Age-related changes in the hamster epididymis

Reproductive ability is decreased in aged animals and in men. Little is known about the changes taking place in the epididymis, and the possible influence on the loss of sperm quality. We studied the age-related alterations in the epididymis and in epididymal spermatozoa of hamsters. Adult (6-month-old), middle-aged (18-month-old), and aged (24-month-old) hamsters were used. Serum samples were obtained to determine testosterone levels. Testes and epididymides were removed and studied by light and electron microscopy. Epididymal sperm was also obtained and the motility, position of cytoplasmic droplet, and concentration were evaluated. Measurements of the height of the epithelium, length of stereocilia, external tubular diameter, and thickness of the muscular wall were performed. The proliferative activity was also studied. An ANOVA analysis was used to compare quantitative differences between epididymal zones and age groups. Aged hamsters presented involutive changes in the epididymis. A decrease in tubular diameter was found in cauda; principal cell ultrastructure showed changes including the appearance of damaged mitochondria, bundles of filaments, and the accumulation of lipofuscin. Some clear cells showed an unusual morphology by the presence of large electrondense vacuoles. A reduction in sperm quality was also observed, including a decrease in sperm motility and concentration, and alterations in the migration of sperm cytoplasmic droplet. Testosterone levels and cellular proliferative activity did not change. Aging causes a morphological alteration of hamster epididymis (mainly in the cauda), and a decrease in sperm quality.     

Age‐related changes in the hamster epididymis

Reproductive ability is decreased in aged animals and in men. Little is known about the changes taking place in the epididymis, and the possible influence on the loss of sperm quality. We studied the age‐related alterations in the epididymis and in epididymal spermatozoa of hamsters. Adult (6‐month‐old), middle‐aged (18‐month‐old), and aged (24‐month‐old) hamsters were used. Serum samples were obtained to determine testosterone levels. Testes and epididymides were removed and studied by light and electron microscopy. Epididymal sperm was also obtained and the motility, position of cytoplasmic droplet, and concentration were evaluated. Measurements of the height of the epithelium, length of stereocilia, external tubular diameter, and thickness of the muscular wall were performed. The proliferative activity was also studied. An ANOVA analysis was used to compare quantitative differences between epididymal zones and age groups. Aged hamsters presented involutive changes in the epididymis. A decrease in tubular diameter was found in cauda; principal cell ultrastructure showed changes including the appearance of damaged mitochondria, bundles of filaments, and the accumulation of lipofuscin. Some clear cells showed an unusual morphology by the presence of large electrondense vacuoles. A reduction in sperm quality was also observed, including a decrease in sperm motility and concentration, and alterations in the migration of sperm cytoplasmic droplet. Testosterone levels and cellular proliferative activity did not change. Aging causes a morphological alteration of hamster epididymis (mainly in the cauda), and a decrease in sperm quality. Anat Rec 256:335–346, 1999. © 1999 Wiley‐Liss, Inc.     

Changes in the plasma membrane proteins of stallion spermatozoa during maturation in the epididymis

The present paper reports modifications in the electrophoretic and cytochemical characteristics of mature and immature stallion spermatozoa. Some sperm surface glycoproteins (36, 32, 29, 21, 20, 18 kDa) detected in cauda epididymidis spermatozoa, were either absent or present in a very low relative concentration in immature sperm cells. A major 14 kDa protein band, observed in sperm extracts obtained from ductus efferentes, progressively decreased along the epididymal ductus. The nature and distribution of carbohydrate residues on the sperm membrane, during epididymal maturation, was also studied by use of lectin probes. Some protein bands bound concanavalin A while others, as the 36, 32 and 20 kDa proteins, exhibited higher affinity for WGA lectin. The distribution and relative density of mannose-, galactose-, N-acetylglucosamine-, N-acetylgalactosamine-, fucose- and sialic acid-containing macromolecules showed a characteristic pattern depending on the sperm membrane domain and on its origin. Some sperm surface domains displayed affinity for more than one lectin, indicating a diversity in their exposed carbohydrate residues, whereas others bound only one or no lectin. The passage of spermatozoa through the epididymidis was accompanied by changes in the accessibility or abundance of lectin ligands. Some lectins (UEA, WGA, LPA) gave stronger reaction in mature spermatozoa, while others (RCA, WFH, PNA) stained better immature spermatozoa. This remodeling of sperm surface molecules is probably a consequence of interactions between spermatozoa and the epididymal secretions, and may reflect addition or adsorption of new molecules, space configurations changes or biochemical modifications of pre-existing compounds. Our results suggest that the distribution and density of terminal oligosaccharidic residues on the sperm plasma membrane have species-specific characteristics. These post testicular developmental changes may be of significance in the overall understanding of the stallion fertility.     

Binding of secreted glycoproteins to spermatozoa in the mammalian epididymis: a fine-structure autoradiographic study

Mouse and guinea pig epididymal tissues have been investigated by light and electron microscopic autoradiography after long intervals ranging from 24 h to 5 days postinjection (p.i.) of the glycoprotein precursors, L-fucose-6-3H or D-glucosamine-1-3H. Using modified fixations to enhance glycoprotein preservation in situ, we found intense labelling of luminal contents in at least some of the epididymal segments after all the intervals investigated. At 24 h p.i., the label in guinea pig was associated with spermatozoa during remodelling of the acrosome in segment II, and at 3 days p.i., radioactivity was trapped within sperm head associations ("rouleaux") in segment IV of the epididymis. At this time, similar rouleau labelling extended from segment IV to segment VIII. In mouse, the luminal contents of the cauda epididymis were still intensely labelled at 5 days p.i.; analysis of the electron microscopic autoradiograms showed that relative grain concentration over the spermatozoa was twice that of the epididymal plasma. This concentration was especially elevated in the region of the sperm head. These findings taken together were interpreted as the binding of secreted epididymal glycoproteins to spermatozoa during sperm transit through the epididymis. In contrast to luminal contents, the labelling of the epididymal epithelium was generally lower, except on the clear cells which showed more pronounced labelling than the neighboring principal cells in mouse cauda epididymis at 5 days p.i. This label probably originated from the resorption of luminal glycoproteins.     

锌缺乏导致小鼠睾丸游离锌离子和附睾精子数量减少

目的研究锌缺乏对小鼠睾丸游离锌离子和附睾精子数量的影响。方法锌缺乏饲养小鼠5周后,应用ZnSe金属自显影技术对小鼠睾丸和附睾进行染色,观察睾丸和附睾锌离子的分布,同时计数附睾精子数量,并与对照组小鼠的精子数量做对比。结果缺锌喂养后的小鼠睾丸游离锌离子明显减少,并且睾丸生精小管管腔狭小,生殖细胞层厚度增加,此外,附睾精子数量也明显低于对照组。结论睾丸和附睾内游离锌离子减少是锌缺乏小鼠精子数量下降的原因,本结果有助于改善男性生殖健康的状况。     

Changes in intramembranous particle distribution in the plasma membrane of didelphis virginiana spermatozoa during maturation in the epididymis

Structural specializations in the plasma membrane of opossum spermatozoa obtained from different levels of the epididymis have been analyzed in thin sections and freeze-fracture replicas. The maturation process was accompanied by a redistribution of intramembranous particles in the flagellar midpiece region. Caput epididymal spermatozoa are immotile, and freeze-fracture replicas of the midpiece plasma membrane reveal a random arrangement of intramembranous particles. As spermatozoa transit the corpus epididymis, the intramembranous particles in the midpiece plasma membrane are redistributed from a random arrangement to an organized packing pattern. This redistribution apparently involves the formation of chains of intramembranous particles which gradually increase in length, orient parallel to the flagellar long axis, and ultimately form numerous parallel rows, each three to five particles wide. In cauda epididymal spermatozoa the intramembranous particles within the rows are packed in an organized manner, and few free intramembranous particles are noted between rows. Analysis of thin sections revealed that the reorganization of intramembranous particles is accompanied by the deposition of a mat of amorphous material at the cytoplasmic face of the membrane. No striking changes in intramembranous particle distribution during epididymal maturation were found in other flagellar segments or in the plasma membrane overlying the sperm head.     

Maturation antigen of the mouse sperm flagellum: II. Origin from holocrine cells of the distal caput epididymis

During epididymal transit, the mouse sperm flagellum acquires a surface glycoprotein (SMA4) from epididymal fluid that functions as a sperm antiagglutinin. To determine the origin of this molecule, testes and epididymides of male mice were sectioned for light microscopy and stained with wheat germ agglutinin (WGA)-peroxidase, a probe that has been used previously to examine the biology of SMA4. WGA reactivity was localized to the cytoplasm in a small population of cells in the distal caput epididymis. Testis cells and principle cells of the caput were nonreactive with WGA, while stereocilia were stained on principle cells in the corpus and cauda. The WGA-positive cells in the distal caput were identified as holocrine cells on the basis of morphology, distribution, and PAS + reaction. At high magnification, intense WGA reactivity was due to the presence of numerous apical granules in the cytoplasm. The location of the cells in distal caput coincided exactly with the region of tubule in which sperm first acquired SMA4 on their flagellae. These data suggest that holocrine cells near the junction of caput and corpus epididymis are the source of the sperm antiagglutinin SMA4.     

Purification of GP-83, a glycoprotein secreted by the human epididymis and conjugated to mature spermatozoa

Epididymal secretions are critical for mammalian spermatozoa to acquire both forward motility and an ability to recognize and penetrate oocytes. Previous studies identified two glycoproteins, GP-83 and GP-39, which were secreted by the human epididymis and may be related to maturation of sperm function. In this study, GP-83 was purified from human seminal fluid by DEAE-ion exchange, gel filtration chromatography and preparative gel elution. The isoelectric point (pI) of purified GP-83 was 6.57. Monospecific antiserum to GP-83 was induced in male New Zealand rabbits and confirmed on immunoblots. GP-83 was found in fluid, tissue and sperm extracts of corpus and cauda epididymis, but not in the caput. Immunohistochemical localization identified GP-83 in the luminal contents and in the supranuclear region and cell membrane of principal cells of the corpus and cauda epididymis. GP-83 was found on the anterior acrosome in ejaculated spermatozoa, and shifted to the equatorial region after capacitation and the acrosome reaction.     

Delayed mutation in Chinese hamster cells

The possibility was examined that mutational events can be delayed for more than one or two cell divisions following treatment of Chinese hamster cells with the DNA alkylating agent ethyl methane sulfonate. If mutations in mammalian cells are delayed, the proportion of mutant cells in colonies grown from single mutagen-treated cells will reflect the cell division at which the mutation is genetically fixed, i.e., a first division mutation yields a 1/2 mutant colony, a fifth division mutation produces a 1/32 mutant colony, etc. In the present study, replating of cells from single colonies grown for six to seven days after mutagen treatment resulted in the discrete ratios of glucose-6-phosphate dehydrogenase (G6PD)-deficient mutant to wild-type colonies expected for a delayed mutational process which produces mutations over at least 8–10 cell generations. Further, when cells from 7- to 10-day colonies, grown from ethyl methane sulfonate (EMS)-treated cells were replated into selective medium containing 6-thioguanine (6TG), the number of 6TG-resistant colonies obtained per flask was distributed over a very wide range, consistent with a mutational delay process. These results could not be explained by differences in the number of cells per colony or plating efficiency in selective medium. Assuming that the relative number of 6TG-resistant colonies per flask reflects the time of mutation, EMS treatment produced two groups of mutational events: one which occurred within the first five cell generations and another uniformly distributed over at least the next eight to nine divisions. These results support the conclusion that EMS induces mutants for at least 10–14 cell generations after treatment and raise the possibility that current methods to assess the mutagenic potential of an agent might lead to significant underestimation. The role of delayed mutation in the phenomenon of “mutation expression time” is also discussed.     

The relative viability of human spermatozoa from the vas deferens, epididymis and testis before and after cryopreservation

Testicular and epididymal spermatozoa are routinely used with in-vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) to achieve pregnancies. In addition, excess cryopreserved spermatozoa can be thawed and used for ICSI. However, information on the recovery of epididymal and testicular spermatozoa after freeze-thaw is lacking. This is important to determine the feasibility of using previously cryopreserved aspirated spermatozoa for ICSI. We prospectively compared the viability of fresh and frozen-thawed spermatozoa from the vas deferens, epididymis and testicle by several measures. Testis spermatozoa were obtained from men with non-obstructive azoospermia (n = 5), epididymal spermatozoa from men with obstructive azoospermia (n = 8), and vasal spermatozoa from fertile men by vasal irrigation at vasectomy (n = 5). The viability of fresh spermatozoa was assessed by motility, two vital stains (carboxyfluorescein, 0.08 mg/ml and propidium iodide, 20 mg/ml) and the hypo-osmotic swelling assay (HOS; 100 mmol/l citrate and fructose). After cryopreservation, spermatozoa were thawed and all viability measures repeated. Although fresh vasal spermatozoa were the most motile, testicular spermatozoa exhibited similar, high viability (91 and 86% respectively) by vital stain. Spermatozoa from testis, epididymis and vas deferens survived cryopreservation equally well by vital stain, but not by motility. As a selection measure, the HOS assay identified significantly more viable epididymal and testicular spermatozoa than did motility in both fresh and frozen-thawed populations. It appears feasible to use frozen-thawed extracted spermatozoa for ICSI when motility and a selection measure such as the HOS assay are used. With fresh testis spermatozoa, selection methods may not be necessary prior to ICSI, as cell viability is high.     

The effect of morphology on the ability of human spermatozoa to penetrate zona-free hamster oocytes

The effect of morphology on the ability of human spermatozoato penetrate zona-free hamster oocytes was investigated usingfractions of individual semen samples enriched for morphologicallynormal or abnormal spermatozoa, after separation by buoyantdensity centrifugation on selfgenerating Percoll gradients.Using this separation technique, the percentage of spermatozoaof normal morphology is greater in the lowermost 1 ml of thePercoll column (layer 1) than in the fraction recovered fromhigher up the column. Thus it was possible to compare two populationsof spermatozoa from each semen sample in their ability to penetratehamster oocytes. After normalizing the concentration of motilespermatozoa the mean hamster oocyte penetration rate was significantlyhigher with spermatozoa recovered from layer 1 (39 ? 4%) comparedwith those recovered from layer 7 (21 ? 4%), which suggeststhat morphologically abnormal spermatozoa do not have the samefertilizing capacity as those with normal morphology.     

Pentoxifylline-stimulated capacitation and acrosome reaction in hamster spermatozoa: involvement of intracellular signalling molecules.

We investigated the role of cAMP/cGMP, protein kinases and intracellular calcium ( [Ca2+]i) in pentoxifylline-stimulated hamster sperm capacitation and the acrosome reaction (AR) in vitro. Treatment with pentoxifylline (0.45 mM) initially increased sperm cAMP values 2.8-fold, compared with untreated controls (396 +/- 9.2 versus 141 +/- 6.0 fmoles/10(6) spermatozoa; mean +/- SEM, n = 6) after 15 min, although by 3 h, cAMP values were similar (503-531 fmoles/10(6) spermatozoa). cGMP values ( approximately 27 fmoles/10(6) spermatozoa) were the same in treated and control spermatozoa. Both sperm capacitation and the AR, determined from the absence of an acrosomal cap, were stimulated by pentoxifylline; these were almost completely inhibited by a Cl-/ HCO3- antiporter inhibitor (4,4-diisothiocyanato-stilbene-2,2 disulphonic acid; 1 mM) defined from the degree of sperm motility and by a protein kinase A inhibitor (H89; 10 microM). A protein kinase C inhibitor (staurosporine, 1 nM) did not affect pentoxifylline-stimulated capacitation but inhibited the AR by 50%. A protein tyrosine kinase inhibitor (tyrphostin A-47, 0.1 mM) had no effect on either pentoxifylline-stimulated capacitation or AR. A phospholipase A2 inhibitor (aristolochic acid, 0.4 mM) markedly inhibited the pentoxifylline-stimulated AR but not capacitation. When intracellular sperm calcium [Ca2+/-]i was measured using fura-2-AM, there was an early rise (271 nM at 0.5 h) in pentoxifylline-treated spermatozoa; this appeared to be due to intracellular mobilization rather than to uptake. In the absence of extracellular Ca2+, sperm motility was maintained in the presence of pentoxifylline, but capacitation did not occur; spermatozoa exhibited a low level of hyperactivated motility and had a poor rate of AR (20.5 +/- 2.3%). These results suggest that: (i) the pentoxifylline-stimulated early onset of sperm capacitation may be mediated by an early rise in cAMP and [Ca2+/-]i and involves protein kinase A activity; and (ii) pentoxifylline-stimulated AR may require phospholipase A2 and protein kinase C activity.     

Acetaldehyde-induced aneuploidy in cultured Chinese hamster cells

In this paper we report the cytogenetic activity of ethanol(EA) and its main metabolic derivative, acetaldehyde (AA) incultured Chinese hamster cells. AA induced an increase of aneuploidyand chromosome breaks and exchanges. EA only induced an increaseof achromatic lesions (gaps). However, following treatment withAA, the most notable effect was an increase of hypodiploid cellsand a parallel increase of multinucleated interphase cells,the frequency of hyperdiploid cells being considerably lower.A strong correlation (r = 0.93, P < 0.01) has been foundon comparing the frequency of hypodiploid cells to the frequencyof hyperdiploid plus multinucleated interphase cells. We concludethat there exists a higher chance of hypodiploid cells reachingthe second mitosis after treatment and that the main effectof AA is the induction of hypodiploidy rather than hyperdiploidyand chromosome aberrations. ¹To whom correspondence should be addressed     

Osmotic stress variants in Chinese hamster cells

Stable variants resistant to hypertonic stress have been obtained from V79 cells by one-step selection in media supplemented with graded concentrations of NaCl. Such variants retain a potential for resistance when isolated and propagated in isotonic media. On replating in graded NaCl, a family of dose-response curves is obtained, rising in level of resistance according to the degree of hypertonicity used to isolate the variants initially. In hybrids between variants and sensitive cells, phenotypic expression of resistance to hypertonic NaCl is recessive. Stable variants can also be isolated by one-step selection in media made hypertonic with D-mannitol. Clonal sublines selected with mannitol, as well as those obtained with NaCl, are resistant to both types of hypertonic media. Fluctuation tests in media supplemented with NaCl show that resistance arises spontaneously and at random, with measured rates of variation that depend on the concentration of NaCl used for selection. Treatment of sensitive cells with 5-azacytidine increases the frequency of resistant variants in assays with high levels of added NaCl but is less effective when selection is performed at lower concentrations. Exposure to ethyl methane sulfonate has little or no effect on variant frequency.     

Valinomycin-induced apoptosis in Chinese hamster ovary cells

Accumulating evidence endorses that excessive K(+) efflux is an ionic mechanism underlying apoptosis both in neuronal and non-neuronal cells. K(+) channels play important roles in mediating the pro-apoptotic K(+) efflux. Chinese hamster ovary (CHO) cells have been widely used for gene transfection experiments. These cells lack detectable endogenous voltage-gated K(+) channels. We were interested in knowing whether the absence of endogenous K(+) channels would render wild-type CHO cells more resistant to apoptotic death. We also wished to determine if direct stimulation of K(+) efflux would trigger apoptosis in these cells. Exposing CHO cells to hypoxia (1% O(2)) or to a typical apoptotic insult of serum deprivation for up to 24h did not affect cell survival. On the other hand, the K(+) ionophore valinomycin caused substantial cell death within 12h of its application. Valinomycin-treated CHO cells underwent several apoptotic events, including phosphatidylserine (PS) membrane translocation, caspase-3 activation, and mitochondrial membrane depolarization during the first few hours of exposure. Reducing K(+) efflux by elevating extracellular K(+) concentrations noticeably attenuated valinomycin-induced cell death. This study reinforces a K(+) efflux-mediated apoptotic mechanism in CHO cells and may help to explain the unique feature of their higher tolerance to apoptosis.