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Physiological action of oestradiol on the acrosome reaction in human spermatozoa

Physiological action of oestradiol on the acrosome reactionin human spermatozoaP. Vigil, A. Toro & A. Godoy Unidad de Reproduccio´n y Desarrollo, Departamento de Ciencias Fisiolo´gicas, Facultad de Ciencias Biolo´gicas, Pontificia Universidad Cato´lica deChile, Santiago, Chile Acrosome reaction—cervical mucus—humanspermatozoa—oestradiol—progesterone The acrosome is a secretory vesicle located in the sperm head. The acrosomereaction consists in the fusion of the sperm plasma membrane with the exter- nal acrosomal membrane. It has been observed that this reaction does not take Prof. Dr. Pilar Vigil, Unidad de Reproduccio´n y place in spermatozoa incubated in cervical mucus, hydrogel that contains high concentrations of oestradiol in the peri-ovulatory period. The objective of the Fisiolo´gicas, Facultad de Ciencias Biolo´gicas, present study was to analyse the influence of oestradiol on the acrosome reac- Pontificia Universidad Cato´lica de Chile, Av.
Alameda B. O’Higgins 340, Santiago, Chile.
tion in human spermatozoa to evaluate the possible inhibitory effect of this hormone. Spermatozoa were incubated in progesterone (10.1 nmol l)1); oestra- diol plus progesterone (oestradiol at 840 pmol l)1 and progesterone at10.1 nmol l)1), oestradiol (840 pmol l)1) and control (without steroidal hor- mones) for 30 min, 60 min, 240 min and 24 h. The acrosome reaction wasevaluated by stain with Hoechst 33258 and fluorescein isothiocyanate-conju-gated Pisum sativum agglutinin lectin. Progesterone-incubated spermatozoashowed the highest percentage of acrosome reaction (P < 0.05). Spermatozoaincubated with oestradiol and oestradiol plus progesterone showed the lowestpercentage of acrosome reaction. The present study demonstrates the inhibitoryrole of oestradiol on the acrosome reaction, stimulated by progesterone inhuman spermatozoa under physiological conditions.
Hammerstedt, 1997) and acquisition of the ability to fuse with the plasma membrane, over the principal and equa- The acrosome reaction (AR), a pre-requisite for mamma- torial segment of the acrosome, with the oocyte plasma lian fertilisation, is an exocytotic event, involving the fusion and fenestration of the sperm plasma membrane The sperm cell’s ability to undergo the AR depends on with the outer acrosomal membrane of the principal seg- the presentation of the proper molecules, the assembly of ment of the acrosome (Morales et al., 1992; Baldi et al., macromolecular signal transduction and fusion complex 1998; Ohmura et al., 1999). Reaction of the equatorial (or complexes), the proper ionic milieu and destabilisa- segment has also occasionally been observed (Vigil, 1989; tion of the plasma membrane, among other factors Morales et al., 1992; Ohmura et al., 1999). This process (Nolan & Hammerstedt, 1997). Some of these changes, occurs following contact of the spermatozoa with the which take place before the AR and prepare the sperma- zona pellucida (Baldi et al., 1998) and leads to a series of tozoa for this process, form part of sperm capacitation events such as releases of acrosin and other acrosomal enzymes, which favour the passage of spermatozoa The participation of several factors has been suggested through the zona pellucida (Llanos et al., 1993; Moreno to affect the occurrence of the AR. Among the substances & Alvarado, 2006), exposition of the internal acrosomal that induce the AR is progesterone, which is present in membrane as the new plasma membrane (Nolan & the follicular fluid (Morales et al., 1992). The action of Journal Compilation ª 2007 Blackwell Publishing Ltd Æ Andrologia 40, 146–151 Physiological action of oestradiol on the AR this hormone, mediated by the activation of a sperm membrane receptor, is dose-dependent in character andleads to intracellular signalling cascades (Morales et al., Semen specimens from normozoospermic men were 1992; Baldi et al., 1998). Other elements that stimulate obtained after 3–5 days of sexual abstinence and left at the AR are certain zona pellucida glycoproteins (Morales 37 °C to completely liquefy before being processed.
et al., 1992; Nolan & Hammerstedt, 1997). There are also Semen analysis was performed within 1.5 h after ejaculate some compounds that reduce or delay the occurrence of collection and according to the Laboratory Manual of the the AR, such as interleukin-6 (Carver-Ward et al., 1997), World Health Organization (1999). Semen samples with clathrin, a seminal bovine protein, which modulates the normal parameters of volume, sperm count, motility, transport of calcium (Clark et al., 1993) and zinc, which vitality and morphology, according to WHO were pooled is present in high concentrations in semen (de Lamirande and included in this study. Ejaculates showing sperm agglutination or abnormal viscosity were discarded.
Several investigations have shown that hormones like oestrogen act, in a rapid/nongenomic pathway in several distinct cell types, including spermatozoa (Baldi et al.,1998; Luconi et al., 2004). This process takes place After liquefaction, normal semen samples were pooled and through two subtypes of membrane receptors for this subjected to centrifugation through a two-step Percoll gra- hormone: alpha and beta, and a calcium influx probably dient (P-1644; Sigma Chemical Co., St Louis, MO, USA) mediates this nongenomic effect (Aquila et al., 2004; (Yudin et al., 1988). The two steps of the Percoll gradient Luconi et al., 2004). Although the relationship between were 80% in the lower layer and 40% in the upper layer.
oestradiol and the AR is not fully elucidated, it could be Briefly, 1–2 ml aliquots of semen were placed over the related to the fertilising ability of the spermatozoa and upper layer of the Percoll gradient and then centrifuged probably to the occurrence or absence of the AR (Luconi for 20 min at 300 · g. The pellet was then washed with et al., 1999; Baldi et al., 2000; Adeoya-Osiguwa et al., Biggers, Whitten and Whittingham (BWW) (3.5% HSA; A-1653; Sigma Chemical Co.) medium (Biggers et al., The first barrier that human spermatozoa encounter in 1971) supplemented with BSA (0.4%; A-2153; Sigma the female reproductive tract is cervical mucus (Barros Chemical Co.), by centrifugation for 10 min to 300 · g.
et al., 1983). During a period of up to 7 days, the sper- Subsequently, spermatozoa were capacitated in BWW matozoa are exposed to the action of this hydrogel, which medium for 2 h at 37 °C in an atmosphere containing 5% participates in the transport and selection of the sperma- CO2. The cells were resuspended in fresh medium and the tozoa in the female genital tract (Barros et al., 1983; Mor- sperm concentration was adjusted to 10 · 106 cells ml)1.
ales et al., 1993; Vigil et al., 1999). It has also beenobserved that the AR does not take place in spermatozoa incubated in cervical mucus in vitro; however, the factorresponsible for this action has not been determined (Bar- The suspensions of spermatozoa were incubated for ros et al., 1983). On the other hand, the concentrations different time intervals with BWW medium (3.5% HSA) of steroid sex hormones in cervical mucus have been plus progesterone (10.1 nmol l)1; P-0130; Sigma Chemi- studied and the presence of oestradiol in high concentra- cal Co.), BWW medium (3.5% HSA) plus oestradiol tions has been detected, especially in the period close to (840 pmol l)1; E-2885; Sigma Chemical Co.), BWW med- ovulation (Adamopolous et al., 2000).
ium (3.5% HSA) plus oestradiol (840 pmol l)1) and pro- The objective of this study was to analyse the possible gesterone (10.1 nmol l)1) and as control, BWW medium inhibitory role of oestradiol on progesterone-induced (3.5% HSA) devoid of steroidal hormones at 37 °C in 5% acrosome reaction in human spermatozoa under physio- CO2 and 95% air. The hormonal concentrations used are the ones described for the peri-ovulatory period, whichcorrespond 840 pmol l)1 for oestradiol (Adamopolous et al., 2000).
For each of the treatments, five different experimentswere carried out and the AR was evaluated after 30 min, 60 min, 240 min and 24 h of incubation. After the differ- The present study protocol was approved by the Funda- ent incubation times, an aliquot was taken in which a cio´n Me´dica San Cristo´bal Bioethics Committee. Each minimum of 100 spermatozoa were counted for each of subject gave written informed consent to participate in the experimental conditions described above, discarding all the dead cells identified by Hoechst 33258 (B-2883; ª 2007 The AuthorsJournal Compilation ª 2007 Blackwell Publishing Ltd Æ Andrologia 40, 146–151 Physiological action of oestradiol on the AR Fig. 1 Hoechst H33258 and FITC-PSA fluorescence in human spermatozoa, incubated in BWW medium (3.5% HSA) plus progesterone(10.1 nmol l)1), showing: (a) A principal and equatorial segment acrosome reacted spermatozoa (arrow), acrosome-intact spermatozoa (arrowhead) and principal segment acrosome reacted spermatozoa, with intact equatorial segment (line) stained with FITC-PSA. (b) Live (arrow) anddead (arrow head) spermatozoa under H33258 stain.
Sigma Chemical Co.) staining under microscopic fluores- ing of the acrosome with lectin. The AR was considered as such if spermatozoa were: (i) viable according to theresults of staining with Hoechst and (ii) when the follow-ing patterns of staining were observed: partial or patchy staining of the principal segment of the acrosome with Hoechst dye (1.9 lmol l)1) was used to determine the complete staining of the equatorial segment; the staining live/dead status of human spermatozoa (Green et al., of the equatorial segment only and faint staining of the 1996) with the purpose of identifying dead spermatozoa whole spermatozoon, that is, when there was staining nei- which could have presented a loss in acrosomal content.
ther in the principal nor in the equatorial region because Samples were incubated at 37 °C and 5% of CO2 for the AR was complete (Cross et al., 1986). The rate of sper- 15 min before being washed by centrifugation through matozoa which had reacted was expressed with respect to 350 ll of 2% polyvinylpyrrolidone (PVP40; Sigma Chem- ical Co.) in phosphate-buffered saline (PBS) at 800 · gfor 5 min. The supernatant was removed and the pellet was resuspended in ethanol and kept for at least 30 minin the freezer.
The differences between treatments were analysed by two-way anova (Sokal & Rohlf, 1995). For this analysis, Germany) sperm pellets were thoroughly suspended by time was included as a block. In all cases, the results were repeated pipetting. Aliquots of 10–20 ll were dropped onto considered significant if P < 0.05. To compare the clean glass slides and the ethanol was allowed to evaporate averages for each treatment, the Newman-Keuls test was in a slide warmer. Each slide was covered with a 20 ll drop used (Sokal & Rohlf, 1969). The program statistica 6.0 of fluorescence isothiocyanate-conjugated Pisum sativum (StatSoft Inc, Tulsa, OK, USA) was used for all the statis- agglutinin (100 lg ml)1; FITC-PSA; Vector Laboratories, Inc., Burlingame, CA, USA) and placed in a dark humidi-fied chamber at room temperature for 10 min. Excess stain was removed by gently immersing the slide in ultra purewater approximately 15 times and allowing it to dry. The The AR percentages expressed in terms of mean ± standard smear was then mounted under mounting fluid (Trinity deviation (SD) for the different incubation conditions, this Biotech, Wicklow, Ireland) before examining it under fluo- is: BWW medium plus progesterone (10.1 nmol l)1), rescence microscopy using an oil immersion objective.
BWW medium plus oestradiol (840 pmol l)1), BWWmedium plus oestradiol (840 pmol l)1) and progesterone(10.1 nmol l)1), BWW medium as control (3.5% HSA) at 30 min, 60 min, 240 min and 24 h are shown in Hoechst H33258 and FITC-PSA fluorescence was exam- ined using an Olympus BH2 fluorescence microscope The higher percentage of AR was obtained in progester- (Olympus Optical Co., Ltd., Japan). The acrosome was one-incubated spermatozoa. Using two-way anova (Sokal considered to be intact when there was a complete stain- & Rohlf, 1995), significant differences (P < 0.05) were Journal Compilation ª 2007 Blackwell Publishing Ltd Æ Andrologia 40, 146–151 Physiological action of oestradiol on the AR Table 1 Percentage of AR (mean ± SD) for each treatment in the BWW + P), BWW medium plus oestradiol (840 pmol l)1) and proges- The moment when the AR occurs is fundamental to terone (10.1 nmol l)1) (BWW + E + P), BWW medium as control, achieve successful fertilisation. During the AR, proteolytic (BWW) and BWW medium plus oestradiol (840 pmol l)1; BWW + E) enzymes are liberated, which are necessary for the passage evaluated at 30 min, 60 min, 240 min and 24 h of incubation of spermatozoa through the zona pellucida and the spermplasma membrane of the equatorial and post-equatorial % AR (mean ± SD) at different time intervals segments acquires the fusogenic capacity that is necessary for gamete membrane fusion (Vigil, 1989). Although ithas been proved that the AR is a phenomenon that can 30.4 ± 2.70 49.2 ± 1.92 58.2 ± 0.84 69.8 ± 2.78 BWW + E + P 25.4 ± 0.55 27.4 ± 0.55 30.8 ± 0.84 36.2 ± 0.58 occur spontaneously in spermatozoa incubated in vitro 23.8 ± 0.84 26.6 ± 0.55 29.0 ± 0.71 32.6 ± 0.55 (Green et al., 1999), previous studies have shown the 22.2 ± 0.84 24.6 ± 0.55 26.4 ± 0.55 29.4 ± 0.55 existence of different factors that would take part in themodulation of the occurrence of such reaction (Morales The values are the mean ± SD of five different experiments.
et al., 1992, 2000; Clark et al., 1993; Carver-Ward et al., *Significant difference (P < 0.05).
1997; de Lamirande et al., 1997; Nolan & Hammerstedt,1997; Cross, 2007). Currently, factors such as progester-one are known to be stimulators of the AR (Moraleset al., 1992, 2000; Luconi et al., 1999).
In the female genital tract, spermatozoa interact with cervical mucus (Barros et al., 1983). Previous laboratorystudies have shown that spermatozoa incubated in cervi-cal mucus maintain an intact acrosome (Barros et al.,1983). It has been determined that peri-ovulatory mucuspossesses high concentrations of oestradiol (Adamopolouset al., 2000). The lower percentage of AR obtained in thepresent study when incubating spermatozoa in the pres-ence of oestradiol plus progesterone in comparison withthe percentage of the AR obtained in spermatozoa incu-bated in progesterone, shows that oestradiol has an inhib-itory role in the occurrence of the AR. This inhibitoryeffect could explain the absence of AR observed in previ-ous studies when spermatozoa were incubated in vitro incervical mucus (Barros et al., 1983) and suggests thepossibility that this inhibitory role of oestradiol may con- Fig. 2 Percentage of AR in presence of BWW medium plus proges- stitute a physiological mechanism tending to enhance the terone (10.1 nmol l)1; BWW + P; —), BWW medium plus oestradiol possibilities of fertilisation in humans.
(840 pmol l)1) and progesterone (10.1 nmol l)1) (BWW + E + P; Á Á Á), Other authors (Luconi et al., 1999; Baldi et al., 2000) BWW medium as control (BWW; - - -), and BWW medium plus oes- have shown that spermatozoa have membrane receptors tradiol (840 pmol l)1; BWW + E; Æ–Æ–). Error bars denote SD (n = 5).
for oestrogen and progesterone and that oestrogen recep-tors may play a role in the modulation of the nongenomic found between the results obtained for this steroid and the action of progesterone in sperm cells mediated by a mech- per cent of AR obtained under the other experimental anism in which calcium influx would participate (Luconi conditions described above (Fig. 2). Despite the lower et al., 1999; Baldi et al., 2000). Future studies should AR percentage observed in the oestradiol-incubated sper- be performed using antagonists of the oestrogen and matozoa, the differences were not statistically significant progesterone receptors to elucidate the mechanisms by regarding the control treatment and oestradiol plus pro- which steroidal hormones influence the AR of human The results of the Newman-Keuls test (Sokal & Rohlf, Although it has been shown that oestradiol and proges- 1969) indicate that each one of the AR percentages terone have an influence on the AR (Luconi et al., 1999; observed for BWW plus progesterone incubated sperma- Baldi et al., 2000; Aquila et al., 2003), this study shows for tozoa was significantly different from those observed in the first time that these steroidal hormones – oestradiol the other treatments after 60 min of incubation.
and progesterone – might exert antagonic roles with respect ª 2007 The AuthorsJournal Compilation ª 2007 Blackwell Publishing Ltd Æ Andrologia 40, 146–151 Physiological action of oestradiol on the AR to the AR. The opposite actions of oestradiol and proges- embryology. Daniel JC (ed) Freeman, San Francisco, terone would work in the sense that the first contact of the spermatozoa with oestradiol present in the cervical mucus Carver-Ward JA, Einspenner M, Quinn P, Lydic M, Brody SA inhibits the occurrence of such reaction in the uterine (1997) Effects of cytokines on the human sperm acrosome cervix. This mechanism could prevent the occurrence of a reaction: A possible explanation for infertility caused by premature AR. Later on, when the spermatozoa continue hostile cervical mucus. Fertil Steril 68(Suppl):S186.
their passage through the female genital tract, at the Fallo- Clark EN, Corron ME, Florman HM (1993) Caltrin, the pian tube they would be exposed to high concentrations of calcium transport regulatory peptide of spermatozoa, progesterone coming from the follicular fluid (Morales modulates acrosomal exocytosis in response to the egg’szona pellucida. J Biol Chem 268:5309–5316.
et al., 1992). These high progesterone concentrations Cross NL (2007) Effect of pH on the development of acroso- would induce the AR assuring a successful fertilisation.
mal responsiveness of human sperm. Andrologia 39:55–59.
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ª 2007 The AuthorsJournal Compilation ª 2007 Blackwell Publishing Ltd Æ Andrologia 40, 146–151


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