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Biology of Reproduction 61, 757-763 (1999)
©Copyright 1999 Society for the Study of Reproduction, Inc.

Articles

Quantification of Prolactin Messenger Ribonucleic Acid, Pituitary Content and Plasma Levels of Prolactin, and Detection of Immunoreactive Isoforms of Prolactin in Pituitaries from Turkey Embryos During Ontogeny1

G. Bédécarratsa, D. Guémenéb, C. Morvanb, U. Kühnleina, and D. Zadworny2,a

a Department of Animal Science, McGill University, Ste Anne de Bellevue, Québec, Canada H9X–3V9 b Institut National de Recherche Agronomique, Centre de Tours-Nouzilly, Station de Recherches Avicoles, 37380 Nouzilly, France


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The content of prolactin mRNA as well as total prolactin content and type of isoforms of prolactin were measured in single pituitary glands from turkey embryos and poults. Levels of mRNA and pituitary content of prolactin remained low until 5 days before hatching, while plasma concentrations remained low until 2 days before hatching. Levels of prolactin mRNA then increased until the day of hatch, stayed stable during the 3 first days of age, and significantly increased until 2 wk of age. Similar changes were observed in pituitary content and plasma levels of prolactin. Two immunoreactive bands of apparent molecular masses of 24 and 27 kDa, corresponding to the nonglycosylated and glycosylated form of prolactin, respectively, were visualized on Western blots. In pituitary glands from embryos at 22 days of incubation, 31.5% of the protein was glycosylated, whereas in embryos at 27 days of incubation and poults at 1 and 7 days of age, 48.6%, 48.0%, and 56.0% of prolactin was glycosylated, respectively. The results indicate that the increases in the synthesis and the release of prolactin occur mainly around and after the time of hatching in the turkey embryo. Higher percentages of glycosylated isoforms were associated with increasing levels of total prolactin in the pituitary gland. Thus, the synthesis of prolactin and its post-translational modifications may be important factors involved in the physiologic changes occurring around the time of hatching.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Prolactin (PRL) is known to be involved in numerous biological actions in vertebrates, including osmoregulation, growth and development, metabolism, reproduction, behavior, and immunoregulation [1, 2]. In murine embryos, PRL receptors (PRLRs) are widely distributed, high levels have been detected in numerous tissues [3, 4], and evidence suggests that PRL may play key roles during embryogenesis [5–7]. Similarly, several biological actions have been attributed to PRL during embryogenesis in avian species. A stimulatory role upon T-cell maturation [8] and possibly on thymus development [9] was reported in the chicken. In addition, thyroid hormones, which appear to be important for the survival of the embryo during hatching [10], can be influenced by PRL [11–13]. Furthermore, PRL also seems to be involved in the control of osmoregulation by stimulating the reabsorption of NaCl from the fetal urine by the metanephric kidneys [14, 15].

In mammals, maternal PRL also acts on fetal development via transfer across the fetal placental unit [16] and during early postpartum development via the milk [17]. In oviparous animals, however, embryogenesis proceeds independently of maternal influences, although maternal hormones can be incorporated into the ovum during follicular accretion and egg formation. Hence, birds can provide an advantageous system for examining the ontogeny of the endocrine system. For example, in chicks the differentiation of lactotroph cells in the pituitary gland occurs by 17 days of incubation [18], and about this time circulating levels of PRL begin to increase [19, 20], as well as levels of PRL mRNA [21] in the cephalic lobe of the adenohypophysis [22]. The day after hatching, levels of PRL mRNA decrease and then stay stable, whereas the circulating levels of PRL decrease the first day and then increase during the first week [21]. Similarly in the turkey, plasma levels of PRL increase during the first 2 or 3 wk of age [23, 24].

In vertebrates, PRL is present in multiple isoforms resulting from post-translational modifications [25]. Glycosylation represents a major mechanism for modification of PRL; glycosylated (G)-PRLs have different binding and biological characteristics [25], and the relative proportion of PRL isoforms can vary with the status of the animal [26, 27]. In turkey hens, PRL is present in the pituitary gland in 3 different isoforms, 1 nonglycosylated (NG)-PRL and 2 G-PRLs, which comigrate on SDS-PAGE [28]. The ratio of G-PRL to NG-PRL has been shown to fluctuate with the reproductive status of the animal [29]. Higher percentages of G-PRL are associated with high levels of total PRL (during incubation behavior), and higher percentages of NG-PRL are associated with low levels of total PRL (during photorefractoriness and molting). Moreover, G-PRL and NG-PRL have been shown to be released by the pituitary gland in vitro in the same relative proportion as that observed in pituitary extracts [30].

Changes in the expression of the PRL gene and the type of PRL isoforms present in the pituitary gland of turkey embryos are still unknown. In order to investigate the possible roles played by PRL during embryogenesis in the turkey, we determined the timing of the expression of the PRL gene and the release of PRL in turkeys from 18 days of incubation to 2 wk of age. We developed a competitive coamplification polymerase chain reaction (PCR) assay following reverse transcription (RT) for the quantification of PRL mRNA in individual pituitary glands. The pituitary content as well as the plasma concentrations in PRL were measured by RIA, and the type and ratio of PRL immunoreactive isoforms were determined by Western blotting.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Pituitary Sampling

Eggs from 2 commercial strains of medium white turkey hens (British United Turkeys, Chester, England; and BETINA, Saint Nolff, Elven, France) were artificially incubated at 37.7°C, 54.5% humidity for 25 days. They were then placed in a hatcher at 37.0°C, 78% humidity. After hatching, the poults were raised in a single floor pen under standard conditions, and water and commercial starter feed were provided ad libitum. Pituitary glands from developing embryos (at 18, 20, 22, 24, 25, 26, and 27 days of incubation) and poults (at 1, 2, 3, 7, and 14 days of age) were collected after decapitation, snap-frozen in liquid nitrogen, and stored at -70°C. The sex of the animals was also determined at necropsy.

PRL mRNA Analysis

RNA extraction The RNA was extracted using the Total RNA Isolation (TRIzol) reagent (Gibco-BRL Life Technologies, Rockville, MD) protocol, which is an adaptation of the single-step RNA isolation method developed by Chomczynski and Sacchi [31]. The pituitary glands were homogenized individually in 800 µl of a monophasic phenol and guanidine isothiocyanate solution by sonicating on ice twice for 30 sec. Chloroform was added (200 µl) to the homogenate, and the different phases were separated by centrifugation at 12 000 x g for 15 min. The RNA was precipitated from the aqueous phase with 500 µl of isopropanol and washed with 75% ethanol. The total RNA was dissolved in 8 µl (18–24 days of incubation), 13 µl (25 days of incubation), and 23 µl (26 days of incubation to 14 days of age) of diethyl pyrocarbonate (DEPC)-treated water. The quality and the amount of total RNA in each extract were estimated by spectrophotometry at wavelengths of 260 and 280 nm. For the development and the validation of the assay, the RNA from the pituitary gland of an adult egg-laying turkey hen was also extracted as described above and further diluted in 55 µl DEPC-treated water.

Oligonucleotides Sense and antisense primers were designed to amplify an internal fragment of 183 base pairs (bp) from the turkey (t) PRL mRNA. The sequence and orientation of these primers are indicated in Figure 1. Both primers are 19 bases long with a GC content of 42% and a melting temperature of 54°C.



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  		FIG. 1. Sequence of the tPRL cDNA fragment used in the PCR assay. The positions of the sense and antisense primers are indicated by the boxes, whereas the 8-bp deletion in the competitor is underlined.

Construction of the competitive template An internal standard (competitor) was constructed by deletion of an 8-bp fragment (4% of the PCR product length) located in between two MboI restriction sites at positions 290 and 298 on the PRL cDNA [32] (Fig. 1). In brief, 1 µg of wild-type PCR product (183 bp) from tPRL cDNA was purified on 2% agarose, digested with MboI (Amersham Pharmacia Biotech Inc., Piscataway, NJ), then re-ligated with T4 DNA Ligase (Amersham Pharmacia Biotech Inc.). The ligation product was purified on acrylamide, and the band corresponding to the internal standard (175 bp) was excised. This purified fragment was further amplified by PCR, cloned in a TA vector (TA Cloning kit; Invitrogen Corp., Carlsbad, CA), and sequenced.

For further relative quantification, a stock solution of competitor templates was made and was given a value of 109 arbitrary unit (A.U.) per ml. From that stock solution, serial dilutions to 0.1 A.U were made.

RT The RT was carried out in a final volume of 10 µl containing 5 µl of the total RNA solution previously heat-denatured at 65°C for 10 min, 7 U of Moloney Murine Leukemia Virus reverse transcriptase (Amersham Pharmacia Biotech Inc.), 10 mM dithiothreitol (DTT), 50 mM Tris-HCl (pH 8.3), 8 mM MgCl2, and 0.5 mM each dNTPs, 15 U RNAguard (Amersham Pharmacia Biotech Inc.), and 40 pmol of antisense primer. The RT reaction was conducted at 37°C for 1 h; then the reaction mix was heated at 90°C for 5 min in order to inactivate the reverse transcriptase.

Competitive PCR parameters The PCR parameters were optimized for the amplification of tPRL cDNA from embryo and adult turkey hen pituitaries. No amplification was observed on genomic DNA; thus coamplification of potential DNA contaminants of the cDNA samples was excluded. The volume of each reaction was 12.5 µl and contained PCR buffer (10 mM TrisCl pH 9, 50 mM KCl, 1.5 mM MgCl2, 1 mM DTT), 0.05 mM of each dNTP, 10 pmol of each primer, 0.12 mM spermidine, and 0.312 U of Taq polymerase (Amersham Pharmacia Biotech Inc.). PCR reactions were soaked at 94°C for 10 min and then amplified for 25 cycles. A regular cycle was denaturation at 94°C for 1 min, annealing at 57°C for 2 min, and extension at 72°C for 1 min.

Each reaction mixture contained 2 µl of RT reaction and 2 µl of an appropriate dilution of competitor (total volume of 4 µl). Sense primers were end-labeled with {gamma}P32 (ICN Biomedicals Inc., Costa Mesa, CA) using polynucleotide kinase (Amersham Pharmacia Biotech Inc.) at 37°C for 1 h. PCR products (2.5 µl) were heat-denatured in 95% formamide and separated on a 6% sequencing polyacrylamide gel (40 cm long for 3 h at 55°C). The gels were dried and exposed to x-ray film (Kodak, Rochester, NY). After multiple time exposures, the x-ray films were digitalized with a scanner, and the relative intensity of the amplified products were analyzed by densitometry using Gel Printing Tool Box 3.0 software (BioPhotonics Corporation, Dexter, MI).

In order to validate the assay, two competitive PCRs were performed with the cDNA produced from the adult pituitary gland and serial amounts of competitor. Dilutions (1/10 and 1/100) of cDNA were coamplified with amounts of competitor ranging from 0.62 to 1000.00 A.U. and 0.10 to 50.00 A.U., respectively.

Analysis and quantification In order to estimate the equivalence point between the relative amount of competitor and the amount of PRL mRNA in pituitaries from embryos at different stages, a set of samples was assayed using a range of competitor concentrations.

The log10 of the ratio between competitor product (C) and target product (T) was plotted as a function of the log10 of the initial amount of competitor (Co) added. At the equivalence point, the ratio between competitor and target (C:T) is 1, so the log10 of this ratio is 0, and the amount of target present in the reaction (To) is identical to the corresponding amount of competitor. For the determination of the original amount of cDNA in the amplification reaction, the following formula was used: Log (To) = Log (T/C) + Log (Co).

RIA Measurement of Concentration in PRL

The concentrations of PRL in individual pituitary glands and in plasma from embryos and poults from the BETINA strain at different developmental stages were measured using a homologous tPRL RIA [33]. Plasma samples were collected by intra-cardiac puncture for embryos and poults before 1 wk of age, and from the wing vein for all the birds thereafter; they were then kept at -20°C until assayed. Single pituitary glands were collected after decapitation and homogenized in 300 µl of PBS by sonication (3 bursts of 10 sec). The homogenates were further diluted with 500 µl of PBS and centrifuged at 10 000 x g for 10 min. The supernatant was kept at -20°C until assayed.

Analysis of PRL Immunoreactive Isoforms

Preparation of pituitary extracts Pituitary glands from embryos at 22 (n = 22) and 27 (n = 29) days of incubation and from poults at 1 (n = 31) and 7 (n = 16) days of age were collected after decapitation and snap-frozen into liquid nitrogen. For each developmental stage, 4–6 pituitaries were pooled and homogenized in 45 µl of 10 mM Tris-HCl (pH 8.0), 1 mM EDTA, and 0.5% Tween 20 by sonication for 30 sec on ice. Thus, five different pools were made at 22 days of incubation, 1 day before hatch, and 1 day after hatch; and four different pools were made at 1 wk of age. The protein concentration of each pool was measured using the Bradford method [34]. An aliquot of the pool was mixed with an equal volume of 10 mM Tris-HCl (pH 8.0) containing PMSF (0.1 mM), pepstatin (1 µM), and leupeptin (1 µM) and used for PRL immunoblotting.

Western blotting Aliquots of 100 µg protein from each pool were separated on 12% gels (0.75 mm thick, 8 cm long) by SDS PAGE [35]. Prestained broad-range (Mr 6.5–175 [x 10-3]) molecular weight markers (New England Biolabs LTD, Beverly, MA) were included in each gel. A 10-µg protein sample from pooled pituitary extracts from adult turkey hens at different physiological stages [29] was also included as positive control. After electrophoresis, the proteins were electrotransferred onto a polyvinylidene fluoride (PVFD) membrane (Immobilon-P, 0.45 µm; Millipore Corp., Bedford, MA) at constant voltage of 100 V for 1 h at 4°C with constant stirring in 25 mM Tris, 192 mM glycine, and 20% methanol, pH 8.3. The membranes were blocked in Tris-buffered saline pH 7.5 (TBS: 50 mM Tris base, 150 mM NaCl), 5% nonfat dry milk. All the membranes were incubated overnight with anti-recombinant PRL antibody [33] at a 1/2000 dilution in TBS, 0.5% nonfat dry milk. The membranes were washed 6 times (10 min each time) with TBS, 0.5% Tween 20; incubated for 1 h with 106 cpm/membrane (in 5 ml of TBS) 125I-radiolabeled protein A (specific activity > 30 µCi/µg (ICN Biomedicals Inc., Costa Mesa, CA); washed 4 times (5 min each time) with TBS, 0.05% Tween 20; and finally exposed to x-ray film (Kodak, Rochester, NY) for autoradiography. The proportion of each immunoreactive band was calculated by densitometry using GP Tools 3.0 software (BioPhotonics Corp.) after digitalization of the x-ray film.

Statistical Analysis

All statistical analyses were done with Statview (Abacus Concepts, Calabasas, CA) on Macintosh. The changes in the amount of mRNA, in the concentrations of PRL, and in the ratio of isoforms were analyzed using a nonparametric Kruskal-Wallis test followed by a Mann-Whitney U test (P < 0.05).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Competitive RT-PCR Assay

The competition between the two concentrations of adult cDNA and decreasing amounts of competitor is shown on Figure 2, whereas the logarithmic regression of the data is shown on Figure 3. Before densitometric analysis, multiple exposure times were necessary to obtain an image of each coamplification not saturated on the x-ray film. Both curves were linear with a slope close to 1 and were almost parallel. The intersection points of the curves with the x-axis (Log Co) were 1.16 and 0.10, respectively, and the corresponding calculated dilution factor was 11.48 (101.01).



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  		FIG. 2. Competition between two dilutions (1/100 and 1/10) of cDNA from the pituitary gland of an adult egg-laying turkey hen and decreasing amounts of competitor. The lane labeled Co represents an amplification of competitor only. The values indicated in lanes correspond to the amount of competitor (in A.U.) added for the coamplification.



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  		FIG. 3. Logarithmic regression of the product of the competition between two dilutions (1/10 and 1/100) of cDNA from an adult egg-laying turkey hen pituitary (W) and decreasing amounts of competitor (C) as a function of the initial amount of competitor (Co).

Levels of PRL mRNA in Individual Pituitary Glands

No sex or strain effect was associated with the pituitary content in PRL mRNA. The changes in the expression of the PRL gene in the pituitary gland during embryogenesis and the first 2 wk of age are presented on Figure 4A. The levels of PRL mRNA remained low (about 30 A.U./pituitary) until 5 days before hatching. The mRNA levels then increased consistently until the end of the experiment, with a plateau between the day of hatch (1083 ± 332 A.U./pituitary) and 3 days of age (1925 ± 822 A.U./pituitary). A significant increase was then observed at 4 and 7 days of age with 3052 ± 491 and 5290 ± 1126 A.U./pituitary, respectively.



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  		FIG. 4. Changes in the expression of the PRL gene (A) and the content of PRL (B) in individual pituitary glands, and in the concentration of PRL in plasma (C) from embryos and poults at different developmental stages (mean ± SEM). Stages with common letters do not significantly differ in the amount of PRL mRNA, pituitary, or plasma content in PRL.

Pituitary Content and Plasma Levels of PRL Measured by RIA

The changes in the pituitary content and in the plasma concentrations of PRL are shown in Figure 4, B and C, respectively. No sex effect was detected at any stage for pituitary content or for plasma concentrations in PRL.

The pituitary content of PRL remained low (about 15 ng/pituitary) until 5 days before hatching, then increased consistently until the end of the experiment, with a plateau between 2 days before hatching (226 ± 46 ng/pituitary) and 4 days of age (280 ± 73 ng/pituitary). The amount of PRL measured in pituitary glands then significantly increased at 14 days of age (1240 ± 90 ng/pituitary; Fig. 4B).

Plasma levels of PRL remained low until 2 days before hatching (< 10.00 ng/ml) and then significantly increased (36.44 ± 3.62 ng/ml) the day before hatching. During the 4 days after hatching, the levels of PRL remained constant, then significantly increased to 48.12 ± 7.32 and 78.12 ± 6.27 ng/ml by 7 and 14 days of age, respectively (Fig. 4C).

The correlation between the three parameters measured were 0.94, 0.91, and 0.95 for, respectively, mRNA versus pituitary content in PRL, mRNA versus plasma concentrations in PRL, and pituitary content in PRL versus plasma concentrations in PRL.

Immunodetection of PRL Isoforms

The analysis of pooled pituitary extracts obtained from turkey embryos (22 days of incubation; 1 day before hatching) and poults (1 and 7 days of age) by Western blotting is presented on Figure 5. Two immunoreactive bands with apparent molecular masses of 27 and 24 kDa migrating at the same position as a pooled sample of pituitary extracts from adult turkey hens (Fig. 5, lane A) were detected. The percentage of the 27-kDa band for each developmental stage is presented on Table 1.



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  		FIG. 5. SDS PAGE followed by Western blots of pooled pituitary extracts of turkey embryos at 22 (D-5) and 27 (D-1) days of incubation and poults at 1 (D+1) and 7 (D+7) days of age. Lane A represents a pooled sample of pituitary extracts from adult turkey hens [29]. Lanes 1–6 represent the analysis of different pools of pituitary extracts.


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  		TABLE 1. Percentage of total turkey PRL as a 27-kDa PRL immunoreactive band, and corresponding ratio of 27 kDa: 24 kDa immunoreactive bands in pooled pituitary extracts of turkey embryos at 22 (D - 5) and 27 (D - 1) days of incubation and poults at 1 (D + 1) and 7 (D + 7) days of age.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The use of competitive PCR for the quantification of specific RNA is now commonly used by many laboratories [36]. In this study, we have designed a reaction to semiquantify the expression of the PRL gene in single pituitary glands from turkey embryos. Our coamplification was performed with an 8-bp-deleted competitor (4% difference in length) to ensure equivalent amplification efficiencies between the wild type and competitor templates. Appropriate dilutions of the total RNA after extraction were made to maintain the PCR within the exponential phase of the amplification [37]. The problem of detection resulting from the small difference in size and the low number of cycles was overcome by radiolabeling the sense primer and by separating the PCR products on a sequencing gel. The coamplification of 2 different dilutions of reverse-transcribed RNA from the adult pituitary gland and serial amounts of competitor led to 2 parallel logarithmic regression curves with slopes close to 1 and separated by 1.06, in close agreement to the expected log of the dilution factor [37]. Thus, our assay appears to be reliable for the semiquantification of PRL mRNA in the pituitary gland of turkeys.

The rise in PRL mRNA observed 3 days before hatching is consistent with what was previously reported in the chicken [21, 22]. The latter measurements [21, 22] were performed on pooled samples using Northern blotting, whereas our measurement was done on single pituitary glands, which led to a better estimate of the variability. Conversely to what was reported by Ishida et al. [21], the levels of PRL mRNA significantly increased in our study during the first week of age. As we expressed our results in A.U. per pituitary gland instead of cpm per µg of RNA, this difference could result from the increase in size of the pituitary gland and thus in the increase of total RNA during the first weeks of life. The pituitary content of PRL was highly correlated (0.94) with the levels of mRNA as reported by Ishida et al. [21] and Kansaku et al. [22]. The increase in the synthesis of PRL observed between 5 and 3 days before hatching is consistent with the differentiation of lactotrophs observed during embryonic development in the chicken [18]. The plasma levels of PRL significantly increased the day before hatching, then plateaued during the following days, and significantly increased at 1 wk of age. An increase in plasma levels of PRL before hatching has also previously been reported in chickens [19, 21] and during the first 3 wk of life in turkeys [23, 24]. The two-day delay observed between the increase in pituitary content and plasma levels of PRL suggests that PRL is not immediately released after synthesis but is probably accumulated and/or processed in the pituitary gland.

Around the time of hatching, several critical physiological changes occur [38, 39]: initiation of breathing with a progressive change from chorio-allantoic to lung gas exchange, initiation of egg pipping (several hours after the initiation of breathing) preceded by the development of the complexus musculus (markedly developed just before hatch) and accompanied by a large increase in energy use in order to pip the shell, and maturation of the immune system in preparation for the new environment and antigen exposure. As PRL is thought to modulate a multiplicity of actions in vertebrates [2, 40], changes in PRL mRNA, pituitary content, and plasma concentrations in PRL observed before and after hatching might have an effect on at least one if not all of the physiological parameters mentioned above. Thyroid hormones are involved and are important for the survival of the embryo during hatching [10, 39]. Since PRL was reported to act on thyroid hormone metabolism in chicken embryo via specific receptors in the liver [11–13], and PRLR mRNA is present in the thyroid and the liver of adult turkey hens [41], the variations in PRL observed in our study about the time of hatching may therefore act on thyroid hormones by modulating their concentrations and/or activation. Osmoregulation is also an important factor around the time of hatching [39], and a balance between water [42] and concentration of ions is essential. In the chick, injection of PRL to the chorioallantoic membrane reduced the concentrations of Na+ and Cl- in allantoic fluid [14, 15]. Since the injection of PRL also increased the Na+-K+-ATPase activity in metanephros [14] without changing the allantoic volume [15], the increase in levels of PRL observed before hatching may therefore act on the embryonic kidney by stimulating the reabsorption of NaCl from the fetal urine. Moreover, the production of 1,25-dihydroxyvitamin D3 by chick kidney is stimulated by PRL in vitro [43]. Thus, the large increase in PRL observed during the first 2 wk of age may result in an increase of calcium absorption, which is necessary for the development of the poult. Evidence also suggests that PRL may affect the development of the immune system in chickens. During development, the thymus and the bursa of Fabricius are essential for the maturation of lymphocytes [44]. The partial decapitation of the chicken embryo delays the development of the lymphoid organs and in particular the thymus [9]. In decapitated embryos, an accumulation of large lymphoid cells, which are precursors of T lymphocytes, can be observed in the cortex of the thymus [9]. Furthermore, a general delay in T lymphocyte maturation is observed [8]. The injection of a single dose per day of PRL into the chorio-allantoic membrane induced a significant recovery in T-cell maturation, and the same result is obtained when a pituitary gland is grafted onto the chorio-allantoic membrane [8]. Furthermore, it has been shown that the injection of recombinant tPRL into the embryo induces an increase in the lymphocyte population [45]. The development of the immune system before hatching is critical for the survival of the poults. Thus, a positive action associated with the increase in PRL observed before hatching and during the first weeks of life on the immune system must be taken into consideration. Conversely, poults born from vasoactive intestinal peptide-immunized turkey hens survived during hatching and through a 3-wk experimental period with significantly lower circulating levels of PRL [24]. However, data associated with the hatchability of the eggs or subsequent growth parameters were not provided in the study.

In vertebrates, PRL is known to be synthesized as a number of molecular variants whose ratio fluctuates with different physiological and pathological states [25]. For the first time in avian species, different immunoreactive forms of PRL have been detected in the pituitary gland of embryos and poults during development. The isoforms of PRL detected by Western blotting correspond to those previously detected in pituitary gland from adult turkey hens [29]. These isoforms are consistent with the G- and NG-PRL isoforms described by Corcoran and Proudman [28] with the 27-kDa and 24-kDa forms corresponding to the G- and NG-PRL forms, respectively. In our experiment, the proportion of G- versus NG-PRL appeared to fluctuate during embryonic development. The NG-PRL isoform was predominant 5 days before hatching, then the ratio G-PRL:NG-PRL was close to 1 around hatching, and finally the G-PRL appeared to be predominant at 1 wk of age. We previously reported that, in pituitary glands from adult turkey hens, higher percentages of G-PRL were associated with high levels of total PRL, and lower percentages were associated with low levels of total PRL content [29]. In addition, by using an in vitro perifusion system, we demonstrated that both G- and NG-PRL isoforms are released by the pituitary gland from adult turkey hens in the same relative proportion as observed in the pituitary extracts [30]. Thus, the increase in synthesis and secretion of PRL by the pituitary gland of turkey embryos around the time of hatching and of poults during the first week of life probably corresponded to an increase in the levels of G-PRL released in the blood. Similar results were reported in pigs by Sinha et al. [26, 27], who observed an increased proportion of G-PRL in pituitary glands of fetuses at the end of gestation and higher levels of G-PRL in plasma from piglets during the first year of life. The association between higher levels of glycosylation of PRL around the time of hatch suggests that G-PRL may have specific physiological effects different from those of NG-PRL. Notably, in mammals, G-PRL appears to have an altered affinity for PRLRs and thus frequently presents a lower activity in biological tests [25]. However, this effect may be dependent on the target tissue assessed. For example, Vitt et al. [46] examined the effects on ovarian development of 3 isoforms of FSH that varied in the degree of sialation, and noted that the different isoforms had variable effects depending on the end point measured. Each isoform had different effects on preantral growth of follicles and on estradiol synthesis. Similarly, the effects of glycosylation on PRL may be to partition its effects to different target tissues.

As the list of direct and indirect effects of PRL during embryogenesis is not exhaustive, its actions on the development of diverse organs could be correlated with changes in the expression of the PRL gene in the pituitary gland, and with the synthesis and the release of the different PRL isoforms. Now that the PRLR has been cloned and a competitive PCR has been developed to quantify the expression of the PRLR gene [41], a further step will be to detect and quantify the expression of the PRLR in different embryonic tissues in the turkey.


   FOOTNOTES
 
1 This research was funded by the National Science and Engineering Research Council of Canada (NSERC) and the Institut National de la Recherche Agronomique (INRA) France. Back

2 Correspondence: D. Zadworny, McGill University, Macdonald Campus, Dept. Animal Science, 21,111 Lakeshore Road, Ste Anne de Bellevue, PQ, Canada H9X–3V9. FAX: 514 398 7964. zadworny{at}agradm.lan.mcgill.ca Back

Accepted: April 29, 1999.

Received: March 11, 1999.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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