Expression and processing of the neuroendocrine protein secretogranin II in benign and malignant pheochromocytomas.

The aim of the present study was to compare the expression levels of secretogranin II (SgII), prohormone convertases (PC)1 and PC2, and the proteolytic processing of SgII in benign versus malignant pheochromocytomas. Quantitative (Q)-PCR experiments indicated that SgII, PC1, and PC2 mRNAs were overexpressed in pheochromocytoma compared to non-tumoral chromaffin cells (P<0.001) and in benign compared to malignant tumors (P<0.01). Western blot analysis using a human SgII antiserum revealed the occurrence of a 97-kDa band corresponding to the expected size of SgII, with significantly higher quantities in benign than in malignant tumors (P<0.05). Using antisera directed against sequential regions of SgII (N-terminal, secretoneurin [SN], EM66, internal, and C-terminal sequences), we observed distinct processing profiles between benign and malignant pheochromocytomas. In contrast, using PC1 and PC2 antisera no differences between the two types of tumors were found. RIA measurement showed that EM66 median values between benign and malignant chromaffin cell tumors were significantly different (128.5 vs. 6.3 ng/mg protein, respectively; P<0.001). Taken together, these results indicate that, in pheochromocytoma, malignancy is associated with reduced PC1, PC2, and SgII mRNA expression and decreased levels of processing products of SgII, in line with the low concentrations of EM66 that occur in malignant tumors. These data support the notion that SgII-processing products, such as EM66, could represent prognostic markers of pheochromocytomas.

CLPH, a novel casein kinase 2-phosphorylated disordered protein, is specifically associated with postmeiotic germ cells in rat spermatogenesis.

In a recent proteomic study of rat spermatogenesis, we identified CLPH (for Casein-Like PHosphoprotein), a new testis-specific protein expressed exclusively in postmeiotic germ cells. In situ hybridization showed that the CLPH transcript was mainly present in round spermatids, whereas the protein was specifically detected by immunohistochemistry in elongated spermatids and in residual bodies. Electron microscopy showed the protein to be mostly cytoplasmic, but also frequently associated with the mitochondrial inner membrane during the last steps of spermatid differentiation. The Clph gene was found to be present solely in mammalian genomes, in a chromosomal region syntenic to the mammalian cluster of secretory calcium-binding phosphoprotein (SCPP) genes. CLPH has several distinctive properties in common with SCPPs: calcium overlay experiments showed that CLPH was a calcium-binding protein, whereas trypsin digestion assay, circular dichroism and fluorescence experiments demonstrated its intrinsically disordered structure. We also showed that CLPH was phosphorylated in vitro and in vivo by casein kinase 2, an enzyme critical for spermatid elongation. Given the specific and strong production of CLPH during rat spermiogenesis, together with the particular biochemical properties of this protein, we suggest that CLPH is involved in the extremely complex structural rearrangements occurring in haploid germ cells during spermiogenesis.

Identification, molecular cloning, and cellular distribution of the rat homolog of minichromosome maintenance protein 7 (MCM7) in the rat testis.

As part of a program to decipher the rat testicular proteome, we studied spermatogonia and identified numerous proteins including the human homolog of the Minichromosome Maintenance Protein 7 (MCM7). MCM7 has been implicated in DNA replication in various species, but had not been detected in the testis. Here we describe the cellular distribution of MCM7 transcripts and protein, and their testicular ontogenetic expression. The full-length coding region of the rat MCM7 was also characterized. Northern blot analyses showed that MCM7 transcripts are more abundant in the testis than other organs and confirmed the presence of the 2.4 kb MCM7 transcript at all ages studied. Interestingly, two additional transcripts of 3.2 and 1.6 kb were found from 26 days post partum onwards, when spermatocytes and spermatids accumulate within the tubules. This was confirmed in isolated cell types: the three MCM7 transcripts were observed in meiotic and post-meiotic germ cells. The 3.2 kb isoform has an extended 5' untranslated region (UTR) and the 1.6 kb transcript is the result of alternative splicing of five exons. Western blot and immunohistochemistry experiments evidenced abundant MCM7 in proliferating gonocytes and Sertoli cells in the fetal testis. In the adult testis, an intense signal was observed in spermatogonia and primary spermatocytes. We conclude that the Mcm7 is one example of genes that are differently transcribed and translated in somatic and spermatogenetic cells in mammals. Further work is required to determine the roles of MCM7 in spermatogonia and germ lineage.

The making of "transgenic spermatozoa".

The processes of making transgenic animals by microinjecting DNA into the pronucleus of a fertilized oocyte or after the transfection of embryonic stem cells are now well established. However, attempts have also been made, with varying degrees of success, to use spermatozoa as a vector for transgenesis in mammals and other vertebrates during the last decade. A number of different approaches for making transgenic spermatozoa have been developed. These include directly incubating mature, isolated spermatozoa with DNA or pretreating mature, isolated spermatozoa before assisted fertilization. Microinjection procedures have also been established to transfect male germ cells directly in vivo within the seminiferous tubules or to reimplant previously isolated male germ cells submitted to in vitro transfection into a recipient testis. The latter two techniques present the advantage of being able to create transgenic progeny simply by mating with wild-type females, which avoids the possibility of interference or damage as a result of assisted fertilization or the manipulation of embryos. The different aspects of sperm-mediated transgenesis are presented.

Impaired gametogenesis in mice that overexpress the RNA-binding protein HuR.

A series of experiments, using cell culture models or in vitro assays, has shown that the RNA-binding protein HuR increases the half-life of some messenger RNAs that contain adenylate/uridylate-rich decay elements. However, its function in an integrated system has not yet been investigated. Here, using a mouse model, we report that misregulation of HuR, due to expression of an HuR transgene, prevents the production of fully functional gametes. This work provides the first evidence for a physiological function of HuR, and highlights its involvement in spermatogenesis.

The testicular form of hormone-sensitive lipase HSLtes confers rescue of male infertility in HSL-deficient mice.

Inactivation of the hormone-sensitive lipase gene (HSL) confers male sterility with a major defect in spermatogenesis. Several forms of HSL are expressed in testis. HSLtes mRNA and protein are found in early and elongated spermatids, respectively. The other forms are expressed in diploid germ cells and interstitial cells of the testis. To determine whether the absence of the testis-specific form of HSL, HSLtes, was responsible for the infertility in HSL-null mice, we generated transgenic mice expressing HSLtes under the control of its own promoter. The transgenic animals were crossed with HSL-null mice to produce mice deficient in HSL in nongonadal tissues but expressing HSLtes in haploid germ cells. Cholesteryl ester hydrolase activity was almost completely blunted in HSL-deficient testis. Mice with one allele of the transgene showed an increase in enzymatic activity and a small elevation in the production of spermatozoa. The few fertile hemizygous male mice produced litters of very small to small size. The presence of the two alleles led to a doubling in cholesteryl ester hydrolase activity, which represented 25% of the wild type values associated with a qualitatively normal spermatogenesis and a partial restoration of sperm reserves. The fertility of these mice was totally restored with normal litter sizes. In line with the importance of the esterase activity, HSLtes transgene expression reversed the cholesteryl ester accumulation observed in HSL-null mice. Therefore, expression of HSLtes and cognate cholesteryl ester hydrolase activity leads to a rescue of the infertility observed in HSL-deficient male mice.