Lack of correlation between fertility and sperm numbers in male rats treated with histrelin, a potent LHRH agonist

Adult male rats were treated daily for up to 8 weeks with histrelin, [(ImBzl)-D-His6, Pro9-NEt]LHRH, to study the antifertility effects of this LHRH agonist. Although serum testosterone concentrations and testicular sperm numbers were significantly decreased by weeks 2 and 4 respectively, a reduction in fertility, as judged by the mean number of fetuses per mated female, was not observed until the sixth week of treatment. Furthermore, the number of spermatozoa in the cauda epididymidis of treated rats did not decrease below initial control levels at any time during the study and full fertility returned within 4 weeks after histrelin treatment was stopped. Thus, the lack of correlation between fertility and testicular and epididymal sperm numbers suggests that the antifertility effects of LHRH agonists are not due solely to reduced sperm numbers, but also result from androgen deficiency.     

Acute effects and long-term sequelae of 1,3-dinitrobenzene on male reproduction in the rat. I. Sperm quality, quantity, and fertilizing ability

Groups of eight adult male rats were given a single oral dose of 0 or 48 mg/kg of 1,3-dinitrobenzene and sacrificed at 1, 2, 4, 8, 16, 24, 32, 72, and 175 days posttreatment. The groups killed at 175 days were bred to untreated females during weeks 3, 4, 6, 9, 13, and 24. Decreased testis weight and testicular sperm numbers were observed by day 4; decreased cauda sperm reserves and epididymis weight occurred by day 8 and day 16, respectively. Reduced numbers of motile spermatozoa and abnormal sperm morphology were seen in spermatozoa from the cauda epididymidis by day 16. Fertilizing ability, as indicated by the presence of two pronuclei and a sperm tail in eggs flushed from the oviducts of inseminated females, was slightly reduced by week 4 and declined to zero by week 6. Group means for reproductive organ weights, sperm production, and sperm reserves failed to return to control levels although some individual animals approached full recovery. Normal fertilizing ability was restored in most animals by week 13, but two of seven remained infertile. Occlusion of some efferent ductules was observed in three of seven animals at 175 days. This study indicates that 1,3-dinitrobenzene is a potent testicular toxicant in the rat, capable of producing marked testicular damage, infertility, and possibly sterility from a single exposure.     

Sperm protamine levels as indicators of fertilising potential in sexually mature male rats

We have earlier reported that administration of cyproterone acetate, fluphenazine decanoate, tamoxifen citrate, oestradiol valerate to adult male rats, at doses of 50, 5.77, 0.71, 0.28 micromol kg(-1) body weight given for periods of 15, 60, 60, 10 days, respectively, partially suppressed/reduced availability of one or more reproductive hormones viz. LH, FSH, testosterone and reduced their siring ability. The reduction in epididymal sperm counts was not considerable after treatment with these drugs, but conventional methods of assessment of spermatozoa quality viz. sperm chromatin structure assay (SCSA), nuclear chromatin decondensation (NCD) assay, monobromobimane (mBBr) uptake, had shown quantifiable changes in caput sperm chromatin compaction and reduced the testicular levels of protamine 1. The present follow-up study attempts to quantify changes in caudal sperm chromatin which has undergone compaction in the epididymis, in the altered hormonal microenvironment of rats treated with cyproterone acetate, tamoxifen citrate, fluphenazine decanoate, oestradiol valerate, at doses of 50, 5.77, 0.71, 0.28 micromol kg(-1) body weight respectively given for periods of 15, 60, 60, 10 days, with a view to correlating these changes to reduction in their fertilising potential. During the androgen-dependent transit of spermatozoa from caput to cauda epididymis, thiol group oxidation and tyrosine phosphorylation of protamine occurs in maturing sperms concomitant with development of fertilising ability. The results indicate that conventional methods viz. SCSA, NCD, mBBr uptake fail to detect changes induced by hormone deficits in sperm chromatin condensation, as a result of maturation during transit from caput to cauda epididymis. Absence of protamine 1 in epididymal sperm was observed in either testosterone or FSH deficient rats that correlated with reduced fertilising potential. The study suggests that changes in LH/T or FSH affect a hitherto unknown common molecular mechanism in the testis, underlying the protamination of rat spermatozoa. In conclusion, loss of P1 occurs in adult male rats deprived of T or FSH and is a reliable detectable change in epididymal sperm indicative of chromatin condensation defects associated with endocrine imbalance and poor fertility status.     

Functional role of hepatocyte growth factor receptor during sperm maturation

Mammalian spermatozoa acquire motility and fertilizing capacity during their transit through the epididymis. Hepatocyte growth factor (HGF) is a pleiotropic cytokine with potent motogenic capacities that has been identified in different organs, including the mammalian male genital tract. In mice, HGF is present in the testis and, in large amounts, in the distal part of the epididymis. In prepuberal rats, we have demonstrated that HGF is synthesized by the peritubular myoid cells and in men, HGF is present in significant quantities in seminal plasma. It has been suggested that in mice, HGF has a role in initiating sperm motility, whereas in men, no significant correlations between HGF concentration and sperm motility have been found. In the present paper we report that in rats, HGF receptor, c-met, is expressed in testicular and epididymal spermatozoa. Through immunocytochemistry, we have found that c-met is exclusively localized on the head in testicular sperm. A different localization of c-met has been found in sperm isolated from caput and cauda epididymidis. Cells isolated from epididymal caput show a c-met localization exclusively restricted to the head in most cells. In a minority of caput epididymis spermatozoa the receptor is localized both in the cell head and along the flagellum. Spermatozoa isolated from the epididymal cauda were quite homogeneous, showing the receptor localized along the entire cell surface. We also report that HGF is synthesized and secreted by the rat epididymis as indicated by the scatter effect of epididymal cell homogenate and culture medium on MDCK cells. To clarify whether HGF is involved in the acquisition of sperm motility in the epididymis, its maintenance, or both, spermatozoa isolated from caput epididymidis have been cultured in medium alone or supplemented with HGF. The results obtained indicated that HGF has a positive effect on the maintenance of sperm motility which, in the absence of HGF, significantly decreases during the first hour of culture, whereas it is maintained for at least 3 hours when HGF is present in the culture medium. We also report that HGF does not influence spermatozoa viability as indicated by the cytometrical analysis of propidium iodide-labeled sperm; an equal number of dead cells appeared in control and in HGF-treated preparations. In conclusion, our data strongly support the hypothesis that HGF positively influences sperm motility maintenance during sperm transit through the epididymis, indicating that c-met receptor and its ligand, HGF, have a role in male fertility.     

The current perspective on genetic and epigenetic factors in sperm maturation in the epididymis

Male infertility affects approximately 30% of infertile couples. As spermatozoa mature in the epididymal lumen, their potential for mobility increases, and their protein, lipid and small RNA (sRNA) content changes, whereas capacitation and fertilisation take place in the female reproductive tract. Both of the latter processes are affected by maturation, because impaired maturation causes premature capacitation and fertilization. The epididymis produces a suitable environment for sperm maturation via ion transport, vesicle secretion and protein matrix formation. The microenvironment for sperm maturation varies in three broad segments: the caput, the corpus and the cauda epididymis. Epididymosomes transfer proteins, lipids and sRNAs from the epididymal epithelium to spermatozoa and genetic alterations of epididymal genes can lead to decreased sperm motility, morphological abnormalities of spermatozoa and subfertility. Genetic factors are involved in all aetiological categories in male infertility. However, studies conducted on the genes involved in epididymal functions are limited. The sRNA content of spermatozoa changes during epididymal migration, and these sRNAs play a role in embryo development and epigenetic inheritance. This review aims to clarify the role of the epididymal epithelium in the maturation of spermatozoa in light of the current molecular genomic knowledge.     

Effect of dehydroleucodine on the reproductive tract of male mice

The effects of a sesquiterpene lactone, dehydroleucodine, on the reproductive tract were investigated using adult male mice. Dehydroleucodine was dissolved in tap water and administered as drinking water for 30 days. All the parameters were compared with a control group that received only vehicle. Animals were killed by decapitation and the trunk blood, the testes and the epididymes were collected. Plasma concentrations of testosterone and oestradiol, and testicular weight and concentration of spermatids did not change by dehydroleucodine. Nevertheless, in epididymal cauda dehydroleucodine treatment caused a diminution in sperm number, a decrease in the amount of tubular fluid and a reduction in the activity of the hydrolytic enzyme N‐acetyl‐β‐d ‐glucosaminidase. However, the sperm motility was not altered by dehydroleucodine treatment, although sperm binding to zona‐free oocytes increased significantly. These results suggest that dehydroleucodine, which has been implicated in the inhibition of aromatase P450, does not affect the plasma concentration of testosterone and oestradiol or testicular activity, whereas altering several epididymal parameters. The epididymis is thus a more sensitive target for dehydroleucodine action.     

Posttesticular antifertility action of triptolide in the male rat: evidence for severe impairment of cauda epididymal sperm ultrastructure

A variety of active diterpene epoxides, including the triptolide (isolated from Tripterygium wilfordii) have been reported to cause infertility in male rats. Previously, we showed that oral administration of triptolide at a dosage of 100 microg/kg per body weight for 70 days completely inhibited fertility in male rats, with little or no demonstrable detrimental effect on spermatogenesis and Leydig cell function as determined by testicular light microscopic appearance and serum and intratesticular testosterone levels. Despite the apparent absence of effects on the testes, cauda epididymal sperm were abnormal, with complete cessation of sperm motility and some reduction in sperm numbers. This study was undertaken to provide additional insight into the subcellular sites and possible mechanisms of action of this compound using ultrastructural analysis of the testes and epididymidis. The most striking effect of triptolide treatment was observed in sperm in the epididymis. In rats rendered infertile with 100 microg/kg per body weight of triptolide daily for 70 days, virtually all cauda epididymal sperm exhibited complete absence of plasma membrane over the entire middle and principal piece, premature decondensation of the nuclei, and disorganization of the mitochondrial sheath with many vacuolated mitochondria. No ultrastructural differences in the epididymal epithelium were observed between control and triptolide-treated rats. The testes appeared to be mildly affected after triptolide treatment but exhibited only subtle ultrastructural defects in the germ cells. The findings of severe impairment of cauda epididymal sperm ultrastructure, along with minimal discernible abnormalities in the fine structural cytology of the testes, further suggest that the site of action of this compound is posttesticular and may be confined to the cauda epididymal sperm. However, we cannot rule out an effect of triptolide that occurs during germ cell maturation but is delayed in its manifestation or triggered at the rete testis and epididymal level.     

The Effect of Epididymal and Testicular Fluids on the Fertilising Capacity of Testicular and Epididymal Spermatozoa

It is possible that the fertilising capacity of spermatozoa in the epididymis is influenced by the epididymal secretion. We have studied this problem by obtaining spermatozoa before entry into the epididymis and after passage through it, incubating both types of spermatozoa in fluids from the rete testis and cauda epididymidis and then checking their fertilising capacity. While spermatozoa from the rete testis were infertile, rete testis fluid did not decrease the fertilising capacity of epididymal sperm from the cauda epididymidis. Fluid from the cauda epididymidis did not promote the fertilising capacity of testicular spermatozoa. These results are discussed in the light of the current understanding of epididymal physiology.     

Effects of tripchlorolide on the epididymides and testes of rats

Aim: To further evaluate the antifertility effects of tripchorolide, a derivative of triptolide produced at the extraction pro-cedure of Tripterygium wilfordii Hook. f., in male rats and to investigate its sites and possible mechanisms of action.Methods: In male rats, tripchlorolide was given by oral garage at a dose of 50 ug.kg～(-l).d～(-1) for 5 weeks, fertility wasassessed by mating tests, and biochemical indices and light microscopic observation of the epididymides and testes werealso performed. Results: Administration of tripchlorolide at 50 ugg.kg～(-l)-d～(-1) for 3 weeks did not influence the fertilityof male rats, but 5-week treatment rendered the rats infertile. The density and motility of spermatozoa collected fromcauda epididymides were reduced significantly. The epididymal weights, as well as the L-carnitine concentration and α-glucosidase content in the epididymal fluid were decreasd. There were no significant differences in α-glucosidase andacid phosphatase (ACP) in caput epididymal ho     

Effects of α-Interferon on Sperm Production, Concentration, and Motility in the Rat

Background :
We evaluated possible effects of α-interferon (α-IFN) on testicular spermatogenesis and epididymal sperm quality in the nude rat.
Methods :
Nude male rats were administered subcutaneous injections of human α-IFN daily for 3 months. The luminal content of the cauda epididymidis was collected by micropuncture. Daily sperm production was determined by Amann's method and sperm concentrations were determined by microassay. Progressive motility was judged by evaluating the linear distance traveled by the sperm in a diluent. Serum levels of testosterone, luteinizing hormone (LH), and follicle stimulating hormone (FSH) were also measured at the end of the experiment.
Results :
Daily sperm production and epididymal sperm concentrations were significantly increased after administration of α-IFN, while progressive motility of the spermatozoa was not altered. α-IFN significantly increased serum testosterone levels, while it decreased serum LH levels and left serum FSH levels unchanged.
Conclusion :
α-IFN may improve testicular spermatogenesis and increase the epididymal sperm concentration in the rat. These promising results with α-IFN may pave the way for a new approach to treating male infertility.     

Effects of antifertility drugs on epididymal protein secretion, acquisition of sperm surface proteins and fertility in male rats

The possibility that α-chlorohydrin, 6-chloro-6-deoxyglucose (6CDG) and cyproterone acetate (CPA) might affect epididymal protein secretion or acquisition of sperm surface proteins as the cause of their antifertility action in male rats was investigated. Daily administration of 9 mg/kg α-chlorohydrin for 7–14 days and 24 mg/kg 6CDG for 14–21 days induced sterility in male rats and impaired the capacity of the cauda epididymal spermatozoa to initiate motility. Treatment with CPA (30 mg/kg/day) for 21–28 days, however, was found to have no effect on fertility and initiation of sperm motility, although the epididymis of the treated animals underwent a loss in weight. The antifertility effects of α-chlorohydrin or 6CDG did not seem to be attributed to an interference with epididymal protein secretion. The cauda epididymal fluids of the α-chlorohydrin, 6CDG and CPA treated animals have similar protein patterns compared to those of the control animals. However, when the surface proteins of the spermatozoa were labelled with radioactive iodine, the sperm surface proteins of the α-chlorohydrin and 6CDG treated animals were found to differ from those of the control animals. Two peaks (MW 32 000 and 70 000) and one peak (70 000) were significantly reduced in the α-chlorohydrin treated and 6CDG treated animals, respectively. Additional bands appeared on the surface of the treated (infertile) animals. In contrast, CPA treatment did not affect the surface protein pattern of the epididymal spermatozoa. It was concluded that the antifertility affects of α-chlorolydrin and 6CDG are not due to an interference with epididymal secretion of specific proteins but to an intervention of the subsequent acquisition of these proteins by epididymal spermatozoa. This results in a decrease in the capacity of the epididymal sperm to initiate motility and hence a loss of fertilizing capacity.     

The effect of p-nonylphenol on the fertility potential of male rats after gestational, lactational and direct exposure

There is growing concern that abnormalities in male reproductive health are becoming more frequent. The most fundamental change has been the striking decline in sperm counts and semen quality. The effect of maternal exposure of rats to the oestrogenic environmental substance p-nonylphenol (p-NP) was determined in this study. Exposure to p-NP for the experimental period impaired general growth. The lower testicular mass indicated a direct toxic effect on the testis in animals exposed to p-NP during foetal life, the postnatal period and after weaning until termination at 10 weeks of age. The epididymal mass was also negatively affected by p-NP; this was supported by the decrease in the epididymal ratio. The total cauda epididymal sperm count was significantly lower in the 250 mg kg-1 p-NP dosage group compared to the control and 100 mg kg-1 p-NP groups. The overall lower sperm count with increased p-NP concentrations corresponded with the decreased testicular and epididymal masses. This emphasized the toxicity of p-NP on both testis and epididymis. Seminiferous tubule diameter, lumen diameter and seminiferous epithelium thickness were smaller in the exposed groups, even at the low dose level. These histological measurements further supported the finding of a low testicular mass. In spite of the measurements being smaller, p-NP had no effect on the stages of spermatogenesis except for one animal with disrupted spermatogenesis in some tubules, while others were normal.     

Effects of putative epididymal osmolytes on sperm volume regulation of fertile and infertile c-ros transgenic Mice

Volume regulation by spermatozoa has been demonstrated to be crucial in both mice and men for transport in the female tract. In order to determine the nature of osmolytes used by spermatozoa, they were released from the cauda epididymis of fertile c-ros heterozygous mice into incubation medium of uterine osmolality (representing an osmotic challenge), containing increasing concentrations of compounds that are major epididymal fluid components and known osmolytes in somatic cells. This should nullify the concentration gradients for osmolytes that mediate volume regulation, prevent osmolyte efflux, and lead to swelling. Of the osmolytes tested, K(+) caused the most rapid and extensive volume increases; glutamate, taurine, L-carnitine, and myo-inositol also were effective, but glycerophosphocholine was not. Such effects were not observed in cauda sperm from the infertile knockout mice, demonstrating a defect in normal volume regulation. K(+) concentrations in cauda epididymal fluid were 21 mM higher in the knockout than the heterozygous mice, but no differences were found in caudal fluid glutamate, carnitine, or myo-inositol. The carnitine content of cauda sperm from knockout males was not different from that of fertile males, but lower amounts of glutamate and inositol were found that could explain the poor volume regulation. In heterozygous mice, cauda but not caput sperm responded to the K(+) channel blocker quinine by swelling, demonstrating development of volume regulation during epididymal transit, whereas knockout cauda sperm showed no response, as with the osmolytes. Major epididymal secretions could serve as osmolytes in murine spermatozoa for volume regulation in response to physiological osmotic challenge in the normal fertile mice; the reduced sperm content of inositol and glutamate in the c-ros knockout mice might reflect maturational abnormalities in volume regulation.     

荧光酶法测定大鼠附睾精子ATP含量

参照ＬＫＢ公司新的ＡＴＰ测定法，测定了大鼠附睾精子ＡＴＰ含量，并比较了大鼠附睾头、尾部精子ＡＴＰ含量的差别及活动精子百分率的关系。结果表明，附睾尾部活动精子百分率和ＡＴＰ含量均明显高于附睾头部的精子     

Chromatin condensation in cat spermatozoa during epididymal transit as studied by aniline blue and acridine orange staining

Summary.  Chromatin stability and DNA-resistance to acidic denaturation was evaluated by acidic aniline blue and acridine orange staining of cat sperm from different regions of the epididymis. The results were related to conventional sperm parameters. The percentage of aniline blue-stained spermatozoa (persisting histones) decreased significantly from the caput to the cauda region (31.8% and 7.8%, respectively; P <0.0001). The percentage of stained heads of cauda sperm was much lower in populations of morphologically normal forms than in those with abnormal forms (4.1% and 13.8%, respectively, P <0.0001). Among spermatozoa with abnormalities, the percentage of stained heads was significantly higher in cells with head abnormalities than in sperm with only tail abnormalities (87.1% and 10.3%, respectively; P >0.0001). With acridine orange fluorescence staining, 86.5% of cauda epididymal sperm were found with well-condensed chromatin, stabile against acid-induced denaturation. Chromatin stability increased significantly from the caput epididymal region (51.1%) to the cauda epididymal region (86.5%). The percentage of cauda epididymal sperm with normal condensed chromatin was neither linked to testicular sperm count, motility nor to age of the cats. The parameter of chromatin condensation and stability can be a valuable index of sperm quality, reflecting the possible disorders of spermatogenesis and epididymal sperm maturation, frequently observed in feline species.     

The automated analysis of rat sperm motility following subchronic epichlorohydrin administration: methodologic and statistical considerations

The automated analysis of sperm motion endpoints is potentially useful in identifying male reproductive toxicants and ultimately in predicting fertility in humans. The present study was designed to evaluate the automated analysis of rat sperm motility characteristics following subchronic administration of epichlorohydrin. This type of validation is a prerequisite for inclusion of sperm motion measurements in the process of reproductive risk assessment. In the present studies videotapes were made of cauda epididymal spermatozoa from Long-Evans rats, both untreated and treated with epichlorohydrin. From analysis of videotapes of control epididymal spermatozoa, the relationship of various sperm motion endpoints and settings of the CellSoft computer-assisted sperm motion analysis system (Cryo Resources, Ltd., New York, NY) is described. Optimal settings of the system for analysis of rat spermatozoa are detailed. Employing data from both control and epichlorohydrin-treated animals, a statistical methodology is described that evaluates: (1) the distributions of CellSoft generated sperm motion endpoints, (2) the correlations between these endpoints, and (3) techniques for detection of dose-related effects.     

葡萄糖调节蛋白前体GRP78和肿瘤排斥抗原GP96为仓鼠附睾头部精子特有

哺乳动物睾丸中的精子经过“附睾成熟”期,由静止状态转变成为运动状态。该过程中精子从附睾头部向附睾尾部移动,同时精子发生了一系列的形态、生理和生化改变,如蛋白组成和蛋白修饰的改变可能会影响精子获能的潜能。本实验使用基质辅助激光解吸／电离串联质谱（MALDI-MS／MS）法分析仓鼠睾丸头部和尾部精子的蛋白组学,成功发现了113个蛋白质点。对113个蛋白质点进一步对照比较发现30个蛋白质点（对应20个蛋白）的密度发生了显著改变,其中附睾尾部精子5个蛋白的密度增加,11个蛋白密度减少;此外,葡萄糖调节蛋白前体GRP78和肿瘤排斥抗原GP96为仓鼠附睾头部精子特有,而纤维蛋白原样蛋白1为附睾尾部精子所特有。几个蛋白密度增加可能与附睾成熟过程中精子代谢和ATP产生相关。一些蛋白如ERp57,GRP78,GP96,Hsp60,Hsp70和二氢硫辛酰胺S-乙酰转移酶的密度改变通过免疫印迹法得到验证。本研究首次报道了仓鼠精子的蛋白质组学研究,全面展示了仓鼠精子附睾成熟过程中的蛋白结构改变。     

Decreased fertility and motility of spermatozoa from rats immunized with a prealbumin epididymal-specific glycoprotein

This study investigated the effects on the progressive motility, zona-binding capacity, and fertility of spermatozoa from the cauda epididymidis of adult male rats that were actively immunized against an acidic glycoprotein secreted by the epididymis. The percentage of motile spermatozoa was less than 5% in nine of ten rats that received the epididymal antigen, and 40 to 50% in eight of the 10 control rats. In animals immunized against the antigen, there was a dramatic decrease, but not a complete suppression, in the capacity of epididymal spermatozoa to bind the zona pellucida as compared with the nonimmunized controls. Fertility was decreased two weeks after the end of the treatment, but partial restoration of fertility was observed 6 months later.     

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.     

Sperm structural and motility changes during aging in the Brown Norway rat

The Brown Norway rat provides a useful model to study aging of the male reproductive tract because of the selective age-dependent pathological changes that are found in the testis, epididymis, and prostate. In the testis, there is a clear age-dependent decrease in both steroidogenesis and spermatogenesis. In the epididymis, some striking segment-specific changes occur at the histological and biochemical levels prior to the major loss of spermatogenesis. We hypothesized that formation of spermatozoa in the testis and maturation of spermatozoa in the epididymis (ie, acquisition of motility and loss of the cytoplasmic droplet) may be altered during aging. Changes in the morphology of spermatozoa were assessed by light and electron microscopy. Using computer-assisted sperm analysis, the motility parameters of spermatozoa obtained from the caput and cauda epididymidis of young and old Brown Norway rats were compared. In old animals, we also compared the motility of spermatozoa from epididymides adjacent to regressed testes with those from epididymides adjacent to nonregressed testes. There was a marked increase with age in the number of spermatozoa with abnormal flagellar midpieces; the nature of these defects did not change with age. In caput epididymidis, the percentage of motile sperm was similar in young and old rats. In contrast, the percentage of motile spermatozoa was significantly decreased in cauda epididymidis of old rats; spermatozoa from the regressed testis side had altered motility characteristics. Furthermore, in the cauda epididymidis on the regressed testis side of aged Brown Norway rats, the proportion of spermatozoa that retained their cytoplasmic droplet was markedly elevated. Some of these effects are likely due to changes taking place in spermatozoa during the process of spermatogenesis in the testis (eg, formation of the flagellum), whereas others could occur during sperm maturation in the epididymis (eg, acquisition of motility). The multiple effects of aging on sperm morphology, the acquisition of motility, and the shedding of the cytoplasmic droplet clearly indicate that the quality of spermatozoa is affected by aging.