The Employment of Strontium to Activate Mouse Oocytes: Effects on Spermatid-Injection Outcome

REPRODUCTIONRESEARCH

The employment of strontium to activate mouse oocytes: effects on spermatid-injection outcome

Jean Loren and Orly Lacham-Kaplan Monash Immunology and Stem Cell Laboratories, Level 3, STRIP 1- Building 75, Monash University, Wellington Rd., Clayton, Australia, 3800 Correspondence should be addressed to O Lacham-Kaplan; Email: [email protected]

Abstract The present research investigated the effects of various strontium concentrations, in combination with different incubation periods, on mouse parthenogentic oocyte activation and blastocyst development. The results for blastocyst development showed a trend indicating that 10 mM strontium for 3 h was the optimal strontium protocol. Ethanol, an agent that incites oocyte activation via a monotonic rise in calcium, was employed as a control. The outcome of blastocyst formation arising from parthenogenic ethanol activation was significantly less (P < 0.001) than that achieved by the optimal strontium protocol. To assess the impact of strontium oocyte activation on embryo viability following fertilization with immature germ cells, the protocol of 10 mM strontium for 3 h was applied to oocytes injected with round spermatids and then compared with other protocols. The results indicate that following round-spermatid injection the benefits derived from strontium artificial oocyte activation are evident during both pre- and post-implantation development. However, in order to adjust the protocol to the most effective round-spermatid injection in relation to the oocyte cell cycle, injection was done 1.5 h after strontium activation followed by another 1.5 h activation in strontium. The implementation of round-spermatid injection in combination with this oocyte-activation protocol led to live-birth outcomes not significantly different to those outcomes obtained by mature spermatozoa. Reproduction (2006) 131 259–267

Introduction intracellular calcium oscillates continuously throughout oocyte activation (Cuthbertson & Cobbold 1985, Miyazaki Oocyte activation is not a sole event but a series of occur- et al. 1992), continuing until pronucleus formation (Jones rences that initiate the progression of the oocyte from a et al. 1995). It has been suggested that calcium oscil- haploid fertilized cell to a zygote. Mammalian oocyte lations coordinate the many events that occur throughout activation, in nature triggered by fertilization, allows the oocyte to resume meiosis and proceed to embryonic fertilization (Ben-Yosef & Shalgi 2001) and that each cal- development (Carroll 2001). Meiosis is completed with cium oscillation incrementally propels the events of the extrusion of the second polar body and the resulting oocyte activation (Ducibella et al. 2002). Oocyte acti- haploid chromosomal state of the oocyte. vation can, however, occur with a single rise in calcium, Artificially recreating the events of oocyte activation has so the full role of calcium oscillations is unclear (Swann & become an important component of assisted reproduction Ozil 1994). An association has been identified between technology. To allow for the release of the oocyte from calcium oscillations and superior blastocyst composition metaphase II (MII) arrest, effective artificial oocyte acti- (Bos-Mikich et al. 1997). Additionally, the pattern, fre- vation is critical unto itself but it is also critical because of quency, and amplitude of early calcium oscillations the impact it has on later developmental events (Ozil & impact on post-implantation development (Ozil & Huneau Huneau 2001). Suboptimal artificial oocyte activation is 2001). Prevention of calcium oscillations completing their thought to contribute to disappointing outcomes experi- full course during fertilization will retard pronucleus for- enced by many procedures for assisted reproduction tech- mation (Lawrence et al. 1998) and calcium oscillations of nology (Alberio et al. 2001). abnormal frequencies will induce development arrest The earliest notable event in oocyte activation, in all (Gordo et al. 2002). studied species, is an increase in the level of intracellular The paucity of knowledge surrounding the events of calcium (Swann & Ozil 1994). In mammals, the levels of oocyte activation present some challenges for artificial

q 2006 Society for Reproduction and Fertility DOI: 10.1530/rep.1.00894 ISSN 1470–1626 (paper) 1741–7899 (online) Online version via www.reproduction-online.org Downloaded from Bioscientifica.com at 09/25/2021 03:41:55AM via free access 260 J Loren and O Lacham-Kaplan oocyte activation. As calcium is recognized as the most the Australian National Health and Medical Research known fundamental factor of oocyte activation, many Council (NIH & MRC) code of practice for the care and approaches to artificially induce oocyte activation have use of animals for scientific purposes. focused on recreating increases in intracellular calcium. Methods to induce this include the direct injection of cal- Oocyte collection cium into the oocyte (Machaty et al. 1996), an electrical pulse that through phospholipid destabilization creates Hybrid F1 female mice (C57BL female £ CBA male) at 4–6 pores for the influx of extracellular calcium (Sasagawa & weeks of age were superovulated by subcutaneous Yanagimachi 1996), isolation and injection of the sperm injection of 10 IU pregnant mares’ serum gonadotrophin factor thought to be responsible for oocyte activation at (Intervet, Sydney, Australia) followed by subcutaneous fertilization (Fissore et al. 1998), employment of adeno- injection of 10 IU human chorionic gonadotrophin (Inter- vet) 48–50 h later. Female mice were killed by cervical dis- phostin A, an inositol trisphosphate (InsP3) receptor ago- nist, (Brind et al. 2000), promoting of extracelluar calcium location 13–14 h after injection of human chorionic entry via the use of ethanol (Cuthbertson & Cobbold gonadotrophin. The oocytes were released from the ovi- 1985), and inducing calcium oscillations by employing ducts into M2 handling medium containing 100 IU/ml hya- the divalent cation, strontium (Kline & Kline 1992). Alter- luronidase (type IV-S; Sigma Chemical Co., St Louis, MO, natively, protein inhibitors such as cycloheximide bypass USA) for no longer than 5 min to remove cumulus cells. an intracellular rise in calcium and directly induce meio- Morphologically normal MII oocytes were washed in and sis by undermining cyclin B and therefore maturation transferred to warm and equilibrated M16 culture medium promoting factor (MPF) (Bos-Mikich et al. 1995). While and placed into incubation under 5% CO2 in air at 37 8C these may be credible methods for re-enacting oocyte for 20 min before they were used for experiments. activation, none are able to achieve this with the effi- ciency provided by spermatozoa. Oocyte activation with strontium In the mouse, strontium has been successfully employed The amounts of SrCl (Sigma) that were required for differ- in many studies to induce artificial oocyte activation. The 2 ent-strength solutions were dissolved in sterile deionized ability of strontium to provoke calcium oscillations water (JDH Biosciences, Lenexa, KS, USA) to give 10 £ appears to be more physiologically sound than alternative stock solution. For each experiment, fresh strontium culture methods of oocyte activation that produce a monotonic medium was prepared. A volume of 0.1 ml prepared stock rise in calcium. Calcium oscillations induced by strontium solution was placed into an Eppendorf tube (Greiner lead to improved blastocyst composition (Bos-Mikich et al. Labortechnik, Frikenhaussen, Germany) containing 0.99 ml 1997) and superior pre- implantation development calcium-free M16. Microdroplets (20 ml) were arranged (Lacham-Kaplan et al. 2003). The aptitude of strontium onto a 35 mm Petri dish (Falcon, Franklin Lakes, NJ, USA), has lent itself to satisfactory outcomes in a range of mouse covered with mineral oil (Sigma) and incubated under 5% reproductive technologies; however, there is a paucity of CO in air at 37 8C for 30 min. For activation, oocytes were research that has investigated how strontium is best 2 washed twice, cultured in equilibrated strontium solution, applied. Therefore, the potential to exploit the benefits of and left for the allotted amount of time. Following the strontium may be thwarted by a lack of insight concerning oocyte activation period, oocytes were washed twice in its optimal employment. þ Ca2 /M16 where they remained for culture under mineral The aim of the present study was to compare various oil in an atmosphere of 5% CO in air at 37 8C. strontium protocols with the intention of revealing an opti- 2 mal strontium strength and incubation time combination. The research also intended to compare strontium acti- Oocyte activation with ethanol vation with ethanol, an agent that activates oocytes A50ml droplet of 8% ethanol (BDH, Poole, Dorset, through the production of a monotonic rise in calcium. England) in M2 handling medium was placed onto a The development of artificially activated oocytes was com- 35 mm Petri dish (Falcon) and covered with mineral oil. pared with that of in vitro-fertilized oocytes. In addition, Oocytes were transferred into 8% ethanol for 5 min exactly we examined the effects of these activation protocols on Once removed oocytes were washed three times in pre- pre- and post-implantation embryo development following equilibrated M16 in which they remained for culture under injection of immature male germ cells into MII oocytes. mineral oil in an atmosphere of 5% CO2 in air at 37 8C.

Sperm and round-spermatid collection Materials and Methods Hybrid F male mice (C57BL female £ CBA male) at 8–12 Ethics 1 weeks of age were killed by cervical dislocation. The testes All experiments were approved by the Monash University and cauda epididymis were dissected out from the rest of Animal Ethics Committee under application number the reproductive tract. An incision was made in the testis 2003/05. Experiments were conducted in accordance with capsule to allow for the removal of the seminiferous

Reproduction (2006) 131 259–267 www.reproduction-online.org

Downloaded from Bioscientifica.com at 09/25/2021 03:41:55AM via free access Strontium in the activation of mouse oocytes 261 tubules into 2 ml previously equilibrated modified T6 were also used to compare the total number of pups (MT6) medium. The content of the seminiferous tubules resulting from embryo transfer. were liberated by mechanical agitation with forceps, leav- ing a suspension of cells including round spermatids, Experimental design which were used immediately for the experiments. For spermatozoa, a small incision was made in the For all experiments there was a minimum of five repeats, cauda before placing it in 2 ml of equilibrated MT6 with each group being assigned a total of 100 oocytes. medium. The tissue was removed 1 h later. Spermatozoa Following treatment, oocytes underwent two or three 2þ were incubated under 5% CO2 in air at 37 8C and left washes in Ca /M16 20 ml droplets under mineral oil, undisturbed for 2 h for capacitation before they were used before incubation under 5% CO2 in air at 37 8C. Once for in vitro fertilization (IVF) or intracytoplasmic sperm activation and fertilization were established the numbers injection (ICSI). of two-cell, four-cell, morulae and blastocyst embryos were recorded up until day 5. Development was assessed every 24 h at a time dictated by the time of oocyte acti- IVF vation or fertilization on day 1. Approximately 20 oocytes were introduced into a sperm The experiments in this study were as follows. Initially, solution of a concentration of (3–5) £ 106 sperm/ml. After oocytes were exposed to strontium – 1.74, 5, 10 or 2.5 h of co-incubation with sperm, the oocytes were 15 mM – and cultured for 1.5, 3, 6 or 12 h; resulting in 16 removed from the MT6 medium, washed three times and different strontium protocols for comparison. The compari- cultured in equilibrated M16 medium at 37 8Cinan sons were done between different concentrations for each atmosphere of 5% CO2 in air. Oocytes were assessed time point. Within each time frame, three control groups approximately 6 h following IVF treatment for the presence were implemented: an ethanol-treatment group, an IVF of a second polar body and two pronuclei. The presence group and a vehicle control of Ca2þ/M16 containing no of such confirmed fertilization. Zygotes were isolated and strontium. Following activation treated oocytes were incu- their development assessed every 24 h up to 5 days. bated in Ca2þ/M16 for further development. Oocyte activation and development through to the blastocyst stage were assessed every 24 h from activation and fertilization. ICSI and round-spermatid injection (ROSI) Based on the outcome of the first experiments, oocytes A microinjection procedure followed that described in were exposed to a series of strontium concentrations. The detail that by Lacham-Kaplan et al. (2003). Oocytes were oocytes were exposed to a high concentration of stron- injected using a fine glass capillary with a 5 mm internal tium, followed by culture in a lower concentration of diameter, attached to a Piezo microinjection system strontium. The following combinations that were (PiezoDrill; Burleigh Instruments, Burleigh Park, Fishers, employed were (a) 10 mM strontium for 1.5 h followed by NY, USA). Before injection, the heads of the sperm were 1.74 mM strontium for 1.5 h, (b) 10 mM strontium for 3 h separated from the tails by applying a strong Piezo pulse followed by 1.74 mM strontium for 3 h and (c) 10 mM on the neck region. Sperm heads were aspirated and strontium for 3 h followed by 1.74 mM strontium for 6 h. injected individually into the oocytes. Round spermatids The best outcome from the first experiments was used as a were identified by size and morphology as described by control for these protocols. Following activation treat- Kimura & Yanagimachi (1995). The selected cells were ments oocytes were incubated in Ca2þ/M16 for a further individually drawn into an injection pipette and through development. Oocyte activation and development through repeated aspiration the nucleus was isolated from the rest to the blastocyst stage were assessed every 24 h from acti- of the cell before injected into the oocytes. Following vation and fertilization. microinjection, oocytes which survived the injection were Effective activation protocols identified were used in into M16 for incubation under 5% CO2 in air at 37 8C. combination with ROSI. It was anticipated that the Confirmation of fertilization and embryo development of implementation of ROSI following second polar-body oocytes following ICSI or ROSI was undertaken as extrusion might necessitate an interruption to the employ- described above for IVF. ment of strontium culture. The effect of this interruption on blastocyst development was explored. Firstly, the tim- ing of a second polar-body extrusion following artificial Statistical analysis oocyte activation was investigated. Oocytes were acti- The data of activation, fertilization and embryo develop- vated by the optimal strontium protocol, or by ethanol, ment to the blastocyst stage were analysed by x2 test and examined for the presence of a second polar body at using Yates’ correction and analysis of variance tests as 30, 60, 90 and 120 min from the commencement of acti- appropriate. The total number of fertilized and activated vation. Following second polar-body extrusion, oocytes oocytes and blastocysts were compared between different were removed from the strontium culture medium and treatment groups. If the P value was less than or equal to placed into Ca2þ/M2 for 30 min, mimicking the break 0.05 the difference was regarded as significant. x2 tests required for manipulation. Oocytes were then placed www.reproduction-online.org Reproduction (2006) 131 259–267

Downloaded from Bioscientifica.com at 09/25/2021 03:41:55AM via free access 262 J Loren and O Lacham-Kaplan back into strontium culture for the time required to give a Results total time in the best concentration of strontium as identified in experiments 1 and 2. As a control, a group of For all experiments, there was a minimum of five repeats, with each group being assigned a total of 100 oocytes. oocytes was activated using the most optimal protocol Percentages of blastocysts are indicated from the num- identified in experiments 1 and 2 with no interruption. þ ber of oocytes activated or fertilized (Table 1, Fig. 1). Each Following treatment, oocytes were washed in Ca2 /M16 of the strontium and ethanol treatment groups produced and incubated under 5% CO in air at 37 8C. Develop- 2 high rates of oocyte activation and no statistical difference ment was assessed until day 5. Once the impact of a break between treatments were found. Development to blasto- in strontium culture was established, oocytes prior to, or cysts in the control group following IVF was significantly following, ROSI were subjected to different activation higher (P , 0.001) than all oocyte-activation treatments. protocols. Each protocol amounted to an overall time of Progression to the blastocyst stage was not significantly the best protocol and the optimal concentration identified. different between strontium treatment groups within any These protocols were as follows: (a) ROSI in Ca2þ/M2 of the time groups. Overall, incubation in 10 mM stron- followed by incubation in 10 mM Sr2þ/M16 for 3 h; 2þ tium for 3 h produced the highest blastocyst rate of 39%. (b) exposure to 10 mM Sr /M16 for 1.5 h followed by This group was therefore used as a control group for fol- ROSI in Ca2þ/M2 followed by incubation in 10 mM 2þ 2þ lowing experiments. Sr /M16 for 1.5 h; (c) ROSI in Ca /M2 followed by acti- The results show that the higher the concentration of vation in 8% ethanol for 5 min; (d) exposure to 8% ethanol 2þ strontium used the lower exposure time is required to for 5 min followed by ROSI in Ca /M2 1 h later; (e) ICSI obtain the highest development to blastocysts. For (control). Following treatment, oocytes were washed and example, when oocytes were activated for 1.5 h with 2þ cultured in Ca /M16. Fertilization was assessed 6 h later strontium the highest blastocyst development resulted and development was assessed until day 5. from 15 mM strontium, while 1.74 mM strontium attained To identify the effects strontium activation has on in the lowest blastocyst rate (Table 1, Fig. 1A). Following vivo embryo development, embryos at the two-cell stage 12 h strontium treatment, 1.74 mM strontium resulted in were transferred to pseudopregnant female on day 1 of the highest blastocyst development of 11% (Table 1, Fig. pregnancy. The most favourable strontium and ethanol 1D). The lowest blastocyst development from strontium protocols from experiment 3 as indicated by blastocyst activation was 2% produced by 15 mM strontium. outcomes were implemented to activate oocytes to pro- In all activation groups investigated, the decline in duce two-cell ROSI embryos for transfer. Pregnant development was evident between the four-cell to the females were allowed to give birth. The number of pups eight-cell/morula stage (Fig. 1). Whereas activation with born was recorded and their development up to 4 weeks strontium produced high activation followed with a gra- after birth was monitored. A random sample of pups, two dual reduction in development similar in all strontium- of each sex, from each of the strontium ROSI, ethanol treatment groups, ethanol activation found less reliable. ROSI and ICSI groups, were used for fertility assessment. Development to the two- and four-cell stages was not con- At 6 weeks of age, F2 female offspring were allowed to sistent even though the method used was similar for all mate with 8-week-old F1 male mice. At 8 weeks of age experiments. Nonetheless, development to blastocysts was F2 male offspring were allowed to mate with 6-week-old lower than that obtained by strontium at any concen- F1 females. The number of foetuses was examined on tration regardless of whether the development to two- and day 15 of pregnancy. four-cell stages was high.

Table 1 Blastocyst development following activation of mouse oocytes in different strontium concentrations for different periods of time.

Blastocyst development

Strontium

Time (h) 1.74 mM 5 mM 10 mM 15 mM Ethanol IVF Not treated

1.5 Activated 90 (90%) 97 (97%) 97 (97%) 99 (99%) 91 (89%) 98 (98%) 2 (2%) Blastocysts from activated 16a (18%) 20a (21%) 26a (26%) 28a (28%) 4b (4%) 94c (96%) 0 (0%) 3 Activated 77 (77%) 89 (89%) 96 (96%) 96 (96%) 94 (94%) 97 (97%) 1 (1%) Blastocysts from activated 12a (16%) 23a (26%) 37a (37%) 32a (32%) 4b (4%) 87c (87%) 0 (0%) 6 Activated 83 (83%) 92 (92%) 97 (97%) 98 (98%) 89 (89%) 98 (98%) 0 (0%) Blastocysts from activated 19a,e (23%) 12a (13%) 9d,f (9%) 6d,g (6%) 3b (3%) 91c (93%) 0 12 Activated 97 (97%) 98 (98%) 97 (97%) 96 (96%) 89 (89%) 97 (97%) 1 (1%) Blastocysts from activated 11a (11%) 7a (7%) 5a (5%) 2a (2%) 2a (2%) 88c (91%) 0 (0%)

Statistical analyses were done for groups in each time period: a is significant to b (P , 0.01); a, b, d are significant to c (P , 0.001); e is significant to f(P , 0.01) and to g (P , 0.001).

Reproduction (2006) 131 259–267 www.reproduction-online.org

Downloaded from Bioscientifica.com at 09/25/2021 03:41:55AM via free access Strontium in the activation of mouse oocytes 263

A 100 1.74 80 5 60 10 40 15

% embryos 20 Ethanol 0 IVF Activated 2C 4C 8C/Morulae blastocysts Developmental stage

B 100 1.74 80 5 60 10 40 15 20 Ethanol % embryos 0 IVF Activated 2C 4C 8C/Morulae blastocysts Developmental stage

C 100 1.74 80 5 60 10 40 15

% embryos 20 Ethanol 0 IVF Activated 2C 4C 8C/Morulae blastocysts Developmental stage

D 100 1.74 80 5 60 10 40 15

% embryos 20 Ethanol 0 IVF Activated 2C 4C 8C/Morulae blastocysts Developmental stage Figure 1 Overall embryo developmental outcomes following oocyte activation in different strontium concentrations (mM) for different time periods. (A) 1.5 h, (B) 3 h, (C) 6 h, (D) 12 h C, cells.

Exposing the oocytes to high strontium concentrations 36 (86%), of oocytes had extruded their second polar followed by lower ones did not have any positive effect body by 90 min and all by 120 min. Ethanol activation on blastocyst development (Table 2). Hence treatment of resulted in 28 out of 30 (93%) oocytes extruding the oocytes for 3 h in 10 mM strontium was still superior to second polar body by 60 min and all by 90 min. Based on any combination of two different strontium this information, from the 100 oocytes which were concentrations. exposed to strontium but had a break in the exposure for An interruption to strontium culture was implemented 30 min, 98 extruded their second polar body and 92 at 1.5 h due to the observation that this was the time of (94%) from these cleaved to two cells. A total of 27 (28%) second polar-body extrusion and therefore may be an embryos from activated oocytes reached the blastocyst appropriate time at which to conduct ROSI in Ca2þ/M16. stage. From the 100 oocytes that were exposed to 10 mM Extrusion of the ﬁrst polar body following oocyte acti- strontium continuously for 3 h, 95 extruded a second vation varied between strontium and ethanol treatments. polar body, 92 (97%) from these cleaved to two cells and Following 10 mM strontium culture the majority, 31 out of 42 (44%) developed to blastocysts. These results indicate www.reproduction-online.org Reproduction (2006) 131 259–267

Downloaded from Bioscientifica.com at 09/25/2021 03:41:55AM via free access 264 J Loren and O Lacham-Kaplan

Table 2 Blastocyst development of oocytes activated using bi-phase strontium protocols.

Blastocyst development

Strontium protocol... 10 mM 1.5 h ! 1.74 mM 1.5 h 10 mM 3 h ! 1.74 mM 3 h 10 mM 3 h ! 1.74 mM 6 h 10 mM 3 h (control)

Activated 95 (95%) 98 (98%) 98 (98%) 95 (95%) Two-cell from activated 92 (97%) 95 (97%) 92 (94%) 92 (97%) Four-cell from activated 71 (75%) 83 (85%) 82 (84%) 84 (88%) Morula from activated 32 (34%) 31 (32%) 36 (37%) 46 (48%) Blastocyst from activated 29 (31%)b 28 (29%)b 33 (34%)b 42 (44%)a

Significance difference is indicated from blastocyst development between the different protocols: a is significant to b (P , 0.05). that the blastocyst outcome is significantly affected led to four live pups from 49 that were transferred to five (P , 0.05) when the activation of oocytes by strontium is females following ROSI. The number of pups resulting interrupted. Nonetheless, this protocol was the most sui- from ICSI was 21 (32%) out of 66 that were transferred to ted to the needs of ROSI as injection of the oocytes in five females. The number of pups born from ICSI embryos Sr2þ medium was detrimental to their survival. From a was significantly higher (P , 0.001) than that of embryos total of 62 oocytes that were incubated in 10 mM stron- transferred following ethanol activation but not to tium for 1 h and injected in Sr2þ/M2 medium, none sur- embryos transferred after strontium activation. vived. In addition, when 20 oocytes were exposed to Randomly selected offspring were mated to F1 animals 10 mM strontium for 1 h and then underwent ROSI in cal- born following natural mating. A total of two females and cium M2, followed by incubation in 10 mM strontium for two males from each of the newborn groups were mated. 2 h, none developed to the blastocyst stage. These results All females fell pregnant and all males were able to pro- indicate that manipulation during the time where oocytes duce pregnancies. The average litter sizes were 8, 7.5, 6.5, are in the process of moving into telophase and extruding 8.5, 7 and 7.5 for females obtained from strontium acti- the polar body may affect embryo development, possibly vation, males obtained from strontium ROSI activation, due to disruption of the microtubule. females obtained from ethanol activation, males obtained The results from ROSI using strontium activation and from ethanol activation, ICSI females and ICSI males ethanol activation before and after extrusion of the polar respectively. No statistical differences were found between the groups. body are summarized in Table 3. Development to the blastocyst stage was higher when ROSI was performed after the extrusion of the polar body. This was 1 h after ethanol activation and 1.5 h after strontium activation. The best Discussion blastocyst development of 58% was achieved when ROSI The purpose of this study was to identify the optimal stron- was performed after the oocytes extruded their polar body. tium protocol to activate mouse oocytes. To explore this, In this protocol oocytes were activated for 1.5 h followed different protocols that encompassed a range of strontium by ROSI and a continuation of activation in 10 mM stron- dosage and time-exposure combinations were used. tium for a further 1.5 h. This group was also the only group, Assessment of oocyte activation as the presence of a which was not significantly different to the ICSI group. second polar body and a single pronucleus indicated that Details of live-birth outcomes for all groups are indi- each of the groups explored demonstrated a similar ability cated in Fig. 2. From a total of 52 embryos transferred to to activate oocytes. It has been suggested, however, five females the strontium-activation protocol led to 10 that proper indication of oocyte activation extends live pups following ROSI. The ethanol activation protocol beyond second polar-body and pronucleus formation

Table 3 Blastocyst development following ROSI using different activation protocols.

Two-cell Four-cell Morula Blastocyst Protocol Oocyte no. Survived Fertilized (from fertilized) (from fertilized) (from fertilized) (from fertilized)

ROSI ! 3 h in 10 mM Sr2+ 69 51 (75%) 33 (63%) 32 (97%) 29 (91%) 27 (82%) 13a,c (39%) 1.5 h in 10 mM Sr2+ ! ROSI ! 159 100 (63%) 53 (53%) 49 (92%) 45 (85%) 40 (75%) 31c (58%) 1.5 h in 10 mM Sr2+ ROSI ! 8% Ethanol 47 34 (72%) 18 (53%) 18 (100%) 16 (89%) 3 (72%) 8a,c (44%) 8% Ethanol ! ROSI 1 h after 161 100 (62%) 64 (64%) 56 (88%) 49 (77%) 44 (69%) 32a,c (50%) ICSI 178 100 (56%) 80 (80%) 75 (94%) 70 (88%) 67 (84%) 51b,c (71%) No activation 67 50 (75%) 9 (18%) 7 (78%) 6 (67%) 6 (67%) 2d (22%)

Significance was analysed for blastocyst development between the different protocols: a is significant to b (P , 0.01); c is significant to d (P , 0.001).

Reproduction (2006) 131 259–267 www.reproduction-online.org

Downloaded from Bioscientifica.com at 09/25/2021 03:41:55AM via free access Strontium in the activation of mouse oocytes 265

70 ROSI Ethol Ethanol activation induces a high incidence of aneuploidy a 60 ROSI Strontium in haploid embryos following artificial oocyte activation ICSI with ethanol (O’Neill & Kaufman 1989) due to promoting 50 premature postovulatory aging resulting in spindle organ- 40 ization errors and thereby chromosome malsegregation. a Data from the present study also show that embryonic 30 development to the two- and four-cell stages following b 20 ethanol activation is not consistent, contributing to the b fact that ethanol is not beneficial for mouse oocyte 10 activation. 0 The minimum dosage required to induce calcium oscil- % pregnant % pups from total % pups from lations in mouse oocytes is unknown. While Cheek et al. females 2-c transfers pregnant females (1993) reported that 8 mM strontium is not enough to induce calcium oscillations in mouse oocytes, Kline & Figure 2 Births following embryo transfers of two-cell (2-c) embryos from ROSI embryos resulted from strontium activation and from etha- Kline (1992) identified 4.6 mM strontium as able to induce nol (Ethol) activation, and from ICSI. Letters above the bars: a is sig- oocytes oscillations and 10 mM strontium for 2 h to induce nificant to b (P , 0.001). calcium oscillations that were similar to those induced by spermatozoa but lower in frequency and amplitude. Bos- Mikich et al. (1995) also suggested that 10 mM strontium (Lacham-Kaplan et al. 2003). Therefore, development to is the optimal dosage to induce calcium oscillations in the blastocyst stage was examined as a more complete mouse oocytes to result in optimal embryo development. indicator of oocyte activation capability and to help In the present study calcium oscillations were not distinguish between treatments. measured, and therefore it cannot be certain which stron- It appears that blastocyst development reached an opti- tium concentrations were able to stimulate calcium oscil- mal time exposure for each strontium concentration and lations at sufficient levels to support optimal embryo declined once this optimal time exposure was exceeded development By using ROSI we have been able to explore (Fig. 1). For the lowest strontium concentrations explored whether the findings of 10 mM for 3 h exposure can be (1.74 mM), 6 h was optimum while for moderate dosages extrapolated into improved pre-implantation developmen- (5 and 10 mM) of strontium a 3 h time period was opti- tal outcomes in a diploid model and whether this will mum and for high concentrations (15 mM) a 1.5 h time translate to have a positive impact on post-implantation period was optimal, with the best outcome resulting from development. exposure of oocytes to 10 mM strontium for 3 h. Nonethe- Round spermatids are the most immature germ cells to less, the different combinations of time and concentration have acquired a haploid set of chromosomes (Lacham- treatments revealed that exposure of mouse oocytes to Kaplan & Trounson 1997). Their DNA status is identical strontium for 1.5 h is not an effective treatment to result in to mature spermatozoa, making them attractive replace- optimal blastocyst development and longer than 6 h found ments for spermatozoa. Aslam et al. (1998) has reported detrimental to embryo development. Exposure to calcium that of the 648 ROSI attempts worldwide only nine nor- transients (Ozil et al. 2005) for durations of between 15 mal deliveries have occurred, indicating that while poten- and 50 min was not different in inducing activation and tially viable, this is also a highly problematic technique. development to blastocysts when electropermeabilization This has led to the advocacy of complete dismissal of at 2-min intervals was used. Strontium promotes calcium ROSI (Silber et al. 1997) and to the procedure being oscillations through InsP3 receptors (Zhang et al. 2005), completely banned in the UK (Ezeh et al. 1999). In the which take longer time to activate, and hence require context of disappointing outcomes, critiques of ROSI’s longer periods of time to be effective. This may also failings have ensued (Devroey 1998, Silber & Johnson explain why polar-body extrusion after strontium treatment 1998, Vanderzwalmen et al. 1998), with focus being occurred later (1.5 h) than that observed following ethanol placed upon oocyte activation inadequacies in addition activation (1 h). Following the exposure to ethanol, an to genetic concerns. intracellular rise in calcium is attained by facilitating an Suboptimal oocyte activation may be responsible for increase in oocyte membrane permeability to calcium and the low success rates of clinical ROSI (Yanagimachi thereby permitting entry of extracellular calcium into the 2001). Human round spermatids are generally capable of cell (Cuthbertson & Cobbold 1985). Mobilization of intra- inducing effective oocyte activation but the employment cellular calcium is also believed to contribute to the of round spermatids originating from men with spermio- monotonic rise in calcium by ethanol activation (Shiina genesis failure leads to impaired oocyte activation (Tesarik et al. 1993). Thus it is likely that both extracellular and et al. 1998). This could be owing to a low concentration, intracellular calcium similar to electropermeabilization or absence, of the sperm cytosolic factor (Palermo et al. are drawn upon to reach the intracellular calcium 1997, Aslam et al. 1998), or due to the impermeability of threshold level required to activate quicker than strontium. the round-spermatid membrane inhibiting the release of www.reproduction-online.org Reproduction (2006) 131 259–267

Downloaded from Bioscientifica.com at 09/25/2021 03:41:55AM via free access 266 J Loren and O Lacham-Kaplan sperm factor (Palermo et al. 1996) responsible for oocyte Aslam I, Fishel S, Green S, Campbell A, Garratt L, McDermott H activation (Swann 1990). Saunders et al. (2002) proposed et al. 1998 Can we justify spermatid microinjection for severe male factor infertility? Human Reproduction 4 213–222. that the sperm-specific phospholipase-z is the elusive Ben-Yosef D & Shalgi R 2001 Oocyte activation: lessons from human sperm factor. In somatic cells, phospholipase generates infertility. Trends in Molecular Medicine 7 163–169. InsP3 (Berridge 1993) and is likely to do so in the oocyte. Berridge M 1993 Inositol trisphosphate and calcium signaling. Nature 361 315–325. Calcium oscillations within oocytes are attributed to InsP3 activity (Parrington et al. 1998, Halet et al. 2003). Hence, Bos-Mikich A, Swann K & Whittingham D 1995 Calcium oscillations and protein synthesis inhibition synergistically activate mouse strontium, which promotes calcium oscillations through oocytes. Molecular Reproduction and Development 41 84–90. InsP3 receptors in oocytes (Zhang et al. 2005), compensates Bos-Mikich A, Whittingham D & Jones K 1997 Meiotic and mitotic for the lack of cytosolic factor activity in round spermatids. Ca2þ oscillations affect cell composition in resulting blastocysts. Kimura & Yanagimachi (1995) demonstrated that Developmental Biology 182 172–179. superior outcomes could be achieved by injecting round Brind S, Swann K & Carroll J 2000 Inositol 1,4,5-trisphosphate receptors are downregulated in mouse oocytes in response to spermatids into an activated oocyte at the telophase stage sperm or adenophostin A but not to increases in intracellular Ca2þ of the cell cycle. It has been demonstrated that round or egg activation. Developmental Biology 223 251–265. þ spermatids achieve the highest fertilization rates when the Carroll J 2001 The initiation and regulation of Ca2 signalling at fer- oocyte is activated 1 h before ROSI (Kimura & Yanagima- tilization in mammals. Seminars in Cell & Developmental Biology 12 37–43. chi 1995). In the present study, a means to accommodate Cheek T, McGuinness O, Vincent C, Moreton R, Berridge M & this information, while still adhering to the 3-h strontium Johnson M 1993 Fertilization and thimerosal stimulate similar protocol was explored through various protocols. calcium spiking patterns in mouse oocytes but by separate mech- We hypothesized that conducting ROSI before second anisms. Development 119 179–189. polar-body extrusion may have unwittingly inflicted Cuthbertson K & Cobbold P 1985 Phorbol ester and sperm activate mouse oocytes by inducing sustained oscillations in cell Ca2þ. meiotic-spindle disturbance, which has been previously Nature 326 541–542. demonstrated to be damaging to oocytes development Devroey P 1998 Clinical application of new micromanipulative tech- after fertilization (Hardarson et al. 2000). Although it has nologies to treat the male. Human Reproduction 13 (Supplement 3) been identified that mouse oocytes maintain the ability to 112–122. result in a normal embryo if activated 60–80 min before Ducibella T, Huneau D, Angelichio E, Xu Z, Schultz R, Kopf G et al. 2002 Egg-to-embryo transition is driven by differential responses to and after ROSI (Kishigami et al. 2004) in the present Ca2þ oscillation number. Developmental Biology 250 280–291. study, activation of oocytes before introducing the round Ezeh U, Taub N, Moore H & Cooke I 1999 Establishment of pre- spermatid into the cytoplasm was superior to all other pro- dictive variables associated with testicular sperm retrieval in men tocols. This resulted in a pup rate not significantly different with non-obstructive azoospermia. Human Reproduction 14 than ICSI. In addition, the offspring obtained from ROSI 1005–1012. Fissore R, Gordo A & Wu H 1998 Activation of development in were fertile as already identified by others (Tamashiro et al. mammals: is there a role for a sperm cytosolic factor? Theriogenol- 1999). The present study also indicates the importance of ogy 49 43–52. a competent oocyte activation protocol to promote devel- Gordo A, Rodrigues P, Kurokawa M, Jellerette T, Exley G, Warner C opment outcomes from round spermatids that are equival- & Fissore R 2002 Intracellular calcium oscillations signal apoptosis rather than activation in in vitro aged mouse eggs. Biology of ent to those from mature spermatozoa. As clinical ROSI Reproduction 66 1828–1837. has been largely abandoned due to safety concerns, ani- Halet G, Marangos P, FitzHarris G & Carroll J 2003 Calcium oscil- mal models such as used here may provide valuable lations and the 5th UK calcium signalling conference. Biochemi- insights. The present research suggests that clinical ROSI cal Society Transactions 31 907–911. may benefit from the application of an optimal oocyte- Hardarson T, Lundin K & Hamberger H 2000 The position of the metaphase II spindle cannot be predicted by the location of the activation technique that needs to be explored, particu- first polar body in the human oocyte. Human Reproduction 15 larly one that promotes calcium oscillations in human 1372–1376. oocytes with no toxic effect on pre- and post-implantation Jones K, Carroll J, Merriman J, Whittingham D & Kono T 1995 Re- 2þ development. petitive sperm-induced Ca transients in mouse oocytes are cell cycle dependent. Development 121 3259–3266. Kimura Y & Yanagimachi R 1995 Mouse oocytes injected with testicular spermatozoa or round spermatids can develop into normal offspring. Development 121 2397–2405. Acknowledgements Kishigami S, Wakayama S, Thuan N & Wakayama T 2004 Similar The authors declare that there is no conflict of interest that time restriction for intracytoplasmic sperm injection and round spermatid injection into activated oocytes for efficient offspring would prejudice the impartiality of this scientific work. production. Biology of Reproduction 70 1863–1869. Kline D & Kline J 1992 Thapsigargin activates a calcium influx path- way in the unfertilized mouse egg and suppresses repetitive cal- References cium transients in the fertilised egg. Journal of Biomedical Chemistry 267 17624–17630. Alberio R, Zakhartchenko V, Motlik J & Wolf E 2001 Mammalian Lacham-Kaplan O & Trounson A 1997 Fertilization and embryonic oocyte activation: lessons from the sperm and implications for developmental capacity of epididymal and testicular sperm and nuclear transfer. International Journal of Developmental Biology 45 immature spermatids and spermatocytes. Reproductive Medicine 797–809. Reviews 6 55–68.

Reproduction (2006) 131 259–267 www.reproduction-online.org

Downloaded from Bioscientifica.com at 09/25/2021 03:41:55AM via free access Strontium in the activation of mouse oocytes 267

Lacham-Kaplan O, Shaw J, Sanchez-Partida L & Trounson A 2003 fertilized and activated mouse oocytes. Journal of Reproduction and Oocyte activation after intracytoplasmic injection with sperm Fertility 97 143–150. frozen without cryoprotectants results in live offspring from inbred Silber S & Johnson L 1998 Are spermatid injections of any clinical and hybrid mouse strains. Biology of Reproduction 69 1683–1689. value? Human Reproduction 13 509–515. Lawrence Y, Ozil J & Swann K 1998 The effects of a Ca2þ chelator Silber S, Nagy Z, Devroey P, Tournaye H & Van Steirteghem A and heavy-metal-ion chelators upon Ca2þ oscillations and acti- 1997 Distribution of spermatogenesis in the testicles of azoos- vation at fertilization in mouse eggs suggest a role for repetitive permic men: the presence or absence of spermatids in the Ca2þ increases. Biochemical Journal 335 335–342. testes of men with germinal failure. Human Reproduction 12 Machaty Z, Funahashi H, Day B & Prather R 1996 Effects of inject- 2422–2428. ing calcium chloride into in vitro-matured porcine oocytes. Bi- Saunders C, Larman M, Parrington J, Cox L, Royse J, Blayney L ology of Reproduction 54 316–322. et al. 2002 PLC zeta: a sperm-speciﬁc trigger of Ca2þ oscillations Miyazaki S, Yuzaki M, Nakada K, Shirakawa H, Nakanishi S, in eggs and embryo development. Development 129 3533–3544. Nakeade S & Mikoshiba K 1992 Block of Ca2þ wave and Ca2þ os- Swann K 1990 A cytosolic sperm factor stimulates repetitive calcium cillation by antibody to the inositol 1,4,5-trisphosphate receptor in increases and mimics fertilization in hamster eggs. Development fertilised hamster oocytes. Science 257 251–255. 110 1295–1302. O’Neill G & Kaufman M 1989 Cytogenetic analysis of ethanol- Swann K & Ozil J 1994 Dynamics of the calcium signal that triggers induced parthenogenesis. Journal of Experimental Zoology 249 mammalian egg activation. International Reviews of Cytology 152 182–192. 182–222. Ozil J & Huneau D 2001 Activation of rabbit oocytes: the impact of Tamashiro K, Kimura Y, Blanchard R, Blanchard D & Yanagimach R the Ca2þ signal regime on development. Development 128 1999 Bypassing spermiogenesis for several generations does not 917–928. have detrimental consequences on the fertility and neurobehavior Ozil JP, Markoulaki S, Toth S, Matson S, Banrezes B, Knott JG, of offspring: A study using the mouse. Journal of Assisted Repro- Schultz RM, Nuneau D & Ducibella T 2005 Egg activation events duction and Genetics 16 315–324. 2þ are regulated by the duration of a sustained [Ca ]cyt signal in the Tesarik J, Sousa M, Greco E & Mendoza C 1998 Spermatids as mouse. Developmental Biology 282 39–54. gametes: indications and limitations. Human Reproduction 3 Palermo G, Schlegel P, Colombero L, Zaninovic N, MoyF& 89–107. Rosenwaks Z 1996 Aggressive sperm immobilization prior to intra- Vanderzwalmen P, Nijs M, Stecher A, Zech H, Bertin G, Lejeume B cytoplasmic sperm injection with immature spermatozoa improves et al. 1998 Is there a future for spermatid injections? Human fertilization and pregnancy rates. Human Reproduction 8 Reproduction 13 (Suppl 4) 71–84. 1023–1029. Yanagimachi R 2001 Gamete manipulation for development: new Palermo G, Avrech O, Colombero L, Wu H, Wolny Y, Fissore R & methods for conception. Reproduction Fertility and Development 13 Rosenwaks Z 1997 Human sperm cytosolic factor triggers Ca2þ os- 3–14. cillations and overcomes activation failure of mammalian oocytes. Zhang D, Pan L, Yang LH, He XK, Huang XY & Sun FZ 2005 Stron- Molecular Human Reproduction 3 367–374. tium promotes calcium oscillations in mouse meiotic oocytes and Parrington J, Brind S, De Smedt H, Gangeswaran R, Lai F, early embryos through InsP3 receptors, and requires activation of Wojcikiewicz R & Carroll J 1998 Expression of inositol 1,4,5-tri- phospholipase and synergistic action of InsP3. Human Reproduc- sphosphaste receptors in mouse oocytes and early embryos: the tion 20 3053–3061. type I isoform is upregulated in oocytes and downregulated after fertilisation. Developmental Biology 203 451–461. Sasagawa I & Yanagimachi R 1996 Comparison of methods for activat- ing mouse oocytes for spermatid nucleus transfer. Zygote 4 Received 20 July 2005 269–274. First decision 26 August 2005 Shiina Y, Kaneda M, Matsuyama K, Tanaka K, Hiroi M & Doi K 1993 Revised manuscript received 6 September 2005 Role of the extracellular Ca2þ on the intracellular Ca2þ changes in Accepted 26 September 2005

www.reproduction-online.org Reproduction (2006) 131 259–267

Downloaded from Bioscientifica.com at 09/25/2021 03:41:55AM via free access