Progesterone treatment of boar spermatozoa improves male pronuclear formation after intracytoplasmic sperm injection into porcine oocytes

Boar spermatozoa were prepared for intracytoplasmic sperm injection (ICSI) by two different treatments to facilitate sperm chromatin decondensation and improve fertilisation rates after ICSI in pigs: spermatozoa were either frozen and thawed without cryoprotectants, or treated with progesterone. Morphological changes of the sperm heads after the treatments were examined and then the activation of oocytes and the transformation of the sperm nucleus following ICSI were assessed. After freezing and thawing, the plasma membrane and acrosomal contents over the apical region of sperm head were lost in all the spermatozoa. Following treatment with 1 mg/ml progesterone, the acrosome reaction was induced in 61% of spermatozoa. After injection of three types of spermatozoa, non-treated spermatozoa and progesterone-treated (i.e. acrosome-reacted) spermatozoa induced oocyte activation, but frozen-thawed spermatozoa induced oocyte activation at a significantly lower rate. Sixty-two per cent of sperm heads remained orcein-negative for 6 h, however, resulting in delayed sperm chromatin decondensation and low male pronuclear formation in the oocytes injected with a non-treated spermatazoon. Since the treatments of freezing and thawing and progesterone for spermatozoa accelerated the initial change in sperm chromatin and the latter treatment induced oocyte activation earlier, it is considered that the delay in oocyte activation and decondensation of sperm chromatin after injection of non-treated spermatozoa is caused by the existence of the sperm plasma membrane. These results show that progesterone treatment efficiently induces the acrosome reaction in boar spermatozoa without destroying their potency for oocyte activation, and the induction of the acrosome reaction results in the promotion of male pronuclear formation after ICSI.     

A combined treatment of ionomycin with ethanol improves blastocyst development of bovine oocytes harvested from stored ovaries and microinjected with spermatozoa

Regardless of the presence of sperm-borne oocyte-activating factors, activation of bovine oocytes with exogenous activation stimuli is required for further development after intracytoplasmic sperm injection (ICSI). The current study was designed to develop a new activation regimen for improving the blastocyst yield after ICSI of bovine oocytes harvested from ovaries stored at 10 to 12 °C for 24 h. After ICSI, oocytes were treated with 5 μM ionomycin for 5 min, 7% ethanol for 5 or 10 min, ionomycin followed by ethanol (5 or 10 min), ionomycin followed by 10 μg/mL cycloheximide for 5 h, or ionomycin followed by 1.9 mM 6-dimethylaminopurine for 3 h. Across the activation regimens, the cleavage rates of ICSI oocytes (45% to 77%) were higher than those of parthenogenetically activated oocytes (11% to 21%; P < 0.05). Activating the ICSI oocytes with ionomycin plus ethanol improved the blastocyst yield (29% to 30%) compared with that of nontreated oocytes (12%; P < 0.05), but the other regimens did not improve the blastocyst yield (9% to 18%; P > 0.05). Higher blastocyst yields were due to increasing the proportion of ICSI oocytes that passed through the early postfertilization events until cleavage. None of the regimens have any adverse effect on the quality of the blastocysts regarding the total cell number or the proportion of the inner cell mass cells. Thus, a new activation regimen using two triggers for single calcium increase effectively improved blastocyst yield after bovine ICSI using oocytes harvested from stored ovaries.     

Changes in subpopulations of boar sperm defined according to viability and plasma and acrosome membrane status observed during storage at 15 degrees C

Four boar ejaculates were preserved for 42 d at 15 °C to examine the changes produced in the quality of sperm membranes according to their response to a combined short hypoosmotic swelling test (sHOST) plus viability test designated the sHV test. Every 1 or 2 d, a sample from each ejaculate preserved in long-term extender was subjected to sperm motility determination and the sHV test. Through simultaneous examination by phase contrast and fluorescence microscopy, three subpopulations of sperm were identified according to their response to sHOST challenge and their viability status. In the subpopulations scoring positive in the sHOST, a further four sperm subpopulations were defined according to their viability and acrosome status. All the sperm subpopulations differed in terms of changes in their proportions produced during the course of preservation and individual differences among ejaculates were detected in terms of relationships shown among subpopulations. The combined sHOST/viability test was able to identify sperm subpopulations with the strongest plasma and acrosome membranes as well as a subpopulation of sperm that had undergone a true acrosome reaction.     

In vitro characteristics of frozen-thawed, sex-sorted bull sperm after refreezing or incubation at 15 or 37 °C

The objective was to determine the in vitro characteristics of frozen-thawed dairy bull sperm after sex-sorting and refreezing and thawing (0, 2, and 4 h post-thaw; 37 °C) or post-sort incubation at 15 or 37 °C for 30 and 24 h, respectively. These sperm were compared with nonsorted frozen-thawed sperm (control) and with nonsorted sperm undergoing two cryopreservation procedures (FF; 0, 2, and 4 h). Frozen-thawed sex-sorted (FS) sperm maintained at 15 or 37 °C had higher (P < 0.001) progressive motility (PM), velocity, mitochondrial function, viability, and acrosome integrity than that of control sperm but similar total motility at 0 and 2 h of incubation. Frozen-thawed sex-sorted sperm incubated at 15 °C maintained high levels of motility (66.5 ± 1.6%) and viability/acrosome integrity (64.9 ± 1.2%) at 24 h incubation and, after rewarming and further 6 h incubation at 37 °C, acceptable levels of motility (35.8 ± 1.6%) and viability/acrosome integrity (51.2 ± 1.2%) were maintained. Frozen-thawed sex-sorted sperm maintained at 37 °C had lower levels of motility, integrity, mitochondrial respiration, and velocity from 4 h of incubation onward than that of those incubated at 15 °C. However, when frozen-thawed sex-sorted sperm were refrozen (FSF), motility and velocity were depressed at all hours compared with levels exhibited by control sperm, but membrane viability/acrosome integrity and mitochondrial respiration were similar at 0 and 2 h post-thaw. Acrosome integrity of sperm in all groups undergoing sorting was exceptionally high at 0 h (≥90%), even after re-cryopreservation and 4 h of incubation (77.5 ± 1.3%). Double frozen-thawed nonsorted sperm (FF) had similar motility to FSF sperm at 0 and 2 h post-thaw but at all time points had the lowest (P < 0.001) levels of acrosome intact/viable sperm and mitochondrial respiration and the lowest velocity at 0 h. In conclusion, whereas sex-sorting improved the functionality of frozen-thawed sperm, refreezing depressed motility, viability, and velocity but not acrosome integrity after extended incubation compared with that of control sperm. Furthermore, frozen-thawed, sex-sorted sperm may be stored for transport at 15 °C for up 24 h without detrimental effects on in vitro sperm characteristics.     

Fertilization of C57BL/6 mouse sperm collected from cauda epididymides after preservation or transportation at 4 °C using laser-microdissected oocytes

The C57BL/6 mouse is commonly used to produce transgenic and knockout strains for biomedical research. However, the motility and fertility of its sperm decrease markedly with freezing. Short-term preservation of sperm without freezing can avoid this. Furthermore, such samples can be transported safety without the special skills or equipment needed for the transportation of live animals or frozen products. We evaluated the motility and fertility of sperm collected from cauda epididymides after preservation or transportation at 4 °C. Oocytes with the zona pellucida subjected to laser-microdissection were used to assist fertilization in vitro. Although the motility of sperm gradually decreased with storage (P < 0.05), no disruption of the sperm plasma membrane was seen. The proportion of zona-intact oocytes fertilized with sperm preserved for 0, 24, 48 and 72 h were 70, 14, 5 and 1%, respectively. On the other hand, 45, 20 and 14% of laser-microdissected oocytes were fertilized by sperm preserved for 24, 48 and 72 h, respectively (P < 0.05). The fertility of sperm collected from cauda epididymides of two transgenic strains after transportation at 4 °C were also significantly increased using laser-microdissected oocytes rather than zona-intact oocytes (57 and 68% vs. 5%, P < 0.05). Efficient production of offspring from sperm preserved or transported at 4 °C was achieved using laser-microdissected oocytes. Thus the fertility of sperm preserved or transported at 4 °C could be maintained, although motility gradually decreased with storage. Laser-microdissected oocytes will contribute to the efficient production of embryos and offspring using such preserved sperm samples.     

Effects of oocyte activation and sperm preparation on the development of porcine embryos derived from in vitro-matured oocytes and intracytoplasmic sperm injection

The objective was to determine the effects of various methods of oocyte activation and sperm pretreatment on development of porcine embryos derived from in vitro-matured oocytes and intracytoplasmic sperm injection (ICSI). The second polar body was extruded in the majority (>78.4%) of in vitro-matured (IVM) oocytes 4h after electrical pulse activation. In embryos generated by ICSI and sham-ICSI, a combination of an electrical pulse, with various chemical activators 4 h later, improved (P < 0.05) blastocyst formation rate compared to activation only with a pulse. Treatment with 6-dimethylaminopurine (DMAP) after electrical activation significantly increased the oocyte activation rate. The effects of exposure of sperm to repeated freeze-thaw cycles (without cryoprotectant) on oocyte activation and the effects of sperm pre-incubated with dithiothreitol (DTT) or Triton X-100 on early embryo development were also examined. Blastocyst formation rates after ICSI did not differ between motile sperm and those rendered immotile by one-time freezing and thawing without cryoprotectant. However, sperm rendered immotile by three cycles of freezing/thawing without cryoprotectant had a significantly lower blastocyst formation rate. Although oocytes injected with sperm pre-incubated with Triton X-100 had a higher normal fertilization rate than those pre-incubated with DTT or one-time frozen/thawed sperm, rates of blastocyst formation and cell numbers were similar among the three groups. In conclusion, various methods of oocyte activation and sperm preparation significantly affected the developmental capacity of early porcine embryos derived from IVM and ICSI.     

Factors affecting fertilization of porcine oocytes following intracytoplasmic injection of sperm

The objective of this study was to optimize intracytoplasmic sperm injection (ICSI), and to assess the effects of membrane-damaged sperm on development of porcine oocytes following ICSI. For optimization of development following the ICSI process, sperm injected oocytes were activated 0.5-1.0 hr after ICSI with 1 x 30 micros pulse of 1.2, 1.7, 2.2, and 2.7 kV/cm DC in experiment 1. After 7-days of culture ICSI oocytes activated with [x1]2.2 kV/cm produced more blastocyts ([x2]34.4%, P < 0.05) than other treatment groups. In experiment 2, oocytes were activated with 1 x 30 micros pulse of 2.2 kV/cm at either 0, 1.5, 3, or 6 hr after ICSI. Oocytes activated 1.5 hr after [x3]ICSI yielded more blastocysts (27.6%)[M4] than in other treatments. In experiment 3, sperm were briefly exposed to 0.1% Triton X-100 to induce membrane damage. Live-dead staining of Percoll-sorted untreated spermatozoa (frozen-thawed) used in this study showed that over 96% were "alive" whereas none were "alive" after Triton X-100 treatment. The rate of development to blastocyst of oocytes injected with Triton X-100 treated sperm combined with electrical activation (EA) at 1 x 30 micros pulse of 2.2 kV/cm (EA, 40.0%) was the best, when compared with those injected with untreated sperm plus EA (P < 0.05). In experiment 4, the development rate of oocytes to the blastocyst stage ([x5]32.1%) following injection of a sperm head only was not significantly different from that of oocytes injected with whole sperm (31.0%). In conclusion, we found that an intact membrane and tail structures of pig spermatozoa are not essential for embryo development by ICSI, and furthermore, dead porcine spermatozoa, at an early stage of necrosis caused by plasma membrane damage, support better embryo development than do live non-damaged sperm.     

Intracytoplasmic sperm injection in bovine: effects of oocyte activation, sperm pretreatment and injection technique

Intracytoplasmic sperm injection (ICSI) is a very important technique for treating male subfertility and for basic research. The efficiency of ICSI in bovine is very limited because of the necessity for additional oocyte activation before or after the ICSI procedure. In this study, we compared the effects of seven different protocols on activation and fertilization rates of bovine oocytes after ICSI and on their subsequent development under in vitro conditions. The protocols include 1) different chemical activation of oocytes, 2) pretreated or nonpretreated sperm, and 3) conventional or Piezo-driven injection techniques. In all three groups, ICSI, sham-injected, and noninjected, the highest activation rates were obtained after treatment of oocytes with ionomycin followed by 6-dimethylaminopurine (6-DMAP). Using this treatment for oocyte activation, 59% of oocytes were activated and 31% of oocytes were fertilized using dithiothreitol (DTT) pretreated spermatozoa and Piezo-driven injection. Using the protocols with the same oocyte activation or activation with calcium ionophore (Ca-I) and cycloheximide (CHX), nonpretreated sperm, and conventional injection technique, early cleavage rate (79.6% and 77.6%, respectively) were significantly (P <0.01) higher when compared with all other protocols. The latter protocol resulted in 8% blastocyst and 90% of the obtained blastocysts were found to be diploid. Our results demonstrate that activation of oocytes, sperm treatment, and injection technique separately or together could improve the success of bovine ICSI.     

Generation of porcine diploid blastocysts after injection of spermatozoa grown in nude mice

It is anticipated that the utilization of spermatogonia through testicular xenografting will open new avenues for the conservation of male gametes. With the aim of establishing this new technique for genetic preservation of pigs, we used it in combination with intracytoplasmic sperm injection (ICSI). Testicular tissues derived from neonatal piglets, which contained seminiferous cords consisting of only gonocytes/spermatogonia, were transplanted under the back skin of castrated nude mice. Between 125 and 192 d after xenografting, sperm (morphologically similar to epididymal sperm) were recovered from 41 of the 65 host mice (63.1%). Testicular spermatozoa from adult boars were used as a positive control. A single spermatozoon was injected into an in vitro matured porcine oocyte, and the oocytes were electro-stimulated and cultured (graft-ICSI and testis-ICSI, respectively). Blastocyst rates in both ICSI groups (24.9% and 37.4%, respectively) were higher (P < 0.05) than those without the injection procedure (parthenogenetic; 12.7%) and after injection of a small amount of injection buffer (sham; 13.0%). Rates of diploid blastocysts in both graft-ICSI and testis-ICSI groups (48.9% and 60.6%) were higher (P < 0.05) than those in the parthenogenetic and sham groups (13.5% and 28.0%). Therefore, we demonstrated that porcine oocytes injected with xenogeneic sperm have in vitro developmental ability to the blastocyst stage.     

Viability of ICSI oocytes after caffeine treatment and sperm membrane removal with Triton X-100 in pigs

Non-adequate decondensation of injected sperm nucleus is one the main problems of intracytoplasmic sperm injection (ICSI) in porcine. With the aim of improving pronuclear formation, the effects on activation and embryo development rates of 0.1% Triton X-100 (TX) sperm pre-treatment for membrane removal and/or 5 mM Caffeine (CAF) addition in oocyte manipulating and culture medium for 2 h after ICSI or artificial activation were studied. The effects of 4 different Ca²⁺ concentrations contained in the injection medium on embryo development after sham injection were also analysed. In Experiment 1, no significant effect on cleavage or blastocyst rate was detected independently of Ca²⁺ concentration contained in the injection medium. In Experiment 2, oocytes injected with TX pre-treated sperm showed a significant higher rate of male pronuclear formation in comparison with oocytes from control group (2PN; 54.1 vs 36.6%). However, no differences on in vitro embryo development, cleavage or blastocyst rates were observed. In Experiment 3, oocytes treated with CAF during and after micromanipulation and injected with sperm pre-treated with TX had a significantly lower oocyte activation rate than any other experimental groups (25.7 vs 56.3–66.3%). No differences were observed in cleavage rates among different experimental groups. However, the CAF group showed a higher blastocyst rate significantly different from TX+CAF group (12.0 vs 1.9%, respectively). In a second approach, the effect of electric field strengths and CAF treatments on oocyte activation was studied. In Experiment 4, oocytes submitted to 0.6 kV/cm showed significant higher activation rates than 1.2 kV/cm ones regardless of the caffeine treatment (83.7 vs 55.9% and 75.7 vs 44.3%; in control and caffeine groups, respectively). No effect of caffeine treatment was observed in any experimental group. In conclusion, TX sperm treatment before ICSI without an additional activation procedure improved male pronuclear formation, but did not improve embryo development until blastocyst stage. No significant effect of caffeine was found when sperm was not treated with TX, although in membrane absence caffeine avoided oocyte activation and embryo development. Finally, caffeine had no effect on female pronuclear formation regardless of electric field strengths applied to the parthenogenetic activation.     

Viability of ICSI oocytes after caffeine treatment and sperm membrane removal with Triton X-100 in pigs

Non-adequate decondensation of injected sperm nucleus is one the main problems of intracytoplasmic sperm injection (ICSI) in porcine. With the aim of improving pronuclear formation, the effects on activation and embryo development rates of 0.1% Triton X-100 (TX) sperm pre-treatment for membrane removal and/or 5 mM Caffeine (CAF) addition in oocyte manipulating and culture medium for 2 h after ICSI or artificial activation were studied. The effects of 4 different Ca²⁺ concentrations contained in the injection medium on embryo development after sham injection were also analysed. In Experiment 1, no significant effect on cleavage or blastocyst rate was detected independently of Ca²⁺ concentration contained in the injection medium. In Experiment 2, oocytes injected with TX pre-treated sperm showed a significant higher rate of male pronuclear formation in comparison with oocytes from control group (2PN; 54.1 vs 36.6%). However, no differences on in vitro embryo development, cleavage or blastocyst rates were observed. In Experiment 3, oocytes treated with CAF during and after micromanipulation and injected with sperm pre-treated with TX had a significantly lower oocyte activation rate than any other experimental groups (25.7 vs 56.3-66.3%). No differences were observed in cleavage rates among different experimental groups. However, the CAF group showed a higher blastocyst rate significantly different from TX+CAF group (12.0 vs 1.9%, respectively). In a second approach, the effect of electric field strengths and CAF treatments on oocyte activation was studied. In Experiment 4, oocytes submitted to 0.6 kV/cm showed significant higher activation rates than 1.2 kV/cm ones regardless of the caffeine treatment (83.7 vs 55.9% and 75.7 vs 44.3%; in control and caffeine groups, respectively). No effect of caffeine treatment was observed in any experimental group. In conclusion, TX sperm treatment before ICSI without an additional activation procedure improved male pronuclear formation, but did not improve embryo development until blastocyst stage. No significant effect of caffeine was found when sperm was not treated with TX, although in membrane absence caffeine avoided oocyte activation and embryo development. Finally, caffeine had no effect on female pronuclear formation regardless of electric field strengths applied to the parthenogenetic activation.     

“This is where it all started” – the pivotal role of PLCζ within the sophisticated process of mammalian reproduction: a systemic review

Mammalian reproduction is one of the most complex and fascinating biological phenomenon, which aims to transfer maternal and paternal genetic material to the next generation. At the end of oogenesis and spermatogenesis, both haploid gametes contain a single set of chromosomes ready to form the zygote, the first cell of the newly developing individual. The mature oocyte and spermatozoa remain in a quiescent state, during which the oocyte is characterized by nuclear and cytoplasmic arrest, while the spermatozoa necessitates further maturation within the epididymis and female reproductive track prior to egg fertilization. Either in vivo or in vitro, the sperm initiates a series of irreversible biochemical and physiological modifications in the oocyte. The earliest detected signal after fertilization is cytosolic Ca²⁺ oscillations, a prerequisite step for embryo development. These oscillations trigger the release of the oocyte from the second meiosis arrest towards embryogenesis, also known as “oocyte activation”. Phospholipase C zeta (PLCζ) is a unique sperm-soluble protein responsible for triggering the InsP₃/Ca²⁺ pathway within the oocyte, leading to Ca²⁺ oscillations and consequently to embryo development. The specific structure of PLCζ (compared to other PLCs) enables its specialized activity via the preserved X and Y catalytic domains, as well as distinct features such as rapid onset, high sensitivity to Ca²⁺ and cession of oscillations upon zygote formation. The emerging discoveries of PLCζ have stimulated studies focusing on the possible clinical applications of this protein in male infertility evaluation and management during IVF/ICSI. Fertilization failure is attributed to lack of oocyte second meiosis resumption, suggesting that ICSI failure may be related to impaired PLCζ activity. Microinjection of recombinant human PLCζ to human oocytes after ICSI fertilization failure may trigger Ca²⁺ oscillations and achieve successful fertilization, offering new hope for couples traditionally referred to sperm donation. However, more studies are still required prior to the routine implementation of this approach in the clinic. Directions for future studies are discussed.     

Effect of sperm immobilisation and demembranation on the oocyte activation rate in the mouse.

To analyse the effect of the state of the sperm plasma membrane on oocyte activation rate following intracytoplasmic sperm injection (ICSI), three types of human and mouse spermatozoa (intact, immobilised and Triton X-100 treated) were individually injected into mouse oocytes. At 30, 60 and 120 min after injection, maternal chromosomes and sperm nuclei within oocytes were examined. Following human sperm injection, the fastest and the most efficient oocyte activation and sperm head decondensation occurred when the spermatozoa were treated with Triton X-100. Intact spermatozoa were the least effective in activating oocytes. Thus, the rate of mouse oocyte activation following human sperm injection is greatly influenced by the state of the sperm plasma membrane during injection. When mouse spermatozoa were injected into mouse oocytes, the rates of oocyte activation and sperm head decondensation within activated oocytes were the same irrespective of the type of sperm treatment prior to injection. We witnessed that live human spermatozoa injected into moue oocytes often kept moving very actively within the ooplasm for more than 60 min, whereas motile mouse spermatozoa usually became immotile within 20 min after injection into the ooplasm. In 0.002% Triton X-100 solution, mouse spermatozoa are immobilised faster than human spermatozoa. These facts seem to suggest that human sperm plasma membranes are physically and biochemically more stable than those of mouse spermatozoa. Perhaps the physical and chemical properties of the sperm plasma membrane vary from species to species. For those species whose spermatozoa have 'stable' plasma membranes, prior removal or 'damage' of sperm plasma membranes would increase the success rate of ICSI.     

精子和卵母细胞质量控制对山羊卵胞质内精子注射（ICSI）受精的影响

ABSTRACT Effects of sperm and oocyte quality control on the efficiency of ICSI of in vitro matured goat oocytes were studied in this paper. The results showed that when injected intracytoplasmically, spermatozoa from caput, corpus and cauda epididymidis resulted in similar rates of fertilization, cleavage and morulae/blastocysts, but when injected subzonally, spermatozoa from caput and corpus gave rise to significantly lower rates of fertilization and embryo development than spermatozoa from the cauda epididymidis and ejaculates. When dead spermatozoa collected from semen that had been preserved in different ways were used for ICSI, those dead from liquid storage at 20 degrees C for 24 h gave rise to the best, but those dead from liquid storage at 5 degrees C for 15 days produced the poorest fertilization and embryo development. When spermatozoa were treated with different concentrations of Triton X-100 before ICSI, significantly higher rates of fertilization, cleavage and morulae/blastocysts were obtained with 0.0005% Triton X-100 than with other concentrations and manual immobilization. Oocytes were classified as of good and poor qualities by treatment in hypertonic sucrose solution, and rates of fertilization and embryo development were significantly higher in the good than in the poor oocytes after ICSI. Post-injection activation of oocytes with either A23187 or ionomycin/6-DMAP significantly increased the rates of fertilization, cleavage and morulae/blastocysts after ICSI. It is therefore concluded that (i) epididymal maturation mainly endowed spermatozoa with the capacity to fuse with the egg plasma membrane; (ii) different methods of semen storage caused different impairment of sperm fertilizing capacity; (iii) pre-injection treatment of spermatozoa with proper concentrations of Triton X-100 might be used to replace manual immobilization for ICSI; (iv) oocyte quality was a major factor influencing the efficiency of ICSI; (v) post-injection activation treatment of oocytes improved fertilization and embryo development after ICSI.     

Oocyte activation and phospholipase C zeta (PLCζ): diagnostic and therapeutic implications for assisted reproductive technology

ABSTRACT: Infertility affects one in seven couples globally and has recently been classified as a disease by the World Health Organisation (WHO). While in-vitro fertilisation (IVF) offers effective treatment for many infertile couples, cases exhibiting severe male infertility (19-57%) often remain difficult, if not impossible to treat. In such cases, intracytoplasmic sperm injection (ICSI), a technique in which a single sperm is microinjected into the oocyte, is implemented. However, 1-5% of ICSI cycles still fail to fertilise, affecting over 1000 couples per year in the UK alone. Pregnancy and delivery rates for IVF and ICSI rarely exceed 30% and 23% respectively. It is therefore imperative that Assisted Reproductive Technology (ART) protocols are constantly modified by associated research programmes, in order to provide patients with the best chances of conception. Prior to fertilisation, mature oocytes are arrested in the metaphase stage of the second meiotic division (MII), which must be alleviated to allow the cell cycle, and subsequent embryogenesis, to proceed. Alleviation occurs through a series of concurrent events, collectively termed 'oocyte activation'. In mammals, oocytes are activated by a series of intracellular calcium (Ca2+) oscillations following gamete fusion. Recent evidence implicates a sperm-specific phospholipase C, PLCzeta (PLCζ), introduced into the oocyte following membrane fusion as the factor responsible. This review summarises our current understanding of oocyte activation failure in human males, and describes recent advances in our knowledge linking certain cases of male infertility with defects in PLCζ expression and activity. Systematic literature searches were performed using PubMed and the ISI-Web of Knowledge. Databases compiled by the United Nations and World Health Organisation databases (UNWHO), and the Human Fertilization and Embryology Authority (HFEA) were also scrutinised. It is clear that PLCζ plays a fundamental role in the activation of mammalian oocytes, and that genetic, molecular, or biochemical perturbation of this key enzyme is strongly linked to human infertility where oocyte activation is deficient. Consequently, there is significant scope for our understanding of PLCζ to be translated to the ART clinic, both as a novel therapeutic agent with which to rescue oocyte activation deficiency (OAD), or as a prognostic/diagnostic biomarker of oocyte activation ability in target sperm samples.     

Feasibility of sex-sorting sperm from the white and the black rhinoceros (Ceratotherium simum, Diceros bicornis)

The objective of these studies was to investigate the practicality of flow cytometric sex-sorting for spermatozoa from the white and the black rhinoceros (Ceratotherium simum, Diceros bicornis). In Experiment 1, four semen extenders were tested regarding their suitability for liquid preservation of spermatozoa before sorting. Dilution in MES-HEPES-based semen extender followed by incubation generated best sperm quality parameters (motility, viability, and acrosome integrity). In Experiment 2, the effect of staining method (15 °C for 4 to 6 h during transport or 37 °C for 1 to 1.5 h) on sort efficiency and sperm quality was investigated. Staining at 15 °C during transport resulted in a higher percentage of sperm samples showing a resolution of X- and Y-chromosome-bearing populations (60%) compared with that for staining at 37 °C after transport (33%) and resulted in superior sperm integrity after staining (43.8 ± 11.3% vs. 19.6 ± 12.1%). Sort rate was 300 to 700 cells/sec and sort purity, determined for one sorted sample, was 94% for X-chromosome-bearing spermatozoa. In Experiment 3, the highly viscous component of rhinoceros seminal plasma, which complicates the process of sperm sorting, was examined by gel electrophoresis and mass spectrometry. Results suggested a 250-kDa glycoprotein (most likely originating from the bulbourethral gland) to be responsible for the characteristic viscosity of ejaculates. In Experiment 4, viscosity of seminal plasma, as measured by electron spin resonance spectroscopy, was significantly decreased after addition of α-amylase or collagenase (0.5 and 3 IU per 100 μL seminal plasma, respectively) by 28% and 21%, respectively, with no negative effect on sperm characteristics. The results of this study demonstrate for the first time that rhinoceros spermatozoa can be successfully sorted into high-purity X- and Y-chromosome-bearing populations. Furthermore, the successful liquefaction of viscous ejaculates provides the means to greatly improve sort-efficiency in this species.     

Equine spermatozoa stored in the epididymis for up to 96 h at 4 °C can be successfully cryopreserved and maintain their fertilization capacity

After injury or death of a valuable male, recovery of epididymal spermatozoa may be the last chance to ensure preservation of its genetic material. The objective of this research was to study the effect of sperm storage, at 4 °C up to 96 h, in the epididymides obtained from castrated horses and its effect on different functional sperm parameters. Aims were to study the effect of (1) sperm storage on viability and chromatin condensation; (2) pre-incubation of recovered epididymal sperm in the freezing extender, prior cryopreservation, on viability and chromatin condensation; and (3) freezing–thawing on viability, chromatin condensation, ROS generation, protein tyrosine phosphorylation and heterologous fertilization rate (ICSI and IVF using bovine oocytes) of sperm recovered from the epididymis up to 96 h post castration. The average volume (720 ± 159 μL) and the concentration (6.5 ± 0.4 × 10⁹ spermatozoa/mL) of sperm recovered from the epididymis were not affected by storage. Sperm viability after refrigeration at 4 °C for up to72 h was similar (P < 0.01). The effect of sperm dilution in the freezing media showed similar values up to 48 h, while viability was preserved up to 72 h (P < 0.01). Cryopreserved spermatozoa show similar viability between different storage times. Chromatin condensation was not affected by storage time; however, incubation for 30 min in freezing medium and freezing–thawing process induced an increase in the chromatin decondensation. ROS generation was not affected by storage up to 96 h. Epididymal storage did not affect sperm protein tyrosine phosphorylation patterns; although the pattern of phosphorylation changed to strong staining of the equatorial segment when the sperm where capacitated in sperm–TALP. Finally, successful and similar pronuclear formation (analyzed by ICSI) and in vitro penetration (evaluated with bovine zone free oocyte) was observed using cryopreserved sperm obtained from prolong epididymal storage at 4 °C. In conclusion, cryopreservation of epididymal stallion sperm stored for up to 72 h in the epididymis at 4 °C, maintain both viability and ability to fertilize in vitro.     

Recovery of mare oocytes on a fixed biweekly schedule, and resulting blastocyst formation after intracytoplasmic sperm injection

Oocytes may be collected from live mares from either the stimulated preovulatory follicle or from all visible immature follicles. We evaluated the yield of mature oocytes, and of blastocysts after intracytoplasmic sperm injection (ICSI), for both follicle types. In Experiment 1, mares were assigned to Progesterone (1.2 g biorelease progesterone weekly) or Control treatments. Transvaginal aspiration of all follicles was performed every 14 d. Overall, 596 follicles were aspirated, with a 54% oocyte recovery rate. There was no difference between treatments in number of follicles punctured (9.0 to 9.1) or oocytes recovered (4.8 to 5.0) per mare per aspiration session. Of 314 oocytes recovered, 180 (57%) matured in culture. Thirty-six mature oocytes were subjected to ICSI; 33% formed blastocysts (63% per mare per aspiration session). In Experiment 2, the preovulatory follicle was aspirated every 14 d for three to four cycles. Prostaglandin F_2α was given on Days 6 and 7 after aspiration. A follicle ≥25 mm in diameter was present on Day 13, the day of deslorelin administration, in 23 of 24 cycles, and ovulatory response (granulosa expansion) was seen in 24 of 25 follicles aspirated. Blastocyst development after ICSI was 41% per injected oocyte, or an estimated 33% per mare per aspiration session. We concluded that both aspiration of immature follicles and aspiration of the preovulatory follicle can be performed effectively every 14 d without monitoring ovarian follicular growth. As performed in these separate experiments, aspiration of immature follicles provided more blastocysts per aspiration session.     

The testicular and epididymal expression profile of PLCζ in mouse and human does not support its role as a sperm-borne oocyte activating factor

Phospholipase C zeta (PLCζ) is a candidate sperm-borne oocyte activating factor (SOAF) which has recently received attention as a potential biomarker of human male infertility. However, important SOAF attributes of PLCζ, including its developmental expression in mammalian spermiogenesis, its compartmentalization in sperm head perinuclear theca (PT) and its release into the ooplasm during fertilization have not been established and are addressed in this investigation. Different detergent extractions of sperm and head/tail fractions were compared for the presence of PLCζ by immunoblotting. In both human and mouse, the active isoform of PLCζ was detected in sperm fractions other than PT, where SOAF is expected to reside. Developmentally, PLCζ was incorporated as part of the acrosome during the Golgi phase of human and mouse spermiogenesis while diminishing gradually in the acrosome of elongated spermatids. Immunofluorescence localized PLCζ over the surface of the postacrosomal region of mouse and bull and head region of human spermatozoa leading us to examine its secretion in the epididymis. While previously thought to have strictly a testicular expression, PLCζ was found to be expressed and secreted by the epididymal epithelial cells explaining its presence on the sperm head surface. In vitro fertilization (IVF) revealed that PLCζ is no longer detectable after the acrosome reaction occurs on the surface of the zona pellucida and thus is not incorporated into the oocyte cytoplasm for activation. In summary, we show for the first time that PLCζ is compartmentalized as part of the acrosome early in human and mouse spermiogenesis and is secreted during sperm maturation in the epididymis. Most importantly, no evidence was found that PLCζ is incorporated into the detergent-resistant perinuclear theca fraction where SOAF resides.     

The role of Ca2+ in oocyte activation during In Vitro fertilization: Insights into potential therapies for rescuing failed fertilization

At fertilization the mature mammalian oocyte is activated to begin development by a sperm-induced series of increases in the cytosolic free Ca²⁺ concentration. These so called Ca²⁺ oscillations, or repetitive Ca²⁺ spikes, are also seen after intracytoplasmic sperm injection (ICSI) and are primarily triggered by a sperm protein called phospholipase Czeta (PLCζ). Whilst ICSI is generally an effective way to fertilizing human oocytes, there are cases where oocyte activation fails to occur after sperm injection. Many such cases appear to be associated with a PLCζ deficiency. Some IVF clinics are now attempting to rescue such cases of failed fertilization by using artificial means of oocyte activation such as the application of Ca²⁺ ionophores. This review presents the scientific background for these therapies and also considers ways to improve artificial oocyte activation after failed fertilization.