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PubMed1993

The cytoplasmic droplet of rat epididymal spermatozoa contains saccular elements with Golgi characteristics.

The cytoplasmic droplet of epididymal spermatozoa is a small localized outpouching of cytoplasm of the tail of unknown significance. EM revealed flattened saccular elements as the near exclusive membranous component of the droplet. Light and electron microscopic immunolabeling for Golgi/TGN markers showed these saccules to be reactive for antibodies to TGN38, protein affinity-purified alpha 2,6 sialyltransferase, and anti-human beta 1,4 galactosyltransferase. The saccules were isolated by subcellular fractionation and antibodies raised against this fraction immunolabeled the saccules of the droplet in situ as well as the Golgi region of somatic epithelial cells lining the epididymis. The isolated droplet fraction was enriched in galactosyltransferase and sialyltransferase activities, and endogenous glycosylation assays identified the modification of several endogenous glycopeptides. EM lectin staining in situ demonstrated galactose and N-acetyl galactosamine constituents in the saccules. Endocytic studies with cationic and anionic ferritin as well as HRP failed to identify the saccules as components of the endocytic apparatus. Epididymal spermatozoa were devoid of markers for the ER as well as the Golgi-associated coatamer protein beta-COP. It is therefore unlikely that the saccular elements of the droplet participate in vesicular protein transport. However, the identification of Golgi/TGN glycosylating activities in the saccules may be related to plasma membrane modifications which occur during epididymal sperm maturation.

Animals↗
PubMed1994

Catecholamine-sensitive lipolysis in the rat: different loci for effect of age on the lipolytic cascade in epididymal vs perirenal fat cells.

The biochemical locus of the decrease of lipolytic responsiveness to catecholamines in the aging rat has not heretofore been completely identified. Although increased sensitivity to the inhibitory action of adenosine is the likely explanation for the decrease during maturation, the nature of the age effect during senescence has been unclear. In order to determine whether the proximal or distal portion of the lipolytic pathway is involved, we have studied the lipolytic effect of the distally acting cyclic AMP analogue, 8-(4-chlorophenylthioadenosine)3'5'-monophosphate (cyclic) (Cl-cAMP) on rat fat cells from both the epididymal and perirenal fat pads of mature (6 mo) and senescent (24 mo) Fischer 344 rats. Using an adenosine (N6-1-2-phenylisopropyl-adenosine; PIA) regulated system, the lipolytic response to epinephrine (glycerol release) was measured simultaneously with that to Cl-cAMP. The effects of age on lipolysis are greatly influenced by the anatomic site of origin of the fat cells. The epididymal cells of the old rats showed no decreased responsiveness to either epinephrine or Cl-cAMP. However, the perirenal cells of the old rats showed a grossly impaired maximal response to both epinephrine (60% decrease relative to young; p < .005) and Cl-cAMP (42% and 58% decrease in 2 sets of experiments; p < .05 and .04, respectively). Although decreased lipolytic response to epinephrine in epididymal cells was not seen in these studies, this has been clearly shown in earlier work, suggesting that diminished response to epinephrine is demonstrable only when the system is not already maximally inhibited by PIA.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Deaminase↗
PubMed1997

Changes in lipids and membrane anisotropy in human spermatozoa during epididymal maturation.

Previously it was demonstrated that immature and immotile human spermatozoa from the caput epididymides developed a good progressive motility after in-vitro stimulation with phosphatidylcholine (PC). In order to define the role of PC and membrane anisotropy in epididymal maturation and to determine the exact lipid composition of human spermatozoa during epididymal maturation, spermatozoa from seven epididymides from patients who underwent orchiectomy because of prostatic cancer were investigated. Lipids were determined by high-performance thin-layer chromatography and gas chromatography. Membrane anisotropy was measured by fluorescence polarization. The ratio between PC and phosphatidylserine (PS) plus phosphatidyl ethanolamine (PE) plus sphingomyelin (SM) was significantly higher in spermatozoa from the cauda compared to those from the caput and corpus. This was due to an increase of PC and a decrease of the concentration of PS plus PE plus SM. With regard to fatty acids, those with saturated chains predominated in caput spermatozoa while the highest concentration of unsaturated long-chain fatty acids was in cauda spermatozoa. A lower membrane anisotropy of cauda spermatozoa compared with caput or corpus spermatozoa was found. In conclusion, during epididymal maturation human spermatozoa integrate lipids, particularly PC, which is strongly associated with the induction of progressive motility. A change in the pattern of fatty acids and a decrease in the cholesterol/phospholipid molar ratio cause a decrease in membrane anisotropy in cauda spermatozoa.

Aged↗
PubMed1998

A follow-up study of children born after intracytoplasmic sperm injection (ICSI) with epididymal and testicular spermatozoa and after replacement of cryopreserved embryos obtained after ICSI.

The aim of this prospective follow-up study of children born after intracytoplasmic sperm injection (ICSI) was to compile data on karyotypes, congenital malformations, growth parameters and developmental milestones in order to evaluate the safety of this new technique. The study design included karyotyping of the parents and their agreement to genetic counselling and prenatal diagnosis and it was based on a physical examination of the child at the Centre for Medical Genetics at the ages of 2 months, 1 year and at 2 years, where major and minor malformations and psychomotor evolution are recorded. Here we describe the first 57 children born from 40 ICSI pregnancies with epididymal spermatozoa (group 1), the first 50 children born from 34 ICSI pregnancies with testicular spermatozoa (group 2) and the first 58 children born from 48 pregnancies after replacement of cryopreserved ICSI embryos (group 3). Parental karyotypes were obtained from only 72/246 (29%) parents and were all normal. Prenatal karyotypes were determined for a total of 70 samples (40%): 21 in group 1, 15 in group 2 and 34 in group 3. In this last group 2 abnormal 47,XXY karyotypes (5.8%) and no structural aberrations were found. This increase in de-novo sex-chromosomal aberrations has already been described with regard to the first 877 children born after ICSI carried out at our Centre and is probably linked directly to the characteristics of the infertile men treated rather than to the ICSI procedure itself. Major malformations, defined as those causing functional impairment or requiring surgical correction, were observed in four children: two born after ICSI with epididymal spermatozoa, one after ICSI with testicular spermatozoa and one after ICSI and cryopreservation. No particular malformation was disproportionally frequent. In the follow-up examinations at 2 months (107/161 or 66.5%) and at 1 year (37/161 or 22.9%), no additional anomalies were observed. Lost for follow-up rate at 2 months was 33.5%. These observations on a limited number of children do not suggest a higher incidence of diseases linked to imprinting, nor do they suggest a higher incidence of congenital malformations. These observations are still limited in number and should be further completed by others and by collaborative efforts. In the meanwhile patients should be told about the available data before any treatment: there appears to be some risk of transmitted chromosomal aberrations, of de-novo, mainly sex-chromosomal aberrations and of transmitting fertility problems to the offspring. Patients should also be reassured that until now there seems to be no higher incidence of congenital malformations in children born after ICSI with epididymal or testicular spermatozoa or after replacement of ICSI embryos.

Adult↗
PubMed2000

Microsurgical epididymal sperm aspiration: aspirate analysis and straws available after cryopreservation in patients with non-reconstructable obstructive azoospermia. MESA/TESE Group Giessen.

Microsurgical epididymal sperm aspiration (MESA) combined with intracytoplasmic sperm injection (ICSI) represents a great advance in the therapy of non-reconstructable obstructive azoospermia. For procedure synchronization, a great number of organizational facilities are needed. Intentional cryopreservation of the aspirate may reduce these problems, therefore the aim of this study was to analyse the amount and quality of aspirate fluid obtained by means of MESA and the quality of the vials after thawing. Furthermore, the available cryopreserved straws were calculated. A total of 93 consecutive MESA procedures were performed and epididymal spermatozoa were obtained in 88 patients. Mean sperm concentration was 40.9 x 10(6) spermatozoa/ml. Global and progressive motility were 24.8 and 7.5% respectively. In one-third of the aspirates, no progressive motile spermatozoa were found. The mean number of straws available was 7.6. In 33 ICSI cycles with frozen-thawed epididymal spermatozoa, a pregnancy rate of 42.4% was achieved. In conclusion, these data show that enough spermatozoa are available for various ICSI cycles following a single MESA procedure in men with non-reconstructable obstructive azoospermia. Furthermore, ICSI with cryopreserved spermatozoa leads to excellent pregnancy rates

Adult↗
PubMed2004

Analysis of chromosomal abnormalities in testicular and epididymal spermatozoa from azoospermic ICSI patients by fluorescence in-situ hybridization.

BACKGROUND: An increased incidence of numerical chromosomal abnormalities has been reported in the ejaculated spermatozoa of infertile patients. However, there are few cytogenetic studies of testicular and epididymal spermatozoa, and their results are still controversial. METHODS: Fluorescence in-situ hybridization (FISH) analysis of chromosomes 13, 18, 21, X and Y was performed on seven testicular samples and two epididymal samples from patients with obstructive azoospermia (OA), and on 13 testicular samples from patients with non-obstructive azoospermia (NOA). Five ejaculated sperm samples from normozoospermic fertile donors were evaluated as a control group. RESULTS: Both epididymal sperm samples showed normal FISH results for the parameters analysed when compared with those of the control group. FISH results were abnormal in 29% (two of seven) of testicular samples from OA patients and in 54% (seven of 13) of those from NOA patients, although this difference was not statistically significant. Testicular samples from OA patients showed a significant increase of disomy for sex chromosomes (P<0.01), whereas NOA patients displayed significantly higher rates of diploidy (P<0.0001) and disomy for chromosomes 13 (P<0.0001), 21 (P<0.001) and sex chromosomes (P<0.0001) than the control group. CONCLUSIONS: Testicular spermatozoa from azoospermic patients present increased rates of chromosomal abnormalities, mainly of the sex chromosomes, which are particularly high in NOA patients.

Adult↗
PubMedDate not supplied

Studies of percutaneous epididymal sperm aspiration (PESA) and intracytoplasmic sperm injection.

Four distinct studies were carried out using two data sets of percutaneous epididymal sperm aspiration (PESA) and intracytoplasmic sperm injection (ICSI) procedures performed from March 1993 to January 1997. In study A, an analysis of 181 ICSI treatment cycles following PESA revealed a successful epididymal sperm retrieval rate of 83%. It confirmed that PESA is an effective sperm retrieval method and the associated ICSI pregnancy rate (35% per embryo transfer) compared favourably with that of other sperm retrieval methods. In study B, the relevance of a prior diagnostic PESA procedure was ascertained by comparing the sperm retrieval rates in two groups of patients having their first ICSI treatment cycle with spermatozoa retrieved through PESA. Group B1 (n = 50) had diagnostic PESA prior to the ICSI treatment cycle PESA procedure, unlike patients in group B2 (n = 64) who did not. The sperm retrieval rate in the treatment cycle procedure was not different at 90 and 82.8% for groups B1 and B2 respectively. However, the discontinuation of diagnostic PESA is fraught with problems including liability to medico-legal sanctions. In study C, analysis of 177 treatment cycles involving PESA and ICSI revealed a successful sperm retrieval rate by PESA of 82% in the first cycle, 93% in the second, 96% in the third and 100% in the fourth cycle. The same trend was evident when sperm retrieval was examined in relation to each of the epididymides. Retrieved spermatozoa were found to be motile in 67-100% of cases and the frequency of samples containing motile spermatozoa did not decrease with increase in the number of PESA attempts. These results show that PESA does not jeopardize future epididymal sperm retrieval. In study D, the outcome of treatment with ICSI using ejaculated spermatozoa (305 cycles) (group D1) was compared with that of ICSI using spermatozoa obtained through PESA (54 cycles) (group D2). The median age of women in the two groups of couples was similar (34 years). In group D1, 70% of metaphase II oocytes were fertilized compared with 61% in group D2 (P < 0.01). The cleavage rate and the median numbers of transferred and cryopreserved embryos were similar in both groups. There was no significant difference between the clinical pregnancy rates (33 and 42% in groups D1 and D2 respectively). Our results show that the outcome of PESA-ICSI treatment compares favourably with that of ICSI using ejaculated spermatozoa.

Adult↗
PubMedDate not supplied

Recent biochemical approaches to post-testicular, epididymal contraception.

Results from recent animal models with implications for putative human male contraceptives acting on the epididymis are reviewed. Inducing sterility by enhancing sperm transport through the epididymis has not been achieved. The induction of infertility in males of several species is easier to achieve by direct actions of drugs on sperm function (e.g. inhibition of sperm-specific isoenzymes of the glycolytic pathway by chloro-compounds) than by indirectly reducing amounts of epididymal secretions normally present in high concentration (e.g. alpha-glucosidase, L-carnitine). The former show promise for the clinic since human spermatozoa are susceptible to inhibition. On the other hand, the infertile male mice of the c-ros knock-out model demonstrate the influence of even a small region of the epididymis on fertility, so that targeting the as yet unknown epididymal factors presumably secreted in limiting amounts by this epididymal segment, is a new lead for a contraceptive. Targeting a specific sperm protein acquired in the testis, but depleted in the epididymis by toxicants that induce rapid infertility, may also lead to the discovery of new contraceptives, but these will require developing new means of organ-specific delivery of contraceptive drugs.

Animals↗
PubMed1983

Effect of selenium on the subcellular distribution of glutathione peroxidase in rat liver, epididymal fat pad and seminal vesicle.

The effect of dietary selenium on the subcellular distribution of selenium-dependent glutathione peroxidase (SeGSH-Px) activity in rat liver, epididymal fat pad, and seminal vesicle was determined. Tissues were fractionated by differential centrifugation, and the subcellular distribution of SeGSH-Px activity was determined by comparison with the distribution of biochemical marker enzymes. Liver SeGSH-Px activity was located in both the cytosol and mitochondria. In epididymal fat pad and seminal vesicle, SeGSH-Px activity was located primarily in the cytosol; association with another subcellular organelle, however, was indicated. In liver and epididymal fat pad, SeGSH-Px activity increased linearly, and, in the seminal vesicle, increased linearly and quadratically, with increasing dietary selenium concentration. Distribution of SeGSH-Px activity among the cellular fractions from the tissues, however, was not affected by dietary selenium supplementation.

Adipose Tissue↗
PubMed1996

Micromanipulation improves in-vitro fertilization results after epididymal or testicular sperm aspiration in patients with congenital absence of the vas deferens.

In all, 58 couples suffering from infertility because of congenital bilateral absence of the vas deferens underwent a total of 67 combined microsurgical epididymal aspiration or testicular sperm extraction (TESE) and in-vitro fertilization (IVF) treatments. The oocytes recovered were inseminated by either the microdroplet IVF technique (N = 20), subzonal insemination (SUZI; n = 10) or intracytoplasmic sperm injection (ICSI; n = 37). Of the ICSI cycles, 12 were performed using spermatozoa obtained by TESE. Fertilization rates for epididymal spermatozoa were significantly higher for SUZI (17.9%, 17/95) and ICSI (34.4%), 137/398) than for microdroplet IVF (5.2%, 18/343) cycles. The proportion of cycles in which fertilization was achieved was higher in the SUZI (80%) and ICSI (95%) cycles than in the IVF cycles (45%). Delivery or an ongoing pregnancy was achieved in one (5%) IVF cycle, two (20%) SUZI cycles and seven (18.95) ICSI cycles. SUZI or ICSI using epididymal or testicular spermatozoa significantly improved the oocyte fertility rate. The ICSI procedure was especially advantageous in patients for whom spermatozoa were obtained from a testicular biopsy.

Cytoplasm↗
PubMed1996

Changes in motility patterns during in-vitro culture of fresh and frozen/thawed testicular and epididymal spermatozoa: implications for planning treatment by intracytoplasmic sperm injection.

The present report describes the motility changes in vitro (percentage motile and progressively motile) of freshly collected testicular and epididymal spermatozoa and following freeze/thaw of the same spermatozoa from a man with obstructive azoospermia. Washed spermatozoa were cultured in micro droplets under paraffin oil or in test tubes using HEPES-buffered or bicarbonate-buffered medium containing 10% human serum. In fresh testicular sperm cultures 60-65% of the sperm cells became motile within 2 days of culture; the motility was maintained for a further 4-5 days before a decline was observed. The progressive motility improved markedly on the third day of culture and it peaked around day 5. Only a small number of frozen/thawed testicular spermatozoa became motile during in-vitro culture (15-20%) and the motility was maintained for only 2-3 days before it declined. Furthermore, only 10-12% of the spermatozoa showed progressive motility. Spermatozoa recovered from micro-epididymal sperm aspiration (MESA) showed a gradual decrease in progressive motility and in 5 days all sperm cells were found to be immotile in both freshly collected and frozen/thawed spermatozoa. All culture systems supported sperm motility. It is clear that testicular spermatozoa, particularly from men with obstructive azoospermia, can be collected and maintained in vitro for up to 1 week before the oocyte retrieval but when frozen testicular or epididymal spermatozoa are used it is more reliable to thaw these spermatozoa on the day of intracytoplasmic sperm injection.

Adult↗
PubMed1995

Epididymal sperm aspiration with assisted reproductive techniques: difference between congenital and acquired obstructive azoospermia?

From July 1992 to May 1994, 31 cases of obstructive azoospermia-induced infertility underwent 35 epididymal sperm aspiration procedures and assisted reproductive techniques. These included in-vitro fertilization (IVF), zygote intra-Fallopian transfer (ZIFT), subzonal insemination (SUZI) and intracytoplasmic sperm injection (ICSI) with embryo transfer or tubal embryos transfer. The motile spermatozoa were retrieved using a gauge 24 Medicut cannula and flush medium. Total oocyte fertilization rate was 23.4% and the clinical pregnancy rate was 12.5% (four out of 32 treatment cycles). Congenital absence of the vas deferens was found in 16 cases (51.6%) and secondary genital duct obstruction was present in 15 cases (48.4%). In all, 29 aspirations were retrieved from the caput of the epididymis and six from the epididymal body or tail. The fertilization rate for the caput spermatozoa was much less than that for other areas of the epididymis (P < 0.05). Though there were no predictable differences in fertilization rates between the congenital and acquired groups, by using epididymal spermatozoa for assisted reproduction, the congenital group seemed to have a stronger tendency to achieve pregnancy (20 versus 5.9%).

Adult↗
PubMed1991

Epididymal secretory function in men with asthenoteratozoospermia.

Epididymal function was evaluated in normozoospermic men and men with asthenoteratozoospermia using alpha-glucosidase as a secretory parameter. No difference was observed in the total quantity of the enzyme secreted into the ejaculate in spite of major differences between the two groups in the proportion of spermatozoa exhibiting progressive motility and normal morphology. In general, the secretion of alpha-glucosidase was positively correlated with the sperm concentration in the normozoospermic group, but not in asthenozoospermic subjects. The total enzyme contents of the ejaculate were significantly greater in men with asthenoteratozoospermia (sperm concentrations, greater than 35 x 10(6)/ml) than in subjects with severe oligoasthenoteratozoospermia (sperm concentrations less than 10 x 10(6)/ml, P less than 0.01) indicating a higher incidence of epididymal occlusion and/or dysfunction in the latter group. Within normozoospermic individuals, extremes in progressive motility or abnormal morphology could not be related to the quantities of enzyme when expressed per million sperm cells. It is concluded that if alpha-glucosidase is a reliable parameter of human epididymal secretory function, variation in the secretory function of the epididymis cannot explain the poor sperm quality observed in asthenoteratozoospermic individuals.

Epididymis↗
PubMed1992

Replacement of frozen embryos generated from epididymal spermatozoa: the first two pregnancies.

This report describes the first two pregnancies which occurred after cryopreservation, thawing and transfer of embryos generated using epididymal spermatozoa from men with congenital absence of the vas deferens. Of the 32 embryos that were thawed, 24 survived (75%) and were replaced in seven transfer cycles resulting in two (29%) pregnancies successfully delivered. The demonstration that excess embryos obtained with the use of epididymal spermatozoa can be safely frozen/thawed, and that their survival is not different from other embryos generated by in-vitro fertilization, represents a great advantage for the reproductive performance of men with congenital absence of the vas deferens undergoing microsurgical aspiration of epididymal spermatozoa.

Cryopreservation↗
PubMed1993

Epididymal sperm aspiration and use of donor spermatozoa when necessary for in-vitro fertilization, and vasoepididymostomy: an acceptable combination in the treatment of obstructive azoospermia.

Infertility due to obstructive azoospermia in 24 men was treated with a combination of scrotal exploration, microsurgical sperm aspiration and vasoepididymostomy, at the same operation. In-vitro fertilization (IVF) and embryo transfer were performed using epididymal spermatozoa. Donor spermatozoa were used if no motile epididymal spermatozoa were obtained. With this combination, emotionally and economically acceptable pregnancy rates were achieved: 24% per aspiration, 43% per embryo transfer, and 25% per couple. One twin pregnancy resulting in the birth of two healthy female infants and one ongoing twin pregnancy were achieved with epididymal spermatozoa; four pregnancies (one twin, two singletons, one abortion) were achieved with donor spermatozoa.

Adult↗
PubMed1993

Cystic fibrosis mutations impair the fertilization rate of epididymal sperm from men with congenital absence of the vas deferens.

One of the limiting factors for the successful treatment of male sterility due to congenital absence of the vas deferens (CAVD) is the low (< 20%) and extremely unpredictable rate of in-vitro fertilization of their epididymal spermatozoa. The recent demonstration that CAVD is a mild form of cystic fibrosis (CF) disease, almost exclusively involving the genital tract, has prompted us to investigate the hypothesis of whether there could be an association between particular CF genotype mutations and the in-vitro performance of epididymal sperm. In this study 63 patients with surgically confirmed diagnosis of CAVD undergoing microsurgical epididymal sperm aspiration (MESA) and in-vitro fertilization (IVF) were evaluated. The genetic screening was carried out on DNA extracted from peripheral lymphocytes and amplified by the polymerase chain reaction. A total of 12 mutations in the cystic fibrosis transmembrane regulator gene (CFTR), representing approximately 90% of the total known CF mutations, were tested. The presence or absence of mutations, as well as the type of mutation found, was correlated with the IVF and pregnancy rates. Of the 63 patients examined, 40 (64%) were positive to the CF screening and 23 were negative. In three cases no spermatozoa were found because of rete testis blockage. The difference in fertilization rates between patients testing positive and negative was highly significant (P = 0.000003), 13 versus 23%, respectively. In order to pinpoint which mutation could account for the most deleterious effect on the IVF results, we analysed these by keeping the patients separated by CF mutations.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed2002

Novel antimicrobial peptide of human epididymal duct origin.

HE2, a gene expressed specifically in human epididymis, gives rise to multiple mRNAs that encode a group of small cationic secretory peptides. Localization of HE2 within the defensin gene cluster and prediction that beta-defensin-like modules exist suggest that these peptides have antimicrobial activity and represent components of the innate epithelial defense system of the epididymal duct. Reverse transcription-polymerase chain reaction analysis confirmed the occurrence of eight human HE2-derived transcripts, including minor mRNA variants, that had previously been shown only in animal species. Employing isoform-specific antibodies against the predicted HE2 products, multiple 4- to 8-kDa peptides were detected in human epididymal epithelium, epididymal fluid, and ejaculate. N-terminal microsequencing has suggested a proteolytic processing of these peptides by a furin-like proprotein convertase, which cleaves a propiece from the longer precursor peptides. HE2alpha and HE2beta1, representing major peptide isoforms in the human epididymis, were recombinantly expressed, and their susceptibility to furin cleavage was demonstrated in vitro and in vivo. Processed recombinant peptides and chemosynthetic fragments were included in antimicrobial tests. In addition to the beta-defensin-like HE2beta1 with its expected antibacterial function, HE2alpha C-terminal fragments showed antibacterial activity against Escherichia coli, although it showed no significant similarity to beta-defensins nor to any other known protein family.

Amino Acid Sequence↗
PubMed2002

Indazole inhibition of cystic fibrosis transmembrane conductance regulator Cl(-) channels in rat epididymal epithelial cells.

Previous studies have shown that two indazole compounds, lonidamine [1-(2,4-dichlorobenzyl)-indazole-3-carboxylic acid] and its analogue AF2785 [(1-(2,4-dichlorobenzyl)-indazol-3-acrylic acid], suppress fertility in male rats. We also found that these compounds inhibit the cystic fibrosis transmembrane conductance regulator chloride (CFTR-Cl(-)) current in epididymal epithelial cells. To further investigate how lonidamine and AF2785 inhibit the current, we used a spectral analysis protocol to study whole-cell CFTR current variance. Application of lonidamine or AF2785 to the extracellular membrane of rat epididymal epithelial cells introduced a new component to the whole-cell current variance. Spectral analysis of this variance suggested a block at a rate of 3.68 micro mol(-1)/sec(-1) and an off rate of 69.01 sec(-1) for lonidamine, and an on rate of 3.27 micro mol(-1)/sec(-1) and an off rate of 108 sec(-1) for AF2785. Single CFTR-Cl(-) channel activity using excised inside-out membrane patches from rat epididymal epithelial cells revealed that addition of lonidamine to the intracellular solution caused a flickery block (a reduction in channel-open time) at lower concentration (10 micro M) without any effect on open channel probability or single-channel current amplitude. At higher concentrations (50 and 100 micro M), lonidamine showed a flickery block and a decrease in open-channel probability. The flickery block by lonidamine was both voltage-dependent and concentration-dependent. These results suggest that lonidamine and AF2785, which are open-channel blockers of CFTR at low concentrations, also affect CFTR gating at high concentrations. We conclude that these indazole compounds provide new pharmacological tools for the investigation of CFTR. By virtue of their interference with reproductive processes, these drugs have the potential for being developed into novel male contraceptives.

Animals↗