Meiotic progression and developmental competence of oocytes collected from juvenile and ewes S. Ledda, L. Bogliolo, P. Calvia, G. Leoni and S. Naitana Department of Animal Biology, University of Sassari, Italy

The complete nuclear maturation and fertilization in vitro of oocytes from 30\p=n-\40-day-old juvenile lambs, and their ability to develop up to the blastocyst stage when transferred into recipient ewes after fertilization in vivo and culture was studied. Cumulus\p=n-\oocytecomplexes were recovered from juvenile ovaries and only those with several cumulus cell layers were selected and compared with oocytes from adult sheep. The rate of meiotic progression was significantly lower (P <0.001) for juvenile oocytes (8%) than for adult oocytes (58%) without gonadotrophins in the culture medium. However, a similar maturation rate was observed in oocytes of both juvenile (76%) and adult sheep (84%) in the presence of gonadotrophins. Eighteen hours after in vitro insemination, the fertilization rates for juvenile oocytes were not significantly different from those of adult oocytes (64% and 72%, respectively). Parthenogenetic activation and polyspermy were higher in juvenile than in adult oocytes (P < 0.001). The proportion of blastocysts produced was lower for juvenile (20% than for in vitro matured adult oocytes (49%) after their transfer into transitory recipients for 5.5 days (P<0.01). However, the viability of blastocysts (67%) derived from in vitro matured juvenile oocytes showed a rate of hatching similar to that obtained from adult oocytes (74%). Pregnancy rates for recipient ewes at 90 days were similar for both juvenile (57%) and adult (61%) oocytes. The results indicate that it is possible to mature and fertilize in vitro matured juvenile oocytes to produce viable .

Introduction to exogenous gonadotrophin until 2—4 weeks after birth (Mansour, 1959; Mauleon, 1969), when granulosa and thecal In mammals, oogénesis starts in the embryonic ovary. After cells become well developed (Kennedy et al, 1974). A further several cycles of mitotic activity primordial germ cells are advantage of using oocytes collected from juvenile animals meiotically arrested as primary oocytes. Near birth, meiosis is would be a shorter generation interval in genetic selection arrested at the dictyate stage of the first meiotic prophase and programmes (Georges and Massey, 1991). female resumes after the onset of in the preovulatory follicles Oocytes obtained from ovaries of juvenile calves as the consequence of the LH surge. The cumulus—oocyte after treatment with pituitary gonadotrophins may be fertilized complex isolated from the antral follicles of most adult dom¬ and develop in vitro to the blastocyst stage at a rate that is estic and rare wildlife species can be matured, fertilized and similar to that of the adult cow (Armstrong et al, 1992; Irvine cultured in vitro, to stages competent for development to birth, et al, 1993). However, oocytes collected from untreated (Palma following transfer into synchronized recipients (see Brackett, et al, 1993) and untreated/treated (Revel et al, 1995) juvenile 1992; Loskutoff and Betteridge, 1992). The acquisition of calves result in a lower rate of development to blastocysts than meiotic competence appears to be possible not only in oocytes do those of adult cows. In juvenile goats, a lower rate of in vitro collected from adult females but also in oocytes from juvenile fertilization was observed when compared with adult animals animals which represent an alternative source of oocytes. A (Martino et al, 1995). More recently, blastocysts have been previous study showed that there are morphological changes in produced using in vitro matured (IVM) and in vitro fertilized the ovaries of juvenile Merino ewes during the first 8 weeks (IVF) oocytes from treated and untreated 60—120-day-old after birth (Kennedy et al, 1974). A significant increase in lambs (Earl et al, 1995). ovarian mass was accompanied by a marked proliferation and The aim of the present study was to examine the meiotic growth of vesicular follicles with accumulation of follicular competence of oocytes obtained from untreated juvenile fluid, although no significant differences were found in mean (30-40 day) and adult ewes under different culture conditions plasma concentrations of pituitary gonadotrophins (Tassell and their fertilization in vitro. The developmental potential of et al, 1978). Antral follicles of prepubertal ewes do not respond in vitro matured oocytes was assessed after transfer to inseminated temporary recipients and to term after transfer of Received 1 May 1996. blastocysts to definitive recipients.

Downloaded from Bioscientifica.com at 09/28/2021 04:38:39PM via free access Materials and Methods motility and 5, vigorous motility. Spermatozoa that showed a motility grade of less them 4 were rejected. Semen samples were pooled and incubated at a concentration of 10 x 10 the oocytes 1 Recovery of ml Coulter Electronics Ltd, in Petri ~ (Coulter Counter, Luton) Ovaries from juvenile (30—40 days of age and body mass of dishes containing a suspension of fresh oviductal epithelial cells 6—12 kg) and from adult sheep were collected from a local and 2 ml TCM 199 + 10% FCS for 3 h at 39°C in 5% C02 in abattoir and transported in Dulbecco's phosphate-buffered air. For assessment of nuclear maturation, 15—20% of total saline (PBS; Sigma, St Louis, MO) containing 50 U penicillin oocytes were fixed for 24 h in acetic acid—ethanol solution and ml-1 and 50 µg streptomycin ml-1 (Sigma) at 20-25°C, stained with Lacmoid (1%). After capacitation, sperm motility arriving at the laboratory within 1 h. Ovaries were washed was checked and the sperm washed twice by centrifugation three times in fresh PBS with penicillin and streptomycin. (5 min at 250 g) in modified Tyrode's medium (TALP) at 35°C. the Ovaries were sliced using a microblade and contents The semen pellet was resuspended in TALP medium at a released in medium TC-199 (plus Earle's salts and bicarbonate; concentration of 1 10 spermatozoa ml" and used at this 1 and Sigma) supplemented with 25 mmol Hepes ~ (Sigma) dilution for insemination. penicillin and streptomycin, and 0.1% (w/v) polyvinyl alcohol After mechanical removal of cumulus cells surrounding the (PVA; Sigma). The cumulus-oocyte complexes (COCs) oocytes with a fine glass pipette, in vitro matured COCs were selected according to morphological criteria (intact COCs with transferred into 50 µ drops of sperm suspension covered by several dense cumulus cell layers and homogeneous cytoplasm) mineral oil and incubated for 18 h at 39°C in 5% C02 in air. were released in the medium and washed three times in the Success of fertilization was determined at 18 h after insemina¬ same medium before in vitro maturation. Oocytes obtained tion after fixation in acetoalcoholic solution for 24 h and either from juvenile or adult ovaries were selected according to staining with Lacmoid. Oocytes with only one pronucleus were their dimensions using an inverted microscope equipped with a evaluated as parthenogenetically activated. Oocytes with male calibrated eyepiece, and small oocytes ( < 135 µ ) and those and female pronuclei were considered fertilized, while those the with signs of atresia were discarded. For each ovary, that showed more than two pronuclei were evaluated as number of oocytes recovered was recorded. polyspermic. Four experiments were performed to compare the com¬ petence of juvenile ovine oocytes with those of adult ewes. Experiment 3: production from oocytes matured in vitro Experiment 1: in vitro maturation after transfer to inseminated recipients The COCs obtained from juveniles were allocated to the In this the ability of juvenile and adult oocytes maturation systems. Juvenile and adult oocytes were experiment following to after their transfer to for cultured in for the first 24 h in medium TC-199 develop temporary recipients parallel fertilization and in vivo for 5.5 days was with 10% (v/v) heat-treated fetal calf serum (FCS; development supplemented ' examined. or FSH in the absence of 10 µg ml ~ and Sigma) presence After 24 h of in vitro maturation, under the conditions LH 25—30 10 mg ml ~ (Pergonal, Serono, Roma). Only described in Expt 1, oocytes were washed with fresh TCM- oocytes were put in a single Petri dish (35 mm) containing 2 ml 199 + 10% FCS and transferred to recipient ewes. culture medium which was maintained on a rotary shaker at surgically For this Sardinian ewes were low during the entire incubation The culture purpose, temporary recipient speed period. with sponges contain¬ conditions were maintained at a of 39°C in a synchronized intravaginal progestagen temperature 40 acetate and inserted for 14 All humidified with 5% in air. At the end of the ing mg fluorogestone days. atmosphere C02 were anaesthetized with 10 ~ body maturation oocytes (30%) from each culture system recipients mg kg weight period, of sodium (Farmaceutici Gellini, in saline. The were fixed for 24 h in acetic acid ethanol solution (1:3; v:v), pentothal Aprilia) abdominal cavity was opened and the genital tract exposed. stained with 1% (w/v) Lacmoid (Sigma), and examined under a Only ewes that did not have corpora lutea were used as phase-contrast microscope at x 200—400. Stages of nuclear recipients. The oviducts were ligated just above the uterotubal maturation of oocytes were evaluated as germinal vesicle (GV) and 20-30 were via the into and vesicle breakdown (GVBD), I (M I), junction oocytes injected ampulla germinal metaphase the oviduct of each in about 15-20 µ of medium and metaphase II (M II) and degenerated (Deg.). recipient 50 µ of spermatozoa (100x10 ml-1) using a Tom-Cat catheter (Sherwood, St Louis, MO) connected to a syringe. The oviducts of recipients were flushed 5.5 days later and all ova 2: sperm and in vitro Experiment capacitation fertilization and embryos were recovered from the flushing medium and This experiment was designed to evaluate the percentage of observed under an inverted microscope to assess their devel¬ in vitro fertilized oocytes obtained from juvenile and adult opmental stage. Embryos that had developed beyond the ovaries matured under the conditions outlined in Expt 1. 16-cell stage were immediately stained in Hoechst 33342 to Fresh semen samples were obtained from two rams of count the number of nuclei using epifluorescent microscopy proven fertility using an artificial vagina. After collection, 20 µ (Nikon-Diaphot 300). Embryos at the blastocyst stage, evalu¬ of sperm suspension was evaluated under 50 magnification ated using morphological criteria such as the formation of a on prewarmed glass slides. They were scored using a qualita¬ blastocoel, were used for assessment of viability in vivo and tive scale of 0-5 in which 0 signified complete absence of in vitro.

Downloaded from Bioscientifica.com at 09/28/2021 04:38:39PM via free access Experiment 4; in vitro and in vivo viability of blastocysts derived from in vitro matured juvenile oocytes The potential of the blastocysts produced in Expt 3 to develop in vitro was evaluated by co-culturing embryos in TCM 199 + 10% FCS on monolayers of ovine oviductal epithelial cells up to the time of their complete hatching from the zona pellucida. All blastocysts were maintained for up to 4 days in the same culture conditions and were checked every 12 h under an inverted microscope. Hatched blastocysts were stained with Hoechst 33342 and the number of nuclei counted. For assessment of the viability in vivo of the blastocysts developed in Expt 3, blastocysts were transferred to mature Sardinian ewes synchronized by the protocol used in Expt 3 and given 500 IU pregnant mares' serum gonadotrophin at the same time of sponge removal. Oestrus was detected using a vasectomized ram. Permanent recipients were anaesthetized as described. The wall one was previously uterine of horn Fig. 1. Different size of juvenile (J) and adult (A) oocytes after being perforated with a needle and the transfer catheter inserted totally denuded of cumulus cells by gentle mechanical pipetting. Scale about 3 cm into the lumen where the blastocyst was deposited. bar represents 100 µ . Pregnancy in recipients was determined on day 30 and confirmed at day 90 after transfer of blastocysts, using an ultrasound scanner (Toshiba, Tochigi) equipped with a 5 MHz observed in the proportion of metaphase II oocytes during transabdominal probe. maturation in the presence of gonadotrophins. Significantly more adult oocytes matured to metaphase II (P < 0.001) in the absence of Statistical analyses gonadotrophins (Table 1). Differences in meiotic progression and in developmental 2 competence of juvenile oocytes were subjected to chi-squared Experiment or were considered analyses the Fisher exact test. Differences After six replicate experiments, the results of in vitro at < 0.05. statistically significant fertilization (Table 2) show no significant difference in the percentage fertilized between juvenile (64%) and adult oocytes (72%). However, polyspermic fertilization rate of oocytes was Results significantly (P < 0.001) higher in juvenile (22%) than in adult ewes (5%). activation 2), assessed by the More oocytes were recovered from ovaries of juvenile lambs Parthenogenetic (Fig. presence of a occurred more (1634 oocytes from 46 ovaries mean 30-40 oocytes per single pronucleus, frequently - (19%) in juvenile oocytes than in adult oocytes (2%; < 0.001). ovary) than were obtained from ovaries of adult ewes (1653 from 189 ovaries mean 5-12). After they were totally denuded of cumulus- cells mechanical by gentle pipetting, Experiment 3 juvenile oocytes were smaller than those recovered from . Juvenile oocytes had a mean outside diameter In four replicate experiments, it was shown that the cleavage (141 ± 2.8 µ ) of about 89% of that of oocytes recovered from rate of oocytes from juveniles (72%) did not differ from that of adults (158 ± 2.6 µ ; Fig.l). adult oocytes (73%). However, the percentage of embryos that reached the blastocyst stage was significantly lower (P < 0.01) for juvenile oocytes (20%) compared with that for adult Experiment 1 embryos (49%). Embryos that did not develop to blastocysts were arrested at 16 this Results from five showed that 131 of early cleavage stages (< cells) and replicate experiments involved 80% and 51% of all and adult total 274 (48%) juvenile oocytes cultured in the absence of juvenile embryos, (Table 3). gonadotrophin were able to resume the meiotic process but respectively most (40%) were arrested in M I (109 of 274) and only a small meiosis to M II of In percentage (8%) completed up (22 274). 4 contrast, of 83% of the adult oocytes that resumed meiosis, Experiment 25% (70 of 280) reached metaphase I and 58% (162 of 280) After 4 days of culture in TCM 199 + 10% FCS and completed nuclear maturation. The addition of FSH and LH to oviductal monolayer no differences in hatching rates were the maturation medium significantly increased the percentage observed between juvenile (65%) and adult (75%) blastocysts. of juvenile oocytes that reached M II (235 of 310; 76%). The The number of embryonic cells was also not significantly same positive influence of the presence of gonadotrophins was different for juvenile (135 ± 22) and adult hatched blastocysts observed in adult oocytes, 84% reached M II. No significant (147+ 13). No difference was observed in the proportion of difference between the juvenile and adult oocytes was blastocysts that developed to day 30 and day 90 of pregnancy

Downloaded from Bioscientifica.com at 09/28/2021 04:38:39PM via free access Table 1. Effect of presence or absence of FSH/LH on meiotic progression of juvenile and adult ewes

Number of Meiotic progression (%) Ewes oocytes FSH/LH GV Metaphase I Metaphase II

Juvenile 274 143 (52) 109 (40) 22 (8)a 310 + 13 (4) 62 (20) 235 (76) Adult 48 (17) 70 (25) 162 (58)b 350 + 4 (1) 52 (15) 294 (84)

GV = germinal vesicle. a Values in the same column with different superscripts are significantly different (P< 0.001).

Table 2. In vitro fertilization of oocytes of juvenile and adult ewes after IVM for 24 h

Parthenogenetic Number of activation Fertilization Ewes oocytes Metaphase II (%) (%) (%) Polyspermy (%)

Juvenile 780 618 (79) 117 (19)a 398 (64) 85 (21)a Adult 820 673 (82) 13 (2)b 486 (72) 26 (5)b

'^Values in the same column with different superscripts are significantly different (P< 0.001).

matured oocytes to the blastocyst stage after transfer to temporary recipients for in vivo fertilization and culture was significantly lower than for adult oocytes. However, no differ¬ ences were observed in viability of blastocysts derived from juvenile oocytes and adult oocytes. They had similar hatching rates in vitro and pregnancy rates in vivo. Although oocytes of juveniles where smaller than those of adults, they are capable of resuming meiosis with a high rate of maturation to met¬ aphase II. Previous observations have indicated that oocytes acquire the ability to proceed to metaphase I when they reach about 80% of their full size, but are unable to progress to metaphase II until growth is completed (Moor and Gandolfi, 1987). Meiotic progression of juvenile oocytes was strongly affected by gonadotrophins in the culture system; the percent¬ age of juvenile oocytes resuming meiosis in the absence of gonadotrophins was lower than for oocytes from adult ewes. Fully grown ovine oocytes collected from antral follicles > 2 mm in diameter resume meiosis spontaneously during in vitro maturation (Szollosi et al, 1988). The reduced matu- rational competence of juvenile oocyte in vitro when compared with those of adults may depend on different factors. Environ¬ activation of assessed the Fig. 2. Parthenogenetic juvenile oocyte by mental conditions may not be optimal before the onset of of a after 18 h of in vitro fertilization. Scale presence single pronucleus puberty. In particular, the absence of a pulsatile pattern of LH bar 20 represents µ . secretion (Foster, 1984) and the absence of progesterone may be responsible for insufficient gonadal maturation (Berardinelli et al, 1980). A small number of on from juvenile (8 of 14) and adult (11 of 18) oocytes gonadotrophin receptors sheep follicular cells or fewer follicular cells can decrease the amount (Table 4). of cell signalling between the oocyte and surrounding cells. Furthermore, maintenance of metabolic coupling between the Discussion oocyte and its companion granulosa cells is required for oocyte development in vitro (Eppig et al, 1994). It is known that The results of this work show that oocytes obtained from gonadotrophin stimulation appears to be beneficial for prepubertal ewes as young as 30-40 days of age can be in vitro maturation of oocytes in adult sheep (Galli and Moor, matured and fertilized in vitro. Development of juvenile in vitro 1991). The positive influence of gonadotrophins on the

Downloaded from Bioscientifica.com at 09/28/2021 04:38:39PM via free access Table 3. Embryo production from oocytes of juvenile and adult ewes after in vitro maturation and in vivo fertilization and development in temporary recipients

Number of oocytes (%) Stage of development (%) Ewes Transferred Recovered Cleaved < 16 cells Blastocyst

Juvenile 270 196 (73) 142 (72) 113 29 (20)a Adult 203 153 (75) 113 (73) 58 55 (49)b

abValues in the same column with different superscripts are significantly different (P < 0.01).

Table 4. Survival rates of blastocysts obtained from in vitro matured and in vivo fertilized/developed oocytes from adult and juvenile ewes

In vitro In vivo Hatched Number of blastocysts Transferred Pregnancy* Ewes blastocysts (%) blastocysts (%)

Juvenile 15 10 (67.0) 14 8(57) Adult 19 14 (74.0) 18 11 (61)

*Confirmed at 90 days. developmental ability of in vitro matured oocytes obtained predominant population in the ovaries of 3-4-week-oId from juvenile animals was also observed after priming the female lambs. with donors in vivo exogenous FSH (Armstrong et al, 1995). The low developmental competence to of The in blastocysts results of vitro and in vivo fertilization rates reported juvenile in vitro matured oocytes after their transfer to transi¬ here show differences between and adult juvenile oocytes. This tory recipients may also indicate an incomplete cytoplasmic is in agreement with other in cattle et reports (Revel al, 1995; maturation caused by some deficiency in the IVM system, or et al, 1996). In Martino et al. a Duby contrast, (1995) reported may be due to an intrinsic absence of key regulators in the lower fertilization rate (24.4%) in prepubertal goat oocytes than juvenile oocytes not only for the meiotic progression but also in ovulated or in vitro matured (62.2%) oocytes (48.7%) from for early embryonic development. The age of juvenile animals adult In their goats. experiment, donors were not primed with can significantly influence oocyte developmental competence FSH before the collection of the oocytes. These authors also in rodents and their oocytes may need to be grown in vitro reported an increase in abnormal fertilization with a delay in before complete maturation competence and subsequent devel¬ male formation. This indicate pronuclear may incomplete opment can be achieved (Eppig and Schroeder, 1989). This may cytoplasmic maturation after culture in vitro. A high incidence explain the smaller number of embryos capable of of fertilized has been development abnormally oocytes also observed after in culture from juvenile than from ewes et al, 1976). In in vitro maturation of (Wright juvenile calf oocytes and this could be cows, there are conflicting results. A lower developmental due to an altered migration of cortical granules and a lower ability of calf oocytes has been reported by Kajihara et al. release of calcium ions after fertiliz¬ Palma et sperm penetration during (1991), al (1993) and Revel et al (1995), while a high ation et al, In the (Duby 1996). culture conditions used in the developmental ability of calf oocytes to the blastocyst stage study here, an increase in was observed and reported polyspermy similar to that of oocytes from adult cows was reported by and this indicate that maturation can occur may incomplete in Armstrong et al. (1994) and Irvine et al (1993). However, a as well. these oocytes It was observed (Lonergan et al, 1994) in direct comparison of these data is difficult because donors of bovine maturing oocytes obtained from adult animals that different ages were used in the experiments and the possibility follicular dimension can be for the important subsequent ability that oocytes obtained from younger or older donors may have of in vitro matured oocytes to develop to blastocysts. A smaller better developmental capabilities cannot be excluded. In sheep, percentage of morula or blastocyst stage embryos was viable fetuses were obtained after in vitro fertilization of obtained with oocytes collected from small follicles. After oocytes recovered from young (8-9 weeks of age) FSH-treated in vitro maturation, fertilization and culture of goat oocytes, juveniles (Armstrong et al, 1995). In this work, a higher developmental competence is acquired progressively during pregnancy rate was obtained using in vivo matured rather than follicular growth (Crozet et al, 1995). The inferior in vitro matured oocytes. Our results showed that mental develop¬ blastocysts competence of juvenile oocytes may be explained by derived from in vitro matured oocytes of non-stimulated small follicle size. In the present study oocytes were collected animals remained viable after their transfer to recipient ewes from follicles 1—2 mm mainly in diameter which is the (about 60%) with comparable rates to blastocysts derived from

Downloaded from Bioscientifica.com at 09/28/2021 04:38:39PM via free access adult In vitro also showed no Galli C and Moor RM (1991) Gonadotrophin requirements for the in vitro oocytes. viability (hatching) their and adult derived maturation of sheep oocytes and subsequent embryonic development significant differences between juvenile 35 the Theriogenology 1083-1093 blastocysts. This result may be due to positive effects Galli C and Lazzari G (1994) Large-scale production of bovine embryos. In exerted by the oviduct environment. At present this culture Progress in Embryo Technology and Genetic Engineering in Cattle and Sheep Krakow system seems best able to support embryonic development for Breeding pp 11-116 Eds M Tischner and KJ Scott. Drukol, the of bovine embryos (Galli and Lazzari, 1994). Georges M and Massey JM (1991) Velogenetics or the synergistic use of production marked assisted selection and germ line manipulation Theriogenology 35 151-159 in translation. 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