Phospholipase Cζ (PLCζ) versus postacrosomal sheath WW domain‐binding protein (PAWP): Which molecule will survive as a sperm factor?

During mammalian fertilization, sperm is fused with the oocyte's membrane, triggering the resumption of meiosis from the metaphase II arrest, the extrusion of the second polar body, and the exocytosis of cortical granules; these events are collectively called 'oocyte activation.' In all species studied to date, the transient rise in the cytosolic level of calcium (in particular, the repeated calcium increases called 'calcium oscillations' in mammals) is required for these events. Researchers have focused on identifying the factor(s) that can induce calcium oscillations during fertilization. Sperm‐specific phospholipase C, i.e., PLC zeta (PLCζ), is a strong candidate of the factor(s), and several research groups using different species obtained evidence that PLCζ is a sperm factor that can induce calcium oscillations during fertilization. However, postacrosomal sheath Tryptophan‐Tryptophan (WW)—domain‐binding protein (PAWP) was recently shown to have a pivotal role in inducing calcium oscillations in some species. In this review, we focus on PLCζ and PAWP as sperm factors, and we discuss this controversy: Which of these two molecules survives as a sperm factor?     

Distribution of protein disulfide isomerase during maturation of pig oocytes

Oocyte maturation in mammals is characterized by a dramatic reorganization of the endoplasmic reticulum (ER). In mice, the ER forms accumulations in the germinal vesicle (GV) stage and distinctive cortical clusters in metaphase II (MII) of the oocyte. Multiple evidence suggests that this ER distribution is important in preparing the oocyte for Ca²⁺ oscillations, which trigger oocyte activation at fertilization. In this study, we investigated the time course and illustrated the possible functional role of ER distribution during maturation of porcine oocytes by immunostaining with protein disulfide isomerase (PDI). PDI forms clusters in the cytoplasm of oocytes. After immunostaining, PDI clusters were identified throughout the cytoplasm from the GV to metaphase I (MI) stage; however, at the MII stage, the PDI formed large clusters (1–2 µm) in the animal pole around the first polar body. PDI distribution was prevented by bacitracin, a PDI inhibitor. Our experiments indicated that, during porcine oocyte maturation, PDI undergoes a dramatic reorganization. This characteristic distribution is different from that in the mouse oocyte. Moreover, our study suggested that formation of PDI clusters in the animal pole is a specific characteristic of matured porcine oocytes.     

Influence of co‐culture with denuded oocytes during in vitro maturation on fertilization and developmental competence of cumulus‐enclosed porcine oocytes in a defined system

Co‐culture of cumulus‐oocyte complexes (COCs) with denuded oocytes (DOs) during in vitro maturation (IVM) was reported to improve the developmental competence of oocytes via oocyte‐secreted factors in cattle. The aim of the present study was to investigate if addition of DOs during IVM can improve in vitro fertilization (IVF) and in vitro culture (IVC) results for oocytes in a defined in vitro production system in pigs. The maturation medium was porcine oocyte medium supplemented with gonadotropins, dbcAMP and β‐mercaptoethanol. Cumulus‐oocyte complexes were matured without DOs or with DOs in different ratios (9 COC, 9 COC+16 DO and 9 COC+36 DO). Consequently; oocytes were subjected to IVF as intact COCs or after denudation to examine if DO addition during IVM would affect cumulus or oocyte properties. After fertilization, penetration and normal fertilization rates of zygotes were not different between all tested groups irrespective of denudation before IVF. When zygotes were cultured for 6 days, no difference could be observed between all treatment groups in cleavage rate, blastocyst rate and cell number per blastocyst. In conclusion, irrespective of the ratio, co‐culture with DOs during IVM did not improve fertilization parameters and embryo development of cumulus‐enclosed porcine oocytes in a defined system.     

Roles of the zona pellucida and functional exposure of the sperm‐egg fusion factor ‘IZUMO’ during in vitro fertilization in pigs

The zona pellucida (ZP) is considered to play important roles in the prevention of polyspermy in mammalian oocytes. However, in pigs we have shown that the presence of the ZP accelerates sperm penetration into the ooplasm during in vitro fertilization (IVF). In the present study, we investigated the effects of the ZP on sperm binding, acrosomal status, and functional exposure of IZUMO, a critical factor involved in sperm‐egg fusion, during IVF in pigs. We evaluated the numbers and acrosomal statuses of sperm binding to the ZP and oolemma, and being present in the ZP and perivitelline space (PVS) using ZP‐intact and ZP‐free oocytes. More sperm bound to the ZP than to the oolemma. The average number of sperm present in the PVS was 0.44?0.51 per oocyte, and all sperm had lost their acrosomes. The proportion of sperm that were immunopositive for anti‐IZUMO antibody was significantly higher after they were passing or had passed through the ZP. Furthermore, addition of anti‐IZUMO antibody to the fertilization medium significantly inhibited the penetration of sperm into ZP‐free oocytes. These results suggest that, in pigs, the ZP induces the acrosome reaction, which is associated with the functional exposure of IZUMO, resulting in completion of fertilization.     

Microtubule assembly crucial to bovine embryonic development in assisted reproductive technologies

Centrosome integrity and microtubule network are crucial to the events around fertilization, including pronuclear development, migration and fusion, and the first mitotic division. The present review highlights the importance of bull spermatozoal centrosomes to function as a microtubule‐organizing center for successful fertilization and the subsequent embryonic development. Spermatozoal centrosomes need to be blended with ooplasmic pericentriolar materials accurately to nucleate and organize the sperm aster. Dysfunction of the spermatozoal centrosomes is associated with fertilization failure, which has been overcome with supplemental stimuli for oocyte activation following intracytoplasmic sperm injection in humans. Even though the spermatozoal centrosomes are functionally intact, abnormal sperm aster formation was frequently observed in vitrified‐warmed bovine oocytes, with delayed pronuclear development and migration. Treatment of the post‐warm oocytes with Rho‐associated coiled‐coil kinase inhibitor or α‐tocopherol inhibited the incidence of the abnormal aster formation, resulting in higher blastocyst yields following in vitro fertilization and culture. Thus, understanding of centrosomal function made it possible to improve the performance of advanced reproductive technologies.     

Growth of primordial oocytes in neonatal and adult mammals

Mammalian ovaries are endowed with a huge number of small oocytes (primordial oocytes) in primordial follicles. A small number of primordial oocytes start to grow, while others remain quiescent. Little is known about the mechanism regulating the activation of primordial oocytes. Recently, we found that primordial follicles in mature cows and prepubertal pigs took longer to initiate growth in xenografts compared with those in neonatal animals. We think that primordial oocytes in adult mammals are different from those in neonatal mammals. In this review, we summarize the results regarding the activation of primordial oocytes in neonatal and adult ovaries of different species and propose a model in which ovaries of neonatal mammals contain a mixed population of both quiescent and activated primordial oocytes, while almost all primordial oocytes are quiescent in adult females. The dormancy of primordial oocytes may be required to reserve the non-growing oocyte pool for the long reproductive life in mammals. FOXO3 is considered one of the molecules responsible for the dormancy of primordial oocytes in adult ovaries. These quiescent primordial oocytes are activated, perhaps by certain mechanisms involving the interaction between stimulatory and inhibitory factors, to enter the growth phase.     

谷胱甘肽对哺乳动物卵母细胞的影响

谷胱甘肽与哺乳动物卵母细胞之间关系密切,谷胱甘肽具有一种还原能力。谷胱甘肽参与保护雌性配子细胞免受氧化物的伤害,还参与维持卵母细胞减数分裂纺锤体形态。卵母细胞中谷胱甘肽浓度可以作为衡量哺乳动物卵母细胞质和核成熟程度的标志。它对于受精后雄原核形成起了积极的作用,并且还有助于早期胚胎发育。卵丘细胞对卵母细胞内的谷胱甘肽合成起重要作用。不同种类的低分子量硫醇复合物可以诱导卵母细胞中谷胱甘肽的合成。     

Birth of normal mice following round spermatid injection without artificial oocyte activation

For fertilization using round spermatid injection (ROSI) in mice, oocytes need to be artificially preactivated because of the lack of oocyte-activating capacity in round spermatids of this species. However, when round spermatids were frozen-thawed before microinjection, 11-71% of injected oocytes developed into 2-cell embryos without any artificial activation. After being transferred into recipient females, 5-27% of these embryos reached term. At least some of the injected oocytes showed intracellular Ca(2+) oscillations, which normally occur after fertilization by mature spermatozoa. Thus, these round spermatids could transmit a sperm-borne oocyte-activating factor, which might have been released from spermatozoa and elongated spermatids in the same suspension by freezing and thawing. This possibility was further supported by activation of intact oocytes following transplantation of the pronuclei from ROSI-generated embryos. Thus, one-step ROSI can be achieved in mice simply by injecting frozen-thawed round spermatids into intact oocytes. Clearly, there is a need for careful interpretation of microinjection experiments when assessing the oocyte-activating capacity of spermatogenic cells, especially when they are derived from frozen-thawed stocks.     

玻璃化冷冻对哺乳动物卵母细胞Ca2+的影响机制

卵母细胞冷冻保存是胚胎生物技术(如体外受精、胞浆内单精子注射、体细胞克隆)的重要组成部分,对优良种畜和濒危动物种质资源保存,加速家畜品种改良进程都具有重要意义。与常规冷冻法相比,玻璃化冷冻具有操作简单、降温速率快、耗时短、冷冻效率高等优点,被越来越广泛地应用于家畜卵母细胞的冷冻保存。然而,与新鲜卵母细胞相比,玻璃化冷冻卵母细胞的受精率及发育能力仍不理想,这严重影响了玻璃化冷冻卵母细胞的应用潜力。玻璃化冷冻会引起卵母细胞Ca²⁺浓度升高及钙振荡模式异常,导致其透明带硬化、受精信号紊乱等问题。综合前人研究进展,作者分析了玻璃化冷冻对胞内Ca²⁺浓度、钙振荡模式的影响和作用机制,并指出胞外Ca²⁺内流和胞内钙库Ca²⁺释放是导致冷冻卵母细胞胞内Ca²⁺浓度升高的主要原因,1,4,5-三磷酸肌醇(IP3)Ⅰ型受体分布异常和线粒体损伤可能是导致冷冻卵母细胞钙振荡模式异常的重要原因,以期为正向调控冷冻卵母细胞Ca²⁺浓度及钙振荡模式提供技术参考,从而进一步提高玻璃化冷冻卵母细胞的受精和后续的发育能力。     

Phosphorylation of inositol 1,4,5-triphosphate receptor 1 during in vitro maturation of porcine oocytes

During fertilization in mammalian species, a sperm-induced intracellular Ca²⁺ signal ([Ca²⁺]_i) mediates both exit of meiosis and oocyte activation. Recently, we demonstrated in mouse oocytes that the phosphorylation levels of inositol 1,4,5 trisphosphate receptor type1 (IP₃R1), the channel responsible for Ca²⁺ release and oscillations during fertilization, changed during maturation and fertilization. Therefore, we examined the expression and phosphorylation of IP₃R1 during in vitro maturation of pig oocytes. Here, our present study shows that expression of IP₃R1 protein did not change during maturation, although the phosphorylation status of the receptor, specifically at an MPM-2 epitope, did. We found that while at the beginning of maturation IP₃R1 lacked MPM-2 immunoreactivity, it became MPM-2 reactive by 24 h and reached maximal reactivity by 36 h. Interestingly, the acquisition of MPM-2 reactivity coincided with the activation of p34^cdc2 kinase and mitogen-activated protein kinase (MAPK), which are involved in meiotic progression. Following completion of maturation, inactivation of MAPK by U0126 did not affect IP₃R1 phosphorylation, although inactivation of p34^cdc2 kinase by roscovitine dramatically reduced IP₃R1 phosphorylation. Neither inhibitor affected total expression of IP₃R1. Altogether, our results show that IP₃R1 undergoes dynamic phosphorylation during maturation and this might underlie the generation of oscillations at fertilization.     

Effects of Different Oocyte Activation Procedures on Development and Gene Expression of Porcine Pre‐Implantation Embryos

Among the factors that affect the efficiency of somatic cell nuclear transfer (SCNT) in pigs, the activation protocol is the most variable among the current SCNT procedures. The aim of this study is focused on defining an efficient activation treatment of porcine oocytes. In Experiment 1, we studied the effects of nine different oocyte activation procedures (including chemical‐ and electrical‐based treatments) on parthenogenetic embryo development. In Experiment 2, we studied the effect of the more efficient activation procedures on the gene expression profile of Oct4 and Igf2r in parthenogenetic blastocysts. In conclusion, ionomycin as a first calcium stimulus is not able to activate porcine oocytes efficiently in comparison with electric procedures. Electrical treatments with 6‐DMAP significantly increased the level of Oct4 expression, whereas the single and double pulse treatments alone maintained the same profile as the IVF group.     

Cryopreservation of immature oocytes of the indigeneous Vietnamese Ban Pig

We report the cryopreservation of oocytes from Ban miniature pigs which are endemic in Vietnam. Immature cumulus‐oocyte complexes were collected from antral follicles of 7–8 mo old female cyclic Ban pigs and vitrified in micro‐drops. Oocyte morphology, lipid content, post‐warming survival, nuclear maturation, and embryo development were compared to those of oocytes from commercially slaughtered Landrace × Large white hybrid pigs. The size of oocytes in the two breeds was similar. However, significantly lower amounts of intracellular lipid were detected in Ban oocytes. There was no difference (p > 0.05) between Ban and Landrace × Large white oocytes in percentages of post‐warming survival (93.1 ± 3.4% vs. 70.7 ± 16.7%, respectively) and nuclear maturation after in vitro maturation (80.4 ± 5.1% vs. 90.0 ± 1.3% respectively). Similarly, cleavage (30.8 ± 7.8% vs. 10.3 ± 6.1%, respectively) and blastocyst development rates (9.4 ± 5.0% vs. 0.79 ± 0.79, respectively) were not different (p > 0.05) between vitrified Ban and Landrace × Large white oocytes after in vitro fertilization and embryo culture. In conclusion, high survival and maturation rates were achieved after vitrification of immature Ban oocytes and their cryo‐tolerance was similar to that of Landrace × Large white oocytes, despite the difference in lipid content. We succeeded to generate reasonable rates of blastocysts from vitrified Ban oocytes by in vitro fertilization.     

Laminin‐111 Inhibits Bovine Fertilization but Improves Embryonic Development in vitro,and Receptor Integrin‐β1 is Involved in Sperm–Oocyte Binding

This study detected the distribution of laminin during embryonic formation by immunofluorescence. To determine the possible function of laminin on developmental ability of in vitro fertilized embryos, the presumptive zygotes were divided and transferred to CR1aa medium supplemented with different concentrations (0 μg/ml, 5 μg/ml, 10 μg/ml and 20 μg/ml) of laminin. To explore the association with sperm–oocyte fusion, oocytes and/or sperm were pre‐incubated with laminin or anti‐β1 antibody before insemination. Laminin was absent in mature oocytes and could be detected first at the 8‐cell stage and then displayed an increasing tendency. Adding 10 μg/ml laminin to the culture medium improved embryonic development including cleavage rate, blastocyst rate, total cell numbers in the blastocyst and cell numbers in the inner cell mass. Laminin inhibited sperm–oocyte fusion when incubated with oocytes and/or sperm before in vitro fertilization, and only integrin‐β1 of sperm was involved in sperm–oocyte binding. Inhibition may be caused by blocking β1, but why laminin inhibits fertilization is still unknown. The results suggest that laminin plays an important role during embryonic formation and has a negative function in sperm–oocyte fusion, but improves embryonic development. However, only integrin‐β1 is involved in sperm–oocyte binding.     

Function of the Cumulus Oophorus Before and During Mammalian Fertilization

Fertilization encompasses a series of different steps which have to be performed in a well‐orchestrated way to create a new individual. They include sperm capacitation, sperm binding and penetration of the zona pellucida, traversing the perivitelline space, binding and fusion with the oolemma, activation of the oocyte and decondensation of the sperm head to form the male pronucleus. In most mammalian species, cumulus cells surround the oocyte at the time of fertilization. Removal of the cumulus oophorus at this point of time often leads to a drop in fertilization rates. It is not yet known how cumulus cells interact with the oocyte or with spermatozoa to promote fertilization. There are different possibilities:

• 1 cumulus cells cause mechanical entrapment of spermatozoa and guide hyperactivated spermatozoa towards the oocyte, while preventing abnormal spermatozoa to enter the cumulus matrix;
• 2 cumulus cells create a micro‐environment for the spermatozoa which favours their capacitation and penetration into the oocyte;
• 3 cumulus cells prevent changes in the oocyte which are unfavourable for normal fertilization; these changes can be located in the zona pellucida or in the cytoplasm.

In this review, studies in several species are listed to prove the importance of these three cumulus cell functions and the current lines of research are highlighted. Moreover, different ways to improve in vitro fertilization of bovine cumulus‐denuded oocytes are discussed.     

Oocyte and Embryo Quality: Effect of Origin, Culture Conditions and Gene Expression Patterns

In general, the majority of immature bovine oocytes fail to develop to the blastocyst stage following maturation, fertilization and culture in vitro. The evidence suggests that while culture conditions during in vitro embryo production can impact on the developmental potential of the early embryo, the intrinsic quality of the oocyte is the key factor determining the proportion of oocytes developing to the blastocyst stage. In addition, evidence suggests that the period of post‐fertilization embryo culture is the most critical in determining blastocyst quality. This paper reviews the current literature, with emphasis on the bovine model, demonstrating evidence for an effect of oocyte origin and/or in vitro maturation conditions on the developmental capacity and gene expression patterns in the oocyte. Furthermore, the well‐documented effects of post‐fertilization culture environment on embryo gene expression and quality are highlighted.     

犬科动物繁殖生物技术研究进展

犬科动物繁殖生物技术的研究远远落后于其他哺乳动物,因为犬科动物为季节性单次发情,其生殖生理有独特性,这使得犬科动物繁殖生物技术面临着一些困难,尤其是卵子和胚胎在体外培养及发育的效率很低。本文综述了犬科动物卵母细胞的成熟培养(IVM)、体外受精(IVF)和体外胚胎生产(IVP)、精液冷冻和人工授精、胚胎移植及克隆等的研究进展。     

Vitrification procedure decreases inositol 1,4,5‐trisphophate receptor expression,resulting in low fertility of pig oocytes

Although cryopreservation of mammalian oocytes is an important technology, it is well known that unfertilized oocytes, especially in pigs, are highly sensitive to low temperature and that cryopreserved oocytes show low fertility and developmental ability. The aim of the present study was to clarify why porcine in vitro matured (IVM) oocytes at the metaphase II (MII) stage showed low fertility and developmental ability after vitrification. In vitro matured cumulus oocyte complexes (COCs) were vitrified with Cryotop and then evaluated for fertility through in vitro fertilization (IVF). Although sperm‐penetrated oocytes were observed to some extent (30–40%), the rate of pronuclear formation was low (9%) and none of them progressed to the two‐cell stage. The results suggest that activation ability of cryopreserved oocytes was decreased by vitrification. We examined the localization and expression level of the type 1 inositol 1,4,5 trisphosphate receptor (IP₃R1), the channel responsible for Ca²⁺ release during IVF in porcine oocytes. Localization of IP₃R1 close to the plasma membrane and total expression level of IP₃R1 protein were both decreased by vitrification. In conclusion, our present study indicates that vitrified‐warmed porcine COCs showed a high survival rate but low fertility after IVF. This low fertility seems to be due to the decrease in IP₃R1 by the vitrification procedure.     

猪体外受精中钙调控机理研究

本研究探讨了猪精子体外获能、卵母细胞成熟和受精过程中钙调控机制,并用X射线微分析仪,测定了钙的含量变化和分布状态.结果发现,猪精子获能后,质膜表面钙含量降低,而顶体内部钙升高,并诱发顶体反应,发生囊泡化;中段线粒体基质内的钙较获能前高;A类卵母细胞经体外成熟培养后,其质膜上钙含量升高,而B、C类卵母细胞钙变化却降低.卵子受精后,质膜上钙含量明显升高,分布状态也发生相应变化,受精后20h,质膜上的钙呈集团分布.     

Glycine ameliorates mitochondrial dysfunction caused by ABT-199 in porcine oocytes

Mitochondria play an important role in controlling oocyte developmental competence. Our previous studies showed that glycine (Gly) can regulate mitochondrial function and improve oocyte maturation in vitro. However, the mechanisms by which Gly affects mitochondrial function during oocyte maturation in vitro have not been fully investigated. In this study, we induced a mitochondrial damage model in oocytes with the Bcl-2-specific antagonist ABT-199. We investigated whether Gly could reverse the mitochondrial dysfunction caused by ABT-199 exposure and whether it is related to calcium regulation. Our results showed that ABT-199 inhibited cumulus expansion, decreased the oocyte maturation rate and the intracellular glutathione (GSH) level, caused mitochondrial dysfunction, which was confirmed by decreased mitochondrial membrane potential (ΔΨm) and the expression of mitochondrial function-related genes PGC-1α, and increased reactiveoxygenspecies (ROS) levelsand the expression of apoptosis-associated genes Bax, Caspase-3, and Cyto C.More importantly, ABT-199-treated oocytes showed an increase in the intracellular free calcium concentration ([Ca²⁺]i) and had impaired cortical type 1 inositol 1,4,5-trisphosphate receptors (IP₃R1) distribution. Nevertheless, treatment with Gly significantly ameliorated mitochondrial dysfunction, oxidative stress, and apoptosis, and Gly also regulated [Ca²⁺]i levels and IP₃R1 cellular distribution, which further protects oocyte maturation in ABT-199-induced porcine oocytes.Taken together, our results indicate that Gly has a protective action against ABT-199-induced mitochondrial dysfunction in porcine oocytes.     

Sucrose assists selection of high‐quality oocytes in pigs

We investigated whether high‐quality in vitro matured (IVM) oocytes can be distinguished from poor ones based on the morphological changes after treatment with hyperosmotic medium containing 0.2 mol/L sucrose in pigs. We hypothesize that IVM oocytes maintaining round shape have higher quality than mis‐shapened oocytes following dehydration. Oocyte quality was verified by determining embryonic developmental competence using in vitro fertilization, nuclear transfer and parthenogenetic activation. In all cases, the round oocytes had greater (p < .05) developmental competence than that of mis‐shapened oocytes in terms of blastocyst rate and total cell number in blastocysts obtained after 6 days of in vitro culture. We also confirm that round aged oocytes are higher in quality than mis‐shapened aged oocytes. In an attempt to find out why high‐quality oocytes maintain a round shape whereas poorer oocytes become mis‐shapened following sucrose treatment, we examined the arrangement of actin microfilaments and microtubules. Abnormal organization of these cytoskeletal components was higher (p < .05) in mis‐shapened oocytes compared to round oocytes after 52 hr of IVM. In conclusion, sucrose treatment helps selection of high‐quality oocytes, including aged oocytes, in pigs. Abnormal cytoskeleton arrangements partly explain for low developmental competence of mis‐shapened oocytes.