Filippo Maritato

Intracytoplasmic sperm injection (ICSI) versus conventional IVF on abattoir-derived and in vitro-matured equine oocytes.

Conventional IVF as well as several assisted microfertilization techniques have shown limited success in the horse. After recent positive results achieved with intracytoplasmic injection of a single spermatozoon (ICSI) in human IVF, we chose to try the method in the horse. We compared conventional IVF to ICSI by fertilization rates of oocytes with compact and expanded cumuli and by developmental potential of the resulting embryos. Cumulus-oocyte complexes (COCs) were obtained by aspirating the follicular fluid from the ovaries of slaughtered mares. Complexes showing complete cumulus investment, either compact or expanded, were randomly assigned to IVF or ICSI trials and separately cultured for IVM. Frozen-thawed stallion spermatozoa were prepared for IVF with a swim-up procedure conducted in Talp-Hepes with heparin or for ICSI in Earles balanced salt solution (EBSS) supplemented with human serum albumin (HSA). Oocytes for IVF were partially decumulated by pipetting, whereas those for ICSI were totally denuded with 80 UI/ml hyaluronidase. Oocytes were fixed, stained and examined for signs of fertilization the day after IVF or ICSI. The percentage of normally fertilized oocytes showing 2 pronuclei or cleavage was significantly higher with ICSI than IVF (29.8%, 17/57 vs 8.7%, 9/103 ; P < 0.01). Significantly higher fertilization rates were observed in oocytes retrieved with an expanded cumulus when submitted to ICSI procedure as compared with IVF (52.2%, 12/23 vs 17.1%, 6 35 ; P < 0.01), whereas in oocytes recovered with a compact cumulus, fertilization rates were low (14.7%, 5/34 with ICSI and 4.4%, 3 68 with IVF; NS). Embryonal development did not occur after culture following IVF, as indicated by absence of cleavage in any of the 93 inseminated oocytes. Following ICSI, 7 of 55 injected oocytes cleaved, 5 of which had shown expanded cumuli; of the 5, 2 were at the 16-cell stage and one each at the 8-, 3- and 2-cell stage, respectively. The other 2 fertilized oocytes, originating from compact cumuli, reached 4- and 8- cell stages, respectively. These results indicate that ICSI can be applied successfully to in-vitro matured equine oocytes to increase the fertilization rates. In addition, it seems that in vitro cytoplasmic maturation of oocytes issuing from a compact cumulus may not be complete enough to lead to a successful fertilization and that ICSI may be a tool to evaluate ooplasmic maturation.

Toxic effects of the mycotoxin zearalenone and its derivatives on in vitro maturation of bovine oocytes and 17β-estradiol levels in mural granulosa cell cultures

Moulds parasites of livestock foodstuffs alter the quality of grains by synthesizing mycotoxins. Zearalenone (ZEA) and its derivatives (alpha- and beta-zearalenol, zeranol, taleranol and zearalanone) are produced by fungi of the genus Fusarium and, after ingestion via contaminated cereals, may lead to fertility disturbances and other reproductive pathologies. Zearalenone, alpha-zearalenol and zearalanone were tested, at levels ranging from 0.3 to 30 microg/ml, in order to evaluate the effect on the in vitro maturation (IVM) rate of bovine oocytes and on the formation of 17 beta-estradiol in supernatants of mural granulosa cells (GC) cultures. These compounds induced dose-dependent oocyte maturation delay and chromatin abnormalities. Maturation of oocytes to metaphase II (M II) was inhibited in oocytes cultured in the presence of 30 microg/ml ZEA, alpha-zearalenol or zearalanone, with a significant increase in chromatin abnormalities occurring in the presence of ZEA (P<0.05) and alpha-zearalenol (P< 0.001). In preliminary trials on 17 beta-estradiol formation, at the same testing concentration, higher levels of 17 beta-estradiol were found in the presence of alpha-zearalenol (mean value 1.6 ng/ml) with respect to ZEA and zearalanone (mean estradiol concentrations of 0.06 and 0.5 ng/ml, respectively). These data demonstrate a negative effect of ZEA and its derivatives on meiotic progression of bovine oocytes, possibly attributable to a toxic mechanism not related to the binding affinity of these compounds to estrogen receptor sites, and support previous observations that alpha-zearalenol acts as a stronger estrogenic inducer than the original molecule (ZEA).

In vitro maturation and fertilization of equine oocytes recovered during the breeding season

The aim of this study was to develope an efficient and reproducible procedure for in vitro maturation (IVM) and fertilization (IVF) in the horse. Cumulus-oocyte complexes (COCs) recovered from the ovaries of mares slaughtered during the breeding season were morphologically evaluated, and those showing a compact cumulus and homogeneously appearing cytoplasm were selected for culture. Effects on the maturation of estrous mare serum (EMS) versus estrous cow serum (ECS) as medium supplement were also evaluated (Experiment 1). In Experiment 2, the fertilization of in vitro matured oocytes with frozen-thawed semen separated by swim-up and treated with heparin was carried out to determine the effects on fertilization of 1) increasing sperm concentrations (1x 10(6), 5 x 10(6) and 1 x 10(7)sperm cells/ml), 2) IVM medium supplementation with EMS or ECS and 3) partial cumulus mass removal before insemination. Forty-nine percent of the collected oocytes (335 683 ) showed a compact cumulus and homogeneous ooplasm and thus were selected for culture. In Experiment 1, high nuclear maturation rates were observed in both EMS (82%,32 39 ) and ECS (87.5%,56 64 ) groups, with no statistically significant difference. In Experiment 2, the percentage of normal fertilization (2 polar bodies, 2 pronuclei and sperm tail) was similar for all 3 tested sperm concentrations (12.9%,4 31 ; 15.2%,9 59 and 15.5%,9 58 ). No advantage in using the homologous serum in IVM medium was noted in terms of fertilization (12.2%, 5 41 with EMS vs 12.9%, 4 31 for ECS). However, significantly higher fertilization rates were obtained after partial cumulus removal compared with that of oocytes fertilized with a whole cumulus (32.6%, 14 43 vs 12.2%, 5 41 ; P < 0.05). The incidence of polyspermic fertilization was low under all culture conditions (0 to 2.4%). In a replicate in which the oocytes fertilized after the cumulus removal were further cultured for 72 h two embryos, one at the 2-cell stage and the other at the 4-cell stage, could be obtained. These results indicate that, in the horse, the cumulus can be partially removed to increase the fertilization of compact-cumulus oocytes recovered during the breeding season using frozen-thawed, heparin-treated semen.

Cumulus expansion, nuclear maturation and connexin 43, cyclooxygenase-2 and FSH receptor mRNA expression in equine cumulus-oocyte complexes cultured in vitro in the presence of FSH and precursors for hyaluronic acid synthesis

The aim of this study was to investigate cumulus expansion, nuclear maturation and expression of connexin 43, cyclooxygenase-2 and FSH receptor transcripts in equine cumuli oophori during in vivo and in vitro maturation in the presence of equine FSH (eFSH) and precursors for hyaluronic acid synthesis. Equine cumulus-oocyte complexes (COC) were cultured in a control defined medium supplemented with eFSH (0 to 5 micrograms/ml), Fetal Calf Serum (FCS), precursors for hyaluronic acid synthesis or glutamine according to the experiments. After in vitro maturation, the cumulus expansion rate was increased with 1 microgram/ml eFSH, and was the highest with 20% FCS. It was not influenced by precursors for hyaluronic acid synthesis or glutamine. The expression of transcripts related to cumulus expansion was analyzed in equine cumulus cells before maturation, and after in vivo and in vitro maturation, by using reverse transcription-polymerase chain reaction (RT-PCR) with specific primers. Connexin 43, cyclooxygenase-2 (COX-2) and FSH receptor (FSHr) mRNA were detected in equine cumulus cells before and after maturation. Their level did not vary during in vivo or in vitro maturation and was influenced neither by FSH nor by precursors for hyaluronic acid synthesis. Results indicate that previously reported regulation of connexin 43 and COX-2 proteins during equine COC maturation may involve post-transcriptional mechanisms.

Meiotic Competence of Equine Oocytes and Pronucleus Formation after Intracytoplasmic Sperm Injection (ICSI) as Related to Granulosa Cell Apoptosis

Abstract Follicle atresia and granulosa cell apoptosis may be related to oocyte meiotic and developmental competence. We analyzed the relationships among granulosa cell apoptosis, initial cumulus morphology, oocyte nuclear maturation in vitro, and pronucleus formation after intracytoplasmic sperm injection (ICSI) in the horse. For each follicle, the size was measured and granulosa cells were used for DNA laddering analysis. Oocytes were evaluated for cumulus morphology, cultured for in vitro maturation, and submitted to ICSI. Apoptosis was categorized as absent, intermediate, or advanced according to the relative concentrations of two DNA fragments at 900 and 360 base pairs (bp). In 98 oocyte-follicle pairs, 52 oocytes were classified as expanded (Exp), 39 as compact (Cp), and 7 as having a partial (P) cumulus. Advanced apoptosis was detected in 55% (54/98) of follicles; 37% (36/98) of follicles showed an intermediate level of apoptosis; and 8 follicles (8%) were nonapoptotic. Follicle size was not significantly correlated with granulosa cell apoptosis (P > 0.05). Significantly more Exp than Cp oocytes originated from follicles with advanced apoptosis (P < 0.001). The proportion of oocytes maturing in vitro was significantly higher in oocytes issuing from apoptotic follicles than in oocytes issuing from healthy follicles (P < 0.05). The proportion of normally (two pronuclei) or abnormally fertilized oocytes (one or greater than two pronuclei, or partially decondensed sperm) did not differ in relation to granulosa cell apoptosis. We conclude that, in the mare, granulosa cell apoptosis is related to cumulus expansion and an increase in oocyte meiotic competence but has no effect on the proportion of meiotically competent oocytes that activate after ICSI. These results provide selection criteria for horse oocytes used in assisted reproductive techniques so that embryo production may be maximized.

Expression and localization of the µ‐opioid receptor (MOR) in the equine cumulus–oocyte complex and its involvement in the seasonal regulation of oocyte meiotic competence

The µ‐opioid receptor (MOR) was identified in equine oocytes, cumulus and granulosa cells. By RT‐PCR, a 441bp fragment was observed. By immunoblotting, a 65 kDa band was detected in samples of winter anestrous whereas in cells recovered in breeding season, two bands, 65 and 50 kDa, were found. The 65 kDa band was significantly more intense in winter anestrous specimens. In samples recovered in the breeding season, this band significantly decreased with the raise of follicle size and was heavier in compact oocytes and cumulus cells. The protein was localized on the oolemma and within the cytoplasm of oocytes and cumulus cells. In vitro oocyte maturation rate (MR), analyzed by confocal microscopy for nuclear chromatin, microfilaments and microtubules, was reduced after the addition of 3 × 10−8 M β‐endorphin in medium without additional hormones. Inhibitory effects of 10−3 M Naloxone in oocytes collected in anestrous and spring transition were observed, both in presence and absence of hormones added to culture medium. Increased MRs were observed in oocytes collected in anestrous and cultured in presence of 10−8 M Naloxone. The exposure to 10−3 M Naloxone induced significant intracellular calcium increases in cumulus cells recovered all over the year. β‐Endorphin 3 × 10−8 M induced significant calcium increases only in cumulus cells recovered in fall transition and anestrous. Naloxone 10−8 M did not induce intracellular calcium modifications. We conclude that the MOR is differentially expressed in equine cumulus–oocyte complexes in the different seasons of the year and plays a role in the seasonal regulation of meiotic competence of equine oocytes. Mol. Reprod. Dev. 75: 1229–1246, 2008.

rDNA and Acrocentric Chromosomes in Man I. rDNA Levels in a Subject Carrier of a 8p/13p Balanced Translocation and in His Unbalanced Son

A malformed female infant was found to have a 46,XX complement with a chromosome 8 shorter than normal with a secondary constriction and satellites on the short arm. Chromosome studies on the clinically normal father showed a balanced translocation between chromosome 8 and 13, i.e., 46,XY,t(8;13) (p21 p12). The proposita, carrier of the unbalanced form of the translocation, resulted partially monosomic for short arm of chromosome 8 (8p-) and partially trisomic for short arm of chromosome 13. The levels of DNA complementary to rRNA (normal in the father who had 10 NOR and increased in the proposita who had 11 NOR) confirmed our interpretation of the rearrangement.

Unstable telocentric chromosome produced after centric misdivision of a 21q/21q translocated element.

SummaryAn unstable telocentric chromosome was found in an individual with Downs syndrome and an unusual chromosomal mosaic, 46,XX, t(21q21q)+,21-/46,XX,21p-/45,XX,21-. As the 21q/21q chromosome was of paternal origin, based on the characteristics of its centromeric heterochromatin and on the characteristics of both 21 chromosomes of the father, it was concluded that its formation involved centric breakage and loss of centromeric material. The cell line with the 21p- chromosome may have originated from the translocation by an asymmetric misdivision of the reduced centromeric material. Of the two telocentrics produced by this fracture, one, possessing the smaller amount of centromeric apparatus, would be immediately lost; the other would be retained, but complete activity of its centromere would not be restored. It would therefore be unstable and might be lost.

rDNA and acrocentric chromosomes in man

SummaryA malformed female infant was found to have a 46,XX complement with a chromosome 8 shorter than normal with a secondary constriction and satellites on the short arm. Chromosome studies on the clinically normal father showed a balanced translocation between chromosome 8 and 13, i.e., 46,XY,t(8;13) (p21 p12). The proposita, carrier of the unbalanced form of the translocation, resulted partially monosomic for short arm of chromosome 8 (8p-) and partially trisomic for short arm of chromosome 13.The levels of DNA complementary to rRNA (normal in the father who had 10 NOR and increased in the proposita who had 11 NOR) confirmed our interpretation of the rearrangement.