Impact of heat stress on prolactin-mediated ovarian JAK-STAT signaling in postpubertal gilts

Abstract

Heat stress (HS) compromises almost every aspect of animal agriculture including reproduction. In pigs, this infecundity is referred to as seasonal infertility (SI), a phenotype including ovarian dysfunction. In multiple species, HS-induced hyperprolactinemia has been described; hence, our study objectives were to characterize and compare HS effects on circulating prolactin (PRL) and ovarian Janus kinase/signal transducer and activator of transcription (JAK-STAT) signaling during the follicular (FOL) or luteal (LUT) phases of the estrous cycle in postpubertal gilts. Gilts were estrus synchronized using altrenogest and environmental treatments began immediately after altrenogest withdrawal. For the FOL study: postpubertal gilts were allocated to constant thermoneutral (TN; n = 6; 20 ± 1.2 °C) or cyclical HS (n = 6; 25 to 32 ± 1.2 °C) conditions for 5 d. In the LUT study: postpubertal gilts were assigned to either TN (n = 7; 20 ± 2.6 °C) or cyclical HS (n = 7; 32 to 35 ± 2.6 °C) conditions from 2 to 12 days postestrus (dpe). Blood was collected by jugular venipuncture for PRL quantification on day 5 in the FOL and on day 0 and day 12 in the LUT gilts. Ovaries and corpora lutea (CL) were obtained from euthanized FOL and LUT gilts on day 5 and day 12, respectively. Western blotting was performed to quantify prolactin receptor (PRLR) and JAK/STAT pathway protein abundance. In the FOL phase, no difference (P = 0.20) in circulating PRL between thermal groups was observed. There was no effect (P ≥ 0.34) of HS on PRLR, signal transducer and activator of transcription 3 (STAT3), signal transducer and activator of transcription 5α (STAT5α), and phosphorylated signal transducer and activator of transcription α/β tyrosine 694/699 (pSTAT5α/βTyr694/699) abundance and Janus kinase 2 (JAK2), phosphorylated janus kinase 2 tyrosine 1007/1008 (pJAK2Tyr1007/1008), STAT1, phosphorylated signal transducer and activator of transcription 1 tyrosine 701 (pSTAT1Tyr701), phosphorylated signal transducer and activator of transcription 1 serine 727 (pSTAT1Ser727), and phosphorylated signal transducer and activator of transcription 3 tyrosine 705 (pSTAT3Tyr705) were undetectable in FOL gilt ovaries. Ovarian pSTAT5α/βTyr694/699 abundance tended to moderately increase (4%; P = 0.07) in FOL gilts by HS. In the LUT phase, circulating PRL increased progressively from 2 to 12 dpe, but no thermal treatment-induced difference (P = 0.37) was noted. There was no effect (P ≥ 0.16) of HS on CL abundance of PRLR, pJAK2Tyr1007/1008, JAK2, STAT1, pSTAT1Tyr701, pSTAT1Ser727, pSTAT3Tyr705, STAT5α, or pSTAT5α/βTyr694/699. In LUT phase, CL STAT3 abundance was increased (11%; P < 0.03) by HS. There was no impact of HS (P ≥ 0.76) on levels of pJAK2Tyr1007/1008 and pSTAT5α/βTyr694/699 in LUT gilts; however, the CL pSTAT3Tyr705:STAT3 ratio tended to be decreased (P = 0.10) due to HS. These results indicate an HS-induced estrous cycle-stage-dependent effect on the ovarian JAK/STAT pathway, establishing a potential role for this signaling pathway as a potential contributor to SI.

Keywords: JAK-STAT; gilts; heat stress; ovary; prolactin; seasonal infertility.

Figure 1.

Impact of heat stress on circulating plasma PRL level. (A) Gilts were exposed to TN (20 ± 1.2 °C) or cyclic HS (25 to 32 ± 1.2 °C) during the follicular phase for 5 d. (B) Gilts were exposed to TN (20 ± 2.6 °C) or diurnal HS (32 to 35 ± 2.6 °C) during the luteal phase from 3 to 12 dpe. The concentration of circulating PRL (ng/mL) ± SEM is presented. Abbreviations: dpe, days postestrus; HS, heat stress; PRL, prolactin; TN, thermoneutral.

Figure 2.

Heat stress effect on STAT3 and pSTAT3^Tyr705. (A) Gilts were exposed to TN (20 ± 1.2 °C) or cyclic HS (25.4 to 32 ± 1.2 °C) during the follicular phase for 5 d. (B, C) Gilts were exposed to TN (20 ± 2.6 °C) or diurnal HS (32 to 35 ± 2.6 °C) during the luteal phase from 3 to 12 dpe. Western blots with positive staining for STAT3 or pSTAT3^Tyr705 or ACTB proteins are presented. Bar charts represent the relative STAT3:ACTB or pSTAT3^Tyr705:ACTB protein abundance ± SEM. *P < 0.05. Abbreviations: ACTB, beta-actin; dpe, days postestrus; HS, heat stress; pSTAT3^Tyr705, phosphorylated signal transducer and activator of transcription 3 tyrosine 705; STAT3, signal transducer and activator of transcription 3; TN, thermoneutral

Figure 3.

Heat stress effect on STAT5α and pSTAT5α/β^Tyr694/699. (A, B) Gilts were exposed to TN (20 ± 1.2 °C) or cyclic HS (25.4 to 32 ± 1.2 °C) during the follicular phase for 5 d. (C, D) Gilts were exposed to TN (20 ± 2.6 °C) or diurnal HS (32 to 35 ± 2.6 °C) during the luteal phase from 3 to 12 dpe. Western blots with positive staining for STAT5α or pSTAT5α/β^Tyr694/699 or ACTB proteins are presented. The abundance of (A, C) STAT5α:ACTB and (B, D) pSTAT5α/β^Tyr694/699:ACTB protein ± SEM is presented; ^†P < 0.10. Abbreviations: ACTB, beta-actin; dpe, days postestrus; HS, heat stress; pSTAT5α/β^Tyr694/699, phosphorylated signal transducer and activator of transcription α/β tyrosine 694/699; STAT5α, signal transducer and activator of transcription 5α; TN, thermoneutral.

Figure 4.

Effect of heat stress on the ratio of phosphorylated JAK-STAT pathway proteins. The relative ratio of (A) pJAK2^Tyr1007/1008:JAK2, (B) pSTAT3^Tyr705:STAT3, (C) pSTAT5α/β^Tyr694/699:STAT5α during the luteal phase and (D) pSTAT5α/β^Tyr694/699:STAT5α during the follicular phase ± SEM is presented; ^†P < 0.10. Abbreviations: pJAK2^Tyr1007/1008, phosphorylated janus kinase 2 tyrosine 1007/1008; pSTAT3^Tyr705, phosphorylated signal transducer and activator of transcription 3 tyrosine 705; pSTAT5α/β^Tyr694/699, phosphorylated signal transducer and activator of transcription α/β tyrosine 694/699; STAT3, signal transducer and activator of transcription 3; STAT5α, signal transducer and activator of transcription 5α.