Evaluation of the Hypothalamic Kisspeptin System Throughout the Estrous Cycle in Gilts.

doi:10.21203/rs.3.rs-700168/v1

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Evaluation of the Hypothalamic Kisspeptin System Throughout the Estrous Cycle in Gilts.

https://doi.org/10.21203/rs.3.rs-700168/v1

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Background: Kisspeptin has been demonstrated to affect reproductive cyclicity and the attainment of puberty in multiple species, presumably through its actions on gonadotropin releasing hormone and luteinizing hormone. Kisspeptin administration causes increased plasma concentrations of LH in pigs, sheep, and rats. The objective of this experiment was to evaluate changes in the hypothalamic kisspeptin system throughout the estrous cycle in gilts.

Methods: Forty crossbred gilts (191 d, 121 kg) were administered an intramuscular injection of PG600. Twelve days after the administration of PG600, gilts were fed 15 mg of altrenogest daily for 15 d to synchronize estrus. Following estrus synchronization, estrus detection was performed by exposing gilts to a mature boar and the first day gilts stood immobile was denoted d 1 of the estrous cycle. Blood samples were collected via jugular venipuncture on d 1, 4, 7, 9, 14, 16, and 19 of the estrous cycle. Ten animals were slaughtered on d 1, 9, 14, and 21 of the estrous cycle when medial basal hypothalami, anterior pituitary glands, and blood were collected. Relative expression of hypothalamic kisspeptin (KISS1), kisspeptin receptor (KISS1R), estrogen receptors-α, anterior pituitary gland GnRH receptor and -β, β-actin and GAPDH was determined using real-time reverse transcriptase PCR. Fold changes in relative expression were determined using the Relative Expression Software Tool.

Results: Relative expression of KISS1 was increased (P=0.006) 3.2 fold on d 1 versus d 21 and 2.3 fold (P=0.003) on d 9 versus d 21 of the estrous cycle. Relative expression of KISS1 was not different (P>0.05) among the remaining days of the estrous cycle. Relative expression of estrogen receptor-b was decreased (P=0.05) 0.8 fold on d 9 versus d 21 and (P=0.005) 0.7 fold on d 14 versus d 21. Relative expression of anterior pituitary gland GnRH receptor was increased (P<0.01) on d 1 and 21 versus d 9 and 14. Relative expression of estrogen receptor-b was not different (P>0.05) among the remaining days. Relative expression of KISS1R and estrogen receptor-a were each not different (P>0.05) among days of the estrous cycle.

Conclusions: These data support the notion that medial basal hypothalamic expression of KISS1 changes throughout the estrous cycle and may influence reproductive cyclicity in the gilt.

Animal Science

Biotechnology and Bioengineering

Kisspeptin

Gilts

Estrous cycle

GnRH

Gonadotropin releasing hormone (GnRH) is a decapeptide hormone essential to reproduction through its actions on the release and synthesis of gonadotropins (1–3). Circulating concentrations of gonadotropins fluctuate throughout the estrous cycle and stimulate ovarian follicular development, while lutenizing hormone (LH) in particular is luteotrophic in many species and is responsible for causing ovulation. It is widely accepted that progesterone and estradiol − 17β control the release of gonadotropins through positive and negative feedback, however, exact mechanisms by which this occurs are not clear. It would be expected that GnRH neurons bear estrogen receptors, however, data remains equivocal as to the presence of estrogen receptors on the GnRH neurons (4–6). Thus, the release of GnRH may be controlled by other hormones, one of which is kisspeptin.

Kisspeptin may elicit the direct release of GnRH because it has been found that kisspeptin neuronal axons are associated with the dendrites of GnRH neurons (5). It has also been shown that the kisspeptin receptor, GPR54, is expressed by GnRH neurons and is directly stimulated by kisspeptin to cause the release of GnRH (7).

Recently, research has been performed to ascertain the relationship between the expression of hypothalamic kisspeptin (KISS1) and plasma concentrations of estradiol-17β (E₂) (8, 9). An increase in circulating concentrations of E₂ caused an increase in the expression of anteroventral periventricular nucleus (AVPV) KISS1 but decreased the expression of arcuate nucleus (ARC) KISS1 in the female mouse (9, 10). In the rat, expression of AVPV KISS1 peaks at a time coincident with the pre-ovulatory LH surge and KISS1 neurons express c-Fos induction at a coincident time (10, 11). Clarkson et al. (12) reported that mice bearing deletions in the kisspeptin receptor appear to lack the capacity to exhibit an LH surge versus mice that were ovariectomized and then treated with estrogen and progesterone. Hence, it is plausible that an increase in the expression of hypothalamic KISS1 may contribute to the pre-ovulatory GnRH/LH surge in the pig.

Kisspeptin has been demonstrated to play a critical role in the regulation of reproduction through multiple endocrine pathways, however, the changes in kisspeptin and related reproductive hormones throughout the estrous cycle in the gilt are yet to be determined. Therefore, the objective of this research was to determine serum concentrations of estradiol-17β and progesterone, anterior pituitary gland (AP) concentrations of LH, kisspeptin, and insulin like growth factor-I (IGF-I), medial basal hypothalamus (MBH) concentrations of kisspeptin and MBH expression of KISS1, kisspeptin receptor (KISS1R), estrogen receptor alpha (ER-α), estrogen receptor beta (ER-β) and AP GnRH receptor (GnRHR) throughout the porcine estrous cycle.

Animals

Forty crossbred (Duroc x Large White x Landrace) gilts of similar age (191 ± 11.6 d) and weight (120.7 ± 8.8 kg) were used in this experiment. Gilts were penned (1.83 m x 2.44 m) into groups of four and received ad libitum access to a corn-soy based diet that contained 3.4 Mcal ME/kg, 18% protein, and 0.9% lysine and water. Each gilt was administered an intramuscular injection of PG600 (Merck Animal Health, Summitt, NJ) two days after they were penned into groups of four. This product contains 400 IU equine chorionic gonadotropin and 200 IU human chorionic gonadotropin. Twelve days after the administration of PG600 gilts were orally administered 15 mg of altrenogest (Matrix; Merck Animal Health, Kenilworth, NJ) each day for 15 d to synchronize estrus. Gilts were exposed to a mature boar twice daily beginning the fourth day after the cessation of the altrenogest treatment and continuing for 4 d to detect estrus. The first day gilts stood immobile in the presence of the boar was designated as d 1 of the estrous cycle.

Ten gilts were slaughtered at the South Dakota State University Meat Lab on days 1, 9, 14, and 21 of the estrous cycle at which time blood samples, anterior pituitaries (AP), MBH, and reproductive tracts were collected. Blood samples (10 mL) were collected from all gilts on d 1 of the estrous cycle and remaining gilts that had not yet been slaughtered on days 4, 7, 9, 14, 16, and 19 of the estrous cycle via jugular venipuncture. Blood samples were allowed to clot overnight at 4º C then serum was collected by centrifugation (1,500 x g for 30 minutes at 4º C) and stored at -20º C. Anterior pituitary glands and MBH were trimmed of connective tissue, bisected midsaggitally, wrapped in aluminum foil, snap frozen in liquid nitrogen, and stored at -80º C. All experimental procedures were reviewed and approved by the Institutional Animal Care and Use Committee at South Dakota State University.

Estradiol-17β

Serum concentrations of estradiol-17β were determined in duplicate in all blood samples by radioimmunoassay (RIA). Estradiol-17β (E8875; Sigma Life Science, St. Louis, MO) was the standard and radioiodinated estradiol-17β (#07138228; MP Biomedicals, Solon, OH) was the tracer. Antisera (GDN#244 anti-estradiol-17β-6-BSA; Fort Collins, CO) was used at a dilution of 1:425,000. Sera (250-µL) were extracted with a 4-mL volume of methyl tert-butyl ether. Recovery of [125I]estradiol-17β added to porcine serum before extraction averaged 96 ± 2%. Inhibition curves of increasing amounts of sample were parallel to standard curves. Intra-assay and inter-assay coefficients of variation were 9.2% and 18.6%, respectively. Sensitivity of the assay was 0.2 pg/tube.

Progesterone

Serum concentrations of progesterone (P₄) were determined in duplicate in all blood samples by RIA. Progesterone (P0130; Sigma Life Science; St. Louis, MO) was the standard and radioiodinated progesterone (#07-170126; MP Biomedicals, Solon, OH) was used as the tracer. Antisera (#111.2C7.3; Enzo Life Sciences, Farmingdale, NY) was used at a dilution of 1:700,000. Samples were diluted 1:10 prior to assay. Inhibition curves of increasing amounts of sample were parallel to standard curves. Intra-assay coefficient of variation was 12.1%. Sensitivity of the assay was 0.33 ng/tube.

Insulin-Like Growth Factor-I

Anterior pituitary gland concentrations of IGF-I were determined in duplicate by RIA (13, 14). One half of each anterior pituitary gland was homogenized in a 15-mL polypropylene tube with 2-mL of homogenization buffer (1% cholic acid, 0.1% SDS, 200 µM phenylmethylsulfonylfluoride, 100 µM EDTA, 1 µM leupeptin, and 1 µM pepstatin) and homogenized on ice with a T25 Ultra-Turrax tissue dispenser (IKA Works, Wilmington, NC) for 30 s at 20,500 rpm. Anterior pituitary glands were then diluted to 100 mg of AP tissue/mL with homogenization buffer. Homogenates were centrifuged at 12,000 x g for 10 min at 4º C and the supernatant was removed and stored at -20º C. Protein content of the AP homogenates (1:20 dilution) was determined by the Bradford method using reagents provided by Bio-Rad (Hercules, CA). Insulin-like growth factor binding proteins were extracted from all homogenized anterior pituitary gland samples with a 1:17 ratio of sample to acidified ethanol (12.5% 2 N HCl:87.5% absolute ethanol; (15)). Recombinant human IGF-I (GF-050; Austral Biological, San Ramon, CA) was used as the standard and radioiodinated antigen. Antisera (UB2-495; National Hormone and Peptide Program, NIDDK) was used at a dilution of 1:62,500. Recovery of [125I]IGF-I added to porcine serum before extraction averaged 91 ± 3.2%. Inhibition curves of increasing amounts of sample were parallel to standard curves. Intra-assay coefficient of variation was 14.8%. Sensitivity of the assay was 10.9 pg/tube.

Luteinizing Hormone

Anterior pituitary gland concentrations of LH were determined in triplicate by RIA (16). Porcine LH (AFP3881A; National Hormone and Peptide Program, NIDDK) was used as the radioiodinated antigen and standard. Luteinizing hormone antiserum (AFP15103194; National Hormone and Peptide Program, NIDDK) was used at a dilution of 1:200,000. Anterior pituitary homogenates were diluted 1:25,000 in 0.01 M PBS-0.1% gelatin prior to assay. Inhibition curves of increasing amounts of sample were parallel to standard curves. Intra-assay coefficient of variation was 9.9%. Sensitivity of the assay was 0.12 ng/tube.

Kisspeptin

Hypothalamic and AP concentrations of kisspeptin were determined in duplicate by RIA. Human kisspeptin (1443; Tocris Bioscience, Ellsville, MO) was used as the radioiodinated antigen and standard. Kisspeptin antiserum (GQ2; provided by Waljit Dhillo, Imperial College, London, England, UK) was used at a dilution of 1:50,000. The GQ2 kisspeptin antibody cross reacted 100% with human kisspeptin-54, kisspeptin-14, and kisspeptin-10 and less than 0.01% with any other related human RF amide protein (17). Hypothalami and AP homogenates were diluted 1:30 in 0.01 M PBS-0.1% gelatin prior to assay. Inhibition curves of increasing amounts of sample were parallel to standard curves. Inter-assay coefficients for MBH and AP assays were 9.4% and 11.2%, respectively. Intra-assay coefficients of variation for MBH and AP assays were 4.6% and 8.0%, respectively. Sensitivity of the assay was 9.7 pg/tube.

Real-Time PCR

Total RNA was isolated from one half of each MBH and AP using TriReagent (TR118, Molecular Research Company, Cincinnati, OH). Purity of RNA was determined by measuring the A260/A280 ratio. The ratio of all samples ranged from 1.8 to 2.0. The integrity of RNA was confirmed by agarose gel electrophoresis. Samples were treated with DNase according to the manufacturer’s protocol (TurboDNA-free kit, Applied Biosystems, Foster City, CA, USA). Reverse transcriptase PCR was used to measure the abundance of each specific mRNA relative to the abundance of porcine β-actin and porcine GAPDH in the total RNA isolated from MBH tissue. Expression of β-actin and GAPDH did not differ among days. Two micrograms of total RNA were reverse transcribed using random hexamer primers (High Capacity cDNA Reverse Transcription Kit, Applied Biosystems, Foster City, CA) to produce cDNA. One hundred micrograms of cDNA were used in each reaction. Twenty-five-microliter PCR reactions were performed using RT2 SYBR Green/ROX qPCR Master Mix (SuperArray Bioscience Corp., Foster City, CA). Primer pairs used for specific amplification of MBH expression of KISS1, KISS1R, ER-α, ER-β, AP GnRHR, β-actin and GAPDH are listed in Table 1. Reactions were measured using the Stratagene MX3000P quantitative real-time PCR instrument (Agilent Technologies, Foster City, CA) using thermal cycling conditions recommended by the manufacturer (40 cycles of 30 sec at 95˚C, 1 min at 55˚C and 1 min at 72˚C). Concentrations of forward and reverse primers used for the genes of interest were 300 nM, except KISS1R, in which concentrations of forward and reverse primers were used at 800 nM due to its low abundance. A linear response was obtained when these concentrations of primer pairs were used with increasing amounts of cDNA. Dissociation curve analysis was performed after each real time PCR run and confirmed that a single amplicon of appropriate melting temperature was present. Additionally, all amplicons were electrophoresed through a 2% agarose gel and stained with ethidium bromide to visualize that only amplicons of the appropriate size were present in each sample.

Statistical Analysis

The effect of day of the estrous cycle on serum concentrations of estradiol-17β, and progesterone, was analyzed by the Proc Mixed procedure with repeated measures using the JMP 8.0 program (Statistical Analysis System [SAS], Cary, NC, USA). The effect of day of the estrous cycle on AP concentrations of IGF-I, LH, kisspeptin and MBH concentrations of kisspeptin was analyzed by one-way analysis of variance of SAS. Fold changes in relative expression of MBH ERα, ERβ, KISS1, KISS1R, and AP GnRHR were determined using the Relative Expression Software Tool (18).

Mean serum concentrations of E₂ were greatest (P < 0.05) on d 19 of the estrous cycle and were not different (P > 0.05) on days 4, 7, 9, and 14 of the estrous cycle (Fig. 1A). On d 1 of the estrous cycle, mean serum concentrations of estradiol-17β were greater (P < 0.05) than on days 4 and 21 of the estrous cycle. Mean serum concentrations of estradiol-17β were greater (P < 0.05) on d 16 compared to d 21 of the estrous cycle. Mean serum concentrations of progesterone followed the typical pattern observed throughout the estrous cycle in gilts (Fig. 1B). Mean serum concentrations of P₄ increased from d 1 through 14 and were greatest (P < 0.05) at d 14 of the estrous cycle. From d 14 to d 21 of the estrous cycle mean serum concentrations of P₄ decreased (P < 0.05).

Mean AP concentrations of IGF-I were greater (P < 0.05) on d 21 compared to d 1 of the estrous cycle (Fig. 2A). No other differences among days of the estrous cycle were detected (P > 0.05). Mean AP concentrations of LH were greatest (P < 0.05) on day 14 of the estrous cycle (Fig. 2B). Mean AP concentrations of LH were not different (P > 0.05) on days 1 and 9, and 1 and 21 of the estrous cycle, however mean AP concentrations of LH were greater (P < 0.05) on day 9 versus 21 of the estrous cycle.

No differences were detected (P > 0.05) in mean AP concentrations of kisspeptin throughout the estrous cycle (Fig. 3A). No differences were detected (P > 0.05) in mean MBH concentrations of kisspeptin throughout the estrous cycle (Fig. 3B).

Mean relative expression of MBH ER-α did not differ (P > 0.05) in gilts throughout the estrous cycle (Fig. 4A). Mean relative expression of MBH ER-β was up-regulated (P < 0.05) on days 9 and 14 compared to day 21 of the estrous cycle (Fig. 4B). Mean relative expression of AP GnRHR was up-regulated (P < 0.01) on d 1 and 21 versus d 9 and 14.

Mean relative expression of MBH KISS1 was greatest (P < 0.05) on d 21 of the estrous cycle (Fig. 5A). Mean relative expression of MBH KISS1 was down-regulated (P < 0.05) on days 1 versus 14 and 21 and 9 versus 21 of the estrous cycle. Mean relative expression of MBH KISS1R (Fig. 5B) did not differ (P > 0.05) in gilts throughout the estrous cycle.

Kisspeptin, a protein produced by most mammalian species, has been found in many tissues throughout the body (19). Kisspeptins are small, neurohormones encoded by the KISS1 gene (20). After ICV and intra-ARC infusion of kisspeptin antibodies there was a profound decrease in serum concentrations of LH in the rat (21). Kisspeptin has been shown to elicit the release of LH via ICV, intramuscular, and intravenous infusion in a number of mammalian species (22–24). Kisspeptin-54 has also been demonstrated to stimulate the hypothalamo-pituitary gonadal axis in humans (17, 25). Although these studies demonstrated that exogenous kisspeptin can cause the release of LH, it is also thought that endogenous kisspeptin released by the hypothalamus of mammals can cause the release of LH.

In the present study, estradiol-17β and progesterone followed patterns that were indicative of a normal porcine estrous cycle. Henricks et al. (26) demonstrated that during the luteal phase (from d 4 to 16 of the estrous cycle) plasma concentrations of estrogen were low (< 20 pg/mL/animal). Following that, plasma concentrations of estrogen started to increase and reached their peak (> 50 pg/mL/animal) between 1 and 2 days before estrus (26). Mean plasma concentrations of progesterone increased dramatically from d 2 to 6 of the estrous cycle and continued until they peaked by d 12 of the estrous cycle (26). In the present study, serum concentrations of estrogen started to increase only after plasma concentrations of progesterone started to decrease, which was temporally related with the next expected LH surge.

Insulin-like growth factor-I has been shown to be positively associated with estrogen, in that E₂ caused an increase in peripheral and AP concentrations of IGF-I in the pig (27, 28). Estrogens are synthesized from androgens via aromatase enzymes (29). Anastrozole is a non-steroidal aromatase-inhibiting compound that has been shown to dramatically reduce the synthesis of estrogens and reduce plasma concentrations of estrogen by 86% in women (30, 31). Hilleson-Gayne and Clapper (27) demonstrated that serum concentrations of IGF-I were greater in boars that were untreated versus boars that were treated with anastrozole. Estrogen has also been demonstrated to affect concentrations of AP IGF-I in the pig as well. Rempel and Clapper (28) reported that estrogen implanted barrows had greater AP concentrations of IGF-I versus untreated barrows and boars. These data support the notion that E2 could also affect the AP concentrations of IGF-I during various days of the estrous cycle. Gilts slaughtered on the last day of the estrous cycle and at a time directly following greater serum concentrations of E2 (day 19) had greater AP concentrations of IGF-I versus gilts that had lesser serum concentrations of E2 directly following a standing estrus on d 1 of the experiment. Insulin-like growth factor-I has been demonstrated to stimulate the release of LH from cultured AP cells in rats and pigs (32, 33). Adam et al. (34) also demonstrated that peripheral administration of IGF-I can acutely stimulate an increase in plasma concentrations of LH in sheep. Therefore, it is plausible that IGF-I may affect the AP LH system and work in concert with kisspeptin to cause a greater release of LH.

Mean AP concentrations of LH were greatest on d 14 of the estrous cycle and least on d 1 and 21; a time coincident with a standing estrus and a time temporally related with the next expected estrus, respectively. These data coincide with the patterns of E₂ and P₄. Henricks et al. (26) demonstrated that serum concentrations of LH decreased after a standing estrus until just before the subsequent estrus when LH increased to its peak. During the peak in LH, serum concentrations of estrogen were increased and serum concentrations of progesterone remained decreased (26). Additionally, Baird and Scaramuzzi (35) found that basal peripheral concentrations of LH decreased to a level similar to peripheral concentrations of LH during the luteal phase of the estrous cycle following subcutaneous P₄ implantation in ewes. In the present study, it is plausible that increased P₄, in the face of decreased E₂, was providing negative feedback to the MBH and AP during the luteal phase of the estrous cycle causing a buildup of AP LH prior to the pre-ovulatory LH surge.

Past research has shown that there is a correlation between circulating concentrations of sex steroids and hypothalamic concentrations of kisspeptin and expression of KISS1 (9, 36, 37). In the present study, there was no difference in MBH and AP concentrations of kisspeptin-54 throughout various days of the estrous cycle, however there was an increase in relative MBH KISS1 gene expression on the first day of the estrous cycle compared to the last day of the estrous cycle. Increased relative MBH KISS1 gene expression occurred at a time coincident with greater serum concentrations of E₂ and greater AP concentrations of LH. Cui et al. (38) performed an experiment measuring KISS1 and GnRH gene expression and protein concentration in multiple areas of the hypothalamus in ovariectomized (OVX) rats treated with E₂. During the onset of puberty, kisspeptin-immunoreactive cells increased in the ARC, periventricular nucleus, and preoptic areas (38). Levels of both hypothalamic KISS1 and GnRH gene expression were greater in OVX + E₂ and/or intact + E₂ compared to OVX and intact animals that were not treated with E₂ (38). Furthermore, hypothalamic expression of KISS1 mRNA during estrus was decreased versus other stages of the estrous cycle in the rat (39). These data support the notion that an increase in hypothalamic KISS1 mRNA and hypothalamic kisspeptin concentration play a role in modulating the activity of estrogen during the time an animal expresses estrus. Though the timing of sample collection impacts the outcome greatly, the present study parallels with past research. During expression of estrus, when serum concentrations of E₂ were greater, there was a decrease in hypothalamic expression of KISS1 compared to the last day of the estrous cycle when serum concentrations of E₂ were less.

In the present study relative expression of AP GnRHR was increased during the periovulatory period of the estrous cycle as has been previously documented (40). This, coupled with the fact that MBH KISS1 expression was increased at a coincident time, may reflect a mechanism by which kisspeptin and GnRH receptors work together to cause the preovulatory LH surge. Kisspeptin could cause the increased release of GnRH and, in turn, the increased GnRH receptors could respond to cause a surge release of LH to cause ovulation.

Kisspeptins bind to their cognate receptor, GPR54, with high affinity (41). There is evidence that GnRH neurons possess kisspeptin receptors and after kisspeptin binds to GPR54 there is an increase in hypothalamic concentrations of GnRH (41). The KISS1R gene has been demonstrated to be highly abundant in multiple tissues in the body, including the hypothalamus and anterior pituitary gland (19). In the present study, hypothalamic KISS1R gene expression was measured on various days of the estrous cycle but no differences among days were found. Kisney-Jones et al. (42) reported that there was a down-regulation of hypothalamic expression of KISS1R during a stress-induced suppression of LH in the female rat. Although there were no differences among days of the estrous cycle, it is plausible that variations in the expression of the KISS1R gene and the number of kisspeptin receptors may influence the GnRH pathway and endocrine status of the animal at a time coincident with a standing estrus.

No differences were detected in mean relative expression of MBH ER-α, however mean relative expression of MBH ER-β was decreased on d 21 of the estrous cycle compared to d 9 and 14 of the estrous cycle and tended to be less on d 21 compared to d 1. In the rat, selective blockade of ER-α in gonadally intact, cyclic females decreased the release of LH in response to kisspeptin, inhibited the preovulatory LH surge, and blocked ovulation (43). Roa et al. (43) also demonstrated that after selective activation of ER-α there was an increase in LH secretion in response to kisspeptin. This was not the case when ER-β was activated (43). Additionally, when rats were treated with an ER-β antagonist the preovulatory LH surge still occurred, however there was a decrease in acute LH responses to kisspeptin (43). Although there are alterations in the MBH expression of ER-α and ER-β in the present study, GnRH neurons may not be direct targets for the actions of E₂ (4, 44). Kisspeptin is a potent secretagogue of GnRH (17, 45). Additionally, kisspeptin possess estrogen receptors (9, 10, 46), and AVPV expression of KISS1 is up-regulated at a time coincident with the LH surge (10, 11). Therefore, the hypothalamic kisspeptin system and ER-α and –β are closely integrated and alterations in hypothalamic expression of ER-α and –β may cause increased release of kisspeptin, the subsequent GnRH/LH surge, and ovulation in mammalian species.

In conclusion, kisspeptin has been demonstrated to cause the release of LH in vivo and in vitro in a number of mammalian species, including the gilt (22–24). Recent research has also described the relationship between increased peripheral concentrations of E₂ and increased hypothalamic concentrations of KISS1 (8–10). In the present study, there were variations in hypothalamic KISS1 gene expression at key time points in the estrous cycle; particularly just before gilts expressed estrus. Kisspeptin may be an important hormone driving normal reproductive cyclicity, however the timing at which samples are collected is critical and needs to be further investigated.

Acknowledgements

Not applicable

Authors’ contributions

The experiment was designed and conducted by Jeffrey Clapper and Eric Jolitz. Experimental data were collected analyzed and the manuscript prepared by Jeffrey Clapper and Eric Jolitz. Development of the kisspeptin radioimmunoassay and kisspeptin antibody was performed by Waljit Dhillo.

Funding

Funding was provided by the South Dakota State University Agricultural Experiment Station through the use of HATCH funds.

Availability of data and materials

All data generated or analyzed during this study are included in this manuscript.

Ethics approval and consent to participate

All experimental procedures involving animals were approved by the South Dakota State University Institutional Animal Care and Use Committee.

Consent for publication

Not applicable

Competing interests

There are no conflicts to declare.

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Table 1 Forward and reverse primers for Real-Time PCR for porcine mRNA

^aporcine KISS1; ^bporcine KISS1 type 1 receptor; ^cporcine estrogen receptor-beta; ^dporcine estrogen receptor-alpha; ^eporcine GnRHR; ^fporcine glyeraldehyde 3 – phosphate dehyrdogenase; ^gporcine beta-actin

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Evaluation of the Hypothalamic Kisspeptin System Throughout the Estrous Cycle in Gilts.

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