J Physiol Sci (2016) 66:207–212 DOI 10.1007/s12576-015-0419-y MINI-REVIEW Non-genomic regulation and disruption of spermatozoal in vitro hyperactivation by oviductal hormones 1 1 2 Masakatsu Fujinoki • Gen L. Takei • Hiroe Kon Received: 21 July 2015 / Accepted: 14 October 2015 / Published online: 5 November 2015 Ó The Physiological Society of Japan and Springer Japan 2015 Abstract During capacitation, motility of mammalian Keywords Amine Á Amino acid Á Hyperactivation Á Non- spermatozoon is changed from a state of ‘‘activation’’ to genomic regulation Á Spermatozoa Á Steroid ‘‘hyperactivation.’’ Recently, it has been suggested that some hormones present in the oviduct are involved in the regulation of this hyperactivation in vitro. Progesterone, Introduction melatonin, and serotonin enhance hyperactivation through specific membrane receptors, and 17b-estradiol suppresses In the oviduct, mammalian spermatozoa fertilize the this enhancement by progesterone and melatonin via a oocyte. Before fertilization, however, spermatozoa must membrane estrogen receptor. Moreover, c-aminobutyric be capacitated [1–4]. Capacitation is a qualitative change acid suppresses progesterone-enhanced hyperactivation in the spermatozoa that is needed for fertilization of the through the c-aminobutyric acid receptor. These hormones oocyte. Capacitated spermatozoa exhibit two reactions dose-dependently affect hyperactivation. Although the associated with capacitation. One is an acrosome reaction complete signaling pathway is not clear, progesterone that occurs at the head of a spermatozoon. This reaction is activates phospholipase C and protein kinases and enhan- a specialized exocytosis that is required for penetration of ces tyrosine phosphorylation. Moreover, tyrosine phos- the zona pellucida (ZP) and for binding to the oocyte [1, phorylation is suppressed by 17b-estradiol. This regulation 2, 4, 5]. The other is hyperactivation that occurs at the of spermatozoal hyperactivation by steroids is also dis- flagellum. Hyperactivation induces a specialized flagellar rupted by diethylstilbestrol. The in vitro experiments movement that creates the driving force for swimming in reviewed here suggest that mammalian spermatozoa are the oviduct and for penetrating the ZP [1–4]. Moreover, it able to respond to effects of oviductal hormones. We has been shown that the ability of spermatozoon to be therefore assume that the enhancement of spermatozoal hyperactivated correlates with the success of in vitro hyperactivation is also regulated by oviductal hormones fertilization [6]. in vivo. By use of a specific culture medium, capacitation is also made to occur in vitro. During in vitro capacitation, spermatozoa show motility change, such as from ‘‘acti- vation’’ to ‘‘hyperactivation.’’ Just after swim up in a Electronic supplementary material The online version of this specific culture medium, spermatozoa are activated article (doi:10.1007/s12576-015-0419-y) contains supplementary material, which is available to authorized users. (movies 1, 3, and 5). In many animals, activated sper- matozoa show a small bend amplitude in flagellar & Masakatsu Fujinoki movement and swim linearly. After incubation for some [email protected] hours (for example, 3–4 h in hamster and mouse sper- 1 Department of Physiology, School of Medicine, Dokkyo matozoa and 4–5 h in rat spermatozoa), most spermatozoa Medical University, Mibu, Tochigi 321-0293, Japan show hyperactivated motility (movies 2, 4, and 6). 2 Laboratory Animal Research Center, School of Medicine, However, the movement pattern of hyperactivated sper- Dokkyo Medical University, Mibu, Tochigi 321-0293, Japan matozoon basically depends on animals [1]. In hamster 123 208 J Physiol Sci (2016) 66:207–212 and mouse (movies 2 and 4), hyperactivated spermatozoa 4.2–7.4 lg/ml in the follicular fluid, and was show a large amplitude and a large asymmetric beating 44.04–175.06 ng/ml in the oviductal fluid [41]. Therefore, pattern in flagellar movement. Sometimes, their sperma- it seems that the progesterone present in the follicular fluid tozoa writhe and swim in the form of eight characters. In induces the acrosome reaction and that the progesterone rat spermatozoa (movie 6), many hyperactivated sperma- present in the oviductal fluid increases ZP penetration and tozoa show the large amplitude of head, the arched enhances hyperactivation. Although progesterone regulates movement of middle peace of flagellum, and the decrease cell functions through genomic signals in somatic cells, it of progressive movement although identification of their regulates the acrosome reaction, ZP penetration, and movement pattern is difficult. hyperactivation through non-genomic regulation in mam- During spermatozoal capacitation, hyperactivation malian spermatozoa [23, 24, 30, 42]. In human spermato- occurs spontaneously and time-dependently [1–3, 7–9]. zoa, progesterone stimulates an influx of Ca2? associated The first stimulation for capacitation/hyperactivation is the with CatSper activation, tyrosine phosphorylation, chloride removal of cholesterol from a spermatozoal plasma mem- efflux, and cAMP increase, and subsequently induces the brane by albumin [10–13]. The next step is Ca2? influx and acrosome reaction and hyperactivation [24, 30, 42–45]. In - cAMP production stimulated by HCO3 . Stimulation by hamster spermatozoa, progesterone enhances hyperactiva- 2? - Ca and HCO3 activates certain protein kinases and tion together with tyrosine phosphorylation [11]. Although phosphorylates proteins [14–19]. Additionally, the sup- the traditional genomic progesterone receptor (PR) does pression of protein phosphatases induces hyperactivation not exist in spermatozoa, a novel non-genomic PR exists at and protein phosphorylation [20]. Tyrosine phosphoryla- the plasma membrane of spermatozoa [23, 24, 27, 28, 42]. tion is well known as a capacitation-associated intracellular Moreover, it has been suggested that progesterone binds to signal [14, 15, 19]. The most popular tyrosine phosphory- the acrosome region where PR are localized in human and lation molecule is an 80-kDa protein, which was identified hamster spermatozoa [11, 46]. Downstream of the sper- as an A-kinase anchoring protein (AKAP) [21]. These matozoal PR, phospholipase C (PLC) [47] and/or protein stimulations and signal transductions are associated with kinase A (PKA) [48] are involved in the progesterone- regulation of capacitation, and induce the acrosome reac- induced acrosome reaction in mouse and human sperma- tion and hyperactivation. tozoa. In hamster spermatozoa, PLC, PKA, and protein After the 1980s, it has been reported that several kinase C (PKC) are involved in the progesterone-enhanced molecules that are found from the oviductal and follicular hyperactivation downstream of the PR [11, 49]. fluids affect spermatozoal acrosome reaction and hyper- It is well known that tyrosine phosphorylation sites, activation [11–13, 22–39]. Progesterone and serotonin are including AKAP, are associated with the regulation of well-known classical effectors of the acrosome reaction spermatozoal capacitation/hyperactivation [1, 2, 14, 15, [22, 40]. Recently, it has been suggested that proges- 19]. In several cases [11, 20], tyrosine phosphorylation is terone, melatonin, and serotonin act as inducers or enhanced when spermatozoal hyperactivation is induced by enhancers of the acrosome reaction and hyperactivation molecules present in the oviduct. Although it is not clear [11–13, 29–31, 33], and that 17b-estradiol acts as a which kinases cause tyrosine phosphorylation of sperma- suppressor for these processes [32, 34]. Moreover, it has tozoal proteins, it has been reported that tyrosine phos- been reported that c-aminobutyric acid (GABA) acts as an phorylation is regulated through Ca2? signals associated inducer of the acrosome reaction and hyperactivation in with an inositol 1,4,5-tris–phosphate (IP3) receptor-gated humans, rams, and rats [35–38], although it acts as a Ca2? store located at the base of the flagellum and suppressor of hyperactivation in hamsters [39]. In the calmodulin-dependent protein kinase [7, 9, 16, 17, 50]. present article, we review effects of the above molecules Moreover, it has also been reported that tyrosine phos- on hyperactivation. phorylation is regulated through cAMP-PKA signals [1, 14, 15, 19]. Because PLC, IP3 receptor, PKA, and PKC are involved in enhancement of hyperactivation in hamster Non-genomic regulation of hyperactivation spermatozoa [11, 49], it seems that progesterone enhances by progesterone spermatozoal hyperactivation through binding to PR and activation of PLC. This binding results in the production of 2? Progesterone was found to be an inducer of the acrosome IP3 and diacylglycerol, release of intracellular Ca from 2? reaction in human follicular fluid [22]. Additionally, in an IP3 receptor-gated Ca store, activation of PKC, acti- hamsters, 20 ng/ml of progesterone increased ZP penetra- vation of adenylate cyclase, production of cAMP, activa- tion and enhanced hyperactivation [11, 25]. In hamsters, tion of PKA, and enhancement of tyrosine phosphorylation moreover, the concentration of progesterone was (Fig. 1). 123 J Physiol Sci (2016) 66:207–212 209 Fig. 1 Hypothesized Ca P mechanism for the regulation of hyperactivation enhancement by progesterone and estradiol. AC adenylate cyclase, ATP Plasma membrane PR adenosine triphosphate, CAMK AC calmodulin-dependent protein PI/PC cAMP kinase, cyclic adenosine PLC monophosphate, DAG Ca diacylglycerol, E estradiol, ER cAMP ATP IP3