Effect of single-chain ovine gonadotropins with dual activity on ovarian function in sheep

Abstract

We examined the half-life and biological activity of two single-chain proteins that combined portions of ovine FSH and LH. We proposed the hypothesis that these chimeric proteins would display LH and FSH activities and would promote follicle maturation in ewes. Estrus activity was synchronized using progestogen-impregnated vaginal pessaries. To negate the impact of endogenous LH and FSH, animals received serum-containing antibodies against GNRH 1 day before pessary removal (PR). At PR sheep (five animals per group) received a single injection (10 IU/kg, i.v.) of either the ovine-based (oFcLca) gonadotropin analog, an ovine-based analog containing oLHb truncated at the carboxyl terminus (oFcL(DT)ca), or a human-based gonadotropin analog (hFcLca). Control animals received a comparable amount of gonadotropin-free protein. Ovulation was induced 3 days after PR using human chorionic gonadotropin (1000 IU, i.v.). Ovaries were collected 11 days after PR. Neither estradiol (E 2 ) or progesterone (P 4 ) production, development of preovulatory follicles or corpora lutea (CL) were noted in control animals receiving gonadotropin-free protein. Significant increase in the synthesis of E 2 and P 4 was noted in sheep receiving the dually active gonadotropin analogs. The number of CLs present 11 days after PR was significantly increased in sheep receiving the chimeric glycoproteins compared with control animals. The magnitude of the secretory and ovarian responses did not differ between hFcLca and oFcLca or between oFcLca and oFcL(DT)ca. Immunoactivity of LH and FSH was low in control animals, but was significantly elevated in sheep receiving the gonadotropin analogs. In conclusion, ovine-based gonadotropin analogs are functionally active in sheep and a single injection is adequate to induce the development of multiple ovulatory follicles.

Follicle-Stimulating Hormone (FSH) Action on Spermatogenesis: A Focus on Physiological and Therapeutic Roles

“…Such dual-active analogs could be relevant for the treatment of HH and infertile men. These constructs included FSHβ-CTP-CGβ-α and FSHβ-CTP-LHβ-CTP-α, and, when injected in sheep, elicited increased serum estradiol concentration, ovarian weight, and formation of corpora lutea [177,180,181]. The clearance rate of the dual-active FSHβ-CTP-LHβ-CTP-α was also significantly improved compared with endogenous ovine gonadotropin (1 to 2 h).…”

FSH for the Treatment of Male Infertility

“…These analogues had comparable or improved biological activity in vivo compared to native FSH, half-lives were also improved presumably because O-linked glycosylation sites of the CTP reduced hepatic clearance rates [156][157][158]. Some analogues ( Figure 2) presenting dual FSH and LH activities (FSHβ-CTP-CGβ-α and FSHβ-CTP-LHβ-CTP-α) increased serum estradiol, ovarian weight and the formation of corpora lutea when injected in sheep [158][159][160]. To date, no single-chain FSH or dually active gonadotropin analogue has reached the clinical phase of development.…”

Trends in recombinant protein use in animal production

“…To obtain a functional hormone, both subunits must be assembled together [14, 20] posing an important bottleneck for its recombinant production. However, and because the interaction between subunits is not an absolute requirement for receptor activation ([21] and references therein), functional single-chain gonadotropin analogs have been successfully developed recombinantly by merging their α and β subunit genes in a single sequence [21–24]. Nevertheless, and even if the steroidogenic response is achieved, the structural differences in the hormone analogs commonly imply differences in steroid secreted levels [21].…”

“…The CG has a longer region because of a peptide called carboxyl terminal peptide (CTP) that provides the CG with more glycosylation places and prolong CG half-life by reducing its renal clearance [56]. These extra glycosylation sites have been fused to FSH [6, 19, 32–34] and LH [21, 24, 57–59], which do not naturally contain this sequence, to achieve long-acting hormones. This allowed the reduction in the number of injections for superovulation treatments [13, 36, 40].…”

“…Both dimer- [64, 65] and single-chain forms [21, 24, 57–59, 66, 67] of LH and CG have been studied in CHO expression system. Also, human recombinant CG was obtained by CHO cell expression for the construction of a chimera hCG-boCTP used to study the potential of a CTP-like sequence present but not expressed in the β subunit of bovine (among other mammal species) LH [68].…”