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Role of Calcium and Magnesium in capacitation/acrosome reaction

Role of Calcium and Magnesium in capacitation/acrosome reaction


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During the acrosome reaction, the activation enzyme present in the sperm dissolves the corona radiata and zona pellucida enabling the sperm to reach the plasma membrane of the egg. Also calcium and magnesium ions play an important role in acrosome reaction [ref]. What I fail to understand is how exactly do calcium and magnesium ions play a key role in acrosome reaction?


The process is not clearly understood. Calcium helps in capacitation: During capacitation calcium enters the spermatozoa through ion channels; blocking these channels inhibits the acrosome reaction but what calcium does inside the spermatozoa is not very clear. This paper says that calcium activates a certain tyrosine kinase in the spermatozoa which in turn activates a protein called p32. This they say occurs "concomitantly with capacitation". Other papers cited by this article talk about involvement of calcium dependent adenylate cyclase.

This article also says that protein tyrosine phosphorylations are important for capacitation but doesn't describe how.

I could not find a more precise information on this topic. I shall update the answer if I find one.


Cellular and molecular biology of capacitation and acrosome reaction in spermatozoa

A comparative account is given of advances in cellular and molecular biology of capacitation and acrosome reaction in spermatozoa by comparing and contrasting their biochemical and physiological changes in response to various factors in vivo and in vitro. It can now be stated that phenomena of sperm capacitation and acrosome reaction are endogenous molecular events occurring at the membrane level which can be modulated by external environmental factors. The molecular mechanisms and the signal transduction pathways mediating the process of capacitation and acrosome reaction are only partially defined and appear to involve modification of intracellular Ca2+ and other ions, lipid transfer, and phospholipid remodeling in the sperm plasma membrane as well as changes in protein phosphorylation. Evidences for the involvement of cAMP-dependent kinase pathway in the acrosome reaction are discussed. The mediation of one or more external signals by the sperm plasma membrane appears to activate this pathway after or simultaneously with the influx of Ca2+. Concurrent with or following entry of Ca2+, adenylate cyclase is activated, leading to increased concentrations of cAMP-activation of cAMP-dependent kinase and protein phosphorylation the identity of such proteins and their role in the acrosome reaction must be determined. The roles of biological effectors of the acrosome reaction, such as ZP3 and follicular fluid are still to be defined at the molecular level. The gaps in our knowledge about the cellular and molecular aspects of capacitation and acrosome reaction are emphasized.


Materials and Methods

Materials

BSA (Fraction V), FITC-Phalloidin, monoclonal antiphosphotyrosine (clone PT-66), and all chemicals were purchased from Sigma (St. Louis, MO). Horseradish peroxidase (HRP)-linked goat anti-mouse IgG was from Bio-Rad Laboratories (Hercules, CA).

Sperm Preparation

Ejaculated bull spermatozoa were obtained using an artificial vagina. The semen was washed three times by centrifugation (780 × g, 10 min at 25°C) in NKM buffer containing 110 mM NaCl, 5 mM KCl and 10 mM N-morpholinopropanesulfonic acid (pH 7.4). The washed cells were suspended in NKM buffer to a concentration of 10 9 cells/ml and were maintained at room temperature until use. Investigations were conducted in accordance with the Guide for the Care and Use of Agricultural Animals.

Capacitation and Acrosome Reaction

In vitro capacitation of bull sperm was induced by the method of Parrish et al. [ 25]. Briefly, sperm pellets were resuspended to a final concentration of 10 8 cells/ml in glucose-free Tyrode medium (TALP) containing 100 mM NaCl, 3.1 mM KCl, 1.5 mM MgCl2, 25 mM NaHCO3, 0.29 mM KH2PO4, 21.6 mM sodium lactate, 0.1 mM sodium pyruvate, 2 mM CaCl2, 20 mM Hepes (pH 7.4), 30 μg/ml BSA, 10 U/ml penicillin and 20 μg/ml heparin. The cells were incubated in this capacitation medium for 4 h at 39°C with 5% CO2.

Whole Cell Lysates

Washed sperm cells (10 9 cells) were solubilized in SDS-lysis buffer consisting of 125 mM Tris (pH 7.5), 4% SDS, 1 mM sodium orthovanadate, 1 mM benzamidine, and 1 mM PMSF added just before use. Cells were lysed for 10 min at room temperature and centrifuged at 12 930 × g for 5 min at 4°C. The supernatant was supplemented with 0.05% bromophenol blue, 5% glycerol, and 2% β-mercaptoethanol and boiled for 5 min.

Immunoblot Analysis

For immunoblotting, proteins derived from equivalent cell numbers were separated on 7.5% SDS-polyacrylamide gels and then electrophoretically transferred to nitrocellulose membranes (200 mAmp 1 h), using a buffer composed of 25 mM Tris (pH 8.2), 192 mM glycine, and 20% methanol. For Western blotting, nitrocellulose membranes were blocked with 5% BSA in Tris-buffered saline, pH 7.6, containing 0.1% Tween 20 (TBST), for 30 min at room temperature. The membranes were incubated overnight at 4°C with the antibody diluted 1:10 000. Next, the membranes were washed three times with TBST and incubated for 1 h at room temperature with specific HRP-linked secondary antibody diluted 1:10 000 in TBST. The membranes were washed three times with TBST and visualized by enhanced chemiluminescence (Amersham, Little Chalfont, UK).

Measurement of Intracellular cAMP

Spermatozoa (10 8 cells/ml) were incubated for 1.5 h in capacitation medium without NaHCO3, CaCl2, and heparin. The cAMP-dependent phosphodiesterase (PDE) inhibitor 3-iso-butyl-methylxantine (IBMX) (100 μM) was added during the last 15 min of the incubation period. Then the inducers were added according to the assay for 15 min. The cells were diluted in NKM buffer and centrifuged at 500 × g for 10 min. The cell’s pellets were resuspended and the amount of cAMP produced in the cells was determined after lysis using a nonradioactive enzyme immunoassay kit (RPN 255 Amersham) according to the manufacturer’s instructions.

Statistical Analysis

Statistical analyses were performed using the ANOVA test and t-test with multiple comparisons. Statistical significance is indicated in the figure legends.


Introduction

Intracellular Ca 2+ has a regulatory role in the control of sperm motility, capacitation, and the acrosome reaction. The use of Ca 2+ by the cell as an intracellular messenger requires precise regulation of its intracellular concentration [ 1]. Proposed regulatory sites of sperm intracellular calcium include both the plasma membrane [ 2] and the mitochondria [ 3– 5]. The fact that sperm intracellular Ca 2+ is maintained at a very low level (< 0.1 μM) in a medium containing millimolar Ca 2+ supports the concept that the plasma membrane is the primary regulatory site. However, though several groups have described systems involved in sperm Ca 2+ secretion [ 6, 7] and influx [ 2], little is known about the role of intracellular membranes in regulating Ca 2+ in spermatozoa.

It has been shown that thapsigargin, a highly specific inhibitor of the microsomal Ca 2+ pump, can induce elevation of intracellular free calcium [Ca 2+ ]i [ 8, 9] and initiate the acrosome reaction (AR) [ 9, 10] in human and bovine spermatozoa. Putative sites for thapsigargin-sensitive intracellular Ca 2+ stores include the cytoplasmic droplet, the sperm nucleus, and the acrosome. In a more recent study, it was shown that inositol 1,4,5-tris-phosphate receptors (IP3-R) are selectively localized to the acrosomes of rat, hamster, mouse, and dog sperm, suggesting that the acrosome is an intracellular Ca 2+ store [ 11]. Moreover, working with isolated acrosomal membranes, we recently observed that these membranes possess an ATP-dependent Ca 2+ pump that is inhibited by thapsigargin [ 12].

Walensky and Snyder [ 11] studied permeabilized rat sperm treated with sodium azide to follow ATP-dependent Ca 2+ loading into nonmitochondrial Ca 2+ stores. However, using azide to block the mitochondrial electron transport chain does not prevent the buildup of a proton gradient in the mitochondria due to ATP hydrolysis, resulting in active Ca 2+ transport into the mitochondria.

In the present study, we used a mitochondrial uncoupler to prevent the buildup of a proton gradient therefore no active accumulation of Ca 2+ in the mitochondria occurred. Thus, this is the first definitive demonstration of the presence of an intracellular nonmitochondrial ATP-dependent Ca 2+ pump in mammalian spermatozoa. A role for this internal calcium store in capacitation and in the AR is suggested.


Endocrinology

3.16.3.7 The Role of “Mating Factors” in Tick Reproduction

The sperm in any organism comprises the gametes plus secretions from various male accessory glands. Some of these male accessory gland secretions enhance fecundity and inhibit receptivity often these are the same material in a given species (review: Gillott, 2003 ). A fecundity-enhancing substance is one that increases egg production, oviposition, or egg fertility. A receptivity-inhibiting substance is one that renders the female less likely to copulate with further males, and hence reduces their chances for paternity. Because such substances are produced by one individual in order to influence the physiology of another, strictly speaking they fall into the category of pheromones rather than hormones. Nevertheless, these mating factors merit a brief mention here because of their influence on reproductive endocrinology.

Although male ticks appear not to transfer a receptivity inhibiting substance to the female (relying instead on proximate mate-guarding for assuring paternity), a number of mating factors have been identified in ticks which enhance fecundity (review: Kaufman, 2004 ), as summarized below.

3.16.3.7.1 A “sperm capacitation factor” from argasid and ixodid ticks

Spermatozoa must undergo capacitation before they are capable of fertilizing eggs. In both argasid and ixodid ticks, a 12.5 kDa protein originating from the male accessory gland stimulates capacitation following transfer to the female ( Shepherd et al., 1982 ). The sperm capacitation factor from argasid ticks is not active on the sperm of ixodid ticks and vice versa.

3.16.3.7.2 A “vitellogenesis-stimulating factor” from argasid ticks

When virgin O. moubata feed, they begin vitellogenesis but then resorb the yolk within 3 months (abortive vitellogenesis) ( Connat et al., 1986 ). If mated at this time, vitellogenesis resumes and oviposition occurs, an effect caused by a “vitellogenesis-stimulating factor” (VSF). One or two large proteins (100 and 200 kDa), originating from within the spermatozoon itself, constitute VSF ( Sahli et al., 1985 ). Whether VSF acts directly on the fat body to stimulate Vg synthesis or on the neuroendocrine system is not known.

3.16.3.7.3 A “male factor” from ixodid ticks

SG degeneration occurs in A. hebraeum which have exceeded the critical weight, a process regulated by 20E (see Section 3.16.3.5 ). Whereas the process takes only 4 days in mated females, it takes 8 days in virgins ( Lomas and Kaufman, 1992a ). This difference is due to a protein (20–100 kDa) from the testis/vas deferens, and is responsible for a number of other physiological and behavioral changes in the female which enhance fecundity ( Kaufman and Lomas, 1996 ).

3.16.3.7.4 An “engorgement factor” from ixodid ticks

Pappas and Oliver (1972 ) showed that female Dermacentor variabilis require a protein from the male gonad in order to feed to repletion. The authors recently extended these observations to A. hebraeum. Using a differential cross-screening approach to identify feeding-induced genes in the testis/vas deferens, 35 feeding-induced transcripts were identified ( Weiss et al., 2002 ). Recombinant proteins (recproteins) were produced from these transcripts, and engorgement factor was identified among them using a specific bioassay. None of the other recproteins had engorgement factor activity ( Weiss and Kaufmann, 2004 ). The latter authors present strong circumstantial evidence that engorgement factor and male factor are the same substance. The site of action of these factors in the female has not been determined, but the working hypothesis is that it either stimulates the synganglion to release the ecdysteroidogenic neuropeptide, or it acts directly on the epidermis to release E. It is also conceivable that the engorgement factor effect occurs at the synganglion and the male factor effect at the epidermis.


The Role of Glucose in Supporting Motility and Capacitation in Human Spermatozoa

University Division of Obstetrics & Gynaecology, St Michael's Hospital, Bristol, United Kingdom.

University Division of Obstetrics & Gynaecology, St Michael's Hospital, Bristol, United Kingdom.

University Division of Obstetrics & Gynaecology, St Michael's Hospital, Southwell Street, Bristol BS2 8EG, United Kingdom (e-mail: [email protected] ).Search for more papers by this author

University Division of Obstetrics & Gynaecology, St Michael's Hospital, Bristol, United Kingdom.

University Division of Obstetrics & Gynaecology, St Michael's Hospital, Bristol, United Kingdom.

University Division of Obstetrics & Gynaecology, St Michael's Hospital, Southwell Street, Bristol BS2 8EG, United Kingdom (e-mail: [email protected] ).Search for more papers by this author

Abstract

ABSTRACT: Glucose has been reported to be beneficial to human sperm for optimal capacitation and fertilization, although it is unclear whether glucose is required for providing extra metabolic energy through glycolysis, or for generating some other metabolic product. In this study, the effects of sugars on human sperm capacitation, motility, and energy production were investigated. The glucose concentration that supported the greatest number of acrosome reactions was 5.56 mmol L −1 . Compared with incubations with no added sugar, this concentration of glucose, fructose, mannose, or galactose appeared to slightly increase the number of acrosome reactions occurring after 18 hours of capacitation, or following induction by 2 μmol A23187 + 3.6 mmol pentoxifylline L −1 , but only glucose had a statistically significant effect. Glucose supported increased penetration of zona-free hamster oocytes, but its advantage was not statistically significant. The addition of 5.56 mmol glucose or fructose L −1 to sugar-free medium immediately increased the adenosine triphosphate (ATP) concentration and motility of sperm. These parameters were then stable for 3 hours, but declined markedly after 18 hours. In the absence of a glycolysable sugar, motility began to decline in the first hour and only 2% or 3% of sperm remained motile after 18 hours. Glucose or fructose was required to support hyperactivated motility. 2-Deoxyglucose was detrimental to the ATP concentration and motility of sperm, and supported fewer spontaneous or progesterone-stimulated acrosome reactions than were observed in the absence of a sugar. We conclude that glycolytic ATP production is required for vigorous motility and hyperactivation in human sperm. Other products of glucose metabolism are not essential to support capacitation, but they may have a small, enhancing effect.


Semen Evaluation

Acrosomal Integrity and Function of Spermatozoa

The acrosome reaction allows the sperm by exocytotic release of acrosomal enzymes to penetrate the zona pellucida of the egg. 101,111,142 The species-specific zona pellucida protein that serves as a sperm receptor also stimulates a series of events that lead to fusion between the plasma membrane and outer acrosomal membrane. 157,158 Membrane fusion and vesiculation expose the acrosomal contents, leading to leakage of acrosomal enzymes from the sperm’s head. As the acrosome reaction progresses and the sperm passes through the zona pellucida, more and more of the plasma membrane and acrosomal contents are lost. By the time the sperm traverses the zona pellucida, the entire anterior surface of its head, down to the inner acrosomal membrane, is denuded. Stallion sperm that lose their acrosomes or have undergone spontaneous acrosome reaction prematurely immediately after semen collection, after cooled storage, or after freezing and thawing are unable to bind to the zona pellucida and thereby are no longer capable of fertilization. Assessment of acrosomal integrity of sperm is one main target of semen analysis. Therefore, assays to analyze acrosomal status were developed in two ways either by measurement of the initial percentage of acrosome reacted sperm in the test sample or by assays testing the ability to undergo acrosome reaction in response to adequate stimuli.

Most methods available to assess acrosomal integrity are based on the use of dyes or fluorescent markers. These methods are inadequate for evaluating the existence of primary acrosomal defects (e.g., knobbed acrosomes). Intact acrosomes will be labeled with the acrosome-specific dye or fluorophore, whereas sperm with reacted acrosomes will not this relationship could be determined either by microscopical counting or, in the case of fluorophores, by using a flow cytometer. 112,133

In general, fluorescent staining of acrosomes can be achieved by two methods: (1) using spermatozoa with alcohol-permeabilized plasma and acrosomal membranes, 116,159 which allow fluorescent-labeled lectins to enter and stain intact acrosomes, or (2) using viable non-permeabilized spermatozoa. The ideal standard acrosomal stain is using fluorescently labeled agglutinins from peas (PSA) or peanut (PNA) plants. The most commonly used method to analyze acrosomal integrity is with a plant lectin labeled by a fluorescent probe (lectins conjugated with fluorescein isothiocyanate [FITC]). PNA (Arachis hypogea agglutinin) is a lectin from the peanut plant that binds to β-galactose moieties, which are exclusively associated with the outer acrosomal membrane thus, FITC-PNA (excitation/emission 488/515 nm wavelength) has been used successfully to determine the acrosomal status of acrosome-intact stallion sperm. 115,116 PSA (Pisum sativum agglutinin) is a lectin from the pea plant that binds to α-mannose and α-galactose moieties of the acrosomal matrix. Since PSA cannot penetrate an intact acrosomal membrane, only acrosome-reacted or damaged spermatozoa will stain, which is commonly assessed in combination with fluorescence microscopy. 113,117 Permeabilization of spermatozoal membranes has the disadvantage that acrosomal integrity and viability cannot be assessed simultaneously. However, PNA is believed to display less non-specific binding to other areas of the sperm, thus some laboratories favor this over PSA. FITC-PSA or FITC-PNA labeling of stallion spermatozoa is used in order to determine whether there is an association between the acrosome reaction and the incidence of subfertility in stallions. Acrosome-intact sperm will fluoresce evenly across the entire acrosomal cap. Under a fluorescent microscope, sperm in the process of acrosome reaction show patchy fluorescence over the acrosome, whereas those that have acrosome reacted most typically do not fluoresce or fluoresce only over the equatorial segment (see Fig. 6-6 ). Acrosome-intact and reacted sperm can be counted manually using a fluorescent microscope, or alternatively, flow cytometry can be used to count larger numbers of sperm.

Monoclonal antibodies specific for an acrosomal antigen can be used to evaluate integrity of acrosomal membranes in combination with indirect immunolabeling techniques. The antigen is localized at the inner surface of the outer acrosomal membrane. Only cells with damaged plasma and acrosomal membranes will bind primary antibody and demonstrate fluorescence after exposure to a secondary antibody (anti-mouse IgG-FITC) when viewed by epifluorescence microscopy. 160

The acrosome reaction in equine sperm can be induced by exposing capacitated sperm to suitable inducers such as progesterone, calcium ionophore A23187, heparin, and PC12. 62,143,161–164 Induction of the acrosome reaction with calcium ionophore (A23187) is rapid and does not require capacitation of sperm. Ionophores (e.g., A23187) are non-physiological stimuli that induce influx of calcium into the cell. This will result in the acrosome reaction in the major number of intact sperm. It has been suggested that sperm from some infertile/subfertile stallions have a <20% acrosome reaction rate when exposed to ionophore treatment. Others have reported that the drastically reduced ability of spermatozoa to acrosome react when exposed to ionophore A23187 for up to 3 hours was the sole abnormal spermatozoal characteristic in a group of severely subfertile stallions. Acrosome reaction in this group was 6%, compared with 84% in normal stallions. 165 Demonstration of reduced acrosomal responsiveness in sperm of stallions has been a major breakthrough in shedding light on the reason of subfertility in specific candidates. A possible explanation for this condition lies on the fact that the molar ratio of cholesterol-to-phospholipid was 2.5 times greater in the seminal plasma and 1.9 times greater in whole sperm of subfertile stallions compared with fertile stallions. 166 Furthermore, the discovery of an alteration in the cholesterol-to-phospholipid ratio in the sperm of affected stallions may allow further elucidation of the underlying acrosomal problem, and this finding may lead to therapeutic dietetic options in affected stallions. 167 However, prevalence of both phenomena (acrosomal reaction rate, cholesterol-to-phospholipid ratio in the sperm) needs further confirmation on larger numbers of stallions.

Induction of the acrosome reaction with progesterone is the more physiological option. 150,168 There is evidence that the initial percentage of spontaneous acrosome reactions may be higher in subfertile than in fertile stallions. On the other hand, the incidence of a progesterone-induced acrosome reaction was significantly lower in subfertile (6%) compared with fertile (17%) stallions, suggesting that assessment of the induced acrosome reaction may be a useful parameter to assess fertility. 169,170 In addition, the percentage of spermatozoa with exposed progesterone receptors has been reported to correlate highly with fertility of stallions. 171 Therefore, it seems obvious that this approach could serve as an excellent diagnostic assay. On the other hand, this assay is not easy to run and requires that the sperm be capacitated in vitro.

The golden standard to evaluate acrosomal status in the stallion is to perform electron microscopy. 34,91 However, this approach is limited by costs of the microscope, the need for trained personnel, and the limited number of sperm examined per sample. Although this technique has limited use in routine practice, electron microscopical evaluation of acrosomal status can be performed at referral centers.

Regardless of which assay is chosen to evaluate initial acrosomal status or the ability of sperm to acrosome react, normal values have not been validated for large numbers of fertile or subfertile stallion populations. Thus, if any of the tests are to be used in a clinical setting, positive controls from known fertile animals must be run simultaneously.

Oocyte Binding of Spermatozoa

The physiological inducer of the acrosome reaction is the zona pellucida. 158,168 For sperm to bind to the zona pellucida, it must possess an intact acrosome and have capacitated sufficiently to expose their zona binding proteins. Therefore, the ideal test of a sperm’s ability to acrosome react is to expose sperm to intact or hemizonae.

Those assays have been developed to test the ability of sperm to bind to homologous 170,172,173 and heterologous 158,174 zona pellucida, and the results had been correlated well with the fertility of some candidate stallions. Unfortunately, this assay is even more difficult, costly, and time consuming than the progesterone-induced acrosome reaction assay. Similar to the progesterone assay, sperm can undergo the zona-induced acrosome reaction only if they have previously been capacitated. In addition, the zona assay requires that a reasonable number of intact or hemizonae be available. Isolation of equine zonae is difficult and costly, and maturation state of the oocytes will have an impact on the results. Furthermore, assays that evaluate the ability of sperm to fertilize oocytes are carried out in vitro 175 under nonphysiological conditions, therefore making these assays of limited practical use.


After ejaculation, as the sperm enters the vagina, the acidic environment and secretions of the female reproductive tract prompt a chemical change in the sperm. The head of the sperm (the acrosome) becomes more permeable, allowing the release of the enzymes that will allow the sperm to bind with the egg during the process of fertilization.

This is part of the physiological maturation process the sperm must complete and only occurs once the sperm enters the female reproductive tract. These changes are essential to allow the sperm to penetrate the outer layer of the egg to successfully complete fertilization.

Two major changes in the sperm occur during capacitation:

  1. The removal of the outer protein layer on the head of the sperm. Removing this layer releases enzymes that allow the sperm to penetrate the outer layer of the egg (called the acro.
  2. The tail of the sperm moves in a whipping motion with larger movements, increasing sperm motility and allowing the sperm to travel to the egg.

When trying to achieve pregnancy through intercourse, the capacitation process occurs in the female reproductive tract. During in vitro fertilization (IVF), this process is completed artificially by placing the collected sperm in an activating fluid before combining them with the collected eggs.


Male Reproduction

John E. Schjenken , . Sarah A. Robertson , in Encyclopedia of Reproduction (Second Edition) , 2018

Secretions of the Seminal Vesicle

The fluid secreted by the seminal vesicle is rich in a wide range of bioactive moieties ranging from inorganic salts and micronutrients such as potassium and zinc, carbohydrates particularly reducing sugars such as fructose, lipid derivatives including prostaglandins and phosphorylcholine, and a unique array of glycoproteins notably lactoferrin, proteinase inhibitors and cell–cell signaling molecules known as cytokines ( McGraw et al., 2015 ). The factors secreted by the seminal vesicles have functional roles in semen coagulation, sperm motility and capacitation and female immune regulation.

There are limited studies aimed at assessing the proteomic composition of human seminal vesicle fluid, primarily due to difficulties associated with the collection of clinical samples. Some studies have utilized aspirated seminal vesicle fluid collected during surgery to identify the presence of a group of basic proteins termed seminal vesicle specific antigens ( Lee et al., 1989 Rui et al., 1984 ). These proteins are produced by the glandular epithelium and are now identified as members of the Semenogelin family, which are amongst the most abundant seminal vesicle secreted proteins.

In rodents, these proteins play a crucial role in copulatory plug formation ( Lee et al., 1989 Duncan and Thompson, 2007 ). Other copulatory plug proteins, including the seminal vesicle secretion 2 (SVS2) protein, fibronectin, lactoferrin, and kallikrein are also produced at high levels by the seminal vesicle ( Kawano et al., 2014 Lilja et al., 1987 ). Transglutaminase IV is essential for normal plug formation in mice, and its deficiency leads to infertility despite normal sperm count, sperm motility, and reproductive morphology since without a plug, few sperm are retained in the female tract after mating ( Dean, 2013 ). SVS2 has been shown to be essential for normal fertility in mice through a role in sustaining sperm survival in the uterus ( Kawano et al., 2014 ).

Considered as markers of human seminal vesicle function by the World Health Organization, fructose and prostaglandins are secreted at high levels by the seminal vesicle ( WHO, 2010 ). Fructose is the primary sugar produced by the seminal vesicles with the levels of fructose reflecting the rate of production of secretory exudate ( WHO, 2010 Mann and Lutwak-Mann, 1976 ). Fructose is consumed by sperm following ejaculation and is used as the primary source of energy by spermatozoa as they traverse the female tract ( Gonzales, 2001 ).

Prostaglandins are present in the seminal plasma of most vertebrate species ( Schjenken and Robertson, 2014 Gonzales, 2001 ). Prostaglandins are found in all accessory sex glands, but in human semen are predominantly derived from the seminal vesicles ( Gerozissis et al., 1982 ). These hormones are thought to enhance sperm motility as well as play a role in immune regulation in the female reproductive tissues, with prostaglandins being present in the seminal plasma of a number of species ( Schjenken and Robertson, 2014 Gonzales, 2001 ).

Other seminal vesicle secreted proteins, including MHS-5 and protein C inhibitor (PCI) have also been used to assess seminal vesicle function ( Calderon et al., 1994 Kise et al., 2000 ). For example, PCI has been used as a marker of seminal vesicle obstruction or agenesis ( Kise et al., 2000 ).


Proceedings of the 18th ICAR

Veronica Maillo , . Dimitrios Rizos , in Theriogenology , 2016

2 Anatomical and morphological characteristics of the bovine oviduct

The oviduct maintains and modulates the fluidic milieu for sperm capacitation, transport and fertilization of the mature oocyte, and early embryonic development [7,8] . The first stages of bovine embryo development occur in the oviduct, where the embryo spends around 4 days [9] . As shown in Figure 1 , the infundibulum receives the ovulated oocyte with dynamic movements, whereas the ciliated cells guide it to the lumen of the ampulla where the final events of oocyte maturation and the fertilization take place [10] . The oviductal isthmus functions as a sperm reservoir where sperm adhere transiently to the epithelium and are released at the time of ovulation [11] . After fertilization in the ampullary-isthmic junction (AIJ), the developing embryo passes through the isthmus, supported by ciliary activity and muscular contractions, until it reaches the uterus at about the 16-cell stage on Day 4 (for review see [12] ).

Fig. 1 . Schematic representation of the bovine oviduct, its anatomical parts, and oocyte-embryo developmental stages.

The oviductal epithelium is composed of two different cell types ciliated and secretory. During transport, the cilia exhibit a synchronized movement leading to a directed flow of fluids [13] . Secretory cells possess microvilli on their apical side and secrete macromolecules including oviduct-specific glycoprotein (OVGP1) and growth factors, usually by exocytosis, associated with the first days of the estrous cycle, which contribute to the development of the early embryo [14] .

The conditions of the oviductal environment are reflected in the oviductal fluid (OF). The OF is generated by (i) selective passage of constituents from the plasma into the oviductal lumen together with (ii) the secretion of substances synthesized by the secretory cells [15] . Oviductal fluid composition is very complex, containing simple and complex carbohydrates, ions, lipids, phospholipids, and proteins [16] . Some of these components are metabolic substrates, such as lactate, pyruvate, amino acids, and glucose, whose concentrations differ from those present in uterine fluid and the serum [17,18] . During preimplantation development, bovine embryos obtain energy by oxidative phosphorylation (especially pyruvate and amino acid oxidation), whereas at compaction and blastulation glucose is a more prominent substrate, a high proportion of which is converted to lactic acid by glycolysis [19] . Oviduct-specific glycoprotein is a component of the OF identified in many species in a highly conserved form, which binds to the zona pellucida (ZP) of the oocyte and early embryo suggesting a role in early development.


Watch the video: Calcium and Magnesium ion concentration determination with EDTA titration (February 2023).