Molecular determinants and mechanisms involved in ovarian follicular growth, ovulation, and luteinization are not well understood. The objective of this study was to identify genes expressed in bovine granulosa cells (GC) of dominant follicles (DF) and downregulated after hCG-induced ovulation, using the suppression subtractive hybridization (SSH). GC were collected from DF at Day 5 of the estrous cycle and from ovulatory follicles (OF) obtained 23 h following injection of hCG. A subtracted cDNA library (DF-OF) was generated and screened using unsubtracted (DF, OF) and subtracted (DF-OF, OF-DF) cDNAs as complex (32)P-probes. A total of 32 nonredundant cDNAs were identified: 23 cDNAs matched with sequences of known biological function and 9 cDNAs with complete or partial sequences of undefined biological function. Detection of genes known to be downregulated during the periovulatory period in the bovine species, such as CPD, CYP11A1, CYP19A1, FSHR, LRP8/ ApoER2, and SERPINE2, validated the physiological model and analytical techniques used. For a subset of genes, such as ARFGAP3, CYP11A1, CYP19A1, FSHR, FST, GJA1, IDH3, INHBA, LHCGR, LHCGR lacking exon 10, PRC1, PRG1, RPA2, SCD, and TRIB2, gene expression profiles were compared by virtual Northern blot or reverse transcriptase-polymerase chain reaction from follicles obtained at different developmental stages. Results confirmed a downregulation of the respective mRNAs in GC of OF compared with that of DF. We conclude that we have identified novel genes that are downregulated by hCG in bovine GC of DF during the periovulatory period, which may contribute to follicular growth, ovulation, and/or luteinization.

In mammals, sex development is a genetically and hormonally controlled process that begins with the establishment of chromosomal or genetic sex (XY or XX) at conception. At approximately 6 to 7 weeks of human gestation or embryonic day e11.5 in the mouse, expression of the Y chromosome-linked sex determining gene called SRY (described in detail in this chapter) then initiates gonadal differentiation, which is the formation of either a testis (male) or an ovary (female). Male sex differentiation (development of internal and external reproductive organs and acquisition of male secondary sex characteristics) is then controlled by three principal hormones produced by the testis: Mullerian inhibiting substance (MIS) or anti-Mullerian hormone (AMH), testosterone, and insulin-like factor 3 (INSL3). In the absence of these critical testicular hormones, female sex differentiation ensues. This sequential, three-step process of mammalian sex development is also known as the Jost paradigm. With the advent of modern biotechnologies over the past decade, such as transgenics, array-based gene profiling, and proteomics, the field of mammalian sex determination has witnessed a remarkable boost in the understanding of the genetics and complex molecular mechanisms that regulate this fundamental biological event. Consequently, a number of excellent reviews have been devoted to this topic. The purpose of the present chapter is to provide an overview of selected aspects of mammalian sex determination and differentiation with an emphasis on studies that have marked this field of study. (c) 2005 Elsevier Inc.

Angiotensin IV (Ang IV) is a metabolite of the potent vasoconstrictor angiotensin II (Ang II). Because specific binding sites for this peptide have been reported in numerous tissues including the brain, it has been suggested that a specific Ang IV receptor (AT4) might exist. Bolus injection of Ang IV in brain ventricles has been implicated in learning, memory, and localized vasodilatation. However, the functions of Ang IV in a physiological context are still unknown. In this study, we generated a transgenic (TG) mouse model that chronically releases Ang IV peptide specifically in the brain. TG mice were found to be hypertensive by the tail-cuff method as compared with control littermates. Treatment with the angiotensin-converting enzyme inhibitor captopril had no effect on blood pressure, but surprisingly treatment with the Ang II AT1 receptor antagonist candesartan normalized the blood pressure despite the fact that the levels of Ang IV in the brains of TG mice were only 4-fold elevated over the normal endogenous level of Ang peptides. Calcium mobilization assays performed on cultured CHO cells chronically transfected with the AT1 receptor confirm that low-dose Ang IV can mobilize calcium via the AT1 receptor only in the presence of Ang II, consistent with an allosteric mechanism. These results suggest that chronic elevation of Ang IV in the brain can induce hypertension that can be treated with angiotensin II AT1 receptor antagonists.

Development of antral follicles beyond 3 to 4 mm in cattle appears as a wave pattern that occurs two to three times during the estrous cycle. Each wave presents a cyclic recruitment of multiple follicles at the 3- to 4-mm stage, followed by the selection of a single follicle that becomes the dominant follicle (DF). The molecular determinants involved in the follicular dominance process remain poorly understood. The objective of the current study was to compare gene expression in granulosa cells (GCs) between growing dominant follicles from Day 5 of the estrous cycle and nonselected small follicles (less than or equal to 4 mm) using the suppression subtractive hybridization (SSH) approach to identify candidate genes differentially expressed in GCs of the DF. Small follicle cDNAs were subtracted from DF cDNAs (DF-SF) and used to establish a DF GC-subtracted cDNA library. A total of 42 nonredundant cDNAs were identified. Detection of previously identified genes such as CX43, CYP19, INHBA, and SERPINE2 supported the validity of our experimental model and the use of SSH as the method of analysis. For selected genes such as ApoER2, CPD, CSPG2, 14-3-3 epsilon, NR5A2/SF2, RGN/SMP30, and SERPINE2, gene expression profiles were compared by virtual Northern blot or reverse transcriptase-polymerase chain reaction, and results confirmed an increase or induction of their mRNA in GCs of dominant follicles compared with that of small follicles. We conclude that we have identified novel genes (known and unknown) that are up-regulated in bovine GCs that may affect follicular growth, dominance, or both.

Silversides, D W; Thibault, G; Touyz, R M; Reudelhuber, T L; Lochard, N Author Information

BioTechniquesVol. 34, No. 1 BenchmarksOpen AccessRapid Genomic Identification of Transgenic Integration Sites for Mouse Insertional MutantsA. Boyer, M. Paquet, D. Raiwet, I. Daneau, W. Harrison & D.W. SilversidesA. BoyerUniversity of Montreal, St-Hyacinthe, Québec, Canada, M. PaquetUniversity of Montreal, St-Hyacinthe, Québec, Canada, D. RaiwetUniversity of Montreal, St-Hyacinthe, Québec, Canada, I. DaneauUniversity of Montreal, St-Hyacinthe, Québec, Canada, W. HarrisonUniversity of Montreal, St-Hyacinthe, Québec, Canada & D.W. Silversides*Address correspondence to Dr. David W. Silversides, Faculty of Veterinary Medicine, University of Montreal, St-Hyacinthe, Québec, Canada. e-mail: E-mail Address: silverdw@medvet.umontreal.caUniversity of Montreal, St-Hyacinthe, Québec, CanadaPublished Online:24 Sep 2018https://doi.org/10.2144/03341bm03AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInReddit FiguresReferencesRelatedDetails Vol. 34, No. 1 Follow us on social media for the latest updates Metrics Downloaded 117 times History Published online 24 September 2018 Published in print January 2003 Information© 2018 Author(s)PDF download

To study the process of mammalian sex determination and in particular to further understand the mechanisms of transcriptional regulation of the SRY gene, we have isolated a 4.5-kilobase (kb) pig SRY 5' flanking sequence. To facilitate the in vitro analysis of these sequences, we have generated a porcine genital ridge (PGR) cell line (9E11) that expresses SRY as well as SOX9, steroidogenic factor-1 (SF-1), and DAX1. Via primer extension analysis on RNA from this cell line, a transcription start site for porcine SRY was identified at -661 base pairs (bps) 5' from the translation initiation site. Deletion studies of the SRY 5' flanking sequences in PGR 9E11 cells demonstrated that -1.4 kb of 5' flanking sequences retained full transcriptional activity compared with the -4.5 kb fragment, but that transcriptional activity fell when further deletions were made. Sequences downstream of the transcriptional start site are important for promoter activity, because deleting transcribed but not translated sequences eliminated promoter activity. Sequence analysis of the -1.4 kb fragment identified two potential binding sites for SF-1, at -1369 and at -290 from the ATG. To address the role of SF-1 transactivation in SRY promoter activity, mutagenesis studies of the potential SF-1 binding sites were performed and revealed that these sites were indeed important for SRY promoter activity. Cotransfection studies in a heterologous cell system (mouse CV-1 cells) demonstrated that pig SF-1 was able to transactivate the pig SRY promoter. Gel shift assays confirmed that the upstream site was recognized by mouse SF-1 protein. We conclude that two sites for SF-1 transactivation exist within the pig SRY promoter, at -1369 bp and at -290 bp, and that the site at -1369 bp is quantitatively the most important.

Mice deficient for angiotensinogen (AGT), or other components of the renin-angiotensin system, show a high rate of neonatal mortality correlated with severe renal abnormalities including hydronephrosis, hypertrophy of renal arteries, and an impaired ability to concentrate urine. Although transgenic replacement of systemic or adipose, but not renal, AGT in AGT-deficient mice has previously been reported to correct some of these renal abnormalities, the tissue target for this complementation has not been defined. In the current study, we have used a novel transgenic strategy to restore the peptide product of the renin-angiotensin system, angiotensin II, exclusively in the brain of AGT-deficient mice and demonstrate that brain-specific angiotensin II can correct the hydronephrosis and partially correct renal dysfunction seen in AGT-deficient mice. Taken together, these results suggest that the renin-angiotensin system affects renal development and function through systemically accessible targets in the brain.