Fetuin: a tool to study the block to polyspermy

Fetuina: um instrumento para estudar o bloqueio da polispermia

FETUIN: A TOOL TO STUDY THE BLOCK TO POLYSPERMY¹

FETUINA: UM INSTRUMENTO PARA ESTUDAR O BLOQUEIO DA POLISPERMIA

Paulo Bayard Gonçalves² Charles Graves³

SUMMARY

The effect of fetuin, an a₁-glycoprotein that has protease inhibitor activity, on the biock to potyspermy was determined. Cumulus-oocyte complexes from eCG primed mice were matured in vitro in the presence of 0, 0.01, 0.1, l and 10mg/ml of fetuin in modifica TCM 199. Both in vivo and in vitro matured oocytes were fertilized in the presence of fetuin and incubated for 6 and 24h. Fetuin present in a concentration of 1 mg/ml in the ferlilization but not in the maturation medium was able to induce polyspermy in 52.4% of the eggs. There was a positive relationship between concentration of fetuin in the fertilization medium and the proportion of polyspermic eggs (p<0.05). A significant interaclion between 0.1 and l mg/ml of fetuin during maturation and fertilization was observed (p<0.05). The results of these experiments demonstrate the inhibition of the block to polyspermy using a protease inhibitor during the fertilization.

Key words: murine, block to polyspermy, fetuin, supernumerary sperm

RESUMO

No presente experimento, foi determinado o efeito da fetuina, uma a₁-glicoproteina com capacidade de inibir protease, no bloqueio da polispermia. Complexos cumulus-oócitos de murideos superovulados com eCG foram maturados in vitro na presença de 0, 0,01. 0,1, lê Idmg/ml de fetuina em TCM 199 modificado. Oócitos maturados in vivo e in vitro foram fecundados na presença de fetuina e incubados por 6 e 24 horas. Fetuina presente em uma concentração de 1 mg/ml durante a fecundação. mas não no meio de maturação, foi capaz de induzir polispermia em 52,5% dos óvulos. Houve uma relação positiva entre u concentração de fetuina no meio de fecundação e a percentagem de óvulos polispérmicos (p<0.05). Uma interação significativa entre 0,1 e l,0mg/ml de fetuina durante a maturação e fecundação foi observada (p<0.05). Os resultados destes experimentos demonstram a inibição do bloqueio da polispermia utilizando um inibidor de protease durante a fecundação.

Palavras-chave: murino, bloqueio da polispermia. fetuina, espermatozóide supernumerário.

INTRODUCTION

A complex sequence of events is required to obtain successfül fertilization and embryonic development. To prevent the penetration of more than one sperm, the egg undergoes changes at the level of the zona pellucida (zona reaction) and viteiline membrane (viteiline block). The zona reaction is believed to be mediated mainly by a protease and glycosidase from the cortiça Granules (WASSARMAN, 1987; YANAGIMACH1, 1994). These substances from the cortical granules are released into the periviteiline space after sperm attachment to the vitellus. The contents of the cortiçagranules seem to cause hydrolysis of the glycoproteins of the zona pellucida. The changes of these glycoproteins result in termination of the spenn receptors, termination of the ability to induce na acrosome reaction and a block to the spenn binding (BLEIL and WASSARMAN, 1980; DUCIBELLA et al., 1990).

A precocious block to sperm penetration is observed when mouse oocytes are cultured in vitro without serum (DE FELICI and SIRACUSA, 1982). Fetuin was identifíed as the compound present in serum that is able to preveni hardening of the zona pellucida (SCHROEDER et al., 1990). If fetuin inhibits the exocitosis of the cortical granules during fertilization, this a a₁-glycoprotein can be used to study the mechanisms of the block to polyspermy. For this reason, the present work investigates the effect of fetuin on the inhibition of the block to polyspermy during oocyte maturation and/or fertilization.

MATERIALS AND METHODS

Cumulus-oocyte complexes were isolated from 22-to 34-day-old ICR mice. The mice received intra-abdominally 5IU of equine chorionic gonadotropin (eCG) a 44 to 52 hours prior to colilecting the oocytes to be matured in vitro or prior to injecting 5IU of human chorionic gonadotropin (hCG) when collecting oocytes matured in vivo. For in vitro maturation, the oocytes were isolated from the folheies. The ovaries were removed from 5 mice for each replication and placed into 8ml of modifíed TCM 199 medium containing l mg/ml of fetuin covered by 4ml of silicon oil in a tissue culture dish. The TCM 199 with glutamine was modified by adding sodium pyruvate (0.2mM) and 100 units of penicillin and 50mg of streptomycin/ml of medium. After isolation, the cumulus-oocyte complexes were divided randomiy into different groups, washed 2 times in 200ml and transferred to 200ml of maturation médium. The oocytes were matured in modified TCM 199 in which fetuin was added at levels from 0.0 to l0.0mg/ml in according to the experimental design. The incubation period was 15.5 hours and the oocytes were maintained under a temperature of 37°C a humidity of 100% and an atmosphere of 5% CO₂ in air. To obtain in vivo matured oocytes, the females were euthanized by cervical dislocation 14 to 16 hours after injection of hCG and the oocytes matured in vivo were released by puncturing the ampulla of the oviduct with two sterile 30 G1/2 needies. Cumuius cell-oocyte complexes matured in vivo or in vitro were fertilized in vitro using a method previousiy described by HOPPE and PITTS(1973).

To determine the frequency of polyspermy, the eggs were stained with aceto orcein (0.75% of orcein in 45% of glacial acetic acid in PBS; DONAHUE, 1968) after the incubation period. The number of sperm heads decondensed in the ooplasm or the number of pronuclei was observed under phase microscopy (40-2000x).

The study of the inhibition of the block to polyspermy using fetuin during oocyte maturation was designed as a randomized complete block. Five treatment groups were investigated simultaneousiy in 3 to 5 replications. Each time that the oocytes in each treatment were matured and fertilized simultaneousiy was treated as one replication or block. Oocytes were matured in the presence of 0.01, 0.1, 1 and 10mg/ml of fetuin. For the control group, the oocytes were matured in vivo. After 6 hours of incubation with sperm, the eggs were washed and either fixed in 10% formalin for staining or cultured in M-16 for na additional 18 hours to determine the embryo development to the two cell stage. The percentage of embryos that cleaved to two cells were calculated based on the total number of penetrated eggs.

The inhibition of the physiological barrier to polyspermy was aiso verified using fetuin during the fertilization period. Oocytes matured in vivo were randomiy divided into tive different treatments. The oníy difference among treatments was the level of fetuin in the fertilization medium. The fetuin concentrations examined were 0, 0.01, 0.1, l and l0mg/ml. The eggs were incubated with sperm for 6 hours. Following incubation with sperm, the eggs were washed and fixed in 10% formalin for staining or cultured in M-16 for a total of 24 hours. These two experiments (6 or 24 hours incubation period) with tive treatments were replicated four different times.

Two experiments designed by randomized complete block with three crossed factors were conducted to investigate the degree of inhibition of the block to polyspermy using fetuin during oocyte maturation and fertilization. The factors studied were block, concentration of fetuin during maturation and concentration of fetuin during fertilization. In one experiment, the eggs were washed and fixed for staining after 6 hours of incubation with sperm. In the other experiment, the eggs were washed and cultured in M-16 for a total 24 hours after the same period of incubation with sperm. In both experiments, oocytes were matured and fertilized in 0.1 and 1mg/ml of fetuin.

The percentage of penetration, polyspermy and embryo development to the two cell stage was transformed by PROC RANK (CONOVER and IMAN, 1981) m the SAS statistical program to use parametric tests. The relationship between independent (concentrations of fetuin or time of egg incubation) and dependent variables (percentage of eggs penetrated, polyspermic or developing to the two cell stage) was tested using a polynomial regression.

RESULTS

The optimal level of fetuin during oocyte maturation was 1mg/ml (Table 1). However, the percentage of polyspermic eggs increased while the number of embryos that reached two cells decreased when the concentration of fetuin was l0mg/ml in the course of oocyte maturation (p<0.05 in relation to the control group). Most of the penetrated eggs matured in the presence of high levels of fetuin had more than one sperm head decondensed in the ooplasm or more than one male pronuclei.

Using a protease inhibitor (fetuin) during fertilization, the percentage of polyspermic eggs increased in a dose dependent manner (Table 2).

A positive linear relationship was observed between concentration of fetuin and percentage of polyspermic eggs at 6 hours (F=19.69; p=0.0004) and 24 hours (F=18.72; p=0.0005) of culture. At the end of the 6-hour egg-sperm incubation period, many sperm were still bound to the zona pellucida. At concentration of Img/ml of fetuin during fertilization, the percentage of polyspermic eggs decreased to levels statistically similar to the control group (in vivo matured) when they were cultured for 24 hours. However, the number of polyspermic eggs was constantiy high following 6 and 24 hours of culture and cleavage to two cells when fetuin was present in a concentration of l0mg/ml.

The effect of enzymatic inhibition during both oocyte maturation and fertilization is demonstrated in Table 3. In a dose dependent manner, fetuin inhibited the block to polyspermy oníy when present during fertilization. Significant numbers of polyspermic eggs were observed only when the concentration of 1mg/ml of fetuin was used during fertilization. The level of 0.1 mg/ml of fetuin was not enough to inhibit the physiological barrier to polyspermy even when higher concentrations of protease inhibitor were used during oocyte maturation.

DISCUSSION

Results of the present study indicate that a serine protease inhibitor (fetuin) in the fertilization medium is able to inhibit the physiological barrier to polyspermy in a considerable number of mouse eggs. There is evidence that the release of protease and glycosidase by cortical granules during exocitosis after sperm penetratíon catalyze the conversion of ZP2 and ZP3 to ZP2_f and ZP3_f respectively, causing hardening of the zona pellucida and inactivation of sperm receptors (BLEIL et al., 1981; WASSARMAN, 1987; WASSARMAN, 1990). Therefore, the presence of fetuin in the fertilization médium seems to inhibit temporarily the so called zona reaction or secondary block to polyspermy.

In the course of mouse oocyte maturation, cortical granules appear also to release proteases resulting in a premature block to polyspermy (zona pellucida hardening) when oocytes are matured in vitro in the absence of serum or fetuin (DUCIBELLA et al., 1990; SCHROEDER et al., 1990). In the present experiment, fetuin was able to allow penetration in a dose dependent manner when used in the maturation medium. The ideal concentration of fetuin to obtain penetration of the zona pellucida by the sperm, considering the range and the levels tested in this work, was lmg/ml of fetuin. This concentration of fetuin is identical with that observed by SCHROEDER et al (1990) to impede the hardening of the zona pellucida. In the study performed by SCHROEDER et al. (1990), the fírst cleavage was observed in a few eggs when fetuin was in a concentration of l0mg/ml in the maturation médium, which led them to infer that at this level fetuin causes a toxic effect. These results are not inconsistent with the data observed in the present study. However, the percentage of eggs penetrated in the l0mg/ml of fetuin groups was similar to the in vivo control group when eggs were cultured for 24 hours in the current work. At the level under consideration, polyspermy was the most importam abnormality noticed. Therefore, there are at least two possibilities for the significant level of polyspermy when high concentrations of fetuin is in the oocyte maturation médium. On the one hand, a toxic effect could cause a total disorder in the ooplasm resulting in an inability of the cortical granules to respond to the sperm penetration. On the other hand, a residual effect of fetuin cannot be discarded to affect the block to polyspermy. The results of many studies involving fetuin usage during oocyte maturation and fertilization could be affected if fetuin hás a residual effect in inhibiting the block to polyspermy. In this regard, a factorial experiment was designed to test the interaction between different concentrations of fetuin duringmaturation and fertilization. If fetuin hás a residual effect in the protease activity of the cortical granules during maturation, lower levels of fetuin would be required during fertilization to inhibit the barrier to polyspermy. This was not the case in the situation in which these experiments were performed. A significant interaction was detected when oocytes were matured and fertilized in the presence of 0.1 and l mg/ml of fetuin. The percentage of polyspermic eggs was significantiy high oníy when Img/ml of fetuin was used in the fertilization médium. Aiso, the concentration of fetuin during maturation did not predispose the oocytes to be more sensitive to low levels of fetuin at the concentrations tested in this study.

In 1974, GALEMBECK and CANN reported that fetuin is a trypsin-like proteases inhibitor, which maintains its activity upon desialicization similar to human a,-antítrypsin. Therefore, it is expected that fetuin would be able to inhibit the block to polyspermy considering that proteases play an important role in the physiological barrier to polyspermy (BLEIL et al, 1981; WASSARMAN, 1987; WASSARMAN, 1990). This expected result was observed in the present work when fetuin was present in the fertilization médium. The percentage of polyspermy increased in a positive linear relationship with the fetuin concentration. The results of the-present study demonstrate that fetuin inhibits the exocitosis of the cortical granules during fertilization. Therefore, this glycoprotein is a powerfül tool to study the mechanisms of the block to polyspermy, enabling to use zona intact eggs.

² Universidade Federal de Santa Maria, Departamento de Clínica de Grandes Animais, 97119-900 – Santa Maria – RS. E-mail: [e-mail]. Author for correspondence.

³ University of Illinois,Urbana, USA.

Recebido para publicação em 16.08.96. Aprovado em 25.09.96

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¹

This work was supported in part by funds from the Agricultural Experiment Station, University of Illinois at Urbana-Champaign.

Publication Dates

• Publication in this collection
  19 May 2008
• Date of issue
  Mar 1997

History

• Received
  16 Aug 1996
• Accepted
  25 Sept 1996