Cytoplasmic lipid droplets and mitochondrial distribution in equine oocytes: Implications on oocyte maturation, fertilization and developmental competence after ICSI

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Theriogenology 71 (2009) 1093–1104 www.theriojournal.com

Cytoplasmic lipid droplets and mitochondrial distribution in equine oocytes: Implications on oocyte maturation, fertilization and developmental competence after ICSI§ B. Ambruosi a,*, G.M. Lacalandra a, A.I. Iorga a, T. De Santis a, S. Mugnier b, R. Matarrese a, G. Goudet b, M.E. Dell’Aquila a b

a Department of Animal Production, University of Bari, Strada Provinciale per Casamassima km 3. 70010 Valenzano, Bari, Italy INRA, UMR85 Physiologie de la Reproduction et des Comportements, CNRS, Universite´ de Tours, Haras Nationaux, F-37380 Nouzilly, France

Received 30 July 2008; received in revised form 4 December 2008; accepted 5 December 2008

Abstract Lipid droplets (LDs) and mitochondria in the ooplasm are essential for energy production required for maturation, fertilization and embryo development. This study investigates the correlations between cytoplasmic LDs polar aggregation and: (1) nuclear maturation (Experiment 1); (2) mitochondrial (mt) distribution pattern and localization (Experiment 2); (3) fertilization and embryonic development after intracytoplasmic sperm injection (ICSI; Experiment 3) in equine oocytes recovered from slaughtered mares and matured in vitro. Morphologically normal oocytes were selected after culture and categorized as having polar (P) aggregation or uniform (U) distribution of LDs. In Experiment 1, the maturation rate was significantly higher in P compared with U oocytes (69%, 40/58 vs. 32%, 13/41; P < 0.001). In Experiment 2, it was observed that P and U oocytes showed heterogeneous mt distribution at comparable rates (68%, 25/37 vs. 50%, 2/4 for P and U respectively; NS). Moreover, only in 8/25 (32%) of P oocytes, LDs overlapped with mt aggregates in the area containing meiotic spindle. In Experiment 3, normal fertilization (51%, 19/37 vs. 60%, 6/10, for P and U) and cleavage rates (83%, 20/24 vs. 67%, 4/6, for P and U) did not differ between groups, also in oocytes with LDs located nearby the polar body. Overall, P aggregation of LDs was related to cumulus expansion at collection. In conclusion, in equine matured oocytes, P aggregation of LDs is related with cumulus expansion and nuclear maturation. However, it is not related with heterogeneous mt distribution and cannot be considered a predictive indicator for normal fertilization and embryo development. # 2009 Elsevier Inc. All rights reserved. Keywords: Equine; Oocyte; Cytoplasmic lipid droplets; Mitochondrial distribution; Intracytoplasmic sperm injection (ICSI)

1. Introduction

§ Part of this work was presented at the 16th International Congress of Animal Reproduction (ICAR), Budapest, Hungary, 13–17 July 2008. * Corresponding author at: Department of Animal Production University of Bari, Str. Prov. Casamassima Km 3 - 70010 Valenzano-Bari, Italy. Tel.: +39 080 4679888; fax: +39 080 4679883. E-mail address: [email protected] (B. Ambruosi).

0093-691X/$ – see front matter # 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2008.12.002

The oocyte, the largest cell in the female mammal, is known to contain considerable endogenous energy stores to use during meiotic maturation, fertilization and preimplantation development. This is particularly evident in oocytes of domestic mammals, where very high levels of lipids have been reported [1], the majority of them in form of triglycerides [2] assembled in lipid droplets (LDs) some times surrounded by a phospholipid

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monolayer [3,4]. Lipid content in oocytes differs among species, as reported in studies performed in porcine [5– 7], bovine [8,9] and canine oocytes [10] in which LDs number, size and distribution have been analyzed. In the horse, Grondahl et al. [11,12] described the rearrangement of membrane-bound vesicles and LDs from an even distribution, observed in oocytes at the germinal vesicle (GV) stage, to an often semilunar domain adjacent to meiotic spindle giving the ooplasm a polarized appearance, as observed in metaphase II (MII) oocytes. Carnevale and Maclellan [13], reviewing most important criteria for selection of equine oocytes for clinical use, confirmed that as maturation is complete, the ooplasm can have different shades of gray, often with a polarized appearance, caused by uneven distribution of LDs and organelles. Lipids play important roles in energy metabolism during oocyte maturation, fertilization and early embryonic development. Their amount and distribution have interesting implications on the sensitivity of oocytes and embryos to freezing procedures. Triglycerides are metabolised by b-oxidation and the TCA cycle within the mitochondrial matrix [14]. Thus it was hypothesized that, in the oocyte cytoplasm, mitochondria and LDs should reside in close proximity. This has been proven only in sheep [15], bovine [8,16] and porcine [6,7,17] where mitochondria and LDs are seen to associate, forming ‘metabolic units’ which tend to accumulate at the edge/cortex of the oocyte. To our knowledge, no studies were reported to date on the functional relationships between LDs and mitochondria. It is known that the distribution of mitochondria within the oocyte is indicative of the energy and ion requirement through various key events during oocyte maturation, fertilization and early embryonic development [17] and that mitochondrial (mt) dysfunctions or abnormalities in the oocyte may be a critical determinant in embryonic developmental competence. Mitochondrial dysfunction in the oocyte may not only compromise developmental processes, by inducing chromosomal defects, but also trigger apoptosis in the embryo [18–21]. In the mouse, Calarco [22] first reported, by using specific fluorescent probes and confocal laser scanning microscopy, mt distribution changes, from homogeneous (small granules diffused throughout the cytoplasm) to pericortical/perinuclear (P/P), occurring during meiotic maturation. Since then, mt distribution and activity have been analyzed in oocytes of several species, such as mouse [23–25], hamster [26], pig [17,20,27,28], bovine [29,30], goat [31] and humans [19,23,32,33]. In the horse, two studies

have been reported on mt distribution patterns in equine in vivo [34] and in vitro matured oocytes [35]. Functional studies on the physiological relevance of LDs aggregation on oocyte meiotic competence have been reported to date only in porcine in which the presence of large amounts of LDs is a striking characteristic. Sturmey and Leese [36] reported a significant fall in intracellular triglyceride content in porcine oocytes occurring during in vitro maturation revealed by oxygen consumption measurements. In a more recent study, by fluorescence resonance energy transfer (FRET) methods it was demonstrated that, in porcine oocytes, these organelles are colocalized as they lie within 6–10 nm from each other, indicating an association on a molecular scale [14]. To our knowledge, no studies have been reported to date on the biological role of LDs polar aggregation on the subsequent steps of maturation, fertilization and embryo development in any mammalian species. This was considered as a particularly interesting aim in the horse, in which cytoplasmic LDs polar aggregation is an easily detectable morphological parameter in living matured oocytes. Thus, the aim of the present study was to evaluate whether cytoplasmic LDs polar aggregation, in matured equine oocytes, could be considered as a predictive indicator of oocyte nuclear and cytoplasmic maturation, suitable energy status and competence to sustain normal fertilization and embryonic development. 2. Materials and methods All chemicals were purchased from Sigma–Aldrich (Milano, Italy) unless otherwise indicated. 2.1. Collection of oocytes The study was conducted in Southern Italy (418 North parallel). Ovaries from mares of unknown reproductive history obtained at two local abattoirs, located at a maximum distance of 20 km (30 min) from the laboratory, were transported and processed for the scraping procedure as previously described [37]. Cumulus-oocyte complexes (COCs) were recovered from medium size follicles (0.5–2.5 cm in diameter), identified in the collected mural granulosa cells by using a dissection microscope and only healthy COCs, classified as having an intact, compact (Cp) or expanded (Exp), cumulus investment [37,38] were selected for culture; degenerating oocytes (having shrunken, dense or fragmented cytoplasm) were recorded and discarded. The time between follicle scraping and beginning of

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oocyte culture was less than 1 h. Total time between slaughter and culture ranged between 2 and 4 h. 2.2. In vitro maturation In vitro maturation (IVM) was performed following the procedure by Dell’Aquila et al. [37]. Medium TCM199 buffered with Earle’s salts and 4.43 mM HEPES and supplemented with 0.1 g/L L-glutamine, 33.9 mM sodium bicarbonate, 2 mM sodium pyruvate, 2.92 mM calcium-L-lactate penthahydrate (Fluka 21175 Serva Feinbiochem GmbH & Co. Heidelberg, Germany No. 29760) and 50 mg/mL gentamicin was used. After preparation, pH was adjusted to 7.18 and the medium was filtered through 0.22-mm filters (No. 5003-6, Lida Manufacturing Corp., Kenosha WI, USA) and further supplemented with 20% (v/v) Fetal Calf Serum (FCS). Then, gonadotrophins (10 mg/mL ovine FSH and 20 mg/mL ovine LH) and 1 mg/mL 17b Estradiol were added. The medium was filtered again and allowed to equilibrate for 1 h under 5% CO2 in air before being used. Compact and expanded COCs were washed three times in the culture medium and groups of up to 10 COCs with the same cumulus morphology were placed in 400 mL of medium/well of a four-well dish (Nunc Intermed, Roskilde, Denmark), covered with preequilibrated lightweight paraffin oil and cultured for 28–30 h at 38.5 8C under 5% CO2 in air. After IVM culture, oocytes underwent cumulus and corona cells removal by incubation in TCM 199 with 20% FCS containing 80 IU hyaluronidase/mL and aspiration in and out of finely drawn glass pipettes. Oocyte morphology after denuding was assessed by observation under a Nikon SMZ 1500 stereomicroscope (60– 110 magnification). Those oocytes showing an intact zona pellucida, regular-shaped perivitelline space intact oolemma and regular ooplasmic shape and texture (no vacuoles) classified as morphologically normal [11– 13], were further categorized as having uniform (U) distribution or polar (P) aggregation of LDs within the cytoplasm and assigned to trials. Cytoplasmic LDs polarization was assessed by rotating the oocytes with the aid of finely drawn glass pipettes. 3. Experiment 1 3.1. Nuclear chromatin evaluation Oocytes were stained with 2.5 mg/mL Hoechst 33258 in 3:1 (v/v) glycerol/PBS, mounted on microscope slides, covered with cover slips, sealed with nail polish and kept at 4 8C in the dark until observation.

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Oocytes were evaluated in relation to their meiotic stage under an epifluorescence microscope (Nikon Eclipse 600 equipped with B-2 A (460 nm excitation/346 nm emission) filter as previously described [39,40]. Nuclear chromatin status was classified as follows: germinal vesicle (GV) when a fluorescent nucleus with diffused, fibrillar or compact chromatin was observed; metaphase to telophase I (MI-TI) includes all stages between metaphase I and telophase I; metaphase II (MII) with the second metaphase plate and the first polar body (PB) extruded. Oocytes with multipolar spindle, irregular chromatin distribution or absence of chromatin were considered as abnormal. 3.2. Statistical analysis For each cumulus morphology, the maturation rates of oocytes showing either P or U LDs distribution were compared by Chi-square test with the Yates correction for continuity. The Fisher’s exact test was applied in all cases in which at least one cell contained a value less than 5. Differences with P < 0.05 were considered statistically significant. 4. Experiment 2 In this experiment, oocytes showing the 1st PB extruded after IVM and denuding, were observed under an inverted microscope (TE 2000-U Nikon) equipped with Hoffmann differential interferential optics (400 magnification) with the aid of micromanipulation needles (with rotating movements), in order to visualize LDs distribution, if either U (Fig. 1A) or P (Fig. 1B–D) and their localization in respect to the position of the 1st PB, if either corresponding (Fig. 1B) or opposite (Fig. 1C) or longitudinal (Fig. 1D). This procedure was also performed on oocytes of Experiment 3. 4.1. Nuclear and mitochondrial staining Oocytes were washed three times in PBS with 3% bovine serum albumin (BSA) and incubated for 30 min in the same medium containing 280 nM MitoTracker Orange CMTM Ros (Molecular Probes M-7510, Oregon, USA) at 38.5 8C under 5% CO2. This cell-permeant probe is readily sequestered only by actively respiring organelles depending on their oxidative activity [27,41]. After incubation, oocytes were washed three times in prewarmed PBS without BSA and fixed overnight at 4 8C with 2% paraformaldehyde solution in PBS. Oocytes were then stained with 2.5 mg/mL Hoechst 33258 in 3:1 (v/v) glycerol/PBS and mounted on microscope slides as

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Fig. 1. Equine oocytes evaluated for their LDs aggregation status and localization while observed under an inverted microscope equipped with Hoffmann differential interferential optics. (A) an oocyte with U distribution of LDs within the cytoplasm is shown. (B–D) show oocytes having P aggregation of cytoplasmic LDs localized in corresponding (B), opposite (C) and longitudinal (D) position in respect to PB. Scale bar represents 50 mm.

previously described. Particular attention was paid to avoid sample exposure to the light during staining and fixing procedures in order to reduce photobleaching. 4.2. Mitochondrial evaluation by confocal laser scanning microscopy Oocytes which showed the 2nd metaphase plate and the first PB (MII + PB) were observed at 600 magnification in oil immersion with a laser scanning confocal microscope (C1/TE2000-U Nikon). A helium/ neon laser ray at 543 nm and the G-2 A filter (576 nm exposure and 551 nm emission) was used to point out the MitoTracker Orange CMTM Ros. Scanning was conducted with 25 optical series from the top to the bottom of the oocyte with a step size of 0.45 mm to allow three-dimensional mt distribution analysis. Parameters related to fluorescence intensity were maintained at constant values for all measurements. General criteria for mt pattern definition were adopted on the basis of previous studies in equine oocytes [34,35] as well as in other species (see References cited in Section 1). Thus, homogeneous/even distribution of small mt granules throughout the cytoplasm was considered as an indication of immature cytoplasmic condition. Hetero-

geneous/uneven distribution of small and/or large mt granules indicated metabolically active ooplasm [22]. In particular, accumulation of active mitochondria in the peripheral cytoplasm (pericortical mt pattern) and/ or around the nucleus (perinuclear and pericortical/ perinuclear mt pattern, P/P) were considered as characteristic of full cytoplasmic maturation. 4.3. Statistical analysis For each group of oocytes, if either showing P or U LDs distribution, the rates of oocytes showing P/P mt distribution pattern were compared, between oocytes showing P or U LDs distribution, by Chi-square test with the Yates correction for continuity. The Fisher’s exact test was applied in all cases in which at least one cell contained a value less than 5. Differences with P < 0.05 were considered statistically significant. 5. Experiment 3 5.1. Semen preparation for ICSI Sperm cells for ICSI were prepared by the swim-up procedure in Earle’s balanced salt solution (EBSS)

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supplemented with 0.4% BSA, 50 mg/mL gentamicin as described by Dell’Aquila et al. [37,39]. Semen samples from a mature stallion with a reproductive history of normal fertility were used for the first set of trials aimed to evaluate embryonic developmental rate performed in the reproductive season (February to July). The stallion was located in the reproductive centre Pegasus (Department of Animal Production, University of Bari, Southern Italy) and was routinely used in artificial insemination programs. Semen was collected by using Missouri artificial vagina with an inline gel filter, extended with INRA 96 (IMV Technologies, Piacenza, Italy) at the concentration of (20–25)  106 sperm cells/mL and used immediately. Semen samples (0.4 mL/straw) from a single ejaculate frozen at a concentration of 1  108 sperm cells/mL were used for the second set of trials aimed to evaluate the fertilization rate performed in fall transition (September to October). Sperm cells were rapidly thawed (30 s) in a water bath at 37 8C. Total motility after thawing was 70%, with 50–60% progressive motility. 5.2. ICSI procedure Intracytoplasmic sperm injection was carried out as previously reported by Dell’Aquila et al. [37,39]. All procedures were performed at 38.5 8C in Life Global medium (The ART Media Company, USA). Each injected oocyte was then transferred to a single 25 mL drop of fresh Life Global medium covered by lightweight paraffin oil and incubated at 38.5 8C for 18–20 h under 5% CO2 in air. 5.3. Assessment of fertilization Eighteen to twenty hours after ICSI, oocytes were assessed for signs of fertilization by evaluating the extrusion of the 2nd PB. Presumptive zygotes were then fixed in 3.8% formaldehyde solution in PBS, stained with Hoechst and observed under epifluorescence microscopy. Normal fertilization was defined by the presence of two PBs with two pronuclei (PN). Presence of MII + PB with the swollen sperm head, a single PN with signs of the sperm cell in the cytoplasm, tripronucleate zygotes with a single PB extruded, were considered to represent retarded, arrested or abnormal fertilization, respectively, and were classified and grouped as abnormal. Oocytes with one PN with intact sperm cell were regarded as activated oocytes. Oocytes showing MII + PB with an intact sperm cell were classified as unfertilized.

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5.4. Embryo culture and evaluation In five replicates, oocytes were allowed to further develop in vitro for 72 h in the same medium. Embryo quality was graded as follows: type a = blastomeres of equal size with <10% cytoplasm fragmentation; b = unequal blastomeres with 10–40% fragmentation; c = unequal blastomeres with >40% fragmentation. The uncleaved ova were removed from culture, fixed and evaluated as described previously. Fertilization rates in these replicates included the oocytes that developed further into embryos as well as those that were found uncleaved but with evident signs of fertilization after staining. 5.5. Statistical analysis For each cumulus morphology, the proportions of matured, survived after ICSI, normally or abnormally fertilized, activated and cleaved oocytes were compared between groups (P vs. U) by Chi-squared analysis. The Fisher’s exact test was applied in all cases in which at least one cell contained a value less than 5. Differences were considered statistically significant at P < 0.05. 6. Results 6.1. Experiment 1 6.1.1. Correlation between cytoplasmic LDs polar aggregation and maturation rate In Experiment 1, the ovaries of 35 mares were processed. One hundred and thirty-six oocytes were recovered (1.9 oocytes/ovary), 69 surrounded by a Cp cumulus and 67 with an Exp cumulus and cultured for IVM in seven trials performed in the reproductive season. After culture, 99 oocytes (73%), 47 Cp and 52 Exp, were found as morphologically normal, further evaluated as P or U and analyzed for nuclear maturation. In the group of Cp oocytes, the maturation rate was significantly higher in P than in U oocytes (P < 0.001) and in the group of Exp oocytes, a trend was observed (Chi-square = 0.89, P-value <0.5). Overall (Cp + Exp oocytes), the maturation rate was significantly higher in P that in U oocytes (P < 0.001; Table 1). 6.2. Experiment 2 6.2.1. Correlation between cytoplasmic LDs polar aggregation and mitochondrial distribution Based on the results obtained in Experiment 1, our subsequent aim was to analyze, within each oocyte: (1)

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Table 1 Correlation between cytoplasmic lipid droplets polar aggregation and maturation rate in equine oocytes after in vitro culture. Cumulus morphology

No. (%) of oocytes showing Compact

Experiment 1 Normal morphology after IVM Lipid droplets distribution Metaphase II with 1st polar body Overall experiment 1 + 2 + 3 Normal morphology after IVM Lipid droplets distribution Metaphase II with 1st polar body

Expanded

Total

47/69 (68) Polar 26/47 (55) 17/26 (65)a

Uniform 21/47 (45) 2/21 (9)b

52/67 (78) Polar 32/52(62) 23/32(72)

Uniform 20/52 (38) 11/20 (55)

99/136 (73) Polar 58/99 (59) 40/58(69)a

Uniform 41/99 (41) 13/41 (32)b

154/226 (68) Polar 82/154(53)c 64/82(78)a

Uniform 72/154(47) 20/72(28)b

167/227 (74) Polar 113/167(68) d 92/113(81) a

Uniform 54/167(32) 24/54(44)b

321/453 (71) Polar 195/321(61) 156/195(80)a

Uniform 126/321(39) 44/126(35)b

Chi-square test with the Yates correction for continuity and Fisher’s exact test, 2  2 contingency table, 1 d.f.:

LDs and mt distribution patterns and (2) whether LDs and mitochondria were overlapped and localized in the same area containing meiotic apparatus. The ovaries of 41 mares were processed. One hundred and nine oocytes were recovered (1.3 oocytes/ovary), 49 with Cp cumulus and 60 with Exp cumulus, cultured for IVM in two trials during the reproductive season and evaluated. Ninety-five morphologically normal oocytes were obtained after IVM culture. Of them, 41 MII + PB (13 Cp + 28 Exp) oocytes were analyzed. Thirty-seven of the oocytes (11 Cp and 26 Exp) showed P aggregation (37/41, 90%) and only 4 oocytes (2 Cp and 2 Exp) showed U distribution of LDs. Both types of P and U oocytes displayed P/P mt distribution in the area corresponding to the meiotic apparatus at comparable rates (25/37, 68% vs. 2/4, 50% for P and U, respectively; NS). The remaining oocytes had homogeneous mt distribution of small granules throughout the cytoplasm (Fig. 2). In P oocytes, the LDs semilunar domain was not always associated with mt aggregates in the area corresponding to meiotic spindle. Instead, it was observed in three different positions, as corresponding (C; 32%), opposite (O; 28%) and longitudinal (L; 40%,) as regards to PB position (Table 2; NS). In Fig. 2, representative micrographs of the mt distribution observed in P and U oocytes at the MII stage are shown. Fig. 3 shows a representative 25 optical planes analysis of the oocyte in Fig. 2(A–C). Oocytes with homogeneous mt pattern showed uniform distribution of small granules in all serial planes over the 25 optical planes (data not shown). As a control, a group of oocytes in this experiment was analyzed immediately after retrieval. Thirty-two oocytes were recovered from the ovaries of 12 mares (1.3 oocytes/ovary), 15 with Cp cumulus and 17 with

a,b

P < 0.001;

c,d

P < 0.01.

Exp cumulus and evaluated. Twenty-three morphologically normal oocytes (13 Cp + 10 Exp) were selected and analyzed for LDs and mt distribution. In both groups (Cp and Exp), all oocytes showed U distribution of LDs and, after fixing and staining, showed the GV nucleus and homogeneous granular mt pattern (data not shown). 6.3. Experiment 3 6.3.1. Correlation between cytoplasmic LDs polar aggregation and fertilization/cleavage rate In Experiment 3a, five consecutive IVM/ICSI trials were performed in the reproductive season with the aim to evaluate the correlations between LDs polar aggregation within the cytoplasm and the developmental potential of equine oocytes. The ovaries of 32 mares were processed and 87 oocytes were recovered (1.4 oocytes/ovary), 53 surrounded by a Cp cumulus and 34 with an Exp cumulus. After culture and cumulus removal, 63 oocytes (72%), 22 Cp and 41 Exp, were found as morphologically normal, further evaluated as P or U, analyzed for maturation (1st PB extrusion), submitted to ICSI and allowed to develop in vitro for 72 h after sperm injection. In Exp oocytes, the maturation rate was significantly higher in P compared with U oocytes (27/31, 87% vs. 4/ 10, 40%; P < 0.05). The proportion of oocytes showing normal fertilization did not differ between P or U oocytes. There was also no statistically significant differences between groups with respect to the percentages of oocytes abnormally fertilized or activated. The embryo cleavage rates (2-, 4–8 and 8–16 cells stages) did not statistically differ between U and P oocytes (Table 3). The rates of embryos showing normal morphology also did not differ between groups. In fact,

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Fig. 2. Representative photomicrographs of mitochondrial distribution patterns observed in P and U MII oocytes. (A) shows an oocyte with P aggregation of LDs. The same oocyte is shown for its nuclear chromatin configuration, the 2nd metaphase plate and the 1st PB (B) and for its consistent amount of mitochondria beneath the oolemma and in the hemisphere containing meiotic apparatus (peripheral/perinuclear mt pattern, C). (D) shows an oocyte with U distribution of LDs. The same oocyte is shown for its nuclear chromatin configuration (E) and for its mt distribution in small granules diffused throughout the cytoplasm (F). Scale bar represents 60 mm.

18 out of the 20 embryos obtained from P oocytes and 2 out of the 3 embryos obtained from U oocytes were categorized as grade a. The remaining embryos (2/20 P and 1/3 U) were graded as category b.

Considering the lack of significance in embryo cleavage rates between P and U oocytes, Experiment 3b was performed with the aim to evaluate if any difference in the fertilization rates between groups. The ovaries of

Table 2 Correlation between cytoplasmic lipid droplets polar aggregation and mitochondrial distribution in equine oocytes after in vitro maturation (Experiment 2). Cumulus morphology

No. (%) of oocytes showing Compact

Normal morphology after IVM

Expanded

Total

Lipid droplets distribution Metaphase II with 1st PB

44/49 (90) Polar 18/44(41) 11/18(61)a

Uniform 26/44 (59) 2/26(8)b

51/60 (85) Polar 34/51 (67) 26/34(76)a

Uniform 17/51 (33) 2/17(12)b

95/109 (87) Polar 52/95 (55) 37/52(71)a

Uniform 43/95 (45) 4/43(9)b

Mitochondrial distribution P/P Homogeneous

8/11 (73) 3/11 (27)

1/2 (50) 1/2 (50)

17/26 (65) 9/26(35)

1/2 (50) 1/2 (50)

25/37 (68) 12/37 (32)

2/4 (50) 2/4 (50)

Lipid droplets/1st PB position (*) Corresponding Opposite Longitudinal

3/8 (38) 3/8 (38) 2/8 (25)

– – –

5/17 (39) 4/17 (24) 8/17 (47)

– – –

8/25 (32) 7/25 (28) 10/25 (40)

– – –

IVM = in vitro maturation; PB = polar body; P/P = pericortical/perinuclear; (*) only for oocytes with P distribution of LDs. Chi-square test with the Yates correction for continuity and Fisher’s exact test, 2  2 contingency table, 1 d.f.: a,bP < 0.001.

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Fig. 3. Mitochondrial distribution pattern and localization on 25 serial optical sections in an equine MII oocyte. The sample, which is the same as in Fig. 2C, is representative of confocal investigations performed on all examined oocytes. The section circled in white indicates the equatorial plane. In the lower (planes 1–3) and upper (planes 20–25) parts of the oocyte, a labelling in clumps/clusters, representing the subplasmalemmal mt foci, can be seen, whereas in planes nearer the equatorial position (planes 4–19), the perinuclear mt aggregation in the hemisphere containing the MII together with an intense fluorescence beneath the oolemma can be observed. Scale bar represents 60 mm.

35 mares were retrieved and processed in four consecutive IVM/ICSI trials in fall transition. One hundred and twenty-one oocytes were recovered (1.7 oocytes/ovary), 74 surrounded by a Cp cumulus and 47 with an Exp cumulus. After IVM culture and cumulus removal, 64 oocytes (53%), 41 Cp and 23 Exp, were found as morphologically normal, further categorized as P or U, submitted to ICSI and removed from culture 20 h after sperm injection. For Cp oocytes, the maturation rate was significantly higher in P compared with U oocytes, (26/26, 100% vs. 8/15, 53%; P < 0.001). The proportion of oocytes with normal fertilization after ICSI did not differ between P and U oocytes. There was also no statistically significant differences between groups with respect to

the percentages of oocytes abnormally fertilized or activated (Table 4). Furthermore in P oocytes, Cp and Exp, the rate of normally fertilized oocytes was not different in relation to the position of LDs to the PB position, both in the group of Cp (4/8, 50%; 6/10, 60%; 4/5, 80%) and Exp oocytes (1/3, 33%; 2/7, 29%; 2/4, 50%) and in overall samples (5/11, 45%; 7/15, 47%, 7/ 11, 64%) for C, O and L type oocytes, respectively. 7. Discussion This study provides new insights on the significance of cytoplasmic LDs polar aggregation as a predictive marker of oocyte meiotic competence, energy status, ability to be fertilized and develop into embryos.

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Table 3 Correlation between cytoplasmic lipid droplets polar aggregation and embryo cleavage rates in equine oocytes matured in vitro and submitted to ICSI (Experiment 3a). Cumulus morphology

No. (%) of oocytes showing Compact

Normal morphology after IVM

Expanded

Total

Lipid droplets distribution 1st polar body extruded Survival after ICSI Normal fertilization

22/34 (65) Polar 12/22(55) 10/12(83) 10/10 (100) 6/10 (60)

Uniform 10/22 (45) 8/10(80) 8/8 (100) 4/8 (50)

41/53 (77) Polar 31/41 (76) 27/31(87) a 27/27(100) 18/27 (67)

Uniform 10/41 (24) 4/10(40)b 4/4 (100) 2/4 (50)

63/87 (72) Polar 43/63 (68) 37/43(86)a 37/37 (100) 24/37 (65)

Uniform 20/63 (32) 12/20(60)b 12/12(100) 6/12 (50)

No. of embryos cleaved at the 2 cell stage 4–8 cell stage 8–16 cells stage Activation Abnormal fertilization

6/6 (100) 6/6 (100) 0/6 (0) 0/10 (0) 1/10 (10)

4/4 3/4 0/4 2/8 0/8

14/18(100) 12/14 (86) 2/14 (14) 2/27 (7) 0/27 (0)

0/2 0/2 0/2 1/4 1/4

20/24 (83) 18/20 (90) 2/20 (10) 2/37 (5) 1/37 (3)

4/67 (67) 3/6 (50) 0/6 (0) 3/12 (25) 1/12 (8)

(100) (75) (0) (25) (0)

(0) (0) (0) (25) (25)

Chi-square test with the Yates correction for continuity and Fisher’s exact test, 2  2 contingency table, 1 d.f.:

Previous studies in the horse [11–13] reported a detailed description of the rearrangement of LDs from an even distribution, observed in oocytes at the GV stage, to a semilunar domain adjacent to meiotic spindle observed in MII oocytes. However, to our knowledge, LDs functional role in subsequent steps of maturation, fertilization and embryo culture has not been analyzed. In Experiment 1, LDs distribution was strongly related to nuclear maturation (P < 0.001). This was confirmed by overall data (Table 1, summary data of Experiment 1 + 2 + 3). By grouping data of all experiments, it comes out that the majority of oocytes with normal morphology after IVM showed P aggregation of LDs. Overall maturation rate was significantly higher in P compared with U oocytes, both in Cp and Exp groups (P < 0.001). Furthermore, P aggregation of LDs was related with cumulus expansion at recovery time. In fact, the

a,b

P < 0.05.

percentage of Exp oocytes with P LDs was significantly higher than that of Cp oocytes (P < 0.01). These observations, about different competence of Cp and Exp oocytes, are in agreement with previous studies. Oocytes with Cp cumuli were reported as having a lower meiotic competence [42,43], a slower rate of maturation [43–45], reduced ability to respond to an activation stimulus [46] and reduced male pronucleus formation rate after ICSI [47] when compared to oocytes with Exp cumuli. Expanded oocytes fertilized by ICSI and transferred into the oviducts of recipient mares, showed 85% cleavage and development to an average of 16 cells at 96 h after transfer, equivalent to normal development in vivo [48]. In Experiment 2, the correlation between the aggregation patterns of two cytoplasmic organelles, LDs and mitochondria, involved in energy metabolism of the oocyte, was analyzed. Our results showed that

Table 4 Correlation between cytoplasmic lipid droplets polar aggregation and fertilization rates in equine oocytes matured in vitro and submitted to ICSI (Experiment 3b). Cumulus morphology

No. (%) of oocytes showing: Compact

Normal morphology after IVM Lipid droplets distribution 1st polar body extruded Survival after ICSI Normal fertilization Activation Abnormal fertilization

41/74 (55) Polar 26/41(63) 26/26(100)a 23/26 (88) 14/23 (61) 1/23 (4) 2/23 (9)

Expanded Uniform 15/41 (37) 8/15(53)b 4/8 (50) 3/4 (75) 0/4 (0) 0/4 (0)

23/47 (49) Polar 16/23 (70) 16/16(100) 14/16 (88) 5/14 (36) 1/14 (7) 3/14 (21)

Total Uniform 7/23 (30) 7/7(100) 6/7 (86) 3/6 (50) 1/6 (17) 0/6 (0)

Chi-square test with the Yates correction for continuity and Fisher’s exact test, 2  2 contingency table, 1 d.f.:

64/121 (53) Polar 42/64 (66) 42/42(100) c 37/42 (88) 19/37 (51) 2/37 (5) 5/37 (14) a,b

P < 0.01;

Uniform 22/64 (34) 15/22(68) d 10/15 (67) 6/10 (60) 1/10 (10) 0/10 (0)

c,d

P < 0.05.

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LDs polar aggregation is not related with P/P distribution of active mitochondria in equine oocytes. Mitochondrial patterns observed in our studies are in line with previous observation in equine oocytes [34,35]. Caillaud et al. [34] first described mt patterns in equine oocytes matured in vivo and reported the heterogeneous mt pattern as an indicator of high cytoplasmic maturity compared with the homogeneous mt pattern indicative of low cytoplasmic maturity. Torner et al. [35] described three mt patterns types in equine oocytes recovered by ultrasound-guided follicle aspiration and examined at collection or after IVM. Mitochondrial patterns in their study were defined as: fine, crystalline and granulated. Granulated pattern (Type 3 pattern) in the study by Torner et al. was the dominant aggregation type of mitochondria observed in Cp and Exp horse oocytes after 24 and 32 h of IVM. This pattern can be considered as assimilable to our perinuclear mt pattern, even considering different methods and equipment for image capture and analysis. In previous studies in other species, P/P mt distribution was also observed in mature oocytes. In the mouse, Calarco [22] described a cortical aggregation of mitochondria present as a remnant and marking the former location of the first meiotic spindle in most mature oocytes. The same authors also reported nonspindle mitochondria accumulated in several large foci within the egg and found in cortical and interior locations in matured oocytes. Of great interest was the finding that nearly all non-spindle mitochondria were polarized to one hemisphere of the mature oocyte, a hemisphere defined by the spindle at the proximal pole and extending distally over roughly half the oocyte. Van Blerkom et al. [23] reported that clusters of apparently high-polarized mitochondria occur in the pericortical cytoplasm of the murine and human oocyte and never far from interconnected networks of smooth endoplasmic reticulum. They hypothesized that high-polarized pericortical mitochondria may have a role in the acquisition of oocyte competence. Dell’Aquila et al. [49] reported that higher rates of MII oocytes showing perinuclear distribution are recovered from women treated with gonadotropin releasing hormone agonists compared with antagonists, for pituitary down regulation. The finding of P/P mt distribution in matured oocytes is in agreement with the described physiological role of mitochondria during last phases of meiosis. In human oocytes mitochondrial clustering around the spindle and in peripheral cytoplasm could be important for controlling local intracellular pH and influencing protein function and cytoskeletal and cytoplasmic organization [18]. Observations of a perinuclear

distribution of mitochondria may relate to their hypothesized role in providing energy for chromosome migration. In the mouse oocyte, mitochondria translocate, during in vitro maturation, to the perinuclear region along microtubular arrays extending from perinuclear micotubular organizing centres [50]. Mitochondria form a sphere of organelles that encloses the condensing bivalent chromosomes and, later, the nascent metaphase I and II spindles [51,52]. Observations of a peripheral distribution of mitochondria may relate to the fate of the mitochondria within the preimplantation embryo, all of which are derived from the oocyte with de novo mitochondrial synthesis not occurring until the blastocyst stage [53]. Blastomeres deficient in mitochondria fail to divide and lyse during culture [54]. The second part of Experiment 2 was performed with the aim to observe, in each individual oocyte, LDs and mt distribution in respect to the PB position. Only 32% of P oocytes (8/25) showed LDs and mitochondria localized in the same area containing meiotic spindle. This finding was not expected considering previous studies in other species in which researchers reported that mt and LDs should reside in close proximity [6– 8,15–17] in the place where chromosome migration occurs. Our data could be explained considering that, in the equine oocyte, lipids forming clearly visible aggregates at a pole of the ooplasm, could not be directly involved in mitochondrial metabolism but just represent an energy store at the vegetative pole. In the study by Sturmey and Leese [36] has been reported that triglycerides content of pig oocytes decreases during IVM. This finding could explain the polar aggregation of LDs in the equine oocyte as a loss of the triglyceride content occurring during IVM and the remaining triglycerides could be a surplus that gives to the ooplasm a polarized appearance. In Experiment 3, it was shown that LDs P aggregation is not a predictive parameter for normal fertilization and embryo development, since no statistical difference was found between the two groups (P and U) for fertilization and developmental rates as well as for embryo quality. However, it is necessary to consider that, since conventional IVF reaches only limited success in equine species [47,55,56] the ICSI technology was applied in this study. Thus, our experimental system does not allow us to examine the different ability of P or U oocytes in sustaining sequentials steps of sperm penetration. Moreover our experiments allowed us to evaluate only events occurring during 96-h post-injection. Further studies are needed to evaluate the correlation between LDs and

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