Visualization of Gene Expression of Short and Long Forms of Prolactin Receptor in Rat Reproductive Tissues

BIOLOGY OF REPRODUCTION 49, 528-536 (1993) Visualization of Gene Expression of Short and Long Forms of Prolactin Receptor in Rat Reproductive Tissues ALLAL OUHTIT,2 GERARD MOREL, 1' 2 and PAUL A. KELLY3 CNRS URA 1454,2 Neuroendocrinologie,Facult de Mdecine Lyon-Sud, 69921, Oullins Cedex, France INSERM Unite 344,3 Endocrinologie Moleculaire, Facult de Medecine Necker 75730 Paris Cedex 15, France Prolactin receptor gene expression was visualized in various tissues by in situ hybridization. Probes specific to the intracellular domains of the short and long form of receptor were prepared. The specificity of these signals was controlled by competition with excess unlabeled homologous probes or heterologous probes; moreover, some tissues, such as penis and vagina, show no expression of either form of receptor mRNA. Macroautoradiogram signals (optical density) were quantified and expressed in arbitrary units. The long form of receptor mRNA was preferentially expressed in testis, epididymis, prostate, seminal vesicle, and mammary gland from lactating animals, whereas the expression of the two forms of mRNA was equivalent in ovary, uterus, and pregnant mammary gland. Signals were also localized at the light microscopic level to individual cells. This approach has permitted the precise localization of prolactin receptor mRNAs in reproductive tissues. Actions of prolactin have not been demonstrated in all tissues expressing receptor transcripts; thus it will be interesting to correlate the expression of long and short forms of receptor with specific functions. INTRODUCTION MATERIALS AND METHODS Prolactin (PRL) has long been associated with reproductive effects in male and female rats. A stimulatory effect of prolactin on LH receptor levels, has been demonstrated in adult [1-3] and hypophysectomized rat [4-5] and mice [6] testes. Prolactin has a similar effect in immature rats [7] and mice [8-9]. Prolactin has also been shown to induce LH receptors in rat ovary [10-11]. In secondary sexual organs in both males and females, although prolactin receptors have clearly been identified [12-13] and characterized, specific actions associated with this hormone in many of these organs have not been clearly shown. In addition to autoradiographic localization [14], PRL receptors have been identified by immunocytochemistry [15] in testes [16], mammary gland [17], ovary [18], and prostate [19-21]. The cDNA encoding a short form of PRL receptor in the rat was first identified [221; subsequently a long form of receptor encoded by a separate mRNA was identified [2324]. Although expression of mRNA transcripts encoding these two forms of receptor have been determined by Northern analysis in certain tissues, in situ hybridization offers a means of directly visualizing mRNAs within specific regions of tissues and even within single cells. In order to clarify which tissues of the male and female reproductive system express mRNAs specific to each form of receptor, we have performed a series of studies in tissues of male and female rats. Animal and Tissue Preparation Four groups of adult Wistar rats (IFFA CREDO; St. Germain sur L'arbresle, France) were used in experiments: the first group consisted of normal males (n = 6; 220-250 g) and the second of normal females (n = 5; 180-200 g); the third group comprised 3 pregnant females that were killed on the 20th day of gestation; the fourth group consisted of 3 lactating females killed on the 15th day of lactation. Animals were first stunned and then killed by decapitation. The male reproductive tissues (testis, epididymis, prostate, seminal vesicle, and penis) were rapidly dissected from the animals of the first group, and the female reproductive tissues (ovary, uterus, and vagina) were rapidly dissected from the animals of the second group; only mammary glands were dissected from the animals of the third and the fourth groups. Tissues were rinsed in 0.1 M PBS (0.1 M phosphate buffer, 0.15 M NaCl, pH 7.4), fixed by immersion for 8 h in 4% paraformaldehyde made in 0.1 M PBS, washed in 0.1 M PBS containing 15% sucrose for 34 h at 4°C, and frozen in liquid nitrogen, in which they remained until use. Sections (10 Fim) were obtained with cryostat (Microm, Francheville, France), placed in 3-aminopropyl-triethoxysilan [25]-coated microscope slides, and stored at -20°C. Oligonucleotide Probes and Labeling Specific probes complementary to different forms of prolactin receptor mRNAs were synthetic oligonucleotides of 30 bases synthesized by solid-phase phosphoramidite chemistry from rat PRL receptor sequences as follows. Two types of oligoprobes were used: the first (oligonucleotides 1 and 2) consisted of Accepted April 18, 1993 Received January 6, 1993. 'Correspondence: Dr G6rard Morel, CNRS URA 1454, Neuroendocrinologie, Facult6 de Medecine Lyon-Sud, BP 12, 69921 Oullins Cedex, France. FAX: (33) 78 51 61 73. 528 ABSTRACT PRL RECEPTOR IN REPRODUCTIVE TISSUES Probe 1 (1060-1089): 5'-GGCCAGGTACAGATCCACCTTGTAT'ITGCT-3 ' Probe 2 (1150-1179): 5'-AGAACCCATTITGAGAGGCTCCTATTTGAG-3' complementary to sequences encoding the intracellular section of the short form of the prolactin receptor. The second type (oligonucleotides 3 and 4) consisted of complementary to sequences encoding the intracellular section of the long form of PRL receptor (Fig. 1). These oligonucleotides were radiolabeled by tailing of the 3' ends with 35S-dATP (3000 Ci /mmol). Briefly, 10 pmol of oligonucleotide was incubated for 1 h at 37 0C with 25 U of terminal desoxynucleotide transferase (Boehringer Mannheim, Meylan, France) in a final volume of 50 lI. The labeled probes were purified by ethanol precipitation to a specific activity of 99.1 + 1.7 Ci /mmol. In Situ Hybridization (ISH) ISH was carried out as previously described [26]. Briefly, sections were washed and postfixed in 4% paraformaldehyde for 10 min, before and after proteolytic digestion with proteinase K (1 Rg /ml) for 20 min at 37°C. Sections were ethanol dehydrated and air dried. Hybridization was performed overnight at 37 0C in 50% deionized formamide, 4strength SSC (standard sodium citrate; single-strength SSC = 0.015 M sodium citrate and 0.15 M NaCl, pH 7.0), 10% dextran sulfate, single-strength Denhardt's solution (50strength = 1% BSA, Ficoll 400, polyvinylpyrrolidone), 200 [Lg/ml tRNA, and 2.5 pmol of each type of probe (1 and/ or 2; 3 and/or 4)/ml of hybridization buffer, which represents a saturating concentration for each 35 S-labeled oligoprobe of each pair. The data reported represent a mix- | Short Long 5'-| \.,, s' \., _ _ I :.: -U-w ,_ _ _ ;3' _, I I K/'i n= I. \ ,-I--- 3 4 FIG. 1. Schematic representation of cDNAs encoding short and long forms of PRL receptor in the rat. Figure shows the position of oligonucleotide probes complementary to sequences encoding the cytoplamic domain specific to the short form (probes 1 and 2) and the cytoplasmic domain specific to the long form (probes 3 and 4). The t (...truncated)