POLYSPERMY& HOW TO REDUCE ITR. Taufiq Purna Nugraha

Program Studi Biologi ReproduksiSekolah Pasca Sarjana Institut Pertanian Bogor2011

IntroductionNormal Fertilization : MonospermicIn vivo condition : regulate by oviduct, oocytePolyspermic : multiple sperm fertilize 1 eggDiploid : two copy of each parent chromosome, Polyspermic : three or more copy : Inviable zygotWhen more than one sperm manages to enter the ovum (dispermy= 2; triploidy= 3), the fetus nearly always aborts

Polyspermica secretion reaction, the "cortical reaction" modifies the extracellular coat of the egg

Polyspermic

PolyspermicHigh incidence of polyspermic in-vitro ZP and membrane blocks to polyspermy is not fast enough to prevent entry of the excess spermatozoa in vitro compared to the in vivo situationHuman : incidence of polyspermy during IVF ranges from less than 3% to over 30% Cattle range from 5% to 45%for in vitro systemsSheep and goats : almost 20%Coy & Aviles 2010

PolyspermicPig : high incidence of polyspermic during IVF, 14% to 93% (with percentages of penetration ranging from 19 to 95%, respectively)higher than that of in vivo matured and fertilized oocytes : 5%

Polyspermic in PigIVF : high concentrations of spermatozoa are usually introduced to microdrops in which oocytes have been placedExcess spermatozoa and suboptimal IVF conditions are the major reasons for polyspermy in pig oocytesDead Sperm also introduced : detrimental factor ?Reducing sperm number decreased polyspermic penetration, also reduced sperm penetration rates

Li et al 2003

Polyspermic in PigQuality of SemenHigh variation between individual maleVariation between BreedDifficulty of cryopreservation of boar semenQuality of the matured oocytes in the pigSuzuki et al 2003

Polyspermic in PigMonospermic zygote,paternal and maternal microtubule domains unified during PN apposition. Interaction with microfilaments may be important for merging of two microtubule domains.Polyspermic Multiple microtubule domains in the polyspermic zygote became merged, but some delay or interruption was noted in the oocyte showing a higher degree of polyspermy (usually over trispermy).

Effort to Reduce PolyspermicIn pigs, decreasing the sperm concentration during IVF induces a parallel reduction in penetration frequency, decreasing polyspermy but not eliminating it

Method to Reduce PolyspermicIVF in the strawReduced Polyspermic Penetration in Porcine Oocytes Inseminated in a New In Vitro Fertilization (IVF) System: Straw IVF, Li et al 2003Pretreatment with adenosine & co-incubation with caffeineReduction of the incidence of polyspermic penetration into porcine oocytes by pretreatment of fresh spermatozoa with adenosine and a transient co-incubation of the gametes with caffeine, Funahashi & Romar 2004Modified Swim up methodA modified swim-up method reduces polyspermy during in vitro fertilization of porcine oocytes, Park et al 2008EncapsulationBoar Sperm Encapsulation Reduces In Vitro Polyspermy, Faustini et al 2010

IVF in the StrawTry to mimic oviduct conditionSterile 0.25-ml embryo cryopreservation straws Plugs in the straws were cut off with scissors.One end of the straw was connected to a 1-ml syringe through a hosepipeLoaded in the following sequences. straw with air columns :1 cm medium with spermatozoa, 5 cm insemination medium, 1 cm medium with 60 oocytes, 1 cm air, and 1 cm paraffin oil were loaded. Then the hosepipe was connected to the other end of the straw, 1 cm air and 1 cm paraffin oil were loaded. In the strawwithout air columns :1 cm medium with spermatozoa, 7 cm insemination medium, 1 cm medium with 60 oocytes, and 1 cm paraffin oil were loaded. Then the hosepipe was connected to the other end of the straw, and 1 cm paraffin oil was loaded. After being loaded, the strawsPut on the adhesive tape holders horizontally, incubated for 6 h at 398C in an atmosphere of 5% CO2 in air.Li et al 2003

IVF in the Straw

IVF in the Straw

IVF in the Straw

IVF in the StrawAir columns in the straws had no obvious effect on fertilization parameters, especially on the rates of normal fertilized oocytes when oocytes were inseminated in the straws. The rate of cleavage (2- to 4-cell embryos) was significantly higher in the straws with air columns than in straws without air cAir columns for air equilibration when the columns incubated in CO2

IVF in the StrawIn microdrop sperm concentration higher (3.5x) but penetration lower than straw methodDead sperm also introduced detrimental to oocytes, thus affecting subsequent embryo development

Adenosin Pretreatment + Co-incubation in CaffeineCaffeine inhibits cyclic nucleotide phosphodiesterase, resulting in an increase in intracellular cyclic adenosine 30,50-monophosphate and induction of capacitation and spontaneous acrosome reactions of boar spermatozoalonger duration of co-culture of gametes in the presence of caffeine increase the chance for more spermatozoa bind to the zona pellucidaAdenosine and fertilization-promoting peptide (FPP, which is a tripeptide, pGlu-Glu-ProNH2) induce capacitation of both fresh and frozenthawed boar spermatozoa in vitro but prevent a spontaneous acrosome reaction via the adenylyl cyclase/cAMP pathway, even in the presence of caffeine Supplementation with adenosine and FPP during co-culture of oocytes with frozenthawed spermatozoa increased the proportion of monospermic penetrationFunahashi & Romar 2003

Adenosin Pretreatment + Co-incubation in Caffeine5 mmol caffeine/l supplemented during periods of co-culture and additional culture periods until 8 h after inseminationShortened co-incubation period of gametes from 30 to 5 min increased the monospermy rate Spermatozoa binding to the zona surface lower in oocytes co-cultured for 5 min than 8 h Limited exposure of gametes to 5 mmol caffeine/l only during a transient co-culture period for 5 or 30 min significantly reduced sperm cells that penetrated into the oocyte. 5 min Transient exposure of spermatozoa to caffeine increased the percentage of capacitated cells but not acrosome-reacted cells, compared with a whole exposure treatment.

Adenosin Pretreatment + Co-incubation in CaffeinePreincubation of spermatozoa with 10mmol adenosine/l for 90 min increased the incidence of capacitated cells and monospermy ratePreincubation of fresh spermatozoa with adenosine before the transient co-incubation IVF can also improve the monospermy rateAsynchrony in the morphology of sperm nuclei in polyspermic oocytes was reduced by the pretreatment with adenosine and a brief exposure to caffeine.

Adenosin Pretreatment + Co-incubation in Caffeine

Modified Swim Up MethodSemipermiable 70 m pore sized cell strainer separate the sperm pellet placed at the bottom of a tube from the mature oocytes placed within the upper region. To ensure that only motile sperm gained access to the oocytesPark et al 2003

The sperm pellet, diluted to a concentration of 1.0109 cells/ml in IVF medium, was added to the bottom of each test tube.Immediately following insemination, a 70m pore sized cell strainer gently inserted into the upper region of the test tubeOocytes were then carefully distributed evenly across the surface of the cell strainer, and the tube was incubated at 39 C for 6 h in an atmosphere containing 5% CO2 and 5% O2 at 100% humidity.

swim-up method reduce the incidence of polyspermy and hence yield more high quality embryos with a higher incidence of chromosome normalityThe cell strainer, through the limitation of sperm-binding sites on the zona pellucida may also further prevent polyspermic penetration.

Encapsulation Boar sperm encapsulation is an emerging technique to solve several problems linked to spermatozoa preservation and artificial insemination proceduresAble to fertilize in vitro and in vivoMaintains high percentages of non-reacted acrosomes at 18oC over 72 h of storage

Faustini et al 2010

4.4 x 108 cells ml; motility 95%; average velocity 65 m s and secondary anomalies

medium (Brackett & Oliphantsmedium with 12% foetal calf serum and 7 mg ml caffeine) to a concentration of 1 . 106 sperm ml50 matured denuded oocytes transferred in 500 l of sperm-containing IVF medium. After 1 h of co-incubation,transferred in fresh IVF medium.

Oocytes collected 18 h after exposure to spermatozoa and fixed in acetic acid : ethanol (1 : 3 v v) for 24 h, stained with 1% (w v) lacmoid solution

Encapsulation On average, the POLY rate lower in encapsulated spermatozoa than that of diluted semen after 24 and 48 h of storageMONO rate was higher for all time of storage if encapsulatedPOLY rate diminished from 24 to 48 h and increased from 48 to 72 h of storage time for both treatments : ageingIRR = 0.766

Encapsulation Encapsulated spermatozoa have lesser damage of sperm membranes and preserved specific ligand or ligands for the oolemma receptor.morphological and functional alterations during semen processing influences the penetration ratio and the incidence of polyspermy.

ConclusionPolyspermic high in in-vitroNo oviduct role to prevent polyspermicEffort to prevent polyspermic mostly to mimic in-vivo condition

IVF in the Straw

Berbagai faktor diduga sebagai penyebab tingginya polyspermia di babi diantaranya *The first was the traditional IVF in microdrops. Matured oocytes were freed from cumulus cells in the maturation medium containing 0.1% (w/w) hyaluronidase obtained from bovine testis (type I-S, H-3506) and then washed three times with the insemination medium (mBO). Thereafter, groups of 40 oocytes were transferred into a 50-l droplet of mBO medium covered with paraffin oil. The dishes were kept in a CO2 incubator until spermatozoa were added for insemination. For IVF, one 0.1-ml frozen semen pellet, was thawed at 39oC in Dulbecco PBS (DPBS) containing 1 mg/ml BSA (fraction V, A-8022) and antibiotics. The semen was collected from three fertile boars tested in a farm by artificial insemination and mixed together after collection (before cryopreservation). Two different frozen batches of semen were used. After being washed three times, spermatozoa were resuspended with mBO medium containing 2 mM caffeine to give a concentration of 5 x 106 cells/ml, and 50 l of sample was added to 50 l of the fertilization drops containing the oocytes. The oocytes and sperm were cocultured for 6h at 39oC in an atmosphere of 5% CO2 in air.

*(30 denuded oocytes in 100ml modified Medium 199-suspended spermatozoa at 2.5 x 105 sperm/ml) **A saturated BaCl2 solution added to semen to reach a Ba2+ ion concentration of 25 mmollSuspension was added dropwise into a 0.5% (wv) sodium alginate solutionExtrusion with a peristaltic pump : forced through a needle (25GX5 8 in.) flow rate of 60 droplets min. The height of the needle above the solution was 120 mmIons diffuse out of the droplets and react with the alginate by ionic interaction : formation of barium alginate around the semen dropletAfter 1 h, the capsules were collected by filtration, rinsed twice with a swine sperm extender Re-suspended in the same solution (capsule : extender volumetric ratio of 1 : 0.3). IVF tests were performed at 24, 48 and 72 h

(aqueous solution containing glucose 60 g l and NaHCO3 1.2 g l, pH 7.2) An aliquot of the same ejaculate was diluted with the same extender in 1 : 10 ratio. Diluted and encapsulated semen was preserved at 18 oC; three

Ovaries collected in an abattoir from cyclic sows weremaintained in physiological saline at 3035C andforwarded to the IVF laboratory within 2 h. A totalnumber of 1288 cumulus-oocyte complexes (COCs) wereaspirated from follicles with 46 mm diameter by usingsyringe. Under a stereomicroscope, intact COCs wereselected and transferred into a Petri dish pre-filled with2 ml of modified phosphate-buffered saline supplementedwith 0.4% bovine serum albumin. After fourwashes in maturation medium (NCSU 37 with5.0 mg ml insulin, 0.6 mM cysteine, 1.0 mM glutamine,10 ng ml EGF and 50 lM mercaptoethanol), groups of50 COCs were cultured in 500 ll of the same medium at39C in a humidified atmosphere of 5% CO2 95% air.For the first 24 h of maturation, the medium wassupplemented with 1.0 mM db-cAMP, 10 IU ml eCG(Folligon, Intervet, Peschiera Borromeo, Milan, Italy)and 10 IU ml hCG (Corulon, Intervet). After culturingfor 24 h, COCs were transferred to fresh maturationmedium and cultured for further 24 h.

*Encapsulated spermatozoa were aspirated from capsules and suspended in IVF medium (Brackett & Oliphantsmedium with 12% foetal calf serum and 7 mg mlcaffeine) to a concentration of 1 . 106 sperm ml

*