Prolactin Is a Strong Candidate for the Regulation of Luteal Steroidogenesis in Vizcachas (Lagostomus maximus)

Abstract

Prolactin (PRL) is essential for the maintenance of the corpora lutea and the production of progesterone (P4) during gestation of mice and rats, which makes it a key factor for their successful reproduction. Unlike these rodents and the vast majority of mammals, female vizcachas (Lagostomus maximus) have a peculiar reproductive biology characterized by an ovulatory event during pregnancy that generates secondary corpora lutea with a consequent increment of the circulating P4. We found that, although the expression of pituitary PRL increased steadily during pregnancy, its ovarian receptor (PRLR) reached its maximum in midpregnancy and drastically decreased at term pregnancy. The luteinizing hormone receptor (LHR) exhibited a similar profile than PRLR. Maximum P4 and LH blood levels were recorded at midpregnancy as well. Remarkably, the P4-sinthesizing enzyme 3β-HSD accompanied the expression pattern of PRLR/LHR throughout gestation. Instead, the luteolytic enzyme 20α-HSD showed low expression at early and midpregnancy, but reached its maximum at the end of gestation, when PRLR/LHR/3ß-HSD expressions and circulating P4 were minimal. In conclusion, both the PRLR and LHR expressions in the ovary would define the success of gestation in vizcachas by modulating the levels of 20α-HSD and 3ß-HSD, which ultimately determine the level of serum P4 throughout gestation.

Figure 1

Luteal levels of PRLR, LHR, and 3ß-HSD increased in pregnant-ovulating vizcachas. Representative photomicrographs of luteal cells in ovary cross sections immunostained for PRLR, LHR, and 3ß-HSD, at early pregnancy (EP), midpregnancy (MP), and term pregnancy (TP) of vizcachas. Immunoreactivity is shown in brown and nuclei counterstained with hematoxylin. Immunohistochemical scoring was determined using a 4-point scale: “+” = weak, “++” = moderate, “+++” = strong, and “−” = negative reactivity. LLC: large luteal cells; SLC: small luteal cells; O: nonovulated oocyte into a secondary corpus luteum. Scale bar represents 25 μm.

Figure 2

Transcription of ovarian PRLR and LHR sharply increased in pregnant-ovulating vizcachas. qPCR analysis of mRNA abundance in ovaries revealed that both PRLR (a) and LHR (b) exhibit their highest levels of transcripts at MP. Values expressed as the mean ± SEM. Significant differences were determined by a one-way ANOVA (p < 0.05, n = 5 per group). Different letters indicate significant differences. GAPDH was used as housekeeping gene. (NP) non-ovulating non-pregnant, (EP) early pregnant, (MP) midpregnant, (TP) term pregnant, and (LCT) lactating nonpregnant groups.

Figure 3

Transcription of ovarian 3ß-HSD and 20α-HSD exhibited opposite patterns during midgestation and term gestation. qPCR analysis of 3ß-HSD mRNA abundance revealed the highest levels at MP (a) while 20α-HSD transcription remained low (b). Such relation was reversed at term pregnancy, when 20α-HSD mRNA levels reached its maximum and 3ß-HSD mRNA abundance dropped low close to the recorded basal values (NP). Values expressed as the mean ± SEM. Significant differences were determined by a one-way ANOVA (p < 0.05, n = 5 per group). Different letters indicate significant differences. GAPDH was used as housekeeping gene. (NP) non-ovulating non-pregnant, (EP) early pregnant, (MP) midpregnant, (TP) term pregnant, and (LCT) lactating nonpregnant groups.

Figure 4

Strongest colocalization of PRLR and 3ß-HSD occurred in luteal cells of pregnant-ovulating vizcachas. Representative confocal photomicrographs of ovary sections of pregnant vizcachas stained for PRLR (green) and 3ß-HSD (red) using double-labeled immunofluorescence. Yellow stained represents coexpression of PRLR and 3ß-HSD. Scale score: “+” = weak, “++” = moderate, and “+++” = strong. Scale bar: 50 μm. CL: corpus luteum; FL: follicle; O: nonovulated oocyte into a secondary corpus luteum. EP, MP, and TP stand for early pregnancy, midpregnancy, and term pregnancy, respectively.

Figure 5

Strongest colocalization of PRLR and LHR occurred in luteal cells of pregnant-ovulating vizcachas. Representative confocal photomicrographs of ovary sections of pregnant vizcachas stained for PRLR (red) and 3ß-HSD (green) using double-labeled immunofluorescence. Yellow stained represents coexpression of PRLR and LHR. Scale score: “+” = weak, “++” = moderate, and “+++” = strong. Scale bar: 50 μm. CL: corpus luteum; FL: follicle; O: nonovulated oocyte into a secondary corpus luteum. EP, MP, and TP stand for early pregnancy, midpregnancy, and term pregnancy, respectively.

Figure 6

Pituitary expression of PRL increased during pregnancy. Representative photomicrographs of pituitary gland cross sections of adult non-ovulating non-pregnant (NP), early pregnant (EP), midpregnant (MP), term pregnant (TP), and lactating non-pregnant (LCT) vizcachas immunostained for PRL. Immunoreactivity is shown in black (color modification of diaminobenzidine precipitation by addition of nickel) and is highlighted by arrows. “s” indicates a capillary sinusoidal space. Scale score: “+” = weak, “++” = moderate, and “+++” = strong. Scale bar represents 50 μm. Bottom right qPCR analysis of pituitary PRL mRNA abundance revealed that TP group exhibits the highest level of transcripts. After parturition and while nurturing the litter (LCT), PRL mRNA quantity decreases although is still significantly higher than NP, EP, and MP (p < 0.0005, n = 5 per group, one-way ANOVA). Different letters indicate significant differences. GAPDH was used as housekeeping gene.