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Role of the ionic environment and internal pH

on sperm activity

Samir Hamamah

1

3

and Jean-Luc Gatti

2

^nite de Biologie de la Reproduction, Departement de Gynecologie-

Obstetrique, Faculte de Medecine, CHU Bretonneau, 37044 Tours cedex,

and

2

URA INRA -CNRS 1291, INRA -Station de Physiologie de la

Reproduction des Mammiferes Domestiques, 37380 Nouzilly, France

3

To whom correspondence should be addressed at: IVF Center, A. Beclere Hospital,

157 rue Porte de la Trivaux, 92141 Clamart, France

In most species, once formed in the testis, spermatozoa are bathed in a fluid

where they remained immobile and with a very low level of metabolism.

This immotile status is understandable in view of the need to preserve the

sperm energy reserve and to decrease the risk of alteration to membranes,

internal structures and biochemical compounds by endogenous oxidizing

agents produced by mitochondrial activity. This quiescent phase can be of

different lengths and finishes when the semen is released into the external

environment where the spermatozoa become motile and metabolically active.

For invertebrates, and some fish, sexual activity is generally seasonal and

fertilization is external. Spermatozoa, once differentiated in the gonad,

remain there completely quiescent until they are released into the external

medium, which is either fresh water or sea water. Dilution of the testicular

fluid surrounding the spermatozoa allows the initiation of motility and

metabolism. In fact, this seminal fluid has an inhibitory effect on sperm

activity. For birds and mammals (including humans), the situation is much

more complex. In these species, sperm production is almost continuous

although for some of them, seasonal variations occur. When spermatozoa

are released from the Sertoli cells, they are rapidly exported from the testis

to the epididymis where the composition of the surrounding medium is

profoundly modified. For most species, the spermatozoa remain immobile

in the lower part of the epididymis, even though they have gained the

capability to be fully motile as shown by dilution in an adequate medium.

In

vivo,motility is activated when the spermatozoa are mixed with secretions

from the different accessory glands during ejaculation. This paper will

review the role played by environm ental factors , such asions,in the activation

of sperm motility and metabolism of different species of invertebrates and

vertebrates. Special attention is given to changes in sperm internal pH, its

regulation and role in the activation of sperm axonemal movement.

Key words:

human/internal pH/mammal/motility/spermatozoa

Introduction

The activation of sperm motility occurs in response to changes in the external

medium. Among the external factors, ions and particularly internal pH (pHj)

20 Europe an Society for Human Reproduc tion and Embry ology Hum an Reproduction Volume 13 Supplement 4 199H

byguestonSeptember17,2015

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Sperm internal pH

seem to play a pivotal role in sperm physiology in invertebrates, lower vertebrates

and, to some extent, in mammals. Over the past 20 years, an increasing amount

of data have been collected on the regulation of pH

{

in a large number of cell

types. These studies have been conducted to determine the mechanisms involved

in this pH, regulation. These mechanisms include: plasma membrane Na

+

/H

+

and K

+

/H

+

exchange, NaCl-HCO

3

co-transport, H

+

ATPases, etc. One of the

reasons for the rapid progress in this area is the fact that somatic cells are large

enough to be impaled without damage by microelectrodes during electro-

physiological studies; sperm cells are too small for such studies to be carried out

easily, although several attempts have been made, with variable results. In these

studies, sperm degradation produced by the microelectrode was difficult to

evaluate. The main progress in our understanding of sperm pHj regulation and

of other internal ions, is the result of analysis, either by the accumulation of

radioactive probes or fluorescent indicators. One of the main drawbacks of these

methods is the difficulty of determining the participation of each of the

several compartments forming the spermatozoa (i.e. acrosomal vesicle, nucleus,

mitochondria, cytoplasm) in the measurements. This leads to an average pH

;

, ion

concentration or changes in concentration of the cell popu lation. This last problem

can be partially resolved by using video-microscopy coupled with computer

image analysis, but this type of approach is time-consuming and is not completely

exempt from the effects of compartmentation.

Sea urchin sperm activation and pHj regulation

Sea urchin semen is the oldest and the best demonstrated example of the tight

relationship between external ionic conditions, pH

i5

motility and metabolic

activation (Lillie, 1919). Sea urchin spermatozoa are immotile and show no

oxygen consumption in the coelomic fluid after the induction of spawning. In

the field, their motility as well as their metabolism are turned on after spawning

in sea water, in which spermatozoa can be active for several hours. Under normal

conditions, sperm activation is accompanied by a release of H

+

ions. Measurement

of sperm pHj was carried out using [

31

P]-nuclear magnetic resonance (NMR), by

the fluorescent probe 9 amino-acridine, and also by the distribution of radioactive

probes (Christen

et al,

1982, 1983a,b; Lee

et al,

1983). These studies have

shown that the dilution of spermatozoa in artificial sea water containing sodium,

produces a pH; increase of 0.4-0.5 pH units. The activation of motility as well

as the pHj increase are inhibited when the external media is acidified (pH < 6.5),

lacks sodium ions or when the K

+

concentration is >30 mM (Christen

et al,

1982, 1983a,b; Lee et al, 1983). Under these inhibitory conditions, addition of

a diffusible base-like NH

4

C1 in the external medium produces an activation of

motility and metabolism by raising the pHj over a critical threshold value of 7.5

(Christen

et al,

1982, 1983; Lee

et al,

1983).

Proton release and, as a result, pHj increase, occurs by the activation of a

plasma membrane sodium-proton exchange. This Na

+

/H

+

exchange is controlled

21



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S.Hamamah and J.-L.Gatti

by the plasma membrane potential and blocked by depolarization when the

external K

+

concentration is >30 mM (Christen et al, 1986). In coelom ic fluid,

a high CO

2

level which decreases the pHj and an external concentration of

potassium of ~30 mM may explain why spermatozoa are not activated, even

though a there is a high NaCl concentration (Christen et al., 1983a; Johnson

et al.,

1983). It is interesting to note that, in the presence of sodium and low

potassium, the pHj is maintained at an almost constant value of 7.2-7.3, within

the external pH (pH

e

) range of 6.9-8.3 while, in the absence of sodium, the pHj

increases linearly from 6.1 to 7.3 and the difference between the pH

e

and the

pHj remains almost constant at ~0.8-1.0 pH units (see Table I in Christen

et al,

1982). This suggests that, in the absence of sodium, the protons are distributed

passively and no other pHj regulatory mechanism is functional.

How does pHj act on motility and metabolism? The activity of the outer arm

dynein-ATPase from sea urchin sperm axonemes shows a strict pH dependence.

This key enzyme for flagellar beating is almost inactive at pH 7.2 and its activity

increases at pH 7.4-8.0 (Christen et al, 1982). However, the action of pH on

regulatory mechanisms involving the cAMP pathway and protein phosphorylation

also seems likely. In inactive sea urchin spermatozoa, the ATP level is high and

oxygen consumption is negligible. The turn-on of the dynein-ATPases by pH

produces ADP and then increases mitochondrial activity (Christen et al, 1982,

1983a,b, 1986). Mitochondrial activity then produces H

+

ions which are exported

from the cell by Na

+

/H

+

exchange, and then a compensatory efflux of Na

+

by

the Na

+

/K

+

-ATPase is necessary to maintain a stable internal ionic environment

(Gatti and Christen, 1985).

Motility and metabolism are not the only steps in sea urchin sperm physiology

in which ions are involved. Changes in pHj and calcium influx also play a pivotal

role in the acrosome reaction and in the responses to egg-released factors (for

review, see Ward and

Kopf

1993).

Fish sperm activation

Teleost fish reproduction has also been studied for a long time because of their

econom ic interest, particularly salmonids, and cyprinids (Stoss, 1983). Both species

have external fertilization and spawning occurs in fresh water, but activation of

sperm motility is dependent upon different external factors: for salmonid fishes it

is external ions (H

+

, K

+

and Ca

2 +

), while for cyprinids, osmotic pressure is the

most important factor. O smotic pressure activation is also found in sea water fishes

although interaction w ith external ions has also been suggested.

Trout sperm activation and pH

t

regulation

Trout spermatozoa are immobile in testicular fluid. Under natural conditions,

trout spermatozoa are released into the fresh water where they are active for

< 1 min. Since the spermatozoa are released at the same time as the eggs at a



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Sperm internal pH

sperm/egg ratio of ~10

9

, almost 100% of eggs are fertilized. Meanwhile, for

artificial insemination, simplified media isosmotic to seminal plasma have been

made and, in these media, the presence of high potassium concentrations

(>30 mM) and low pH (1.0 pH units (pHj


5/11

S.Hamamah and J.-L.Gatti

flow cytometry (Marian et al., 1997). This study has shown that the pHj of the

spermatozoa increases slightly by 0.2-0.3 pH units at activation by a Na

+

/H

+

-

dependent mechanism which is amiloride sensitive. This Na

+

/H

+

mechanism is

inactivated by high osmolarity and the change in the pHj is not the trigger of

motility, although the value of the pH; can influence the length of the motility

phase. The decrease in pHj observed at dilution may be partially explain by the

production of protons during the utilization of ATP by the axonemal dynein-

ATPase activity (Perchec et al., 1995).

Bird sperm motility activation and pH

regulation

In birds, spermatozoa formed in the testis are expelled continuously in the

epididymis. The spermatozoa stay in the epididymis for ~1 day. Only a small

percentage of the spermatozoa from the upper part of epididymis are able to

move after dilution, while >70% spermatozoa from the lower part are capable

(Clulow and Jones, 1982). Sperm motility after dilution is dependent upon the

temperature: they are immotile at 30C and motile at 40C (Ashizawa

et al.,

1989a,b). This temperature inhibition can be removed by increasing the pH

e

or

increasing the external Ca

2 +

concentration. Measurement of the pH; using a

9 amino-acridine fluorescent dye shows that pH; is dependent upon the pH

e

(Ashizawa

et al.,

1989a,b). At 30C, external pH and pHj are equilibrated for

pH

e

of 6.0-10.0. At 40C, a difference of 0.2-0.3 pH units (pHj


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Sperm internal pH

Table I.

Internal pH (pH

;

) of human spermatozoa under different ionic conditions: sodium

(NaM), and potassium (KM) at different external pH (pH

e

). Values are given as mean SD

(data modified from Hamamah

et al,

1996)

PH

6

7.2

7.4

7.8

8.2

NaM

(140 mM Na

+

)

pH,

6.8 0.2

7.0 0.1

7.3 0.1

7.6 0.1

KM

(140 mM K

+

)

pHi

7.2 0.1

7.3 0.1

7.6 0.1

7.9 0.1

indicators (Florman et al, 1989; Vredenburgh-W ilberg and Parrish, 1995; Brook

et al,

1996; Hamamah

et al,

1996; Cross and Razy-Faulkner, 1997), [

31

P]-NMR

(Smith et al, 1985; Robitaille et al, 1987; Hamam ah et al, 1995) and also by

the distribution of a radioactive amine (Gatti

et al,

1993a; Ham amah, 1996).

It has been proposed that inhibition of motility of the spermatozoa of hamster,

rat, bull and dog in the caudal epididym is is due to an acidic pH, maintained by the

presence of lactate (Babcock, 1983; A cott and Carr, 1984; Carr

et al,

1985; Turner

and Reich, 1985). However, the concentration of this compound

in vivo

is not

sufficient by itselftodecrease the pH;. Meanwhile, the pH of the epididymal cauda

fluid is acidic in most species and this pH

e

acts directly on pHj (Mann, 1964; Gatti

et al, 1993a). Rat spermatozoa are immobile in epididymal cauda fluid and their

motility initiation is accompanied by an intracellular alkalization mediated m ay be

by a sodium-proton exchange mechanism (Wonget al, 1981;Wong andLee,1983)

but calcium may also be an important factor (A rmstrong

et al,

1992).

Babcocket al.(1983) showed that anp jalkalization stimulated both metabolism

and motility of epididymal bull spermatozoa. Protein phosphorylation, dependent

upon changes in bull sperm pH, have also been reported (Carr and Acott, 1989).

pH

e

also acts on the mobility of spermatozoa from the epididymal cauda sperm ato-

zoa ram and boar (Gattiet al, 1993b). In both species, spermatozoa are activated

by dilution in an alkaline medium while, after 1 h, a peak of m otility around

pH

e

7-8 is found.

Recently, we analysed the effect of pH

e

on the human sperm motility in a medium

supporting in-vitro fertilization. We observed that motility parameters, in particular

percentage motility, were slightly affected by alkaline pH values (pH

e

= 8.0) after

a long period of incubation. Optimum pH

e

would be ~7.2 (Table I).

Only a few studies have addressed the question of the mechanism implicated

in the regulation of the sperm pH;. In ram spermatozoa, external potassium has

been shown to act on pHj through a complex mechanism involving calcium

(Babcock and Pfeiffer, 1987; Rigoni

et al,

1987). Analysis of the pH

{

of ram

and boar spermatozoa diluted in different ionic conditions (with or without

sodium and potassium ions) has also shown that the pH

;

is strongly dependent

upon the pH

e

and that it increases linearly with pH

e

(Gatti et al, 1993a). In this

study, the presence or absence of sodium in the dilution media had almost no

effect on the pHj value. The slightly more alkaline pHj of ~0.1-0.2 pH units

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S.Hamamah

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J.-L.Gatti

Table II.

Effect of different external pH (pHe) on the percentage of motile ejaculated washed

spermatozoa as a function of time. Motility was evaluated by computer-assisted sperm analysis

(CASA).Values are given as mean SD

(n

= 6)

Time of incubation

pH

e

30 min 150 min pHj

7.2 60 2 1 61 13 6.8

7.4 53 27 55 23 7.0

7.8 65 1 4 49 22 7.3

8.2 55 3 0 43 16 7.6

pH

;

= internal pH.

which was observed in potassium media may be due to the dissipation of the

plasma membrane potential gradient. The same type of measurement was

conducted with ejaculated human spermatozoa (Hamamah

et al.,

1996) and it

was also found that pHj is highly dependent upon pH

e

: a quasi-linear relationship

existing between pH

e

and pH; in the presence of potassium and sodium (Table II).

The pHj values were also slightly lower in presence of sodium than in presence

of potassium; and probably for the same reason. Recently, a study on the

mechanism of pHj regulation was carried out on mouse cauda epididymal

spermatozoa (Zeng

et al.

1996). After dilution, two regulation mechanisms were

found: one appears to be a NaCl/HCO

3

exchange which seems specific to the

sperm cell, the other mechanism, which allows the cell to recover after

alkalinization and acidification, was ion independent and not precisely defined.

These differing results suggest that the mammalian sperm membrane is

permeable to protons, and that large a change in pH

e

produces changes in pHj

that can not be overridden by the pHj regulatory mechanisms which may exist.

In mam mals, ejaculated spermatozoa, w hile motile, are unable to fertilize. They

must undergo a capacitation step that takes placein vivo in the female genital tract

and which is the prelude to the acrosome reaction in the egg zona pellucida (for

review s, see Yanagimachi,1994;Breitbarte?al.,1997).External calciumisrequired

for both capacitation and the acrosome reaction, but several other factors, e.g. the

pH of the medium , and the presence of glucose, external potassium and bicarbonate,

are also involved. Sperm pH

i5

the Na

+

/K

+

-ATPase, a proton ATPase, and Ca-

ATPase are also cellular mechanisms that have been implicated as factors in this

phenomenon (for reviews, see Fraser, 1993; Kopf

et al.,

1994; Breitbart

et al,

1997).

R ecent studies have also suggested that

the

increase of pH during capacitation

is more likely to be an accompanying phenomenon rather than the trigger of this

physiological step (Uguz

et al.,

1994). Progesterone, a component of follicular

fluid, and some ofitsderivatives are able to induce sperm capacitation and prom ote

the acrosome reaction via an influx of calcium (Hyne et al., 1981; Thomas and

Meizel, 1988; Blackmore et al., 1990). The effect of the addition of progesterone

and human follicular fluid (HFF) on sperm pHj was analysed by Hamamah

et al.

(1996). Addition of progesterone, oestradiol-17(3 or 20% HFF to spermatozoa

incubated did not induce any rapid

pH ;

variation. A slight change in pH (~0.2 units)

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Sperm internal pH

Figure 1. Hypothetical pathway of the sperm internal pH change during the acrosome reaction (AR). See

text for explanation.

occurred with progesterone after 15 min. Florman

et al.

(1989) suggested that

this slow increase in pHj could be interpreted by the recruitment of spermatozoa

undergoing the acrosome reaction . Since the acrosomal vesicle represents an acidic

compartment ofthespermatozoa (Christen

e t al,

1983; Thomas and Meizel, 1988;

Florman

et al.,

1989, 1995). This compartment lowers the overall pH, of the cell.

When this vesicle loses its containment, and hence when its pH equilibrates with

the pH

e

, an increase in sperm pHj occurred (see Figure 1).This is mainly due to the

fact that the acrosome represents a small volume with a large pH gradient (see

1996).

Conclusions

Eukaryotic cells have powerful membranes mechanisms that control the pHj

(exchange of Na

+

/H

+

, K

+

/H

+

, NaCl/HCO

3

etc). Invertebrate and vertebrate

spermatozoa have a different type of mechanism or lack apparent large regulatory

mechanisms to maintain their pHj at a constant value.

In the sea urchin, the only mechanism is a Na

+

/H

+

exchange regulated by the

plasma membrane potential. In trout spermatozoa, pHj is mainly dependent upon

pH

e

.Inthesecells,the plasma membrane potentialisregulated by both the potassium

and proton gradients. It seems that for fish sperm atozoa w ith external fertilization,

and particularly those spawning in fresh water, regulation of the internal milieu

after release is not a priority since the spermatozoa are rapidly altered by the effects

of osmotic pressure, and the egg can be fertilized only for a short period.

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Mamm alian sperm pHj regulation has not been fully investigated yet. In simple

media, the value of pH

;

is mainly dependent on the value of the pH

e

, suggesting

that the membrane is permeable to protons. No important regulatory mechanisms,

such as N a

+

/H

+

or K

+

/H

+

exchange, have been found. This may be due to the fact

that after ejaculation, mam malian spermatozoa are in a physiological m ilieu where

the pH is well maintained. The different mechanisms described appear to be able

to regulate only to a very sm all degree the different physiological processes before

fertilization or to preserve the sperm pH; during these different steps. Further

research is required to gain a better understanding of sperm ionic regulation, with

a view to obtaining be tter media for short- or long-term preservation and to improve

conditions for the spermatozoa during in-vitro fertilization.

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