Roles of Intracellular Cyclic AMP Signal Transduction in the Capacitation and Subsequent Hyperactivation of Mouse and Boar Spermatozoa

It is not until accomplishment of a variety of molecular changes during the transit through the female reproductive tract that mammalian spermatozoa are capable of exhibiting highly activated motility with asymmetric whiplash beating of the flagella (hyperactivation) and undergoing acrosomal exocytosis in the head (acrosome reaction). These molecular changes of the spermatozoa are collectively termed capacitation and promoted by bicarbonate, calcium and cholesterol acceptors. Such capacitation-promoting factors can stimulate intracellular cyclic AMP (cAMP) signal transduction in the spermatozoa. Meanwhile, hyperactivation and the acrosome reaction are essential to sperm fertilization with oocytes and are apparently triggered by a sufficient increase of intracellular Ca²⁺ in the sperm flagellum and head, respectively. Thus, it is necessary to investigate the relationship between cAMP signal transduction and calcium signaling cascades in the spermatozoa for the purpose of understanding the molecular basis of capacitation. In this review, I cover updated insights regarding intracellular cAMP signal transduction, the acrosome reaction and flagellar motility in mammalian spermatozoa and then account for possible roles of intracellular cAMP signal transduction in the capacitation and subsequent hyperactivation of mouse and boar spermatozoa.     

Signalling Events and Associated Pathways Related to the Mammalian Sperm Capacitation

Capacitation is a biological phenomenon occurring prior to fertilization and is a multiple event process. Many physiological and biochemical changes takes place during the process; these changes are related to lipid composition of membrane, intracellular modulation of ion concentration, protein phosphorylation, sperm movement and membrane permeability. These events occur when the sperm is exposed to the new environment of ion concentration in the female reproductive tract. Ions such as bicarbonate and calcium facilitate capacitation by activating adenylyl cyclase, thus initiating protein kinase A (PKA) signalling cascade. Extracellular‐regulated kinase pathway is activated by ligand binding to the membrane receptors and intracellular activation by reactive oxygen species (ROS). Activation of these pathways leads to the phosphorylation of different proteins, which is associated with events such as capacitation, hyperactivation and acrosome reaction that are essential for successful fertilization. Extensive studies were carried out on protein phosphorylation in relation to capacitation, but its role still remains ambiguous.     

Physiology and endocrinology symposium: evidence that oviduct secretions influence sperm function: a retrospective view for livestock

The mammalian oviduct has long been recognized as an organ essential for successful reproduction. Bovine, ovine, porcine, and equine animal models have offered clear advantages for oviduct study related to gamete physiology, fertilization, and early embryonic development. Livestock species are amenable to surgical alteration of the reproductive tract, estrous cycle manipulation, gamete cryopreservation, and AI, as well as in vitro fertilization and embryo production. Although most reproductive technology developed for livestock was intended to benefit production animal agriculture, these techniques are a treasure trove of tools for researchers to better understand how the oviduct influences gamete function. Oviduct secretions obtained from in vitro tissue cultures or via indwelling oviduct catheters have been used for analyses to define the protein, lipid, carbohydrate, enzyme, and electrolyte compositions of the secretions during the estrous cycle or in response to hormone treatment. Oviduct secretions or components purified from them have also been used in in vitro assays to assess their ability to bind to sperm, influence sperm viability, motility, sperm capacitation, the acrosome reaction, sperm-egg binding, and egg penetration, as well as subsequent embryonic development. Compelling data have emerged which show that the composition of secretions differs during the estrous cycle and that their composition differs whether they originate from the ampullary or isthmic regions of the oviduct. These differences in composition are functionally relevant and associated with different responses by sperm. Evidence indicatess that oviduct-specific glycoproteins, glycosaminoglycans, carbohydrates, norepinepherine, catecholamines, heat-shock protein, and osteopontin are components of the oviductal milieu that have the capacity to modulate sperm function. Future research on the livestock oviduct will likely define the role that oviduct secretions have in modulating sperm function and how these modifications ultimately affect fertilization and embryo development.     

Calmodulin is involved in the occurrence of extracellular Ca2+-dependent full-type hyperactivation in boar ejaculated spermatozoa incubated with cyclic AMP analogs

In mammals, hyperactivation is essential for sperm fertilization with oocytes in vivo. Two types of hyperactivation “full-type and nonfull-type patterns” can be observed in the spermatozoa from boars, bulls, and mice. We have a hypothesis that the full-type hyperactivation is a physiological (in vivo) pattern and are elucidating its molecular bases. The aims of this study were to detect calmodulin in boar sperm flagella by Western blotting and indirect immunofluorescence and to investigate effects of extracellular Ca²⁺ and calmodulin antagonists “W-7 and W-5 (W-5; a less potent antagonist)” on the occurrence of full-type hyperactivation in boar spermatozoa. Calmodulin was specifically detected as the 17-kDa antigen in the flagella and postacrosomal region of the heads. Full-type hyperactivation could be induced effectively in the samples incubated with 3.42 mM CaCl₂ for 120–180 min, and it was significantly reduced in the concentration-dependent manners of W-7 and W-5. Suppressing effects of W-7 on the full-type hyperactivation were stronger than those of W-5. These observations indicate that flagellar calmodulin is involved in the occurrence of extracellular Ca²⁺-dependent full-type hyperactivation in boar spermatozoa. This is the first indication of the intracellular Ca²⁺-sensing molecule which can function in the full-type hyperactivation.     

酪氨酸磷酸化在精子获能过程中的作用

在精子细胞发生顶体反应并使卵母细胞受精之前,获能是一个重要的生理先决条件。获能是精子在雌性生殖道中进一步成熟的复杂现象,其赋予精子以增强活性的能力,使精子能够与卵母细胞透明带（ZP）相互作用,进行顶体反应并与卵母细胞质膜融合,进而完成受精过程。然而精子获能的分子机制十分复杂,目前还未完全明确,但获能后的精子会有诸多结构及生化方面的变化,如蛋白酪氨酸磷酸化、精子膜胆固醇外流、活性氧的产生及精子膜超极化。蛋白质通过磷酸化或去磷酸化调节精子获能和顶体反应等一些重要的现象,这是精子到达、结合、穿透和融合卵母细胞所必需的过程。因此蛋白磷酸化是获能的一个非常重要的过程,尤其是在酪氨酸残基处的磷酸化是获能过程中发生的最重要的事件之一,且酪氨酸磷酸化可能是细胞中信号转导途径的主要甚至是唯一的指标。作者主要针对蛋白磷酸化、精子中酪氨酸磷酸化的发现、作用和定位,以及影响获能过程中酪氨酸磷酸化的几个因素进行阐述。     

Formation of a Reservoir of Sperm in the Oviduct

A reservoir of sperm in the initial segment of the oviduct has been found in several species of domestic and laboratory mammals. Evidently, the reservoir serves to ensure successful fertilization by providing the appropriate number of sperm in the appropriate physiological state for fertilizing oocytes soon after they enter the oviduct. Recent evidence indicates that sperm are trapped in the reservoir by binding to specific carbohydrate moieties on the surface of the mucosal epithelium of the oviduct. A bovine seminal plasma protein has been identified that associates with sperm and confers on them the capacity to bind to the carbohydrate moiety.     

The bovine oviduct and its role in reproduction: a review of the literature

The bovine oviduct provides the environment for sperm transport and capacitation, oocyte transport and maturation, fertilization and early embryonic cleavage. Gamete interactions in the tube occur in contact with both the tubal epithelium and the oviduct fluid secreted by these cells. Current research continues to reveal the active role of the oviduct and its products play in normal fertilization and embryo development. This paper reviews the anatomy and physiology of the oviduct of the cow, including the specific events of reproduction which occur in this organ.     

兔体外受精的研究

为完善家兔体外生产胚胎技术，采用FSH＋HCG法处理48只母兔，获得卵母细胞.采用获能的精子与卵母细胞进行体外受精，使用TCM199为基础液进行受精卵的体外培养.结果显示：平均每只供体兔可获得30.69枚可用卵母细胞.屠宰法获得的精子获能以后与卵母细胞进行体外受精，卵裂率达65.51%，囊胚发育率18.04%.自然射出法获得的精子获能以后，与卵母细胞进行体外受精，卵裂率达66.33%，囊胚发育率17.83%.屠宰法与自然射出法获得的精子获能以后卵裂率和囊胚发育率差异不显著.     

Changes in fertilization medium viscosity using hyaluronic acid impact bull sperm motility and acrosome status

The female reproductive tract, in particular the composition of the uterine and oviduct fluids, is responsible, at least in part, for triggering sperm cell modifications, essential for the acquisition of fertilization ability. Hyaluronic acid (HA) is a glycosaminoglycan present in these fluids, and its role in the fertilization process and sperm functionality is still barely understood. This work was designed to (a) determine the rheological characteristics of the fertilization medium by the addition of HA and (b) determine the HA influence on sperm motility and functional status. To that end, the in vitro fertilization medium was supplemented with 4 doses of HA (6, 60, 600 and 6,000 µg/ml) and analysed for viscosity and adhesion strength characteristics. Then, thawed semen from 6 bulls were incubated in these media and assessed at 4 different moments for morphological and functional parameters (plasma and acrosomal membrane integrities, mitochondrial membrane potential, capacitation, acrosomal reaction, and motility). The rheological evaluation showed that the addition of HA was able to increase both the viscosity and the adhesion strength of the fertilization medium, especially in the 6,000 µg/ml group in which the effect was more pronounced. No influence of HA could be observed on mitochondrial potential, and acrosomal and plasma membrane integrities. However, HA supplementation, at lower doses, led to an increase in the number of reacted sperm, as well as changes in motility parameters, with increase in the number of motile, rapid and progressive spermatozoa. In conclusion, the addition of HA alters the rheological properties of the fertilization medium and leads to the improvement of the properties related to sperm motility and capacitation, without compromising other functional aspects of the cell.     

Sperm interaction with the female reproductive tract

Sperm transit in the female tract is a critical event for the success of fertilization. From their deposition in the vagina to final migration in the oviduct, sperm pass through the different compartments of the genital tract in which they encounter different environments. The cervix and the uterotubal junction (UTJ) are two barriers with different relative importance according to the species. The protein composition, the degree of glycosylation and the hydration of the cervical mucus change during the oestrous cycle. Several sperm surface proteins are associated with their migration through the cervical mucus and the UTJ. Data regarding the interaction of sperm with secretions of the epithelial tissue lining the different compartments of the female genital tract during the sperm transit are reviewed, with a particular emphasis on the migration of sperm through the cervix.     

Effect of human tubal fluid medium and hyperactivation inducers on stallion sperm capacitation and hyperactivation

Conventional in vitro fertilization has not yet been implemented in the equine species. One of the main reasons has been the inability to develop a culture medium and incubation conditions supporting high levels of stallion sperm capacitation and hyperactivation in vitro. Although different culture media have been used for this purpose, human tubal fluid (HTF) medium, widely used in the manipulation of human and mice gametes, has not been reported so far in stallion sperm culture. The first part of this study aimed to compare HTF and Whitten's media on different stallion sperm quality and capacitation variables. Additionally, the effect of procaine, aminopyridine and caffeine in both media was evaluated on sperm motility parameters at different incubation times. Integrity and destabilization of the plasma membrane were evaluated by merocyanine 540/SYTOX Green (MC540), mitochondrial membrane potential (?Ψm) using tetramethylrhodamine methyl ester perchlorate (TMRM), acrosome membrane integrity by PNA/FITC and tyrosine phosphorylation by P‐tyrosine mouse mAb conjugated to Alexa Fluor® by flow cytometry. Motility parameters were evaluated using the integrated semen analysis system (ISAS®). We found no differences between Whitten's and HTF media and incubation time in terms of sperm viability, uninduced acrosome membrane damage or mitochondrial membrane potential at 30‐ and 120‐min incubation. Membrane fluidity (MC540) increased in both media at 30‐ and 120‐min incubation compared to noncapacitating conditions. Similarly, tyrosine phosphorylation increased in both media in capacitating conditions at 2‐ and 4‐hr incubation compared to noncapacitating conditions. Although procaine showed the best result in terms of sperm hyperactivated motility in both media, aminopyridine also showed parameters consistent with the hyperactivation including an increase in curvilinear velocity and decrease in straightness. In conclusion, HTF medium and aminopyridine equally support capacitation‐related parameters in stallion sperm.     

牛卵母细胞体外受精技术研究

本研究从3方面进行了牛卵母细胞的体外受精试验,即用不同离心法、不同温度上浮法、不同精子浓度等方法对COCs进行体外受精,测定其体外受精率。结果表明,两种离心法中,以上清液二次离心法的精子获能效果最好,受精率最高,该法首次采用低速离心技术,突破了离心法的传统方法,离心效果明显提高;不同温度条件下的精子上浮效果以CO2培养箱恒温（38.5℃）上浮法最好,受精率最高;对于选择受精时获能精子的浓度,宜控制在1.0×106～1.5×106个/ml,不仅能提高受精率,还可避免或降低多精受精现象。     

Influence of canine cumulus oophorus on homologous sperm motility

Sperm ejaculated by 8 beagle dogs and the cumuli oophori collected from 3 estrous beagle bitches were co-incubated, and penetration of the sperm into cumuli was observed to investigate the influence of cumuli on homologous sperm.The percentages of hyperactivated sperm and acrosome-reacted sperm were calculated after incubation with homogenized cumuli. The hyaluronic acid content of the incubated cumuli was measured, and hyperactivation and the acrosome reaction of the sperm were evaluated in medium containing hyaluronic acid. The mean percentage of hyperactivated sperm (33.0%) and number (3.0) of sperm that had penetrated a cumulus among sperm incubated for 7 hr were significantly higher than the values for sperm incubated for 0.5 hr (P<0.01). Almost all sperm that had penetrated the cumuli had intact acrosome, as though they were hyperactivated. The percentages of motile sperm (77.3%) and hyperactivated sperm (23.6%) after 2 hr incubation in the medium containing homogenized cumuli were significantly higher than in control medium (P<0.01), but there was no difference between cumulus and control media in the percentages of acrosome-reacted sperm. The hyaluronic acid content of a cumulus increased after 24 hr incubation. After 2 and 4 hr of incubation the percentages of hyperactivated sperm in the medium containing hyaluronic acid were significantly higher than in the control medium (P<0.01). These results suggest that canine hyperactivated sperm with intact acrosome can penetrate homologous cumuli and that the sperm are able to pass through the cumulus because the hyperactivated movement is maintained by hyaluronic acid secreted by the cumulus cells.     

Addition of D-penicillamine,hypotaurine, and epinephrine (PHE) mixture to IVF medium maintains motility and longevity of bovine sperm and enhances stable production of blastocysts in vitro

The present study aimed to establish an efficient system for bovine embryo production by in vitro fertilization (IVF) that can achieve stable normal fertilization and blastocyst developmental rates in any bull without optimization of the sperm concentration in IVF medium. We examined the effects of a PHE mixture (20 μM D-penicillamine, 10 μM hypotaurine and 1 μM epinephrine), theophylline (2.5 mM), and sperm concentration (1, 2 or 5 × 10⁶ cells/ml) on fertilization and blastocyst developmental rates. High cleavage rates (78.3 to 92.4%) and blastocyst developmental rates (31.9 to 62.0%) at day 7 were obtained in the presence of PHE and theophylline in IVF medium with a sperm concentration of 2 × 10⁶ cells/ml using sperm from 9 bulls. In addition, the synergistic effect of PHE and theophylline on normal fertilization (2 pronuclei) was clarified at 12 h after IVF with a sperm concentration of 1 × 10⁶ cells/ml. Moreover, high linearity, high flagellar beat cross frequency, and low amplitude of lateral head of motile sperm were found by computer-assisted sperm analysis. In conclusion, the combination of the PHE mixture and theophylline synergistically accelerates sperm motility and sperm penetration of bovine oocytes. Theophylline activates sperm motility with increasing intracellular cAMP. However, PHE prevents an excessive increase of cAMP and maintains sperm motility without hyperactivation. When the combination of PHE and theophylline is added to IVF medium at a sperm concentration of 2 × 10⁶ cells/ml, we can achieve stable normal fertilization and blastocyst development in any bull.     

犬卵母细胞体外成熟及其体外受精研究进展

犬科动物胚胎对于改进犬科珍贵物种及保护濒危犬种十分重要,因此对犬科动物体外受精(IVF)以及辅助生殖(ART)的研究逐渐变得必要.通过体外受精和核移植已经成功获得能够发育的犬科动物胚胎,克隆的犬胚胎移植到受体母犬能成功产仔,但是这一技术的效率仍然很低.主要原因是犬科动物胚胎在体外发育能力很低.犬卵母细胞早期发育和其他动物不同,犬卵巢排出的卵母细胞处于生发泡(GV)期,在输卵管中恢复减数分裂.犬卵母细胞核难以观测并且不容易确定排卵时间,所以很难确定精子入卵的确切时间,并且多精受精的情况时常发生.回顾犬卵母细胞体外成熟(IVM)和体外受精取得的进展,对于提高其胚胎生产效率很有必要.     

In vitro assessment of semen for the prediction of fertility

The prediction of fertility is a primary goal in the field of reproductive medicine. The aim of the present paper is to describe the value of conventional and modern sperm analysis systems considering the process of fertilization. The classical assessment of motility and morphology enables the rough estimation of semen quality in order to select ejaculates for the use in artificial insemination. Recent methods for sperm diagnosis, such as fluorescent marking for the detection of sperm plasma membrane integrity, the hypoosmotic swelling test, and computer assisted semen analysis allow for the evaluation of a large number of spermatozoa and the assessment of sperm dynamics under in vitro-fertilization conditions. The oocyte penetration test investigates the ability of spermatozoa for capacitation, hyperactivation and acrosome reaction in vitro. The amount of specific seminal plasma proteins is related to fertility and thereby provides an additional semen evaluation method. For the use of a given semen test the specific in vitro condition has to be considered. In addition, the evaluated criteria relevant for the process of fertilization need to be defined. The combination of selected semen tests gives a higher accuracy for the prediction of fertilizing capacity compared with a single test.     

Fertilization and Early Embryonic Development in the Porcine Fallopian Tube

Fertilization and early embryo development relies on a complex interplay between the Fallopian tube and the gametes before and after fertilization. Thereby the oviduct, as a dynamic reproductive organ, enables reception, transport and maturation of male and female gametes, their fusion, and supports early embryo development. This paper reviews current knowledge regarding physiological processes behind the transport of boar spermatozoa, their storage in and release from the functional sperm reservoir (SR), and of the interactions that newly ovulated oocytes play within the tube during their transport to the site of fertilization. Experimental evidence of an ovarian control on sperm release from the SR is highlighted. Furthermore, the impact of oviductal secretion on sperm capacitation, oocyte maturation, fertilization and early embryo development is stressed.     

Fertilizing Ability of Electro-Ejaculated Cryopreserved Semen in the Domestic Cat

Semen collection and AI in the cat are still not routine procedures. The correlation between semen quality and fertility under natural conditions is a relatively unknown field in the cat. In the present study, functional in vitro tests, such as the ability to bind and penetrate the zona pellucida or to fertilize in vitro, were used to determine fertilizing ability of sperm cryopreserved with a practical and efficient freezing protocol previously developed in our laboratory. Semen was collected by electroejaculation, evaluated for motility and diluted with Tris-glucose-citrate egg-yolk extender supplemented with Equex STM paste (0.5% v/v). After equilibration and loading into 0.25 ml straws, semen was frozen at 3.85 degrees C/min. Frozen-thawed semen was co-cultured with in vitro matured cat oocytes. Penetration rate was recorded 30 h after in vitro fertilization and cleaved zygotes were cultured in vitro until day 7. A correlation was found between sperm motility index (SMI) after thawing and semen fertilizing ability (p<0.05). In conclusion, it was demonstrated that the post-thaw motility quality, expressed as SMI, of spermatozoa frozen using the protocol mentioned above can be considered an index of the sperm ability to penetrate in vitro matured oocytes.