Bovine luteal prolactin receptor expression: Potential involvement in ...

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Dec 4, 2014 - ment of PRLR, especially s-PRLR, in the regulation of progesterone secretion and metabolism during the bovine estrous cycle and pregnancy.
Published December 4, 2014

Bovine luteal prolactin receptor expression: Potential involvement in regulation of progesterone during the estrous cycle and pregnancy1 I. M. Thompson, M. Ozawa, J. W. Bubolz, Q. Yang, and G. E. Dahl2 Department of Animal Sciences, University of Florida, Gainesville 32611

ABSTRACT: In the present study, we performed quantitative reverse-transcription PCR (qPCR) to examine changes in gene expression of prolactin receptor (long form: l-PRLR; short form: s-PRLR) and 20α-hydroxysteroid dehydrogenase (20α-HSD; EC 1.1.1.149) in the bovine corpus luteum (CL) throughout the estrous cycle and pregnancy. Western blotting was used to determine protein abundance. Bovine CL were collected and luteal stages (n = 6/stage) were classified by macroscopic observation as early (d 1 to 4 after ovulation), mid (d 5 to 10), late (d 11 to 17), and regressing (d 18 to 20). A CL of pregnancy (n = 6) was determined by the presence of conceptus (d 28 to term). The mRNA for both forms of PRLR were expressed at all the luteal stages. Expression of s-PRLR and l-PRLR mRNA was less (P < 0.01) during early and regressing luteal stages compared with mid and late stages. Expression of s-PRLR mRNA in CL of pregnancy was greater (P < 0.01) than early, mid, and

regressing CL and did not differ from late luteal stage expression. A greater (P < 0.01) expression of l-PRLR mRNA was observed in pregnant vs. early and regressing CL. In addition, qPCR showed the presence of 20α-HSD mRNA during all luteal stages of the estrous cycle, with the greatest (P < 0.01) expression observed in the regressing luteal stage. Western blotting revealed protein abundance of both PRLR isoforms during all luteal stages and pregnancy, with a predominance of the s-PRLR protein. Densitometry analysis indicated that protein abundances of s-PRLR were greater (P < 0.05) than l-PRLR during early, mid, and late luteal stages and did not differ during the regressing luteal stage. Protein abundances of 20α-HSD were least (P < 0.05) during the early luteal stage. In conclusion, results of the current study suggest a possible involvement of PRLR, especially s-PRLR, in the regulation of progesterone secretion and metabolism during the bovine estrous cycle and pregnancy.

Key words: corpus luteum, estrous cycle, 20α-hydroxysteroid dehydrogenase, pregnancy, prolactin receptor ©2011 American Society of Animal Science. All rights reserved. J. Anim. Sci. 2011. 89:1338–1346 doi:10.2527/jas.2010-3559

INTRODUCTION Prolactin (PRL) is a polypeptide hormone synthesized and secreted by the anterior pituitary gland and various extrapituitary tissues (Bole-Feysot et al., 1998). As a multifunctional hormone, PRL is involved in the regulation of a variety of physiological processes such as mammary tissue development, immune function, and reproduction. The effects of PRL are initiated by its binding to a specific membrane receptor. The PRL receptor (PRLR) is a transmembrane protein belonging to the cytokine receptor superfamily. Long and short isoforms of PRLR are expressed by alternative splicing

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The authors thank Peter Hansen for providing the ovaries and Alan Ealy (Department of Animal Sciences, University of Florida, Gainesville) for allowing us to perform quantitative reverse-transcription PCR and Western blotting in his laboratory. 2 Corresponding author: [email protected] Received September 29, 2010. Accepted December 28, 2010.

of a single PRLR gene and differ by the lengths of the carboxyl-termini of their cytoplasmic domains (Bignon et al., 1997; Schuler et al., 1997). Prolactin exerts luteotropic action in rodents by promoting adequate corpus luteum (CL) development, stimulating progesterone secretion, and inhibiting luteal catabolism of progesterone. Expression of both forms of PRLR in rodent CL varies throughout the estrous cycle and pregnancy (Clarke et al., 1993; Clarke and Linzer, 1993). Mice carrying a null mutation of the PRLR gene are sterile, show absence of pseudopregnancy, and egg development is arrested immediately after fertilization (Ormandy et al., 1997). The PRLR maintains luteal progesterone concentrations via downregulation of luteal 20α-hydroxysteroid dehydrogenase (20α-HSD) gene expression and synthesis (Albarracin et al., 1994; Grosdemouge et al., 2003). In the ovary of rats and mice, 20α-HSD promotes functional luteolysis by catalysis of progesterone to its inactive form, 20α-hydroxyprogesterone. Although it is generally not considered as luteotrophic in the cow or ewe (Kalten-

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bach et al., 1967, 1968; Hoffmann et al., 1973), there is evidence that PRL contributes to maintenance of early pregnancy in heifers (Wettemann and Hafs, 1973). Therefore, to elucidate the role of PRL in bovine CL, we examined changes in PRLR (long form: l-PRLR; short form: s-PRLR) and 20α-HSD gene expression and protein abundance throughout the estrous cycle and pregnancy.

MATERIALS AND METHODS All procedures and tissue collections were approved by the University of Florida Institutional Animal Care and Use Committee. Ovaries with CL were collected at a local abattoir, and luteal stages were classified as early (d 1 to 4 after ovulation), mid (d 5 to 10), late (d 11 to 17), and regressing (d 18 to 20), according to macroscopic appearance of the CL (n = 6/stage), as described previously (Ireland et al., 1980). In addition, a CL of pregnancy (n = 6) was determined by the presence of conceptus (d 28 to term). Immediately after collection and classification of luteal stages, CL were frozen in liquid nitrogen and stored at −80°C for further total RNA and protein extractions.

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3′-GCACTAAGGGTCACTCTTTCT-5′, respectively. In addition, GAPDH gene forward and reverse primer sequences were 5′-ACCCAGAAGACTGTGGATGG-3′ and 3′-GTGAGGGTTGCACAGACAAC-5′, respectively. Standard curves generated from serial dilutions of bovine liver RNA were used to validate the amplification efficiency. In addition, a dissociation curve analysis (60 to 95°C) was used to verify the amplification of a single product. Quantitative PCR was completed with SYBR green as the indicator and the ABI 7300 Real Time PCR System (Applied Biosystems). Amplification of specific genes was obtained by running a mixture of 2.5 μL of the cDNA product for each specific primer (l-PRLR, s-PRLR, 20α-HSD, or GAPDH) and 22.5 μL of SYBRGreen PCR Master Mix (Applied Biosystems) in the reaction. Triplicate reactions were completed for each sample, and a fourth reaction containing RNA sample not exposed to reverse transcriptase was included as a negative control. After an initial activation/ denaturation step (60°C for 2 min; 95°C for 10 min), 40 cycles of a 2-step amplification protocol (95°C for 15 s and 60°C for 1 min) were completed. The abundances of l-PRLR, s-PRLR, and 20α-HSD mRNA were calculated relative to GAPDH mRNA (endogenous control) using the following formula: 2–Ct(target gene)/2–Ct (GAPDH), where Ct is the cycle threshold.

RNA Extraction and Quantitative ReverseTranscription PCR Analyses

Protein Extraction and Western Blotting

The abundances of PRLR (l-PRLR: NM_001039726.1, and s-PRLR: NM_174155.2) and 20α-HSD (S54973.1) transcripts in bovine CL during all luteal stages and pregnancy were determined by quantitative reversetranscription PCR (qPCR). Total RNA was extracted from CL using Tri reagent (Sigma, St. Louis, MO) with the PureLink RNA Mini kit (Invitrogen, Carlsbad, CA), according to instructions provided by the manufacturer. Subsequently, concentration and purity of isolated RNA were assessed using a NanoDrop 2000 spectrophotometer (Thermo Scientific, Rockford, IL). All samples were incubated with DNase (Applied Biosystems, Foster City, CA) for 30 min at 37°C to remove genomic DNA and subsequently heat-denatured at 75°C for 15 min. Total RNA (250 ng/reaction) was reverse transcribed to complementary DNA using the high-capacity cDNA Reverse Transcription Kit (Applied Biosystems) following manufacturer’s instructions. Gene-specific primer sets for the l-PRLR, s-PRLR, 20α-HSD, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) genes were designed. Glyceraldehyde 3-phosphate dehydrogenase was used as the reference gene. Sequences of the l-PRLR forward and reverse primers were 5′-GCCATCCTTTCTGCTGTCAT-3′ and 3′-CAAGTAGACGACCTCTTCCC-5′, respectively. The s-PRLR forward primer was the same one used for l-PRLR; however, a specific reverse primer sequence was designed for the s-PRLR: 3′-ATAGTGTCGGAAGAGCGGAA-5′. Sequences of the 20α-HSD forward and reverse primers were 5′-GACCTTGGGTACCGTCACAT-3′ and

Frozen CL tissue was homogenized in RIPA buffer (Pierce, Rockford, IL) with Halt protease inhibitor cocktail (100 µL/10 mL of lysate; Thermo Scientific). The homogenates were centrifuged (at 12,000 × g for 15 min at 4°C), resulting supernatants were isolated, and protein concentrations were determined using a BCA Protein Assay (Pierce). Samples (20 μg) were boiled for 5 min in Lane Marker Reducing Sample Buffer (Thermo Scientific). Denatured proteins were separated by SDS-PAGE using a 10% (wt/vol) polyacrylamide gel. A prestained protein ladder was included in each gel (Benchmark Pre-stained Protein Ladder, Invitrogen). Electrophoresed proteins were transferred to 0.45-μmpore polyvinylidene fluoride membranes (Immobilon-P Transfer Membranes, Millipore, Billerica, MA) using a semi-dry blotter. Membranes were blocked with 5% nonfat dried milk in Tris-buffered saline containing 0.1% (vol/vol) Tween-20 at room temperature for 1 h. A monoclonal antibody (U5, 1 μg/mL; Pierce) that recognizes a common extracellular epitope of both short and long PRLR was used to determine PRLR protein abundance. A polyclonal anti-AKR1C1 antibody (1 μg/mL; Sigma) was used to determine 20α-HSD protein content. Liver tissue was used as a positive control. Each antibody was diluted in Tris-buffered saline containing 0.1% (vol/vol) Tween-20 containing 3% BSA and exposed to blots overnight at 4°C. Membranes were then incubated with horseradish-peroxidase (HRP)linked secondary antibody (anti-mouse or anti-rabbit IgG HRP conjugate; Cell Signaling Technology, Bev-

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erly, MA; 0.2 μg/mL) at room temperature for 1 h. Enhanced chemiluminescence and X-OMAT film (Kodak, Carestream Health Inc., Rochester, NY) was used to visualize reactive protein bands (ECL Plus, GE Healthcare Bio-Sciences Corp., Piscataway, NJ). Protein loading in each lane was determined using an anti-β-actin monoclonal antibody (rabbit monoclonal antibody, HRP conjugate; Cell Signaling Technology; 1 μg/mL).

Densitometry The relative protein contents of long and short PRLR and 20α-HSD protein bands were quantified by densitometry analyses using ImageJ Software (National Institutes of Health, Bethesda, MD). Relative intensities of bands were corrected based on the signal intensity of β-actin.

Statistical Analysis Analyses of qPCR and densitometry were completed by ANOVA using the general linear model (PROC GLM) procedure (SAS Inst. Inc., Cary, NC). Differences in mean values were obtained from comparison of LSMEANS by the PDIFF option of PROC GLM, which provide a table of P-values for all possible pairwise comparisons. Data had a normal distribution, and variance within each population was the same.

RESULTS qPCR Specific mRNA for both forms of PRLR were expressed in the bovine CL at all luteal stages. Expression of s-PRLR and l-PRLR mRNA was less during early and regressing luteal stages compared with mid and late stages (P < 0.01; Figures 1 and 2). Expression of l-PRLR mRNA did not differ between pregnant and mid (P = 0.16) and late luteal CL stages (P = 0.52). However, greater expression (P < 0.01) of l-PRLR mRNA was observed in CL of pregnancy vs. early and regressing CL (Figure 1). Expression of CL of pregnancy mRNA of the s-PRLR was greater (P < 0.01) than early, mid, and regressing CL and did not differ from late luteal stage expression (P = 0.19; Figure 2). In addition, qPCR results showed the presence of 20α-HSD mRNA during all luteal stages of the estrous cycle, with the greatest expression (P < 0.01) observed during the regressing luteal stage (Figure 3).

Western Blotting Protein expression of both PRLR was observed during all luteal stages and pregnancy, with a predominance of the s-PRLR protein. Western blotting and densitometric analysis results for both PRLR are depicted in Figure 4. Densitometric analysis indicated a decrease in l-PRLR protein during the regressing luteal stage

compared with early (P < 0.01) and late (P < 0.05) luteal stages and CL of pregnancy (P < 0.05). The least s-PRLR protein abundance was observed during the regressing luteal stage compared with early (P < 0.01), mid (P < 0.01), and late (P < 0.01) luteal stages and CL of pregnancy (P < 0.01). Moreover, early stage CL had the least s-PRLR protein abundance compared with the late luteal stage (P < 0.05) and CL of pregnancy (P < 0.05). Additionally, s-PRLR protein quantities were greater than l-PRLR protein during early (P < 0.05), mid (P < 0.05), and late (P < 0.01) luteal stages, but the 2 did not differ during the regressing luteal stage (P = 0.99). Western blotting and densitometry results for 20α-HSD protein abundance are illustrated in Figure 5. Protein quantities of 20α-HSD were least (P < 0.05) during the early luteal stage, but 20α-HSD protein amounts did not differ between other stages of the cycle or pregnancy (P > 0.36; Figure 5).

DISCUSSION Although PRL is generally not considered as luteotrophic in the cow (Hoffmann et al., 1973), mRNA abundance of both short and long forms of PRLR changed in the bovine CL throughout the estrous cycle and pregnancy. In addition, Western blotting revealed that proteins of both PRLR were present throughout the estrous cycle and pregnancy, with greater abundance of s-PRLR protein during early, mid, and late luteal stages and in CL of pregnancy relative to the regressing CL. Interestingly, Bartosik et al. (1967) infused PRL in bovine luteal ovaries estimated to be in the early to midluteal phases of the estrous cycle and documented an increase in progesterone secretion rate, the rate of acetate-1-14C incorporation into progesterone, and the percentage of acetate-1-14C conversion into progesterone. Indeed, midluteal ovaries had a 116% increase in the secretion rate of progesterone after PRL infusion. However, the stimulatory effect of PRL on progesterone secretion occurred only in the ovaries with a CL and was reduced in contralateral ovaries. It was concluded that the stimulatory effect of PRL on progesterone secretion may depend on an interaction between the luteal cells and the ovarian tissue. Therefore, there is evidence that PRL contributes indirectly, or possibly directly, to luteal function in the cow. It is well documented that luteal mRNA expression and protein PRLR isoforms vary throughout the estrous cycle in different species. In rodents, PRLR mRNA is expressed during all luteal phases, with l-PRLR being expressed in greater quantities than the s-PRLR (Clarke et al., 1993). Moreover, PRLR expression is observed in the rodent CL throughout pregnancy (Clarke and Linzer, 1993). Oxberry and Greenwald (1982) used autoradiography to assess variation of PRLR protein and ligand binding in hamster CL throughout the estrous cycle. Results showed that PRL bound moderately to CL at d 1 (i.e., ovulation) of the estrous cycle and binding sharply increased and peaked at d 2. A

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Figure 1. Expression of the long form of the bovine prolactin receptor (l-PRLR) mRNA in the corpus luteum (n = 6/stage) during all luteal stages and pregnancy (preg) presented as least squares means ± SEM. a,bMeans without a common letter differ (P < 0.01). GAPDH = glyceraldehyde 3-phosphate dehydrogenase.

marked reduction in PRL binding was observed on d 3, and light binding was documented on d 4 (Oxberry and Greenwald, 1982). Additionally, immunohistochemis-

try and reverse-transcription PCR analysis of sheep ovary showed differences in localization and expression of both s-PRLR and l-PRLR throughout the estrous

Figure 2. Expression of the short form of the bovine prolactin receptor (s-PRLR) mRNA in the corpus luteum (n = 6/stage) during all luteal stages and pregnancy (preg) presented as least squares means ± SEM. a–cMeans without a common letter differ (P < 0.01). GAPDH = glyceraldehyde 3-phosphate dehydrogenase.

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Figure 3. Expression of 20α-hydroxysteroid dehydrogenase (20α-HSD) mRNA in the bovine corpus luteum (n = 6/stage) during all luteal stages and pregnancy (preg) presented as least squares means ± SEM. a,bMeans without a common letter differ (P < 0.01). GAPDH = glyceraldehyde 3-phosphate dehydrogenase.

cycle, with l-PRLR being particularly localized in stromal cells surrounding primordial and primary follicles, whereas both PRLR isoforms were found in granulosa cells of preantral follicles and luteal cells within the CL (Picazo et al., 2004). Stable mRNA expression of the sPRLR throughout the estrous cycle of sheep was found, but l-PRLR mRNA expression increased at the time of estrus and decreased during the mid-luteal phase and at the onset of the follicular phase (Picazo et al., 2004). Similarly, in the present study, we observed expression of both s-PRLR and l-PRLR transcripts and protein throughout the estrous cycle in the cow; however, the patterns of expression differed from those observed in sheep. Furthermore, in a study of differential gene expression in the primate CL during the menstrual cycle, it was observed that PRLR mRNA was significantly greater through the mid and late luteal stages, and PRLR protein abundance was greatest during the mid to late to late stages (Bogan et al., 2008). Thus, results of the present study are in accord with previous studies performed in various species, which report greater PRLR mRNA expression and protein quantity during the mid and late luteal phases of the estrous cycle and in CL of pregnancy. However, PRLR mRNA expression in red deer CL did not change during the estrous cycle (Clarke et al., 1997), which indicates that PRL function in the CL tissue most likely differs among species. Many studies have shown that expression of both short and long forms of PRLR and its possible roles

vary in a tissue-specific manner. An early study (Nagano and Kelly, 1994) examined the expression of both forms of PRLR mRNA in 16 tissues of adult female rats at 2 stages of the estrous cycle (proestrus and diestrus I), and in the mammary gland of 20-d pregnant and 7-d lactating rats. All tissues examined expressed both forms of PRLR, but mRNA expression was altered by hormonal environment related to stage of the estrous cycle, pregnancy, and lactation. Interestingly, differences in both forms of the PRLR mRNA expression and protein levels were also observed in various bovine tissues (Schuler et al., 1997). Changes in both s-PRLR and l-PRLR mRNA expression and protein abundance were associated with alterations in developmental and physiological status of different tissues. In the present study, increases in mRNA expression of both forms of PRLR during the mid and late luteal stages, and subsequent decreases during the regressing luteal stage, were consistent with the concept that variation in PRLR gene expression is associated with alterations in developmental and hormonal status of the CL. Further, the same pattern was observed for protein levels during the luteal phase and pregnancy in the present study, especially for s-PRLR. Recent data (Binart et al., 2003; Halperin et al., 2008; Devi et al., 2009b) have revealed distinct physiological roles of PRL in mice expressing only the s-PRLR. Overexpression of the s-PRLR rescued mammary gland development and function in heterozygous PRLR knock-

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Figure 4. Western blot (panel A) of the long form (l-PRLR) and short form (s-PRLR) of the bovine prolactin receptor in the corpus luteum during the estrous cycle (n = 6/stage) and pregnancy (preg). Liver samples were used as a positive control, and β-actin (42 kDa) was the normalization control. Densitometry measures (panel B) are presented as least squares means ± SEM. A,BWithin l-PRLR protein, means without a common letter differ (P < 0.05). a–cWithin s-PRLR protein, means without a common letter differ (P < 0.01). *P < 0.05, ***P < 0.01.

out female mice (Binart et al., 2003). Mice expressing only the s-PRLR showed a decrease in mRNA expression of Forkhead box O3 and galactose-1-phosphate uridyltransferase, 2 molecules involved in normal ovarian development, which resulted in early follicular recruitment followed by profound follicular death and premature ovarian failure (Halperin et al., 2008). In a study using both in vivo and in vitro techniques, s-PRLR gene expression in ovary and decidua of mice repressed a specific transcription factor and activated a signaling pathway distinct from that of the l-PRLR (Devi et al., 2009b). Furthermore, another study that used a combined protein/DNA array approach in mice expressing only the s-PRLR gene analyzed the role of PRL on transcription factor activity in ovary and decidua (Devi et al., 2009a). It was found that PRL affected activation or inhibition of DNA binding activity of multiple transcription factors involved in glucose metabolism, growth and differentiation, immune response, development, and steroidogenesis. Additionally, rat ovarian PRLR mRNA expression was examined after induc-

tion of ovulation by PMSG-hCG treatment (Kinoshita et al., 2001), and it was concluded that the 2 forms of PRLR serve different roles in the rat ovary. For example, PMSG increased l-PRLR mRNA expression, suggesting a possible involvement of l-PRLR in folliculogenesis, whereas hCG treatment stimulated mRNA expression of the s-PRLR, indicative of involvement in CL formation and maintenance. Although in the present study there was no difference in mRNA expression between the 2 forms of PRLR, substantially greater amounts of s-PRLR protein were observed by Western blotting during both the estrous cycle and pregnancy, suggesting a possible involvement of s-PRLR in bovine CL development and maintenance. The 37-kDa protein 20α-HSD is an enzyme responsible for catabolism of progesterone to an inactive progestin, 20α-hydroxyprogesterone (Penning, 1997). In the rodent CL, 20α-HSD is involved in decreasing secretion of progesterone (i.e., functional luteolysis) without the need of complete structural luteolysis. Additionally, early studies (Mares et al., 1962; Hafs

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Figure 5. Western blot (panel A) of 20α-hydroxysteroid dehydrogenase (20α-HSD) in the bovine corpus luteum (n = 6/stage) during the estrous cycle and pregnancy (preg). Liver samples were used as a positive control, and β-actin was the normalization control. Densitometry measures (panel B) are presented as least squares means ± SEM. a,bMeans without a common letter differ (P < 0.04).

and Armstrong, 1968) reported that the bovine CL produces 20 β-hydroxyl-pregn-4-ene-3-one. Both 20βand 20α-HSD are involved in clearance of progesterone. Thus, in the present study, we analyzed 20α-HSD as one potential method of progesterone catabolism during estrous cycle and pregnancy. Of relevance to the present study, in rodents, PRL exerts an inhibitory effect on 20α-HSD by downregulating its mRNA expression, protein synthesis, and enzyme activity (Albarracin and Gibori, 1991; Albarracin et al., 1994). To test the effect of PRL on 20α-HSD gene expression, deletion of CL PRLR was performed in female mice (Ormandy et al., 1997). Deletion of CL PRLR resulted in an increase in 20α-HSD mRNA expression, demonstrating a PRL inhibitory effect on 20α-HSD. In addition to increased 20α-HSD mRNA expression, absence of PRLR in female mice results in infertility due to reduced ovulation, fertilization, and arrest of preimplantation development (Ormandy et al., 1997). Therefore, PRL is essential for adequate ovarian func-

tionality and pregnancy maintenance in rodents. At the end of pregnancy, a decline in PRLR in the CL results in an increase in 20α-HSD mRNA expression and a decrease in serum progesterone concentrations (Telleria et al., 1997). In many tissues, binding of PRL to PRLR activates JAK 2/Stat5, causing tyrosine phosphorylation and subsequent activation/deactivation of numerous enzymatic pathways (Bole-Feysot et al., 1998). Results of a study developed to examine the mechanisms of PRL silencing of the 20α-HSD gene indicated that the inhibition of 20α-HSD by PRL does not involve tyrosine kinase phosphorylation (Zhong et al., 1997), but rather is associated with the s-PRLR intracellular domain (Duan et al., 1996). Interestingly, in the present study, an increase in 20α-HSD mRNA expression during the regressing luteal stage was associated with a decrease in both forms of PRLR. Moreover, CL of pregnancy had the least 20α-HSD mRNA expression relative to both forms of PRLR during the same period.

Bovine luteal prolactin receptor expression

Whereas the foregoing evidence of PRLR expression in the bovine CL does not indicate an absolute requirement of PRL for CL development and function, it is useful to consider the potential physiological significance of altered PRL signaling at the CL. Environmental factors such as heat stress have profound negative effects on production, reproduction, and health of dairy cows. High temperature exposure increases circulating PRL (Collier et al., 2008), but the increased PRL decreases PRLR mRNA expression in hepatic (do Amaral et al., 2009) and immune tissue (do Amaral et al., 2011). Thus, extreme increases of PRL may feed back at the cellular level to diminish responsiveness to PRL. Indeed, exposure of lactating cows (Wilson et al., 1998b) or heifers (Wilson et al., 1998a) to heat stress during the second half of the estrous cycle compromises follicle development and function and alters plasma progesterone concentrations and luteolysis. It has been reported that plasma progesterone concentrations decreased in cows subjected to chronic heat stress and increased in cows exposed to acute heat stress (Howell et al., 1994). Interestingly, heat shock protein gene expression, which acts by cytoprotecting during heat stress, has been showed to be stimulated by PRL in rodent luteal cells (Stocco et al., 2001), indicating that greater concentrations of circulating PRL during heat stress has an effect in cellular response to thermal stress. Therefore, PRLR expression in bovine CL may be part of a protective mechanism for adequate reproductive function during periods of increased temperature. In summary, the relationships among 20α-HSD and PRLR mRNA expression and encoded protein quantities in the CL throughout the different stages of bovine estrous cycle and pregnancy were examined. Although there is little direct evidence of a role for PRL in regulation of bovine CL, the present observations suggest a possible involvement of PRLR in CL development and maintenance of progesterone concentrations during the bovine estrous cycle and pregnancy.

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Bogan, R. L., M. J. Murphy, R. L. Stouffer, and J. D. Hennebold. 2008. Systematic determination of differential gene expression in the primate corpus luteum during the luteal phase of the menstrual cycle. Mol. Endocrinol. 22:1260–1273. Bole-Feysot, C., V. Goffin, M. Edery, N. Binart, and P. A. Kelly. 1998. Prolactin (PRL) and its receptor: Actions, signal transduction pathways and phenotypes observed in PRL receptor knockout mice. Endocr. Rev. 19:225–268. Clarke, D. L., B. J. Arey, and D. I. Linzer. 1993. Prolactin receptor messenger ribonucleic acid expression in the ovary during the rat estrous cycle. Endocrinology 133:2594–2603. Clarke, D. L., and D. I. Linzer. 1993. Changes in prolactin receptor expression during pregnancy in the mouse ovary. Endocrinology 133:224–232. Clarke, L. A., D. C. Wathes, and H. N. Jabbour. 1997. Expression and localization of prolactin receptor messenger ribonucleic acid in red deer ovary during the estrous cycle and pregnancy. Biol. Reprod. 57:865–872. Collier, R. J., J. L. Collier, R. P. Rhoads, and L. H. Baumgard. 2008. Invited review: Genes involved in the bovine heat stress response. J. Dairy Sci. 91:445–454. Devi, Y. S., A. Shehu, J. Halperin, C. Stocco, J. Le, A. M. Seibold, and G. Gibori. 2009a. Prolactin signaling through the short isoform of the mouse prolactin receptor regulates DNA binding of specific transcription factors, often with opposite effects in different reproductive issues. Reprod. Biol. Endocrinol. 7:87–98. Devi, Y. S., A. Shehu, C. Stocco, J. Halperin, J. Le, A. M. Seibold, M. Lahav, N. Binart, and G. Gibori. 2009b. Regulation of transcription factors and repression of Sp1 by prolactin signaling through the short isoform of its cognate receptor. Endocrinology 150:3327–3335. do Amaral, B. C., E. E. Connor, S. Tao, J. Hayen, J. Bubolz, and G. E. Dahl. 2009. Heat-stress abatement during the dry period: Does cooling improve transition into lactation? J. Dairy Sci. 92:5988–5999. do Amaral, B. C., E. E. Connor, S. Tao, J. Hayen, J. Bubolz, and G. E. Dahl. 2011. Heat stress abatement during the dry period influences hepatic gene expression and improves immune status during the transition period of dairy cows. J. Dairy Sci. 94:86–96. Duan, W. R., D. I. Linzer, and G. Gibori. 1996. Cloning and characterization of an ovarian-specific protein that associates with the short form of the prolactin receptor. J. Biol. Chem. 271:15602–15607. Grosdemouge, I., A. Bachelot, A. Lucas, N. Baran, P. A. Kelly, and N. Binart. 2003. Effects of deletion of the prolactin receptor on ovarian gene expression. Reprod. Biol. Endocrinol. 1:12–27. Hafs, H. D., and D. T. Armstrong. 1968. Corpus luteum growth and progesterone synthesis during the bovine estrous cycle. J. Anim. Sci. 27:134–141. Halperin, J., S. Y. Devi, S. Elizur, C. Stocco, A. Shehu, D. Rebourcet, T. G. Unterman, N. D. Leslie, J. Le, N. Binart, and G. Gibori. 2008. Prolactin signaling through the short form of its receptor represses forkhead transcription factor FOXO3 and its target gene galt causing a severe ovarian defect. Mol. Endocrinol. 22:513–522. Hoffmann, B., D. Schams, R. Bopp, M. L. Ender, and H. Karg. 1973. Experiments with LH and prolactin antisera and a prolactin inhibitor to further elucidate the luteotropic properties of LH and prolactin in the bovine. Acta Endocrinol. Suppl. (Copenh.) 173:39. Howell, J. L., J. W. Fuquay, and A. E. Smith. 1994. Corpus luteum growth and function in lactating Holstein cows during spring and summer. J. Dairy Sci. 77:735–739. Ireland, J. J., R. L. Murphee, and P. B. Coulson. 1980. Accuracy of predicting stages of bovine estrous cycle by gross appearance of the corpus luteum. J. Dairy Sci. 63:155–160. Kaltenbach, C. C., B. Cook, G. D. Niswender, and A. V. Nalbandov. 1967. Effect of pituitary hormones on progesterone synthesis by ovine luteal tissue in vitro. Endocrinology 81:1407–1409.

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