Cathine, an amphetamine-related compound, acts on mammalian spermatozoa via beta1- and alpha2A-adrenergic receptors in a capacitation state-dependent manner.

BACKGROUND: Mammalian spermatozoa have been shown to have beta(1,2,3)- and alpha(2A)-adrenergic receptors, the former functioning only in uncapacitated spermatozoa and the latter only in capacitated cells. Cathine, an amphetamine-related metabolite of a compound found in Catha edulis leaves, accelerates capacitation and inhibits spontaneous acrosome loss by regulating cAMP production. This study tested the hypothesis that adrenergic receptors are involved in these responses. METHODS: Uncapacitated and capacitated mouse sperm suspensions were incubated with cathine +/- specific antagonists for alpha(2)- and beta-adrenergic receptors for 35 min, then assessed using chlortetracycline fluorescence. Reversibility of receptor accessibility was assessed by depleting suspensions of endogenous decapacitation factor (DF) and then adding crude DF with/without cathine and antagonists. Effects on tyrosine phosphorylation and calcium requirements for both ligand binding and biological responses were also evaluated. RESULTS: Cathine's acceleration of capacitation was blocked by a beta(1)-antagonist, whereas an alpha(2)-antagonist blocked inhibition of acrosome reactions. Cathine accelerated capacitation in decapacitated cells, a response inhibited by a beta(1)-antagonist; cathine also stimulated tyrosine phosphorylation. Although calcium was not required for binding, it was needed for responses. CONCLUSIONS: Cathine acts at beta(1)-adrenergic receptors in uncapacitated spermatozoa and at alpha(2A)-receptors in capacitated cells; biological activity requires calcium but binding does not. Adrenergic receptor-binding sites can be made reversibly accessible/inaccessible by changing the capacitation state of spermatozoa. These results suggest that amphetamine-related compounds might enhance chances of fertilization in vivo.

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.

17beta-Estradiol and environmental estrogens significantly affect mammalian sperm function.

BACKGROUND: Compounds with estrogenic activity can affect reproductive function in mammals. This study investigated possible effects of 17beta-estradiol (E(2)) and three weakly estrogenic environmental estrogens on mammalian sperm capacitation and fertilizing ability in vitro. METHODS: Uncapacitated and capacitated mouse sperm suspensions were incubated for 30 min in the presence of E(2), genistein (Gen), 8-prenylnaringenin (8-PN) and nonylphenol (NP), and then assessed using chlortetracycline (CTC) fluorescence analysis. In addition, treated uncapacitated sperm suspensions were tested for changes in fertilizing ability. RESULTS: In uncapacitated cells, E(2) at >or=1 micromol/l and Gen, 8-PN and NP at >or=0.001 micromol/l, significantly stimulated capacitation and acrosome reactions. Hydroxytamoxifen (an estrogen antagonist) did not inhibit responses to any of these compounds. In capacitated cells, E(2) had no effect, but the other three compounds significantly stimulated acrosome reactions. Added to uncapacitated suspensions, 10 micromol/l E(2), 0.1 micromol/l Gen and 0.1 micromol/l 8-PN all significantly stimulated sperm fertilizing ability ( approximately 76% oocytes fertilized) compared with untreated control sperm ( approximately 36%). CONCLUSIONS: This study provides the first evidence that E(2) and environmental estrogens can significantly stimulate mammalian sperm capacitation, acrosome reactions and fertilizing ability, with the environmental estrogens being much more potent than E(2). The inability of hydroxytamoxifen to block these responses suggests that classical estrogen receptors may not be involved. Whether these responses have effects on fertility in vivo remains to be determined, along with the mechanisms of action involved.

Fertilization promoting peptide--a possible regulator of sperm function in vivo.

Fertilization promoting peptide (FPP), a tripeptide related to thyrotrophin releasing hormone (TRH), is found in seminal plasma. Recent evidence obtained in vitro suggests that FPP may play an important role in regulating sperm fertility in vivo. Specifically, FPP initially stimulates nonfertilizing (uncapacitated) spermatozoa to "switch on" and become fertile more quickly, but then arrests capacitation so that spermatozoa do not undergo spontaneous acrosome loss and therefore do not lose fertilizing potential. These responses are mimicked, and indeed augmented, by adenosine, known to regulate the adenylyl cyclase (AC)/cAMP signal transduction pathway. Both FPP and adenosine have been shown to stimulate cAMP production in uncapacitated cells but inhibit it in capacitated cells, with FPP receptors somehow interacting with adenosine receptors and G proteins to achieve regulation of AC. These events affect the tyrosine phosphorylation state of various proteins, some being important in the initial "switching on," others possibly being involved in the acrosome reaction itself. Calcitonin and angiotensin II, also found in seminal plasma, have similar effects in vitro on uncapacitated spermatozoa and can augment responses to FPP, suggesting that all four molecules may be involved in regulating availability of cAMP. It is plausible that these molecules have similar effects in vivo, affecting fertility by stimulating and then maintaining fertilizing potential. Either reductions in the availability of FPP, adenosine, calcitonin, and angiotensin II or defects in their receptors could contribute to male infertility. These exciting results may provide new approaches for diagnostic tests and treatments of certain categories of male infertility.

Fertilization promoting peptide and adenosine, acting as first messengers, regulate cAMP production and consequent protein tyrosine phosphorylation in a capacitation-dependent manner.

Fertilization promoting peptide (FPP) and adenosine have been shown to act as first messengers, regulating availability of the second messenger cAMP by initially stimulating cAMP production in uncapacitated spermatozoa and then inhibiting it in capacitated cells. This study investigated possible capacitation-related changes in protein tyrosine phosphorylation in response to FPP and adenosine. Time-dependent changes in phosphorylation of proteins of approximately 30-140 kDa were observed in both uncapacitated and capacitated suspensions, the general level of phosphorylation being markedly greater in capacitated cells. In the presence of FPP, phosphorylation was stimulated in uncapacitated but inhibited in capacitated spermatozoa, compared with untreated control samples. Adenosine, cholera toxin, and CGS-21680, a stimulatory A(2a) adenosine receptor agonist, also stimulated phosphorylation in uncapacitated spermatozoa, while Gln-FPP, a competitive inhibitor of FPP, blocked responses to FPP. In capacitated cells, FPP's inhibition of phosphorylation was abolished when cells were treated with FPP in the presence of pertussis toxin. Consistent with the capacitation-dependent effects of FPP and adenosine on cAMP production, these results support the hypothesis that FPP and adenosine modulate sperm function by regulating the AC/cAMP signaling pathway and, consequently, protein tyrosine phosphorylation. Of particular significance is the identification of several phosphoproteins showing FPP-induced alterations in phosphorylation. In uncapacitated spermatozoa, proteins of approximately 116, 95, 82, 75, 66, 56, and 42 kDa showed increased phosphorylation, while in capacitated cells, phosphoproteins of approximately 116, 95, 82, 75, 70, 66, 56, and 50 kDa showed decreased phosphorylation. This suggests that these particular proteins may be involved in stimulation and arrest of capacitation, respectively.

FPP modulates mammalian sperm function via TCP-11 and the adenylyl cyclase/cAMP pathway.

Fertilization promoting peptide (FPP; pGlu-Glu-ProNH2), which is found in seminal plasma, promotes capacitation but inhibits spontaneous acrosome loss in mammalian spermatozoa in vitro. Adenosine, known to modulate the adenylyl cyclase (AC)/cAMP pathway, elicits these same responses whereas FPP + adenosine produces an enhanced response, leading to the hypothesis that FPP and adenosine modulate the same signal transduction pathway but act via different receptors. TCP-11, the product of a t-complex gene, is the putative receptor for FPP: Fab fragments of anti-TCP-11 antibodies have the same effect as FPP on mouse spermatozoa and Gln-FPP, a competitive inhibitor of FPP, also competitively inhibits responses to the Fab fragments. In the present study, specific binding of 3H-FPP to sperm membranes was significantly inhibited by 200 nM Gln-FPP and anti-TCP-11 Fab fragments (1/25 dilution), thus confirming that FPP, Gln-FPP, and Fab fragments compete for the same binding site. In addition, spermatozoa treated with A23187 to induce the acrosome reaction bound significantly less 3H-FPP than untreated cells, suggesting that a large proportion of the FPP binding sites are associated with the acrosomal cap region; TCP-11 is located in this region. In other experiments, 100 nM FPP significantly stimulated cAMP production in mouse sperm membranes, permeabilized cells and intact cells. Furthermore, Gln-FPP inhibited production of cAMP in response to FPP but not to adenosine (10 microM) or its analogue NECA (100 nM), supporting the involvement of two different receptors. Finally, anti-TCP-11 Fab fragments (1/25 dilution) significantly stimulated cAMP production, whereas low Fab (1/200; nonstimulatory when used alone) plus adenosine (10 microM) significantly enhanced the stimulation of capacitation by adenosine. These results support the hypotheses that TCP-11 is the receptor for FPP and that FPP<-->TCP-11 interactions modulate AC/cAMP.

Evidence for Ca(2+)-dependent ATPase activity, stimulated by decapacitation factor and calmodulin, in mouse sperm.

Membrane preparations from mouse sperm heads and tails were used in a gamma 32P-ATP hydrolysis assay to investigate Ca(2+)-dependent ATPase activity. In membranes from sperm heads, but not tails, a Ca2(+)-dependent ATPase that was further stimulated by calmodulin was detected. The addition of partially purified mouse sperm decapacitation factor (DF) to head membrane preparations significantly stimulated Ca(2+)-ATPase activity, this effect being further increased in the presence of DF plus calmodulin; in contrast, no response was observed when the same treatment was applied to tail membranes. Sperm preincubated in the presence of trifluoperazine (TFP), a calmodulin antagonist, were significantly more fertile than cells from the same males incubated in the absence of TFP, indicating that inhibition of calmodulin accelerates capacitation. When sperm cells were preincubated briefly, then gently centrifuged to remove DF and resuspended in medium containing 45Ca2+ +/- DF, their ability to accumulate 45Ca2+ was significantly lower in the early stages after resuspension in the presence of DF than in its absence. These data correlated with chlortetracycline analysis of the sperm functional state. When cells were centrifuged and resuspended in medium only, there was a noticeable shift from the F pattern (characteristic of uncapacitated cells) to the B pattern (characteristic of capacitated cells), but the reintroduction of DF caused a significant reversion to the F pattern. Finally, using a monoclonal antibody to somatic cell Ca2(+)-ATPase, we have obtained evidence that the enzyme is particularly localized to the postacrosomal region of the mouse sperm head; specific binding was observed only in permeabilized cells, indicating that the epitope involved in the binding has an intracellular location. Based on these various pieces of evidence, we propose that when present on mouse sperm, DF stimulates calmodulin-sensitive Ca(2+)-ATPase activity and thus ensures maintenance of a low intracellular Ca2+ concentration. As capacitation proceeds, DF is lost and Ca2(+)-ATPase activity declines, allowing intracellular Ca2+ to rise and promoting capacitation-related changes. The fact that inhibitors of Ca(2+)-ATPase and calmodulin appear to accelerate capacitation in several mammalian species, as determined by chlortetracycline analysis, suggests that Ca(2+)-ATPase activity may play an important role in modulating capacitation in many or even all mammals.

A biphasic pattern of 45Ca2+ uptake by mouse spermatozoa in vitro correlates with changing functional potential.

Mouse sperm capacitation in vitro, leading to hyperactivated motility, acrosomal exocytosis and rapid fertilization, takes approximately 120 min in a medium containing sufficient Ca2+. During that period, spermatozoa incubated in 45Ca2+ exhibited a biphasic pattern of Ca2+ uptake, with the first and lower peak occurring from 10 to 50 min and the second and higher peak from 60 to 90 min. When the exogenously supplied glucose was reduced from 5.56 mmol l-1 to 5.56 mumol l-1, the latter supporting capacitation but not fertilization, only the first peak of 45Ca2+ uptake was observed. Increasing the glucose to a millimolar concentration produced a second peak of uptake. We therefore propose that the first phase of 45Ca2+ uptake is associated with capacitation and the second phase with acrosomal exocytosis, which are both necessary prerequisites for fertilization. In micromolar glucose the rate of 45Ca2+ uptake during the first 30 min was 47% higher than in millimolar glucose, suggesting that the former conditions might promote a precocious rise in the intracellular Ca2+ concentration ([Ca2+]i) and hence accelerate capacitation. This hypothesis was confirmed by demonstrating both significantly accelerated transition from the uncapacitated F pattern of chlortetracycline (CTC) fluorescence to the capacitated B and AR patterns and significantly higher fertility in vitro in suspensions preincubated for 30 min in micromolar glucose, compared with those maintained continuously in millimolar glucose. These results suggest that an ATP-dependent mechanism, for example a Ca(2+)-ATPase, may be involved in maintaining a low [Ca2+]i. In micromolar glucose, available ATP would be limited and hence the ATPase activity would decline, allowing [Ca2+]i to rise.(ABSTRACT TRUNCATED AT 250 WORDS)