Altrenogest affects the development and endocrine milieu of ovarian follicles in prepubertal and mature gilts†

Abstract

Altrenogest with gonadotropins is commonly used to synchronize the estrous cycle, but it can also lead to follicular cyst formation, especially in prepubertal gilts. Here, we aimed to investigate how maturity and altrenogest treatment affect the development, endocrine milieu, and molecular control of ovarian follicles. Crossbred prepubertal and mature gilts were challenged or not (control) with altrenogest, and ovaries were collected in the morning on the first day of behavioral estrus. In prepubertal gilts, altrenogest decreased the percentage of primordial and atretic small follicles, but increased large antral follicles when compared to controls. In mature gilts, altrenogest reduced the percentage of primary follicles and elevated the total number of antral follicles. Maturity affected the estradiol level in the follicular fluid of preovulatory follicles, LH-stimulated cAMP generation, and LH receptor mRNA expression in granulosa. Moreover, cytochrome P45017A1 (CYP17A1) mRNA levels in the theca layer were affected and correlated with follicular androstendione and estradiol concentration. Altrenogest negatively affected follicular fluid progesterone concentration and decreased levels of prostaglandin (PG) E2 in prepubertal gilts and PGF2α metabolite in mature gilts. LH-stimulated cAMP release in granulosa cells of mature gilts as well as hCG- and forskolin-induced cAMP were also affected. In addition, altrenogest down-regulated CYP17A1 mRNA in the prepubertal theca layer and PGF2α synthase expression in the granulosa and theca layer of mature gilts. To the best of our knowledge, this is the first study to report multiple effects of maturity and altrenogest on the endocrine milieu and molecular regulations governing ovarian follicle development in gilts.

Endocrine and molecular milieus of ovarian follicles are diversely affected by human chorionic gonadotropin and gonadotropin-releasing hormone in prepubertal and mature gilts

“…In porcine granulosa and theca cells, recombinant (r)-LH and r-hCG had similar effects in vitro, mirrored in identical cAMP generation dynamics. However, altrenogest treatment decreased hCG-stimulated cAMP production in the theca layers of prepubertal but not mature gilts 68 . Thus, we hypothesized that hCG can initiate earlier androgenization of 17-OH-pregnenolone and increase progesterone production (especially in the follicles of prepubertal gilts), whereas GnRH administration, causing LH release, can ensure better (estrogenic) endocrine milieu for the onset of undisturbed ovulation.…”

“…Crossbred gilts at 165 days of age were contacted with mature boar every day for 14 days and then at approximately 180 days of age were used in two trials to create experimental groups. The detailed procedures are described in our recent paper 68 . Briefly, gilts considered as being in the first natural estrus formed a set of future sexually mature (M) gilts, which were recruited at 185–195 days of age.…”

“…Immunohistochemical analysis was performed for antral preovulatory follicles collected from prepubertal and mature gilts at the slaughterhouse to localize transferrin (TF) and vimentin (VIM). Preovulatory follicle walls were fixed, sectioned, and mounted for immunohistochemistry, as previously described by Ziecik et al 68 . Subsequently, sections were incubated in 0.3% (v/v) hydrogen peroxide in Tris-buffered saline (TBS, 0.1 M Tris and 150 mM NaCl; pH 7.4) for 30 min at room temperature to block endogenous peroxidase activity and treated with 5% (v/v) normal goat serum (for TF) or 5% (v/v) normal horse serum (for VIM) at room temperature for 30 min to block nonspecific binding sites.…”

“…RNA isolation and expression analysis were performed as previously described 68 . Briefly, total RNA was isolated from walls of preovulatory follicles using a mirVana microRNA Isolation Kit (Invitrogen, Thermo Fisher Scientific) and genomic DNA was removed by DNAse I (Invitrogen), according to the manufacturer’s instructions.…”

“…Thus, special attention should be paid to confirm ovulation, as too early hormonal treatment may lead to development of follicular cysts in prepubertal gilts 66 . Our recent studies have indicated that in addition to its known effects on ovarian folliculogenesis and development of antral follicles, altrenogest causes multiple changes in the endocrine milieu of follicles in prepubertal and mature gilts 68 .…”