Progesterone and estradiol regulate sperm hyperactivation and in vitro fertilization success in mice.

Progesterone (P) and 17ß-estradiol (Eß) form the well-known hormone pair that regulates sperm capacitation. Here, we examined the regulatory effects of P and Eß on sperm hyperactivation in mice and evaluated the in vitro fertilization (IVF) success. Although P enhanced hyperactivation, Eß dose-dependently suppressed the P-enhanced hyperactivation. Moreover, P increased IVF success, whereas Eß suppressed the P-induced increase in IVF success in a dose-dependent manner. Thus, P and Eß competitively regulate hyperactivation and IVF success in mice. Since P and Eß concentrations generally change during the estrous cycle, sperm are speculated to capacitate in response to the oviductal environment and fertilize the oocyte.

Enhancement of rat spermatozoal hyperactivation by progesterone.

Progesterone (P) is a well-known enhancer of hyperactivation which is associated with the success of in vitro fertilization (IVF). In this study, we examined whether P-enhanced hyperactivation affected IVF success in rats. When rat spermatozoa were exposed to 10, 20, and 40 ng/ml P, 20 ng/ml P enhanced hyperactivation via the membrane progesterone receptor. In addition, the enhancement of hyperactivation by 20 ng/ml P was regulated by phospholipase C, transmembrane adenylate cyclase, and protein kinase A. However, 20 ng/ml P did not affect IVF success. These results suggest that 20 ng/ml P enhances rat spermatozoal hyperactivation through non-genomic pathways. Because the concentration of P changes during the estrous cycle, it seems that rat spermatozoa are hyperactivated in response to the oviductal environment. However, the effect of 20 ng/ml P does not seem to fully capacitate spermatozoa.

Progesterone increases the success of in vitro fertilization via enhanced sperm hyperactivation in mice.

Progesterone (P) enhances spermatozoal hyperactivation, a capacitation event. Hyperactivation is associated with successful in vitro fertilization (IVF). In this study, we examined the effects of P on hyperactivation and IVF in mice. P enhanced spermatozoal hyperactivation and increased IVF success rate in a dose-dependent manner. Moreover, P affected spermatozoal hyperactivation and IVF through the membrane progesterone receptor of the spermatozoal head. These results show that P regulates spermatozoal capacitation and fertilization in mice. The concentration of P changes during the estrous cycle, indicating that spermatozoa are capacitated in response to the oviductal environment and subsequently fertilize the oocyte.

Vasopressin V1a receptor and oxytocin receptor regulate murine sperm motility differently.

Specific receptors for the neurohypophyseal hormones, arginine vasopressin (AVP) and oxytocin, are present in the male reproductive organs. However, their exact roles remain unknown. To elucidate the physiological functions of pituitary hormones in male reproduction, this study first focused on the distribution and function of one of the AVP receptors, V1a. In situ hybridization analysis revealed high expression of the Avpr1a in Leydig cells of the testes and narrow/clear cells in the epididymis, with the expression pattern differing from that of the oxytocin receptor (OTR). Notably, persistent motility and highly proportional hyperactivation were observed in spermatozoa from V1a receptor-deficient mice. In contrast, OTR blocking by antagonist atosiban decreased hyperactivation rate. Furthermore, AVP stimulation could alter the extracellular pH mediated by the V1a receptor. The results highlight the crucial role of neurohypophyseal hormones in male reproductive physiology, with potential contradicting roles of V1a and OTR in sperm maturation. Our findings suggest that V1a receptor antagonists are potential therapeutic drugs for male infertility.

Effects of aging and oviductal hormones on testes, epididymides, and sperm of hamster.

Purpose: Aging is a major cause of decreased fertility. Using hamster, we examined the effects of aging on testes, epididymides, and sperm. Additionally, we examined whether progesterone (P4), melatonin (Mel) and 5-hydroxytryptamine (5-HT) mitigated effects of aging on sperm. Methods: Young (10-16 weeks), Adult (5-7 months), Aged (13-15 months), and Old (19-22 months) hamsters were used. Weights of bodies, testes, and epididymides were measured. Testes and epididymides were studied by histological microscopy. Sera were obtained to determine testosterone concentrations. Sperm were analyzed by video-microscopy. Results: By aging, body weights increased but weights of testes and epididymides decreased. Most hamsters were normozoospermia, although several old hamsters were azoospermia. In testes and epididymides, desquamation and structures resembling residual bodies (SRRBs) were observed. Although desquamation was not always related to aging, SRRBs occurred by aging. Testosterone concentrations were not changed in normozoospermic hamsters, but it was significantly reduced in old azoospermic hamster. Aging significantly reduced sperm ability to exhibit hyperactivation. Additionally, aging significantly increased the straight-line velocity (VSL). P4, Mel, and 5-HT lessened the reduction in sperm hyperactivation and the increasing of VSL. Conclusion: Aging reduces qualities of testes, epididymides, and sperm, and P4, Mel, and 5-HT recover reduced quality of sperm.

Serotonergic signals enhanced hamster sperm hyperactivation.

In the present study, we investigated the regulatory mechanisms underlying sperm hyperactivation enhanced by 5-hydroxytryptamine (5-HT) in hamsters. First, we examined the types of 5-HT receptors that regulate hyperactivation. Hyperactivation was significantly enhanced by 5-HT2A and 5-HT4 receptor agonists. Moreover, the results of the motility assay revealed that 5-HT2A, 5-HT3, and 5-HT4 receptor agonists significantly decreased the velocity and/or amplitude of sperm. Under 5-HT2 receptor stimulation, hyperactivation was associated with phospholipase C (PLC), inositol 1,4,5-trisphosphate (IP3) receptor, soluble adenylate cyclase (sAC), and protein kinase A (PKA). In contrast, under 5-HT4 receptor stimulation, hyperactivation was associated with transmembrane adenylate cyclase (tmAC), sAC, PKA, and CatSper channels. Accordingly, under the condition that sperm are hyperactivated, 5-HT likely stimulates PLC/IP3 receptor signals via the 5-HT2A receptor and tmAC/PKA/CatSper channel signals via the 5-HT4 receptor. After sAC and PKA are activated by these stimulations, sperm hyperactivation is enhanced.

Effects of 5-hydroxytryptamine on spermatozoal hyperactivation and in vitro fertilization in mice.

In this study, we examined the effects of 5-hydroxytryptamine (5-HT) on the motility and hyperactivation of mouse spermatozoa. In addition, we examined whether 5-HT increases the success of in vitro fertilization (IVF) in mice. Interestingly, 5-HT and agonists of the 5-HT2, 5-HT3, 5-HT4, and 5-HT7 receptors significantly increased the percentage of hyperactivated spermatozoa but did not affect the percentage of motile spermatozoa. Moreover, agonists of the 5-HT2, 5-HT3, and 5-HT4 receptors significantly affected the velocities, linearity, straightness, wobbler coefficient, amplitude and/or frequency of spermatozoa. In particular, the improvement of hyperactivation by 5-HT was strongly inhibited by antagonists of the receptors 5-HT4 and 5-HT7 and was completely inhibited by a mixture of the four 5-HT-receptor antagonists. The increase in hyperactivation by the agonists was significantly inhibited by the corresponding 5-HT-receptor antagonist. Moreover, 5-HT significantly increased the percentage of two-cell embryos. The increase in the IVF success rate by 5-HT was significantly inhibited by a 5-HT4-receptor antagonist. These results suggest that 5-HT increased hyperactivation through the 5-HT receptors and increased the success of IVF in mice.

Regulatory mechanisms of sperm flagellar motility by metachronal and synchronous sliding of doublet microtubules.

STUDY QUESTION: What is the role of metachronal and synchronous sliding in sperm flagellar motility? SUMMARY ANSWER: Both metachronal and oscillatory synchronous sliding are essential for sperm flagellar motility, while the change in mode of synchronous sliding between the non-oscillatory synchronous sliding of a specific pair of the doublet microtubules and the oscillatory synchronous sliding between most pairs of doublet microtubules modulates the sperm flagellar motility. WHAT IS KNOWN ALREADY: Metachronal and synchronous sliding of doublet microtubules are involved in sperm flagellar motility and regulation of these sliding movements controls flagellar bend formation. STUDY DESIGN, SIZE, DURATION: To study the regulatory mechanisms of metachronal and synchronous sliding in flagellar movement of golden hamster spermatozoa, changes in these sliding movements during hyperactivation were examined by measuring the angle of the tangent to the flagellar shaft with reference to the central axis of the sperm head (the shear angle) along the flagellum. Golden hamster spermatozoa were obtained from the caudal epididymis of five sexually mature golden hamsters. Results from three experiments were averaged. The number of spermatozoa analyzed is 15 activated sperm, 22 hyperactivated sperm and 20 acrosome-reacted sperm. PARTICIPANTS/MATERIALS, SETTING, METHODS: For detailed field-by-field analysis, an individual flagellar image was tracked automatically using the Autotrace module of image analysis software. The coordinate values of the flagellar shaft were used to calculate the shear angle, which is proportional to the amount of microtubule sliding at any given position along the flagellum. The maximum shear angles of metachronal and synchronous sliding were obtained from the mean shear angles between the maximum shear angles of pro-hook bends and the absolute values of the minimum shear angles of anti-hook bends, which represent the amplitude of a set of successive shear angle curves, with 3-12 shear curves covering one beat cycle of sperm flagellar movement. Asymmetry of flagellar waves was expressed by the mean shear angle between the maximum shear angle of pro-hook bends and the minimum shear angle of anti-hook bends at 100 µm from the head-midpiece junction. MAIN RESULTS AND THE ROLE OF CHANCE: The asymmetrical flagellar movements observed in the activated (non-hyperactivated) and hyperactivated spermatozoa were characterized by the non-oscillatory synchronous sliding of a specific pair of the doublets; the large asymmetrical flagellar movement in the hyperactivated spermatozoa was generated by the large non-oscillatory synchronous sliding. Both the metachronal and synchronous sliding increased during the hyperactivation; however, the large symmetrical flagellar movement of the acrosome-reacted spermatozoa was characterized by the oscillatory synchronous sliding between most pairs of doublets. These results demonstrated that the metachronal and synchronous sliding are involved in generation and modulation of sperm flagellar motility; however, two types of synchronous sliding, non-oscillatory and oscillatory sliding, modulate the sperm flagellar motility by enhancing the sliding of a specific pair of the doublets or the sliding between most pairs of the doublets. LARGE SCALE DATA: None. LIMITATIONS, REASONS FOR CAUTION: This is an indirect study of the metachronal and synchronous sliding of doublet microtubules. Studies based on the direct observation of behavior of dynein are needed to clarify the sliding microtubule theory of flagellar movement of spermatozoa. WIDER IMPLICATIONS OF THE FINDINGS: Both the metachronal and oscillatory synchronous sliding of doublet microtubule generate and modulate sperm flagellar motility, while the change in mode of synchronous sliding between the non-oscillatory synchronous sliding and oscillatory synchronous sliding modulates the sperm flagellar motility. The coordination between these sliding leads to various types of flagellar and ciliary motility, including the asymmetrical beating in flagellar and ciliary movement and planar or helical beating in sea urchin spermatozoa. Moreover, the finding that the metachronal sliding and two types of synchronous sliding generate and modulate the flagellar motility will open a new avenue for quantitative analysis of flagellar and ciliary motility. STUDY FUNDING AND COMPETING INTEREST(S): The authors have no conflict of interest and no funding to declare.

γ-Aminobutyric acid suppresses enhancement of hamster sperm hyperactivation by 5-hydroxytryptamine.

Sperm hyperactivation is regulated by hormones present in the oviduct. In hamsters, 5-hydroxytryptamine (5HT) enhances hyperactivation associated with the 5HT2 receptor and 5HT4 receptor, while 17ß-estradiol (E2) and γ-aminobutyric acid (GABA) suppress the association of the estrogen receptor and GABAA receptor, respectively. In the present study, we examined the regulatory interactions among 5HT, GABA, and E2 in the regulation of hamster sperm hyperactivation. When sperm were exposed to E2 prior to 5HT exposure, E2 did not affect 5HT-enhanced hyperactivation. In contrast, GABA partially suppressed 5HT-enhanced hyperactivation when sperm were exposed to GABA prior to 5HT. GABA suppressed 5HT-enhanced hyperactivation associated with the 5HT2 receptor although it did not suppress 5HT-enhanced hyperactivation associated with the 5HT4 receptor. These results demonstrate that hamster sperm hyperactivation is regulated by an interaction between the 5HT2 receptor-mediated action of 5HT and GABA.

Regulation of hamster sperm hyperactivation by extracellular Na.

Mammalian sperm motility has to be hyperactivated to be fertilization-competent. Hyperactivation is regulated by extracellular environment. Osmolality of mammalian semen is higher than that in female reproductive tract; however, the effect of them on hyperactivation has not been investigated. So we investigated the effect of osmotic environment on hyperactivation using hamster spermatozoa at first. Increase in the osmolality of the media (∼370 mOsm) by increasing the concentration of NaCl (∼150 mmol/L) caused the delay of the expression of hyperactivation. When NaCl concentration varied in the same range (75-150 mmol/L) whereas the osmolality was fixed at 370 mOsm by adding mannitol, the delay of hyperactivation occurred dependent on NaCl concentration. Increase in NaCl concentration also caused suppression of curvilinear velocity, bend angle, and sliding velocity of the flagellum at the onset of incubation, suggesting that NaCl concentration affect both activation and hyperactivation in hamster spermatozoa. Hamster sperm intracellular Ca(2+) concentration decreased as extracellular NaCl concentration increased, whereas membrane potential and intracellular pH were unaffected by extracellular NaCl concentration. SN-6 and SEA0400, inhibitors of Na(+)-Ca(2+) exchanger (NCX), increased intracellular Ca(2+) and accelerated hyperactivation in the presence of 150 mmol/L NaCl. Tyrosine phosphorylation on fibrous sheath proteins was unaffected by extracellular NaCl concentration. These results suggest that extracellular Na(+) suppresses hamster sperm hyperactivation by reducing intracellular Ca(2+) via an action of NCX in a tyrosine phosphorylation-independent manner. It seems that the removal of suppression by extracellular Na(+) leads to the expression of hyperactivated motility.

Non-genomic regulation and disruption of spermatozoal in vitro hyperactivation by oviductal hormones.

During capacitation, motility of mammalian spermatozoon is changed from a state of "activation" to "hyperactivation." Recently, it has been suggested that some hormones present in the oviduct are involved in the regulation of this hyperactivation in vitro. Progesterone, melatonin, and serotonin enhance hyperactivation through specific membrane receptors, and 17ß-estradiol suppresses this enhancement by progesterone and melatonin via a membrane estrogen receptor. Moreover, γ-aminobutyric acid suppresses progesterone-enhanced hyperactivation through the γ-aminobutyric acid receptor. These hormones dose-dependently affect hyperactivation. Although the complete signaling pathway is not clear, progesterone activates phospholipase C and protein kinases and enhances tyrosine phosphorylation. Moreover, tyrosine phosphorylation is suppressed by 17ß-estradiol. This regulation of spermatozoal hyperactivation by steroids is also disrupted by diethylstilbestrol. The in vitro experiments reviewed here suggest that mammalian spermatozoa are able to respond to effects of oviductal hormones. We therefore assume that the enhancement of spermatozoal hyperactivation is also regulated by oviductal hormones in vivo.

Estrogen suppresses melatonin-enhanced hyperactivation of hamster spermatozoa.

Hamster sperm hyperactivation is enhanced by progesterone, and this progesterone-enhanced hyperactivation is suppressed by 17ß-estradiol (17ßE2) and γ-aminobutyric acid (GABA). Although it has been indicated that melatonin also enhances hyperactivation, it is unknown whether melatonin-enhanced hyperactivation is also suppressed by 17ßE2 and GABA. In the present study, melatonin-enhanced hyperactivation was significantly suppressed by 17ßE2 but not by GABA. Moreover, suppression of melatonin-enhanced hyperactivation by 17ßE2 occurred through non-genomic regulation via the estrogen receptor (ER). These results suggest that enhancement of hyperactivation is regulated by melatonin and 17ßE2 through non-genomic regulation.

Suppression of progesterone-enhanced hyperactivation in hamster spermatozoa by γ-aminobutyric acid.

It has been recently shown that mammalian spermatozoa were hyperactivated by steroids, amines and amino acids. In the present study, we investigated whether hyperactivation of hamster sperm is regulated by progesterone (P) and γ-aminobutyric acid (GABA). Although sperm hyperactivation was enhanced by P, GABA significantly suppressed P-enhanced hyperactivation in a dose-dependent manner. Suppression of P-enhanced hyperactivation by GABA was significantly inhibited by an antagonist of the GABAA receptor (bicuculline). Moreover, P bound to the sperm head, and this binding was decreased by GABA. Because the concentrations of GABA and P change in association with the estrous cycle, these results suggest that GABA and P competitively regulate the enhancement of hyperactivation through the GABAA receptor.

Regulation and disruption of hamster sperm hyperactivation by progesterone, 17ß-estradiol and diethylstilbestrol.

PURPOSE: Hyperactivation of hamster sperm is dose-dependently enhanced by progesterone (P) and 17ß-estradiol (E). In the first part of the present study, enhancement of hyperactivation in response to the concentrations of P and E was examined in detail and in the second part, it was examined whether enhancement of hyperactivation by P and E was disrupted by diethylstilbestrol (DES). METHODS: Hamster spermatozoa were hyperactivated by incubation in modified Tyrode's albumin lactate pyruvate medium with P, E and/or DES. After spermatozoa were recorded using a video-microscope, observations were quantified by manually counting the numbers of total, motile and hyperactivated spermatozoa. RESULTS: Hyperactivation was enhanced in response to the concentrations of P and E. When spermatozoa were exposed to DES with E, moreover, DES significantly and strongly suppressed P-enhanced hyperactivation by accelerating the effect of E, but DES itself only weakly suppressed P-enhanced hyperactivation. CONCLUSIONS: Enhancement of hyperactivation was regulated by the concentrations of P and E, suggesting that in vivo hamster spermatozoa are hyperactivated through "monitoring" these concentrations in the oviduct. DES in combination with E suppressed P-enhanced hyperactivation, suggesting that DES significantly disrupts hyperactivation by acting as an accelerator of the effect of E.

Progesterone-enhanced sperm hyperactivation through IP3-PKC and PKA signals.

PROPOSE: The present study examined whether regulation of progesterone-enhanced hyperactivation of spermatozoa is associated with the production of inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) by phospholipase C (PLC) and cyclic adenosine monophosphate (cAMP) by adenylate cyclase (AC), as well as activation of protein kinase C (PKC) and protein kinase A (PKA). METHODS: Hamster spermatozoa were hyperactivated by incubation for 4 h in modified Tyrode's albumin lactate pyruvate (mTALP) medium. In order to examine the effects of IP3 receptor (IP3R), PKC and PKA on progesterone-enhanced hyperactivation, their inhibitors (xestospongin C, bisindolylmaleimide 1 and H-89) were used. RESULTS: Progesterone-enhanced hyperactivation was significantly suppressed by the inhibitors of IP3R, PKC and PKA. CONCLUSIONS: The results suggest that progesterone-enhanced sperm hyperactivation occurs through two signal pathways. One is an intracellular Ca2+ signal through production of IP3 and DAG by PLC, binding of IP3 to IP3R and activation of PKC by DAG and Ca2+. The other is a cAMP-PKA signal through production of cAMP by AC and activation of PKA by cAMP.

Characterization of an alternative chromatin remodeling to parasperm in a cottid fish, Hemilepidotus gilberti.

The dimorphic sperm of Hemilepidotus gilberti, i.e., haploid eusperm and diploid parasperm, have different morphologies corresponding to their own roles in fertilization. To estimate how these specific sperm morphologies were established, we focused on the nuclear morphologies and examined their changing processes in dimorphic spermiogenesis. Electron microscopic observation revealed that, in euspermatids, chromatin condensation first appeared as a mosaic pattern of moderate electrodense material in the peripheral region of the round nucleus. Those materials spread across the whole area to form a uniformly condensed nucleus. Chromatin condensation began similarly in paraspermatids to that in euspermatids. These became localized to one side of a nucleus and further condensed to form strong electrodense chromatin clusters, which are a specific feature of parasperm. From the remodeled nuclei of eusperm and parasperm, we found five and three kinds of sperm-specific basic proteins (SBPs), respectively, substituted to histones. The N-terminus amino acid sequences of the SBPs suggest that, in parasperm, one major SBP and two minor ones were distinct from each other. In eusperm nuclei, two kinds of specific SBPs were detected in addition to the homologs of parasperm SBPs. The specific SBPs had homologous amino acid sequences with huge arginine clusters, and one of them was most dominant among the five kinds of SBPs. The different combinations of SBPs in the eusperm and parasperm may cause a specific pattern of chromatin condensation in the dimorphic sperm nuclei of H. gilberti.

Serotonin-enhanced hyperactivation of hamster sperm.

The effects of serotonin on reproductive function were examined using hamster spermatozoa. When serotonin at concentrations from 1 fmol/l to 1 µmol/l was added to modified Tyrode's albumin lactate pyruvate (mTALP) medium, hyperactivation was significantly enhanced. Agonists and antagonists of 5-hydroxytryptamine hydrochloride (5-HT) receptors (5-HT(2) and 5-HT(4) receptors) were added to the medium. Both 5-HT(2) and 5-HT(4) receptor agonists significantly enhanced hyperactivation, although the effect was greater than the former. However, both 5-HT(2) and 5-HT(4) receptor antagonists significantly suppressed serotonin-enhanced hyperactivation, with the former suppressing stimulation by a lower concentration of serotonin than the latter. These results indicate that serotonin enhances hyperactivation via 5-HT(2) and/or 5-HT(4) receptors in a dose-dependent manner.

Suppression of progesterone-enhanced hyperactivation in hamster spermatozoa by estrogen.

In this study, I examined whether sperm hyperactivation in hamster is regulated by steroid hormones such as estrogen (estradiol, E(2)) and progesterone. Although sperm hyperactivation was enhanced by progesterone, 17beta-estradiol (17betaE(2)) itself did not affect sperm hyperactivation. However, 17betaE(2) suppressed progesterone-enhanced hyperactivation in a concentration-dependent manner through non-genomic pathways when spermatozoa were exposed to 17betaE(2) at the same time or before exposure to progesterone. When spermatozoa were exposed to 17betaE(2) after exposure to progesterone, 17betaE(2) did not suppress progesterone-enhanced hyperactivation. Moreover, 17alpha-estradiol, an inactive isomer of E(2), did not suppress progesterone-enhanced hyperactivation. Observations using a FITC-conjugated 17betaE(2) showed that it binds to the acrosome region of the sperm head. Binding of 17betaE(2) to spermatozoa was not inhibited by progesterone, although 17betaE(2) did not suppress progesterone-enhanced hyperactivation when spermatozoa were exposed to 17betaE(2) after exposure to progesterone. On the other hand, binding of progesterone to spermatozoa was also not inhibited by 17betaE(2) even if progesterone-enhanced hyperactivation was suppressed by 17betaE(2). Although tyrosine phosphorylations of sperm proteins were enhanced by progesterone, enhancement of tyrosine phosphorylations by progesterone was suppressed by 17betaE(2). Moreover, tyrosine phosphorylations were inhibited by 17betaE(2) when only 17betaE(2) was added to the medium. From these results, it is likely that 17betaE(2) competitively suppresses progesterone-enhanced hyperactivation through the inhibition of tyrosine phosphorylations via non-genomic pathways.

Regulation of hyperactivation by PPP2 in hamster spermatozoa.

It has been widely accepted that serine/threonine protein phosphatases (PPPs) are associated with the regulation of sperm hyperactivation. In the present study, we examined the types of PPPs associated with the regulation of hamster sperm hyperactivation. Protein phosphatases PPP1CA, PPP1CC, PPP2, and PPP3 are present in hamster sperm. In the experiments using several inhibitors, sperm hyperactivation was enhanced when PPP2 was inhibited at least, although inhibition of PPP1 also enhanced sperm hyperactivation. Interestingly, sperm were hyperactivated after PPP2 became an inactive form. And then, PPP1CA became an active form after sperm were hyperactivated. It has also been widely accepted that tyrosine phosphorylation is closely associated with the regulation of sperm hyperactivation. When PPP2 was inhibited, tyrosine phosphorylation was not enhanced at all. On the other hand, inhibition of PPP1 enhanced tyrosine phosphorylation. From the results, it is likely that PPP2 is closely associated with the regulation of sperm hyperactivation, although it is not associated with the regulation of tyrosine phosphorylation.

Non-genomic regulation of mammalian sperm hyperactivation.

Although it has been suggested that the acrosome reaction is induced through non-genomic regulation in a ligand-dependent manner, it is not known whether hyperactivation is similarly regulated. Progesterone and melatonin have been identified as ligands that regulate hyperactivation, the former through non-genomic regulation with phospholipase C and the latter most likely through a reactive oxygen species-mitogen activated protein kinase cascade. Both may be involved in spontaneous regulation of hyperactivation via tyrosine phosphorylation. The concentration of many hormones changes according to environmental conditions and biological rhythms, which will modulate ligand-dependent regulation of hyperactivation.