Acrosomal Loss Research Articles

SAMMA Induces Premature Human Acrosomal Loss by Ca2+Signaling Dysregulation

SAMMA is licensed for development as a contraceptive microbicide. Understanding mechanisms of its biological activity is prerequisite to designing more active second generation products. This study examined Ca(2+) involvement in SAMMA-induced premature acrosomal loss (SAL) in noncapacitated human spermatozoa. SAMMA causes acrosomal loss (AL) in a dose-dependent manner (ED(50) = 0.25 microg/mL). SAL requires extracellular Ca(2+) (ED(50) = 85 microM). SAL is inhibited by verapamil (nonspecific voltage-dependent Ca(2+) channel blocker; IC(50) = 0.4 microM), diphenylhydantoin and NiCl(2) (T-type [Ca(v)3.x] channel blockers; IC(50) 210 microM and 75 microM, respectively). Verapamil blockade of L-type (Ca(v)1.x) channels is use-dependent; activated channels are more sensitive to inhibition. However, verapamil inhibition of SAL does not increase after repeated SAMMA stimulation. SAL is unaffected by 10 microM nifedipine (selective L-type channel blocker). This contrasts to 40% inhibition (P < .001) of AL induced by 1 microM thapsigargin (Ca(2+)-ATPase inhibitor; releases intracellular Ca(2+) stores, promotes capacitative Ca(2+) entry). SAL is unaffected by 1 microM BAPTA-AM (intracellular Ca(2+) chelator), and 50 microM 2-APB (blocks InsP3 receptors and store-operated channels). This contrasts with thapsigargin-induced AL, inhibited nearly 65% by BAPTA-AM (P < .005) and 91% by 2-APB (P, .001). The results suggest that SAL is mediated by Ca(2+) entry through channels pharmacologically similar to the T-type (Ca(v)3.2) class. This process appears distinct from that caused by physiological stimuli such as progesterone or zona pellucida-derived proteins. SAMMA's contraceptive activity may be caused by induction of premature AL through dysregulation of Ca(2+) signaling.

Influence of tris(4-chlorophenyl)methanol, non-ortho PCB 77 and gamma-hexachlorocyclohexane on human sperm function in vitro

In recent years, there has been growing concern that environmental pollutants in general, and organochlorines in particular, adversely affect male fertility. Therefore, we investigated the effects of tris(4-chlorophenyl)methanol (TCPM), non-ortho PCB 77 and gamma-hexachlorocyclohexane (gamma-HCH, lindane) on human sperm functions in vitro. Human spermatozoa from healthy donors were washed in human tubular fluid medium containing 1% human serum albumin, filtered through glass wool and exposed to different concentrations of TCPM, PCB 77 or gamma-HCH. After incubation for 5 h at 37 degrees C and 5% CO(2), sperm vitality and the percentage of living acrosome-reacted spermatozoa were examined using triple stain technique. Total sperm motility was evaluated by computer-assisted sperm analysis (Stroemberg-Mika) after 5 h. For TCPM, total motility was additionally measured after 18 and 40 h. Different concentrations of PCB 77 and gamma-HCH did not alter the percentage of spontaneous living acrosome-reacted spermatozoa, vitality and total motility. TCPM dose-dependently altered sperm motility, vitality and acrosome reaction. The percentage of living acrosome-reacted spermatozoa was increased at overtly toxic concentrations. Therefore, it is suggested that unspecific acrosomal loss has been induced by degenerative processes. In conclusion, even high concentrations of PCB 77 and gamma-HCH did not affect human sperm functions in vitro. Only very high cytotoxic TCPM concentrations modulated spontaneous acrosome reaction and total motility. Therefore, in vivo effects on human sperm function seem to be unlikely. However, individual susceptibility has to be considered and little is known about additive and possible synergistic effects as other environmental pollutants with similar potencies have been found in the human male and female reproductive tract.

Effects of estrogenic xenobiotics on human and mouse spermatozoa

To investigate human sperm responsiveness to the estrogenic xenobiotic genistein and seek further information regarding the mechanism of action of estrogenic xenobiotics using mouse spermatozoa. Uncapacitated human spermatozoa were incubated with genistein and assessed using chlortetracycline (CTC) fluorescence. CTC was also used to evaluate mouse sperm responses to daidzein and combinations of genistein, 8-prenylnaringenin and nonylphenol. Several steroids were tested to determine structure-function relationships, and possible involvement of cAMP and G proteins in responses was also investigated. Genistein significantly accelerated capacitation and acrosome loss in human spermatozoa, with 1, 10 and 100 nmol/l being equally effective. In mouse spermatozoa, daidzein produced significant responses, and combinations of xenobiotics at low concentrations were more effective than used singly. The compounds appear to act at the cell surface, and responses to three different steroids were nonidentical. A protein kinase-A inhibitor blocked responses to xenobiotics, while genistein and nonylphenol significantly stimulated cAMP production. Pertussis toxin and dideoxyadenosine blocked responses, suggesting involvement of inhibitory G proteins and membrane-associated adenylyl cyclases. Human and mouse sperm responses to genistein are very similar, but human gametes appear to be even more sensitive. The mechanism of action may involve unregulated stimulation of cAMP production, leading to significant acrosome loss, undesirable because already acrosome-reacted cells are nonfertilizing. Xenobiotics were even more effective in combination. Since simultaneous exposure to low concentrations of multiple xenobiotics is likely to occur in animals and humans, further investigation is needed to determine whether this could impair fertility.

Regulation of mammalian sperm capacitation by endogenous molecules

Capacitation in vitro in mammalian spermatozoa can be regulated by a number of first messengers, including fertilization promoting peptide, adenosine, calcitonin and angiotensin II, all of which are found in seminal plasma. The responses appear to involve several separate signal transduction pathways that have a common end point. These seminal-plasma derived first messengers can bind to specific receptors and directly or indirectly modulate the activity of membrane-associated adenylyl cyclase isoforms and production of the second messenger cAMP. Responses to all of these except angiotensin II involve initial acceleration of cAMP production and capacitation followed by inhibition of both cAMP production and spontaneous acrosome loss, resulting in maintenance of fertilizing potential. Appropriate G proteins and various phosphodiesterase isoforms also appear to be involved. The transition from stimulatory to inhibitory responses involves loss of decapacitation factors (DF) from receptors (DF-R) on the external surface; a DF-R present on both mouse and human spermatozoa has recently been identified as phosphatidylethanolamine-binding protein 1. The presence/absence of DF appears to cause changes in the plasma membrane that then alter the functionality of various membrane-associated proteins, including receptors. Since spermatozoa contact these first messengers at ejaculation, it is plausible that their actions observed in vitro also occur in vivo, allowing these molecules to play a pivotal role in enhancing the chances of successful fertilization.

303 FERTILIZATION-RELATED PARAMETERS OF FROZEN - THAWED SPERMATOZOA FROM SUBFERTILE JAPANESE BLACK CATTLE

The subfertility derived from male factors is a problem of concern in domestic animals, because it could cause a disintegration of the breeding system and large economic losses, particularly when the subfertility affects genetically superior male animals. Therefore, it is urgent that causal factors of male subfertility be determined. Recently, an increasing number of subfertile bulls have been found among Japanese Black cattle, which is a representative breed of Japanese beef cattle. The purpose of the present study was to elucidate causal factors of male subfertility in Japanese Black cattle. Frozen–thawed spermatozoa from 8 subfertile (S1-S8) and 7 fertile (F1–F7, control) bulls were used for the assessment of fertilization-related parameters. The data obtained from each subfertile bull in the following experiments were individually compared with the mean values of the fertile bull group. In Experiment 1, sperm motility was observed in samples that were frozen-thawed and subsequently washed in PBS. Many spermatozoa (higher than 65%) exhibited flagellar movement in all samples from fertile and subfertile bulls. However, the percentages of progressively motile spermatozoa from 2 subfertile bulls were significantly lower (S2: 6%; S7: 7%; P &amp;lt; 0.05, ANOVA and Tukey&amp;apos;s multiple range tests) than those from fertile bulls (average: 37&amp;percnt;). Moreover, rapidly progressive movement was not observed in the spermatozoa from 4 subfertile bulls (S1, S2, S6, and S7). These data suggest abnormality in the motility system of sperm flagella in these 4 subfertile bulls. In Experiment 2, the capacitation&amp;ndash;acrosome reaction state of frozen&amp;ndash;thawed spermatozoa was examined by the CTC-staining assay. More than 50&amp;percnt; of the frozen&amp;ndash;thawed spermatozoa from 4 subfertile bulls (S5&amp;ndash;S8) were prematurely progressing in the capacitation state immediately after washing and resuspension in the medium lacking CaCl2. Moreover, the addition of CaCl2 to the medium induced acrosomal loss in these sperm samples (percentages of spermatozoa without the acrosome: 36&amp;ndash;49&amp;percnt;). These findings indicate the occurrence of premature capacitation and a spontaneous acrosome reaction in spermatozoa from these 4 subfertile bulls. In Experiment 3, the in vitro fertilizing ability of frozen&amp;ndash;thawed spermatozoa was evaluated by the IVF test. The percentages of fertilized eggs with both male and female pronuclei or developmental rates of fertilized eggs to the 2-cell or 4-cell stage were significantly lower in the spermatozoa from S6 to S8 bulls than in those from fertile bulls (P &amp;lt; 0.05, chi-squared tests). This may suggest that spermatozoa from these 3 subfertile bulls hardly accomplish the normal fertilization process. In summary, low progressive motility and low in vitro fertilizing ability because of premature capacitation were found in the spermatozoa from subfertile bulls. It is therefore possible that these are causal factors for the subfertility of male Japanese Black cattle.

Effect of Cryoprotective Additives-Reduced Glutathione, Acetyl-L-Carnitine on Sperm Membrane Lipid Peroxidation, DNA Integrity and Recovery of Motile Human Sperm

Cryopreservation induces lethal and sub-lethal sperm damage that lead to loss of viability, acrosome loss, DNA fragmentation and poor fertilization. Reactive oxygen species, lipid peroxidation and loss of antioxidant activity such as reduced glutathione (GSH). are considered as mediators of sub-lethal cryodamage to human sperm during cryopreservation. Improvements in semen parameters have been reported by in vivo use of carnitine and acetyl-L-carnitine (ALC), possibly due to its key role in energy metabolism and antioxidant activity.

239 CAPACITATION OF BUFFALO SPERMATOZOA IN VITRO

The efficiency of in vitro embryo production (IVEP) in buffalo is hampered by the poor cleavage rate. The quality of the frozen semen may affect fertilization efficiency, due to damages of the male gamete that occur following cryopreservation. However, it is not possible to rule out that the process of capacitation, required by spermatozoa to acquire the fertilizing ability, is impaired in the IVF system. Although several agents have been proven to induce sperm capacitation in vitro, heparin is still the most efficient method in most of the domestic species. The aim of the study was to evaluate the efficiency of buffalo estrus serum (BES) and follicular fluid (FF) to induce buffalo sperm capacitation in vitro, indirectly assessed by estimating the capability of spermatozoa to acrosome react. Frozen semen from a bull previously tested for IVF, thawed at 37°C for 40 s in water, was treated by swim-up in order to select only the motile population. Spermatozoa (n = 1546) were assessed immediately after swim-up separation, to evaluate the incidence of acrosomal loss in nontreated cells (time 0). The remaining spermatozoa were incubated in the presence of 0.01 mM heparin (control; n = 3531), 20% BES (n = 2442) and 20% FF (n = 1419), the latter recovered from a pool of dominant follicles, for 1, 2, and 3 hours. Sperm was then exposed for 15 min to 60 μg mL−1 of lysophosphatidylcholine, an agent known to induce acrosome reaction only on capacitated spermatozoa. Trypan blue was used to differentiate live from dead spermatozoa, and the dried smears were then fixed in 37% formaldehyde and stained with Giemsa for acrosome evaluation by microscopic examination. The proportion of acrosome-reacted spermatozoa in each group was used to assess the efficiency of capacitation under different incubation conditions. Differences between groups were analyzed by χ2. No dead spermatozoa were found in all groups. Acrosomal loss was observed in only 15.3% of the sperm population at time 0; it may be accounted for either by damages preceding cell death or by freezing-induced capacitation. No differences were found between incubation times within each treatment group. Interestingly, sperm treatment with both BES and FF resulted in a significantly higher incidence of acrosome reaction compared with heparin (84.3, 94.5 vs. 50.1%, respectively; P &lt; 0.001), the capacitating agent currently used in the IVF system, and, in particular, FF showed the highest percentage of acrosome reaction at all incubation times, even when compared with BES (P &lt; 0.01). It is likely that factors derived by BES and FF, present in the oviduct environment around fertilization, play a critical role in processing the male gamete in vivo. These preliminary results show the possibility of significantly improving the efficiency of sperm capacitation in vitro in buffalo species with BES and FF and strongly suggest investigating the effects of these factors also on the fertilizing capability of buffalo spermatozoa. The authors thank to Dr. O. Paciello for his technical assistance.

Cathine and norephedrine, both phenylpropanolamines, accelerate capacitation and then inhibit spontaneous acrosome loss

Cathinone, released when Catha edulis leaves (khat) are chewed, has euphoric, stimulatory properties. It is metabolized to the phenylpropanolamines (PPAs) cathine and norephedrine. This study investigated whether PPAs affect mammalian sperm function, using primarily mouse, but also human, spermatozoa. Uncapacitated sperm suspensions were treated with cathine, norephedrine, adrenaline and noradrenaline, then assessed using chlortetracycline (CTC) fluorescence. Cathine and adrenaline were also evaluated using in vitro fertilization. Capacitated suspensions were treated with PPAs+/-progesterone and+/-pertussis toxin. Finally, cAMP production was evaluated in uncapacitated and capacitated suspensions. In uncapacitated mouse spermatozoa, cathine, norephedrine, adrenaline and noradrenaline all significantly accelerated capacitation; uncapacitated human spermatozoa responded similarly to cathine. Consistent with these results, cathine- and adrenaline-treated suspensions were significantly more fertile than controls. In capacitated spermatozoa, both PPAs inhibited spontaneous acrosome reactions (ARs) but progesterone could over-ride this inhibition. Pertussis toxin abolished cathine's inhibition of ARs, suggesting G protein involvement. Finally, cathine and adrenaline significantly stimulated cAMP production in uncapacitated suspensions, but significantly inhibited it in capacitated suspensions. This is the first demonstration that PPAs can directly affect mammalian sperm function, accelerating capacitation and inhibiting spontaneous ARs. These responses correlated with initial stimulation and subsequent inhibition of cAMP production. Adrenaline/noradrenaline elicited similar responses, suggesting the presence of adrenergic receptors. Therefore, regulation of adenylyl cyclase/cAMP in a G protein-mediated fashion by PPAs may possibly involve adrenergic receptors. These results suggest that PPAs, at appropriate doses, might provide a novel approach to enhance natural fertility.

A unique mechanism for cyclic adenosine 3',5'-monophosphate-induced increase of 32-kDa tyrosine-phosphorylated protein in boar spermatozoa.

A cAMP-induced increase of tyrosine-phosphorylated proteins is involved in the expression of fertilizing ability in mammalian spermatozoa. We (Harayama, 2003: J Androl 24:831-842) reported that incubation of boar spermatozoa with a cell-permeable cAMP analog (cBiMPS) increased a 32-kDa tyrosine-phosphorylated protein (TyrP32). The purpose of this study is to characterize the signaling cascades that regulate the cAMP-induced increase of TyrP32. We examined effects of tyrosine kinase inhibitor (lavendustin A), tyrosine phosphatase inhibitor (Na3VO4), cell-permeable calcium chelator (BAPTA-AM), and cholesterol acceptor (methyl-beta-cyclodextrin: MBC) on the increase of TyrP32 and the change and loss of acrosomes in boar spermatozoa. The spermatozoa were used for detection of tyrosine-phosphorylated proteins by Western blotting and indirect immunofluorescence and for examination of acrosomal integrity by Giemsa staining. At least eight tyrosine-phosphorylated proteins including TyrP32 exhibited the cAMP-dependent increase during incubation with cBiMPS. In many proteins of them, this increase was reduced by lavendustin A but was enhanced by Na3VO4. In contrast, the cAMP-induced increase of TyrP32 was abolished by Na3VO4 but was hardly affected by lavendustin A. Giemsa staining showed that the increase of spermatozoa with weakly Giemsa-stained acrosomes (severely damaged acrosomes) or without acrosomes was correlative to the cAMP-induced increase of TyrP32. Moreover, the lack of calcium chloride in the incubation medium or pretreatment of spermatozoa with BAPTA-AM blocked the change and loss of acrosomes and the increase of TyrP32, suggesting these events are dependent on the extracellular and intracellular calcium. On the other hand, incubation of spermatozoa with MBC in the absence of cBiMPS could mimic the change and loss of acrosomes and increase of TyrP32 without increase of other tyrosine-phosphorylated proteins. Based on these results, we conclude that the cAMP-induced increase of TyrP32 is regulated by a unique mechanism that may be linked to the calcium-dependent change and loss of acrosomes.

First messenger regulation of capacitation via G protein-coupled mechanisms: a tale of serendipity and discovery.

When placed in a suitable environment, mammalian spermatozoa begin to capacitate and continue until fully capacitated; in vitro, some will 'over-capacitate' and undergo spontaneous acrosome loss, undesirable since acrosome-reacted cells are non-fertilizing. Seminal plasma contains several molecules able to bind to specific receptors on spermatozoa, thereby activating/regulating important intracellular signalling pathways. Three such 'first messengers' are fertilization promoting peptide (FPP), adenosine and calcitonin, all of which stimulate capacitation and then inhibit spontaneous acrosome reactions by regulating adenylyl cyclase (AC)/cAMP. A recent study has reported the presence in spermatozoa of several membrane-associated AC isoforms, mainly smaller in size than the corresponding ACs in somatic cells, and evidence suggests that more than one of these isoforms may be involved in responses to these first messengers. To regulate AC, FPP receptors appear to interact initially with stimulatory A(2A) adenosine receptors, which function only in uncapacitated cells, and then with inhibitory A(1) receptors, which function only in capacitated cells. In contrast, there appears to be a single population of calcitonin receptors. Responses to cholera and pertussis toxins suggest involvement of G proteins and G(s) plus several G(i) subunits have been identified in both mouse and human spermatozoa. In particular, Galpha(s) and Galpha(i2) are found in the same regions as FPP, adenosine and calcitonin receptors, supporting biochemical evidence for G protein involvement in these responses. In vivo, these first messengers could have a significant effect, helping to maximize the number of capacitated, acrosome-intact (i.e. potentially fertilizing) spermatozoa by regulating what is clearly an important signalling pathway.

Seminal lead concentrations negatively affect outcomes of artificial insemination

Objective To determine the relationships among seminal lead levels, acrosome status, and artificial insemination cycle fecundity (AI f) in semen donors. Design Longitudinal analysis of seminal lead levels, sperm function testing, and fecundity. Setting University medical center andrology and research laboratories. Patient(s) Semen donors (n = 15) participating in a therapeutic donor insemination program. Intervention(s) None Main outcome measure(s) Seminal plasma lead levels, acrosome sensitivity to progesterone (P) and voltage-gated potassium channel inhibitors (e.g., charybdotoxin [CBTx]), and AI f. Result(s) Seminal plasma lead levels and AI f were strongly negatively correlated. Semen donors were divided into three groups by acrosome response to P: normal (CBTx sensitive [Rs] or CBTx resistant [Rr]: responders) and reduced ( nonresponders [NR]) (Rs > Rr >> NR). Seminal lead differed among the three groups (NR > Rr > Rs). Comparison of 330 artificial insemination cycles from four Rs, four Rr, and two NR demonstrated that cycle AI f also differed significantly between groups (Rs >Rr >>NR). Conclusion(s) Measurements of seminal plasma lead, P-stimulated acrosome loss, and sensitivity to CBTx may provide prognostic information on the fertility status of potential donors as well as male infertility patients. Such evaluations may assist in donor acceptance, or in the case of patients, in selection of the appropriate treatment regimen.

Calcitonin acts as a first messenger to regulate adenylyl cyclase/cAMP and mammalian sperm function.

Calcitonin stimulates capacitation in uncapacitated mouse spermatozoa and then inhibits spontaneous acrosome loss in capacitated cells, responses similar to those elicited by fertilization promoting peptide (FPP), a peptide known to regulate the adenylyl cyclase/cAMP pathway. This study investigated the hypothesis that calcitonin also modulates this pathway. Calcitonin significantly stimulated cAMP production in uncapacitated spermatozoa and then inhibited it in capacitated cells; the magnitude of both stimulatory and inhibitory changes was similar to that obtained with FPP but the inhibitory responses to FPP preceded those of calcitonin. This possibly reflects the involvement of two different adenosine receptors in response to FPP compared with one calcitonin receptor. Calcitonin receptors were located on the acrosomal cap and the flagellum, the midpiece having a greater abundance than the principal piece. Although both calcitonin and adenosine receptors are found in the head and flagellum, there was no evidence for cross-talk between them. Chlortetracycline investigations to determine the minimum extracellular Ca(2+) requirement for responses to calcitonin revealed that calcitonin significantly stimulated capacitation in Ca(2+)-deficient medium but FPP did not. Calcitonin also significantly stimulated cAMP production under these conditions, and similarly preincubated suspensions, when diluted into +Ca(2+) medium, were significantly more fertile in vitro than untreated controls. These results indicate that calcitonin, like FPP, acts as a first messenger to regulate the production of cAMP and mammalian sperm function, but the differences in Ca(2+) requirements suggest that calcitonin and FPP may regulate different isoforms of adenylyl cyclase.

Viable piglets generated from porcine oocytes matured in vitro and fertilized by intracytoplasmic sperm head injection.

Intracytoplasmic sperm injection (ICSI) of a nonmotile cell into the ooplasm for assisted fertilization is a highly specialized procedure for producing the next generation. The production of piglets by ICSI has succeeded when in vivo-matured oocytes have been used as recipients. Our objective was to generate viable piglets by using porcine oocytes matured in vitro and fertilized by ICSI after evaluating the efficacy of using donor spermatozoa in which the acrosome had been artificially removed by treatment with calcium ionophore A23187 (Ca-I). The rate of acrosomal loss in spermatozoa was increased significantly as the duration of treatment with 10 micro M Ca-I was prolonged for 30-120 min (Ca-I treated; 55.6-78.6%), whereas the rate was not different as the duration of incubation without Ca-I was prolonged for 30-120 min (control; 45.3-58.4%). On the sixth day of in vitro culture after injection of the sperm head and subsequent stimulation with an electrical pulse, the rates of blastocyst formation were not significantly different between the two groups: the rates for oocytes injected with Ca-I-treated sperm heads (incubated for 120 min) and for those injected with control sperm heads were 8.6% and 4.0%, respectively. The mean cell numbers of the blastocysts were not significantly different between the two groups (25.6 and 22.7, respectively). Within 2 h after the stimulation, the injected oocytes were transferred to estrous-synchronized recipients. The three recipients that received oocytes injected with Ca-I-treated sperm heads (77-150 oocytes per recipient) were not pregnant, whereas two of the four recipients given oocytes injected with control sperm heads (55-100 oocytes per recipient) were pregnant. One of these farrowed three (a male and two female) healthy piglets. The results demonstrate clearly that in vitro-matured oocytes injected with sperm heads are developmentally competent and can produce viable piglets. They also suggest that removal of the acrosome from the spermatozoon before injection does not affect the development of the blastocyst in vitro. This might not also improve the production of piglets in vivo.

EFFECTS OF FERTILIZATION PROMOTING PEPTIDE, ADENOSINE, AND PENTOXIFYLLINE ON THAWED HUMAN SPERM

Fertilization promoting peptide (FPP) and adenosine were demonstrated to be potential modulators of sperm capacitation in mammals. Both FPP and adenosine, by modulating the adenylate cyclase (AC)/cAMP signaling pathway, elicit similar biphasic responses in mammalian sperm (i.e., stimulating capacitation and inhibiting spontaneous acrosome loss). Pentoxifylline, an artificial sperm stimulant, is clinically used to enhance motility of sperm from infertile men. By inhibiting phosphodiesterase, pentoxifylline increases the intracellular cAMP level of sperm, and thus contributes to capacitation, hyperactivation, and acrosome reaction in animal studies. The effects of FPP, adenosine, and pentoxifylline on thawed human sperm are stressed. Chlortetracycline (CTC) fluorescence assessment revealed that none of the 3 reagents improved fertilization ability of post-thawed sperm. Motility studies with computer-aided sperm analyzer (CASA) showed significantly smaller STR (straight-line velocity) and LIN (linearity) in the FPP-treated group at 4 h of incubation p\leqq. 005, significantly larger VCL in the adenosine-treated group p\leqq. 05, and significantly larger VCL (curvilinear velocity) and smaller LIN in the pentoxifylline-treated group p\leqq. 05. Significant decreases in percentage motility were also noted in both FPP and adenosine-treated groups p\leqq. 05. It would appear that FPP potentiates fertilizing ability and prevents spontaneous acrosome loss, via regulating membrane-bound Na + -K + ATPase, and/or Ca 2+ ATPase, by keeping the sperm intracellular Ca 2+ concentration within the physiological range optimal for fertilization.

Increased seminal plasma lead levels adversely affect the fertility potential of sperm in IVF.

Lead remains in high levels in the environment and is known to reduce fertility in animal models, but a direct link between lead exposures and human infertility has not yet been established. In a prospective, double-blind study of the metal ion levels and sperm function, semen was obtained from partners of 140 consecutive women undergoing their first IVF cycle. Lead in seminal plasma was determined by atomic absorption spectroscopy. Motile sperm populations were assessed for surface receptors for mannose binding, and the ability to undergo premature ('spontaneous'), and free mannose-induced acrosome reactions. Fertile donor (n = 9) sperm were exposed to exogenous lead during capacitating incubations and then assessed for mannose receptor expression and acrosome loss. Lead levels were negatively correlated with IVF rates. Lead levels were negatively correlated to two of the three sperm function biomarkers (mannose receptors, mannose-induced acrosome reactions). Lead levels positively correlated with the spontaneous acrosome reaction. These findings were mimicked by in-vitro exposure of fertile donor sperm to lead. Multiple sperm parameters are affected as lead levels rise. Increased lead levels may contribute to the production of unexplained male infertility.

Effects of fertilization promoting peptide, adenosine, and pentoxifylline on thawed human sperm.

Fertilization promoting peptide (FPP) and adenosine were demonstrated to be potential modulators of sperm capacitation in mammals. Both FPP and adenosine, by modulating the adenylate cyclase (AC)/cAMP signaling pathway, elicit similar biphasic responses in mammalian sperm (i.e., stimulating capacitation and inhibiting spontaneous acrosome loss). Pentoxifylline, an artificial sperm stimulant, is clinically used to enhance motility of sperm from infertile men. By inhibiting phosphodiesterase, pentoxifylline increases the intracellular cAMP level of sperm, and thus contributes to capacitation, hyperactivation, and acrosome reaction in animal studies. The effects of FPP, adenosine, and pentoxifylline on thawed human sperm are stressed. Chlortetracycline (CTC) fluorescence assessment revealed that none of the 3 reagents improved fertilization ability of post-thawed sperm. Motility studies with computer-aided sperm analyzer (CASA) showed significantly smaller STR (straight-line velocity) and LIN (linearity) in the FPP-treated group at 4 h of incubation p<or=005, significantly larger VCL in the adenosine-treated group p<or=.05, and significantly larger VCL (curvilinear velocity) and smaller LIN in the pentoxifylline-treated group p<or=.05. Significant decreases in percentage motility were also noted in both FPP and adenosine-treated groups p<or=05. It would appear that FPP potentiates fertilizing ability and prevents spontaneous acrosome loss, via regulating membrane-bound Na(+)-K(+) ATPase, and/or Ca(2+) ATPase, by keeping the sperm intracellular Ca(2+) concentration within the physiological range optimal for fertilization. adenosinefertilizationFPPpentoxifyllinespermthawing

Relationship between human sperm morphology and acrosomal function.

In this study, we investigated the relationship between functionality of the acrosome and sperm morphology. Acrosome reaction (AR) was separately determined in live and dead sperm and in those with normal, small, and large sized acrosomes by means of the triple stain. Morphology was analyzed according to strict criteria after Papanicolaou stain. AR and morphology correlated regarding detection of large and small sized acrosomes, but not for normal sized acrosomes. Spontaneous AR was significantly influenced by acrosomal size. Sperm with large (11.4%) and normal (9.2%) acrosomes exhibited a significantly higher percentage of life spontaneously acrosome-reacted sperm than those with small acrosomes (4.5%). Sperm with small acrosomes were associated with a higher percentage of cell death. The results indicate that sperm with small acrosomes are more susceptible to cell death and nonphysiological acrosomal loss. Acrosome size reflects the physiological capability of sperm function and therefore male fertility potential.

Use of mandelic acid condensation polymer (SAMMA), a new antimicrobial contraceptive agent, for vaginal prophylaxis

Objective: To assess the contraceptive properties, antimicrobial activity, and safety of mandelic acid condensation polymer (SAMMA). Design: Experimental study of SAMMA’s in vitro and in vivo properties. Setting: Academic research laboratories. Patient(s): Healthy volunteers for semen donation in an academic research environment. Intervention(s): Inhibition of sperm function indicators, conception, sexually transmitted infection-causing pathogens (including HIV), and lactobacilli was evaluated. Safety indicators were studied. Main Outcome Measure(s): Quantitation of SAMMA’s effect on microbial infectivity or multiplication and on sperm function in vitro; evaluation of contraceptive efficacy in vivo; assessment of safety in vitro and in vivo. Result(s): Mandelic acid condensation polymer is not cytotoxic toward lactobacilli, microbial host cells, and spermatozoa. The compound inhibits hyaluronidase and acrosin, induces sperm acrosomal loss, and is contraceptive in the rabbit model. Mandelic acid condensation polymer prevents infectivity of HIV and herpesviruses 1 and 2 and, to a lesser extent, of Chlamydia trachomatis. It inhibits the multiplication of Neisseria gonorrhoeae. Mandelic acid condensation polymer is not mutagenic, has low acute oral toxicity, and is safe in the rabbit vaginal irritation assay. Conclusion(s): Mandelic acid condensation polymer inhibits sperm function, is contraceptive, has broad-spectrum antimicrobial activity, and is highly safe. Further development as a microbicide is warranted.

Preclinical evaluation of sodium cellulose sulfate (Ushercell) as a contraceptive antimicrobial agent.

The spread of sexually transmitted infections (STIs) and limited methods for control of pregnancies presents high risks to the reproductive health of women. Methods controlled by women and directed toward disease prevention and contraception are needed. We report on preclinical studies of the biological properties of sodium cellulose sulfate (Ushercell) currently being developed for use as a topical contraceptive antimicrobial agent. Ushercell was evaluated with tests designed to identify its contraceptive and antimicrobial properties. Ushercell inhibits hyaluronidase (reversible; IC50 = 1.7 mg/mL), impairs sperm penetration of cervical mucus (approximately 70% inhibition at 1 mg/mL), and acts as a stimulus for acrosomal loss (IC50 = 52 ng/mL). It prevents conception in rabbits when added to spermatozoa (approximately 95% inhibition at 1 mg/mL) or when vaginally applied (complete contraception by 45 mg) before insemination. However, up to 50 mg/mL, Ushercell does not irreversibly immobilize spermatozoa, suggesting that Ushercell is not cytotoxic. Ushercell has a broad spectrum of antimicrobial activity in vitro. Inhibited microbes include human immunodeficiency viruses (different laboratory strains and clinical isolates; IC50 values range from 3 to 78 microg/mL), herpes viruses, HSV-1 (IC50 = 59 ng/mL) and HSV-2 (lC50 = 24 ng/mL), Neisseria gonorrhoeae (IC50 = 2 microg/mL), and Chlamydia trachomatis (IC50 = 78 microg/mL). In contrast, Ushercell does not inhibit growth of beneficial vaginal bacteria, Lactobacillus gasseri, at 5 mg/mL. These results suggest that the antimicrobial effects of Ushercell are selective, and not likely mediated by nonspecific cytotoxic mechanisms. These data provide the basis for further clinical development of Ushercell as a vaginal agent to prevent unplanned pregnancy and STIs.

Efficacy and safety of a new vaginal contraceptive antimicrobial formulation containing high molecular weight poly(sodium 4-styrenesulfonate).

Host cell infection by sexually transmitted disease (STD)-causing microbes and fertilization by spermatozoa may have some mechanisms in common. If so, certain noncytotoxic agents could inhibit the functional activity of both organisms. High molecular mass poly(sodium 4-styrenesulfonate) (T-PSS) may be one of these compounds. T-PSS alone (1 mg/ml) or in a gel (2% or 5% T-PSS) completely prevented conception in the rabbit. Contraception was not due to sperm cytotoxicity or to an effect on sperm migration. However, T-PSS inhibited sperm hyaluronidase (IC(50) = 5.3 microg/ml) and acrosin (IC(50) = 0.3 microg/ml) and caused the loss of acrosomes from spermatozoa (85% maximal loss by 0.5 microg/ml). T-PSS (5% in gel) also reduced sperm penetration into bovine cervical mucus (73% inhibition by 1 mg gel/ml). T-PSS (5% in gel) inhibited human immunodeficiency virus (HIV; IC(50)= 16 microg gel/ml) and herpes simplex viruses (HSV-1 and HSV-2; IC(50) = 1.3 and 1.0 microg gel/ml, respectively). The drug showed high efficacy against a number of clinical isolates and laboratory strains. T-PSS (5% in gel) also inhibited Neisseria gonorrhea (IC(50) < 1.0 gel/ml) and Chlamydia trachomatis (IC(50) = 1.2 microg gel/ml) but had no effect on lactobacilli. These results imply that T-PSS is an effective functional inhibitor of both spermatozoa and certain STD-causing microbes. The noncytotoxic nature should make T-PSS safe for vaginal use. T-PSS was nonmutagenic in vitro and possessed an acute oral toxicity of >5 g/kg (rat). Gel with 10% T-PSS did not irritate the skin or penile mucosa (rabbit) and caused no dermal sensitization (guinea pig). Vaginal administration of the 5% T-PSS gel to the rabbit for 14 consecutive days caused no systemic toxicity and only mild (acceptable) vaginal irritation. T-PSS in gel form is worthy of clinical evaluation as a vaginal contraceptive HIV/STD preventative.