Adult Rat Epididymis Research Articles

Effect of early antibiotic treatment on the formation of sperm antibodies in experimentally induced epididymitis.

This investigation was conducted to evaluate whether or not experimentally produced epididymitis could induce the development of cytotoxic sperm antibodies and if effective antibiotic therapy could reverse the development of immunity to sperm. Escherichia coli was injected into the tail of the epididymis in adult Lewis rats to induce epididymitis and was allowed to incubate for 24 h, 72 h, 8 days, or 15 days. Serum titers of cytotoxic sperm antibodies at these time intervals were determined. Sperm antibody titers began to rise 3 days after inoculation, peaked, and plateaued at 8 days. The titers were negligible in the control rats. Two other groups of rats were inoculated with E. coli in a similar manner and were treated with tetracycline 25 mg/kg/day starting at either 24 h or 8 days after inoculation, for 7 days. The antibody titers became negligible in these two treated groups, the results being statistically significant when contrasted with the infected but untreated groups (p < .001 and < .05, respectively, for the 24-h and 8-day groups). However, histological examination of the antibiotic-treated and untreated specimens revealed significant inflammation and infection of the epididymis in both treated groups. Testicular alterations were consistent in both groups. It is concluded that epididymitis consequent to infection with E. coli can induce cytotoxic antibody formation in Lewis rats. Treatment with appropriate antibiotics may suppress the antibody response either through a direct immunosuppressive effect of the antibiotic or through a decrease in the antigenic load of killed sperm secondary to eradication of the infection.

A novel system for the co-culture of epididymal epithelial cells and sperm from adult rats

To study interactions that occur between the epididymal epithelial cells and sperm within the epididymis during sperm maturation, a specialized co-culture system capable of supporting the differentiated function of these cell types must be utilized. A multifaceted approach has been devised to isolate plaques of epithelial cells from the epididymis of adult rats which involves microdissection, two collagenase digestions, and extensive pipetting and washing. The plaques of cells are cultured on a customized extracellular matrix within a primary semipermeable support and form into highly contiguous, but not confluent, regions of polarized epididymal epithelial cells during the first 2 days of culture. On Day 3, immature sperm are recovered from the caput epididymidis and placed within a second semipermeable support resting above the epithelial cells. During 24 h of co-culture, both sperm and epithelial integrity are maintained. Moreover, certain functions requisite to epididymal sperm maturation occur such as protein secretion by the epithelial cells, the association of secreted protein with the sperm, and the acquisition of progressive motility by the sperm. A comprehensive and illustrative description of this novel co-culture methodology is provided.

Differential Patterns of Regulated Gene Expression in the Adult Rat Epididymisa

Specialization among the principal epithelial cells of the epididymal tubule is documented following the analysis of transcriptional activity of four distinct species of mRNA. In situ histochemical analysis revealed a unique pattern of expression for each transcript. This observation supports the concept that region-specific patterns of transcriptional expression along the epididymal tubule serve as the major molecular basis underlying region-specific patterns of luminal proteins within the tubule. Additionally, multiple testicular factors appear to regulate expression of these mRNAs. The transcript encoding peptidyl-prolyl cis-trans isomerase is constitutively expressed. Those encoding the major secretory proteins, protein B/C and protein D/E, are directly regulated by testicular androgen. That encoding the opioid peptide precursor, proenkephalin, is regulated by a non-androgen testicular factor(s), specifically, spermatozoa or a spermatozoa-related factor. Thus, a complex array of nuclear events and signals received by the principal cells serve to determine the transcriptional status of genes expressed within this epididymal cell type.

Isolation and Characterization of Transcripts Induced by Androgen Withdrawal and Apoptotic Cell Death in the Rat Ventral Prostate

A variety of stimuli have been identified which initiate transcription-dependent programmed cell death (apoptosis) in specific target cells. Since the withdrawal of androgens induces regression and apoptosis in rat ventral prostate (RVP) epithelial cells, and it is known that the androgen receptor is a transcriptional regulator, we used subtraction cDNA cloning to isolate differentially expressed transcripts from the RVP of androgen ablated rats. In addition to sulfated glycoprotein-2 and glutathione S-transferase (GST), which had been previously described, several other transcripts were found to be elevated 3- to 8-fold in the regressing RVP. DNA sequencing revealed that two of these cDNA clones encode matrix carboxyglutamic acid and gamma-actin, respectively. A third cDNA contained novel sequence information and was named RVP.1. The RVP.1 transcript is expressed at very low levels in the RVP and epididymis of normal adult rats (less than 0.01% of the total mRNA) and is undetectable in other tissues, such as kidney, liver, and muscle. RVP.1 encodes a putative 280-amino acid protein, which shares no significant homology with previously described protein functional domains. We examined the expression of these transcripts in serum-starved NIH 3T3 cells to determine whether any of them are elevated in cells that are growth arrested. It was found that only GST mRNA levels are increased under these conditions. These data may suggest that induction of some genes, such as RVP.1, could be associated with apoptosis, whereas other transcripts, such as GST, may be up-regulated in response to altered rates of cellular metabolism.

In situ histochemical analysis of region‐specific gene expression in the adult rat epididymis

The epididymal tubule is a dynamic structure, in which spermatozoa undergo distinct physiological and morphological changes. The epithelial cells lining the ductuli vary dramatically in their histochemical and cytological properties according to the region of the tubule in which they are located. Additionally, regional variation is observed regarding the biosynthetic, secretory, and absorptive properties of the epithelial cells. Using in situ histochemical analysis, we document here the region-specific expression of a variety of genes that are transcriptionally active in the adult rat epididymis. Radiolabeled antisense riboprobes were used to localize, within the efferent duct/caput epididymis, transcripts encoding protein B/C, protein D/E (acidic epididymal glycoprotein), sulphated glycoprotein 1, sulphated glycoprotein 2, cellular retinol-binding protein, and the neuroendocrine peptide precursor proenkephalin. Each species of mRNA exhibits a unique pattern of hybridization, revealing that gene transcription within the efferent duct/caput epididymis is also highly region specific. This observation may partially elucidate the molecular basis underlying the phenomenon of regional alterations in the composition of protein factors within the tubule lumen.

Multiple effects of ethane dimethanesulfonate on the epididymis of adult rats

Ethane dimethanesulfonate (EDS), a Leydig cell toxicant which results in transient infertility, was used in a 4 day postexposure experimetal protocol designed to identify any effects this compound might exert on the epididymis. The techniques of efferent duct ligation and testosterone (T) implantation were used to negate the role of testicular effects on the epididymal parameters. Numerous evaluations were performed including light and electron microscopy, computer assisted sperm motion analyses, and electrophoresis of sperm membrane proteins. EDS was shown to affect the epididymis in a dose-dependent fashion. The action of EDS on the epididymis is in part due to Leydig cell cytotoxicity and the resulting decrease in circulating androgen since T implantation prevented some of the changes in sperm proteins and motility. However, neither efferent duct ligation nor T implantation prevented the formation of sperm granulomas in the caput epididymidis, the distinct morphological alterations of the corpus epididymidis, the modification of certain sperm membrane proteins, or the decrease in the progressive motility and velocity of sperm following EDS treatment. Although we cannot prove these effects of EDS are due to a direct action on the epididymis, it is now clear that EDS has a distinct action on the epididymis which is unrelated to circulating T or testicular fluid.

Diverse Secretory Patterns of Clusterin by Epididymis and Prostate/Seminal Vesicles Undergoing Cell Regression after Orchiectomy*

Nucleotide sequence analysis of the complimentary DNAs (cDNA) and N-terminal amino acid sequence analysis have shown that clusterin is equivalent to sulfated glycoprotein-2 (SGP-2), testosterone-repressed prostate protein-2 (TRPP-2), and androgen-repressed protein (ARP) in the rat, as well as serum/seminal plasma protein, SP-40,40, in the human. In view of its widespread presence in various species, a specific RIA was established to quantify the tissue distribution of this protein. Rat clusterin is present in almost all organ tissues examined, including testis, epididymis, serum, liver, prostate, seminal vesicles, and uterus. Displacement curves generated using cytosols prepared from these organs were parallel to those obtained using purified rat clusterin and crude Sertoli cell-enriched culture medium. Immunoreactive clusterin was also visualized in these organ extracts by immunoblots. Studies on the tissue distribution of immunoreactive clusterin using RIA revealed that the concentration of clusterin in the epididymis of adult rats was 6- and 10-fold higher than that in the serum and testis, respectively and is 50- to 100-fold higher in the liver, spleen, kidney, brain, ventral prostate, seminal vesicles, and uterus. A study of the distribution of clusterin in various compartments of the epididymis indicated its concentration in the caput epididymis was almost 3-fold higher than that in the corpus and cauda epididymis. After orchiectomy, the concentrations of clusterin in the ventral prostate and seminal vesicles increased as much as 100- and 10-fold and peaked at day 4 after surgery, respectively; daily injection of dihydrotestosterone (DHT) beginning at day 3 after orchiectomy reduced the concentrations of clusterin and restored them to a normal level. A different pattern was noted in the epididymis after orchiectomy; the concentration of clusterin in the caput epididymis decreased with time; however, daily injection of DHT beginning at day 3 increased the caput epididymal clusterin concentration and restored it to a normal level. The concentration of clusterin was not altered in the corpus or cauda epididymis after castration and/or DHT administration. Also, the serum and liver clusterin levels did not change with time after orchiectomy. These observations suggest that clusterin will be a valuable marker to monitor the diverse effects of androgen withdrawal in the male reproductive tract. We conclude that clusterin may be a multifunctional protein in view of its broad tissue distribution and association with numerous physiological and pathological conditions.

A Spermatozoa-Associated Factor Regulates Proenkephalin Gene Expression in the Rat Epididymis

The gene encoding the opioid peptide precursor preproenkephalin is expressed at high levels in the initial segment of the adult rat epididymis. Expression is localized to principal cells, the secretory epithelial cells lining the epididymal duct. During development, epididymal proenkephalin mRNA levels show a pronounced increase at about 44 days of age, coincident with the initial entry of spermatozoa into the epididymal lumen. Hypophysectomy leads to a 60-fold decrease in epididymal proenkephalin mRNA levels. Testosterone replacement can prevent this decline in a manner consistent with an effect upon spermatogenesis. Castration studies demonstrate that a gonadal factor other than testosterone directly regulates epididymal proenkephalin expression, and the results of efferent duct ligation suggest that this factor must be supplied through an intact connection of the testis and epididymis. Proenkephalin mRNA levels in the epididymis correlate with the decline and reappearance of spermatozoa induced by the alkylating agent busulphan. Thus, the developmental profile of proenkephalin expression, coupled with the results of both surgical and pharmacological manipulations of the reproductive tract, indicate that spermatozoa, or a spermatozoa-associated factor, regulate proenkephalin gene expression in the epididymis.

Localization of immunoreactive testibumin in the testis and epididymis of adult rats.

Testibumin is a glycoprotein previously isolated from the spent media of primary Sertoli cell-enriched cultures prepared from 20-day-old rats. Immunoassayable testibumin is found in the highest concentrations in testis, epididymis and fluids of the male reproductive tract in adult rats. In the present study, light microscopy was used to show that immunostainable testibumin in paraffin sections of rat testis was localized along the base of the seminiferous epithelium and in finger-like projections from the base of the epithelium, corresponding to the position of the Sertoli cells. The immunostaining of Sertoli cells was shown to be specific since either purified testibumin or crude Sertoli cell-enriched culture medium could compete with antibody for binding sites in tissue sections. The observations using light microscopy were confirmed when Sertoli cells were examined by electron microscopy using a pre-embedding immunostaining technique. The epithelium of the epididymis also contained immunoreactive testibumin which was localized in the caput, corpus and cauda. Immunostainable testibumin was also localized in the corpora lutea of the rat ovary and in the epithelium of the uterine endometrium. These observations are consistent with previous reports that immunoreactive testibumin is present in these organs as demonstrated by radio-immunoassays. We conclude that (i) the immunolocalization of testibumin in Sertoli cells adds to a growing list of observations suggesting that it is made in this cell type; (ii) the other sites of testibumin synthesis in the male are uncertain but the ovary and uterus are possible sites in the female; (iii) electron microscopy following pre-embedding immunostaining and epitope selection can be used as an adjunct to conventional immunocytochemistry to localize proteins in Sertoli cells.

Cyclic adenosine 3′,5′-monophosphate-phosphodiesterases in epididymis and prostate of castrate and of aged rats

The activities of the low- K m and high- K m forms of cyclic AMP phosphodiesterase (PDE) were determined in extracts prepared from epididymis and prostate taken from adult (10–12 months) and from aged (32–35 months) rats. The maximum velocity V of low- K m PDE in the epididymis of normal adult rats (approx. 300 pmoles/min per mg protein) was increased after castration by 80%, whereas V of the high- K m form remained unchanged, suggesting that only the low- K m PDE is subject to regulation by the animals' endocrine status. In the epididymis of aged rats V of low- K m PDE was unchanged, whereas V of high- K m PDE (approx. 3000 pmoles/min per mg protein) was decreased by 40%; in the prostate of aged rats V of both low- K m (approx. 225 pmoles/min per mg protein), and of high- K m PDE (approx. 1500 pmoles/min per mg protein) were decreased by 50–60%. The absence of interdependence of the two forms of the enzyme in their response to changes in environmental conditions in each organ suggests different mechanism of control of their activities and adds validity to the reported age-related decline of low- K m-PDE activity in prostate.

The Ontogeny of Biologically Active Androgen-Binding Protein in Rat Plasma, Testis, and Epididymis*

Androgen-binding protein (ABP) can be detected in the blood of sexually immature male rats by its ability to specifically bind [3H]5 alpha-dihydrotestosterone (5 alpha-DHT). Since the androgen-binding site is functional, we consider this ABP to be biologically active. ABP can be detected (641 +/- 107 ng/ml; n = 5) in plasma by the 15th day of postnatal life, it reaches a maximum concentration (1631 +/- 323 ng/ml; n = 5) on day 20 of age, and is no longer detectable after day 40. ABP can be detected in the testes of all age groups studied (15 days to adult). However, no ABP is detectable in the epididymis until the animals are 25 days old. Plasma ABP comigrates on nondenaturing gels with photolabeled ABP from the adult or immature rat epididymis. Serum that had been treated with Affigel blue to remove albumin and with hydroxylapatite to decolorize it was photolabeled using [3H]17 beta-hydroxy-4,6-androstadien-3-one. Photolabeled serum ABP migrated on polyacrylamide gels containing sodium dodecyl sulfate as 60,000 and 48,000 dalton androgen-specific peaks. In contrast, photolabeled adult epididymal ABP exhibited the 47,000 and 41,000 dalton peaks characteristic of ABP subunits. When photolabeled plasma and epididymal ABP were combined and electrophoresed on the same gel under denaturing conditions, prominent 60,000 and 47,000 dalton peaks were obtained, indicating that the two species of ABP retained their identities when combined. Photolabeled epididymal ABP from 25-day-old rats exhibited similar subunit mol wt in the same ratios as ABP from the adult. When epididymal ABP from the two age groups was combined and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the resulting pattern was identical to that obtained when the samples were run individually, except that there was an increase in peak height. These data indicated that there is no significant difference in the subunit mol wt of epididymal ABP from the two age groups.

Androgens in male rat reproductive tract fluids: hypophysectomy and steroid replacement.

The control of androgen concentrations in the intraluminal fluids of the male reproductive tract is not well understood. The present experiments were performed to determine the effects of hypophysectomy, hypophysectomy plus testosterone (T), and hypophysectomy plus pregnenolone treatment on intraluminal androgen concentrations in the adult rat testis and epididymis. T and 5 alpha-dihydrotestosterone (DHT) concentrations were determined in the vascular, interstitial, and intraluminal compartments of the epididymis. Testicular and epididymal morphology also were examined under light microscopy. Hypophysectomy of at least 5 days duration significantly reduced T and DHT concentrations in serum, tissues, and intraluminal fluids of the reproductive tract. T replacement for 14 days, which produced peripheral T concentrations of 5 ng/ml, did not support intraluminal androgen concentrations in the seminiferous tubules equivalent to controls; rete testis androgen concentrations were similar to controls, however. Pregnenolone administration at 2 mg X rat-1 X day-1 for 14 days did not maintain spermatogenesis nor intraluminal T concentrations in the seminiferous tubules equivalent to controls; however, a low level of spermatogenesis continued when intraluminal and tissue androgen concentrations were maintained at 10-20% of controls by either the testosterone and pregnenolone treatments.

Phosphorylated secretory proteins from rat epididymis and their androgenic control.

Slices of the rat epididymis were incubated with [32P]orthophosphate. Analysis of the radioactive proteins in the medium by two-dimensional gel electrophoresis and autoradiography revealed 6-phosphorylated secretory proteins from the epididymis of adult rats: Mr = 62 000; 66 000; 76 000; 68 000; 19 000 and 20 000. Studies of the epididymides of immature and 7-day-castrated adult rats indicated that these phosphorylated secretory proteins were controlled by androgens.

Structural features of cultured epithelial cells from the adult rat epididymis.

Epithelial cells isolated from the caput epididymidis of adult rats were placed in primary culture and examined daily for ten days for changes in external anatomy, reorganization of cytoskeletal components, maintenance of characteristic cytoplasmic features, and response to media formulated to minimize nonepithelial cell proliferation. Significant cell attachment to the substrate began after the first 24 hours of culture. After attachment, the cells underwent a progressive flattening and became closely applied to the substrate. This was accompanied by a redistribution of microvilli on the cell surface and a reorganization of cytoskeletal elements within the cell. After flattening, the cultured cells displayed an extensive array of 10-nm filaments which were associated with the desmosomes attaching adjacent cells. Immunofluorescence studies demonstrated that these were keratin-containing intermediate filaments and 2-D gel electrophoresis of intact cells and cell cytoskeletons revealed that a family of "keratin-like" polypeptides were major components of the cells. Epithelial cell attachment, morphology, and maintenance in the primary culture were unaffected by D-valine, cytosine arabinoside, or both; however, these agents, either individually or in combination, reduced significantly the number of cells incorporating 3H-thymidine. These data show that isolated epithelial cells retain some differentiated structural features that characterize the intact cell and that enriched cultures of epithelial cells can be maintained under conditions where fibroblast proliferation is inhibited.

Influence of hypothyroidism on epididymal enzymes involved in carbohydrate metabolism. Studies in pubertal and adult rats.

The influence of thyroidectomy on key epididymal enzymes of the Embden-Meyerhof and pentose phosphate pathway have been studied in pubertal and adult animals in relation to the serum hormone profile. Age related differences in the response of epididymal segments were observed with respect to hexokinase activity, although the other 2 key enzymes of the Embden-Meyerhof pathway (6-PFK and PK) were suppressed in all regions of the epididymis in both pubertal and adult rats. The enzymes involved in the pentose phosphate pathway (G-6-PDH and 6-PGDH) remained unaltered. The serum hormone profile revealed that while FSH and testosterone titres were reduced, LH and Prl were unaltered. Replacement of T4 in thyroidectomized animals maintained serum hormone levels and the activities of the enzymes studied at control levels. It is inferred that thyroid hormones may be one part of a complex mechanism that controls carbohydrate metabolism in the epididymis.

Rat sperm enzymes during epididymal transit.

The activities of cAMP and cGMP phosphodiesterases (EC 3.1.4.1), adenylate cyclase (EC 4.6.1.1) and protein carboxyl-methylase (EC 2.1.1.24) were measured in the particulate and soluble (105 000 g supernatant) fractions of washed spermatozoa isolated from five segments of the adult rat epididymis. The activities of both phosphodiesterases decreased during epididymal transit, whereas adenylate cyclase and protein carboxyl-methylase underwent a progressive increase, the latter showing the most marked alteration. Both cAMP and cGMP phosphodiesterases as well as the adenylate cyclase were all associated primarily with the particulate fraction, and the extent to which these enzymes were associated with the membranes increased as the spermatozoa passed through the epididymis. Sperm protein carboxyl-methylase activity was, on the other hand, predominantly soluble in all segments of the epididymis. Adenylate cyclase, cAMP phosphodiesterase and protein carboxyl-methylase activities were found predominantly in the sperm tails, whereas cGMP phosphodiesterase was equally distributed between heads and tails. These observations imply that the acknowledged increase in intracellular cAMP levels which occurs in spermatozoa during epididymal transit may be a consequence of both increased synthesis (adenylate cyclase) and reduced hydrolysis (phosphodiesterase).

Membrane specialization in the rat epididymis. II. The clear cell.

AbstractThe clear cell is a characteristic cell type, particularly frequent in the epididymal tail, which has many small vesicles and larger vacuoles in the apical cytoplasm. In freeze‐fracture replicas of adult rat epididymis, we found that the limiting membrane (P‐face) of these vesicles and vacuoles, as well as the luminal plasma membrane (but not the basolateral membrane) contain a high density of prominent intramembrane particles. In contrast, the apical vesicles of the most common cell type, the principal cell, have a much lower density of particles, which are also of smaller size. The unique organization of these membranes in clear cells suggests that this cell type has a special role in epididymal function.

Estradiol-17 β inhibition of androgen uptake, metabolism and binding in epididymis of adult male rats in vivo: A comparison with cyproterone acetate

The effects of estradiol-17β on androgen uptake, metabolism and binding were studied in rat epidymis in vivo in comparison with cyproterone acetate. Steroids (250 ug/100 g body weight) were injected 5 min prior to 3H-testosterone in castrate rats. Estradiol-17β inhibited 3H-testosterone uptake into epididymal cytosol by 58% as compared to 38% by cyproterone acetate. 3H-Testosterone uptake into epididymal nuclei was inhibited 95% by estradiol-17β and 83% by cyproterone acetate. Total bound radioactivity in cytosol fractions was reduced to a greater extent by estradiol-17β than cyproterone acetate when either 3H-testosterone or 3H-dihydrotestosterone was injected. Binding of 3H-dihydrotestosterone to nuclear receptors was completely abolished by estradiol-17β; whereas approximately 20% binding remained in the nuclear extract after cyproterone acetate treatment. Metabolism of 3H-testosterone in vivo was also altered by estradiol-17β, resulting in diminished conversion to 3H-dihydrotestosterone. Cyproterone acetate, on the other hand, did not affect 3H-testosterone metabolism. Estradiol-17β and cyproterone acetate inhibited in vitro binding of 3H-dihydrotestosterone to the intracellular cytoplasmic receptor, but not the intraluminal androgen binding protein (ABP). These data suggest that estradiol-17 β may have a more potent antiandrogenic effect on the epididymis than cyproterone acetate due to inhibition of 5 α reduction of testosterone as well as binding to the androgen receptor.

Regional differences in steroidogenesis and hormone levels in the epididymis and vas deferens of adult rats.

In vivo and in vitro studies with different parts of the epididymis and vas deferens were carried out to determine their inherent capacity to synthesize steroids and to correlate with the endogenous levels with or without the administration of hCG. Incubation with 14C-labelled pregnenolone and testosterone demonstrated that caput epididymidis was more active than other parts in synthesizing testosterone from 14C-pregnenolone and in converting labelled testosterone to 5 alpha-dihydrotestosterone (DHT). The cauda epididymidis and vas deferens accumulated more radioactivity in progesterone and dehydroepiandrosterone (DHEA) than the caput epididymidis. The levels of DHT, testosterone and 4-androstene-3, 17-dione in the caput epididymidis were reduced after ligation of ipselateral efferent ductules indicating the testicular origin of these steroids. The cauda epididymidis and vas deferens had higher levels of progesterone as compared to the other regions of the epididymis, which were decreased after the ligation. Intravenous injection of hCG increased the levels of oestradiol-17 beta in all tissues and markedly in the cauda epididymidis and vas deferens. The high levels of progesterone and oestradiol-17 beta present in these organs may be of importance in maintaining fertilizing ability of spermatozoa stored in the cauda epididymidis and vas deferens and their transport.

Morphological characteristics of cells with apical nuclei in the initial segment of the adult rat epididymis.

The cytology of epithelial cells with apical nuclei in the initial segment of the rat epididymis was studied with the light and electron microscopes. Two types of cells were distinguished and were designated apical cells and narrow cells. The apical cells are more numerous than the narrow cells and closely resemble principal cells except for the location of the nucleus. They probably correspond to the apical cells of Reid and Cleland ('57) and may represent a variation of the principal cell. The narrow cells differ markedly from the apical cells in both light microscopic appearance and fine structure. Narrow cells strain intensely with toluidine blue and are characterized by a slender shape, many mitochodria with tubular cristae, and a large number of apical cup-shaped cytoplasmic vesicles. The possible relationship of narrow cells to other cell types is discussed.