Gail A. Cornwall

Gene Expression and Epididymal Function

The epididymis is a long convoluted tubule through which spermatozoa must pass to acquire the functions of progressive motility and fertility. Since spermatozoa entering the epididymis are, for the most part, synthetically inactive, the process of sperm maturation is thought to involve the interaction of spermatozoa with proteins that are synthesized and secreted by the epididymal epithelium. The epididymal epithelial cells secrete proteins in a highly regulated and regionalized manner such that spermatozoa encounter luminal fluid proteins in a specific sequence. Indeed, each region within the epididymis is its own microenvironment uniquely controlled by the cells’ differential response to extracellular signals and mediated by specific signaling molecules and DNA binding proteins ultimately resulting in region-specific gene expression and secretion of proteins. To understand the complex process of sperm maturation, therefore, not only requires the identification of secretory proteins which interact with spermatozoa but also knowledge of specific signal transduction pathways and regulatory proteins involved in this highly orchestrated series of events. Recently, significant progress has been made towards our understanding of epididymal function by the application of transgenic and gene inactivation approaches to examine gene regulation and function in the epididymis. The objectives of this review are to discuss region-specific expression of genes expressed exclusively or predominantly in the epididymis, the mechanisms regulating this tissue- and region-specific gene expression, and the value of transgenic technology for understanding epididymal function.

A new subgroup of the family 2 cystatins.

The cystatins are a superfamily of cysteine protease inhibitors. Several genes including Cres (cystatin-related epididymal spermatogenic), testatin, and cystatin T, have been identified that are related to the family 2 cystatins but lack critical consensus sites important for cysteine protease inhibition. In addition, these genes are primarily expressed in the reproductive tract suggesting they may have evolved to perform tissue-specific functions distinct from that of the typical cystatins. This review describes the CRES subgroup within the family 2 cystatins including potential new members and their putative functions in the reproductive tract.

DNA Microarray Analysis of Region-Specific Gene Expression in the Mouse Epididymis

Abstract Microarray analysis was carried out to identify genes with enriched expression in the initial segment region of the mouse epididymis. A set of approximately 15 000 clones developed at the National Institutes for Aging and consisting of expressed sequence tags (ESTs) derived from pre- and peri-implantation embryos, Embryonic Day 12.5 female gonad/mesonephros, and newborn ovary were hybridized with probes generated against the initial segment (epididymal region 1) and the remainder of the epididymis (epididymal regions 2–5). The median values for the normalized ratios of region 1 to regions 2–5 from three independent experiments were averaged for each gene/EST using Genespring 5.0 software. The majority of clones showed a ratio of 1.0, suggesting they were expressed at similar levels in all epididymal regions. In addition, 123 clones exhibited 2-fold or higher expression in the initial segment, including Cres3, prostein, lipocalin 2, ALEX3, synaptotagmin-like 4, erm, and milk fat globule factor, whereas 216 clones, including elafin-like 1, lactotransferrin, Sin3B, zinc-finger protein 91, and membrane-type frizzled-related protein, showed 2-fold or higher expression in epididymal regions 2–5. Northern blot analyses of 12 clones predicted by microarray analysis to be either enriched in the initial segment (n = 8), enriched in epididymal regions 2–5 (n = 2), or similar in all regions (n = 2) were carried out. All clones exhibited the expected region-specific expression, thus confirming the microarray results. The studies presented here show a global survey of region-specific gene expression in the epididymis, identifying 15 287 sequences, the majority of which have not previously been shown to be expressed in this organ.

Two novel human X-linked homeobox genes, hPEPP1 and hPEPP2, selectively expressed in the testis.

The PEPP genes are a recently described subfamily of mouse homeobox genes preferentially expressed in reproductive tissues. Pem, the founding member of the PEPP subfamily, has undergone rapid divergence due to positive selection, rendering the identification of its human orthologue difficult. Here we report the isolation and characterization of two human homeobox genes, hPEPP1 and hPEPP2, that are related to Pem and other PEPP family members. We identified these human genes based on their location in Xq24, which is syntenic to the mouse X-chromosome region containing three PEPP genes: Pem, Psx-1, and Psx-2. We found that hPEPP1 and hPEPP2 are selectively expressed in the testis, where the mouse and rat Pem genes are also expressed. However, unlike all mouse PEPP genes, hPEPP1 and hPEPP2 were not expressed in placenta, which suggests the possibility that the regulation of PEPP genes has significantly changed since the split between hominids and rodents. Although hPEPP1 exhibits highly selective expression in normal tissues, it is aberrantly expressed in tumor cell lines from several different organs, analogous to the expression pattern of mouse and rat Pem but not mouse Psx-1 or Psx-2. We conclude that we identified two human homeobox genes from the PEPP subfamily that are good candidates to encode transcription factors that regulate downstream genes and biological events in the human testis.

Gene and Protein Expression in the Epididymis of Infertile c-ros Receptor Tyrosine Kinase-Deficient Mice

Abstract Transgenic male mice bearing inactive mutations of the receptor tyrosine kinase c-ros lack the initial segment of the epididymis and are infertile. Several techniques were applied to determine differences in gene expression in the epididymal caput of heterozygous fertile (HET) and infertile homozygous knockout (KO) males that may explain the infertility. Complementary DNA arrays, gene chips, Northern and Western blots, and immunohistochemistry indicated that some proteins were downregulated, including the initial segment/proximal caput-specific genes c-ros, cystatin-related epididymal-spermatogenic (CRES), and lipocalin mouse epididymal protein 17 (MEP17), whereas other caput-enriched genes (glutathione peroxidase 5, a disintegrin and metalloproteinase [ADAM7], bone morphogenetic proteins 7 and 8a, A-raf, CCAAT/enhancer binding protein β, PEA3) were unchanged. Genes normally absent from the initial segment (γ-glutamyltranspeptidase, prostaglandin D2 synthetase, alkaline phosphatase) were expressed in the undifferentiated proximal caput of the KO. More distally, lipocalin 2 (24p3), CRISP1 (formerly MEP7), PEBP (MEP9), and mE-RABP (MEP10) were unchanged in expression. Immunohistochemistry and Western blots confirmed the absence of CRES in epididymal tissue and fluid and the continued presence of CRES in spermatozoa of the KO mouse. The glutamate transporters EAAC1 (EAAT3) and EAAT5 were downregulated and upregulated, respectively. The genes of over 70 transporters, channels, and pores were detected in the caput epididymidis, but in the KO, only three were downregulated and six upregulated. The changes in these genes could affect sperm function by modifying the composition of epididymal fluid and explain the infertility of the KO males. These genes may be targets for a posttesticular contraceptive.

Cystatin-Related Epididymal Spermatogenic Protein Colocalizes with Luteinizing Hormone-β Protein in Mouse Anterior Pituitary Gonadotropes1

The CRES (cystatin-related epididymal spermatogenic) protein, a member of the cystatin superfamily of cysteine protease inhibitors, exhibits highly restricted expression in the mouse testis and epididymis, suggesting roles in reproduction. Considering the well-established relationship that exists between the gonads and the neuroendocrine system, the present studies were undertaken to determine whether the CRES messenger RNA and protein are expressed in the anterior pituitary gland and, if so, whether the expression is regulated by hormones. RT-PCR analysis of whole pituitary gland RNA preparations, and Northern blot analyses of pituitary gland cell lines, demonstrated that the CRES gene is expressed in the male and female anterior pituitary gland gonadotropes. Furthermore, Western blot analysis demonstrated that CRES protein was present in whole mouse pituitary glands and was synthesized and secreted by the LβT2 gonadotrope cell line. Interestingly, whereas the predominant CRES proteins present in epididy...

Isolation and Proteomic Characterization of the Mouse Sperm Acrosomal Matrix

A critical step during fertilization is the sperm acrosome reaction in which the acrosome releases its contents allowing the spermatozoa to penetrate the egg investments. The sperm acrosomal contents are composed of both soluble material and an insoluble material called the acrosomal matrix (AM). The AM is thought to provide a stable structure from which associated proteins are differentially released during fertilization. Because of its important role during fertilization, efforts have been put toward isolating the AM for biochemical study and to date AM have been isolated from hamster, guinea pig, and bull spermatozoa. However, attempts to isolate AM from mouse spermatozoa, the species in which fertilization is well-studied, have been unsuccessful possibly because of the small size of the mouse sperm acrosome and/or its fusiform shape. Herein we describe a procedure for the isolation of the AM from caput and cauda mouse epididymal spermatozoa. We further carried out a proteomic analysis of the isolated AM from both sperm populations and identified 501 new proteins previously not detected by proteomics in mouse spermatozoa. A comparison of the AM proteome from caput and cauda spermatozoa showed that the AM undergoes maturational changes during epididymal transit similar to other sperm domains. Together, our studies suggest the AM to be a dynamic and functional structure carrying out a variety of biological processes as implied by the presence of a diverse group of proteins including proteases, chaperones, hydrolases, transporters, enzyme modulators, transferases, cytoskeletal proteins, and others.

Identification and Characterization of Cystatin-Related Epididymal Spermatogenic Protein in Human Spermatozoa: Localization in the Equatorial Segment

Abstract Our earlier studies in mouse have shown that the cystatin-related epididymal spermatogenic (CRES) protein is highly expressed in elongating spermatids in the testis and is present in mouse sperm acrosomes, suggesting specific roles in sperm function, fertilization, or both. However, whether the human CRES gene is similar to that of the mouse and is expressed in germ cells has not yet been determined. Therefore, the present study was undertaken to characterize the human ortholog of mouse Cres. Northern blot and in situ hybridization experiments showed that CRES is highly expressed in the human testis, specifically within clusters of round spermatids. Furthermore, reverse transcription-polymerase chain reaction detected CRES mRNA in the epididymis. Western blot analysis of protein lysates prepared from human testis and ejaculated spermatozoa showed a predominant 19-kDa protein and a minor 14-kDa protein. However, in contrast to the acrosomal localization of CRES protein in mouse spermatozoa, indirect immunofluorescence of human spermatozoa treated with methanol/acetic acid using anti-human CRES antibodies revealed that CRES was strictly localized to the equatorial segment. Furthermore, the same staining was observed in both capacitated and acrosome-reacted spermatozoa. To determine whether CRES was associated with the plasma membrane, live spermatozoa were incubated with CRES antibody after capacitation and acrosome reaction. Only acrosome-reacted spermatozoa showed a weak but specific equatorial staining. Taken together, these studies show that CRES protein is present in the sperm equatorial segment and becomes accessible to the extracellular environment during fertilization.

B-Myc, A Proximal Caput Epididymal Protein, Is Dependent on Androgens and Testicular Factors for Expression

Abstract The myc family of transcriptional regulators carries out critical roles in the control of cellular proliferation, differentiation, apoptosis, and tumorigenesis. The B-myc gene is a recently identified myc family member that has not been well characterized. Previously, we have shown that B-Myc inhibits the ability of c-Myc to transform cells and can inhibit cellular proliferation. Because B-myc is primarily expressed in hormonally regulated tissues with predominant expression in the epididymis, we examined in greater detail B-myc expression in the epididymis to ultimately understand potential roles B-myc may play in this and other hormonally regulated tissues. Herein we demonstrate that, in contrast to c-myc, B-myc mRNA and protein expression are highly regionalized with expression predominantly in the proximal caput epididymal region. Furthermore, in situ and immunohistochemical analyses show that within the epididymis B-myc mRNA and protein are specifically expressed by the epithelial cells and that B-Myc protein is localized to both the nuclear and cytosolic compartments. Castration and hormone replacement studies further show that expression of the B-myc mRNA is highly dependent on the presence of androgens and testicular factors. Finally, mRNA turnover studies demonstrate that the B-myc mRNA is relatively unstable with a half-life of 3.5 h. Taken together, the highly restricted and regulated expression of the B-myc gene suggests it may play important regulatory roles in the epididymis and perhaps other hormonally regulated tissues.

Role of Posttranslational Protein Modifications in Epididymal Sperm Maturation and Extracellular Quality Control

The epididymal lumen is a complex microenvironment in which spermatozoa acquire motility and fertility. Spermatozoa are synthetically inactive and therefore the maturation process requires their interaction with proteins that are synthesized and secreted in a highly regionalized manner by the epididymal epithelium. In addition to the integration of epididymal secretory proteins, posttranslational modifications of existing sperm proteins are important for sperm maturation and acquisition of fertilizing potential. Phosphorylation, glycosylation, and processing are several of the posttranslational modifications that sperm proteins undergo during epididymal transit resulting in changes in protein function and localization ultimately leading to mature spermatozoa. In addition to these well-characterized modifications, protein aggregation and cross-linking also occur within the epididymal lumen and may represent unique mechanisms for controlling protein function including that for maturation as well as for extracellular quality control.