In many mammals, the concentration of myo-inositol in the fluid of the seminiferous tubules is dramatically higher than levels found in serum. Two enzymes involved in myo-inositol synthesis: myo-inositol-1-phosphate synthase (ISYNA1) and myo-inositol monophosphatase-1 (IMPA1), are known to have high activity in the testes. ISYNA1 is an isomerase that catalyzes the conversion of glucose-6-phoshate to myo-inositol-1-phosphate. IMPA1 then hydrolyzes the phosphate group to produce myo-inositol. Although no physiological role for the high concentration of myo-inositol has yet to be elucidated, it has been suggested that it could be involved in osmoregulation. Previous research on these enzymes in the testis has focused on enzyme activity. The objective of this study was to evaluate the expression of these genes and the myo-inositol transporter, Slc5a3, within the testis. Using Northern blot analyses, we found that all three genes, Impa1, Isyna1, and Slc5a3 are expressed in Sertoli cells. Isyna1 is highly expressed in two types of germ cells, pachytene spermatocytes and round spermatids. IMPA1 was expressed in round spermatids. Slc5a3 expression is upregulated when Sertoli cells are treated with 0.1 mM dibutyryl cAMP. When Sertoli cells were cultured in a hypertonic medium, there was an increase in the expression of Isyna1 and Slc5a3. We postulate that this upregulation is a result of the capability of the Sertoli cell to sense and then react to a change in osmolarity by increasing the transport and production of the osmolyte myo-inositol.

The epididymis is an androgen-responsive tissue where spermatozoa mature and gain motility. The three major regions of the epididymis, caput, corpus, and cauda, are known to have different functions and exhibit varied gene expression. Specific genes within the different regions of the epididymis have been identified to be under the influence of androgens. The goal of this study was to begin to elucidate the profile of androgen-responsive genes that may be important for sperm maturation using the Affymetrix MGU74Av2 GeneChip oligonucleotide microarray platform. Adult mice (B6/129 strain) were castrated and treated 6 days after castration with two injections of 5 mg of dihydrotestosterone (DHT) or oil over a 48-h period. The mice were killed 48 h later and total RNA was purified from the caput, corpus, and cauda regions of the epididymis. Using GeneSpring 5.0 (Silicon Genetics) software, transcripts were identified that were upregulated 2-fold or more by DHT in the caput (33 transcripts), the corpus (8 transcripts), and the cauda (9 transcripts).

Spermatozoa are highly specialized cells that, when mature, are capable of navigating the female reproductive tract and fertilizing an oocyte. The sperm cell is thought to be largely quiescent in terms of transcriptional and translational activity. As a result, once it has left the male reproductive tract, the sperm cell is essentially operating with a static population of proteins. It therefore is theoretically possible to understand the protein networks contained in a sperm cell and to deduce its cellular function capabilities. To this end, we performed a proteomic analysis of mouse sperm isolated from the cauda epididymis and confidently identified 2850 proteins, which to our knowledge is the most comprehensive sperm proteome for any species reported to date. These proteins comprise many complete cellular pathways, including those for energy production via glycolysis, beta-oxidation and oxidative phosphorylation, protein folding and transport, and cell signaling systems. This proteome should prove a useful tool for assembly and testing of protein networks important for sperm function.

Gonadotropin-releasing hormone (GnRH), a hypothalamic neurohormone, regulates transcription of Lhb in gonadotrophs indirectly through transient induction and accumulation of EGR1, a zinc finger transcription factor. AlphaT3 and LbetaT2 cell lines model gonadotrophs at two distinct stages of development, prenatal and postnatal expression of Lhb. Although GnRH induces EGR1 in both cell lines, the levels of the DNA-binding protein are lower and disappear more quickly in alphaT3 than in LbetaT2 cells. Herein we show that overexpression of Egr1 in alphaT3 cells rescues activity of a transfected LHB promoter-reporter, suggesting that its transcription is dependent on EGR1 crossing a critical concentration threshold. We also show that Csda, a gene that encodes an RNA-binding protein and is a member of the cold-shock-domain (CSD) family, is expressed at higher levels in LbetaT2 compared to alphaT3 cells. Transient expression studies indicate that at least one Csd element, residing in the 3' untranslated region of Egr1 mRNA, increases activity of a chimeric pGL3 luciferase reporter vector in LbetaT2 cells. Additional experiments indicate that CSDA physically interacts with Egr1 mRNA. Furthermore, siRNA-mediated reduction of endogenous Csda mRNA attenuates GnRH regulation of a transiently transfected LHB reporter vector. Taken together, these studies suggest that CSDA contributes posttranscriptionally to GnRH-regulated expression of Egr1, thereby enabling the transcription factor to cross a critical concentration threshold necessary for maximal accumulation of Lhb mRNA in response to the neurohormone.

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