Characterization of the melatonin receptor gene MT1 in mouflon (Ovis Gmelini Musimon) and its relationship with reproductive activity.

Wild small ruminants living at temperate latitudes reproduce
during decreasing photoperiods, resulting in spring
lambing and thus a greater possibility of newborn survival.
This process depends on the nocturnal pineal secretion
of melatonin, which plays an essential role in the
control of seasonal reproduction as a humoral encoding
of photoperiodic information. Melatonin exerts its seasonal
effect by acting on specific receptors. Of these receptors,
MT1 seems to be the only one involved in the regulation
of reproductive activity. Some polymorphisms have been
found in the MT1 gene in sheep. In particular, using MnlI
endonuclease, the homozygous genotype for the absence
of the cleavage site at position 328 of MT1 exon II was
associated with the seasonal anovulatory activity in ewes
(Pelletier et al., 2000).
The goals of the present research were to characterize
the MT1 gene in mouflon, identify possible polymorphisms,
and facilitate a better understanding of their relationship with
reproductive activity. Blood samples from 80 mouflon (35
females and 15 males living wild in restocking areas on land
belonging to Sardinia’s Regional Forestry Commission and
another 25 females and 5 males kept in a fenced outdoor
environment) were collected and used for genomic DNA
extraction to allow amplification of the main part of exon II of
the MT1 gene (Messer et al., 1997). The amplicons from
30 samples were randomly chosen, cloned and sequenced,
and then aligned to the ovine MT1 sequence (GenBank
accession number U14109) to evaluate any difference to
the sheep sequence (Carcangiu et al., 2009). All 80 samples
were genotyped using MnlI endonuclease which identifies
anA (allele, cleavage site absent) to aG(þallele, cleavage
site present) substitution at position 328 (GenBank
FJ801038). Allele and genotype / frequency (0.66 and
0.50, respectively) were predominant compared to allele þ
and genotype þ/þ (0.34 and 0.18, respectively). Sequencing
permitted the characterization of MT1 exon II in mouflon
(GenBank FJ801038). Five mutations were shown: four
silent (T169G, C322T, A328G, T607C) and one leading to
an amino acid substitution (A422G!Ile141Val). The presence
of an A at position 328 was always found to be
associated with the presence of a T at positions 169 and
607 and an A at position 422. Lambing dates of the
25 females kept in the outdoor environment were recorded.
The comparison of reproductive activity with genotype in this
latter group showed that the five animals with þ/þ genotype
lambed earlier than the 12 with / genotype (P<0.05)
(Table 1). In conclusion, the present research provides the
partial sequence of theMT1exon II in the wild sheep mouflon
and shows that / genotype is more frequent than the
others. This genotype’s link to reproductive activity strongly
influenced by photoperiod results in late lambing. This presumably
contributes to a greater possibility of newborn
survival as suggested by the high frequency of the /
genotype.