Effect of Adrenalectomy and Corticosterone Replacement on Epididymal Carbohydrate Metabolism - Studies on Mature Male Rats

The effect of adrenalectomy and corticosterone replacement on epididymal enzymes involved in obligatory steps of glycolysis and pentose phosphate pathway were studied along with serum hormonal profiles. Adrenalectomy was found to elevate serum prolactin while the gonadotropins and testosterone were unaltered. In caput epididymal tissue enzymes of the pentose phosphate pathway. Glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activities were increased after adrenalectomy. However, in corpus epididymal tissue the key enzymes viz. hexokinase, 6-phosphofructokinase and pyruvatekinase of the glycolytic pathway were elevated leaving the pentose phosphate pathway unaffected. Adrenalectomy was also found to favour glycolysis of the epididymal spermatozoa. The possible direct effect of prolactin is discussed to explain the enzymatic changes in epididymis. Corticosterone replacement was found to maintain the enzyme activities along with serum prolactin and corticosterone at control levels. In conclusion, it is suggested that the adrenalectomy induced changes in enzyme activities could be due to the direct effect of prolactin.     

Effect of thyroxine treatment on epididymal carbohydrate metabolism in the pubertal rat

The influence of thyroxine treatment on key enzymes involved in the glycolytic and HMP shunt pathways was studied in the caput, corpus and cauda epididymides of pubertal rats, and related to the serum hormone profile. The activity of 6-phosphofructokinase and pyruvate kinase was significantly increased in all 3 segments of the epididymis, but the HMP shunt pathway was suppressed. Thyroxine treatment was found to depress the serum levels of testosterone, LH and FSH, although an increase in prolactin levels was observed. Withdrawal of hormone treatment resulted in the restoration of normal activity of 6-phosphofructokinase and pyruvate kinase and restoration of normal serum hormone levels. However, the activity of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase increased following withdrawal of treatment. It is concluded that hyperthyroidism exerts an influence on epididymal enzymes involved in carbohydrate metabolism.     

The presence of α-glucosidase, glycerophosphocholine and carnitine in the epididymis and ejaculate of the vervet monkey (Cercopithecus aethiops) and the Chacma baboon (Papio ursinus)

Summary.  The epididymis is the site of post-testicular sperm maturation in the male genital tract. Studies on human epididymides are hampered by the practical inaccessibility of epididymides of healthy men in their reproductive years. The limited use of laboratory animals therefore seems unavoidable. The objective was to establish baseline values of the epididymal markers α-glucosidase, glycerophosphocholine (GPC) and carnitine in the lumen of the caput, corpus and cauda epididymidis and in the ejaculate of adult male Chacma baboons and vervet monkeys. In both primates, α-glucosidase was found throughout the epididymis and in the ejaculate; values did not vary significantly. In monkeys, the highest concentration of GPC was found in the cauda epididymidis, but smaller amounts were found in the other regions and the ejaculate. In baboons, GPC was absent from the caput, but present in the other regions, including the ejaculate. Carnitine concentrations increased significantly from the caput to the cauda in monkeys and from the caput to the corpus in baboons. With this study, the relative concentration ranges in which these markers are present in the epididymides of these primates have been established. In future studies, changes in concentrations of these substances would probably indicate changes in epididymal function.     

The ability of the rat epididymis to concentrate spermatozoa. Responsiveness to aldosterone

Experiments have been performed to determine if aldosterone is involved in the control of water reabsorption from the epididymal lumen in vivo. Micropuncture samples of lumen content were collected from the epididymides of control rats and those receiving aldrenalectomy, adrenalectomy + 25 micrograms aldosterone/day, 10 mg spironolactone/kg body weight/day, 10 mg spironolactone + 1 mg testosterone/kg body weight/day, 5 mg desoxycorticosterone acetate (DOCA)/day, 50 micrograms aldosterone/day, or 0.1 ml vehicle alone. The treatment period was three days. Seminal vesicles weights and testis weights were obtained. Sperm concentrations (SEM) in the caput, corpus, and cauda epididymidis of normal rats were 0.75 +/- 0.05, 1.24 +/- 0.13, and 1.99 +/- 0.15 x 10(9) sperm/ml, respectively. Both inhibition and removal of aldosterone caused significant reduction (P less than .01) of intraluminal sperm concentrations. Sham treatment had no effect. Sperm concentrations were normal in animals receiving aldrenalectomy plus aldosterone replacement. It is concluded that water resorption in the rat epididymis is responsive to aldosterone.     

Effect of corticosterone on rat epididymal lipids

Corticosterone-induced changes in serum hormone profiles and the lipid composition of the caput and cauda epididymidis were studied. The analysis was conducted in both unwashed and washed (free from fluids and spermatozoa) epididymal tissues. Corticosterone treatment significantly depressed serum prolactin and testosterone but gonadotropins were unaltered. In the unwashed caput region, lipid analysis showed a significant decrease in total lipids, as well as in cholesterol, phospholipid, and the phosphatidyl inositol, phosphatidyl choline, and phosphatidyl ethanolamine fractions. However, in the unwashed cauda region, the total lipid and cholesterol content was not altered while total phospholipid and phospholipid fractions were significantly decreased. On the other hand, in the washed caput and cauda regions, corticosterone induced a significant increase in total lipid, glyceride, and the mono, di, and triglyceride fractions, leaving total phospholipid and its fractions unaltered. Following 20-day withdrawal of corticosterone treatment, all lipid classes returned to normal along with serum hormone profiles. Our findings imply that an excess of corticosterone influences epididymal lipids. These changes in the epididymal lipid pattern probably are reflected in fertility disorders in patients with glucocorticoid excess.     

Electrolyte and Water Transport in Rat Epididymis; Its Possible Role in Sperm Maturation

Electrolyte and water transport in different regions of the rat epididymis has been studied using a microperfusion technique. The caput and proximal corpus epididymides were found to absorb NaCl and water and secrete K⁺ at a lower rate than the cauda epididymidis. The secretion rate of protein was the same in both regions. In the caput and proximal corpus, reabsorption of chloride was hypertonic. Reabsorption of sodium could not account for water reabsorption. In contrast, water reabsorption in the cauda epididymidis was dependent upon the intraluminal sodium ions. Amiloride inhibited both the Na⁺ and water reabsorption in this region. It was concluded that in the proximal regions of the rat epididymidis, water reabsorption may be secondary to an active transport of chloride, whereas in the cauda, a net transepithelial transport of sodium ions is the driving force for water reabsorption.
Transport of electrolytes and water across the perfused rat cauda epididymidis has also been studied under various experimental conditions. Treatment of rats with alpha-chlorohydrin (9 mg/kg/day) for 7 days inhibited the rate of sodium and water reabsorption without affecting the secretion of proteins. Ligation of the testicular efferent duct or the corpus epididymidis had no significant effect on the transport functions of the cauda epididymidis. When cyproterone acetate (10 mg/rat/day) was injected into male rats, the rate of sodium and water reabsorption was reduced. This effect was accompanied by a loss of sperm motility. It is concluded that the transport functions of the cauda do not require the normal flow of testicular fluid, but may depend on the supply of circulating androgen in the blood. Alpha-chlorohydrin and cyproterone acetate may affect sperm maturation by disrupting the normal milieu of the epididymal duct.     

Appearance of specific proteins in rat epididymis during postnatal development

In the immature 30-day-old caput and cauda epididymides of the rat, only a single prealbumin protein was visible. Additional epididymis-specific proteins appeared in the caput on day 45, coinciding with the entry of testicular fluid containing first wave of spermatozoa. In the cauda epididymides, additional proteins appeared only on day 50. These results indicate that the functional differentiation of caput precedes that of the cauda epididymidis.     

Effect of adrenalectomy on rat epididymidis

Aim: To investigate the effect of adrenalectomy (ADX) on the epididymidis of Sprague-Dawley rats. Methods: The histological, biochemical (cholesterol protein, zinc, copper, alkaline and acid phosphatase aryl sulphatase, lactic dehydrogenase and leucine amino peptidase) and hormonal (FSH, LH and testosterone) changes of caput and cauda epididymis in ADX rats were observed. Results: Organ wet weight, histological studies and morphometric measurements indicated a cellular degeneration in caput and cauda epididymis of ADX rats. Serum testosterone level was significantly lower in ADX than in sham-operated rats, while the serum FSH and LH were below the detection limit of 1 mIU/mL. The enzymatic activity was higher in ADX than in sham-operated rats. Epididymal zinc level increased whereas copper level decreased in ADX rats compared to the sham-operated. Conclusion: Adrenalectomy leads to degeneration of caput and cauda epididymidis epithelial cells as a result of decreased supply of testosterone. (Asian J And     

Loss of sperm surface sialic acid induces phagocytosis: an assay with a monoclonal antibody T21, which recognizes a 54K sialoglycoprotein

Using a monoclonal antibody T21, we reported that a mouse sperm maturation-associated antigen sialoglycoprotein of 54000 daltons (54K sialoglycoprotein) was secreted at the distal caput to proximal corpus epididymidis and that the 54K sialoglycoprotein had a hidden determinant (cryptodeterminant), which could be eliminated by sialidase treatment (Toshimori et al. (1988): Histochemistry 90:195-200; (1990a): Biol Reprod 42:151-160; (1990b): Arch Histol Cytol 53:339-349). This study evaluated the mouse sperm susceptibility to phagocytosis by macrophage in vitro. Comparisons were made between sperm from the caput epididymidis (caput sperm) incubated in modified Krebs Ringer's solution (MKR) and caput sperm incubated in MKR containing cauda fluid, and between sialylated (sialidase-untreated) sperm from the corpus and cauda epididymidis (corpus/cauda sperm) and desialylated (sialidase-treated) corpus/cauda sperm. The results showed that macrophages were least actively engaged in phagocytosis for caput sperm incubated in MKR containing cauda fluid, and most active for desialylated corpus/cauda sperm. Incubation of caput sperm in MKR containing cauda fluid revealed that the 54K sialoglycoprotein in cauda fluid could be bound to the flagellar surface of caput sperm. These results together with previous findings strongly suggest that the 54K sialoglycoprotein bound to immature sperm during maturation in the epididymis is implicated in the protection of sperm from phagocytosis with the aid of sialic acid residues.     

Two-dimensional gel electrophoretic analysis of rat sperm membrane interaction with cauda epididymal fluid

Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) was used to analyze the polypeptide composition of rat cauda epididymal fluid, blood serum and membrane-enriched fractions of caput, corpus, and cauda epididymal spermatozoa. Several polypeptides were found in both cauda fluid and blood serum, and in both cauda fluid and epididymal spermatozoa. Prominent cauda epididymal fluid polypeptides that were associated with caput, corpus, and cauda sperm membranes were 32 and 33 kDa. Passage of spermatozoa from the caput to the cauda epididymidis was characterized by the loss of three glycopolypeptides of 32, 30 and 29 kDa, and by the addition of a 37-kDa glycopolypeptide. Incubation of intact caput, corpus and cauda spermatozoa with cauda epididymal fluid revealed major changes in the polypeptide maps of the incubation fluid and the membrane-enriched fractions of caput and corpus, but not cauda spermatozoa. The incubation of cauda fluid with caput and corpus sperm cells was characterized by a loss of several polypeptides and the addition of a 24-kDa glycopolypeptide. The most striking change in spermatozoa incubated with cauda epididymal fluid was the addition of two glycopolypeptides of 32 and 33 kDa to the polypeptide maps of caput sperm cells. These data demonstrate that rat spermatozoa undergo surface modifications during epididymal maturation and that these modifications can be influenced by epididymal fluid.     

Further characterization of a secreted epididymal glycoprotein in mice that binds to sperm tails

Sperm maturation antigen 4 (SMA-4) is a surface component of the mouse sperm tail. Previously, immunofluorescence studies indicated that SMA-4 may be secreted by principal cells of the distal caput epididymidis and bound to spermatozoa as they pass through that region of the duct. In the present study, detergent extracts of spermatozoa from the cauda epididymidis were subjected to polyacrylamide gel electrophoresis under reducing and denaturing conditions, transferred to nitrocellulose, and immunostained with a monoclonal antibody against SMA-4. A band of approximately 54,000 molecular weight was revealed. The band was also stained by the periodic acid-Schiff (PAS) procedure. This glycoprotein was not detected in extracts of spermatozoa from the proximal caput epididymidis or of spermatozoa from the cauda epididymidis that were preincubated for 4 hours in an in vitro fertilization environment. Blots of sperm-free fluid from the corpus and cauda epididymidis displayed an immunoreactive and PAS-positive band of about 85,000 molecular weight that was not observed in fluid from the caput epididymidis. The difference in the molecular weights of the antigen in the fluid and that in extracts of cauda spermatozoa suggests that SMA-4 may be modified chemically upon association with the sperm surface.     

大鼠附睾上皮细胞的培养及其功能测定

用酶消化法分离成年雄性ＳＤ大鼠附睾头、体、尾的上皮细胞，贴壁培养。４～１１天后取贴壁细胞进行ＨＥ染色及扫描电镜观察，发现附睾头部和体部大部分为带有微绒毛的主细胞，而尾部细胞较小。并以ＰＮＰＧ为底物测定上清液内α－１．４糖苷酶的活性，结果为体部最高，尾部次之，头部最低。用硫代巴比妥酸法检测上清液中唾液酸的含量，仍以体部最高，但尾部最低。统计学处理均有显著差异。提示大鼠附睾体部可能是精子成熟的关键部位。     

Effect of adrenalectomy on rat epididymidis

Aim:To investigate the effect of adrenalectomy (ADX) on the epididymidis of Sprague-Dawley rats.Methods:The histological,biochemical(cholesterol protein,zinc,copper,alkaline and acid phosphatase aryl sulphatase,lactic dehydrogenase and leucine amino peptidase)and hormonal (FSH,LH and testosterone) changes of caput and cauda epididymis in ADX rats were observed.Results:Organ wet weight,histological studies and morphometric measurements indicated a cellular degeneration in caput and cauda epididymis of ADX rats.Serum testosterone level was significantly lower in ADX than in sham-operated rats,while the serum FSH and LH were below the detection limit of 1 mIU/mL.The enzymatic activity was higher in ADX than in sham-operated rats.Epididymal zinc level increased whereas copper level decreased in ADX rats compared to the sham-operated.Conclusion:Adrenalectomy leads to degeneration of caput and cauda epididymidis epithelial cells as a result of decreased supply of testosterone.     

Modification of the rat sperm flagellar plasma membrane during maturation in the epididymis

Previously, we demonstrated that surface radiolabeling of rat epididymal spermatozoa by lactoperoxidase-catalyzed iodination reveals a major component with an apparent molecular weight of 26,000 to 28,000 daltons (26 kDa) on spermatozoa from the cauda but not the caput epididymidis. To characterize this surface component further, sperm surface constituents radiolabeled by lactoperoxidase-catalyzed iodination were separated by 2-D PAGE. The 26 kDa component was localized by autoradiography and appeared as the major labeled acidic spot on cauda spermatozoa, but neither a radiolabeled spot nor a corresponding stained spot was present on caput spermatozoa. The 26 kDa spot was excised from 2-D gels of plasma membranes from cauda spermatozoa and utilized for immunization. The monospecific antiserum stained a single band of 26 kDa on Western blots of SDS-PAGE-separated plasma membranes from cauda spermatozoa and in a 100,000 X g supernatant fluid of the luminal contents of the cauda epididymidis. Immunohistochemical staining of cauda spermatozoa revealed antigen exclusively on the flagellar domain; the antigen was not seen on caput spermatozoa but first appeared in spermatozoa from the proximal corpus epididymidis. Immunoelectron microscopy confirmed the 26 kDa component was localized to the external face of the flagellar plasma membrane. Immunohistochemical staining of caput spermatozoa incubated in vitro with cauda epididymal luminal fluid revealed the 26 kDa component specifically bound the flagellar domain of immature spermatozoa.     

Glutathione peroxidase activity in cell cultures from different regions of human epididymis

AIM: To study the secretory activity and androgen regulation of glutathione peroxidase (GPx) in epithelial cell cultures from human epididymis. METHODS: Tissue was obtained from patients undergoing therapeutic orchidectomy for prostatic cancer. Epithelial cell cultures were obtained from the caput, corpus and cauda epididymides. Enzymatic activity was measured in conditioned media by colorimetric methods in absence or presence of 1, 10 or 100 nmol/L testosterone. The effect of 1 micromol/L flutamide was also evaluated. RESULTS: GPx activity was higher in cultures from corpus and cauda than caput epididymidis. The presence of different concentrations of testosterone increase enzyme activity in cell cultures from all epididymal regions. Addition of flutamide reverses the androgen dependent increase of GPx activity. CONCLUSION: GPx activity is secreted from human epididymal cells in a region dependent manner and is regulated by androgens.     

Gamma-glutamyl transpeptidase activity in the epididymis during fetal and postnatal development in rats.

The histochemical and biochemical distributions of gamma-glutamyl transpeptidase (gamma-GT) were investigated in the epididymis of rats during fetal and postnatal development. In the epididymal homogenates, gamma-GT activity was detected on the fifth day after birth. A sharp increase was observed after 30 days of life in the caput homogenates. Moderate levels of the enzyme were found in the cauda epididymis. Gamma-GT is histochemically detected from the 15th day of gestation in Wolffian ducts and in 17- to 18-day-old fetuses in newly differentiated epididymal tubules. Enzyme activity, was associated with the plasma membranes (apical, lateral, and basal), was preponderant on the apical part of the epithelial cells. During the first 15 days of the postnatal life, the histochemical reaction intensities were identical from the caput to the cauda epididymidis. From the 18th day onwards, enzyme activity decreased in the corpus and in the cauda, while gamma-GT increased in the caput epididymidis, and a strong activity was found on the apical surface of epithelial cells. Weak or moderate gamma-GT activity of spermatozoa in the caput tubules, increasing steadily from caput to cauda epididymidis, suggests that gamma-GT may be related to the functional maturation of spermatozoa.     

Experimental chlamydial epididymitis

Male Wistar rats were infected with the chlamydial agent of guinea pig inclusion conjunctivitis by inoculation of chlamydiae into the vas deferens. Epididymitis was observed in all infected animals clinically and histologically. Chlamydiae were detected in the epithelium of epididymal tubules by immunohistochemical staining (alkaline phosphatase anti-alkaline phosphatase technique). Inflammation progressed from the cauda to the corpus and caput epididymidis leading to fibrosis of the cauda epididymidis 28 days after infection. Animals responded to the infection with a rise of both serum IgM and IgG antibodies.     

Changes in Porcine Sperm Lactate Dehydrogenase Isoenzymes During Sperm Maturation

The changes in sperm lactate dehydrogenase (LDH-lactate: NAD oxidoreductase, EC 1.1.1.27) activity and the relative occurrence of LDH isoenzymes during sperm maturation were studied using pubertal German improved Landrace boars. The LDH of spermatozoa liberated from the testis, caput epididymis, proximal and distal corpus epididymidis and cauda epididymidis was spectrophotometrically quantified while the LDH isoenzymes were separated on fine cellulose acetate membrane strips with the Sartorius Sartophor system. The LDH content of sperm dropped drastically as they moved from the testis to the caput epididymidis. Thereafter, only little and insignificant changes were observed. Testicular sperm was composed more of the fastest anodically-migrating isoenzyme (LDH1) while with sperm maturation, the least or slowest migrating isoenzymes (LDH4 and 5) became progressively more dominant. This loss in LDH content in sperm and the shifts in the LDH isoenzyme patterns indicate that the development of sperm during maturation is dependent on a delicate balance between lactate and pyruvate, such that the cathodic isoenzymes involved in the anaerobic energy-supplying metabolic processes are sufficiently available for sperm activity and survival.     

Differential effect of hyperthyroidism on rat epididymal glycosidases

The impact of hyperthyroidism on epididymal glycosidases was studied in albino rats. Hyperthyroidism was induced in Wistar rats aged 30 days by daily injection of T4 (25 microg/100 g body weight/day intramuscularly) for 30 or 60 days; control rats were injected with vehicle (alkaline saline, pH 7.8). One set of hyperthyroid rats was reverted to euthyroid status by withdrawing T4 treatment after 30 days of hyperthyroidism. To asses the direct effect of thyroid hormone on epididymal hexosaminidases, caput, corpus and cauda tissues were stimulated with 25, 50 or 100 ng/mL T3 for 24 h, after an initial culture of 24 h. The activity of beta-glucosidase decreased in caput, corpus and cauda epididymis of hyperthyroid rats. beta-Galactosidase activity increased in the caput epididymis irrespective of the duration of hyperthyroidism. While a similar decrease occurred in the corpus and cauda epididymis in the 30 day hyperthyroid group, an opposite trend was observed in 60 day hyperthyroid rats. Caput beta-N-acetylglucosaminidase activities increased at both time points, whereas activity decreased in the corpus and cauda in 30 day, but increased in 60 day hyperthyroid rats. Hyperthyroidism consistently increased caput and corpus beta-N-acetylgalactosaminidase activity irrespective of the duration. Cauda epididymal beta-N-acetylgalactosaminidase activity was decreased in 30 day and increased in 60 day hyperthyroid rats. Hyperthyroidism induced changes in caput beta-galactosidase, beta-N-acetylgalactosaminidases, corpus beta-N-acetylglucosaminidase and cauda beta-N-acetylgalactosaminidase which were irreversible while the remaining actvities were brought back to normal when T4 treatment was withdrawn. In vitro studies showed that T3 stimulates epididymal hexosaminidases (beta-N-acetylglucosaminidase and beta-N-acetylgalactosaminidase) irrespective of the dose. These data suggest that thyroid hormones have a specific and direct influence on glycosidases in specific regions of the epididymis.     

Estimation of daily sperm production rate (DSPR) by quantitative testicular histology in Buffalo-bulls (Bubalus bubalis)

Testes and epididymides from six sexually rested buffalo-bulls were removed during breeding season. The average weight of the testicular parenchyma was 138.62 g, of which the tunica albuginea accounted for 8.45 g. Relative distribution of spermatozoa in caput, corpus, and cauda epididymides averaged 5.42, 0.75, and 11.45 billion, respectively. Total epididymal sperm reserve per bull was 36.2 billion. The efficiency of sperm production was quite uniform and averaged 14.5 x 10(6) sperm per gram of testicular parenchyma per day with a mean of 2.02 x 10(9) sperm per testis. Thus a typical buffalo-bull produces about 4 x 10(9) sperm daily during breeding season.