Pem homeobox gene regulatory sequences that direct androgen-dependent developmentally regulated gene expression in different subregions of the epididymis

The epididymis is a useful model system to understand the mechanisms that govern region-specific gene expression, as many gene products display spatially restricted expression within this organ. However, surprisingly little is known about how this regulation is achieved. Here, we report regulatory sequences from the Pem homeobox gene that drive expression in different subregions of the mouse epididymis in vivo. We found that the 0.3-kb 5'-flanking sequence (region I) from the Pem proximal promoter (Pem Pp) was sufficient to confer androgen-dependent and developmentally regulated expression in the caput region of the epididymis. Expression was restricted to the normal regions of expression of Pem in the caput (segments 2-4), but there was also aberrant expression in the corpus region. This corpus misexpression was extinguished when 0.6 kb of Pem Pp 5'-flanking sequence was included in the transgene, indicating that one or more negative regulatory elements exist between 0.6 and 0.3 kb upstream of the Pem Pp start site (region II). When heterologous sequences were introduced upstream of the Pem Pp, expression was further restricted, mainly to caput segment 3, implying that the Pem Pp has segment-specific regulatory elements. To our knowledge, the regulatory regions we have identified are the shortest so far defined that dictate regionally localized expression in the epididymis in vivo. They may be useful for identifying the factors that regulate region-specific expression in the epididymis, for expressing and conditionally knocking out genes in different subregions of the epididymis, for treating male infertility, and for generating novel methods of male contraception.     

Remarkable expansions of an X-linked reproductive homeobox gene cluster in rodent evolution

Rhox is a recently identified cluster of 12 X-linked homeobox genes in mice. The expression pattern of Rhox genes during postnatal testis development corresponds to their chromosomal position, much like the colinear gene regulation of the Hox gene clusters during animal embryonic development. We here report the identification of 18 additional Rhox genes and 3 pseudogenes in mice. Comparative analyses of the mouse, rat, human, dog, cow, opossum, and chicken genomes suggest that the Rhox cluster originated in the common ancestor of primates and rodents. It subsequently underwent two remarkable expansions, first in the common ancestor of mice and rats and then in mice. Positive selection promoting amino acid substitutions was detected in some young Rhox genes, suggesting adaptive functional diversification. The recent expansions of the Rhox cluster provide an opportunity to study the mechanism and origin of colinear gene regulation, but they may also undermine the utility of mouse models for understanding the development and physiology of the human reproductive system.     

Rhox homeobox gene cluster: recent duplication of three family members

We recently reported the discovery of a homeobox gene cluster on the mouse X chromosome, Rhox, whose 12 members are selectively expressed in specific cell types in reproductive organs. Here we report the existence of 20 additional Rhox homeobox genes in this gene cluster. Most of the newly identified Rhox paralogs retain the same order and relative orientation as three of the originally described Rhox genes, suggesting that they arose from recent duplications of this trimer unit. Many of these new Rhox family members are expressed in the testis and placenta. Analysis of synonymous and nonsynonymous substitutions in their homeodomain region suggests that these new Rhox paralogs duplicated so recently that their encoded proteins have not yet acquired distinct DNA-binding specificities. The existence of these new Rhox genes provides an opportunity to examine the initial stages of gene cluster evolution.     

Region-specific gene expression in the epididymis

The epididymis is responsible for post-testicular sperm maturation, which consists in the acquisition of forward motility and fertilizing ability. This organ is composed of three main anatomical regions - the caput, corpus and cauda epididymidis - which possess distinct gene expression profiles, ensuring different epididymal functions essential to the different steps of sperm maturation. Since many genes display spatially restricted expression in the epididymis, this organ constitutes a model of choice to study the mechanisms that govern region-specific gene expression. Factors such as steroid hormones, lumicrine factors and temperature affect the pattern of gene expression in the epididymis. Recently, the contribution of small RNAs in epididymal gene regulation has been investigated and constitutes a promising avenue for clinical application with regard to male fertility.     

Developmental expression and function of Bmp4 in spermatogenesis and in maintaining epididymal integrity

Bone morphogenetic proteins (BMPs) play essential roles in many aspects of developmental biology. We have previously shown that Bmp7, Bmp8a, and Bmp8b of the 60A class of Bmp genes have additive effects in spermatogenesis and in maintaining the epididymal integrity of the caput and caudal regions. Here we report that Bmp4 of the Dpp class has a unique expression pattern in the developing testis and epididymis. Bmp4 heterozygous males on a largely C57BL/6 background show compromised fertility due to degeneration of germ cells, reduced sperm counts, and decreased sperm motility. More interestingly, some of these males show extensive degeneration of the epididymal epithelium in the corpus region, rather than in the caput and cauda regions as for Bmp7 and Bmp8 mutants. Thus, these genetic data reveal a region-specific requirement of different classes of BMPs for epididymal epithelium to survive and have significant implications on male reproductive health and perhaps birth control.     

New member of the guanosine triphosphatase activating protein family in the human epididymis

The effect of the guanosine triphosphatase activating proteins (GAPs) on spermatogenesis has been studied for years, though no GAPs have been explored in epididymis, an essential organ for sperm maturation. In this study, a new GAP member, designated as MacGAP, was cloned in human epididymis. The MacGAP gene encodes a protein of 618 amino acids with a putative size of 70 kDa and harbors the conserved RhoGAP domain. The N-terminal and C-terminal peptides of MacGAP were expressed and their corresponding antisera were prepared. The antisera against N-terminal peptide could detect antigen as low as 0.3 ng, and its specificity was also confirmed. However, the antisera against C-terminal peptide failed to detect its antigen because of its low sensitivity. Immunohistochemistry showed that the MacGAP protein was dependent on epididymis and had a region-specific expression pattern, with high expression in the epithelial cells'basal section in the caput region. The results have created a foundation for further interpretation of the biological effects of GAPs in sperm maturation.     

The region-specific functions of two ubiquitin C-terminal hydrolase isozymes along the epididymis.

We previously showed that gad mice, which are deficient for ubiquitin C-terminal hydrolase L1 (UCH-L1), have a significantly increased number of defective spermatozoa, suggesting that UCH-L1 functions in sperm quality control during epididymal maturation. The epididymis is the site of spermatozoa maturation, transport and storage. Region-specific functions along the epididymis are essential for establishing the environment required for sperm maturation. We analyzed the region-specific expression of UCH-L1 and UCH-L3 along the epididymis, and also assessed the levels of ubiquitin, which has specificity for UCH-L1. In wild-type mice, western blot analysis demonstrated a high level of UCH-L1 expression in the caput epididymis, consistent with ubiquitin expression, whereas UCH-L3 expression was high in the cauda epididymis. We also investigated the function of UCH-L1 and UCH-L3 in epididymal apoptosis induced by efferent duct ligation. The caput epididymides of gad mice were resistant to apoptotic stress induced by efferent duct ligation, whereas Uchl3 knockout mice showed a marked increase in apoptotic cells following ligation. In conclusion, the response of gad and Uchl3 knockout mice to androgen withdrawal suggests a reciprocal function of the two UCH enzymes in the caput epididymis.     

Evaluation of the 5'-flanking regions of murine glutathione peroxidase five and cysteine-rich secretory protein-1 genes for directing transgene expression in mouse epididymis

Based on strong epididymal expression of the mouse glutathione peroxidase 5 (GPX5) and cysteine-rich secretory protein-1 (CRISP-1) genes, we evaluated whether the 5.0-kilobase (kb)-long GPX5 and 3.8-kb-long CRISP-1 gene 5'-flanking regions could be used to target expression of genes of interest into the epididymis in transgenic mice. Of the two candidate promoters investigated, the CRISP-1 promoter-driven enhanced green fluorescent protein (EGFP) reporter gene was highly expressed in the tubular compartment of the testis in all stages of the seminiferous epithelial cycle between pachytene spermatocytes at stage VII to elongated spermatids at step 16. In contrast to CRISP-1, the 5.0-kb 5' region of the mouse GPX5 gene directed EGFP expression to the epididymis. In the various GPX5-EGFP mouse lines, strongest expression of EGFP mRNA was found in the epididymis, but low levels of reporter gene mRNA were detected in several other tissues. Strong EGFP fluorescence was found in the principal cells of the distal caput region of epididymis, and few fluorescent cells were also detected in the cauda region. No EGFP fluorescence was detected in the corpus region or in the other tissues analyzed. Hence, it is evident that the 5.0-kb 5'-flanking region of GPX5 promoter is suitable for directing the expression of structural genes of interest into the caput epididymidis in transgenic mice.     

Expression of scatter factor/hepatocyte growth factor is regionally correlated with the initiation of sperm motility in murine male genital tract: Is scatter factor/hepatocyte growth factor involved in initiation of sperm motility?

Based upon findings that the scatter factor/hepatocyte growth factor (SF/HGF) has strong mitogenic and motogenic properties, and that the sperm cell acquires its fertilizing capacity and motility in the distal parts of mammalian epididymis, the present study was conducted to investigate the role of SF/HGF in initiation of sperm cell motility. This was investigated by determining the expression of SF/HGF in various regions of the murine male genital tract by scatter and cell tracking assays using MDCK epithelial cells, Western blot procedure, and the immunohistochemical procedure using paraffin sections of various regions of the male genital tract. The findings from all these assays indicate that SF/HGF is differentially expressed in various parts of the male genital tract with slight or no expression in the testes, caput epididymis, and vas deferens, and with the highest expression in cauda and corpus (distal) epididymis followed by expression in the corpus (proximal) epididymis. This region-specific SF/HGF expression pattern coincides with the pattern of acquiring the fertilizing capacity and motility by the sperm cell during its transit through the male genital tract. However, wherever SF/HGF was expressed in the male genital tract, its molecular weight was slightly higher (Mr, 82 kD), compared to the SF/HGF expressed in various other somatic tissues (Mr, 78 kD), indicating that the genital tract SF/HGF may be a different molecular species that shares some immunoreactive epitopes with the somatic cell SF/HGF. Incubation of immotile sperm from caput epididymis with the purified human placental SF/HGF of 78 kD initiated motility in 5–15% of sperm population. These results strongly suggest that the SF/HGF-like activity is expressed in the male genital tract in a region-specific manner, and this activity may have a role in initiation of sperm motility acquired during its transit through the epididymis in mammals. © 1994 Wiley-Liss, Inc.