Daniel J. Ballow

Microarray Analyses of Newborn Mouse Ovaries Lacking Nobox

Abstract Nobox is a homeobox gene expressed in oocytes and critical in oogenesis. Nobox deficiency leads to rapid loss of postnatal oocytes. Early oocyte differentiation is poorly understood. We hypothesized that lack of Nobox perturbs global expression of genes preferentially expressed in oocytes as well as microRNAs. We compared Nobox knockout and wild-type ovaries using Affymetrix 430 2.0 microarray platform. We discovered that 28 (74%) of 38 of the genes downregulated more than 5-fold in the absence of Nobox were preferentially expressed in oocytes, whereas only 5 (15%) of 33 genes upregulated more than 5-fold in the absence of Nobox were preferentially expressed in oocytes. Protein-binding microarray helped identify nucleotide motifs that NOBOX binds and that several downregulated genes contain within putative promoter regions. MicroRNA population in newborn ovaries deficient of Nobox was largely unaffected. Genes whose proteins are predicted to be secreted but were previously unknown to be significantly expressed in early oogenesis were downregulated in Nobox knockouts and included astacin-like metalloendopeptidase (Astl), Jagged 1 (Jag1), oocyte-secreted protein 1 (Oosp1), fetuin beta (Fetub), and R-spondin 2 (Rspo2). In addition, pluripotency-associated genes Pou5f1 and Sall4 are drastically downregulated in Nobox-deficient ovaries, whereas testes-determining gene Dmrt1 is overexpressed. Our findings indicate that Nobox is likely an activator of oocyte-specific gene expression and suggest that the oocyte plays an important role in suppressing expression of male-determining genes, such as Dmrt1.

Lim Homeobox Gene, Lhx8, Is Essential for Mouse Oocyte Differentiation and Survival

Abstract Lhx8 is a member of the LIM-homeobox transcription factor family and preferentially expressed in oocytes and germ cells within the mouse ovary. We discovered that Lhx8 knockout females lose oocytes within 7 days after birth. At the time of birth, histological examination shows that Lhx8-deficient (Lhx8−/−) ovaries are grossly similar to the newborn wild-type ovaries. Lhx8−/− ovaries fail to maintain the primordial follicles, and the transition from primordial to growing follicles does not occur. Lhx8−/− ovaries misexpress oocyte-specific genes, such as Gdf9, Pou5f1, and Nobox. Very rapid loss of oocytes may partly be due to the drastic downregulation of Kit and Kitl in Lhx8−/− ovaries. We compared Lhx8−/− and wild-type ovaries using an Affymetrix 430 2.0 microarray platform. A total of 80 (44%) of 180 of the genes downregulated more than 5-fold in Lhx8−/− ovaries were preferentially expressed in oocytes, whereas only 3 (2%) of 146 genes upregulated more than 5-fold in the absence of Lhx8 were preferentially expressed in oocytes. In addition, the comparison of genes regulated in Lhx8−/− and Nobox−/− newborn ovaries discovered a common set of 34 genes whose expression level was affected in both Lhx8- and Nobox-deficient mice. Our findings show that Lhx8 is a critical factor for maintenance and differentiation of the oocyte during early oogenesis, and it acts in part by downregulating the Nobox pathway.

Expression and localization of the novel and highly conserved gametocyte-specific factor 1 during oogenesis and spermatogenesis

OBJECTIVE To determine the onset of gametocyte-specific factor 1 (Gtsf1) expression in embryogenesis and its relation to Nobox; and to determine its localization during gonadal development and gametocyte maturation. DESIGN Developmental animal study. SETTING University reproductive biology laboratory. ANIMAL(S) Mice ranging in age from embryonic day 12.5 to 8 weeks. INTERVENTION(S) Polymerase chain reaction and quantitative polymerase chain reaction were performed to determine the onset of and relative messenger RNA expression. Western blot was performed to confirm protein expression and antibody specificity. In situ hybridization and immunohistochemistry were used determine localization of expression. MAIN OUTCOME MEASURE(S) Gtsf1 messenger RNA expression levels during embryogenesis through adulthood in wild-type mice and in newborn Nobox knockout mice; GTSF1 expression and localization in postnatal mice. RESULT(S) Gtsf1 functions downstream of Nobox and is highly expressed in embryonic male and female gonads, localizing to germ cells throughout development. GTSF1 expression is confined to the cytoplasm in all stages of postnatal oocyte maturation and to prespermatogonia during early postnatal testicular development. CONCLUSION(S) The expression pattern of Gtsf1 and its high conservation suggests that it may play an important role in germ cell development. Further characterization of Gtsf1 may elucidate mechanisms involved in premature ovarian failure.