Prolactin Binding Sites Research Articles

Central prolactin binding site densities change seasonally in an adult male passerine bird (Junco hyemalis)

Seasonal reproduction is common across temperate zone avian species. In these species, physiological and behavioral adaptations have evolved to change according to day length (i.e., seasonally) in order to maximize reproductive output. The hormone prolactin regulates many aspects of parental care, a critical component of reproductive success. It's secretion in birds has been shown to be under photoperiodic control, with the highest levels measured in the spring and summer months, when birds breed and show parental care. However, to date, no study has tested whether the densities of central prolactin binding sites vary seasonally, which may also account for prolactin’s effect on parental care. To test this, we collected brains from free-ranging adult male dark-eyed juncos, Junco hyemalis, a biparental songbird, in the spring, summer, and fall, and used quantitative in vitro autoradiography to compare the densities of specific prolactin binding sites across 20 different brain regions. Prolactin binding sites were found in regions that regulate parental behavior in other avian species. During the summer, several hypothalamic regions that regulate parental care, including the preoptic area and tuberal nucleus, contained lower densities of prolactin binding sites, suggesting exposure to higher endogenous prolactin levels, than at other times. This observation is consistent with the fact that circulating prolactin is highest during summer, when males would be providing care to young. Overall, these data suggest that prolactin binding sites are relatively conserved in the avian brain and that central prolactin activity supports parental care efforts in juncos and other avian species.

PROLACTIN BINDING SITES IN XENOPUS LAEVIS (DAUDIN) (ANURA PIPIDAE) KIDNEY: AN AUTORADIOGRAPHIC AND IMMUNOCYTOCHEMICAL STUDY

SUMMARY We have studied prolactin binding sites in Xenopus laevis (Daudin) (Anura Pipidae) kidney by autoradiography and immunocytochemistry. By the autoradiographic technique after intracardiac injection of 125I- oPRL labelling is appreciable in Xenopus laevis kidney at the level of the large vessels and capillaries, the renal corpuscle and, to a variable extent, within the lumina and epithelial cells of the proximal convoluted tubules. Compared to the autoradiographic reaction, the immunocytochemical method failed to reveal the hormone when extremely low doses of it were injected but allowed a much more exact localization of prolactin uptake by the epithelial cells of the proximal convoluted tubules. On the basis of the autoradiographs we could not ascertain wheter, or to what extent, the hormone was bound to specific renal cell PRL receptors: in fact, not only insulin and luteinizing hormone, but also unlabelled prolactin, injected as a specific competitor, failed to bring about the recognizable displa...

Endocrine and Molecular Control of Luteal and Placental Function in Dogs: A Review

In the domestic dog (Canis familiaris), the corpus luteum (CL) is the only source of progesterone (P4) in non-pregnant and pregnant animals. The progesterone secretion profiles are almost identical in both conditions until the last third of the luteal phase when the gradual P4 decline turns into a steep drop in pregnant bitches, indicating the onset of parturition. Consequently, the length of the CL-phase in non-pregnant dogs exceeds the luteal lifespan in pregnant animals. The canine CL-function is regulated by many species-specific regulatory mechanisms, the most intriguing of which is the reported independence of gonadotropic support during the first third of dioestrus. Recently, PGE2 has been proposed as one of the most important luteotropic factors acting locally during this time, but afterwards prolactin (PRL) appears to be the main luteotropic factor. Luteal regression/luteolysis occurs, however, in spite of an increased gonadotropic support. Lately, by demonstrating the expression of PRL-receptor (PRLr), a new insight into possible regulatory mechanisms has indicated that the supply of P4 could be controlled upstream of the steroidogenic machinery at the level of PRLr expression and/or function, subsequently leading to the functional suppression of the steroidogenic machinery. An endogenous source of a luteolytic agent is apparently lacking, implicating the luteal regression in non-pregnant bitches as a passive, degenerative process even if the PGF2α-receptor is constitutively expressed in canine CL. This is in contrast to pregnant dogs in which prepartum luteolysis seems to be an active process of CL destruction by PGF2α of utero/placental origin targeting the luteal PGF2α-receptor.

Screening for Colorectal Cancer With a Blood Test: Projected Effectiveness and Cost-Effectiveness of a Novel Plasma Methylated Septin-9 DNA (mSEPT9) Assay

Specific binding sites for prolactin (PRL) have been studied in human peripheral lymphocytes and erythrocytes of normal adult volunteers and of term cord bloods. In erythrocytes from healthy adult subjects of both sexes a very low specific binding of 125I-human PRL was found (0.24%), whereas a higher binding was found in term cord blood (1.1 %). The binding was hormone specific, the binding capacity was 2.6 fmol/4x109 cells and the Kd was 3.4x10−10M. In lymphocytes of both adults and term cord bloods an evident specific binding was observed (male adults: 1.6%; female adults: 1.7%; cord blood: 1.8%). The binding was specific for lactogenic hormones and the binding capacity was 3.7 fmol/2x106 cells and the Kd was 3.9x10−10M. The presence of specific binding sites for PRL on human erythrocytes and lymphocytes could be used to study PRL binding on blood cells of patients in different physiological or pathological situations.

Immunohistochemical Detection of Prolactin and its Receptors in Human Testis/Immunhistochemische Bestimmung von Prolaktin und seinen Rezeptoren im menschlichen Hoden

Prolactin (PRL) and PRL receptors were localized by light microscopic immunohistochemistry in the human testes on interstitial Leydig-like cells. Similar stainings were obtained with both monoclonal antihuman PRL antibodies and polyclonal antibodies raised against rabbit mammary gland PRL receptors. Moreover, the detection of PRL binding sites by radio-receptor assay on the same samples gave a confirmation of these results. This suggests that PRL might act directly on Leydig cells in the man, in the same way as in the rodent.

22- and 20 kDa-human growth hormones bind to different sites within certain cellular receptors.

Human growth hormone 20 kDa (20 kDa-hGH) is a natural variant of the main hGH isoform (22 kDa-hGH). Since some 20- and 22 kDa-hGH biological activities are not identical, we decided to map the prolactin (PRLR) and growth hormone receptor (GHR) binding sites for both isoforms. Monoclonal antibody (MAb) R7B4, directed to both receptors, was employed to estimate the relative proximity between 20- and 22 kDa-hGH receptors binding sites. Results indicated that although both hGH isoforms share the same PRLR present in Nb2-cells and rat liver, MAb R7B4 differently affected hormone binding, suggesting that their receptor binding sites would be close in Nb2-cells and separate in rat liver membranes. Since labelled 20 kDa-hGH did not bind significantly to hGHR, we added to the incubating medium an allosteric MAb anti-hGH that improved 20 kDa-hGH affinity for receptors. Under these experimental conditions MAb R7B4 inhibited 20 kDa-hGH binding to human liver but not to placenta, whereas the Ab impaired 22 kDa-hGH binding to both receptors. Data thus suggested that both hGH isoforms share the same hGHR binding site in liver tissue but bind to different overlapped regions in placenta. Consequently, results presented in this paper indicate that PRLR and GHR binding sites for 22- and 20 kDa-hGH should not be always identical, a fact that could explain some of the isoforms different activities.

Hyperthyroidism and production of precocious involution in the mammary glands of lactating rats

This study investigated the influence of chronic hyperthyroidism on mammary function in lactating rats and the effects on their pups. Thyroxine-treated (10 microg per 100 g body weight per day; hyperthyroid (HT)) or vehicle-treated rats were mated 2 weeks after the start of treatment and killed with their litters on days 7, 14 and 21 of lactation. Serum concentrations of triiodothyronine (T(3)) and tetraiodothyronine (T(4)) increased in thyroxine-treated rats. In HT mothers, serum prolactin decreased on day 7 and day 14 of lactation, whereas insulin-like growth factor I (IGF-I) and progesterone concentrations decreased, and corticosterone increased on day 7 of lactation. In HT pups, T(4) concentration increased on day 7 and day 14 of lactation, whereas T(3) increased only on day 14 of lactation, and growth hormone increased on day 7 of lactation. Mammary prolactin binding sites did not vary, but there was an increase in the binding sites in the liver on day 14 of lactation in thyroxine-treated rats. In an acute suckling experiment, thyroxine-treated rats released less oxytocin, growth hormone and prolactin and excreted less milk than did control rats. Mammary casein, lactose and total lipid concentrations in thyroxine-treated rats were similar to those of control rats on day 14 of lactation. Histological studies of the mammary glands showed an increased proportion of alveoli showing reduced or no lumina and cells with condensed nuclei on day 14 and day 21 of lactation; the TdT-mediated dUTP nick-end labelling (TUNEL) test revealed an increase in apoptosis in alveolar cells on day 21 of lactation in thyroxine-treated rats. Expression of SGP-2, a gene expressed during mammary involution, increased in thyroxine-treated rats on day 14 and day 21 of lactation, whereas expression of insulin-like growth factor binding protein 5, a proapoptotic signal, was unchanged. Bcl-2, which promotes survival of mammary gland epithelial cells was unchanged, whereas expression of IGF-I, which also promotes survival of mammary gland epithelial cells, increased on day 21 of lactation in thyroxine-treated rats. These results indicate that thyroxine treatment produces some milk stasis as a result of impairments in suckling induced release of oxytocin that may initiate the first stage of mammary involution, increasing apoptosis in a gland that is otherwise actively producing and secreting milk.

Hyperthyroidism and production of precocious involution in the mammary glands of lactating rats

This study investigated the influence of chronic hyperthyroidism on mammary function in lactating rats and the effects on their pups. Thyroxine-treated (10 microg per 100 g body weight per day; hyperthyroid (HT)) or vehicle-treated rats were mated 2 weeks after the start of treatment and killed with their litters on days 7, 14 and 21 of lactation. Serum concentrations of triiodothyronine (T(3)) and tetraiodothyronine (T(4)) increased in thyroxine-treated rats. In HT mothers, serum prolactin decreased on day 7 and day 14 of lactation, whereas insulin-like growth factor I (IGF-I) and progesterone concentrations decreased, and corticosterone increased on day 7 of lactation. In HT pups, T(4) concentration increased on day 7 and day 14 of lactation, whereas T(3) increased only on day 14 of lactation, and growth hormone increased on day 7 of lactation. Mammary prolactin binding sites did not vary, but there was an increase in the binding sites in the liver on day 14 of lactation in thyroxine-treated rats. In an acute suckling experiment, thyroxine-treated rats released less oxytocin, growth hormone and prolactin and excreted less milk than did control rats. Mammary casein, lactose and total lipid concentrations in thyroxine-treated rats were similar to those of control rats on day 14 of lactation. Histological studies of the mammary glands showed an increased proportion of alveoli showing reduced or no lumina and cells with condensed nuclei on day 14 and day 21 of lactation; the TdT-mediated dUTP nick-end labelling (TUNEL) test revealed an increase in apoptosis in alveolar cells on day 21 of lactation in thyroxine-treated rats. Expression of SGP-2, a gene expressed during mammary involution, increased in thyroxine-treated rats on day 14 and day 21 of lactation, whereas expression of insulin-like growth factor binding protein 5, a proapoptotic signal, was unchanged. Bcl-2, which promotes survival of mammary gland epithelial cells was unchanged, whereas expression of IGF-I, which also promotes survival of mammary gland epithelial cells, increased on day 21 of lactation in thyroxine-treated rats. These results indicate that thyroxine treatment produces some milk stasis as a result of impairments in suckling induced release of oxytocin that may initiate the first stage of mammary involution, increasing apoptosis in a gland that is otherwise actively producing and secreting milk.

Relative localization of the prolactin receptor binding sites for lactogenic hormones.

A monoclonal antibody termed MAb R7B4, directed to an epitope present in prolactin receptors (PRLRs), was used as a tool to map the receptor binding sites for human growth hormone (hGH), ovine prolactin (oPRL) and human placental lactogen (hPL). Although the three hormones completely inhibited the binding of each other to Nb2 cells or rat liver receptors, MAb R7B4 behaviour was different depending on the hormone tested and the receptor source. According to the MAb effects, PRLR from Nb2 cells would locate both hGH and oPRL close to R7B4 epitope, whereas hPL would bind far from the MAb binding site. On the other hand, PRLR from rat liver should bind hGH close to the R7B4 epitope but oPRL and hPL would be recognized by a separate region of the same receptor. Thus, results presented in this paper suggest that PRLR binding sites for hGH, oPRL and hPL do not exactly overlap in spite of full competition between ligands.

Neuroendocrinology of Maternal Behavior in the Rabbit

Rabbit maternal behavior consists of building an underground nest of straw and body hair during late pregnancy and displaying, with circadian periodicity, a single 3-min nursing bout/day across lactation. Estrogen, androgen, progesterone, and prolactin regulate specific aspects of nest-building and promote the onset of maternal responsiveness. However, the maintenance of this behavior relies on stimuli from the litter: by preventing mother/young contact at parturition or during early lactation maternal responsiveness is altered or abolished. The brain areas controlling the expression of nest-building and nursing were investigated by implanting estradiol, locating the distribution of estrogen and prolactin receptors, quantifying the expression of immediate-early genes, and lesioning structures of the olfactory system. These studies revealed that: (a) estrogen receptor-α, alpha, present in the preoptic region, may mediate the stimulation of nest-building by estradiol; (b) prolactin binding sites, located mainly in periventricular structures, are more abundant in late pregnancy and early lactation; (c) the number of FOS-immunoreactive neurons increases in the lateral septum, but not in the mediobasal hypothalamus, following nursing; (d) the accessory olfactory bulb tonically inhibits the expression of maternal behavior because its removal promotes maternal responsiveness in virgins, which are otherwise unresponsive to daily pup exposure. In summary, rabbits rely on the same hormonal and extrahormonal factors that stimulate maternal behavior in other mammals, yet the way in which such factors promote elaborate nest-building and the unfailing display of circadian nursing is unique to rabbits and warrants future investigation.

Characterization of somatotropin- and prolactin-binding sites on bovine granulosa cells using homologous hormones.

Specific binding of bovine somatotropin (BST) and bovine prolactin (BPRL) to cow granulosa cells from antrum-containing follicles of different diameter was studied. Scatchard analysis of the data revealed a single type of low affinity BST-binding sites on the granulosa cells with dissociation constants similar to those for the BPRL-binding sites. The number of BST-binding sites on the cells decreased with increasing follicle diameter from 3-5 to 6-10 mm. However, the binding capacity to BPRL decreased only in the case of cells from follicles 11-20 mm in diameter. The findings are discussed in relation to the "homologous binding" phenomenon.

GnRH action on luteal steroidogenesis during pregnancy 1

The results of our study presented here establishes that gonadotropin-releasing hormone (GnRH) acts directly on the corpus luteum, leading to suppressed production and release of progesterone and thus disrupting pregnancy. A GnRH-agonist (GnRH-Ag) treatment suppressed the luteal and serum progesterone levels. This suppression is neither mediated by a fall in ovarian testosterone production nor its conversion to estradiol. Although the treatment suppressed the nuclear estradiol-receptor content and binding sites for LH in the corpus luteum, it had no effect on the luteal binding sites for GnRH and prolactin within 24 h. GnRH-Ag augmented the plasma levels of luteinizing hormone, decreased the magnitude of nocturnal surges of prolactin, and had no effect on luteal cyclic adenosine 5′-monotriphosphate levels. Yet, the treatment had no effect on the luteal content of free cholesterol. We have also demonstrated, for the first time, the presence of steroidogenic acute regulatory protein and peripheral benzodiazepine receptor in the rat corpus luteum, and the suppression of these proteins by GnRH-Ag leads to reduced steroidogenesis by the corpus luteum. Concomitantly, P450 side-chain cleavage enzyme, its activity, and its mRNA content and 3β-hydroxy-steroid dehydrogenase content in the corpus luteum decreased. The treatment suppressed the plasma levels of pregnenolone and 20α-dihydroprogesterone. These data suggest that the suppression of luteal steroidogenesis by GnRH-Ag may be due to its inhibitory effect on the cholesterol transport and/or on the enzymes involved in the steroidogenic pathway. Furthermore, based on other observations made in our laboratory, we propose a hypothesis that an endogenous GnRH is present in the corpus luteum/ovary during pregnancy in the rat and that this GnRH may play a physiological role in the regulation, maintenance, and/or termination of pregnancy.

Prolactin receptor localization to the area postrema

The area postrema, which lacks a blood–brain barrier, was examined for the presence of prolactin receptors, which would render it a potential site for vascular prolactin to directly interact with neuronal elements. Using an in vitro autoradiographic technique, frozen sections of New Zealand white rabbit medulla were incubated with radiolabelled ovine prolactin alone (total binding) or radiolabelled ovine prolactin in the presence of excess unlabelled ovine prolactin (non-specific binding). The specificity of the binding was also assessed using excess unlabelled human prolactin or ovine LH. While excess unlabelled ovine and human prolactin caused a statistically significant reduction in radio labeled prolactin binding, unlabelled LH was without effect. Results reveal the presence of specific prolactin binding sites within the area postrema, a previously unknown prolactin target area of the CNS.

The Effect of Exogenous Testosterone on Parental Behavior, Plasma Prolactin, and Prolactin Binding Sites in Dark-Eyed Juncos

Numerous studies have shown that parental behaviors are mediated by prolactin (PRL), while testosterone (T) interferes with their full expression. The limited data available suggest that reduced parental behavior induced by T is not mediated by reduced concentrations of plasma PRL. We hypothesized that T reduces parental behaviors by reducing PRL receptor binding activity at central neural sites that promote the expression of parental behaviors. To test this hypothesis we implanted male dark-eyed juncos (Junco hyemalis) with testosterone-filled or empty implants and measured T and PRL levels, paternal behavior, and specific binding of radiolabeled PRL at selected brain regions that have been implicated in the mediation of parental behaviors. Our findings concurred with previous studies in that T-treated males reduced their parental contributions, had higher levels of T, and had equivalent levels of PRL compared with controls. We found no differences in the capacity to bind125I-oPRL in three brain regions previously implicated in the mediation of parental care in birds, i.e., the preoptic area, ventromedial nucleus of the hypothalamus, and paraventricular nucleus of the hypothalamus. Thus our findings do not support the hypothesis that T interferes with the expression of parental behavior by reducing PRL receptor binding activity at central sites.

Prolactin binding sites in rat brain and liver: effects of long-term ovariectomy and ovarian steroids

The effects of long-term ovariectomy on the levels of brain and liver lactogenic binding sites as well as plasma and liver prolactin (PRL) have been investigated in sham-operated and ovariectomized rats receiving either 17β estradiol (OVX-E), progesterone (OVX-P), or vehicle (OVX-V). The levels of lactogenic binding sites in the parietal and piriform cortices, amygdala, thalamus, hypothalamus, as well as in the liver were significantly decreased after long-term ovariectomy. Moreover, the levels of plasma and liver PRL were also significantly decreased. Exogenous estradiol and progesterone replacement restored the levels of lactogenic binding sites in the parietal cortex and hypothalamus as well as in the liver. However, plasma and liver PRL levels were significantly increased by estradiol but only restored by progesterone. These results suggest that ovarian steroids influence the levels of lactogenic binding sites and prolactin.

Prolactin receptor expression in rat spermatogenic cells.

To identify target cells of prolactin (PRL) in the male gonad, the expression of prolactin receptor (PRL-R) mRNA in adult rat testes was investigated by in situ hybridization using a digoxigenin-labeled cRNA probe. We also investigated PRL binding to testicular cells in vitro. Signals for PRL-R mRNA were detected not only in interstitial cells but also in spermatogenic cells. Although the reaction was positive in all phases of spermatogonia and spermatocytes, it disappeared in early round spermatids. No signals were detected in elongated spermatids or spermatozoa. The signal intensity varied among each phase of spermatogenic cells. PRL-R mRNA was expressed in all stages of the cycle of the seminiferous epithelium. PRL-R were detected on the surface of Leydig cells, Sertoli cells, all phases of spermatogonia and spermatocytes, elongated spermatids, and spermatozoa. Ovine PRL. did not bind to round spermatids. In Leydig cells, pachytene spermatocytes, and spermatozoa, PRL-R were observed in relatively large numbers. There were fewer receptors in other phases of spermatogenic cells. These results indicate that PRL-R mRNA expression is almost consistent with PRL binding sites except for elongated spermatids and spermatozoa, and suggest that PRL may have direct effects on spermatogenic cells.

Prolactin receptor concentrations in the skin of mink during the winter fur growth cycle.

The objectives of this study were to determine: 1) if the skin of mink might be a target organ for prolactin (PRL) by establishing if PRL binding sites (receptors) exist in the cell membranes of skin, and 2) if PRL receptor concentrations change during the onset and progression of the winter fur growth cycle. Skin was collected on October 6, 1992 for characterization of PRL receptors and from July through December 1992 (N = 3 mink/month) to evaluate possible changes in PRL receptor concentrations during the fur growth cycle. PRL receptors were quantified using 125I-oPRL in a validated radioreceptor assay. Scatchard analysis of saturation data revealed a single class of high-affinity (Kd = 5.21 x 10(-11) +/- .84 M), low capacity (Bmax = 27.03 +/- 3.37 fmoles/mg) binding sites. Only oPRL (40% displacement) and to a lesser extent oGH (3% displacement) inhibited the binding of 125I-oPRL to mink skin cell membranes. No inhibition of 125I-oPRL binding to membranes occurred in the presence of a 500-fold excess of bTSH or oLH, indicating that the receptors were hormone specific. Concentrations of 125I-oPRL receptors during the onset and development of winter fur growth (July through November) exhibited no significant change. However, following completion of the winter fur growth cycle (December 1) PRL receptor concentration was significantly higher than all preceding months. The greater binding observed at that time may reflect a change in tissue sensitivity, in preparation for growth of the summer pelage.(ABSTRACT TRUNCATED AT 250 WORDS)

Follicle-Stimulating Hormone, Human Chorionic Gonadotropin, and Prolactin Receptors in Hamster Corpora Lutea or Dispersed Luteal Cells during Pregnancy1

In vitro progesterone (P4) production by hamster luteal cells is stimulated throughout pregnancy by FSH and LH. Prolactin (PRL) by itself, however, increases P4 synthesis only on Day 12; on Day 4, FSH+LH+PRL induces optimal P4 secretion [Biol Reprod 1994; 51:43-49]. In light of these findings, in this study we investigated FSH, hCG, and PRL receptors in hamster CL or dispersed luteal cells on Days 4, 8, and 12 of pregnancy. Scatchard analysis of hamster CL on Days 4 and 8 showed considerably more unoccupied hCG receptors than FSH receptors: on Day 4, there was 9.5 fmol/mg protein for FSH binding sites vs. 1741 fmol/mg protein for hCG binding. Moreover, the binding affinity of hCG was greater than for FSH: the Day 4 Kd was 0.136 nM for hCG vs. 0.308 for FSH. Similar differences were observed on Day 8. Dispersed luteal cells (large+small cells) were incubated for 24 h with or without 10 ng of ovine FSH, LH, and PRL or human recombinant FSH (r-hFSH), alone or in different combinations. The cells were then washed and incubated for 4 h with iodinated hCG, FSH, or PRL with or without 100-fold excess of unlabeled hormones. The number of binding sites per 200,000 luteal cells did not change appreciably for FSH and hCG on Days 4 and 12 of pregnancy, whereas PRL binding sites significantly increased on Day 12.(ABSTRACT TRUNCATED AT 250 WORDS)

Progressive changes in milk protein gene expression and prolactin binding during lactation in the tammar wallaby (Macropus eugenii).

Changes in milk protein gene expression and specific prolactin binding were quantified in mammary tissue from the tammar wallaby (Macropus eugenii) at different stages of lactation. The transition from early (phase 2) lactation to late (phase 3) lactation was characterized by the induction of the gene for late lactation protein, a novel whey protein. During the same period, the levels of beta-lactoglobulin and beta-casein gene expression increased, whereas there was no change in the levels of expression of alpha-lactalbumin and alpha-casein genes. Prolactin binding in the mammary gland doubled during the latter half of phase 2 of lactation but declined significantly during the transition to phase 3 of lactation. These changes in prolactin binding resulted from changes in the number of receptors and not from a change in the affinity of the receptor for prolactin. Treatment of membranes with concanavalin A increased the number of prolactin-binding sites by 40% in membranes from phase 2 mammary tissue but decreased binding by 40% in membranes from phase 3 tissue, indicating that significant changes had occurred in the membranes of cells during this period. The tammar wallaby can secrete phase 2 and phase 3 milk from adjacent mammary glands (asynchronous concurrent lactation) and the developmental changes in milk protein gene expression and prolactin binding observed during lactation were reflected in these individual glands. Taken collectively, these findings suggest that mammary development and milk secretion in the tammar wallaby are regulated by both endocrine and local (intramammary) mechanisms.

Prolactin and Testicular Leydig Cell Function: Characterization of Prolactin Receptors in the Murine MA-10 Testicular Leydig Cell Line

The direct role of prolactin (PRL) in testicular function is still unclear, mostly because of lack of a suitable in vitro model. To establish the suitability of the MA-10 murine tumor Leydig cell line for the study of PRL receptors (PRLR) and effects on steroidogenesis, we initially characterized PRLR on cultured MA-10 cells. The specific binding (Bs) of [125I]human growth hormone (hGH) depends on time, temperature, and Mg2+ ion and protein concentrations, with absolute specificity for the lactogenic hormones hGH and ovine PRL. Bs is saturable and is to a single class of high-affinity (Ka = 3.6 x 10(9) M-1) low-capacity (Bmax = 19.5 fmol/mg protein) binding sites. The molecular weight of PRLR, determined by cross-linking to [125I]hGH, SDS-PAGE and autoradiography, is 35 kDa for the free receptor, suggesting that the short-form PRLR protein, previously described in liver and mammary glands, is that primarily found in MA-10 cells. Thus, the demonstration of specific PRL binding sites on MA-10 Leydig cells, with characteristics similar to primary Leydig cell PRLR, suggests that this cell line can serve as a good model for both the study of PRLR mechanism of action and the role of PRL in Leydig cell function.