Immunologic and biologic characteristics of human decidual prolactin

Immunologic and biologic characteristics of human decidual prolactin K. TOMITA

J. A. McCOSHEN C. S. FERNANDEZ

J. E. TYSON Winnipeg, Manitoba, Canada An increasing and consistent production of prolactin by decidual tissue (dhPRL) he$ been shown duri~ a 24-hour incubation in chernic411y defined buffer: medium. No ~ in ~ release was observed in tiS$~ obtained kom first- and thifd..trlrnester preg&'WIACY. ~ had .neitf'ler a stimulatory nor an inhibitory infl~ on dflPRL r..... although I8leaH of this- poiypeptide by decidl)al tissue incubated with cycloheximide was blOcked. The praCise mechaniSm COi1trolting the produCtion and releaSe of dhPRL is nOt known. :Addition of ~. dibutyfy1 cyclic adenosine monophosphatEi, or Quanosine triphosphate had no effect on dHPRL release diJJing a 4-hciur incublation. In cOntrast; these subStances provoked a statittically sigflificitilt i~ in PAL release from rat hempituitaries co~ in vitro. ~a.crVes kir ~lumen PAL sta.~.ctar-a s-uggest~~-~~~ b.t::wri~·~+..-... The biu'"f-d ~·~Of ~nDAl was next confirmed by means of a new bioes8ay system with a ~ tevet et s8n8itivity of 20 pg/ml. In addition, dis8oeiation of the biolbgic and imrtlunotogic 'actMtV of clt'IPAL has been completed and s~ th8t at least three isohoimones are produEled by htnnan deoidua1 tiSsue. The dhf'RL production rate per gram of decidual tis$ue wet weight is .far below ttlat reported for the pituitary homologue. These studl8$ not only provide confirmatory evid8nce of 1he immiJnologic and biologic activity of dhPRL but also intensify speculation as to its role in human gestation, including that rstated to osrr.ore;t:.iation across hu.-nan amrJcn. {AM. J. Oesrer. GvNeCOL. 142:420, 1982.)

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THE I o EN T I FIcA TI oN of extraglandular prolactin release by human decidua has been reported by several laboratories. 1- 3 The secretory potential of this tissue appears to begin around the twenty-fourth day of the menstrual cycle coincident with the decidualization of secretory endometrium. 4 In the first instance, decidualization occurs in cells proximal to endometrial vascular channels. Prolactin appears to be produced from such cells in increasing concentrations until menstruation. From the Department of Obstetrics, Gynecology and Reproductive Sciences, University of Manitoba, Faculty of Medicine. Supported in part by National Institutes of Health Grant NlCHD 1-RO/-HD 0861-02A2 and tht Sellers Foundati.on. Sponsored by the Society for Gynecologic Investigation. Reprint requests: Dr. ]. E. Tyson, Department of Obstetrics, Gynecology and Reproductive Sciences, University of ManitOba, 59 Emily St., Winnipeg, Manitoba, Canada RJE OWJ'.

420

The secretion of decidual prolactin (dhPRL) increases if conception occurs arid is characterized by a nearly autonomous pattern of secretion throughout pregnancy.;; A slight increase in dbPRL production has been noted in the eighteenth to twenty-fourth week of gestation, a time when amniotic fluid prolactin is elevated. 6 • 7 Mechanisms controlling the production and release of dhPRL remain to be defined. Furthermore, that dhPRL possesses biologic activity has not been sho\,in conclusively. The intracellular mechanisms responsible for the release of pituitary prolactin are, in the main, related to a membrane-bound enzyme activity of adenylate cyclase which generates cyclic adenosine mono phosphate (cAMP). 8 The recent finding of adenylate cyclase activity in human decidua suggests that this enzyme may be associated with the production and release of dhPRL. 9 In the present study we attempted to evaluate factors which regulate dhPRL production and sought to establish whether dhPRL has biologic actiVity similar to that of pituitary PRL 0002-9378/82/040420+07$00.70/0

©

1982 The C. V. Mosby Co.

Volume 142 !'>umber 4

Material and methods

Fresh decidual tissue was obtained by sharp curettage of the uterine cavity in women undergoing elective termination of pregnancy during the first 12 weeks of gestation. Each patient from whom tissue was obtained had received approval for pregnancy termination by an institutional review committee. None of the women was under the personal care of the investigators. Decidual tissue was also obtain~d by similar methodology from the uterine cavity of women undergoing elective repeat cesarean section at term. ·rissue was transported to the laboratory in ice-cold saline, following which it was washed in Gey's buffer containing 0.1 ~(. dextrose.'" Each study was performed in triplicate on decidual tissue samples between I 00 and 300 mg wet weight. Tissue samples were added to 10 ml of buffer, pregassed with 957t oxygen/5% carbon dioxide, and stoppered. Previous studies confirmed that a pH of 7.45 was appropriate for the incubation studies without requirements for readjustment during the incubation period. During incubation at :no C in a shaker bath, 0.4 ml of buffer was obtained at 5, 1.?, and 30 minutes and again at 1. 3, 5, and 24 hours. The remaining buffer was not replenished during the duration of the study. Calculations were adjusted to reflect the decline in total buffer volume following each sampling. In specific studies, sampling was performed at hourly intervals up to 4 hours. Aeration of the buffer mixture was repeated at these intervals as well. The buffer aliquots were frozen at -20° C and stored for the subsequent measurement of dhPRL. In some instances, fresh decidual tissue and samples of that tissue remaining at the end of an incubation were homogenized in 1 ml of phosphate-buffered saline. Tissues were then desiccated in a polytron microprocessor and the resulting suspension was centrifuged at 3,000 xg for 10 minutes. Supernatant was then assayed for dhPRL. In an attempt to influence the release of dhPRL, decidua in the presence of cycloheximide (200 to I ,000 ng/ml of buffer) was incubated over 4 hours. Estradiol (2 p,g/ml) was also incubated with samples of decidual tissue over 4 hours. This concentration of estradiol was approximately l 00 times that observed in maternal plasma at terrr1. To determine if cyclic AMP-dependent processes govern the production of dhPRL, decidual incubates were exposed to 10- 3 M theophylline, I0- 3 M dibutyryl cAMP (dbcAMP), or 10- 4 M guanosine triphosphate (GTP) for a period of 4 hours. A second series of experiments were conducted with the use of these substrates over an incubation period of 24 hours following

Immunology and biology of decidual prolactin

421

the addition of 10% fetal calf serum. Tandem experiments were performed with the same substances on 12 rat hemipituitary glands suspended in 5 ml of buffer. Concentrations of dhPRL were evaluated initially in a homologous radioimmunoassay for human prolactin with the use of the human prolactin standard Friesen i8-5-15 containing ~'2.5 IU/ml. Iodination of hPRL was carried out by means of the lactoperoxidase method of Thorell and Johannsen." The sensitivity of this assay is 2 ng/ml with intra-assay and interassay coefficients of \ariation of~<,{ and ll.ic;{, respectively. Rat prolactin (rPRL) was also measured in a homologous radioimmunoassay with the National Institute of Arthritis, Metabolism, and Digestive Disease kit* and RP-1 rPRL used as standard. Within-assay variance was 6%, 12 and all samples from any one experiment were run in duplicate in the same assay. Additional 24-hour incubates of 3 gm of decidua in 20 ml of buffer were dialyzed against 1Lof distilled water overnight and then subjected to a bed of Sephadex G75 Superfine containing Ampholinet for flat-bed isoe!ectric focusing (IEF) over pH 4.0 to 7.0. IEF was run in a cold room for 16 hours. The dhPRL concentrations for 30 separate zones were determined by RIA. Specific biological activity of dhPRL was determined by using cells from a transplantable lymphoma, designated the Nb2 Node, as described by Tanaka and associates.1:1 This lymphoma arose within lymph nodes of an estrogenized male rat of the Noble ( Nb) strain. Nb2 cells are propagated in a continuous suspension culture in medium supplemented with fetal calf serum and horse serum. The lymphoma cells replicate in response to increasing concentrations of lactogenic hormones. The results of this bioassay are superior to those used previously ,13 The sensitivity of the assay in these experiments was 20 pg/ml. In order to determine the specificity of the assay for dhPRL, antisera to human placental lactogen (hPL) and growth hormone (hGH) in a final concentration of 1 :·I ,300 were added sequentially to samples of buffer to be used in the bioassay. Aliquots of buffer were incubated in culture medium containing known quantities of lymphoma cells for a period of i2 hours at 37° C, following which the cell replication was reevaluated with the use of a Coulter counter. The determinations were performed in duplicate. Statistical analysis of the data was through the use of the unpaired Student'~ t test and regression analysis was performed to evaluate correlations between data sets. *Courtesy of Dr. A. F. Parlow. tPharmacia Fine Chemicals Corporation.

422

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Tomita et al.

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Fig. l. Comparison of mean decidual PRL secretion ± SEM from tissue obtained during first (n = 27) and third (n = 31) trimesters indicated that no significant difference exists between the amounts of PRL released to the medium by decidua from early and late gestation.

Table I. Results of incubation of decidual tissue with theophylline, dbcAMP, and GTP Mean tissue PRL content (ngl100 mg) before incubation

24.5 ± 4.6

Net dhPRL accumulation in medium following 4-hour incubation Control Theophylline dbcAMP GTP

63.8 52.6 59.3 59.4

± 7.6 ± 6.5

± 8.6 ± 6.6

Mean tissue PRL content (ng/100 mg) ajtBr intubation

7.8 ± 0.7 8.9 ± 1.1 7.5 ± 3.1 9.7 ± 0.6

Note: All values are expressed as mean± SEM for six experiments on tissue from two patients performed in triplicate.

Resutts Histologic evaluation of decidual tissue before, during, and following a 24-hour incubation indicated a remarkable stability of the decidual cells with 90% cell viability at 24 hours as measured by the trypan blue exclusion test. The dhPRL accumulated in the buffer medium in increasing concentrations over time. Such concentrations were consistent throughout the various studies. Fig. I displays the accumulation in nanograms per 100 mg of wet weight of tissue. An approximate doubling of dhPRL concentration was observed in the buffer system over the first 3 hours. No statistically significant difference in the amount of dhPRL produced by firsttrimester or term decidual tissue was observed. When tissue from similar stages of gestation was incubated with estradiol-17 {3 in 2 p.g/ml of buffer for 24 hours,

there was also no significant difference in the overall dhPRL increment as evaluated by linear regression analysis. The release of dhPRL into the buffer was inhibited in the presence of cycloheximide: however, the incubation of decidual tissue with theophylline, dbcAMP, and GTP produced no significant change in the amount of dhPRL accumulating in the buffer over a 4-hour incubation (Table I). Moreover, tissue PRL content after incubation with each of the substances was similar. Although theophylline, dbcAMP, and GTP had IlL significant effect on dhPRL release into the buffer medium, this was not the case when these substances were incubated with rat hemipituitaries. Fig. 2 shows the concentration of PRL in buffer containing rat hemipituitaries to which had been added each of the

Immunology and biology of decidual prolactin

Volume 1-12 Number 4

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Fig. 2. Comparison of the effects of w-aM theophylline, I0- 3 M dbcAMP, and 10- 4M GTP on PRL secretion by human decidua (left) and rat pituitary (right). The amounts of rPRL released by the rat pituitary increased following 4 hours' incubation with the addition of these compounds while dhPRL did not change after the same time period (shown) or after 24 hours of incubation (not shown). All values are expressed as mean::!: SEM.

three collli:JOllllds. A statistically significant increase in PRL concentration was observed with all three test substances as compared Lo control values (p < 0.05).

Immunologic and biologic specificity of dhPRL The ~imilarity of dhPRL to pituitary PRL based on its bindiug affinity lor a highly specific human PRL antibody was evaluated through displacement curves representing serial dilutions of decidual incubation buffer. Fig. 3 di~plays the congruity and parallelism between the dhPRL aud the hPRL standard. Replication of Nb2 Node rat lymphoma cells in suspensioll culture has been shown to be stimulated specifically by hC ll, hPL, and pituitary PRL. Similarly, ovine PRL, boviue PRL. and rPRL stimulate replication of lymphoma celb in a dose-dependent fashion. The lower limit of a~~ay sensitivity in this study was 20 pg/ml. Aliyuots of three representative buffers which had l:ontained decidual incubates were used to evaluate the impact of dhl'RL and Nb2 lymphoma cell replication. Concentrations of 32. 52, and 246 ng/ml of dhPRL as measured by radioimmunoassay were added to cell suspen~ions and incubated for a period of 72 hours, after which differential cell counts were performed. Specific lactogenic activity was identified within the buller system~ after the addition of excess antisera to whid1 hGH and hPL had been added. Loss of lymphoma cell replication was observed when an excess

concentration of PRL antisera was added to the system, as shown in Table II. Small disparities in the radioimmunologic and biologic activity of dhPRL were observed when decidual incubation medium was subjected to isoelectric focusing (Fig. 4). Three distinct peaks (pi) in PRL activity were identified by the radioimmunoassay at pi 5.55, 5.98, and 6.17. respectively, with the greatest peak ocCUlTing at 6.17. Biologic activity of each sample was measured in the l\b2 assay. Little biologic activity was identified for peak~ 5.55 and 5.98.

Comment The secretory potential of human decidua was identified initially in studie~ involving the transport of prolactin across human chorioamnion. 10 · 14 In those studies, in addition to the identification of PRL release, the presence of thyrotropin-releasing hormone (TRH) and bromocriptine in the incubation medium was tested and was found to have no effect on the release rate of dhPRL into the buffer.H The presence of PRL in decidua was initially felt to be artifactual and related perhaps to the accumulation of maternal or fetal PRL in decidual cells, a mechanism which is known to occur in certain target tissues exposed to PRL. 1" Subsequently, Riddick and co-workers 1 and Colander and associates 2 confirmed the ability of decidual tissue to synthesize dhPRL. Rosenberg and associates 7 found that dhPRL production varied with the stage of

Februan )',, I!IH:?

424 Tomita et al. .\Jil.

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Fig. 3. The displacement of mi-PRL by serial dilutions of medium obtained from decidual incubations of 24 how·s parallels the displacement of PRL standard isolated from the human pituitarv gland.

Tabl~ II. Effect of antibodies to lactogenic hormones in decidual incubation buffer on Nb2 lymphoma cell growth

Anti-hGH + anti-hPL (1 :4,300) Cell growth with addition of 50 pl

of decidual incubation

Buffer l (32 ng/ml in RIA for hPRL) Buffer 2 (52 ng/ml in RIA for hPRL) Buffer 3 (246 ng/ml in RIA for hPRL)

Anti-hPL + anti-hPRL (1 :4,300)

Anti-hPRL + anti-hGH ( 1 :4,300)

No antibodies

15,678 16,596 21,051

61,766 70,613 76,145

buffer (celL~ x 10/2 ml):

58,015 71,051 77,431

15,803 17,186 24,499

Control (cell growth without incubation

buffer, cells x 10/2 ml) AU figures show the inean of two dishes containing 2 ml of medium. Three samp-les (buffers 1, 2, and 3) of incubation medi~in from which PRL concentrations were determined previously by RIA were tested against Nb2 cell growth. The addition of antihGH + anti-hPL, anti-hPL + anti-hPRL, and anti-hPRL + anti-hGH quantitatively discriminate the effect of each of hPRL, hGH, and hPL on cell growth. All buffers were neutralized to control levels when anti-hPRL was added. Cell growth- without anti-hPRL was the ~me·as that without antibodies. These results indicate that hPKL secretion alone is typ.Kal of decidua and Jl()t. h(}H or hPL. The small degree of cell growth with buffer 3 is related to the failun: of !iR antibodies to neutralize com~ly me hi$h concentration ofPRt. However, since such a high concentration cannot be determined from the standard curve (Fig. 3), the final dilution of l: 4,300 of antisera is appropriate for neutralization.

gestation. These authors suggested that PRL release was augmented in the presence of fetal calf serum although the results of these studies may reflect an alteration in buffer protein concentration leading to alterations in the elution of PRL from the decidual tissue rather than secretion. 1'; Handwerger and associates 17 observed a 40% to 60l/c decrease in dhPRL release during a 4-day incubation with dbcAMP. 17 A decrease of 20% to 32% in dhPRL

release was also observed with the addition of a phosphodiesterase inhibitor, isobutyl methylxanthine. These results are not at variance with the data presented here since Handwerger and associates utilized a different incubation procedure as well as a longer incubation time. However, it is dear that dbcAMP faiis to enhance PRL production by decidua. The current study confirms, however, that -the addition of cellular toxins such as cycloheximide at least

Immunology and biology of decidual prolactin

Volume 142 Number 4

block the release of dhPRL. Buffer content of dhPRL was decreased over that of the control when cycloheximide had been used. Incorporation of 3 H-iabeied amino acids into immunoprecipitable dhPRL appears to support decidua as a primary site for PRL production. However, the overall production rate is exceedingly low on a gram per tissue basis when compared to pituitary content. No significant difference in dhPRL release into buffer was observed after a 24-hour incubation of tissue obtained from first- and third-trimester pregnancies. This is at variance with the results of MasJar and associates 5 and suggests that PRL production may be constant throughout human gestation. Furthermore, the overall dhPRL content of decidual tissue ranged between 300 and 500 ng/gm of decidua. This is significantly lower than the 400 JLg/gm of pituitary tissue which has been quantitated at the time of extraction of the hGI-l from cadaver pituitary (\Vorsley, I.: Personal communication). By our calculation the production rate of decidual tissue in vitro is approximately 1 JLg of dhPRL per gram of tissue per 24 hours. This is indeed low as compared to the overall production rate of hPRL by the maternal pituitary. A failure to observe a significant increment in dhPRL release in the presence of cAMP stimulator was not unexpected. However, methodology used in these experiments was validated in the studies of rat hemipituitaries. Thus, it appears that deciduai tissue. undergoes terminal differentiation and acquires the ability to secrete a polypeptide hormone similar to that secreted by the human pituitary gland. A system of production and secretion may be of a more primitive nature than that usually assigned to more sophisticated endocrine glands such as the pancreas, adrenal, and pituitary. It would suggest, however, that a search be made to identify the specific mechanisms which control dhPRL release. With the use of a sensitive bioassay system, we have confirmed the biologic activity of dhPRL and established that a possible dissociation exists between the measurement of a peptide hormone by radioimmunoassay as compared to bioassay. The impiications of this finding are several. First, radioimmunologic activity may vary with the molecular size of the substance being secreted. Other studies from our laboratory suggest that 75% of dhPRL released in vitro has consistent biologic and radioimmunologic activity (unpublished observations). Our original observation on the potential osmoregulatory role of PRL on water transport across human amnion 10 appears to gain greater significance as a resNlt of the present findings. Indeed, if ~hPRL contributes

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Fig. 4. RIA of decidual PRL collected from each fraction of incubation medium following isoelectric focusing revealed one major peak (pi 6.17; isohormone A) and two minor peaks (pi 5.55 and 5.98; isohormones C and B, respectively). Comparison between Nb2 bioactivity and the immunoactivity of the three isohormones reveals dissociations between isohormones B and C but not A.

to the concentration of amniotic fluid PRL and is transported across fetai membranes,;" the hormone then exerts its effect on the fetal side of the human amnion. \Ve have also been successful in identifying a selectivity by human amnion in localizing hPRL. 19 Recently, Herington and associates 20 presented evidence suggesting that lactogenic receptors exist within the human chorion laeve. They suggest that the presence of such receptors provides more evidence for an osmoregulatory role of PRL on human amnion studies in vitro. Unfortunately, biologic issues raised by Nolin 15 suggest that an intracellular role for PRL may not always be receptor dependent. Therefore, the role of PRL in amniotic osmoregulation need not imply the necessity for a specific identifiable membrane receptor. If decidualization is principally dependent upon the release of prostaglandins, 21 then dhPRL release should be impaired if prostaglandin production is reduced. This has recently been suggested. 22 Unfortunately, that study did not involve the direct administration of prostaglandins to decidual incubates in order to determine the stimulatory effect of these factors on dhPRL release. The present work provides strong support for the concept that gestationally produced PRL is biologically active and similar in many ways to its pituitary

426

Tomita et al.

l·ebruan Arn

homologue. That it is produced by a maternal pelvic reproductive organ is intriguing. The mechanism(s) of dhPRL pwduuion appears to differ fwm that of the pituitary. The failure to observe a difference in dhPRL production bv decidua from early and late gestation suggests an autonomy by decidua that is neither impaired nor enhanced by a changing uterine endocrine environment. The relationship between dhPRL bio-

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logic activity and its role as a regulator of water transport across human amnion remains tu be confin11ed. Dr. Henry G. Friesen of the Departmem of Physiology, Lniversitv of Manitoba, provided PRL anti bod~. access to the ::\lb2 rat lymphoma cell line, and the phvs.ical facilities necessary to perform the bioassays.

REFERENCES I. Riddick. D. H., Luciano, A. A., Kusmik, W. F., and MasJar, I. A.: De novo synthesis of prolactin by human decidua, Life Sci. 23:1913, 1978.

2. Colander, A., Hurley, T., Barrett,]., et al.: Prolactin synthesis by human chorion-decidual tissue: Possible source of prolactin in the amniotic fluid, Science 202:311, 1971-!. :l. Bigazzi, M., Pollicino, G., and Nardi, E.: Is human decidua a specialized endocrine organ? J. Clin. Endocrinol. Metab. 49:847, i979. 4. Maslar, I., and Riddick, D.: Prolactin production by human endometrium during the normal menstrual cycle, AM.j. 0BSTET. GYNECOL. 135:751, 1979. 5. Maslar, I. A., Kaplan, B. M., Luciano, A. A., and Riddick, D. H.: Prolactin production by the endometrium of early human pregnancy. J. Clin. Endocrinol. Metab. 51:7/l, 1980. 6. Tyson, J. E., Hwang, P., Guyda, H., and Friesen, H. G.: Studies-of prolactin secretion in human pregnancy' AM. J. 0BSTET. GYNECOL. 113:14, 1972. 7. Rosenberg, S.M., Maslar, I. A., and Riddick, D. H.: Decidual production of prolactin in late gestation: Further evidence for a decidual source of amniotic fluid prolactin, AM. J. 0BSTET. GYNECOL. 138:681, 1980. 8. Goodyer, C., St. George Hall, C., Guyda, H., et al.: Human fetal pituitary in culture: Hormone secretion and response to somatostatin~ luteinizing hormone releasing factors, thyrotropin releasing factor and dibutyryl cyclic AMP, J. Clin. Endocrinol. Metab. 45:73, 1977. 9. Whittset, J., and Johnsson, C.: Adenylate cyclase from human decidua, AM.j. 0BSTET. GYNECOL.l33:479, 1979. 10. Leontic, E. A., and Tyson, J. E.: Prolactin and fetal osmoregulation: Water transport across isolated human amnion, Am.]. Physiol. 232:124, 1977. II. Thorell,]. I., and Johannsen, B.: Enzymatic iodination of polypeptides to high specific activity, Biochim. Biophys. Acta 215:363, 1971. 12. Rodbard, D.: Statistical quality control and routine data processing for radioimmunoassay and immunoradiometric assays. Clin. Chern. 20:1255, 1974. 13. Tanaka, T., Shiu, R., Gout, P., et al.: A new sensitive and specitic bioassay for lactogenic hormones: Measurement

of prolactin and growth hormone in human serum . .J. Clin. Endocrinol. Metab. 51:1058, !980. 14. Tyson.]. E.: Role of human decidua in the elaboration of polypeptide hormones, in Thiede, H. A., editor: Tran-

15.

16.

17.

18. 19.

20.

script of the Seventh Rochester Trophoblast Conference. 1977, pp. 18-24. :'1/olin, J. M.: Target cell prolactin, 111 McKerns. K. \'\· .. editor: Structure and Function of the Gonadotropins, :\lew York, 1978, Plenum Publishing Corporation, pp. 151-181. Luciano, A. A., :V!aslar, I. A., Kusmik, W., and Riddick, D. H.: Stimulatory activity of serum on prolactin production by human decidua, AM. J. 0BSTET. GYNECOL. 138:665, 1980. Handwerger, S., Colander, A., Barrett. J.. et al.: Differential effect of cyclic AMP and other factors on the secretion of human decidual and pituitary prolactin in vitro, in MacLeod, R. M., and Scapagnini, U.,editors: Central and Peripheral Regulation of Prolactin Function, New York, 1980, Raven Press, pp. 3ll-314. Riddick, D., and Maslar, I.: The transport of prolactin by human fetal membranes, J. Clin. Endocrinol. Metab. 52:220, 1981. McCoshen,J. A .. Tagger, 0., Fernandez, C .. et al.: Selective localization of prolactin by human amniotic epithelium, in Scientific A.bstracts, Twenty-eighth A~nnua! Meet~ ing of the Society for Gynecologic Investigation, St. Louis, Missouri, March 18-21. 1981, p. 61. (Absr. 105.) Herington, A. C .. Graham, j.. and Healy. D. L.: The presence of lactogen receptors in human chorion laeve,J. Clin. Endocrinol. Meta b. 51:1466, 1980.

21. Kennedy, T.: Estrogen and uterine sensitization for rhe

decidual cell reaction: Role of prosraglandins, Bioi Reprod. 23:955, 1980. 22. Masiar, I. A., Rosenberg, S. M., and Riddick, D. H.: Diminished prolactin production by human endometrium exposed to drugs which inhibit prostaglandin synthetase. in Scientific Abstracts, Twenty-seventh Annual Meeting of the Society for Gynecologic Investigation, Demer, Colorado, March 19-22. 1980, p. 164. (Abst. 27i.)