Proximal Caput Epididymidis Research Articles

CUB domains are not required for OVCH2 function in sperm maturation in the mouse epididymis.

Ovochymase 2 (Ovch2) is an epididymis-specific gene that is required for male fertility. While a multitude of reproductive tract-specific genes required for male fertility have been identified, OVCH2 is thus far the first protein required for male fertility that contains Complement C1r/C1s, Uegf, Bmp1 (CUB) domains located in tandem in the C-terminus of the protein. Identifying the functional significance of this unique domain has implications in better understanding fertility and infertility and as a potential contraceptive target. The goals of these studies were to understand the influence and requirement of OVCH2 CUB domains in the localization and functional requirement of OVCH2 in sperm maturation and function. To this end, we performed in vivo localization analysis of OVCH2 and reproductive phenotype analysis of mice containing C-terminal FLAG tag on OVCH2, with either the entire protein intact, or CUB2 or both CUB1 and CUB2 genetically ablated. All mice were generated through the CRISPR/Cas9 gene editing approach. We found that OVCH2 is specifically expressed in the proximal caput epididymidis, and the absence of CUB2 did not affect this localization pattern. Although the absence of both CUB domains significantly reduced sperm motility and progressive motility, this effect was not manifested in a reduction in fertility over a 6-month period mating trial, which showed no significant differences between control and CUB deletant mice. Further, the absence of one or both CUB domains did not affect reproductive organ structure or sperm morphology. Our studies demonstrate that the CUB domains are not required for fertility in male mice, at least under the normal animal housing conditions our mice were tested in, and suggest that the enzymatic activity of the OVCH2 protease, in the absence of its CUB domains, is sufficient for normal sperm processing in the epididymis. Although our findings do not preclude the possibility that OVCH2 CUB domains are required under a yet-identified stress condition, our findings demonstrate that the most likely region for deleterious mutations in men with idiopathic infertility and the most vulnerable site for inhibition of OVCH2 protein function is in its protease domain, and not its CUB domains. Our findings have implications in the genetic screening of infertile men and the development of a novel non-hormonal male contraceptive by honing in on the more critical region of a functionally required protein.

Dose-Response Effects of Epichlorohydrin on Male Reproductive Function in Rats.

Present study was conducted to investigate potential effects of epichlorohydrin on testicular and epididymal function in male rats. The test chemical was administered to adult male rats by gavage at dose levels of 0, 3.125, 12.5, and 50 mg/kg/day for 7 days. Testicular and epididymal function were assessed by measurement of reproductive organ weight, testicular spermatid count, epididymal sperm count, motility and morphology, and histopathology in rats. At 50 mg/kg, a decrease in the sperm motility and an increase in the incidence of sperm abnormalities were observed. Histopatho-logical examinations revealed an increase in the incidence of histopathological changes including cell debris in the ducts, vacuolization of the epithelial cells, oligospermia, and epithelial disruption in the proximal caput epididymidis. At 12.5 mg/kg, an increase in the incidence of histopathological changes of the epididymidis was found. There were no treatment-related effects at 3.125 mg/kg. These results show that 7-day repeated oral administration of epichlorohydrin to male rats results in adverse effects on sperm motility, sperm morphology, and epididymal histology at ≥ 12.5 mg/kg/day.

LXR and ABCA1 control cholesterol homeostasis in the proximal mouse epididymis in a cell-specific manner

Mammalian spermatozoa undergo important plasma membrane maturation steps during epididymal transit. Among these, changes in lipids and cholesterol are of particular interest as they are necessary for fertilization. However, molecular mechanisms regulating these transformations inside the epididymis are still poorly understood. Liver X receptors (LXRs), the nuclear receptors for oxysterols, are of major importance in intracellular cholesterol homeostasis, and LXR(-/-)-deficient male mice have already been shown to have reduced fertility at an age of 5 months and complete sterility for 9-month-old animals. This sterility phenotype is associated with testes and caput epididymides epithelial defects. The research presented here was aimed at investigating how LXRs act in the male caput epididymidis by analyzing key actors in cholesterol homeostasis. We show that accumulation of cholesteryl esters in LXR(-/-) male mice is associated with a specific loss of ABCA1 and an increase in apoptosis of apical cells of the proximal caput epididymidis. ATP-binding cassette G1 (ABCG1) and scavenger receptor B1 (SR-B1), two other cholesterol transporters, show little if any modifications. Our study also revealed that SR-B1 appears to have a peculiar expression pattern along the epididymal duct. These results should help in understanding the functional roles of LXR in cholesterol trafficking processes in caput epididymidis.

Factors Maintaining Normal Sperm Tail Structure During Epididymal Maturation Studied in Gopc−/− Mice1

Gopc (Golgi-associated PDZ- and coiled-coil motif-containing protein)(-/-) mice are infertile, showing globozoospermia, coiled tails, and a stratified mitochondrial sheath. Transmission electron microscope (TEM) images of the spermatozoa were studied quantitatively to analyze disorganization processes during epididymal passage. Factors maintaining straight tail and normal mitochondrial sheath were also studied by TEM and immunofluorescent microscopy. Sperm tails retained a normal appearance in the proximal caput epididymidis. Tail disorganization started between the proximal and the middle caput epididymidis, and the latter is the major site for it. The tail moved up through the defective posterior ring and coiled around the nucleus to various degrees. Tail coiling occurred in the caput epididymidis suggesting it was triggered by cytoplasmic droplet migration. SPATA19/spergen-1, a candidate mitochondrial adhesion protein, remained on the stratified mitochondria, while GPX4/PHGPx, a major element of the mitochondrial capsule, was unevenly distributed on them. From these findings, we speculate GPX4 is necessary to maintain normal sheath structure, and SPATA19 prevents dispersal of mitochondria, resulting in a stratified mitochondrial sheath formation in Gopc(-/-) spermatozoa. The epididymal epithelium was normal in structure and LRP8/apoER2 expression suggesting that tail abnormality is due to intrinsic sperm factors. Three cell structures are discussed as requisite factors for maintaining a straight tail during epididymal maturation: 1) a complete posterior ring to prevent invasion of the tail into the head compartment, 2) stable attachment of the connecting piece to the implantation fossa, and 3) a normal mitochondrial sheath supported by SPATA19 and supplied with sufficient and normally distributed GPX4.

Time-Dependent Rescue of Gene Expression by Androgens in the Mouse Proximal Caput Epididymidis-1 Cell Line after Androgen Withdrawal

Androgens are the primary regulators of epididymal structure and functions. Principal cells are the major cell type of the tissue and are particularly sensitive to androgen removal. To distinguish the direct effects of androgens on this cell type, DNA microarrays were used to identify androgen-regulated genes in the mouse proximal caput epididymidis (PC-1) cell line. PC-1 cells display tissue- and caput-specific gene expression. We examined changes in gene expression occurring 2, 4, and 6 d after androgen deprivation and 2 d after androgen supplementation after being deprived of androgen for 2 or 4 d. Changes in transcript levels were investigated for mediators of androgen action; selected genes were analyzed by real-time RT-PCR, and changes at the protein levels were examined. Four distinct patterns of gene expression were activated after androgen withdrawal; the vast majority of genes displayed an early or late transient increase in expression levels. A differential ability of rescue was seen among androgen-regulated genes, depending on time of androgen supplementation. Many of the genes that were rescued at 4 d were functionally linked by direct interactions and converged on IGF-I. The ability for rescue after 4 d of androgen deprivation was severely compromised in many genes belonging to specific functional gene families (cell adhesion, cell growth, apoptosis, and cell cycle) and may be mediated in part by changes in androgen receptor coregulator expression. These results provide novel insights into the mechanisms of androgen regulation in epididymal principal cells.

Expression and localization of c-ros oncogene along the human excurrent duct.

Compared to other animals, the anatomy of the human epididymis appears unusual. The caput epididymis is composed mostly of efferent ducts with an undefined initial segment. The present study investigates the regionalization of c-ros in human epididymis by real-time quantitative RT-PCR, in situ hybridization and immunohistochemistry studies. C-ros gene encodes a receptor-type protein tyrosine kinase that is expressed in adult mice exclusively in the epithelial cells of the initial segment of the epididymis. Transgenic mice targeted for the c-ros gene lack the initial segment of the epididymis and are infertile. Real-time PCR analysis showed that c-ros mRNA is expressed all along the human epididymis with the exception of the proximal caput epididymidis, where c-ros transcript was undetectable. In situ hydridization revealed that c-ros transcript was strongly expressed by principal cells and to a lower level by basal cells. Immunohistochemical studies showed that c-ros protein distribution was similar to the transcript. These results showed that c-ros expression in the human epididymis differs from that in mice suggesting that the unusual morphology of the human epididymis may reflect species differences in gene expression along the excurrent duct.

On the regulation of Crisp-1 mRNA expression and protein secretion by luminal factors presented in vivo by microperfusion of the rat proximal caput epididymidis.

Synthesis and secretion of certain epididymal proteins are regulated by lumicrine factors from the testis or from upstream regions of the excurrent ducts. Cysteine-rich secreted protein-1 (Crisp-1) is a major androgen regulated protein in the epididymal lumen fluid of the rat and other species. Previous research has demonstrated that disturbance of the luminal microenvironment through obstruction of the tract reduces Crisp-1 synthesis and secretion. The present study was undertaken to determine the influence of the luminal microenvironment on rat proximal caput epididymal Crisp-1 secretion into lumen fluid and on Crisp-1 gene expression in the same tubules. Western blot analysis demonstrated that Crisp-1 protein concentrations were reduced from control levels by perfusion with artificial caput fluid containing no testicular factors and were not increased by perfusion with fluids containing rete testis fluid proteins. Crisp-1gene expression was also reduced by perfusion with artificial caput fluid and not increased by perfusion with rete testis fluid proteins. Perfusion with artificial caput fluid containing 5alpha-dihydrotestosterone did increase one Crisp-1 transcript. This study demonstrates that intraluminal testicular proteins are not important co-regulators with androgens of Crisp-1gene expression or resulting Crisp-1 secretion into the rat proximal caput tubule lumen in vivo.

CCAAT/enhancer binding protein beta regulates expression of the cystatin-related epididymal spermatogenic (Cres) gene.

The CRES protein is a member of the cystatin superfamily of cysteine protease inhibitors with restricted expression in stage-specific germ cells, proximal caput epididymidis, and anterior pituitary gonadotroph cells. To elucidate the molecular mechanisms regulating the highly restricted expression of the cres gene, we have sequenced 1.6 kilobases of mouse cres 5' flanking sequence and performed studies to examine the cres gene promoter. Two putative CCAAT/enhancer binding protein (C/EBP) transcription factor binding motifs exist within the first 135 base pairs of cres promoter. Furthermore, our studies demonstrate that cres mRNA levels are dramatically reduced in the epididymides of C/EBP beta-deficient mice. These data suggest that the C/EBP family of transcription factors, in particular C/EBP beta, plays a role in the regulation of cres gene expression. In support of this finding, Northern blot analysis showed that C/EBP beta is the predominant C/EBP family member expressed in the L beta T2 gonadotroph cell line and the proximal caput epididymidis. Also, gel shift and supershift assays demonstrated that C/EBP beta protein in nuclear extracts from L beta T2 gonadotroph cells and epididymal cells bound to the two C/EBP sites in the cres promoter. Finally, to test the in vivo function of the C/EBP sites in cres gene expression, transfection studies were performed in L beta T2 gonadotroph cells and two heterologous cell systems. These experiments showed a significant reduction of cres transactivation when either C/EBP sites were mutated, and no transC/EBP activation of the cres promoter when both C/EBP sites were mutated. Taken together, these studies demonstrate that the C/EBP beta transcription factor is necessary for high levels of cres gene expression in the proximal caput epididymidis and anterior pituitary gonadotroph cells.

Epididymal specificity and androgen regulation of rat EP2.

In primates, expression of the EP2 gene is androgen-dependent and epididymis-specific. EP2 mRNA expression was investigated in caput, corpus, and cauda regions of rat epididymis and in 15 other rat tissues. Polymerase chain reaction and Northern analyses showed that rat EP2 is expressed predominantly in the proximal caput epididymidis. EP2 mRNA expression was determined in proximal epididymides from castrated, sham-operated, and efferent duct-ligated rats. In castrated rats, EP2 mRNA decreased to <10% of that in sham-operated rats between Days 3 and 4 postcastration, demonstrating the androgen dependence of EP2 expression. In epididymides ligated unilaterally at the efferent ducts, EP2 mRNA levels were approximately equal to those in the unligated contralateral epididymides or in sham-operated rats, indicating that EP2 expression does not depend on testicular factors. In bilaterally castrated rats, immediate and delayed testosterone replacement showed the dependence of EP2 expression on circulating androgens. Injection of testosterone propionate (TP) on Days 0, 1, 2, and 3 postcastration maintained EP2 mRNA levels approximately equal to those in sham-operated rats. Starting at Day 4 postcastration, daily injection of TP for 7 days restored EP2 mRNA to approximately normal levels. These data indicate for the rat that EP2 is expressed specifically in the proximal caput epididymidis and that its expression depends on circulating androgens but not on testicular factors.

Androgen binding protein secretion and endocytosis by principal cells in the adult rat epididymis and during postnatal development.

Androgen binding protein (ABP) has been shown to be secreted by Sertoli cells and to be actively taken up by the efferent ducts and proximal caput epididymidis and, yet, to be present at high concentrations in epididymal fluids. In the present study, ABP was immunolocalized by light microscopy in epithelial cells of the efferent ducts and epididymis of adult rats and during postnatal development and by electron microscopy in specific organelles within these cells. In adults, the efferent ducts actively endocytosed Sertoli cell-derived ABP. In the epididymis, principal cells displayed a variable staining reminiscent of a checkerboardlike pattern, with cells being intensely, moderately, or weakly reactive throughout their cytoplasm or unreactive. In the electron microscope, reactive cells displayed a labeling of their Golgi apparatus and secretory vesicles indicative of an epididymal-secreted form of ABP. However, labeling was also noted over endosomes of principal cells, but only of the initial segment and intermediate zone, which, along with labeling of coated pits and vesicles, indicated that ABP was also endocytosed by principal cells of these regions. The postnatal study revealed that principal cells attained an adultlike staining pattern indicative of secretion in a region-specific manner at different ages, suggesting that ABP secretion is regulated by different factors. Ligation of the efferent ducts of 15-day-old animals revealed no reaction along the entire epididymis in animals sacrificed at later ages, suggesting the importance of luminal testicular factors in its regulation during development. In addition, as in the adult, ABP was also endocytosed by principal cells, but only in the initial segment and intermediate zone. Taken together, the present results indicate that secretion of ABP occurs along the entire epididymis, whereas endocytosis is region specific. The functional role of ABP in the epididymis in relation to sperm maturation is discussed.

ADAM7, a member of the ADAM (a disintegrin and metalloprotease) gene family is specifically expressed in the mouse anterior pituitary and epididymis.

The maturation of spermatozoa in the epididymis is a complex process that requires the active involvement of the epididymal epithelium. The primary focus toward elucidating the role of the epididymis in the maturation process has been the study of epididymal secretory proteins and their interaction with spermatozoa. To date there is a paucity of information regarding epididymal epithelial cell surface proteins, which may also play important roles in epididymal function. Through a subtractive hybridization approach to identify genes specifically expressed in the caput epididymidis, the mouse homologue of a member of the ADAM (a disintegrin and metalloprotease) family of proteins was identified. This rapidly growing gene family encodes cell surface proteins that possess putative adhesion and protease domains. Northern blot analyses demonstrated that the mouse ADAM gene, termed ADAM7, is expressed in the caput region of the epididymis and in the anterior pituitary gonadotropes with no detectable expression in the twenty-six other tissues examined. Furthermore, in situ hybridization experiments revealed that the ADAM7 messenger RNA (mRNA) exhibited an apical localization within the proximal caput epididymal epithelium that may correlate with an unusual sparsely granulated endoplasmic reticulum uniquely present in the proximal region of the epididymidis and to which no known function has been ascribed. Hormonal, surgical, and genetic strategies demonstrated that ADAM7 gene expression requires, in a region-dependent manner, androgens as well as testicular factors for expression. Interestingly, the apical localization of ADAM7 mRNA is dependent upon an intact testis, because in situ hybridization analyses of the proximal caput epididymidis from a testosterone maintained castrate mouse did not show the apical localization of ADAM7 mRNA. Finally, chromosomal mapping demonstrated that the ADAM7 gene maps to the central region of mouse Chromosome 14, approximately 4-5 cM distal from the fertilin beta locus, which encodes another reproductive-specific ADAM protein.

Ultrastructural localization of PGP 9.5 and ubiquitin immunoreactivities in rat ductus epididymidis epithelium

The distribution of protein gene product 9.5 (PGP) and ubiquitin in the spermatozoa and epithelial cells in the different regions of the rat ductus epididymidis (proximal caput, distal caput, corpus and cauda) was studied by Western blotting analyses and electron microscopical immunogold labelling. Western blotting analyses showed that the PGP immunoreactive band was very intense in the caput and cauda epididymidis and almost irrelevant in the corpus, while the ubiquitin immunoreactive band was intense in the distal caput and cauda. No ubiquitin immunoreactive band was observed in the proximal caput and only a very weak band was seen in the corpus. The results of electron microscopical immunogold labelling varied from one epididymal region to another. The proximal caput epididymidis presented immunoreaction to PGP in the rough endoplasmic reticulum, cytosol, mitochondria and microvilli of most principal cells, and in the cytosol, rough endoplasmic reticulum and mitochondria of most basal cells. No ubiquitin immunoreaction was observed in this epididymal region. In the distal caput epididymidis, PGP immunoreactivity was detected in some principal and basal cells in the same intracellular locations as described in the proximal caput. In this region, ubiquitin immunoreactivity appears in the apical cytosol and mitochondria of principal cells. The corpus epididymidis showed no immunoreaction to PGP or ubiquitin. In the cauda epididymidis, immunostaining to PGP was observed in most clear cells and in isolated principal cells. The intracellular location of PGP in both cell types was the cytosol, mitochondria and microvilli. Ubiquitin immunoreactivity was detected in the perinuclear cytosol and mitochondria-but not in the digestive vacuoles-of some clear cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential expression of cathepsins B and D in testis and epididymis of adult rats.

Cathepsins are specific proteases in lysosomes that participate in the degradation of proteins, some of which are derived from endocytosis. In this study we examined the immunocytochemical localization of cathepsin B and D antibodies in cells of rat testis and epididymis, using light and electron microscopic immunocytochemistry. In testis, cathepsin D was immunolocalized over lysosomes of Sertoli cells and Leydig cells and on the acrosome of spermatids. Cathepsin B was found over lysosomes of macrophages. Non-ciliated cells of the efferent ducts revealed intense immunogold labeling over lysosomes with both anti-cathepsin B and D antibodies. In epididymis, cathepsins B and D showed marked variations in expression over the different epithelial cells and regional differences for a given cell type. Anti-cathepsin D antibodies showed intense labeling over lysosomes of principal cells in the corpus and proximal cauda. In contrast, anti-cathepsin B antibodies revealed intensely labeled lysosomes of principal cells of the distal initial segment, intermediate zone, and caput epididymidis, with weaker labeling in other regions. Clear cells of the proximal caput epididymidis revealed intensely labeled lysosomes for anti-cathepsin D antibodies. In the distal caput, clear cells showed a variable reaction pattern from intensely labeled to unreactive. Basal cells of teh intermediate zone and proximal caput region were intensely reactive for anti-cathepsin D antibodies. There was no staining over clear or basal cells with anti-cathepsin B antibodies. Taken together, these results demonstrate cell-specific and regional differences in the distribution of cathepsins B and D in cells of the male reproductive system. Such results suggest substrate specificity with regard to protein turnover within lysosomes of cells of testis and epididymis.

Developmental expression of the Yf subunit of glutathione S‐transferase P in epithelial cells of the testis, efferent ducts, and epididymis of the rat

Glutathione S-transferases (GSTs) are a family of isozymes that catalyze the conjugation of the tripeptide, glutathione, to various electrophilic compounds. The major GST in the pi class is GST-P, a homodimer of the Yf subunit, also known as Yp or rat subunit 7. This subunit is found in high concentrations in the epididymis and has recently been immunolocalized within epithelial principal and basal cells of the epididymis. In the present study we examine in groups of animals fixed in Bouin's fixative for light microscopy and in 4% paraformaldehyde and 0.5% glutaraldehyde in phosphate buffer for electron microscopy, the pattern of immunostaining for the Yf subunit of GST-P in the testis, efferent ducts and epididymis at various ages after birth. In the epididymis, on postnatal days 7 and 15, an immunoperoxidase reaction was localized exclusively to the apical and supranuclear regions of the undifferentiated columnar epithelial cells of the entire epididymis. By day 21, a dramatic change had taken place. In the initial segment, intermediate zone and proximal caput epididymidis, the columnar cells showed a distinct checkerboard-like staining pattern with cells ranging from being intensely reactive to unreactive. In contrast, principal cells of the distal caput, corpus, and proximal cauda epididymidis were weakly reactive. By day 28 the ratio of reactive to unreactive cells in the initial segment, intermediate zone, and proximal caput epididymidis was higher. By day 39, the differentiated columnar epithelial cells, referred to as principal cells, took on their adult staining pattern in the proximal and middle areas of the initial segment as well as the corpus and proximal cauda epididymidis where they were slightly reactive; in the distal initial segment they were strongly reactive. At day 49, principal cells in the intermediate zone and proximal caput became intensely reactive, while showing a distinct checkerboard-like staining pattern in the distal caput; similar observations were made for tissues taken from 56 and 90-day-old animals. Basal cells also showed a variable staining pattern in the different epididymal regions as a function of age. At day 21, when they first appeared, they were unreactive except for an occasional reactive cell in the corpus region. At day 28, only in the corpus epididymidis were many basal cells seen to be reactive. By day 39 the more numerous basal cells of the corpus and proximal cauda epididymidis were intensely reactive and remained so into adulthood. In these regions, basal cells appeared as dome-shaped cells (days 21, 28, 39), but then gradually flattened out and exhibited processes (days, 49, 56, adults) which collectively appeared to envelop the base of each tubule in a mesh-like network. The change in basal cell shape in each region coincided with the arrival of fluid and spermatozoa into the lumen (corpus day 49, proximal cauda day 56). In other epididymal regions, basal cells at day 28 were mostly unreactive. However, there was a gradual increase in the number of reactive basal cells of these regions between day 39 and 56. The present results thus demonstrate a dramatic change in the immunostaining pattern for the Yf subunit of GST-P during postnatal development for both principal and basal cells along the epididymis. Such results suggest that different factors play a role in the regulation of the expression of the Yf protein, not only in different epididymal regions, but also in different cell types during postnatal development.

Immunocytochemical localization of 4-ene steroid 5 alpha-reductase type 1 along the rat epididymis during postnatal development.

Dihydrotestosterone (DHT), the active androgen in many tissues, is synthesized from testosterone by the enzyme 4-ene steroid 5 alpha-reductase (5 alpha-reductase; EC 1.3.1.22). In the epididymis, the maturation and storage of spermatozoa are dependent on the presence of 5 alpha-reduced androgens. The regulation of epididymal 5 alpha-reductase is complex. To date, the regulation of this enzyme has been studied extensively at the level of enzymatic activity and more recently at the mRNA level. Regulation at the level of the protein, however, remains poorly understood. We have raised rabbit polyclonal antibodies to a 24-mer synthetic peptide whose sequence was determined from the predicted amino acid sequence for rat 5 alpha-reductase type 1 to immunolocalize the 5 alpha-reductase type 1 protein in the rat epididymis during postnatal development. Western blot analysis revealed a specific immunoreactive band of 26 kilodaltons in male rat liver, epididymis, and prostate; this apparent molecular size is identical to that obtained when the 5 alpha-reductase type 1 cDNA is expressed in mammalian cells. Furthermore, the relative protein levels, liver > epididymis > prostate, were consistent with the mRNA levels for type 1 rat 5 alpha-reductase. Perfusion-fixed paraffin-embedded epididymal tissue sections were used to immunolocalize type 1 5 alpha-reductase. In the adult rat epididymis, the most intense immunoperoxidase reaction was observed in a discrete lobule of the initial segment of the epididymis. A progressive decrease in staining intensity occurred distally along the tissue to the cauda epididymis. The staining reaction was specific to cytoplasmic elements of epithelial principal cells; no reaction was evident over nuclei. However, specifically in the initial segment, very intense staining was seen in the infranuclear region of the principal cells. In the proximal caput epididymidis, the staining was primarily confined to an oval region above the nuclei, whereas in the remaining epididymal regions, weak staining was seen throughout the cytoplasm. Thus, the intracellular localization of the 5 alpha-reductase type 1 protein changed as one moved down the epididymis. Finally, the pattern of immunolocalization of 5 alpha-reductase type 1 protein was different in the epididymis of rats of different postnatal ages. On day 7, no reactivity was noted; by day 28, a weak apical staining of principal cells was seen throughout the epididymis; by day 47, the adult pattern of staining had been established. Our results revealed that the expression and intracellular localization of the 5 alpha-reductase type 1 protein are both age dependent and epididymal segment specific.

Immunocytochemical localization of 4-ene steroid 5 alpha-reductase type 1 along the rat epididymis during postnatal development

Dihydrotestosterone (DHT), the active androgen in many tissues, is synthesized from testosterone by the enzyme 4-ene steroid 5 alpha-reductase (5 alpha-reductase; EC 1.3.1.22). In the epididymis, the maturation and storage of spermatozoa are dependent on the presence of 5 alpha-reduced androgens. The regulation of epididymal 5 alpha-reductase is complex. To date, the regulation of this enzyme has been studied extensively at the level of enzymatic activity and more recently at the mRNA level. Regulation at the level of the protein, however, remains poorly understood. We have raised rabbit polyclonal antibodies to a 24-mer synthetic peptide whose sequence was determined from the predicted amino acid sequence for rat 5 alpha-reductase type 1 to immunolocalize the 5 alpha-reductase type 1 protein in the rat epididymis during postnatal development. Western blot analysis revealed a specific immunoreactive band of 26 kilodaltons in male rat liver, epididymis, and prostate; this apparent molecular size is identical to that obtained when the 5 alpha-reductase type 1 cDNA is expressed in mammalian cells. Furthermore, the relative protein levels, liver > epididymis > prostate, were consistent with the mRNA levels for type 1 rat 5 alpha-reductase. Perfusion-fixed paraffin-embedded epididymal tissue sections were used to immunolocalize type 1 5 alpha-reductase. In the adult rat epididymis, the most intense immunoperoxidase reaction was observed in a discrete lobule of the initial segment of the epididymis. A progressive decrease in staining intensity occurred distally along the tissue to the cauda epididymis. The staining reaction was specific to cytoplasmic elements of epithelial principal cells; no reaction was evident over nuclei. However, specifically in the initial segment, very intense staining was seen in the infranuclear region of the principal cells. In the proximal caput epididymidis, the staining was primarily confined to an oval region above the nuclei, whereas in the remaining epididymal regions, weak staining was seen throughout the cytoplasm. Thus, the intracellular localization of the 5 alpha-reductase type 1 protein changed as one moved down the epididymis. Finally, the pattern of immunolocalization of 5 alpha-reductase type 1 protein was different in the epididymis of rats of different postnatal ages. On day 7, no reactivity was noted; by day 28, a weak apical staining of principal cells was seen throughout the epididymis; by day 47, the adult pattern of staining had been established. Our results revealed that the expression and intracellular localization of the 5 alpha-reductase type 1 protein are both age dependent and epididymal segment specific.

Protein gene product 9.5 and ubiquitin immunoreactivities in rat epididymis epithelium

A quantitative immunohistochemical study was performed of the distribution of protein gene product 9.5 (PGP, a soluble protein localized in neurons and neuroendocrine cells as well as in some non-nervous cells) and ubiquitin along the rat epididymis. In the ductuli efferents, PGP immunoreaction was observed in the whole cytoplasm of some columnar cells; a smaller number of columnar cells showed ubiquitin immunoreactivity with limited apical and basal cytoplasmic localization. In the proximal caput epididymidis, the whole cytoplasm of all columnar cells showed PGP immunoreactivity, ubiquitin immunostaining was negative in this region. In the middle and distal caput epididymidis and the distal cauda, the apical cytoplasm of some columnar cells and the whole cytoplasm of some basal cells showed immunoreactivity to PGP. In these regions, immunoreactivity to ubiquitin was positive in the supranuclear cytoplasm of some columnar cells but not in the basal cells. No immunoreactivity to PGP or ubiquitin was detected in the corpus epididymis and the proximal cauda. Double immunostaining revealed that all the epididymal ubiquitin immunoreactive cells were also PGP immunoreactive, whereas most PGP immunoreactive cells did not immunoreact to ubiquitin. In ubiquitin-PGP immunoreactive cells, the site of the PGP immunoreaction differed from that of the ubiquitin immunoreaction. PGP-ubiquitin immunoreactive cells also seemed to be immunoreactive to anti-AE1/AE3 keratin antibodies. The spermatozoal heads were immunoreactive to PGP antibodies in the epididymal regions from proximal caput to distal cauda but not in the ductuli efferents. The findings suggest that non-ubiquitinated PGP immunoreactive proteins are secreted in the epididymis, mainly in the proximal caput, and attach to spermatozoa.

Immunocytochemical localization of the Yf subunit of glutathione S-transferase P shows regional variation in the staining of epithelial cells of the testis, efferent ducts, and epididymis of the male rat.

Glutathione S-transferases (GSTs) are a family of isozymes that catalyze the conjugation of glutathione (GSH), a tripeptide found in all mammalian cells; this function plays a protective role, as the addition of GSH to an electrophile generally forms a less toxic product. The pi class of GSTs contains homodimers of the Yf subunit, also known as Yp or rat subunit 7; this subunit is found in high concentrations in the testis and epididymis. The objective of the present study was to localize immunocytochemically the Yf subunit in the testis and in the various regions of the epididymis using light, electron, and confocal microscopy. In the testis, immunoperoxidase staining was localized exclusively to Sertoli and Leydig cells. The low cuboidal epithelial cells of the rete testis and the sparse ciliated cells of the ductuli efferents were also immunoreactive. A distinct pattern of immunostaining for the Yf subunit was observed in the different regions of the epididymis. The proximal area of the initial segment showed intense reactivity localized to epithelial basal cells. Basal cells in the middle area of the initial segment were also reactive, as were a second unidentified population of cells located in the apical region of the epithelium. The epithelium, including both principal and basal cells, in the distal initial segment, intermediate zone, and proximal caput epididymidis showed a weak, moderate, or strong degree of reactivity, respectively. In the distal caput epididymidis, however, principal cells showed a checkerboard-like pattern of immunoreactivity, with some cells being intensely stained or faintly stained, whereas others were unreactive. Strikingly, in the corpus and proximal cauda epididymidis, intense immunostaining was localized exclusively over the epithelial basal cells. As viewed in the light and confocal microscope, the intensely stained basal cells showed extensive processes that covered most of the base of the epididymal tubule. Upon quantitation of the immunogold labeling density (the number of gold particles/microns2) in principal and basal cells of the different regions of the epididymis, we observed a sharp decline in immunogold labeling of principal cells coupled with a dramatic increase in labeling of basal cells as we progressed along the tissue, particularly in the transition from the caput to the corpus epididymidis. This study constitutes the first demonstration of a protein that is selectively expressed in epithelial basal cells of the corpus and proximal cauda epididymidis.(ABSTRACT TRUNCATED AT 400 WORDS)

Epithelial cells of the epididymis show regional variations with respect to the secretion of endocytosis of immobilin as revealed by light and electron microscope immunocytochemistry.

The localization of immobilin, a glycoprotein known to be present and to immobilize spermatozoa in the lumen of the epididymis, was investigated using light and electron microscope immunocytochemistry. In the light microscope, a distinct immunoperoxidase reaction product was observed in the lumen over the brush border of the epithelial nonciliated cells of the efferent ducts, while only a faint reaction was seen over their supranuclear region. In the proximal area of the initial segment of the epididymis no immunoperoxidase staining was observed either over epithelial cells or in the lumen. In the middle area of the initial segment, several epithelial principal cells became intensely immunostained but the majority were unstained; a weak reaction appeared in the lumen. In the distal area of the initial segment, more principal cells became immunostained, and while some were intensely reactive, others were moderately or weakly stained or unreactive. In the intermediate zone and proximal caput epididymidis, the principal cells showed the maximal immunoreactivity with all principal cells being reactive; staining in the lumen also reached its maximal reactivity in these areas. Immunostaining of principal cells gradually decreased along the epididymal duct and disappeared in the cauda epididymidis, however, an intense reaction persisted in the lumen. In the distal area of the cauda epididymidis, clear cells were reactive. In the electron microscope, immunogold labeling of reactive principal cells of the middle and distal areas of the initial segment, intermediate zone, and caput epididymidis was detected over cisternae of endoplasmic reticulum, stacks of Golgi saccules, and spherical electron lucent (200-400 nm in diameter) vesicles. The latter were present on the trans face of the Golgi stack, in the vicinity of th Golgi apparatus, and close to the apical cell surface; they are considered as secretory vesicles involved in the secretion of immobilin. In the distal area of the cauda epididymidis, epithelial clear cells showed an intense immunogold labeling over their endocytic apparatus. Immunogold labeling in the lumen of the epididymis was found over a fine flocculent material dispersed between the sperm. This material was especially abundant in the cauda epididymidis and did not appear to be bound to the surface of the sperm. The present results suggest that principal cells of the epididymis are involved in the secretion of immobilin, but that a differential secretory pattern exists between epididymal segments with maximal secretory activity occurring in the intermediate zone and proximal caput epididymidis, while no secretion takes place in the cauda epididymidis. Excess immobilin appears to be endocytosed for degradation by clear cells of the cauda epididymidis.

Localization of Sulfated Glycoprotein-2 (Clusterin) on Spermatozoa and in the Reproductive Tract of the Male Rat1

Sulfated glycoprotein-2 (SGP-2) is one of the major proteins secreted by rat Sertoli cells and epididymal cells in culture. The disulfide-linked dimeric protein secreted by Sertoli cells and found in seminiferous tubule fluid is composed of monomers of Mr 47 000 and 34 000 whereas the epididymal protein exhibits monomers of Mr 40 000 and 29 000. When both forms were chemically or enzymatically deglycosylated, they yielded proteins of similar molecular weight. No modification of the higher molecular weight testicular form by epididymal cells or fluids could be detected in incubation media. SGP-2 mRNA was localized in epididymal epithelium by in situ hybridization. Northern blot analysis indicated the testicular and epididymal mRNAs were of similar size. These findings suggest that the two forms of the protein occur because of tissue-specific post-translational modifications. The detergent-extracted protein from washed testicular spermatozoa is of the higher molecular weight form while epididymal sperm carry the lower molecular weight form. Immunohistochemical evidence suggests that the testicular form is removed prior to the initial segment of the epididymis and the epididymal form is applied in the proximal caput epididymidis. SGP-2 was immunolocalized to the sperm membrane at the ultrastructural level and was distinctly different from the immunolocalization of outer dense fiber proteins and fibrous sheath proteins.