The highly prolific phenotype of Lacaune sheep is associated with an ectopic expression of the B4GALNT2 gene within the ovary

Abstract

Prolific sheep have proven to be a valuable model to identify genes and mutations implicated in female fertility. In the Lacaune sheep breed, large variation in litter size is genetically determined by the segregation of a fecundity major gene influencing ovulation rate, named FecL and its prolific allele FecL(L) . Our previous work localized FecL on sheep chromosome 11 within a locus of 1.1 Mb encompassing 20 genes. With the aim to identify the FecL gene, we developed a high throughput sequencing strategy of long-range PCR fragments spanning the locus of FecL(L) carrier and non-carrier ewes. Resulting informative markers defined a new 194.6 kb minimal interval. The reduced FecL locus contained only two genes, insulin-like growth factor 2 mRNA binding protein 1 (IGF2BP1) and beta-1,4-N-acetyl-galactosaminyl transferase 2 (B4GALNT2), and we identified two SNP in complete linkage disequilibrium with FecL(L) . B4GALNT2 appeared as the best positional and expressional candidate for FecL, since it showed an ectopic expression in the ovarian follicles of FecL(L) /FecL(L) ewes at mRNA and protein levels. In FecL(L) carrier ewes only, B4GALNT2 transferase activity was localized in granulosa cells and specifically glycosylated proteins were detected in granulosa cell extracts and follicular fluids. The identification of these glycoproteins by mass spectrometry revealed at least 10 proteins, including inhibin alpha and betaA subunits, as potential targets of B4GALNT2 activity. Specific ovarian protein glycosylation by B4GALNT2 is proposed as a new mechanism of ovulation rate regulation in sheep, and could contribute to open new fields of investigation to understand female infertility pathogenesis.

Figure 1. Map of the FecL locus on ovine chromosome 11.

The genes are indicated above the line, markers are indicated by points under the line. The FecL locus (197 kb on OARv3.1, or 194.6 kb, our own sequencing) is flanked by the two closest recombinant markers, g.36910171T>C and g.37107627G>C. Recombinants: white box, zero-recombinant zone; gray boxes, zone with one recombinant; black boxes, at least two recombinants with FecL. N: no Allele sharing between wild-type and carrier animals for the FecL^L allele.

Figure 2. Expression of genes within the minimal FecL locus.

Total RNA from granulosa cells (GC) from small (SF, 1–3 mm), granulosa cells and theca cells (TC) from large (LF, ≥6 mm) follicles, pituitary gland (PG) and hypothalamus (HPT) were reverse-transcribed and submitted to real-time PCR analysis for quantification of B4GALNT2 and IGF2BP1 gene expression. Data are means ± SEM of relative expression to the reference gene RPL19 showed on a log scale. Asterisk indicates significant difference between means (n = 5) from non-carriers (+/+) and homozygous carriers of the FecL^L mutation (L/L), **: p<0.01; ***: p<0.001.

Figure 3. Immunostaining for B4GALNT2 in Lacaune sheep ovary.

Photomicrographs of ovarian sections from +/+ and L/L ewes stained with anti-B4GALNT2 rabbit polyclonal antibody (1/50 dilution). Sections were counterstained with hematoxylin. A black segment indicates the microscopy magnification scale. GC, granulosa cell layer; TC, theca cell layer; Ant, antral cavity.

Figure 4. B4GALNT2 transferase activity revealed by DBA lectin and KM694 antibody staining in Lacaune sheep ovary.

Photomicrographs of ovarian sections from +/+ and L/L ewes and stained either with biotinylated-DBA lectin (500 ng/ml) or KM694 mouse monoclonal antibody (1/1000 dilution). A GalNac treatment (200 µM) was used to compete for DBA staining as specificity control. Sections were counterstained with hematoxylin. A black segment indicates the microscopy magnification scale. GC, granulosa cell layer; TC, theca cell layer; Ant, antral cavity.

Figure 5. B4GALNT2 transferase activity revealed by DBA lectin after in vitro overexpression of B4GALNT2 in ovine granulosa cells.

Primary ovine granulosa cells from +/+ small antral follicles were transiently transfected with either the pCDNA-hB4GALNT2 expressing the human form of B4GALNT2 or the empty pCDNA3.1 vector. Twenty-four hours after transfection, cells were stained with biotinylated-DBA lectin (500 ng/ml). Arrows indicated DBA positive staining only in B4GALNT2 transfected cells. Cells were counterstained with hematoxylin. A black bar indicates the microscopy magnification scale.

Figure 6. Western immunoblotting analysis of B4GALNT2 transferase activity in Lacaune sheep granulosa cells and follicular fluids.

Granulosa cell protein extracts (50 µg) and follicular fluids (200 µg) from +/+ and L/L large antral follicles were precipitated (P) by agarose-DBA lectin or sepharose-protein A-KM694 monoclonal antibody. The resulting purified glycoproteins were separated on SDS-PAGE, transferred on nitrocellulose membrane and revealed after blotting (B) using biotinylated-DBA lectin or KM694 monoclonal antibody.