In vivo regulation of follicle-stimulating hormone receptor by the transcription factors upstream stimulatory factor 1 and upstream stimulatory factor 2 is cell specific.

Pituitary FSH promotes pubertal timing and normal gametogenesis by binding its receptor (FSHR) located on Sertoli and granulosa cells of the testis and ovary, respectively. Studies on Fshr transcription provide substantial evidence that upstream stimulatory factor (USF) 1 and USF2, basic helix-loop-helix leucine zipper proteins, regulate Fshr through an E-box within its promoter. However, despite the strong in vitro support for USF1 and USF2 in Fshr regulation, there is currently no in vivo corroborating evidence. In the present study, chromatin immunoprecipitation demonstrated specific binding of USF1 and USF2 to the Fshr promoter in both Sertoli and granulosa cells, in vivo. Control cells lacking Fshr expression showed no USF-Fshr promoter binding, thus correlating USF-promoter binding to gene activity. Evaluation of Fshr expression in Usf1 and Usf2 null mice further explored USF's role in Fshr transcription. Loss of either gene significantly reduced ovarian Fshr levels, whereas testis levels were unaltered. Chromatin immunoprecipitation analysis of USF-Fshr promoter binding in Usf-null mice indicated differences in the composition of promoter-bound USF dimers in granulosa and Sertoli cells. Promoter-bound USF dimer levels declined in granulosa cells from both null mice, despite increased USF2 levels in Usf1-null ovaries. However, compensatory increases in promoter-bound USF homodimers were evident in Usf-null Sertoli cells. In summary, this study provides the first in vivo evidence that USF1 and USF2 bind the Fshr promoter and revealed differences between Sertoli and granulosa cells in compensatory responses to USF loss and the USF dimeric composition required for Fshr transcription.

Distal regulatory elements are required for Fshr expression, in vivo.

The gonadotropin follicle-stimulating hormone (FSH) is required for initiation and maintenance of normal gametogenesis and acts through a specific, cell-surface receptor (Fshr) present only on Sertoli and granulosa cells in the gonads. Despite extensive examination of the transcriptional mechanisms regulating Fshr, the sequences directing its expression to these cells remain unidentified. To establish the minimal region necessary for Fshr expression, we generated transgenic mice carrying a yeast artificial chromosome (YAC) that contained 413 kilobases (kb) of the rat Fshr locus (YAC60). Transgene expression, as determined by RT-PCR, was absent from immature testis and Sertoli cells, limited to germ cells of the adult testis, and never observed in the ovary. While the data is limited to only one transgenic line, it suggests that the 413kb region does not specify the normal spatiotemporal expression pattern of Fshr. Comparative genomics was used to identify potential distal regulatory elements, revealing seven regions of high evolutionary conservation (>80% identity over 100bp or more), six of which were absent from the transgene. Functional examination of the evolutionary conserved regions (ECRs) by transient transfection revealed that all of the ECRs had modest transcriptional activity in Sertoli or myoid cells with two, ECR4 and ECR5, showing differential effects in expressing and non-expressing cells. These data reveal that distal regulatory regions (outside the 413kb in YAC60) are required for appropriate temporal and spatial Fshr expression and implicate the identified ECRs in transcriptional regulation of Fshr.

Distinct transcriptional mechanisms direct expression of the rat Dmrt1 promoter in sertoli cells and germ cells of transgenic mice.

DMRT1 is a transcription factor expressed only in Sertoli cells and undifferentiated spermatogonia of the postnatal testis, where it is required for proper cellular differentiation and fertility. To elucidate the transcriptional regulatory regions that provide DMRT1's cell-specific expression, transgenic mice containing a LacZ reporter gene driven by variable amounts of rat Dmrt1 5' flanking sequence, 9 kb and smaller, were evaluated. Examination of transgene expression by RT-PCR indicated that multiple promoter regions direct Dmrt1 to the testis and that sequences upstream of 2.8 kb are needed for both Sertoli cell expression and limiting transcriptional influence imposed by surrounding chromatin. Thus, whereas many of the transgenes were expressed in the testis, the ones with smaller promoters were significantly more prone to expression at ectopic sites or to complete silencing. Transgene expression in Sertoli cells and germ cells was assessed by immunohistochemistry and RT-PCR following busulfan treatment to remove germ cells. Both evaluations indicated expression of the 9- and 3.2-kb promoters in Sertoli cells and germ cells, whereas activity of smaller promoters was largely restricted to germ cells. In all, the present study provides in vivo evidence that distinct promoter sequences participate in Dmrt1 regulation in somatic cells and germ cells, with the -3.2 kb/-2.8 kb region directing expression in Sertoli cells and downstream sequences (< or =1.3 kb) directing it in germ cells. Further exploration of the mechanisms restricting Dmrt1 expression to the testis revealed that FOXL2, a transcription factor required for differentiation of the ovary, repressed Dmrt1 promoter through the -3.2 kb/-2.8 kb regulatory region, offering a potential mechanism for Dmrt1 transcriptional silencing in granulosa cells.

Gamendazole, an orally active indazole carboxylic acid male contraceptive agent, targets HSP90AB1 (HSP90BETA) and EEF1A1 (eEF1A), and stimulates Il1a transcription in rat Sertoli cells.

Gamendazole was recently identified as an orally active antispermatogenic compound with antifertility effects. The cellular mechanism(s) through which these effects occur and the molecular target(s) of gamendazole action are currently unknown. Gamendazole was recently designed as a potent orally active antispermatogenic male contraceptive agent. Here, we report the identification of binding targets and propose a testable mechanism of action for this antispermatogenic agent. Both HSP90AB1 (previously known as HSP90beta [heat shock 90-kDa protein 1, beta]) and EEF1A1 (previously known as eEF1A [eukaryotic translation elongation factor 1 alpha 1]) were identified as binding targets by biotinylated gamendazole (BT-GMZ) affinity purification from testis, Sertoli cells, and ID8 ovarian cancer cells; identification was confirmed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry and Western blot analysis. BT-GMZ bound to purified yeast HSP82 (homologue to mammalian HSP90AB1) and EEF1A1, but not to TEF3 or HBS1, and was competed by unlabeled gamendazole. However, gamendazole did not inhibit nucleotide binding by EEF1A1. Gamendazole binding to purified Saccharomyces cerevisiae HSP82 inhibited luciferase refolding and was not competed by the HSP90 drugs geldanamycin or novobiocin analogue, KU-1. Gamendazole elicited degradation of the HSP90-dependent client proteins AKT1 and ERBB2 and had an antiproliferative effect in MCF-7 cells without inducing HSP90. These data suggest that gamendazole may represent a new class of selective HSP90AB1 and EEF1A1 inhibitors. Testis gene microarray analysis from gamendazole-treated rats showed a marked, rapid increase in three interleukin 1 genes and Nfkbia (NF-kappaB inhibitor alpha) 4 h after oral administration. A spike in II1a transcription was confirmed by RT-PCR in primary Sertoli cells 60 min after exposure to 100 nM gamendazole, demonstrating that Sertoli cells are a target. AKT1, NFKB, and interleukin 1 are known regulators of the Sertoli cell-spermatid junctional complexes. A current model for gamendazole action posits that this pathway links interaction with HSP90AB1 and EEF1A1 to the loss of spermatids and resulting infertility.

Sex-specific differences in mouse DMRT1 expression are both cell type- and stage-dependent during gonad development.

Immunohistochemistry was used to examine GCNA1, a germ cell-specific protein, together with DMRT1 (Doublesex and Mab-3-related transcription factor-1), a transcription factor implicated in Sertoli cell and germ cell function, in order to resolve DMRT1's cellular profile during pre- and postnatal gonad development in the mouse. In the indifferent gonad (10.5-11.5 days postcoitus [dpc]), DMRT1 localized to somatic cells and GCNA1(+) germ cells and was indistinguishable in males and females. By 12.5 dpc, a clear sexual preference for DMRT1 in male somatic cells was observed, with male DMRT1 localized to testicular cords and more abundant in Sertoli cells than in germ cells and female DMRT1 diffusely labeled and markedly lower in somatic cells than in germ cells. A male somatic preference continued throughout development, with DMRT1 evident in Sertoli cells at all ages examined and absent in ovarian somatic cells from 13.5 dpc onward. In contrast, expression in primordial germ cells was not sexually distinct, and both sexes showed DMRT1 increasing through 13.5 dpc and absent by 15.5 dpc. Notably, sexual differences in germ cell DMRT1 were detected after birth, when it was detected only in spermatogonia of the testis. Colocalization of DMRT1 with proliferation markers KI67 and proliferating cell nuclear antigen (PCNA) and stem cell markers OCT4 (also known as POU5F1) and NGN3 indicated that, in postnatal testes, DMRT1 was present in both stem and proliferating spermatogonia. Together, the findings implicate opposite functions for DMRT1 in somatic and germ cells of the testis. In Sertoli cells, DMRT1 expression correlated with differentiation, whereas in germ cells, it suggested a role in expansion and maintenance of undifferentiated spermatogonia.