—mu, delta, and kappa—mediate physiological and pharmacological functions of the endogenous opioid peptides and exogenous opioid compounds in the central nervous system (CNS), as well as in peripheral tissues including the immune system. Using reverse transcriptase polymerase chain reaction (RT-PCR) analysis, we show that freshly isolated and highly purified somatic (Sertoli and Leydig) and specific germ (spermatogonia, pachytene spermatocytes, round, and elongating spermatids) cells of the rat testis differentially express the mRNAs for these opioid receptor genes. Furthermore, to identify a functional mechanism for cytokine regulation of testicular opioid receptor gene expression, we employed primary Sertoli cells as a model system. In a semiquantitative PCR analysis using the S16 ribosomal RNA gene as an internal control, we show that interleukin-6 reduces kappa opioid receptor mRNA levels from 6 to 24 h of treatment in primary Sertoli cells. This regulation requires new RNA and protein synthesis and is partially mediated by the protein kinase A pathway. These findings are consistent with a role for the cytokine and opioid signaling pathways in Sertoli cellular function and the interaction that exists between the opioid and the immune systems in the CNS.

FOLLICLE STIMULATING HORMONE AND LEUKEMIA INHIBITORY FACTOR REGULATE SERTOLI CELL RETINOL METABOLISM

Sertoli cells, the somatic epithelial cells of the seminiferous tubules, provide both structural and biochemical support for developing male germ cells. The Sertoli cells are targets of retinoid action in the testis. We have found that FSH, (Bu)2cAMP, and leukemia inhibitory factor elicit substantial changes in the metabolism of[ 3H]retinol (vitamin A) in primary cultures of purified rat Sertoli cells. Addition of (Bu)2cAMP for 2 h or FSH for 6 h results in a 3-fold increase in the metabolism of[ 3H]retinol to [3H]retinoic acid ([3H]RA); the esterification of [3H]retinol to [3H]retinyl esters, especially[ 3H]retinyl palmitate, is also increased by approximately 5-fold. The addition of 1 μm all-trans-RA also elicits changes in [3H]retinol metabolism, but in this case the metabolism of [3H]retinol to[ 3H]RA is inhibited, whereas the metabolism of[ 3H]retinol to [3H]retinyl esters is increased by over 50-fold. Leukemia inhibitory factor increases the esterification of [3H]retinol by 2- to 3-fold. FSH leads t...

SUMOYLATION REGULATES GERMINAL VESICLE BREAK-DOWN AND THE AKT/PKB PATHWAY DURING MOUSE OOCYTE MATURATION

SUMOylation, a process of posttranslational modification of proteins by the small ubiquitin-related modifier (SUMO) family of proteins, is known to be involved in yeast and mammalian somatic cell-cycle regulation. However, the identities of the SUMO-modified oocyte targets are largely unknown and the functional role(s) for SUMOylation during mammalian oocyte maturation remains unclear. On the basis of studies in non-germline cells, protein kinase B/Akt is a potential SUMOylation target in the mouse oocyte, where it plays an essential role in cell-cycle resumption and progression during maturation. This study investigated the temporal patterns and prospective role(s) for interactions between SUMOylation and Akt serine-phosphorylation during oocyte meiotic resumption. Pharmacological inhibition of SUMOylation significantly decreased follicular fluid meiosis-activating sterol-induced cell-cycle resumption in oocytes matured in vitro and negatively affected the phosphorylation and nuclear translocation of Akt. Similarly, nuclear localization of cyclin D1, a downstream target of Akt activation, was significantly decreased following SUMOylation inhibition. Together these data show that SUMO and the posttranslational process of SUMOylation are involved in cell-cycle resumption during murine oocyte maturation and exert a regulatory influence on the Akt pathway during germinal vesicle breakdown.