Co-localisation of gonadotrophins and granins in gonadotrophs at different stages of the oestrous cycle in sheep

Associations between granins (secretogranin II (SgII) and chromogranin A and B (CgA and CgB)) and gonadotrophins (LH and FSH) have been reported in rodents and they may interact to facilitate differential storage and secretion of LH and FSH. This study investigated the relationship between granins and gonadotrophins in sheep at different stages of the oestrous cycle. Thirty-four cycling ewes had their oestrous cycles synchronised, and were divided into late luteal (LL; n=5) and early (EF; n=4), mid (n=3) and late (LF; n=11) follicular stages, and 24-53h (n=5), 80-100h (n=3) and 120-144h (n=3) after the preovulatory LH surge (PS). LHbeta mRNA levels were low in LF ewes (when plasma levels and pulse frequency of LH were high) but had increased by 80-100 h PS. In contrast, FSHbeta mRNA levels decreased during the follicular phase and plasma FSH concentrations followed a similar pattern, to peak at 24-53 h PS due to low plasma oestradiol levels. While alpha-gonadotrophin subunit (alpha-GSU), SgII and CgA mRNA levels did not change, CgB mRNA levels were elevated in EF ewes and had declined in ewes around the surge. Four distinctly sized mRNA transcripts (similar to1-3, 2-0, 2-8 and 3-2 kb) were observed for CgA mRNA, while a double band was observed for LHbeta mRNA that was subsequently reduced to a single band after 3'-poly(A) tail truncation. The long and short LHbeta transcripts were prevalent in follicular and luteal ewes respectively. Numbers of LH+ve/FSH-ve granules stored within gonadotrophs were not different in LL and LF ewes (even though proportions of LH+ve granules were higher in LF ewes), but were reduced at 24-53 h PS. The majority of LH+ve granules also contained SgII, although few CgA(+ve) granules were found. Granule partitioning was evident whereby FSH and CgA were located near the periphery, and LH and SgII throughout the matrix. In conclusion, increases in both storage of LH+ve granules and secretion of LH in LF ewes despite constant LHbeta mRNA levels was facilitated, at least in part, by improved LHbeta mRNA transcript stability. Fewer LH+ve/FSH-ve granules were in storage after the PS, which was mirrored by a reduction in LH pulsatile release. Surprisingly, in view of results in rodents indicating significant changes, SgII and CgA mRNA levels did not change over the oestrous cycle in sheep. Conversely, CgB mRNA levels decreased around the time of PS. These novel results illustrate major differences in granin-gonadotrophin interactions between sheep and rodents.