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Workshop "Pre-implantation embryos development in large animals: oocytes maturation, fertilization, embryo culture and cryopreservation"
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CALCIUM RESPONSES IN SHEEP OOCYTES FERTILIZED BY IVF OR ICSI
No full textCALCIUM RESPONSES IN SHEEP OOCYTES FERTILIZED BY IVF OR ICSI
Luisa Gioia (1), Luca Palazzese (2), Marta Czernik (2), Pasqualino Loi (2)
(1) Università degli Studi di Teramo, Facoltà di Bioscienze e Tecnologie Agro-Alimentari e Ambientali. (2) Università degli Studi di Teramo, Facoltà di Medicina Veterinaria.
Corresponding author: L. Gioia ([email protected])
In mammalian oocytes, at fertilization a rise in the concentration of intracellular free calcium ions (Ca2+) occurs. In particular, repetitive Ca2+ transients initiate shortly after sperm-oocyte fusion and stop around the time of the first mitotic cell cycle. Although the mechanism by which the sperm initiates this calcium response has not been fully clarified, it is widely accepted that this series of Ca2+ oscillations is sustained by the oocyte’s calcium store sensitized by the soluble PLCζ, released from the fertilizing sperm [1], and is necessary for supporting the events of oocyte activation. ICSI, which bypasses fusion of the gametes, has been widely used in several species, with variable efficiency [2]. A great progress has been achieved in humans, making of ICSI the golden standard for embryo production. In this species, it has been also demonstrated that after the injection of spermatozoon, Ca2+ oscillations are evoked with a pattern similar to that of IVF embryos that is able to activate the oocytes. In contrast, despite the efforts of several working groups around the world, the success of this technique has been limited in farm animals, especially in ruminants [3]. Particularly in sheep, ICSI outcomes are very limited and despite the resort to chemical activation the development to blastocyst as well as the number of newborns remain critically low compared to IVF [4]. Our previous reports revealed a discrepancy between the activation rate in sheep ICSI, assessed by pronuclear formation (80%), and the first cleavage (35%). Hoechst staining revealed two pronuclei in presumptive zygotes, indicating in the absence of pronuclear apposition and fusion the limiting factor [5]. Given that pronuclear migration and fusion are calcium-dependent processes, we hypothesized that current activation protocol affects the first calcium spikes, but does not induce the later, crucial ones. To date, there are no reports that compare oscillations patterns of ICSI vs IVF in sheep oocytes; therefore, the aim of this study is to analyse the response of Ca2+ in IVM oocytes fertilized by ICSI or IVF and microinjected with the Ca-sensitive dye Fluo-4. We have optimized the conditions to analyse the fertilized oocytes over the time to cover the time of pronuclei formation until the first cleavage by using time lapse microscopy and maintaining the oocyte in IVC medium at 38.5°C in 5% CO2. We demonstrated for the first time that in sheep oocytes fertilized by ICSI Ca2+ response is different from that observed after IVF. In fact, the majority of oocytes are unable to produce Ca2+ oscillations and, although in some oocytes the initiation of Ca2+ response takes place like in IVF oocytes, altogether a defective Ca2+ pattern is observed. Once identified the specific defective Ca2+ signal occurring after ICSI procedure, it could be interesting to develop ICSI/activation protocols in which the missing Ca2+ rises are artificially provided for as long as necessary, in order to better mimicking the temporal Ca2+ pattern occurring after IVF and improve the impact on embryo development.
[1] Saunders et al. PLCζ: a sperm-specific trigger of Ca2+ o s c i l l ati o ns i n eggs and em bry o dev el o pm ent , Development, 129:3533-3544, 2002.
[2] Salamone et al. Intracytoplasmic sperm injection in domestic and wild mammals, Reproduction, 154:F111 - F124, 2017.
[3] Briski & Salamone. Past, present and future of ICSI in livestock species. Anim Reprod Sci, 19;106925. 2022.
[4] Shirazi et al. Male pronuclear formation and embryo development following intracytoplasmic injection of ovine pretreated sperm. Avicenna J Med Biotechnol (AJMB), 10:41-48, 2018.
[5] Ressaissi et al. The impaired development of sheep ICSI derived embryos is not related to centriole dysfunction, Theriogenology, 159:7-12, 2021
Comparison of slow and rapid freezing in freeze-dry ram spermatozoa
Get PDFSemen lyophilization is an interesting technique that might be a cheap alternative to a long-term storage in liquid nitrogen. The first significant result of this method was achieved by Wakayama and Yanagimachi in the 1998 demonstrating, for the first time, the birth of healthy mouse pups from epididymal freeze-dried (mouse) spermatozoa. Authors follow lyophilisation technique, commonly used in pharmaceutical/food industry, namely deep-freezing, that requires direct immersion of the semen sample into liquid nitrogen before vacuum drying. In this work, we focused on the freezing phase in order to improve and make the technique more reliable. We have compared two different protocols: i) Rapid-freezing, where the semen is plunged directly into liquid nitrogen (LN group); ii) Slow-freezing, where the sample is frozen with a freezing rate of 1°C/min until -50°C degrees (SL group). Then, both frozen samples were lyophilized. Subsequently, after (in a range between 1 – 3 months) dry spermatozoa from LN and SL-groups were used for Intracytoplasmic Sperm Injection (ICSI) and the embryo development was evaluated at 24h (2-Cells stage) and 7 days (expanded blastocyst) post-ICSI. Moreover, a part of the semen, immediately after freezing phase, has been dedicated for the evaluation of acrosome integrity through the Pisum Sativum Agglutinin (PSA) staining. LN-group semen showed the acrosome completely melted, while the SL-group showed a better integrity of the acrosome which was comparable to that of the normal frozen (vital) spermatozoa. ICSI outcomes point out at 24h post-ICSI the SL-group showed a higher number of cleaved embryos than LN-group [42/100 (42%) versus 19/75 (25.3%), P=0.0253, SL and LN respectively]. At 7 days after fertilization the blastocyst rate in SL-group was higher [7/100 (7%)] than in LN-group [2/75 (2.7%), P=0.0238]. Our data shows that lyophilisation can be conveniently achieved in ram spermatozoa with liquid nitrogen – free way, thus simplifying the procedure. This data supports the idea that lyophilisation might be a valuable and cheaper alternative to liquid nitrogen for long-term storage of ram semen
Artificial activation of ovine oocytes is required after ICSI with freeze-dried spermatozoa.
Get PDFObjectives. Freeze-drying allows to store the biological samples in a dry state and represents an interesting alternative low-cost strategy of semen biobanking to save the endangered species. Here, we have established a dry sperm biobank from an endangered Italian sheep breed (Pagliarola) and tested its efficiency through ICSI.
Materials and methods. The motile spermatozoa from ram ejaculates collected with artificial vagina was selected by swim-up in TRIS-based medium (2.42g TRIS, 1.36g citric acid, 1.00g fructose, 100.000 U.I. penicillin G, 100mg streptomycin, in 67.20ml bidistilled water (ddH2O); pH was adjusted to 6.7) for 20 minutes at 38.5°C. The motile spermatozoa were frozen in freeze-drying medium (10mM EGTA and 50mM NaCl in 10mM Tris–HCl buffer; pH was adjusted to 8.4) in a -80°C freezer for 75 minutes and subsequently lyophilized by the freeze-drying apparatus SP Scientific-VirTis, Freeze-dryer 2.0 BenchTop, 20 hours with a condenser temperature of -58°C and vacuum of 20 mTorr). The vials were sealed in glass vials under vacuum and stored in the dark at 4°C for 1-2 months.
Just before the ICSI, the freeze-dried spermatozoa were rehydrated by adding 100μl ddH2O. To evaluate the fertilizing capability of freeze-dried spermatozoa, 108 MII sheep oocytes were subjected to ICSI and allocated to two groups: 56 oocytes were activated by incubation with 5μM ionomycin (ICSI-FDSa); 52 were left un-activated (ICSI-FDSna). Forty-four oocytes injected with frozen spermatozoa (ICSI-FS) and left un-activated, served as control. Pronuclear formation (2PN) and blastocyst development were investigated at 14-16 hours and 7-8 days after ICSI, respectively. Differences were considered statistically significant for p<0.05 (Chi-square test). Data were analyzed using PRISM, software version 5.0; GraphPad.
Results. The freeze-dried spermatozoa were completely immotile after rehydration, however they maintained the capacity to fecund oocytes after ICSI. Two PN were found in 83.3% of ICSI-FDSa, 81.4% of ICSI-FS while only in 14.3% of ICSI-FDSna (p<0.05 ICSI-FDSna vs ICSI-FDSa; p<0.01 ICSI-FDSna vs ICSI-FS). Likewise, the ICSI freeze-dried spermatozoa yielded blastocysts only following artificial activation (ICSI-FDSa: 10.2%; ICSI-FS: 31%; ICSI-FDSna: 0%; p<0.05 ICSI-FDSa vs ICSI-FDSna andICSI-FS; p<0.0001 ICSI-FDSna vs ICSI-FS).
Conclusions. Our finding show that freeze-dried spermatozoa have lost the capacity to trigger oocyte activation but maintained their nuclear viability, whose developmental potential was fully released following artificial activation. Our results support the evidence that freeze-drying effective approach of spermatozoa storage to save endangered species
Freeze-dried sperm: an alternative biobanking solution for endangered farm species
Get PDFIntroduction
An ever-increasing number of domestic species are threatened with extinction. Biobanking of spermatozoa could represent a feasible and efficient way for preserving genetic heritage and to maintain biodiversity. Given the published evidence that lyophilized spermatozoa retain their fertilizing capacity, we have collected semen from an Italian endangered sheep breed (Pagliarola) and created a biobank of cryopreserved and freeze-dried spermatozoa.
Material and Methods
The fertilizing capacity of the all stored semen, cryopreserved and freeze-dried, was evaluated by IVF and ICSI, respectively. To evaluate the activating capability of freeze-dried spermatozoa, 108 MII sheep oocytes were subjected to ICSI, and allocated to two groups: 56 oocytes were activated by incubation with ionomycin (ICSI-FDSa) and 52 were un-activated (ICSI-FDSna). Pronuclear formation (2PN) was investigated at 14-16 hours after ICSI in fixed presumptive zygotes.
Results and Discussion
As expected, the fertilizing capacity of cryopreserved Pagliarola’s spermatozoa was comparable to commercial semen stocks (31.8% vs 29%, respectively). Two PN were observed in 83.3% of ICSI-FDSa while only in 14.3% of ICSI-FDSna. Likewise, only artificially activated oocytes were able to develop to blastocyst after ICSI (10.2% compare to 0% with ICSI-FDSna). Oocytes injected with frozen spermatozoa (ICSI-FS) and left un-activated, served as control (81.4% of 2PN; 31% of blastocyst). In this work, we have demonstrated for the first time that freeze-dried ram spermatozoa could drive blastocyst development following ICSI. Although the developmental potential of embryos derived from lyophilized spermatozoa was significantly lower than cryopreserved ones, sperm lyophilization may be an alternative, low cost storage option, susceptible of improvement of course, to save biodiversity in domestic species
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