ADAMTS-1 (a disintegrin and metalloprotease with thrombospondin motifs-1) is a member of the ADAMTS family of metalloproteases which, together with AD-AMTS-4 and ADAMTS-5, has been shown to degrade members of the lectican family of proteoglycans. AD-AMTS-1 mRNA is induced in granulosa cells of periovulatory follicles by the luteinizing hormone surge through a progesterone receptor-dependent mechanism. Female progesterone receptor knockout (PRKO) mice are infertile primarily due to ovulatory failure and lack the normal periovulatory induction of ADAMTS-1 mRNA. We therefore investigated the protein localization and function of ADAMTS-1 in ovulating ovaries. Antibodies against two specific peptide regions, the prodomain and the metalloprotease domain of ADAMTS-1, were generated. Pro-ADAMTS-1 of 110 kDa was identified in mural granulosa cells and appears localized to cytoplasmic secretory vesicles. The mature (85-kDa prodomain truncated) form accumulated in the extracellular matrix of the cumulus oocyte complex (COC) during the process of matrix expansion. Each form of AD-AMTS-1 protein increased >10-fold after the ovulatory luteinizing hormone surge in wild-type but not PRKO mice. Versican is also localized selectively to the ovulating COC matrix and was found to be cleaved yielding a 70-kDa N-terminal fragment immunopositive for the neoepitope DPEAAE generated by ADAMTS-1 and AD-AMTS-4 protease activity. This extracellular processing of versican was reduced in ADAMTS-1-deficient PRKO mouse ovaries. These observations suggest that one function of ADAMTS-1 in ovulation is to cleave versican in the expanded COC matrix and that the anovulatory phenotype of PRKO mice is at least partially due to loss of this function. ADAMTS-11 (a disintegrin and metalloprotease with thrombospondin-like repeats-1) is a multidomain, multifunctional metalloprotease first cloned from a colon carcinoma cell line (1). Subsequently, ADAMTS-1 has been shown to be induced by luteinizing hormone (LH) in periovulatory follicles of mouse (2), rat (3), and mare.2 Expression of ADAMTS-1 is dependent on LH-induced expression of progesterone and progesterone receptor (PR) in granulosa cells (2, 3). Progesterone receptor null (PRKO) mice fail to ovulate but differentiate normally to form corpora lutea that contain entrapped oocytes (2, 4, 5). AD-AMTS-1 null mice also display impaired ovulation with morphologically abnormal ovaries (6). These observations have indicated that ADAMTS-1 may play a key role in the ovulation process.Ovulation in the mammalian ovary occurs in response to the LH surge that activates several extracellular matrix (ECM) remodeling processes. Dissolution of the connective matrix and cellular layers at the follicular apex and ovarian surface are necessary for release of the oocyte. Based on the anovulatory phenotypes of mice with null mutations of cyclooxygenase-2 (COX-2) (7, 8), prostaglandin E 2 receptor (9, 10), and bikunin (11), it is becoming evident that expansion of the matrix surrounding the cumulus cells and oocyte is ess...

ADAMTS-1 (a disintegrin and metalloproteinase with thrombospondin-like motifs) is a multifunctional protease that is expressed in periovulatory follicles. Herein we show that induction of ADAMTS-1 message in vivo and transcription of the ADAMTS-1 promoter in cultured granulosa cells are dependent on separable but coordinate actions of LH and the progesterone receptor (PR). To analyze the molecular mechanisms by which LH and PR regulate this gene, truncations and site-specific mutants of ADAMTS-1 promoter-luciferase reporter constructs (ADAMTS-1-Luc) were generated and transfected into rat granulosa cell cultures. Three regions of the promoter were found to be important for basal activity, two of which were guanine cytosine-rich binding sites for specificity proteins Sp1/Sp3 and the third bound a nuclear factor 1-like factor. Despite the absence of a consensus PR DNA response element in the proximal ADAMTS-1 promoter, cotransfection of a PRA (or PRB) expression vector stimulated ADAMTS-1 promoter activity, a response that was reduced by the PR antagonist ZK98299. Forskolin plus phorbol myristate acetate also increased promoter activity and, when added to cells cotransfected with PRA, ADAMTS-1 promoter activity increased further. Activation of the ADAMTS-1 promoter by PRA involves functional CAAT enhancer binding protein beta, nuclear factor 1-like factor, and three Sp1/Sp3 binding sites as demonstrated by transfection of mutated promoter constructs. In summary, LH and PRA/B exert distinct but coordinate effects on transactivation of the ADAMTS-1 gene in granulosa cells in vivo and in vitro with PR acting as an inducible coregulator of the ADAMTS-1 gene.

Early growth response factor (Egr-1) is an inducible zinc finger transcription factor that binds specific GC-rich enhancer elements and impacts female reproduction. These studies document for the first time that FSH rapidly induces Egr-1 expression in granulosa cells of small growing follicles. This response is transient but is reinitiated in preovulatory follicles exposed to the LH analog, human chorionic gonadotropin. Immunohistochemical analysis also showed gonadotropin induced Egr-1 in theca cells. The Egr-1 gene regulatory region responsive to gonadotropin signaling was localized within -164 bp of the transcription initiation site. Binding of Sp1/Sp3 to a proximal GC-box at -64/-46 bp was enhanced by FSH in immature granulosa cells but reduced after human chorionic gonadotropin stimulation of preovulatory follicles despite constant protein expression. This dynamic regulation of Sp1 binding was dependent on gonadotropin-regulated mechanisms that modulate Sp1/3-DNA binding activity. Serum response factor was active in granulosa cells and bound a consensus CArG-box/serum response element site, whereas two putative cAMP response elements within the -164-bp region bound cAMP regulatory element (CRE) binding protein (CREB) and a second cAMP-inducible protein immunologically related to CREB. Transient transfection analyses using Egr-1 promoter-luciferase constructs and site-specific mutations show that the serum response element, GC-box, and CRE-131 are involved in gonadotropin regulation of Egr-1 expression in granulosa cells. Specific kinase inhibitors of Erk or protein kinase A antagonized this induction while exogenously expressed Egr-1 enhanced reporter expression. These observations indicate that the Egr-1 gene is a target of both FSH and LH action that may mediate molecular programs of proliferation and/or differentiation during follicle growth, ovulation, and luteinization.

Homeobox genes encode transcription factors that regulate embryonic development and postnatal events. Rhox5 (previously called Pem), the founding member of a homeobox gene cluster that we recently identified on the X chromosome, is selectively expressed in granulosa cells in the ovary and other somatic-cell types in other reproductive organs. In this report, we investigate its regulation in granulosa cells in the rat ovary. We found that Rhox5 expression in the ovary is governed by the Rhox5 distal promoter and is expressed at least as early as Day 5 postpartum. Rhox5 mRNA levels are regulated during the ovarian cycle, peaking before ovulation. Deletion analysis revealed a 25-nt element essential for distal promoter transcription in primary granulosa cells. This distal promoter element contains two ETS and one SP1 transcription-factor family binding sites that mutagenesis analysis indicated were essential for high-level transcription. This element was both necessary and sufficient for transcription, because it activated transcription when placed upstream of a heterologous minimal promoter. Cold competition and electrophoretic mobility shift assay studies demonstrated that SP1, SP3, and the ETS family transcription factor GABP bound this element. Dominant-negative forms of GABP and SP3 repressed distal promoter expression in primary rat granulosa, showing that these factors are crucial for Rhox5 expression. Cotransfection of dominant-negative mutants indicated that Rhox5 expression in granulosa cells is regulated by the c-Jun N-terminal protein kinase (JNK, MAPK8) and RAS pathways, which are known to be upstream of ETS family transcription factors. The discovery that Rhox5 expression in granulosa cells is regulated by MAPK pathways and ETS and SP1 family members provides an opportunity to understand how these regulatory pathways and factors collaborate to regulate gene expression during the ovarian cycle.

The United States is confronting important challenges at both the early and late stages of science education. At the level of K–12 education, a recent National Research Council report (Successful K–12 STEM Education) proposed a bold restructuring of how science is taught, moving away from memorizing facts and emphasizing hands-on, inquiry-based learning and a deeper understanding of the process of science. At higher levels of training, limited funding for science is leading PhDs to seek training and careers in areas other than research. Might science PhDs play a bigger role in the future of K–12 education, particularly at the high school level? We explore this question by discussing the roles that PhDs can play in high school education and the current and rather extensive barriers to PhDs entering the teaching profession and finally suggest ways to ease the entrance of qualified PhDs into high school education.