Stéphane Ferchaud

Influence of sex on the meiotic segregation of a t(13;17) Robertsonian translocation: a case study in the pig

BACKGROUND Comparison of male versus female meiotic segregation patterns for Robertsonian translocation (RT) carriers with similar genetic background has rarely been reported in mammalian species. METHODS The aim of this study was to compare the segregation patterns determined for related males and females carrying a 13;17 RT in an animal model (Sus scrofa domestica L.), using dual colour fluorescence in situ hybridization on decondensed sperm nuclei and metaphases II of in vitro-matured oocytes. RESULTS In males, no association between the trivalent and the XY body was observed in any of the 90 pachytene nuclei studied, and the rate of unbalanced spermatozoa ranged between 2.96% and 3.83%. Female meiotic segregation analyses were carried out on 83 metaphase II oocytes. The rate of unbalanced gametes was higher in females than in males (28.91% versus 3.21%, P < 0.001). This difference was due to higher rates of diploid gametes (12.04% versus 0.05%) and unbalanced gametes produced by the adjacent segregation (16.86% versus 3.16%). CONCLUSIONS This study is a new scientific contribution to the comparison of segregation patterns in related males and females carrying an identical chromosomal rearrangement. It allows a better understanding of the meiotic behaviour of RTs. It also clearly illustrates the relevance of swine as an animal model for such meiotic studies.

Meiotic Recombination Analyses of Individual Chromosomes in Male Domestic Pigs (Sus scrofa domestica)

For the first time in the domestic pig, meiotic recombination along the 18 porcine autosomes was directly studied by immunolocalization of MLH1 protein. In total, 7,848 synaptonemal complexes from 436 spermatocytes were analyzed, and 13,969 recombination sites were mapped. Individual chromosomes for 113 of the 436 cells (representing 2,034 synaptonemal complexes) were identified by immunostaining and fluorescence in situ hybridization (FISH). The average total length of autosomal synaptonemal complexes per cell was 190.3 µm, with 32.0 recombination sites (crossovers), on average, per cell. The number of crossovers and the lengths of the autosomal synaptonemal complexes showed significant intra- (i.e. between cells) and inter-individual variations. The distributions of recombination sites within each chromosomal category were similar: crossovers in metacentric and submetacentric chromosomes were concentrated in the telomeric regions of the p- and q-arms, whereas two hotspots were located near the centromere and in the telomeric region of acrocentrics. Lack of MLH1 foci was mainly observed in the smaller chromosomes, particularly chromosome 18 (SSC18) and the sex chromosomes. All autosomes displayed positive interference, with a large variability between the chromosomes.

Studies of male and female meiosis in inv(4)(p1.4;q2.3) pig carriers

Inversions are well-known structural chromosomal rearrangements in humans and pigs. Such rearrangements generally have no effect on the carriers phenotype. However, the presence of an inversion may impair spermatogenesis and lead to the production of recombinant gametes, responsible for early miscarriages, stillbirth, or congenital abnormalities. This is the first report on meiotic segregation and pairing behavior of the inv(4)(p1.4;q2.3) pericentric inversion in pigs. Despite the very large size of the inverted fragment (76% of the chromosome), SpermFISH results showed that only 4.08% of the gametes produced by male heterozygotes were unbalanced. This low proportion could be explained by the particular behavior of normal and inverted SSC4 chromosomes during the initial stages of meiosis. Indeed, immunohistochemistry analyses revealed that heterosynapsis occurred in 92% of the cells, whereas synaptic adjustment was detected in a few spermatocytes only. Unexpectedly, the proportion of unbalanced gametes produced by female heterozygotes, estimated by FISH on metaphase II oocytes, was also very low (3.69%) and comparable to that in males. According to previous results for male and female meiotic processes, different proportions of recombinant gametes in the two genders would have been expected. Complementary studies should be carried out to further document the meiotic behavior of inversions in pigs.

Sperm DNA Methylation Analysis in Swine Reveals Conserved and Species-Specific Methylation Patterns and Highlights an Altered Methylation at the GNAS Locus in Infertile Boars

ABSTRACT Male infertility is an increasing health issue in todays society for both human and livestock populations. In livestock, male infertility slows the improvement of animal selection programs and agricultural productivity. There is increasing evidence that epigenetic marks play an important role in the production of good-quality sperm. We therefore screened for specific or common epigenetic signatures of livestock infertility. To do so, we compared DNA methylation level in sperm DNA from fertile and infertile boars. We evaluated first the global level of sperm DNA methylation and found no difference between the two groups of boars. We then selected 42 loci of interest, most of them known to be imprinted in human or mice, and assessed the imprinting status of five of them not previously described in swine tissues: WT1, CNTN3, IMPACT, QPCT, and GRB10. DNA methylation level was then quantified in fertile and infertile boars at these 42 loci. Results from fertile boars indicated that the methylation level of the selected loci is highly conserved between pig, human, and mice, with a few exceptions, including the POU5F1 (OCT4) promoter and RTL1. Comparison between fertile and infertile boars revealed that one imprinted region, the GNAS locus, shows an increase in sperm DNA methylation in three out of eight infertile boars with low semen quality. This increase in DNA methylation is associated with an altered expression of the genes belonging to the GNAS locus, suggesting a new role for GNAS in the proper formation of functional gametes.

Non integrative strategy decreases chromosome instability and improves endogenous pluripotency genes reactivation in porcine induced pluripotent-like stem cells

The pig is an emerging animal model, complementary to rodents for basic research and for biomedical and agronomical purposes. However despite the progress made on mouse and rat models to produce genuine pluripotent cells, it remains impossible to produce porcine pluripotent cell lines with germline transmission. Reprogramming of pig somatic cells using conventional integrative strategies remains also unsatisfactory. In the present study, we compared the outcome of both integrative and non-integrative reprogramming strategies on pluripotency and chromosome stability during pig somatic cell reprogramming. The porcine cell lines produced with integrative strategies express several pluripotency genes but they do not silence the integrated exogenes and present a high genomic instability upon passaging. In contrast, pig induced pluripotent-like stem cells produced with non-integrative reprogramming system (NI-iPSLCs) exhibit a normal karyotype after more than 12 months in culture and reactivate endogenous pluripotency markers. Despite the persistent expression of exogenous OCT4 and MYC, these cells can differentiate into derivatives expressing markers of the three embryonic germ layers and we propose that these NI-iPSLCs can be used as a model to bring new insights into the molecular factors controlling and maintaining pluripotency in the pig and other non-rodent mammalians.

Selection for residual feed intake in growing pigs: effects on sow performance in a tropical climate.

The aim of the study was to evaluate the consequences of a divergent selection for residual feed intake (RFI) during growth in a temperate environment (TEMP) on sow performance in a tropical environment (TROP). Sows came from a selection experiment conducted at INRA in which 2 lines were selected for larger (RFI(+)) or smaller (RFI(─)) feed intake than predicted from performance. In the first analysis, a subsample of data obtained in TROP conditions (49 lactations) was compared to those obtained in TEMP on their sibs mated with the same boars (54 lactations). In the second analysis, data obtained in the TROP environment (82 lactations) were analyzed for testing the effect of season (warm vs. hot) and line on sow performance. Except for the lactation length, the interaction between line and climatic environment was not significant for the others traits (P > 0.05). The ADFI expressed per kilogram of litter BW gain tended to be higher in the RFI(+) line bred in the TROP environment (P = 0.080), together with piglet BW at weaning, which tended to be lower (P = 0.080). The ADFI was lower in TROP than in TEMP (4.56 vs. 5.86 kg/d; P = 0.003), with negative consequence on litter BW gain and maternal BW loss. The RFI(-) sows tended to eat less feed than RFI(+) sows during lactation (4.55 vs. 5.86 kg/d; P = 0.099). Litter BW at weaning was higher in the RFI(─) line. The RFI(─) sows ate significantly less feed to produce 1 kg of litter than the RFI(+) sows and tended to lose a larger amount of BW during lactation than the RFI(+) sows (2.40 vs. 3.02 kg/kg and -0.66 vs. -0.39 kg/d, respectively, P < 0.10). Whatever the line, ADFI was reduced by about 21% in the hot season (P < 0.05). Litter BW gain was depressed (P < 0.05) in the hot season (1.72 vs. 2.08 kg/d in the warm season; P = 0.023). Lactation maternal BW loss tended to increase in the hot season (1.10 vs. 0.71 kg/d in the warm season; P = 0.016), but back fat loss remained constant (P = 0.295). In the TROP environment, the amount of feed required to produce 1 kg of litter was not influenced by the line in the warm season (2.53 kg/kg on average; P = 0.99), but it tended to be lower in the RFI(─) line when compared to the RFI(+) line in the hot season (2.06 vs. 3.45 kg/kg; P = 0.050). This higher apparent efficiency in RFI(─) sows was mainly related to greater maternal body reserve mobilization (i.e., BW and back fat losses). In conclusion, selection for low RFI during growth in a TEMP environment did not impair sow and litter performance in tropical conditions.

Cytogenetic analysis of somatic and germinal cells from 38,XX/38,XY phenotypically normal boars

Many chromosomal abnormalities have been reported to date in pigs. Most of them have been balanced structural rearrangements, especially reciprocal translocations. A few cases of XY/XX chimerism have also been diagnosed within the national systematic chromosomal control program of young purebred boars carried out in France. Until now, this kind of chromosomal abnormality has been mainly reported in intersex individuals. We investigated 38,XY/38,XX boars presenting apparently normal phenotypes to evaluate the potential effects of this particular chromosomal constitution on their reproductive performance. To do this, we analyzed (1) the chromosomal constitution of cells from different organs in one boar; (2) the aneuploidy rates for chromosomes X, Y, and 13 in sperm nuclei sampled from seven XY/XX boars. 2n = 38,XX cells were identified in different nonhematopoietic tissues including testis (frequency, <8%). Similar aneuploidy rates were observed in the sperm nuclei of XY/XX and normal individuals (controls). Altogether, these results suggest that the presence of XX cells had no or only a very limited effect on the reproduction abilities of the analyzed boars.

Erratum to: Meiotic pairing and gene expression disturbance in germ cells from an infertile boar with a balanced reciprocal autosome-autosome translocation

Individuals carrying balanced constitutional reciprocal translocations generally have a normal phenotype, but often present reproductive disorders. The aim of our research was to analyze the meiotic process in an oligoasthenoteratospermic boar carrying an asymmetric reciprocal translocation involving chromosomes 1 and 14. Different multivalent structures (quadrivalent and trivalent plus univalent) were identified during chromosome pairing analysis. Some of these multivalents were characterized by the presence of unpaired autosomal segments with histone γH2AX accumulation sometimes associated with the XY body. Gene expression in spermatocytes was studied by RNA-DNA-FISH and microarray-based testis transcriptome analysis. Our results revealed a decrease in gene expression for chromosomes 1 and 14 and an up-regulated expression of X-chromosome genes for the translocated boar compared with normal individuals. We hypothesized that the observed meiotic arrest and reproductive failure in this boar might be due to silencing of crucial autosomal genes (MSUC) and disturbance of meiotic sex chromosome inactivation (MSCI). Further analysis revealed abnormal meiotic recombination (frequency and distribution) and the production of a high rate of unbalanced spermatozoa.

Intraindividual Variation of Meiotic Recombination Parameters in Pig Spermatocytes: A Preliminary Study

Meiotic recombination parameters like crossover (CO) rate or synaptonemal complex (SC) length are known to vary strongly between individuals and between cells from the same individual. The origins of this variability remain elusive, and little is known about the variations that might occur between different samples and/or over time within the same individual. To document this question, pachytene cells from 3 boars of the Large White breed were analyzed twice, at a 1-year interval, using immunocytological techniques. CO rate, SC length, and MLH1 inter-foci distances varied significantly between the 3 individuals. CO rate and SC length differed significantly between the 2 sampling periods for 1 individual. However, no significant differences were observed between the 2 samples for CO distribution and inter-foci distances in the 3 boars studied.

Seminal plasma differentially alters the resistance of dog, ram and boar spermatozoa to hypotonic stress

During ejaculation and the deposition in the female genital tract, spermatozoa undergo hypo-osmotic stress and need to withstand it for optimal fertility. Resistance to hypo-osmotic stress may be affected by the interaction of the spermatozoa with seminal fluid components. The hypo-osmotic resistance of epididymal and ejaculated spermatozoa from dogs, rams and boars was assessed by flow cytometric measurement of sperm viability after incubation in NaCl solutions with osmolalities ranging from 0 to 300 mmol/kg. The hypotonic resistance of epididymal spermatozoa was greater than those of ejaculated spermatozoa in all three species. Among species comparison revealed that ejaculated spermatozoa from dogs were much more resistant than those from rams and boars as 80.4 ± 5.3%, 56.7 ± 4.7 and 9.6 ± 3.6% of live spermatozoa were observed following exposure to an osmolality of 90 mmol/kg in dogs, rams and boars respectively. This can be explained by the fact that dog, ram and boar differ markedly in composition of the seminal plasma owing to the presence (ram, boar) or absence (dog) of seminal vesicles. Hypotonic resistance of epididymal and ejaculated dog spermatozoa was similar whereas ram and boar spermatozoa showed a marked drop in resistance after ejaculation. The in vitro incubation of boar epididymal spermatozoa with raw seminal plasma or the seminal plasma protein fraction induced a similar loss of resistance, suggesting that seminal proteins are involved in the lack of resistance to hypotonic stress of boar ejaculated spermatozoa.