Prolactin messenger ribonucleic acid concentrations throughout the ovine estrous cycle: Assessment relative to prolactin serum and pituitary amounts

Life Sciences, Vol. 48, pp. 1179-1184 Prlnted in the U.S.A.

Pergamon Press

PROLACTIN MESSENGER RIBONUCLEIC ACID CONCENTRATIONS THROUGHOUT THE OVINE ESTROUS CYCLE ASSESSMENT RELATIVE TO PROLACq'IN SERUM AND PITUITARY AMOUNTS Thomas Landefeld, Vasma Rouha, Ten Bagnell, Thamar Ballard and Inna Levltan Department of Pharmacology, Reproductive Sciences Program, The Umverslty of M~chlgan, Ann Arbor, MI 48109-0626 (Received in flnal form January 16, 1991)

Summ~ry Prolactm (PRL) mRNA concentrauons were assessed by nucleic acid hybndazauon assays m pltmtanes of ewes represenung the defined stages of the ovane estrous cycle. Concomitantly, pituitary and serum PRL concentrauons were measured m these ewes using radaolmmunoassays It was observed that PRL serum, pituitary and mRNA concentrauons tended to increase near the ume of the gonadotropm preovulatory surge, parncularly between 24 hrs before behavioral estrus to 5 hours after estrus However, the changes in PRL mRNA, serum and pltu~tary concentrauons were shown not to be statlsucally mgmficant These data suggest that PRL producuon dunng the sheep estrous cycle ~s mmntmned without dramatic changes in synthems or secretion The pattern of secretion of hormones from the pltmtary has been charactenzed throughout the ovlne estrous cycle (1) These hormones (PRL, FSH and LH) all demonstrate a surge prior to ovulauon, with FSH showing a secondary surge, after ovulation, m some studies (2-4) This surge of hormones results m ovulation and is therefore critical to reproductive funcuon The gonadotroplns clearly play an important role m the development of the ovarian events necessary for the release of the ovum However, the role of the PRL surge at this time ~s not as well defined especmlly since PRL has a plethora of effects including cell metabolism. Furthermore, PRL synthems/secreuon regulation ~s qmte complex F~rst, numerous effectors are revolved in secretaon (5-12) Second, PRL gene expression Is also under regulation of several effectors (13-20). Recent work from our laboratory has reported the pattern of gonadotropm subunlt mRNAs throughout the estrous cycle, thereby providing mfonnauon as to the contnbuuon of biosynthesis to the gonadotropm secretory process (21) A chvergent pattern of LHI3 and FSHI3 mRNAs was observed, particularly around the time of the preovulatory surge, providing support for differential regulauon of these subumts at a pretranslanonal level Slmtlar data have not been reported for PRL mRNA concentrauons throughout the ovme cycle; however, a report by Halsenleder et al (22) demonstrated changes m PRL mRNA amounts during the 4-day rat estrous cycle The changes were observed throughout the rat cycle w~th the most s~gmficant (7fold increase) ones seen dunng chestrus Interestingly, this increase was not assocmted with a change m serum PRL At the tame of the surge on proestrus, PRL mRNA amounts rose concomitantly. Thus ~t appeared that different regulatory mechanisms may be involved at chfferent lames of the cycle Smce some studies have reported a rise m serum PRL dunng the ovme estrous cycle (1,5,6,11,23-25) with a particular emphasis on the times surrounchng the gonadotropm surge, this study was designed to assess mRNA changes and as a result, possibly elucidate a 0024-3205/91 $3.00 +.00 Copyright (c) 1991 Pergamon Press plc

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relaaonshlp between PRL synthesis and the serum concentrations observed dunng the estrous cycle. Materials and Methods Ammals: Estrous cycles of Suffolk ewes (n = 5-8/group) were synchronized dunng the breechng season (November) with progesterone implants (26) The ewes were then killed at the selected ames representing the major phases of the cycle (27) Briefly, these included luteal phases (designated as Day 5, 8 and 12), a folhcular phase (1 e behavioral estrus minus 24 hrs), and preovulatory surge ames 0 e behavioral estrus plus 5, 15, 25 and 50 hrs) For purposes of onentaaon relaave to the estrous cycle (~16 days m length), the groups Day 8, Day 12, E-24, E+5, E+15, E+25, E+50 and Day 5 are represented on the X-axis as -8, -4, -1, 0 2, 0 6, 1 0, 2 1 and 5, respecuvely, in Figures 1, 2 and 3 Serum Hormone Analyses" Serum samples (5 ml) were collected by jugular vempuncture every 4 hours beginning on Day 14 and then hourly after the onset of behavioral estrus (26,27) These samples were assayed for PRL m 20-200 lal ahquots m a racholmmunoassay descnbed by Davis et al (24) The senmtlVlty of this assay was 0 3 ng/tube using oPRL (NIH P-S-88) as the standard Pltmtary PRL concentrataons were also assessed m this assay Sample volumes (25100 I.d) of a 1:10,000 chluaon of pltmtary homogenate (15,000 x g supernatant) were used The homogemzaaon buffer included 0 05M Tms-HCI, pH 7 8, 25mM KCI, 5mM MgC12, 7mM l~-mercaptoethanol, and 880mM sucrose RNA Extractions. Glands were removed within 15 minutes of death and frozen m hqmd mtrogen unal RNA extracuons were performed Total p~tmtary RNA was extracted by one of two methods, phenol (27) or guanldlmum lsothlocyanate (21,26) Sxmflar A260/A280 rauos (1 e 1 7-1 9) were observed for the RNA preparations regardless of the method of extraction. cRNA Preoaraaon and Labehn~" PRL cDNA in the pGEM vector (Promega, Ma&son WI) was prepared I~y subclonmg a bovine PRL cDNA fragment (580 bp) into the I-hnc II rote of pGem 3 (Dr R A Maurer, Umvermty of Iowa) The plasmld was hneanzed with Bam HI or Hind III and transcnpaon was performed on the hneanzed DNA using SP6 or T7 RNA polymerases as described previously (21) RNA Ouanlataaon Specific PRL mRNA amounts were assessed m total RNA preparaaons using a soluaon hybn&zaaon assay as prewously described (21) Lmeanty was observed m standard curves (using the sense mRNA strand) between 2 and 30 pg (--0 01-0 20 fmole) with a sensmvlty of ~0 02 fmoles and nonspeclflc binding averaging 2%-5% of total counts Intraassay coefficient of vanaaons ranged from 10%-15%. Concentrauon curves were performed for mchvldual RNAs from each group to test for parallehsm with that obtained for the standard curve Once the opamal amount was determined, samples were assayed m tnphcate. Data were analyzed by one-way analyms of variance Results mRNA Amounts" PRL mRNA concentraaons throughout the ovme estrous cycle are shown m Figure 1. Although there appeared to be a trend for these values to increase near the ame of the onset of estrus (1 e E-24 and E+5), these increases were not staasucally significant A mmflar trend was observed for LHI~ mRNA near the tune of the preovulatory gonadotropm surge (21) Serum PRL Concentrations PRL serum concentrauons (ng/ml) dunng the ovme estrous cycle are shown in Figure 2 Again, an increase in serum PRL appears to occur m the E-24 group (coinciding with the time of the gonadotropm surge, 21,26, and PRL mRNA concentrations, Fig 1), however, due to the large variability associated with this group, the increase is not staustlcally s~gmficant Th~s vanabdlty is most hkely due to chfferences among the ewes relatwe to the changes m serum PRL in each ewe

Vol. 48, No. 12, 1991

PILL mRNA During Sheep Estrous Cycle

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FIG 1 PRL mRNA concentranaons (fmoles/mg total RNA) throughout the ovme estrous cycle Values represent mean 5- S E of each group as descnbed m Methods and Materials Pltulta_rvP R L Concentrauons The concentranaons of P R L m the pltmtanes of the ewes dunng the estrous-cycle are depleted m Fig 3 Again, there are no slgmficant changes m these amounts dunng the cycle, although the values tended to increase between the late folhcular phase (i e E24) and the early stages of the surge 0 e E+5) D18~USSlOn The funcnaon of PRL in the ovme estrous cycle has not been clearly elucidated Based on the metabohc effects of PRL and posmble acnaons m the ovary, a role m reproducnaon is quite feamble. Some ewdence for a nse m PRL near the lame of the preovulatory gonadotropm surge is available, although the data do not support a well-defined peak as seen with the gonadotropms (5-7,11,23,24). As part of the dehneanaon of the role of this hormone, it is important to estabhsh the cellular/molecular mechamsms revolved at this ume and then to examine the effects of individual modulations of PRL synthems and secrenaon In this study, PRL mRNA concentranaons were assessed throughout the ovme estrous cycle at names representanave of the major stages of reproducnaon (1 e. luteal, folhcular and surge), with a parnacular emphams around the preovulatory surge and then examined relanave to PRL p~m]tary and serum concentranaons As such, the overall process of PRL production during the estrous cycle can be mvesugated Although there appeared to be a trend for increasing PRL mRNA concentranaons near the name of the gonadotropm preovulatory surge (1 e 24 hrs before estrus and 5 hrs after estrus), these changes were not stanasnacally slgmficant We had observed stmllar results with LHI~ mRNA concentranaons dunng the cycle, and, m parucular, near this name. Although there are no

PRL mRNA Durlng Sheep Estrous Cycle

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FIG 2 Serum PRL concentrauons (ng/ml) throughout the o v m e estrous cycle represent mean + S E of each group

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pubhshed data on PRL m R N A concentratmns dunng the ovlne cycle, Halsenleder et al (22) reported changes m PRL m R N A amounts dunng the rat estrous cycle The major changes observed m that study were dunng dlestrus where a decrease m the morning was followed by a 7-fold increase by evenmg Relative to secreuon, a statastlcally slgmficant mcrease was not seen for PRL, even near the tame of the gonadotropm surge. However, as with PRL m R N A concentrauons, there was a trend for Increased PRL serum amounts near this ume 0 e 24 hrs before behavioral estrus) It is important to note that the data on serum PRL (F~g 2) do not represent a continuous bleeding protocol, but rather the mean serum PRL concenla'aUons for each group of ewes at the tame of kill. As such, differences among ammals relauve to any changes in PRL will conmbute to the vanabdlty, as evidenced by the SEM for the E-24 group Accordingly these data will influence the stausucal significance of the differences As menuoned earlier, some data m the hterature support a PRL "surge" although it is not well defined The pltmtary PRL content pattern again suggested a trend for an mcrease near the time of estrus (1 e E+5) despite the lack of statistical slgmficance. However, ]t is also possible to interpret the data as a fall m PRL amount dunng the late luteal phase of the cycle, since these values are more than 50% lower than values seen during the umes of the cycle Since there is no prevaously pubhshed data on pltmtary PRL content dunng the sheep cycle, there is no direct barns for comparison However, when one examines the changes m PRL content in the rat cycle (I-Iaasenleder et a l , 1989), an increase does occur shortly before the serum PRL rise This is at 1200 h on proestrus, a u m e that is somewhat mmllar to E+50 In fact, except for the amounts

Vol. 48, No. 12, 1991

PRL mRNA During Sheep Estrous Cycle

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FIG 3 Pituitary PRL concentrations (mg/gm) throughout the estrous cycle represent mean + S E of each group

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seen m the Day 8 group (sheep), the pattern of PRL content between the two spec:es appear quite mmflar. In conclusion, this is the fast pubhshed report on the amounts o f P R L mRNA and p~tmtary PRL content throughout the ovme estrous cycle Furthermore, by measunng serum PRL concentrauons m these ewes, zt zs posmble to examine three parameters that are most dtrectly revolved m the productaon ofPRL 0.e synthems, storage/processmg, secretaon) Generally, all three of these parameters followed mmflar patterns, m that a trend for an increase was suggested near the time of estrus. However stausucal slgmficance was not observed m these groups throughout the estrous cycle. The data suggest that PRL producuon is mmntamed throughout the estrous cycle w~thout major changes in e~ther synthesis or secreuon Acknowledgements The authors are mdebted to Mr Doug Doop for Ins help wxth the anmaals and to Ms Vlrg:e Bnght for her help with the assays. We also want to thank Marjory Falconer and Debble Block for the preparalaon of thin manuscript and Dr Dan Haasenleder for his helpful comments Tins work was supported by NIH grants HD12016 and 18258

REFERENCES 1. 2.

D T BAIRD, A.S MCNEILLY, J Reprod. Fert (Suppl) 30 119-133 (1981) M L'HERMITE, G D NISWENDER, L E REICHERT J R , A R MIDGLEY J R , Biol. Reprod li 325-332 (1972)

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G D NISWENDER, R.T MOORE, A.M AKBAR, T M NETT, M A DIEKMAN, Blol Reprod. 13 381-388 (1975) J R . MCNEILLY, A S MCNEILLY, J S WALTON, F J CUNNINGHAM, J Endocnnol 70 69-79 (1976) G D BRYANT, F C GREENWOLD, G KAHN, J MARTINET, R DENAMUR, J Endocnnol 51405-406 (1971) H M FRASER, A S MCNEILLY, Endocnnology 111 1964-1973 (1982) D L DAVIS, M L BORGER, J Atom Scl 38 795-802 (1984). H ABE, D ENGLER, M E MOLITCH, J BOLLINGER-GRUBER, S REICHLIN, Endocnnology 1!6 1383-1390 (1985) W J DEGREEF, W KLOOTWIJK, B KARELS, T J VISSER, J Endocnnol 105 107-112 (1985) K NIKOLICS, A J MASON, E SZONYI, J RAMACHANDRAN, P H SEEBURG, Nature ~1~ 511-517 (1985) G.B THOMAS, J T CUMMINS, L CAVANAGH, I J CLARKE, J Endocnnol 111 425-431 (1986) C A JOHNSTON, A NEGRO-VILAR, Endocrinology 122 341-350 (1988) R A MAURER, Nature 294 94-97 (1981) R A MAURER, J Blol Chem 257 2133-2136 (1982a) R A MAURER, Endocnnology 110 1507-1514 (1982b) R A MAURER, DNA 4 1-9 (1985) G H MURDOCH, R FRANCO, R M EVANS, M G ROSENFELD, J Blol Chem 258 15329-15335 (1983) B A. WHITE, F C BANCROFT, J Blol Chem 258 4618-4622 (1983) A J CARRILLO, T B. POOL, Z D SHARP, Endocnnology 116 202-206 (1985) J D. SCHULL, J GORSKI, Endocnnology 116 2456-2462 (1985) K LEUNG, K.E KIM, R A MAURER, T D LANDEFELD, Mol Endo 2 272-276 (1988) D J HAISENLEDER, G A ORTOLANO, T D LANDEFELD, S M ZMEILI, J C MARSHALL, Endocnnology 124 2023-2028 (1989) J J REEVES, A ARIMURA, A V SCHALLY, Proc Soc Exp Blol Med 134 938-942 (1970). S.L DAVIS, L E REICHERT, GD NISWENDER, Biol. Reprod 4 145-153 (1971) A S MCNEILLY, D.T BAIRD, J Reprod Fertal 69 559-568 (1983) T.D. LANDEFELD, J KEPA, J Blol Chem. 259 12817-12820 (1984) TD LANDEFELD, J KEPA, F J KARSCH, Proc Nail Acad Scl USA81 1322-1326 (1984).