Caffeine induces sperm detachment from sperm head-to-head agglutination in bull.

Sperm head-to-head agglutination is a well-known known phenomenon in mammalian and non-mammalian species. Although several factors have been reported to induce sperm agglutination, information on the trigger and process of sperm detachment from the agglutination is scarce. Since hyperactivated motility is involved in bovine sperm detachment from the oviduct, we focused on caffeine, a well-known hyperactivation inducer, and aimed to determine the role of caffeine in sperm detachment from agglutination. Agglutination rate of bovine sperm was significantly decreased upon incubation with caffeine following pre-incubation without caffeine. Additionally, we observed that bovine sperm were detached from agglutination only when the medium contained caffeine. The detached sperm showed more asymmetrical flagellar beating compared to the undetached motile sperm, regardless of whether before or after the detachment. Intriguingly, some sperm that detached from agglutination re-agglutinated with different sperm agglutination. These findings indicated caffeine as a trigger for sperm detachment from the agglutination in bull. Furthermore, another well-known hyperactivation inducer, thimerosal, also significantly reduced the sperm agglutination rate. Overall, the study demonstrated the complete process of sperm detachment from sperm head-to-head agglutination and proposed that hyperactivated motility facilitates sperm detachment from another sperm. These findings would provide a better understanding of sperm physiology and fertilization process in mammals.

The comparison of two different extenders for the improvement of large-scale sperm cryopreservation in common carp (Cyprinus carpio).

In our study, a traditionally used (Grayling, already used in cyprinid species) and a newly tested (Pike) extender was tested to avoid sperm agglutination phenomenon following thawing during carp sperm cryopreservation. A large-scale (elevated volume of sperm) freezing method in a controlled-rate freezer using 5 ml straw and 10 ml cryotube was also systematically established. In all experiments, the sperm cryopreserved in using Grayling extender (except only one sample) showed an agglutination phenomenon (damaged and intact cells adhered to each other) after thawing where Pike extender resulted the regular cell suspension. No significant difference was observed between the two cryopreserved groups (Pike and Grayling extender) in all motility parameters using the 0.5 ml straw and the polystyrene box. Similarly, motility parameters did not show a significant difference in the two frozen groups with the 5 ml straw, also in the polystyrene box. A significantly higher progressive motility (pMOT, Grayling: 54% ± 8%, Pike: 37% ± 5%), straight line velocity (VSL, Grayling: 50 ± 5 µm/s, Pike: 39 ± 4 µm/s) and beat cross frequency (BCF, Grayling: 20 ± 1 Hz, Pike: 17 ± 1 Hz) was observed in the case of the grayling extender by the 5 ml straw cryopreserved in a controlled-rate freezer (CRF) compare to the pike extender. A significantly higher VSL (Grayling: 45 ± 3 µm/s, Pike: 38 ± 4 µm/s) was observed by the grayling extender using the 10 ml cryotube than with the pike extender. Despite the randomly occurring differences in a few parameters, our new controlled freezing method using the newly tested Pike extender, the 5 ml straw or the 10 ml cryotube can be a good solution for the preservation of elevated volume of carp sperm.

D-penicillamine prevents ram sperm agglutination by reducing the disulphide bonds of a copper-binding sperm protein.

Head-to-head agglutination of ram spermatozoa is induced by dilution in the Tyrode's capacitation medium with albumin, lactate and pyruvate (TALP) and ameliorated by the addition of the thiol d-penicillamine (PEN). To better understand the association and disassociation of ram spermatozoa, we investigated the mechanism of action of PEN in perturbing sperm agglutination. PEN acts as a chelator of heavy metals, an antioxidant and a reducing agent. Chelation is not the main mechanism of action, as the broad-spectrum chelator ethylenediaminetetraacetic acid and the copper-specific chelator bathocuproinedisulfonic acid were inferior anti-agglutination agents compared with PEN. Oxidative stress is also an unlikely mechanism of sperm association, as PEN was significantly more effective in ameliorating agglutination than the antioxidants superoxide dismutase, ascorbic acid, α-tocopherol and catalase. Only the reducing agents cysteine and DL-dithiothreitol displayed similar levels of non-agglutinated spermatozoa at 0 h compared with PEN but were less effective after 3 h of incubation (37 °C). The addition of 10 µM Cu(2+) to 250 µM PEN + TALP caused a rapid reversion of the motile sperm population from a non-agglutinated state to an agglutinated state. Other heavy metals (cobalt, iron, manganese and zinc) did not provoke such a strong response. Together, these results indicate that PEN prevents sperm association by the reduction of disulphide bonds on a sperm membrane protein that binds copper. ADAM proteins are possible candidates, as targeted inhibition of the metalloproteinase domain significantly increased the percentage of motile, non-agglutinated spermatozoa (52.0% ± 7.8) compared with TALP alone (10.6% ± 6.1).

Receptor mediated amelioration of the detrimental effects of sperm agglutinating factor on sperm parameters.

Sperm agglutinating factor (SAF) isolated from Staphylococcus aureus immobilizes spermatozoa by agglutination and causes sperm death. This interaction of SAF with spermatozoa is receptor mediated and this receptor has been isolated and purified from human spermatozoa. In this study we attempt to study whether the receptor could ameliorate the detrimental effects of SAF on sperm parameters. Receptor was evaluated against SAF mediated compromised sperm parameters such as Mg(2+) dependent ATPase activity, acrosome status and apoptosis, in vitro using fluorescent microscopy and flow cytometry as well as in vivo by studying the impact on fertility in mice. Incubation of SAF (80 µg) with spermatozoa resulted in reduced Mg(2+) dependent ATPase activity and premature acrosomal loss whereas a higher concentration (100 µg), induced apoptosis. However, in the presence of receptor a dose dependent blockage of SAF induced inhibition of Mg(2+) dependent ATPase activity was observed. At higher concentrations 100 and 125 µg, receptor could inhibit both the premature acrosomal loss and apoptosis. In vivo studies showed that receptor (50 µg) could alleviate SAF induced infertility in female Balb/c mice following a single intravaginal application before mating. The work highlights the efficacy of the receptor as a corrective measure against negative influence of SAF on functional parameters of spermatozoa as well as fertility and presents receptor as a potential therapeutic intervention against SAF induced infertility.

Effect of Lomodex-MgSO(4) in the prevention of reperfusion injury following unilateral testicular torsion: an experimental study in rats.

Fertility in patients treated for unilateral testicular torsion has been shown to be significantly reduced in all the reported series to date, implying that the present-day treatment requires further refinement in the form of adjunct pharmacotherapeutic intervention (Lomodex and MgSO(4)) in addition to scrotal exploration. Prepubertal Holtzman strain rats (35 days old) were used for our study. Two sets were formed with six groups of rats in each set. Rats were treated as follows: group 1, sham-operated group; group 2, torsion (4 h); group 3, torsion + detorsion (1 h); group 4, torsion + ATP-MgCl(2) + detorsion; group 5, torsion + Lomodex-MgSO(4) + detorsion; group 6, torsion + normal saline + detorsion. Whereas the first set of animals was sacrificed immediately at the end of experiment, animals in set 2 were sacrificed 8 weeks after the end of the experiment to look for the development of antisperm antibodies. Parameters studied were thiobarbituric acid reductase (TBAR) assay, histology of testicular tissue, and sperm agglutination test. Student's t-test was used for significance. With detorsion (149.95+/-30.68) there was a significant rise in the TBAR values (P<0.05) compared with torsion (57.39+/-14.47). Treatment with both Lomodex-MgSO(4) (40.74+/-6.39) and ATP-MgCl(2) (48.30+/-18.35) yielded TBAR levels comparable to those in the sham group (31.35+/-11.96). Similar injury was also seen on the contralateral testis, with detorsion (114.28+/-10.68) much more detrimental than torsion (40.59+/-15.02) and rescue seen following treatment with Lomodex-MgSO(4) (27.55+/-8.64) as well as ATP-MgCl(2) (38.61+/-12.23). Regarding th histology, with detorsion there was evidence of severe distortion of tubules, with almost all the tubules showing maturation arrest and a few tubules completely devoid of any germinal cells. Treatment with Lomodex-MgSO(4) as well as ATP-MgCl(2) showed preservation of tubular morphology. Our study failed to document the presence of agglutinating antibodies (antisperm antibodies) in any of the groups. Unilateral testicular torsion has bilateral effects and is a form of ischemia-reperfusion injury. Treatment of torsion by detorsion alone does not prevent testicular damage. The results of the present study show that administration of Lomodex + MgSO(4) prior to detorsion results in prolonged testicular salvage with a potential of subsequent improvement in semen quality and fertility and reduction in long-term morbidity. The presence of agglutinating antibodies could not be detected in the present study.

C-kit receptor and its possible function in human spermatozoa.

The presence and role of the c-kit proto-oncogene protein was investigated in the mature sperm of the human. A polyclonal antibody against the c-kit peptide was used to perform immunohistochemical (IHC) staining, electron microscopy (EM) studies, and Western blot analysis. The acrosomal region of fresh sperm specifically stained with the antibody. No acrosomal staining or staining limited to the equatorial region was noted in the acrosome-reacted (AR) sperm. EM studies demonstrated immunogold label on the plasma membrane (PM) of the acrosome, and confirmed the lack of binding following the acrosome reaction. A 150 kDa band was detected by Western blot analysis. This protein was released from the sperm surface during sperm capacitation and the acrosome reaction. Antibody against the c-kit receptor significantly inhibited the acrosome reaction and increased sperm agglutination, but did not significantly inhibit sperm motility. These results suggest that the c-kit receptor protein is present in mature human sperm and is released during capacitation and/or the acrosome reaction. The assessment of the c-kit receptor may also be a useful assay for sperm function in male infertility.

Involvement of cytoplasmic free calcium in boar sperm: head-to-head agglutination induced by a cell-permeable cyclic adenosine monophosphate analog.

When boar spermatozoa are incubated in a medium designed for in vitro fertilization, many of them become agglutinated at the acrosomes. We previously reported that bicarbonate and cyclic adenosine 3',5'-monophosphate (cAMP) promote agglutination. The aim of the present study is to examine the role of cytoplasmic free Ca(2+) in boar sperm agglutination induced by a cell-permeable cAMP analogue. Spermatozoa were collected from five mature boars, washed, and resuspended in a modified Krebs-Ringer-Hepes solution lacking calcium chloride. The sperm suspensions were incubated in a water bath (38.5 degrees C) for 60 minutes and were then used to determine the percentages of head-to-head agglutinated spermatozoa. Percentages of head-to-head agglutinated spermatozoa in the samples rose significantly after incubation, from 28% to 61%-62%, after adding to the medium a cell-permeable, phosphodiesterase-resistant cAMP analogue (cBiMPS, 10 microM) or an adenylyl cyclase stimulator (sodium bicarbonate, 5 mM) plus a cell-permeable phosphodiesterase inhibitor (IBMX, 25 microM). However, the promoting effects of these reagents were blocked when spermatozoa were pretreated with a cell-permeable Ca(2+) chelator (BAPTA-AM, 25 microM), whereas the same pretreatment with a cell-impermeable Ca(2+) chelator (BAPTA, 25 microM) had almost no influence on sperm agglutination. Adding thapsigargin, a potential Ca(2+)-ATPase inhibitor, to the medium raised the percentages of agglutinated spermatozoa in a concentration-dependent manner for concentrations up to 4 microM. When 4 microM thapsigargin and 10 microM cBiMPS were examined for their effects on free Ca(2+) levels in sperm heads by using a cell-permeable Ca(2+) indicator (fluo-3/AM), the samples incubated with both or either of these reagents contained many head-to-head agglutinated cells that exhibited intense fluorescence in the heads. In control samples incubated without these reagents by contrast, most spermatozoa were free (unagglutinated) cells and characterized by almost no or only slight fluorescence in the heads. Moreover, morphological observation of Giemsa-stained preparations revealed that most agglutinated spermatozoa possessed darkly stained acrosomes, which distinguished them from acrosomereacted spermatozoa. This indicated that the sperm agglutination was not a result of the acrosome reaction. Furthermore, with indirect immunofluorescence of Ca(2+)-ATPases, the mouse monoclonal antibody to this enzyme demonstrated high affinity to the acrosomes of permeabilized spermatozoa. Based on these results, we conclude that cytoplasmic free Ca(2+) is involved in sperm head-to-head agglutination induced by a cAMP analogue.

Relationship between bicarbonate and cyclic nucleotide in the promoting effects on head-to-head agglutination in boar spermatozoa.

AIM: To clarify the relationship between bicarbonate and cAMP in the promoting effects on the sperm agglutination. METHODS: Spermatozoa were collected from mature boars, washed and resuspended in a modified Krebs-Ringer HEPES lacking calcium chloride (mKRH). The sperm suspensions were incubated in a water bath (38.5 degrees C) for 60 min and then the percentage of head-to-head agglutinated spermatozoa was determined. RESULTS: Supplementation of the mKRH with sodium bicarbonate (5-10 mM) significantly raised the percentage of head-to-head agglutinated spermatozoa in the samples. The addition of selective inhibitors for calcium/calmodulin-dependent phosphodiesterases (type 1: 8-methoxymethyl-IBMX and vinpocetine, 25-50 micro M) or for cAMP-specific phosphodiesterases (type 4: Ro20-1724 and rolipram, 25-50 microM) enhanced the effect of bicarbonate on sperm agglutination as highly as did the addition of non-selective inhibitors for phosphodiesterases (IBMX and papaverine, 25-50 microM). A calmodulin antagonist (W-7, 2 microM), that potentially blocks the stimulator of the calcium/calmodulin-dependent phosphodiesterases, significantly enhanced the effect of bicarbonate on sperm agglutination. Moreover, a phosphodiesterase-resistant cAMP analogue (cBiMPS, 0.1 mM) markedly induced agglutination in more spermatozoa (76%) after the incubation without bicarbonate and phosphodiesterase inhibitors than did a less potent cAMP analogue (dibutyryl cAMP, 1 mM) (21%), while three kinds of cGMP analogues (0.1-1 mM) had no effect on sperm agglutination. In addition, a cAMP antagonist (Rp-cAMPS, 1 mM) significantly reduced the sperm agglutination resulting from the actions of bicarbonate and IBMX. On the other hand, the effect of bicarbonate was abolished by a change of incubation temperature from 38.5 degrees C to 25 degrees C. CONCLUSION: These findings demonstrate that the bicarbonate-induced agglutination of boar spermatozoa is controlled via the cAMP-mediated, temperature-dependent signaling cascade. This cascade is suppressed by the action of the phosphodiesterase (at least types 1 and 4).

Semen quality of workers occupationally exposed to hydrocarbons.

OBJECTIVE: To determine whether occupational exposure of men to hydrocarbons has adverse effects on the quality of their semen. DESIGN: Comparative study. SETTING: The rubber industry in Mexico City. PATIENT(S): Forty-eight workers who were exposed to hydrocarbons for 2-24 years and 42 unexposed workers. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Environmental hydrocarbon concentrations were determined by continuous air monitoring in all areas of the factory. Analyses of semen samples were performed in accordance with World Health Organization criteria. RESULT(S): Hydrocarbon concentrations were as follows: ethylbenzene, 220.7-234 mg/m3; benzene, 31.9-47.8 mg/m3; toluene, 189.7-212.5 mg/m3; and xylene, 47-56.4 mg/m3. The number of subjects with ejaculates that had normal characteristics was greater in the unexposed group (76%) than in the exposed group (17%). More abnormal characteristics were found in the semen of exposed workers than unexposed workers, including alterations in viscosity, liquefaction capacity, sperm count, sperm motility, and the proportion of sperm with normal morphology. Some abnormal characteristics correlated with the number of years of exposure to the hydrocarbons. CONCLUSION(S): Damage to the spermatogenic process resulting from hydrocarbon exposure was demonstrated by an increased rate of abnormalities in the semen of exposed workers compared with unexposed workers. This information may be useful for conducting future analyses of reproductive risks related to exposure to high concentrations of hydrocarbons.

Role of cyclic adenosine 3',5'-monophosphate and serum albumin in head-to-head agglutination of boar spermatozoa.

It has previously been shown that when boar spermatozoa are incubated in a modified Krebs-Ringer bicarbonate (mKRB), head-to-head agglutination occurs in many cells. The aim of the present study was to investigate the effects of cyclic adenosine 3',5'-monophosphate (cAMP) and serum albumin on sperm agglutination and to discuss a possible mechanism for sperm agglutination. Spermatozoa were collected from four mature boars, washed and incubated in mKRB. After a 1-h incubation, a sample of each sperm suspension was smeared gently on a separate glass slide, dried and stained in a phosphate-buffered solution of Giemsa to assess the percentage of head-to-head agglutinated cells in each suspension. In the samples incubated in mKRB, approximately 50% of the spermatozoa were agglutinated with one another at the acrosome. However, the percentages of head-to-head agglutinated spermatozoa were greatly reduced by a lack of calcium chloride in mKRB, but were recovered by the addition of dibutyryl cAMP (dbcAMP, a cAMP analogue) in a dose-dependent manner between 1 and 1000 microM. Addition of 3-isobutyl-1-methylxanthine (IBMX, 100 and 500 microM) instead of dbcAMP also significantly increased the percentages of head-to-head agglutinated spermatozoa. Moreover, the effects of adding dbcAMP were attenuated by treatment with Rp-adenosine 3',5'-cyclic monophosphorothioate triethylamine salt (0.25-1.0 mM, a cAMP antagonist) or H-89 (5 microM, a protein kinase-A inhibitor), but were enhanced by treatment with okadaic acid (500 nM) and calyculinA (500 nM) (inhibitors of protein serine/threonine phosphatase). In sperm samples incubated in mKRB containing 0.1% polyvinyl alcohol (mKRB-P) or mKRB-P lacking calcium chloride and supplemented with 1 mM dbcAMP, a lack of bovine serum albumin (BSA) resulted in a significant decrease in the percentages of head-to-head agglutinated spermatozoa. Addition of porcine serum albumin (PSA, 1-4 mg mL(-1)) or methyl-beta-cyclodextrin (MBC, 5-10 mg mL(-1)) instead of BSA was as effective as BSA (4 mg mL(-1)) in enhancing sperm agglutination. However, the effects of BSA (4 mg mL(-1)) or MBC (5 mg mL(-1)) were reduced by pre-mixing these reagents with cholesterol 3-sulfate (a cholesterol analogue, 5 microg mL(-1) for BSA and 375 microg mL(-1) for MBC). In addition, a protein 'anti-agglutinin' inhibiting sperm agglutination, was extracted from spermatozoa incubated with serum albumin or MBC and detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting techniques. The obtained Western blots revealed that sperm-bound anti-agglutinin was detected less in the samples incubated with either BSA (4 mg mL(-1)) or MBC (5-10 mg mL(-1)), compared with control samples. Moreover, pre-mixing MBC (5 mg mL(-1)) with cholesterol 3-sulfate (375 microg mL(-1)) reduced this reagent's effects on the loss of sperm-bound anti-agglutinin. Additionally, the assay of sperm agglutination and a chlortetracycline staining assay revealed that the percentages of head-to-head agglutinated spermatozoa were positively correlated with those of spermatozoa classified into B pattern (capacitated spermatozoa). These results are consistent with the following suggestions: (i) an adenylyl cyclase-cAMP-protein kinase system mediates a signalling pathway leading to head-to-head agglutination; and (ii) loss of anti-agglutinin from the spermatozoa may be modulated by changes in the plasma membrane induced by actions of serum albumin or MBC contained in a medium.

Electrophoretic characterization of boar epididymal antiagglutinin.

Boar epididymal antiagglutinin, previously shown to inhibit sperm head-to-head agglutination, was purified from cauda epididymal plasma by precipitation with ammonium sulfate, anion-exchange chromatography, and reverse-phase HPLC, and was characterized by electrophoretic and membrane blotting techniques. Blotting techniques, using the ECL Glycoprotein Detection System (Amersham Life Science, Buckinghamshire, UK) and wheat germ agglutinin (WGA)-peroxidase, established the presence of sialic acid residues on purified antiagglutinin. Removal of sialic acid residues from antiagglutinin greatly reduced its immunoreactivity with the specific antiserum. Further purification by two-dimensional PAGE established the presence of one major and two minor forms that cross-reacted with the antiserum, with only the major form reacting with WGA-peroxidase. Extracts of washed epididymal spermatozoa contained a polypeptide with the same electrophoretic mobility as the major form. Additionally, the antiserum detected cross-reacting material in seminal plasma and in extracts from ejaculated spermatozoa. When spermatozoa were incubated under conditions shown to promote capacitation, the cross-reacting material could not be detected in sperm extracts. These results are consistent with the following conclusions: 1) antiagglutinin contains sialic acid residues that may be related to its immunoreactivity and molecular heterogeneity, and 2) either sperm-bound antiagglutinin is released or its epitope recognized by the antiserum is altered after ejaculation and in vitro capacitation.

Establishment and characterization of a human hybridoma secreting monoclonal antibody with high titers of sperm immobilizing and agglutinating activities against human seminal plasma.

Peripheral blood lymphocytes isolated from an infertile woman possessing strong sperm immobilizing and agglutinating antibodies were stimulated by culturing with poke-weed mitogen (PWM) and spermatozoa from a healthy donor for 5 days. The stimulated lymphocytes were fused with mouse myeloma NS-1 by PEG-1000. Fused growing hybrid cells were observed in 58 of 96 wells, and 22 of these showed the production of human immunoglobulin. Among the 22, one hybridoma clone (H6-3C4) was found to produce human IgM (lambda) with strong sperm immobilizing and agglutinating activities. The supernatant from the culture medium contained approximately 1.5 microgram IgM/ml and the antibody titers were 5000 SI50 units on sperm immobilization and 1:1600 dilutions on sperm agglutination. The hybridoma H6-3C4 has continuously produced high titers of antibody exhibiting sperm immobilizing and agglutinating activities over 8 months and contains chromosomes of acrocentric type from mouse and metacentric type from human. The monoclonal antibody (Mab) H6-3C4 reacted specifically to human seminal plasma, ejaculated spermatozoa and male accessory gland but not to testis, any other somatic tissues, or secreted fluids tested. Immunofluorescence staining indicated that the antigen corresponding to Mab H6-3C4 was present over the surface of ejaculated spermatozoa. The binding of Mab H6-3C4 to human spermatozoa was blocked by the serum of the patient from whom the lymphocytes were obtained for cell fusion.

A comparison of eight techniques for the evaluation of the auto-immune response to spermatozoa after vasectomy.

The tray agglutination tests (TAT), gelatin agglutination test (GAT), side agglutination test (SAT), tube-slide agglutination test (TSAT), sperm immobilization test (SIT), ATP-release cytotoxicity test (ARCT), indirect immunofluorescence technique (IFT) on methanol-fixed, intact spermatozoa, and a lymphocyte transformation test (LTT) were compared using a maximum of 329 blood samples taken from 47 men before and after vasectomy. The TAT, GAT, TSAT, SIT and ARCT discriminated between the pre- and post-vasectomy samples, and the sensitivity for sperm antibodies decreased in that order. The activity in the IFT and the LTT did not change significantly after vasectomy. In the TAT the mode of agglutination varied with serum dilution; the results for the 1:4 dilution showed the best agreement with the SAT results. Almost all TAT activity was detected by a combination of GAT and TSAT. Sperm agglutinins were present in all serum samples positive in the two complement-dependent tests, SIT and ARCT. If improved in sensitivity, the ARCT, which lacks the subjective elements of microscopy, might be suitable for the screening of male sera in clinical work. For the present, we recommend the TAT.

Dexamethasone therapy of infertile men with sperm autoantibodies: immunological and sperm follow-up.

A group of 14 infertile men with significant titres of anti-sperm antibodies in their sera and seminal plasma were treated with dexamethasone acetate at 2 of 3 mg/day for s13 or 9 weeks respectively. Response to the treatment was evaluated by the evolution of seminal and circulating anti-sperm antibodies and semen characteristics. An overall decline in the anti-sperm antibodies was observed. Serum spermotoxicity and seminal agglutinins decreased below detectable levels in 67 and 58% of the men respectively. The decrease in both kinds of antibodies was closely correlated. Serum sperm agglutination titres fell slightly (2 to 3 log2 dilutions) in most cases. The disappearance of antibodies from the semen was accompanied by decreased autoagglutination and increased percentages of progressively motile spermatozoa. In two oligozoospermic men a striking increase in the sperm count was observed. Pregnancies occurred in three couples at times when serum spermotoxic and seminal agglutinating antibodies were undetectable and semen characteristics were normal.

Failure of iron to promote attachment of gonococci to human spermatozoa under physiological conditions.

The effect of iron on the attachment of gonococci to human spermatozoa was investigated using the three iron salts, ferric chloride, ferric nitrate, and ammonium ferric citrate (AFC). Ferric chloride and ferric nitrate were found to be unsuitable for such studies because they were insoluble at physiological pH values, produced a marked decrease in the pH of unbuffered medium (Ringer's solution), and agglutinated spermatozoa. AFC, in contrast, was soluble at physiological pH, did not affect the pH value of Ringer's solution, and did not agglunate spermatozoa. When gonococci and spermatozoa were mixed together in media with and without AFC, the proportion of spermatozoa with adherent gonococci was approximately the same in each case. Thus, in contrast to previous report, we have found that the addition of iron does not increase the attachment of gonococci to human spermatozoa.