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A Susceptibility Locus for Cryptorchidism In the ORL Rat Is Associated with Altered Levels of Muscle- and Hormone Receptor-Specific Transcripts In Fetal Gubernaculum and Cremaster

Saturday, October 20, 2012
Grand Ballroom A/B (Hilton Riverside)
Julia Spencer Barthold, Yanping Wang, Alan Robbins, Joan Pugarelli and Robert E. Akins, A.I. duPont Hospital for Children/Nemours Biomedical Research, Wilmington, DE

Purpose: Altered muscle-specific development in the gubernaculum and/or cremaster is a feature common to human and animal models of cryptorchidism.  To better define the developmental mechanisms of cryptorchidism, we studied the time course and differential expression of hormone receptor and muscle-specific transcripts in the ORL rat.

Methods: In prior genetic linkage analysis, we identified Syne2-Esr2 as a candidate modifier locus, homozygous in ORL and heterozygous in outbred Long-Evans (LE) rats, associated with cryptorchidism susceptibility.  Therefore, we generated genotype-specific timed pregnancies and compared 3 groups: LE-chr6LE, LE-chr6ORL and ORL.  Fetal gubernacula were harvested at gestational days (GD)17, 19 and 21 and cremaster muscle at postnatal day (P)3 and 30.  Total RNA purification, cDNA generation, Taqman-based RT-PCR, and data analysis using the delta-delta threshold cycle method were performed.  Transcript levels of genes encoding androgen receptor (Ar), estrogen receptors alpha (Esr1) and beta (Esr2), relaxin/insulin-like family peptide receptor 2 (Rxfp2), embryonic (Myh3), slow- (Myh7) and fast-IIb (Myh4) myosins; myogenin (Myog), desmin (Des), nesprin-2 (Syne2) and the AR coactivator ARA70 (Ncoa4, located in a second ORL linkage peak) were normalized to Gapdh levels with rat embryonic total RNA as calibrator. Data were log-transformed and analyzed with ANOVA and UNIANOVA using IBM SPSS v. 19.

Results: In LE-chr6LE samples, expression of all 4 hormone receptors, Syne2 and Ncoa4 decreased perinatally; Esr2 expression was low prenatally and undetectable postnatally.  As expected, the early muscle differentiation marker Myog peaked at GD21 during active gubernacular muscle development and Des, Myh3 and Myh7 levels were highest in postnatal cremaster muscle.  Fast twitch Myh4 was not detected until P30 and levels were significantly lower in ORL as compared to both LE-chr6LE and LE-chr6ORL cremaster.  In contrast, Rxfp2, Syne2 and Myh3 transcripts were overexpressed or failed to decrease in ORL fetuses in late gestation, just prior to gubernacular migration. In both ORL and LE-chr6ORL gubernacula, we observed altered trends in Syne2 expression, reduced Esr2 and Ar levels at GD17 and an earlier peak of Myh7 expression compared to LE-chr6LE.   However, other LE-chr6ORL prenatal expression patterns were unique with markedly higher Ncoa4 levels, an earlier rise in Myog and Des and earlier decrease in Rxfp2, Esr2 and Esr1 levels as compared to LE-chr6LE and ORL.  

Conclusion: Allele-specific expression of Syne2 and Esr2 transcripts in ORL and LE-chr6ORL fetuses supports a role for this locus in developmental gubernacular signaling and cryptorchidism.  However, the LE-chr6ORL pattern of hormone- and muscle-associated gene expression is otherwise unique, most notably in marked upregulation of the AR coactivator Ncoa4, which is located within another ORL cryptorchidism susceptibility locusThese data support our hypothesis that multiple genomic loci involved in AR signaling contribute to dysfunctional and/or insufficient gubernacular development and cryptorchidism in the ORL rat.