**4. Experiments**

#### **4.1. Autocrine regulation of prolactin secretion**

*4.1.1. PRL content of electroeluted (EE) PRL variants released from AP regions of lactating non-suckled (NS) and suckled (S) rats* 

The PRL variants released from each AP region of NS and S rats were analyzed previously by SDS-PAGE and by Western blotting (32, 33). In the present study these PRL variants were electroeluted i.e., EE PRL from fractions 1-6 after SDS-PAGE; and the PRL content of each fraction was determined by ELISA, as shown in Fig. 1 A-D, and subsequently, to determine whether their increased and/or decreased release of PRL variants, each electroeluted PRL fraction (EE PRL) was incubated with lactotrophs of each AP region of non-suckled and suckled rat APs i.e., Figs. 2-3 (A-D). The fractions contained bands of 7-23 to 97 kDa under NR conditions, and 7-16 to 42 kDa under R conditions, and between 1 and more than 20 ng/μl of PRL variant protein. However, in spite of these variations, similar amounts of total PRL (about 60 ng/μl right panels), were released from each AP regions of NS and S rat, except for the higher amount (80 ng/μl) released from the central AP region of NS rats.

#### *4.1.2. Effects of electroeluted PRL variants (EE PRL), released from AP regions of lactating non-suckled (NS) rats, upon the in vitro release of PRL variants from lactotrophs of NS rats*

The EE PRL variants that were released from each AP region of NS rats (Figs. 1 A-B) were tested for their effects upon the amount of PRL release by lactotrophs from AP regions of NS rats, and the results are shown in Fig. 2A-B. CM from the PR region of NS rat APs (Fig. 1 A) contained a low concentration of PRL (< 3 ng/μl) in fraction 2 and high (about 10 ng/μl) in fractions 1 and 3-6; after incubation with the EE PRL variants, lactotrophs of the same region, i.e., the peripheral AP region of NS rats, exhibited increased release i.e., 5-7 ng/μl of PRL variants 1, 2, 4, and 6, and decreased release of PRL variants 3 and 5. When incubated with EE fractions from CM of the central AP region of NS rats, lactotrophs from the same region showed increased release of PRL variants in fractions 1 and 6, and lower release in fractions 2, 3, 4 and 5. Thus, except for the amounts of PRL released from fractions 1 and 6, whose levels were higher or similar to those of the other EE PRL variants, the amounts of other PRL variants released were significantly lower than those of the EE variants. When the central AP

#### Autocrine and Paracrine Regulation of Prolactin Secretion by Prolactin Variants and by Hypothalamic Hormones 103

102 Prolactin

**3. Statistical analysis** 

were performed three times (n=3).

*non-suckled (NS) and suckled (S) rats* 

**4.1. Autocrine regulation of prolactin secretion** 

**4. Experiments** 

The PRL concentration was calculated by linear regression and values of PRL concentration obtained by ELISA were averaged for each experimental group. Statistical differences were determined by a one-way analysis of variance (ANOVA), using Dunnett's test, and all treatments were compared versus the control (Earle's medium or Total PRL). Comparisons were analysed with the Graph Pad 5.0 Software, Inc. (San Diego, CA). The significance level was set at p<0.05. Each control or test compound was assayed in duplicate, and the assays

*4.1.1. PRL content of electroeluted (EE) PRL variants released from AP regions of lactating* 

The PRL variants released from each AP region of NS and S rats were analyzed previously by SDS-PAGE and by Western blotting (32, 33). In the present study these PRL variants were electroeluted i.e., EE PRL from fractions 1-6 after SDS-PAGE; and the PRL content of each fraction was determined by ELISA, as shown in Fig. 1 A-D, and subsequently, to determine whether their increased and/or decreased release of PRL variants, each electroeluted PRL fraction (EE PRL) was incubated with lactotrophs of each AP region of non-suckled and suckled rat APs i.e., Figs. 2-3 (A-D). The fractions contained bands of 7-23 to 97 kDa under NR conditions, and 7-16 to 42 kDa under R conditions, and between 1 and more than 20 ng/μl of PRL variant protein. However, in spite of these variations, similar amounts of total PRL (about 60 ng/μl right panels), were released from each AP regions of NS and S rat, except for

*4.1.2. Effects of electroeluted PRL variants (EE PRL), released from AP regions of lactating non-suckled (NS) rats, upon the in vitro release of PRL variants from lactotrophs of NS rats* 

The EE PRL variants that were released from each AP region of NS rats (Figs. 1 A-B) were tested for their effects upon the amount of PRL release by lactotrophs from AP regions of NS rats, and the results are shown in Fig. 2A-B. CM from the PR region of NS rat APs (Fig. 1 A) contained a low concentration of PRL (< 3 ng/μl) in fraction 2 and high (about 10 ng/μl) in fractions 1 and 3-6; after incubation with the EE PRL variants, lactotrophs of the same region, i.e., the peripheral AP region of NS rats, exhibited increased release i.e., 5-7 ng/μl of PRL variants 1, 2, 4, and 6, and decreased release of PRL variants 3 and 5. When incubated with EE fractions from CM of the central AP region of NS rats, lactotrophs from the same region showed increased release of PRL variants in fractions 1 and 6, and lower release in fractions 2, 3, 4 and 5. Thus, except for the amounts of PRL released from fractions 1 and 6, whose levels were higher or similar to those of the other EE PRL variants, the amounts of other PRL variants released were significantly lower than those of the EE variants. When the central AP

the higher amount (80 ng/μl) released from the central AP region of NS rats.

**Figure 1.(A-B).** SDS-PAGE (left panels) and prolactin (PRL) content (ng/μl) of PRL variants (middle and right panels) released from the peripheral (PR) and central (CR) adenohypophyseal

(AP) regions of non-suckled (NS) rats, and electroeluted (EE PRL) from fractions 1-6 of SDS-PAGE. Data are means ± SEM. Letters (a-d) indicates *P* < 0.05 difference between fractions of EE PRL.

Autocrine and Paracrine Regulation of Prolactin Secretion

**Figure 2. (A-B).** Effect of the PRL variants electroeluted (EE PRL) in fractions 1-6 from SDS-PAGE of CM from the peripheral (PR) (panel A), and central (CR) (panel B), AP regions of non-suckled (NS)

by Prolactin Variants and by Hypothalamic Hormones 105

**(C-D).** SDS-PAGE (left panels) and prolactin (PRL) content (ng/μl) of PRL variants (middle and right panels) released from peripheral (PR) and central (CR) AP regions of suckled (S) rats and electroeluted from fractions 1-6 of SDS-PAGE. Data are means ± SEM. Letters (a-d) indicates *P* < 0.05 for the difference of PRL content between electroeluted fractions.

region of NS rats was incubated with the EE PRL variants from the peripheral AP region of NS rats, medium levels (about 10 ng/μl) of PRL variants 1 and 6, and medium to low levels to PRL variants 2, 3, 4, and 5 were released. As a result of these effects, high levels of total PRL were released from the peripheral but not from the central AP region; indeed, total PRL release from the central AP region was significantly depressed, below the initial level.

In figure 2B, the PRL content of the EE control PRL variants released from the central AP region of NS rats was low in fractions 1, 2 and 4 of CM, and high (>10 ng/μl) in fractions 3, 5 and 6. Also, with respect to the effect of incubating lactotrophs from the central AP region of NS rats with the EE PRL variants from the peripheral AP region, increased release occurred only of PRL variants 1 and 6; and low levels occurred to PRL variants 2-5 from the central AP region; and only the PRL variant 6 was above the zero level, i.e., about 10 ng/μl. Overall, significantly lower levels of PRL than those contained both in the EE PRL variants and in those released from the peripheral region, were released from lactotrophs of both the central and peripheral AP regions.

#### *4.1.3 .Effects of EE PRL variants released from AP regions of lactating non-suckled (NS) rats upon the in vitro release of PRL variants from lactotrophs of suckled (S) rat APs*

The effect of incubating lactotrophs from AP regions of S rats with EE PRL variants released from AP regions of NS rats is shown in figures 2 C-D. In figure 2 C the PRL level was low in fraction 2, but medium to high levels, (about 10 ng/ μl) in fractions 1, 3-6; the amount of PRL released from the same AP region of S rats was around zero in fractions 2-5; and low in fractions 1 and 6. Overall, significantly lower amounts of PRL were released from the peripheral AP region of S rats. Also, as shown in figure 2D, the amount of EE PRL released from the central AP region of NS rat APs was low in fractions 1, 2 and 4 and high in fractions 3, 5 and 6; the total amount of released PRL from the peripheral AP region was zero in all fractions, and thus, it was significantly lower than that of the EE PRL. With respect to the amount of PRL released from the central AP region, only fractions 2 and 6 showed high levels and the levels of the other fractions were much lowers (1-3 ng/μl). Thus, as with the effect of CM from the PR of NS rats, the amount of released PRL from the peripheral AP region of S rats, was less than zero, i.e., lower than that of the EE control PRL, and of the still lower amount of PRL released from the central AP region, whose levels also were lower than those of the EE PRL.

PRL.

level.

and peripheral AP regions.

were lower than those of the EE PRL.

(AP) regions of non-suckled (NS) rats, and electroeluted (EE PRL) from fractions 1-6 of SDS-PAGE. Data are means ± SEM. Letters (a-d) indicates *P* < 0.05 difference between fractions of EE

0.05 for the difference of PRL content between electroeluted fractions.

**(C-D).** SDS-PAGE (left panels) and prolactin (PRL) content (ng/μl) of PRL variants (middle and right panels) released from peripheral (PR) and central (CR) AP regions of suckled (S) rats and electroeluted from fractions 1-6 of SDS-PAGE. Data are means ± SEM. Letters (a-d) indicates *P* <

region of NS rats was incubated with the EE PRL variants from the peripheral AP region of NS rats, medium levels (about 10 ng/μl) of PRL variants 1 and 6, and medium to low levels to PRL variants 2, 3, 4, and 5 were released. As a result of these effects, high levels of total PRL were released from the peripheral but not from the central AP region; indeed, total PRL release from the central AP region was significantly depressed, below the initial

In figure 2B, the PRL content of the EE control PRL variants released from the central AP region of NS rats was low in fractions 1, 2 and 4 of CM, and high (>10 ng/μl) in fractions 3, 5 and 6. Also, with respect to the effect of incubating lactotrophs from the central AP region of NS rats with the EE PRL variants from the peripheral AP region, increased release occurred only of PRL variants 1 and 6; and low levels occurred to PRL variants 2-5 from the central AP region; and only the PRL variant 6 was above the zero level, i.e., about 10 ng/μl. Overall, significantly lower levels of PRL than those contained both in the EE PRL variants and in those released from the peripheral region, were released from lactotrophs of both the central

*4.1.3 .Effects of EE PRL variants released from AP regions of lactating non-suckled (NS) rats upon the in vitro release of PRL variants from lactotrophs of suckled (S) rat APs* 

The effect of incubating lactotrophs from AP regions of S rats with EE PRL variants released from AP regions of NS rats is shown in figures 2 C-D. In figure 2 C the PRL level was low in fraction 2, but medium to high levels, (about 10 ng/ μl) in fractions 1, 3-6; the amount of PRL released from the same AP region of S rats was around zero in fractions 2-5; and low in fractions 1 and 6. Overall, significantly lower amounts of PRL were released from the peripheral AP region of S rats. Also, as shown in figure 2D, the amount of EE PRL released from the central AP region of NS rat APs was low in fractions 1, 2 and 4 and high in fractions 3, 5 and 6; the total amount of released PRL from the peripheral AP region was zero in all fractions, and thus, it was significantly lower than that of the EE PRL. With respect to the amount of PRL released from the central AP region, only fractions 2 and 6 showed high levels and the levels of the other fractions were much lowers (1-3 ng/μl). Thus, as with the effect of CM from the PR of NS rats, the amount of released PRL from the peripheral AP region of S rats, was less than zero, i.e., lower than that of the EE control PRL, and of the still lower amount of PRL released from the central AP region, whose levels also

lactating rats upon the *in vitro* release of PRL variants by lactotrophs from AP regions of NS lactating rats. Data are means ± SEM. \*Differences *P* < 0.05 *versus* control (EE PRL). **(C-D).** Effect of the PRL variants electroeluted (EE PRL) in fractions 1-6 from SDS-PAGE of CM from the peripheral (PR) (panel A), and central (CR) (panel B), AP regions of non-suckled (NS) lactating rats upon the *in vitro* release of PRL variants by lactotrophs from AP regions of suckled (S) lactating rats. Data are means ± SEM. \*Differences *P*< 0.05 *versus* control (EE PRL).

Autocrine and Paracrine Regulation of Prolactin Secretion

**Figure 3. (A-B).** Effect of the PRL variants electroeluted (EE PRL) in fractions 1-6 from SDS-PAGE of CM from the peripheral (PR) (panel A), and central (CR) (panel B), AP regions of suckled (S) lactating

by Prolactin Variants and by Hypothalamic Hormones 107

#### *4.1.4. Effects of electroeluted PRL variants released from AP regions of lactating suckled (S) rats upon the in vitro release of PRL variants from lactotrophs of suckled (S) rat APs*

The effect of incubating lactotrophs from AP regions of S rats, with EE PRL variants released from AP regions of S rats is shown in figures 3 A-B. As shown in figure 3A, the EE PRL content from the peripheral AP region (c.f. Fig. 1C-D) showed medium to high levels ( 5>20 ng/μl), in all fractions except fraction 2; the amount of PRL released from the peripheral AP region of S rats was medium to high in fractions 1, 3 and 6 and medium to low in fractions 2, 4 and 5; and with respect to the amount of EE PRL released from the central AP region of S rats, (figure 3B), low and medium levels (2 and 8-10 ng/μl), were found in fractions 1, 2 and 3-6, respectively; and with respect to the effect of incubating lactotrophs from the central AP region of S rats with EE PRL variants from the same AP region of S rats, the levels were low in fractions 1 and 2 and higher (around 10 ng/μl) in fractions 3-6; and after incubation with the EE PRL, medium to low levels (1-6 ng/μl) occurred in fractions 1 and 2, reduced levels (-10 ng) in fraction 4 and higher levels (8-10 ng/μl) in fractions 3, 5 and 6 of PRL variants were released from the peripheral AP region of S rats, below zero levels were released from fraction 4 of the same AP region; and there was only a small stimulatory effect on PRL variants 1, 2, 3, 5, and 6. EE control PRL reduced the release of PRL variants from the central AP region to below zero levels in all fractions. As a result of these effects, the total amount of PRL released from the peripheral AP region, and particularly from the central AP region, was significantly lower than that of both the EE PRL variants and of the amount released from the peripheral AP region.

#### *4.1.5. Effects of electroeluted PRL variants, released from AP regions of lactating, suckled (S) rats, upon the in vitro release of PRL variants from lactotrophs of non-suckled (NS) rat APs*

The effect of PRL variants released from AP regions of suckled rats upon the release of PRL variants from lactotrophs of the peripheral and the central AP regions of non-suckled rats is shown in figures 3 C-D. In Fig. 3C, the PRL content of the EE fractions from the CM of the peripheral AP regions of S rats was low to medium in fractions 2 and 3, medium to high in fractions 1, 4 and 5, and particularly high in fraction 6; and, as a result of incubation, the amount of PRL released from lactotrophs of the peripheral AP region of NS rats was low in fractions 1, 4-6, and high only in fractions 2 and 3; and from the central AP region of these rats, the amount of PRL released was particularly high in fractions 1 and 6; medium in fractions 2 and 3, and low in fractions 1 and 4-5. As a result of these interactions, the amount of total PRL released from both AP regions was significantly lower than the amount electroeluted from SDS-PAGE, but higher than that shown from NS and S rat AP regions, (c.f. Figs. 2 A-B), due to the effect of CM from NS and S rats.

\*Differences *P*< 0.05 *versus* control (EE PRL).

lactating rats upon the *in vitro* release of PRL variants by lactotrophs from AP regions of NS lactating rats. Data are means ± SEM. \*Differences *P* < 0.05 *versus* control (EE PRL). **(C-D).** Effect of the PRL variants electroeluted (EE PRL) in fractions 1-6 from SDS-PAGE of CM from the peripheral (PR) (panel A), and central (CR) (panel B), AP regions of non-suckled (NS) lactating rats upon the *in vitro* release of PRL variants by lactotrophs from AP regions of suckled (S) lactating rats. Data are means ± SEM.

*4.1.4. Effects of electroeluted PRL variants released from AP regions of lactating suckled (S) rats upon the in vitro release of PRL variants from lactotrophs of suckled (S) rat APs* 

the EE PRL variants and of the amount released from the peripheral AP region.

(c.f. Figs. 2 A-B), due to the effect of CM from NS and S rats.

*4.1.5. Effects of electroeluted PRL variants, released from AP regions of lactating, suckled (S) rats, upon the in vitro release of PRL variants from lactotrophs of non-suckled (NS) rat APs* 

The effect of PRL variants released from AP regions of suckled rats upon the release of PRL variants from lactotrophs of the peripheral and the central AP regions of non-suckled rats is shown in figures 3 C-D. In Fig. 3C, the PRL content of the EE fractions from the CM of the peripheral AP regions of S rats was low to medium in fractions 2 and 3, medium to high in fractions 1, 4 and 5, and particularly high in fraction 6; and, as a result of incubation, the amount of PRL released from lactotrophs of the peripheral AP region of NS rats was low in fractions 1, 4-6, and high only in fractions 2 and 3; and from the central AP region of these rats, the amount of PRL released was particularly high in fractions 1 and 6; medium in fractions 2 and 3, and low in fractions 1 and 4-5. As a result of these interactions, the amount of total PRL released from both AP regions was significantly lower than the amount electroeluted from SDS-PAGE, but higher than that shown from NS and S rat AP regions,

The effect of incubating lactotrophs from AP regions of S rats, with EE PRL variants released from AP regions of S rats is shown in figures 3 A-B. As shown in figure 3A, the EE PRL content from the peripheral AP region (c.f. Fig. 1C-D) showed medium to high levels ( 5>20 ng/μl), in all fractions except fraction 2; the amount of PRL released from the peripheral AP region of S rats was medium to high in fractions 1, 3 and 6 and medium to low in fractions 2, 4 and 5; and with respect to the amount of EE PRL released from the central AP region of S rats, (figure 3B), low and medium levels (2 and 8-10 ng/μl), were found in fractions 1, 2 and 3-6, respectively; and with respect to the effect of incubating lactotrophs from the central AP region of S rats with EE PRL variants from the same AP region of S rats, the levels were low in fractions 1 and 2 and higher (around 10 ng/μl) in fractions 3-6; and after incubation with the EE PRL, medium to low levels (1-6 ng/μl) occurred in fractions 1 and 2, reduced levels (-10 ng) in fraction 4 and higher levels (8-10 ng/μl) in fractions 3, 5 and 6 of PRL variants were released from the peripheral AP region of S rats, below zero levels were released from fraction 4 of the same AP region; and there was only a small stimulatory effect on PRL variants 1, 2, 3, 5, and 6. EE control PRL reduced the release of PRL variants from the central AP region to below zero levels in all fractions. As a result of these effects, the total amount of PRL released from the peripheral AP region, and particularly from the central AP region, was significantly lower than that of both

**Figure 3. (A-B).** Effect of the PRL variants electroeluted (EE PRL) in fractions 1-6 from SDS-PAGE of CM from the peripheral (PR) (panel A), and central (CR) (panel B), AP regions of suckled (S) lactating

rats upon the *in vitro* release of PRL variants by lactotrophs from AP regions of suckled (S) lactating rats. Data are means ± SEM. \*Differences *P* < 0.05 *versus* control (EE PRL).

**(C-D).** Effect of the PRL variants electroeluted (EE PRL) in fractions 1-6 from SDS-PAGE of CM from the peripheral (PR) (panel A), and central (CR) (panel B), AP regions of suckled (S) lactating rats upon the *in vitro* release of PRL variants by lactotrophs from AP regions of non-suckled (NS) lactating rats. Data are means ± SEM. \*Differences *P* < 0.05 *versus* control (EE PRL).

Autocrine and Paracrine Regulation of Prolactin Secretion

1 and 4. In the presence of 1.0 μM DA there was inhibition of PRL variants in fraction 3, no effect in fraction 4, and stimulation in fractions 2 and 5, 6. The total amount of PRL released from the peripheral AP region of S rats was decreased only by 1.0 and 1.5 μM DA, but not by the lower dose. The effects of DA upon the release of PRL variants from lactotrophs of

**Figure 4. A-D.** Effect of dose-response of the PRL variants electroeluted in fractions 1-6 from SDS-PAGE of CM from the peripheral (PR) and the central (CR) regions of adenohypophysis (AP) of suckled (S) and non-suckled (NS) lactating rats incubated with 0.5, 1.0 and 1.5 μM of dopamine (DA), upon the *in vitro* release of PRL variants by lactotrophs from AP regions of NS and S lactating rats. Data are

means ± SEM. \*Differences *P* < 0.05 *versus* control (PRL content without DA).

by Prolactin Variants and by Hypothalamic Hormones 109

The PRL content of the electroeluted fractions from the central AP region of S rats was low in fractions 1 and 2, and high in fractions 3-6 of CM (Figure 3D). However, the amount of PRL released mainly from the peripheral, and in part also by the central AP region, was higher by the peripheral than the EE PRL variants in fractions 1, 2 and 5, 6; this level was about the same in fraction 3, and lower in fraction 4; and with respect to the amount of PRL released from the central AP region it was higher than the electroeluted hormone in fractions 1 and 2, lower in fraction 3 and 4, and about the same high level in fractions 5 and 6. As a result of these effects, an increased release of the hormone occurred from both AP regions, particularly from the peripheral region, whose levels, except from that of fraction 3, were significantly higher than those of the EE PRL as well as of that released from the central AP region, whose levels in fractions 1 and 2 were also higher than those of the EE PRL.

#### **4.2. Effects of hypothalamic hormones upon the release of PRL variants from AP regions of NS and S rats**

#### *4.2.1. Dose-response effects of dopamine*

The effects of dopamine (DA) upon the release of PRL variants 1-6 from the lactotrophs of the peripheral and central AP regions of non-suckled (NS) and suckled (S) rats are shown in figures 4 A-D. As shown in Fig. 4 A, as compared with the amount of PRL released without DA, the low dose of DA (0.5 μM) inhibited the release of PRL variant 2 and stimulated PRL variants 3 and 5 of the peripheral AP regions of NS rats, and showed no effect upon the release of PRL variants 4 and 6 from the same AP region. Higher doses of DA increased the release of PRL variants in fractions 3 and 5, but 1.5 μM DA inhibited the release of PRL variants 1 and 6. As a result of these effects, the total amount of released PRL from the peripheral AP region of NS rats was decreased only by the highest dose of DA (1.5 μM) but not by the lower and intermediate doses.

The effects of dopamine upon the release of PRL variants from lactotrophs of the central AP regions of non-suckled rats are shown in figure 4 B. The low dose of DA inhibited the release of the PRL variant 1 and showed no effect upon PRL variants 2 and 4, but it promoted a strong release of PRL variants 3, 5 and 6 from the central AP regions of NS rats; 1.0 μM dopamine provoked decreased release of PRL variant 1, and increased release of PRL variants 2, 3, 5 and 6. With 1.5 μM DA, decreased release occurred in fraction 1, and increased release in fractions 3, 5 and 6.

With respect to the effects of DA upon the release of PRL variants from the peripheral AP region of suckled rats APs, as shown in figure 4 C, at the low dose of DA inhibition occurred in fraction 3, stimulation of PRL in fractions 2, 5 and 6, and no effect on PRL variants in fractions 1 and 4. In the presence of 1.0 μM DA there was inhibition of PRL variants in fraction 3, no effect in fraction 4, and stimulation in fractions 2 and 5, 6. The total amount of PRL released from the peripheral AP region of S rats was decreased only by 1.0 and 1.5 μM DA, but not by the lower dose. The effects of DA upon the release of PRL variants from lactotrophs of

108 Prolactin

rats upon the *in vitro* release of PRL variants by lactotrophs from AP regions of suckled (S) lactating

**(C-D).** Effect of the PRL variants electroeluted (EE PRL) in fractions 1-6 from SDS-PAGE of CM from the peripheral (PR) (panel A), and central (CR) (panel B), AP regions of suckled (S) lactating rats upon the *in vitro* release of PRL variants by lactotrophs from AP regions of non-suckled (NS) lactating rats. Data

The PRL content of the electroeluted fractions from the central AP region of S rats was low in fractions 1 and 2, and high in fractions 3-6 of CM (Figure 3D). However, the amount of PRL released mainly from the peripheral, and in part also by the central AP region, was higher by the peripheral than the EE PRL variants in fractions 1, 2 and 5, 6; this level was about the same in fraction 3, and lower in fraction 4; and with respect to the amount of PRL released from the central AP region it was higher than the electroeluted hormone in fractions 1 and 2, lower in fraction 3 and 4, and about the same high level in fractions 5 and 6. As a result of these effects, an increased release of the hormone occurred from both AP regions, particularly from the peripheral region, whose levels, except from that of fraction 3, were significantly higher than those of the EE PRL as well as of that released from the central AP region, whose

**4.2. Effects of hypothalamic hormones upon the release of PRL variants from AP** 

The effects of dopamine (DA) upon the release of PRL variants 1-6 from the lactotrophs of the peripheral and central AP regions of non-suckled (NS) and suckled (S) rats are shown in figures 4 A-D. As shown in Fig. 4 A, as compared with the amount of PRL released without DA, the low dose of DA (0.5 μM) inhibited the release of PRL variant 2 and stimulated PRL variants 3 and 5 of the peripheral AP regions of NS rats, and showed no effect upon the release of PRL variants 4 and 6 from the same AP region. Higher doses of DA increased the release of PRL variants in fractions 3 and 5, but 1.5 μM DA inhibited the release of PRL variants 1 and 6. As a result of these effects, the total amount of released PRL from the peripheral AP region of NS rats was decreased only by the highest dose of DA (1.5 μM) but

The effects of dopamine upon the release of PRL variants from lactotrophs of the central AP regions of non-suckled rats are shown in figure 4 B. The low dose of DA inhibited the release of the PRL variant 1 and showed no effect upon PRL variants 2 and 4, but it promoted a strong release of PRL variants 3, 5 and 6 from the central AP regions of NS rats; 1.0 μM dopamine provoked decreased release of PRL variant 1, and increased release of PRL variants 2, 3, 5 and 6. With 1.5 μM DA, decreased release occurred in fraction 1, and

With respect to the effects of DA upon the release of PRL variants from the peripheral AP region of suckled rats APs, as shown in figure 4 C, at the low dose of DA inhibition occurred in fraction 3, stimulation of PRL in fractions 2, 5 and 6, and no effect on PRL variants in fractions

rats. Data are means ± SEM. \*Differences *P* < 0.05 *versus* control (EE PRL).

levels in fractions 1 and 2 were also higher than those of the EE PRL.

**regions of NS and S rats** 

*4.2.1. Dose-response effects of dopamine* 

not by the lower and intermediate doses.

increased release in fractions 3, 5 and 6.

are means ± SEM. \*Differences *P* < 0.05 *versus* control (EE PRL).

**Figure 4. A-D.** Effect of dose-response of the PRL variants electroeluted in fractions 1-6 from SDS-PAGE of CM from the peripheral (PR) and the central (CR) regions of adenohypophysis (AP) of suckled (S) and non-suckled (NS) lactating rats incubated with 0.5, 1.0 and 1.5 μM of dopamine (DA), upon the *in vitro* release of PRL variants by lactotrophs from AP regions of NS and S lactating rats. Data are means ± SEM. \*Differences *P* < 0.05 *versus* control (PRL content without DA).

the central AP region of suckled rats are shown in figure 4 D. The low dose of DA inhibited the release of PRL in fraction 1, had no effect upon the release of the PRL variant 3, but it promoted the release of PRL variants 2, 4 and 6; the intermediate dose of DA also inhibited the release of PRL from fraction 1, and promoted the release of fractions 2, 4 and 6; 1.5 μM DA inhibited the release of PRL fractions 1 and 2, but it promoted the release of fractions 3-6.

Autocrine and Paracrine Regulation of Prolactin Secretion

occurred to PRL variant 5; inhibition to PRL variant 1, and no effect upon PRL variants 2, 3, 4 and 6. And with respect to the effect of the same dose of OT i.e., 1.0 μM upon the release of PRL variants from the central AP region of suckled rats, i.e., Fig. 6 D, lower panel, increased release occurred to PRL variants 1, 2, 5 and 6, and no effect upon PRL variants 3 and 4; and with respect to the effect of the same dose of OT upon the release of PRL variants from the

**Figure 5. A-D**. Effect of dose-response of the PRL variants electroeluted in fractions 1-6 from SDS-PAGE of CM from the peripheral (PR) and the central (CR) regions of adenohypophysis (AP) of suckled (S) and non-suckled (NS) lactating rats incubated with 0.1, 1.0 and 10 μM of TRH, upon the *in vitro* release of PRL variants by lactotrophs from AP regions of NS and S lactating rats. Data are means ±

SEM. \*Differences *P* < 0.05 *versus* control (PRL content without TRH).

by Prolactin Variants and by Hypothalamic Hormones 111

#### *4.2.2. Dose-response effects of TRH*

The effects of different doses of TRH, i.e., 0.1, 1.0 and 10 μM, upon the release of PRL variants from AP regions of NS and S rats are shown in Figs. 5 A-D and the values obtained after TRH, are compared with the control values of PRL that were released in the absence of TRH. As compared with control values, without TRH, 0.1 μM TRH provoked increased release, i.e., stimulation of PRL, in fractions 1, 3-6; and inhibition in fraction 2 from the peripheral AP region of NS rats (Fig. 5 A). The effect of 1.0 and 10 μM TRH was to increase the release of all PRL variants 1-6.

The effect of TRH upon the release of PRL variants from the central AP region of NS rats is shown in Fig. 5 B. With 0.1 μM TRH, decreased release of PRL occurred in fraction 1, no change in fractions 2 and 4, and increased release in fractions 3, 5 and 6. Also, with respect to the effect of the higher doses of TRH, increased release occurred to PRL in all fractions 1- 6. Fig. 5 C, shows the effect of the low dose of TRH upon the release of PRL variants from the peripheral AP region of S rats. As shown there, release of PRL variants 1, 4, 5 and 6 was stimulated, and there was no effect on PRL variants 2 and 3; and with respect to the effect of the low dose of TRH upon the release of PRL variants from the central AP region of S rats (Fig. 5 D) a decreased release of PRL variant 1; increased release of PRL variants 3, 4 and 6, and no effect upon PRL variants 2 and 5 were observed.

#### *4.2.3. Dose-response effects of oxytocin*

The dose-response effects of oxytocin (OT) upon the release of PRL variants from NS rat APs are shown in Figs. 6 A-D. With 0.1 μM oxytocin, increased release of PRL, relative to the control, occurred only for PRL variants 3, 5 and 6 of the PR region of NS rat APs, but the release of PRL in fractions 1, 2 and 4 was inhibited. With respect to the effect of 1.0 μM of oxytocin upon the release of PRL variants from the peripheral AP region of NS rats (Fig. 6 A), there was increased release of PRL variants 3, 5 and 6 and reduce release of PRL variants 1, 2 and 4 from lactotrophs of the peripheral AP region. The high dose of OT, i.e., 10 μM resulted in increased release of PRL variants 2, 3, 5 and 6 from the peripheral AP region and to variants 1-6 from the central AP region of NS rats (Fig. 6 B); and with respect to the effect of the 10 μM OT upon the release of PRL from AP regions of S rats, increased release from the peripheral region occurred for PRL variants 1, 2, 5 and 6; whit no effect on release of PRL variants 3 and 4. Finally, with respect to the effect of the high dose of OT upon AP regions of NS rats, there was increased release of fractions 2, 3, 5 and 6 and no effect on fractions 1 and 3 from the peripheral AP region of NS rats; and with the exception of fraction 5 in which there was no effect, increased release occurred in all the other fractions from the central AP region of the NS rats; and upon the effect of the same dose of oxytocin upon the release of PRL variants from the peripheral AP region of S rats, i.e., Fig. 6 C, also, increased release occurred to PRL variant 5; inhibition to PRL variant 1, and no effect upon PRL variants 2, 3, 4 and 6. And with respect to the effect of the same dose of OT i.e., 1.0 μM upon the release of PRL variants from the central AP region of suckled rats, i.e., Fig. 6 D, lower panel, increased release occurred to PRL variants 1, 2, 5 and 6, and no effect upon PRL variants 3 and 4; and with respect to the effect of the same dose of OT upon the release of PRL variants from the

110 Prolactin

*4.2.2. Dose-response effects of TRH* 

the release of all PRL variants 1-6.

and no effect upon PRL variants 2 and 5 were observed.

*4.2.3. Dose-response effects of oxytocin* 

the central AP region of suckled rats are shown in figure 4 D. The low dose of DA inhibited the release of PRL in fraction 1, had no effect upon the release of the PRL variant 3, but it promoted the release of PRL variants 2, 4 and 6; the intermediate dose of DA also inhibited the release of PRL from fraction 1, and promoted the release of fractions 2, 4 and 6; 1.5 μM DA inhibited the release of PRL fractions 1 and 2, but it promoted the release of fractions 3-6.

The effects of different doses of TRH, i.e., 0.1, 1.0 and 10 μM, upon the release of PRL variants from AP regions of NS and S rats are shown in Figs. 5 A-D and the values obtained after TRH, are compared with the control values of PRL that were released in the absence of TRH. As compared with control values, without TRH, 0.1 μM TRH provoked increased release, i.e., stimulation of PRL, in fractions 1, 3-6; and inhibition in fraction 2 from the peripheral AP region of NS rats (Fig. 5 A). The effect of 1.0 and 10 μM TRH was to increase

The effect of TRH upon the release of PRL variants from the central AP region of NS rats is shown in Fig. 5 B. With 0.1 μM TRH, decreased release of PRL occurred in fraction 1, no change in fractions 2 and 4, and increased release in fractions 3, 5 and 6. Also, with respect to the effect of the higher doses of TRH, increased release occurred to PRL in all fractions 1- 6. Fig. 5 C, shows the effect of the low dose of TRH upon the release of PRL variants from the peripheral AP region of S rats. As shown there, release of PRL variants 1, 4, 5 and 6 was stimulated, and there was no effect on PRL variants 2 and 3; and with respect to the effect of the low dose of TRH upon the release of PRL variants from the central AP region of S rats (Fig. 5 D) a decreased release of PRL variant 1; increased release of PRL variants 3, 4 and 6,

The dose-response effects of oxytocin (OT) upon the release of PRL variants from NS rat APs are shown in Figs. 6 A-D. With 0.1 μM oxytocin, increased release of PRL, relative to the control, occurred only for PRL variants 3, 5 and 6 of the PR region of NS rat APs, but the release of PRL in fractions 1, 2 and 4 was inhibited. With respect to the effect of 1.0 μM of oxytocin upon the release of PRL variants from the peripheral AP region of NS rats (Fig. 6 A), there was increased release of PRL variants 3, 5 and 6 and reduce release of PRL variants 1, 2 and 4 from lactotrophs of the peripheral AP region. The high dose of OT, i.e., 10 μM resulted in increased release of PRL variants 2, 3, 5 and 6 from the peripheral AP region and to variants 1-6 from the central AP region of NS rats (Fig. 6 B); and with respect to the effect of the 10 μM OT upon the release of PRL from AP regions of S rats, increased release from the peripheral region occurred for PRL variants 1, 2, 5 and 6; whit no effect on release of PRL variants 3 and 4. Finally, with respect to the effect of the high dose of OT upon AP regions of NS rats, there was increased release of fractions 2, 3, 5 and 6 and no effect on fractions 1 and 3 from the peripheral AP region of NS rats; and with the exception of fraction 5 in which there was no effect, increased release occurred in all the other fractions from the central AP region of the NS rats; and upon the effect of the same dose of oxytocin upon the release of PRL variants from the peripheral AP region of S rats, i.e., Fig. 6 C, also, increased release

**Figure 5. A-D**. Effect of dose-response of the PRL variants electroeluted in fractions 1-6 from SDS-PAGE of CM from the peripheral (PR) and the central (CR) regions of adenohypophysis (AP) of suckled (S) and non-suckled (NS) lactating rats incubated with 0.1, 1.0 and 10 μM of TRH, upon the *in vitro* release of PRL variants by lactotrophs from AP regions of NS and S lactating rats. Data are means ± SEM. \*Differences *P* < 0.05 *versus* control (PRL content without TRH).

same, i.e., peripheral AP region of S rats, increased release occurred to PRL variants 1, 2 and 5, 6; and no effect upon PRL variants 3 and 4. Finally, with respect to the effect of the high dose of OT upon the release of PRL from lactotrophs of the PR region of S rats, increased release occurred to PRL variants 1, 2, 5 and 6, with no change of PRL variants 3 and 4; the high dose of OT resulted in increased release of all PRL variant from lactotrophs of the central AP region.

Autocrine and Paracrine Regulation of Prolactin Secretion

*4.3.1. Effect of conditioned medium from the lateral and central AP regions of S rats upon* 

Cultured sympathetic neurons, previously incubated with Fluo 4, to record variations of intracellular [Ca2+], showed clear increases of [Ca2+] within 60 sec of adding conditioned medium (CM) from lactating rats, thus indicating that these neurons were activated by the CM (Figure 7). These effects did not occurred when the neurons were incubated with

**Figure 7.** Effect of 60 s exposure of conditioned medium from the peripheral region of suckled rat APs

*4.3.2. Effect of conditioned medium from the lateral AP region of suckled and of male rat APs upon electrical activity of male rat lactotrophs, and of astrocytes from the hippocampus* 

As shown in Figures 8 A-D, CM of the peripheral region (CMPR) from male rat APs had no effect upon electrical activity of hippocampal astrocytes (Fig. 8A) whereas application of CM from lactating, suckled rats provoked a cationic inward current, shown as a downward deflection in male lactotrophs (Fig. 8C), as well as in astrocytes from the hippocampus and medial preoptic area (Figs. 8B, D). These responses remained for several seconds after

As shown in Figure 9, CM from lactating, suckled rats (Fig. 9A), but not from male rat APs (Fig. 9B) or from non-suckled rats (data not shown) provoked an increased [Ca2+] in hippocampal astrocytes similar to that induced by application of 120 mM K+ (see Fig. 9C).

*4.3.3. Effect of conditioned medium from lactating and male rat APs upon [Ca 2+]* 

*concentration in astrocytes from the hippocampus* 

(CM PRS).upon intracellular calcium concentration in cultured sympathetic neurons.

**4.3. Electrical activity upon neurons** 

medium from male rat APs (data not shown).

*cultured sympathetic neurons* 

*and medial preoptic area* 

washing CMPR out.

by Prolactin Variants and by Hypothalamic Hormones 113

**Figure 6. A-D.** Effect of dose-response of the PRL variants electroeluted in fractions 1-6 from SDS-PAGE of CM from the peripheral (PR) and the central (CR) regions of adenohypophysis (AP) of suckled (S) and non-suckled (NS) lactating rats incubated with 0.1, 1.0 and 10 μM of oxytocin (OT), upon the *in vitro* release of PRL variants by lactotrophs from AP regions of NS and S lactating rats. Data are means ± SEM. \*Differences *P* < 0.05 *versus* control (PRL content without OT).

#### **4.3. Electrical activity upon neurons**

112 Prolactin

same, i.e., peripheral AP region of S rats, increased release occurred to PRL variants 1, 2 and 5, 6; and no effect upon PRL variants 3 and 4. Finally, with respect to the effect of the high dose of OT upon the release of PRL from lactotrophs of the PR region of S rats, increased release occurred to PRL variants 1, 2, 5 and 6, with no change of PRL variants 3 and 4; the high dose of OT resulted in increased release of all PRL variant from lactotrophs of the central AP region.

**Figure 6. A-D.** Effect of dose-response of the PRL variants electroeluted in fractions 1-6 from SDS-PAGE of CM from the peripheral (PR) and the central (CR) regions of adenohypophysis (AP) of suckled (S) and non-suckled (NS) lactating rats incubated with 0.1, 1.0 and 10 μM of oxytocin (OT), upon the *in vitro* release of PRL variants by lactotrophs from AP regions of NS and S lactating rats. Data are means ±

SEM. \*Differences *P* < 0.05 *versus* control (PRL content without OT).

#### *4.3.1. Effect of conditioned medium from the lateral and central AP regions of S rats upon cultured sympathetic neurons*

Cultured sympathetic neurons, previously incubated with Fluo 4, to record variations of intracellular [Ca2+], showed clear increases of [Ca2+] within 60 sec of adding conditioned medium (CM) from lactating rats, thus indicating that these neurons were activated by the CM (Figure 7). These effects did not occurred when the neurons were incubated with medium from male rat APs (data not shown).

**Figure 7.** Effect of 60 s exposure of conditioned medium from the peripheral region of suckled rat APs (CM PRS).upon intracellular calcium concentration in cultured sympathetic neurons.

#### *4.3.2. Effect of conditioned medium from the lateral AP region of suckled and of male rat APs upon electrical activity of male rat lactotrophs, and of astrocytes from the hippocampus and medial preoptic area*

As shown in Figures 8 A-D, CM of the peripheral region (CMPR) from male rat APs had no effect upon electrical activity of hippocampal astrocytes (Fig. 8A) whereas application of CM from lactating, suckled rats provoked a cationic inward current, shown as a downward deflection in male lactotrophs (Fig. 8C), as well as in astrocytes from the hippocampus and medial preoptic area (Figs. 8B, D). These responses remained for several seconds after washing CMPR out.

#### *4.3.3. Effect of conditioned medium from lactating and male rat APs upon [Ca 2+] concentration in astrocytes from the hippocampus*

As shown in Figure 9, CM from lactating, suckled rats (Fig. 9A), but not from male rat APs (Fig. 9B) or from non-suckled rats (data not shown) provoked an increased [Ca2+] in hippocampal astrocytes similar to that induced by application of 120 mM K+ (see Fig. 9C).

Autocrine and Paracrine Regulation of Prolactin Secretion

This study confirms that PRL variants ranging from 7 to 97 kDa are released from tissue fragment of each AP region of the anterior pituitary gland of non-suckled and suckled lactating rats. When these variants are electroeluted from SDS-PAGE and then incubated with lactotrophs from each AP region of the same type of rats, they exert different effects (promotion, inhibition or no effect) upon the release of PRL variants from lactotrophs of both AP regions of NS and S lactating rats. In support of this was the fact that the immunoprecipitation of PRL contained in the CM from lactating rats, prevented the effects of PRL variants upon PRL release. Thus, these results indicate that autocrine regulatory effects are exerted by PRL variants upon the release of other variants of the hormone from lactating rat APs, and they are in accord with previous studies showing autoregulation of PRL secretion (Nagy et al., 1991; Nagy & Frawley, 1990; Diaz et al., 2002; Spies & Clegg, 1971; Hebert et al., 1979; Melmed et al., 1980). Similar effects on male lactotrophs by CM from pregnant and lactating females and steroid-treated castrated males or females, but not by CM from intact males or by a PRL

standard were reported previously (Huerta-Ocampo et al., 2007; Mena et al., 2010).

& Walker, 2001; Ho et al., 1993; Celotti et al., 1997).

Prior to fractionation the total PRL variants from both, the central and peripheral AP regions of NS rats stimulated the release of PRL from the peripheral region, but they inhibited its release from the central AP region. However, when CM from lactating rats was fractionated by SDS-PAGE, eluates of fractions 5 and 6 containing 23-25 kDa PRL had the greatest effect on PRL release, although weaker immunoreactive bands with lower, or even inhibitory activity, were also detected in the upper gel fractions. In addition, separation by SDS-PAGE and electroelution of PRL variants indicated that CM from the lactating rat pituitary contains 37 to 46 kDa PRL variants as well as 23 to 25 kDa PRL variants, that exert different effects upon the release of other PRL variants from the lactating rat pituitary and from APs of rats in different conditions Huerta-Ocampo et al., 2007; Mena et al., 2010). Therefore, the present study shows that the lactating rat pituitary produces PRL variants that are absent or deficient in the male pituitary gland and in the PRL Standard, even though the AP of male and of other types of rats, do respond to stimulatory factors released from the anterior pituitary of lactating rats (Mena et al., 2010). The results presented here, together with those in our previous study, also indicate that several PRL variants are produced and released by the lactating rat pituitary (Denef, 1988; Sinha 1992; Asawaroengchai et al., 1978); this hormonal heterogeneity may be physiologically very relevant in the context of autoregulatory mechanisms determining the wide range of PRL effects under different physiological conditions (Schwartz & Cherny, 1992; Schwartz, 2000; Sinha, 1992) and upon different structures (Ben-Jonathan et al., 2001; Lorenson

In addition to the regulatory effects of PRL variants from lactating rats upon the release of the hormone, further evidence of these effects was obtained when CM's from lactating rats were treated with phosphatase or with endoglycosidase which increased their ability, i.e., that of the PRL variants present in them to stimulate PRL release from lactating rat APs, similar to the effect shown previously upon male rat lactotrophs (Mena et al., 2010). These effects of dephosphorylation and deglycosylation of CM provide additional evidence that PRL variants in CM are responsible for the effects upon lactating rat lactotrophs. PRL released from the AP of lactating and non-lactating rats is phosphorylated and glycosylated

**5. Conclusions** 

by Prolactin Variants and by Hypothalamic Hormones 115

**Figure 8. (A-D).** Effect of a 1 h incubation with conditioned medium (CM), from the peripheral region (PR) of male rats (A), and from suckled lactating rat APs (B-D) upon hippocampal astrocytes (B) , neurons from the preoptic area (D) and upon male rat lactotrophs (C).

**Figure 9. (A-C).** Effect of conditioned media from the peripheral AP region of suckled lactating rats (CMPR) and of male rats upon intracellular calcium concentration in hippocampal astrocytes (A-B). Arrow in C indicates the application of 120 mM K+. Images in D are from astrocytes in A-C.

#### **5. Conclusions**

114 Prolactin

**Figure 8. (A-D).** Effect of a 1 h incubation with conditioned medium (CM), from the peripheral region (PR) of male rats (A), and from suckled lactating rat APs (B-D) upon hippocampal astrocytes (B) ,

**Figure 9. (A-C).** Effect of conditioned media from the peripheral AP region of suckled lactating rats (CMPR) and of male rats upon intracellular calcium concentration in hippocampal astrocytes (A-B). Arrow in C indicates the application of 120 mM K+. Images in D are from astrocytes in A-C.

neurons from the preoptic area (D) and upon male rat lactotrophs (C).

This study confirms that PRL variants ranging from 7 to 97 kDa are released from tissue fragment of each AP region of the anterior pituitary gland of non-suckled and suckled lactating rats. When these variants are electroeluted from SDS-PAGE and then incubated with lactotrophs from each AP region of the same type of rats, they exert different effects (promotion, inhibition or no effect) upon the release of PRL variants from lactotrophs of both AP regions of NS and S lactating rats. In support of this was the fact that the immunoprecipitation of PRL contained in the CM from lactating rats, prevented the effects of PRL variants upon PRL release. Thus, these results indicate that autocrine regulatory effects are exerted by PRL variants upon the release of other variants of the hormone from lactating rat APs, and they are in accord with previous studies showing autoregulation of PRL secretion (Nagy et al., 1991; Nagy & Frawley, 1990; Diaz et al., 2002; Spies & Clegg, 1971; Hebert et al., 1979; Melmed et al., 1980). Similar effects on male lactotrophs by CM from pregnant and lactating females and steroid-treated castrated males or females, but not by CM from intact males or by a PRL standard were reported previously (Huerta-Ocampo et al., 2007; Mena et al., 2010).

Prior to fractionation the total PRL variants from both, the central and peripheral AP regions of NS rats stimulated the release of PRL from the peripheral region, but they inhibited its release from the central AP region. However, when CM from lactating rats was fractionated by SDS-PAGE, eluates of fractions 5 and 6 containing 23-25 kDa PRL had the greatest effect on PRL release, although weaker immunoreactive bands with lower, or even inhibitory activity, were also detected in the upper gel fractions. In addition, separation by SDS-PAGE and electroelution of PRL variants indicated that CM from the lactating rat pituitary contains 37 to 46 kDa PRL variants as well as 23 to 25 kDa PRL variants, that exert different effects upon the release of other PRL variants from the lactating rat pituitary and from APs of rats in different conditions Huerta-Ocampo et al., 2007; Mena et al., 2010). Therefore, the present study shows that the lactating rat pituitary produces PRL variants that are absent or deficient in the male pituitary gland and in the PRL Standard, even though the AP of male and of other types of rats, do respond to stimulatory factors released from the anterior pituitary of lactating rats (Mena et al., 2010). The results presented here, together with those in our previous study, also indicate that several PRL variants are produced and released by the lactating rat pituitary (Denef, 1988; Sinha 1992; Asawaroengchai et al., 1978); this hormonal heterogeneity may be physiologically very relevant in the context of autoregulatory mechanisms determining the wide range of PRL effects under different physiological conditions (Schwartz & Cherny, 1992; Schwartz, 2000; Sinha, 1992) and upon different structures (Ben-Jonathan et al., 2001; Lorenson & Walker, 2001; Ho et al., 1993; Celotti et al., 1997).

In addition to the regulatory effects of PRL variants from lactating rats upon the release of the hormone, further evidence of these effects was obtained when CM's from lactating rats were treated with phosphatase or with endoglycosidase which increased their ability, i.e., that of the PRL variants present in them to stimulate PRL release from lactating rat APs, similar to the effect shown previously upon male rat lactotrophs (Mena et al., 2010). These effects of dephosphorylation and deglycosylation of CM provide additional evidence that PRL variants in CM are responsible for the effects upon lactating rat lactotrophs. PRL released from the AP of lactating and non-lactating rats is phosphorylated and glycosylated

and thus, it is less bioactive than the dephosphorylated and deglycosylated variants (Ho et al., 1993a; Sinha, 1995, Ho et al., 1993b).

Autocrine and Paracrine Regulation of Prolactin Secretion

In conclusion, the results of the present and previous studies suggest that, in addition to regulation by hypothalamic and other influences, the release of PRL variants from the lactating rat AP is also regulated by autocrine influences exerted upon the gland by the previously released PRL variants; furthermore in parallel and interacting with such autocrine regulation, the effect of the hypothalamic hormones on PRL release is also regulated through the same mechanism i.e., the stimulation or inhibition of the release of PRL variants from the pituitary gland. Moreover, the present results confirm previous findings (Huerta-Ocampo, 2007; Mena et al., 2010; Mena et al., 2011; Möderscheim et al., 2007, Mena et al., 2012a), that CM from lactating rats, contain prolactin variants capable of inducing rapid release of PRL from the untreated male rat pituitary, and that they can also activate of astrocytes and neurons in different areas of the central and peripheral nervous system (Mena et al, 2012b).

**Abbreviations** 

AP Anterior pituitary

CM Conditioned media CR Central region DA Dopamina EE Electroeluted EA Electrical activity

KDa Kilodalton NR Non-reducing NS Non-suckled OT Oxytocin

PRL Prolactin S Suckled

**Author details** 

**Acknowledgement** 

**Funding** 

PR Peripheral region

TRH Thyrotropin releasing hormone

Flavio Mena, Nilda Navarro and Alejandra Castilla

IN206711), and by from CONACyT (No. 128037).

*Departamento de Neurobiología Celular y Molecular, Instituto de Neurobiología, Universidad Nacional Autónoma de México, Campus Juriquilla, Querétaro, México* 

This work was supported by Grants from PAPIIT-DGAPA-UNAM (IN201808 and

We gratefully acknowledge Dr. A.F. Parlow and the National Hormone and Pituitary Program of the National Institute of Diabetes and Digestive and Kidney Diseases for the generous gift of

[Ca2+] Intracellular calcium concentration

ELISA Enzyme-linked immunoabsorbant assay

by Prolactin Variants and by Hypothalamic Hormones 117

In the present study we also analyzed whether the ability of the hypothalamic hormones dopamine, TRH and oxytocin, as established by many previous *in vivo* and *in vitr*o studies (Mena et al., 1989), to regulate the release of AP PRL, would be manifest by their direct action upon the lactotrophs and interaction with the autocrine actions of PRL variants; and whether these effects would finally promote or inhibit the release of PRL variants, thereby regulating the release of the hormone. The results obtained showed that the effects of the hypothalamic hormones were exerted upon the lactotrophs both by interacting with the autocrine actions of the PRL variants, and also by regulating the release of PRL variants from these cells. Thus, when a high dose (1.5 μM) of DA was applied directly upon the lactotrophs of NS and S rats, the secretion of most PRL variants from both AP regions of non-suckled and suckled rats was inhibited, as previously reported by others (Nagy et al., 1991), whereas the lower dose (0.5 μM) slightly stimulated the release of PRL variants, mainly from the central AP region of suckled rats. TRH provoked an increased release of some PRL variants from both the central and peripheral AP regions of NS and S rats, and OT at 1 and 10 μM, showed an intense stimulatory effect, particularly of 23-34 kDa PRL, from both AP regions of non-suckled and suckled lactating rat APs. Thus, these effects of hypothalamic hormones upon the release of PRL variants may also regulate, and thus interact with, the autocrine effects exerted by the PRL variants and lead to an integrative regulation of PRL secretion (Mena et al., 1989; Mena et al., 2011).

This study shows that when pituitaries from male rats are incubated for a short period of time in conditioned media from each pituitary region of lactating rats, either suckled or nonsuckled, there is a significant dose-dependent increase of PRL release from male rat lactotrophs. Also, as shown previously (Mena et al., 2010) our results obtained by Western blotting confirm that CM from lactating rat pituitary contains several prolactin variants, i.e., 37-46 kDa as well as 16-25 kDa, capable of stimulating PRL release from male rat pituitary. In addition, CM obtained from male rat pituitary regions as well as PRL standard have no significant stimulatory effect on PRL secretion of pituitary regions from either lactating or male rats (Huerta-Ocampo, 2007; Mena et al., 2010), this suggests that the male pituitary gland is deficient in the same PRL variants, and of other possible factors, that are released by the lactating rat AP, even though it does contain receptors for stimulating factors released from the anterior pituitary of lactating rats (Mena et al., 2011; Mena et al., 2012a).

Based upon these results, it was of interest to determine whether production and release of pituitary prolactin variants contained in CM from lactating rats under different conditions, and their effects upon PRL release from incubated male AP regions, vary depending on the animal's physiological condition, and whether CM's from lactating or male rats exert effect upon brain structures, i.e., astrocytes in the hippocampus and medial preoptic area, and of sympathetic neurons (Fiordelisio & Hernandez-Cruz, 2002). The results obtained from Electrical recording of these astrocytes showed that astrocytic activation occurred upon exposure to CM from the lateral and central regions of lactating suckled rat APs (Hernández-Morales M & García-Colunga, 2009).

In conclusion, the results of the present and previous studies suggest that, in addition to regulation by hypothalamic and other influences, the release of PRL variants from the lactating rat AP is also regulated by autocrine influences exerted upon the gland by the previously released PRL variants; furthermore in parallel and interacting with such autocrine regulation, the effect of the hypothalamic hormones on PRL release is also regulated through the same mechanism i.e., the stimulation or inhibition of the release of PRL variants from the pituitary gland. Moreover, the present results confirm previous findings (Huerta-Ocampo, 2007; Mena et al., 2010; Mena et al., 2011; Möderscheim et al., 2007, Mena et al., 2012a), that CM from lactating rats, contain prolactin variants capable of inducing rapid release of PRL from the untreated male rat pituitary, and that they can also activate of astrocytes and neurons in different areas of the central and peripheral nervous system (Mena et al, 2012b).

#### **Abbreviations**

116 Prolactin

al., 1993a; Sinha, 1995, Ho et al., 1993b).

regulation of PRL secretion (Mena et al., 1989; Mena et al., 2011).

(Hernández-Morales M & García-Colunga, 2009).

and thus, it is less bioactive than the dephosphorylated and deglycosylated variants (Ho et

In the present study we also analyzed whether the ability of the hypothalamic hormones dopamine, TRH and oxytocin, as established by many previous *in vivo* and *in vitr*o studies (Mena et al., 1989), to regulate the release of AP PRL, would be manifest by their direct action upon the lactotrophs and interaction with the autocrine actions of PRL variants; and whether these effects would finally promote or inhibit the release of PRL variants, thereby regulating the release of the hormone. The results obtained showed that the effects of the hypothalamic hormones were exerted upon the lactotrophs both by interacting with the autocrine actions of the PRL variants, and also by regulating the release of PRL variants from these cells. Thus, when a high dose (1.5 μM) of DA was applied directly upon the lactotrophs of NS and S rats, the secretion of most PRL variants from both AP regions of non-suckled and suckled rats was inhibited, as previously reported by others (Nagy et al., 1991), whereas the lower dose (0.5 μM) slightly stimulated the release of PRL variants, mainly from the central AP region of suckled rats. TRH provoked an increased release of some PRL variants from both the central and peripheral AP regions of NS and S rats, and OT at 1 and 10 μM, showed an intense stimulatory effect, particularly of 23-34 kDa PRL, from both AP regions of non-suckled and suckled lactating rat APs. Thus, these effects of hypothalamic hormones upon the release of PRL variants may also regulate, and thus interact with, the autocrine effects exerted by the PRL variants and lead to an integrative

This study shows that when pituitaries from male rats are incubated for a short period of time in conditioned media from each pituitary region of lactating rats, either suckled or nonsuckled, there is a significant dose-dependent increase of PRL release from male rat lactotrophs. Also, as shown previously (Mena et al., 2010) our results obtained by Western blotting confirm that CM from lactating rat pituitary contains several prolactin variants, i.e., 37-46 kDa as well as 16-25 kDa, capable of stimulating PRL release from male rat pituitary. In addition, CM obtained from male rat pituitary regions as well as PRL standard have no significant stimulatory effect on PRL secretion of pituitary regions from either lactating or male rats (Huerta-Ocampo, 2007; Mena et al., 2010), this suggests that the male pituitary gland is deficient in the same PRL variants, and of other possible factors, that are released by the lactating rat AP, even though it does contain receptors for stimulating factors released from the anterior pituitary of lactating rats (Mena et al., 2011; Mena et al., 2012a).

Based upon these results, it was of interest to determine whether production and release of pituitary prolactin variants contained in CM from lactating rats under different conditions, and their effects upon PRL release from incubated male AP regions, vary depending on the animal's physiological condition, and whether CM's from lactating or male rats exert effect upon brain structures, i.e., astrocytes in the hippocampus and medial preoptic area, and of sympathetic neurons (Fiordelisio & Hernandez-Cruz, 2002). The results obtained from Electrical recording of these astrocytes showed that astrocytic activation occurred upon exposure to CM from the lateral and central regions of lactating suckled rat APs


#### **Author details**

Flavio Mena, Nilda Navarro and Alejandra Castilla *Departamento de Neurobiología Celular y Molecular, Instituto de Neurobiología, Universidad Nacional Autónoma de México, Campus Juriquilla, Querétaro, México* 

#### **Funding**

This work was supported by Grants from PAPIIT-DGAPA-UNAM (IN201808 and IN206711), and by from CONACyT (No. 128037).

#### **Acknowledgement**

We gratefully acknowledge Dr. A.F. Parlow and the National Hormone and Pituitary Program of the National Institute of Diabetes and Digestive and Kidney Diseases for the generous gift of rat PRL, and the corresponding antiserum. Finally, we gratefully acknowledge the help of Martin Garcia Servín Head of the Animal Facilities at INB (Universidad Nacional Autónoma de México), and in particular to Dr. Dorothy Pless for reading and correcting the manuscript.

Autocrine and Paracrine Regulation of Prolactin Secretion

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Mena, F., Navarro, N., Castilla, A., Fiordelisio, T., Morales, T., Hernández-Morales, M., & García-Colunga, J. (2012b). Release of prolactin (PRL) from pituitary lactotrophs of male rats and functional activity of astrocytes from hippocampal and medial preoptic area, and of sympathetic neurons, are stimulated by PRL variants released from the anterior pituitary (AP) of lactating rats. *Neuroendocrinology.* Envoy.

**Chapter 7** 

© 2013 Molik et al., licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

The fundamental feature of all living organisms is the ability to receive and process information about changes in the environment. Succession of physiological changes is synchronized with changes of environmental conditions and conditioned by the activity of

and reproduction in any medium, provided the original work is properly cited.

**The Effect of Physiological and Environmental** 

Light, being an environmental factor, has a significant effect on reproductive functions of animals exhibiting sensitivity to changes of the day length [1]. Among mammals there are many species displaying seasonality of reproduction, and given that, two models of seasonal sensitivity were distinguished. The first one refers to long-day animals (horses), in which reproductive processes are induced by lengthening days, i.e. in the spring. The other model concerns short-day animals, which include sheep, goats and deer; in these animals the reproduction system is stimulated and estrus takes place in the autumn and winter period [2]. In sheep the phenomenon of seasonality relates not only to reproduction but also to lactation. Following the process of mammogenesis in mammals, a mammary gland is developed, which is a complex cutaneous acinotubular gland [3]. The endocrine mechanism of entering and maintenance of lactation in sheep involves a number of hormones, which proves that the process relies basically on the activity of hypothalamus and pituitary gland [4,5,6]. One of the principal hormones conditioning both triggering and maintenance of lactation, synthesis of milk proteins, fat and immunoglobulins is prolactin (PRL), which is secreted chiefly by lactotroph cells of the anterior pituitary gland [7]. Prolactin is also produced locally by the mammary gland of mammals and does not differ immunologically from prolactin produced by the pituitary gland [8]. An important role in the process of mammogenesis and lactogenesis is assigned also to glycocorticoids, insulin and growth

**Factors on the Prolactin Profile in Seasonally** 

**Breeding Animals** 

http://dx.doi.org/10.5772/51465

hormone and estrogens [9].

**1. Introduction** 

Edyta Molik, Tomasz Misztal and Dorota A. Zieba

Additional information is available at the end of the chapter

