Sterility due to inhibition of sperm motility by oral administration of benzene chromatographic fraction of the chloroform extract of the seeds of Carica papaya in rats.

The contraceptive effects of benzene chromatographic fraction of the chloroform extract of the seeds of Carica papaya have been reported in male albino rats at the dose regimens 5 and 10 mg/animal/day; oral for 150 days. The body weight, weight of testis, epididymis, seminal vesicle and ventral prostate remained unaltered during the entire course of the investigation. Total suppression of cauda epididymal sperm motility coincided with a decrease in sperm count, viability and an increase in per cent abnormal spermatozoa during 60-150 days observation period. Minor changes in the germ cell proliferations in the testis and vacuolization and pyknotic nuclei in the few epithelial cells of the cauda epididymis were observed. Histology and biochemical composition of testis and accessory sex organs, haematology and serum clinical biochemistry and serum testosterone levels remained unchanged throughout the course of the investigation. Test for estrogenicity indicated mild estrogenicity. Monthly fertility test showed negative fertility. All the altered parameters returned to normal level following 60 days withdrawal of the treatment. The results suggest that the benzene chromatographic fraction of the chloroform extract of the seeds of Carica papaya exerts antifertility effects in rats without adverse toxicity and that the effects may be directly rendered on the spermatozoa.     

Effects of papaya seeds extract on the sperm characteristics of dogs

The effect of chloroform extracts from seeds of the papaya plant (Carica papaya) on the spermatic characteristics of dogs was evaluated at doses of 50, 100 and 150 mg/kg, Groups 1, 2, and 3, respectively. Structural and ultrastructural changes in sperm cells and testicular parenchyma were also evaluated, and possible side effects were noted. Significant reductions in sperm concentration and motility were observed starting from Days 60 and 75, respectively, in all treated groups (P<0.05), but no azoospermia was noted. A mild osmotic diarrhea occurred in dogs from Group 3 (150 mg/kg), although blood variables were within the normal range of a clinically healthy dog. Arrested spermatogenesis was observed in the seminiferous tubules of all treated groups, and vacuolization and signs of Sertoli cell degeneration were detected in all treated groups, particularly in Group 3 (150 mg/kg). Selective damage to Sertoli cells induced by the extract occurred in all treated groups independently of the extract concentration. Alteration of the epididymal environment may reduce the motility of sperm cells, considering that their structure was normal. Sperm characteristics in treated animals were considered to be similar to those of sub-fertile dogs. However, these effects may be temporary, and dogs may recover normal sperm characteristics when the extract is withdrawn.     

Impact of feeding ethanolic extract of root bark of Cananga odorata (Lam) on reproductive functions in male rats

The 50% ethanolic extract of the root bark of C. odorata administered orally at the dose of 1g/kg body weight/day for 60 days resulted in decreased epididymal sperm motility and sperm count in male albino rats. Morphological abnormalities were also observed in the sperms. The testicular glycogen, the activities of 3beta hydroxy steroid dehydrogenase, glucose 6-phosphate dehydrogenase, malic enzyme, sorbitol dehydrogenase in seminal vesicle, fructose in seminal plasma and serum testosterone were significantly decreased in treated group. While testicular cholesterol level, the concentration of the fecal bile acids, urinary excretion of 17 ketosteroids, the activities of 17beta hydroxy steroid dehydrogenase, epididymal lactate dehydrogenase and that of testicular HMG CoA reductase were increased in treated group when compared to control. The results suggest that the ethanolic extract of C. odorata possesses the spermatotoxic effects in male albino rats.     

Effects of PNU157706, a dual 5alpha-reductase inhibitor, on rat epididymal sperm maturation and fertility

Sperm entering the epididymis gain progressive motility and fertilizing ability in a process termed maturation. The functional dependence of the epididymis on dihydrotestosterone (DHT) is well established, yet few studies have examined the consequences on the epididymis of inhibiting DHT formation. We have shown that inhibition of both isoforms of 5alpha-reductase (types 1 and 2), the enzyme that converts testosterone to DHT, has pronounced effects on epididymal gene expression. In the present study, we investigate whether inhibiting 5alpha-reductase has consequences on epididymal sperm maturation. Rats were treated with vehicle or 10 mg/kg/day PNU157706, a dual-type inhibitor, for 28 days. Fertility and several key facets of sperm maturation were analyzed. Changes in sperm motility were assessed by computer-assisted sperm analysis (CASA). Changes in sperm morphology were assessed by CASA and electron microscopy. The motility of spermatozoa from the cauda epididymidis of treated animals showed a significant decrease in both the percentage of motile and progressively motile sperm as well as altered motion parameters. The morphology of cauda epididymal spermatozoa was also adversely affected by the treatment; the most prominent effect was a markedly elevated proportion of sperm that retained their cytoplasmic droplet. Matings with treated males resulted in fewer successful pregnancies and a higher rate of preimplantation loss. Progeny outcome was unaffected. The compromised sperm motility and morphology likely contribute to the subfertility of inhibitor-treated rats. Our results indicate a role for dual 5alpha-reductase inhibitors in further studies of epididymal physiology and as a potential component of a male contraceptive.     

Cryopreservation of epididymal sperm obtained at necropsy from goats

In the field of transgenic production, the ability to carry a male's genetic contribution beyond its natural life span is remarkably important. The ability to successfully collect and cryopreserve sperm from the epididymis at necropsy may prove to be a useful technique for preserving valuable genes. Thirty-two bucks ranging in age from 13 days to 7 years were examined in this study and 25 had epididymal sperm extracted at necropsy. Seven bucks yielded clear fluid with no spermatozoa; all were under four months of age. Testes were removed from the scrotal sac, small lateral incisions made across the convoluted tubules, pressure applied to the tail of the epididymis and small droplets of sperm pipetted into equilibrated extender. The average initial analysis of wave motion (0 to 5, 5 being rapid wave motion), live/dead sperm percentage and acrosomal integrity of 25 fresh epididymal samples were 5.0, 92%, and 100%, respectively. By comparison, the same parameters obtained from 206 fresh ejaculated samples were 3.0, 86%, and 95%, respectively. After being cryopreserved in liquid nitrogen, one straw from each sample was thawed after 3 to 60 days of cryostorage. Results of post-thaw analysis of 25 cryopreserved epididymal sperm samples for live/dead percentage and acrosomal integrity were 82% and 84%, respectively. By comparison, results of post-thaw analysis of 206 cryopreserved ejaculated sperm samples for live/dead percentage and acrosomal integrity were 60% and 89%, respectively. To assess the competence of the frozen epididymal sperm, IVF and AI were performed. In parallel IVF experiments, 40% of the oocytes showed cleavage patterns, with 6% developing to the blastocyst stage using frozen epididymal sperm, while 37% of the oocytes showed cleavage patterns and 4% developed into blastocysts using frozen ejaculated sperm. One artificial insemination out of 20 resulted in a pregnancy using frozen epididymal sperm, while 7 of 18 artificial inseminations resulted in a pregnancy using frozen ejaculated sperm. This data documents the successful collection and cryopreservation of epididymal sperm from the goat and its use for in vitro fertilization and artificial insemination.     

Effect of tamoxifen treatment on motility related proteins in rat spermatozoa.

The study was undertaken to identify the effect of tamoxifen on the expression and phosphorylation of motility related proteins in the adult male rats. For this purpose, tamoxifen, at a dose of 0.4 mg/kg/day, was administered per os to the male rats for a period of 60 days. Cauda sperms, epididymal fluid and tissue proteins were extracted and analyzed by electrophoresis. Testicular tissues fixed in paraffin wax were analyzed for changes in the immunoexpression of interstitial tissue estrogen receptor alpha. Phosphorylation pattern of sperm proteins was studied in vitro after incubating with 32P-ATP. The expression of dynein and tubulin in sperms, and estrogen receptors in epididymis were analyzed by immunoblotting. Tamoxifen treatment did not alter the protein profile in the cauda sperms, epididymal fluid and tissues. Endogenous phosphorylation pattern of sperm proteins in vitro was also not affected, though it is possible that 32P incorporation observed in the 66 kDa protein could be estrogen receptor. Expression of sperm dynein, tubulin and epididymal estrogen receptors was unchanged as was the expression of testicular estrogen receptors. It was concluded that tamoxifen administration alters forward motility pattern characteristic of cauda sperm without any demonstrable change in the expression or activation of motility related proteins and the phosphorylation of the sperm estrogen receptors may be involved in the regulation of sperm motility.     

Studies on the measurement of phospholipase A2 (PLA2) and PLA2 inhibitor activities in ram semen.

Extraction with Tris-citrate or Tris-NaCl-EGTA improved the yield of phospholipase A2 (PLA2) from ram semen by 40-50 fold over the previously recommended method of extraction by dilute (0.18 N) sulphuric acid. The enzyme activity in the citrate extract deteriorated more rapidly than in Tris-NaCl-EGTA. The semen PLA2 activity was optimum at pH 8.0, heat sensitive at 70 degrees C for 30 min, activated by Ca2+ (although approximately 60% activity was also found in the absence of calcium) and did not exist as a pro-enzyme. The semen PLA2 activity was equally distributed among the sperm and seminal plasma (SP) components of ram semen. However, the low levels of PLA2 activity in the SP of vasectomised rams tend to suggest that PLA2 in the SP fraction may have originated from testicular or epididymal secretions or leakage, from sperm. PLA, in sperm exists as a large molecular weight aggregate, whereas in SP it is present as a smaller aggregate. In addition to PLA2, semen also contained PLA2 inhibitor activities. Inhibition was observed against PLA2s from bee venom, pig pancreas and oviductal extracts. The inhibitory activity is presumed to be due to a large molecular weight protein as the inhibitor activity was not extracted in a chloroform:methanol (2:1; v/v) mixture, it was non-dialysable, precipitated by 10% trichloroacetic acid and destroyed by proteases. The inhibitor activity was distributed in various molecular weight fractions of sperm, SP and SP from vasectomised rams.     

Sperm motility inhibiting activity of a phytosterol from Alstonia macrophylla Wall ex A. DC. leaf extract: a tribal medicine

The role of methanolic extract and n-butanol fraction of A. macrophylla leaves was investigated on the forward motility of goat spermatozoa. The methanol extract (600 micro/g/ml) and one n-butanol fraction (Fraction A; 100 microg/ml) showed marked inhibition of sperm forward motility, tested by microscopic and spectrophotometric methods. Approximately, 50-60% of the spermatozoa lost their motility when treated with 600 microg/ml of methanol extract or 100 microg/ml of Fraction A. The Fraction A at 400 microg/ml concentration showed complete inhibition of sperm forward motility at 0 min. The inhibitory activity increased with the increasing concentrations of the fraction. The motility inhibitory activity of the Fraction A was stable to heat treatment at 100 degrees C for 2 min. The compound showed high inhibitory effect in the pH range 6.7-7.6. Fraction A also showed high efficacy for inhibiting human sperm motility, assessed by the microscopic method. The phytochemical analysis of methanolic extract of A. macrophylla leaves revealed the presence of sterols, triterpene, flavonoid, alkaloid, tannin and reducing sugar, while the Fraction A contains beta-sitosterol, a common phytosterol. The results demonstrate that Fraction A (beta-sitosterol) is a potent inhibitor of sperm motility and thus it has the potential to serve as a vaginal contraceptive.     

ZNF185‐derived peptide induces fertility suppression in mice

Zinc finger protein 185 (ZNF185) belongs to the ZNF family and is involved in male reproduction. However, it is unclear whether ZNF185 may be a target candidate for contraceptive vaccines. In this study, antigenic peptides derived from ZNF185 were prepared, and their immune contraceptive effects were investigated using mice. Results from enzyme‐linked immunosorbent assay (ELISAs) showed that peptide immunization induced an antibody titre increase that reached a peak in week 12. Peptide‐3 and peptide‐4 were then chosen for subsequent experiments. The results of the fertility assays showed that peptide immunization inhibited the mating and fertility rates of the mice, whereas there were no obvious changes in the number of pups per litter. Subsequently, epididymal sperm was analysed. The results demonstrated that the sperm count and sperm motility were significantly decreased in the peptide group, while the amount of abnormal sperm was significantly increased in the peptide‐3 group. The male reproductive organs were also evaluated. There were no obvious differences in testis or epididymal weights, in the diameters of the seminiferous tubules, or in the thicknesses of the seminiferous epithelium between the peptide group and the phosphate buffer saline (PBS) group. In addition, histological analysis indicated that there were no obvious pathologic changes in testis and epididymal histology in the peptide group; however, the number of spermatozoa present in the epididymal lumen of the peptide group was significantly decreased when compared with the PBS group. Our study demonstrates for the first time that peptides derived from ZNF185 may induce fertility suppression in mice without damaging reproductive organs. These peptides have the potential to be used as a male contraceptive vaccine.     

The role of the seed coat in adaptation of dimorphic seeds of the euhalophyte Suaeda salsa to salinity

The role of the seed coat in adaptation of dimorphic seeds of the euhalophyte Suaeda salsa to salinity was investigated during germination and early seedling growth. Black and brown seeds were treated with chloroform for 1 min before the extract was used to analyze waxes and the seeds to investigate the protective role of the seed coat under saline conditions. Waxes in black seed coats were more abundant than those in brown seed coats. Salinity (500 mM NaCl) increased the concentration of Na⁺ and decreased the concentration of K⁺ in both black and brown seeds regardless of chloroform treatment. Chloroform treatment alone (in the absence of NaCl) had no effect on the concentration of Na⁺ or K⁺ in black or brown seeds and in the presence of 500 mM NaCl had no effect on the concentration of Na⁺ or K⁺ in brown seeds. However, chloroform treatment increased Na⁺ and decreased K⁺ in black seeds with 500 mM NaCl. A change of MDA (malondialdehyde) concentration in black and brown seeds treated with or without chloroform was similar to the change of Na⁺ concentration. High salinity (1500 mM NaCl) pretreatment for 40 days had a less adverse effect on germination of black seeds compared with brown seeds after they were transferred to fresh water regardless of chloroform treatment. Similar results were found for seedling emergence. In conclusion, a black seed coat may be more protective than a brown seed coat, probably by shielding the embryo from ion toxicity, because of its higher content of waxes. Thus black seeds can better maintain seed viability than brown seeds for extended periods under hypersaline conditions.     

Effects of papaya seed extract and benzyl isothiocyanate on vascular contraction

To investigate their potentially toxic effects on mammalian vascular smooth muscle, pentane extracts of papaya seeds and the chief active ingredient in the extracts, benzyl isothiocyanate (BITC), were tested for their effects on the contraction of strips of dog carotid artery. BITC and the papaya seed extract caused relaxation when added to tissue strips that had been pre-contracted with phenylephrine (PE). Incubation of the tissue with papaya seed extract or BITC caused inhibition of contraction when the strips were subsequently contracted with KCl or PE. This relaxation and inhibition of contraction did not appear to be endothelium-dependent, as endothelium-denuded rings showed the same degree of relaxation or inhibition of contraction in response to the preparations/drugs as those with the endothelium intact. The effects of both BITC and the extract were irreversible, i.e., the tissue did not recover to normal contractile ability after extensive washing. Exposure of the tissue to the papaya seed extract caused slower relaxation of the tissue, compared to controls, both after contraction with PE and subsequent addition of carbachol (CCh), and after contraction with KCl and then washing. Calcium imaging studies using cultured endothelial cells showed strong influxes of Ca2+ into the cells in response to addition of the papaya seed extract. We conclude that these extracts, when present in high concentration, are cytotoxic by increasing the membrane permeability to Ca2+, and that the vascular effects of papaya seed extracts are consistent with the notion that BITC is the chief bio-active ingredient.     

Exposure to Silica Nanoparticles Causes Reversible Damage of the Spermatogenic Process in Mice

Environmental exposure to nanomaterials is inevitable, as nanomaterials have become part of our daily life now. In this study, we firstly investigated the effects of silica nanoparticles on the spermatogenic process according to their time course in male mice. 48 male mice were randomly divided into control group and silica nanoparticle group with 24 mice per group, with three evaluation time points (15, 35 and 60 days after the first dose) per group. Mice were exposed to the vehicle control and silica nanoparticles at a dosage of 20 mg/kg every 3 days, five times over a 13-day period, and were sacrificed at 15, 35 and 60 days after the first dose. The results showed that silica nanoparticles caused damage to the mitochondrial cristae and decreased the levels of ATP, resulting in oxidative stress in the testis by days 15 and 35; however, the damage was repaired by day 60. DNA damage and the decreases in the quantity and quality of epididymal sperm were found by days 15 and 35; but these changes were recovered by day 60. In contrast, the acrosome integrity and fertility in epididymal sperm, the numbers of spermatogonia and sperm in the testes, and the levels of three major sex hormones were not significantly affected throughout the 60-day period. The results suggest that nanoparticles can cause reversible damage to the sperms in the epididymis without affecting fertility, they are more sensitive than both spermatogonia and spermatocytes to silica nanoparticle toxicity. Considering the spermatogenesis time course, silica nanoparticles primarily influence the maturation process of sperm in the epididymis by causing oxidative stress and damage to the mitochondrial structure, resulting in energy metabolism dysfunction.     

Impact of experimental diabetes and insulin replacement on epididymal secretory products and sperm maturation in albino rats

The present study is aimed to explore the impact of experimental diabetes and insulin replacement on epididymal secretory products, sperm count, motility, and fertilizing ability in albino rats. Prepubertal and adult male Wistar strain rats were made diabetic with a single intraperitoneal injection of streptozotocin (STZ), at 120 and 65 mg/kg body weight for prepubertal and adult rats, respectively. After 3 days of STZ administration, insulin was given to a group of diabetic rats at a dose of 3 U/100 g body weight, subcutaneously and killed after 20 days of treatment. STZ‐diabetes significantly reduced the epididymal tissue concentrations of testosterone, androgen‐binding protein, sialic acid, glycerylphosphoryl choline, and carnitine, suggesting its adverse effects on the secretory activity and concentrating capacity of epididymal epithelium. Impaired cauda epididymidal sperm motility and fertility (in vivo) of STZ‐diabetic rats imply the defective sperm maturation. Insulin replacement prevented these changes either partially or completely. From the above findings, it is evident that STZ‐diabetes has an adverse effect on sperm maturation, which may be due to the decrease in the bioavailability of testosterone and epididymal secretory products. J. Cell. Biochem. 108: 1094–1101, 2009. © 2009 Wiley‐Liss, Inc.     

Characterization and identification of epididymal factors that protect ejaculated bovine sperm during in vitro storage.

The role of secretory epididymal factors on sperm survival and storage in bovine cauda epididymides is poorly understood. Thus, the effects of bovine epididymal epithelium fluid (BEEF) on frozen-thawed bovine sperm motility have been evaluated in vitro. Sperm motion parameters were assessed by computer-assisted sperm analysis. Compared with serum bovine proteins, BEEF efficiently sustained bovine sperm motility after a 6-h incubation period. The positive effect of BEEF on sperm motility was even more apparent using a fractionated BEEF extract (>10 kDa, 2 mg/ml). This beneficial effect was abolished when the BEEF active fraction was heat treated before incubation. A minimal 2-h BEEF preincubation period was necessary to maintain sperm motility activity and to protect sperm against oxidative injury caused by 150 microM hydrogen peroxide. The proteins from the BEEF >10-kDa fractions were biotinylated to identify the proteins that bind to the sperm surface. Five specific sperm-surface-binding proteins were revealed by Western blot analysis probed with avidin-horseradish peroxidase conjugate. These proteins were digested with trypsin for identification by matrix-assisted laser desorption ionization time-of-flight peptide mass spectrometric analyzer. Under reducing conditions, 5 bovine proteins were identified: the beta (36-kDa spot) and alpha (38-kDa spot) chains of clusterin, the beta-adrenergic receptor kinase 2 (48-kDa spot), and the antithrombin-III and the fibrinogen gamma-B chains, both corresponding to a doublet of about 50-52 kDa. These proteins are known to be present at the sperm surface in other species and could play a role in sperm protection in vivo. These results provide new insights to explain how secretory epididymal proteins sustain sperm motility during storage in vitro.     

Ribavirin-induced sperm shape abnormalities in Wistar rat.

Ribavirin (1-beta-D-ribofuranosyl-1,2,4, triazole-3 carboxamide) is a broad-spectrum antiviral drug. This study was aimed to investigate the mutagenicity of ribavirin on germ cells by employing sperm morphology assay. Male Wistar rats were treated with water, cyclophosphamide (CP) 40 mg/kg, and ribavirin 20, 100 and 200 mg/kg (i.p.) for 5 consecutive days at intervals of 24h. Following the last exposure, at 14, 28, 35, 42 and 70 days, the epididymal sperm smears were obtained and stained according to the standard procedure. One thousand sperms per animal were classified into normal and different abnormal types. Both CP and ribavirin-induced anomalies of head and tail of sperm except at 70 days. In CP groups, maximum incidence was observed at 28, 35 and 42 days. Ribavirin 20 mg/kg induced maximum incidence at 14 and 42 days, 100 mg/kg at 28 and 42 days and 200 mg/kg at 28-42 days. These results show that ribavirin is mutagenic to rat germ cells in a transient fashion.     

Analysis of epididymal proteins during sexual maturation in male albino mice.

Androgen dependent epididymal proteins act as antigen to produce autoantibodies and affect normal fertility. In the present study, epididymal proteins were analyzed during the time of sexual maturation and their androgen dependency was studied in male albino mice. Epididymis of 21 days (Pre-pubertal), 45 days (Pubertal), 60 days (Post-pubertal), orchidectomized (15 days after surgery) and orchidectomized with testosterone-treated (15 days after treatment) mice were dissected out and analyzed. Caput, corpus and cauda epididymidis were separated and the protein extract was prepared with 0.1 M PBS for 10% SDS-PAGE analysis. Testosterone assay was performed in the experimental groups except the testosterone treated group. The electrophoretic analysis of proteins in caput, corpus and cauda epididymidis of orchidectomized animals showed the disappearance of several proteins as compared to the adult. However, the disappeared proteins started to reappear in testosterone treated animals. The results suggest that removal of testis depletes the testosterone level and causes significant alteration in epididymal proteins. These proteins need further investigation for the purpose of immunocontraception by using them as antigens.     

Identification of epididymal proteins associated with hamster sperm.

The electrophoretic analysis of the proteins that were extracted from immature caput and mature cauda sperm showed evidence of accumulation of several proteins during the epididymal transit of the sperm. An antiserum, raised against detergent-extracted proteins from mature spermatozoa, immunostained six epididymal proteins with apparent molecular masses of 16, 22.5, 26, 37, 60, and 80 kDa on Western blots of epididymal fluid. Of these proteins, only the 26 kDa protein was significantly immunodetected in proximal caput epididymal fluid. Its biosynthesis by caput epididymis was confirmed by immunoprecipitation of an in vitro translated product of caput poly (A) RNA. The homology of the 26 kDa epididymal protein with the 26 kDa sperm protein was verified by epitope mapping. The other epididymal proteins were found in the fluid of the more distal portions of the organ. Their presence in the epididymal fluid coincided with their detection on the sperm. These epididymal proteins were considered to be sperm-coating proteins.     

Molecular cytogenetic analysis in mouse sperm of chemically induced aneuploidy: studies with topoisomerase II inhibitors

The ability of two topoisomerase II (topo II) inhibitors, etoposide (VP-16) and merbarone (MER), to induce meiotic delay and aneuploidy in mouse spermatocytes was investigated. The progression from meiotic divisions to epididymal sperm was determined by injecting male mice with 5-bromo-2′-deoxyuridine (BrdU) and treating the animals 13 days later with the test chemicals. At 20–24 days after treatment, BrdU-containing sperm were identified with a FITC-labelled anti-BrdU antibody and green fluorescent sperm were scored with a laser scanning cytometer (LSC). It was found that VP-16 (50 mg/kg) treatment induced a meiotic delay of about 24 h. A significant reduction of BrdU-labelled sperm was observed at 22 days compared to the controls (VP-16 group: 14.20%; controls: 41.10%, P<0.001). At 23 and 24 days, there were no significant differences between the VP-16 and the control groups. MER (80 mg/kg) treatment did not cause meiotic delay. To determine the frequencies of hyperhaploid and diploid sperm, male mice were treated with 12.5, 25 and 50 mg/kg VP-16 or 15, 30 and 60 mg/kg MER. Sperm were sampled from the Caudae epididymes 24 days after VP-16 treatment or 22 days after MER treatment. Significant increases above the concurrent controls in the frequencies of total hyperhaploid sperm were found after treatment with 25, 50 mg/kg VP-16 (0.074 and 0.122% versus 0.052%) and after treatment with 60 mg/kg MER (0.098% versus 0.044%). Furthermore, significant increases in the frequencies of diploid sperm were found after treatment of mice with all three doses of VP-16 (0.024, 0.032 and 0.056% versus 0.004 and 0.00%, respectively) and with 30 and 60 mg/kg MER (0.022 and 0.05% versus 0.004 and 0.002%, respectively). All dose responses could be expressed by linear equations. The results indicate that cancer patients may stand transient risk for siring chromosomally abnormal offspring after chemotherapy with these topo II inhibitors.     

度他雄胺对大鼠附睾精子成熟和生育的影响

目的通过度他雄胺对大鼠附睾精子和生育的影响,探索调节雄性生育的睾丸后作用靶点。方法使用度他雄胺20和40 mg/(kg.d)大鼠灌胃给药,连续2周。给药结束后雄雌鼠按1∶2合笼,计算生殖指数;采用计算机辅助精子分析系统分析精子活力和形态;采用SYBR-14和PI双重荧光染色计算精子存活率;采用Elisa法测定大鼠睾酮(T)和双氢睾酮(DHT)血清浓度;采用HE染色法对各组睾丸、附睾进行组织学分析。结果度他雄胺低、高剂量组双氢睾酮浓度均显著下降,分别为0.54和0.28 nmol/L(P<0.01),精子活力明显降低,分别为39.0%和28.7%(P<0.01),畸形率分别增加为10.3%和15.6%(P<0.05),最后受孕率分别降为62.5%和38.4%。而睾酮水平和交配指数均无明显变化(P>0.05),睾丸和附睾亦无明显病理学改变。结论度他雄胺通过抑制DHT生成,影响附睾精子成熟而导致大鼠不育,为今后男性避孕和不育药物研发提供了新思路。     

A time-course study of chronic paternal cyclophosphamide treatment in rats: effects on pregnancy outcome and the male reproductive and hematologic systems

We have found previously that daily treatment of male rats for 11 wk with low doses of the anticancer drug cyclophosphamide had no apparent effect on male reproductive organ weights, epididymal sperm counts, or serum hormones at the end of the treatment period; yet, upon breeding to untreated females, these males produced a high rate of post-implantation loss and fetal anomalies. The present study was designed to investigate the time course and dose response of the effects of chronic cyclophosphamide treatment on the male reproductive and hematologic systems. Male Sprague-Dawley rats were gavage-fed for 1, 3, 6 and 9 wk with saline (control), or 5.1 (low dose) or 6.8 (high dose) mg/kg/day of cyclophosphamide. After each of the treatment periods, males were mated to determine the effect on pregnancy outcome, then killed, and the effects on the male reproductive and hematologic systems were assessed. After 6 wk of treatment, a sharp increase in mortality was found between the 5.1 and 6.8 mg/kg/day doses of cyclophosphamide. The high dose of cyclophosphamide induced higher levels of pre- and post-implantation loss but fewer fetal anomalies than did the low dose. The low dose of cyclophosphamide did not affect reproductive organ weights; in contrast, the high dose caused decreases in epididymal, ventral prostate, and seminal vesicle weights after 3, 6, and 9 wk. Testicular and epididymal sperm counts were decreased in a dose-dependent manner after 3 wk; in addition, the high dose led to a decrease in epididymal sperm counts after 6 wk of treatment. Another rapidly proliferative tissue, the bone marrow, was dramatically affected by both doses of cyclophosphamide at all time points, with leukocyte counts decreasing to 40% of control by 1 wk. After 9 wk of treatment, effects on the male reproductive system were less marked, compared to earlier time points, whereas those on the hematologic system and pregnancy outcome persisted. Thus chronic low-dose treatment of male rats with cyclophosphamide not only had early and striking effects on the bone marrow and the pregnancy outcome but also affected the male reproductive system in a clear time- and dose-dependent manner.