Many lines of evidence in humans and experimental models indicate that changes in the secretion or action of a single hormone are sufficient to affect the integrity of the hypothalamic-pituitary-gonadal axis and thus leading to infertility (22
). We have shown previously that TG hCGβ+ female mice, as a consequence of elevated levels of bioactive hCG, exhibit increased levels of testosterone and progesterone and develop hyperprolactinemia due to pituitary lactotrope adenomas in adult life (23
). These females are overweight, infertile, and anovulatory and have profound alterations in the reproductive endocrine axis (23
). In the present study, we found that hyperprolactinemia was essential for the phenotypic defects of the hCGβ+ females because most of them were reversed by treatment with dopamine agonists with proven efficacy in hyperprolactinemia (16
Prolactin-secreting adenomas are the most common type of pituitary tumors accounting for 30–40% of hyperprolactinemic infertility in women of reproductive age (13
). Several authors have reported the normalization of prolactin secretion and shrinkage or disappearance of macro- or microprolactinomas in patients treated with bromocriptine or cabergoline, with recovery of fertility (17
). In our study, long-term bromocriptine treatment between 2 and 6 months of age succeeded in the control of obesity, pituitary growth and disturbances in the hormone profile of hCGβ+ females (fertility was not tested). Interestingly, the other dopamine agonist cabergoline administered to 5-wk-old hCGβ+ females only for 1 wk, corrected the hyperprolactinemia of the hCGβ+ females, even in the long term, as measured at 2 and 6 months of age. We found concomitant reversal of pituitary overgrowth, normalization of gonadal function, and recovery of fertility of the treated hCGβ+ females. In this regard, previous studies have emphasized the efficiency of cabergoline in normalization of prolactin levels and improvement of amenorrhea or anovulation in humans, with better results than with bromocriptine (16
). The pharmacological approach used herein provided evidence that many of the phenotypic characteristics of the hCGβ+ females were normalized by an early short-term treatment with cabergoline, thus confirming the effectiveness of this drug in the control of hyperprolactinemia. The persistent (at least 5 months) effect of the short-term (1 wk) treatment was an unexpected finding, which depended on the timing because the same treatment administered at 12 wk (3 months) of age failed to rescue the hCGβ+ phenotype. It appeared that the chronically elevated hCG secretion from early stages of sexual maturation induced persistent alterations on the pituitary-gonadal axis that could not be reversed by a short treatment with cabergoline once the dysfunctional phenotype was established in adulthood.
As also shown in the present study, the elevated levels of hCG produced several reproductive alterations, including suppression of pregnancy, anovulation, and estrous cycle defects. The occurrence of constant diestrus has been observed also in other experimental animals with elevated LH, prolactin, or progesterone levels (20
). Because hyperprolactinemia is commonly associated with anovulation both in rodents and humans, the inhibition of prolactin hypersecretion by cabergoline was a logical approach to revert infertility in the hCGβ+ mice. Cabergoline has demonstrated to be efficient and well tolerated, and no deleterious effects on mother or fetus have been observed in treatments of infertility in humans (13
). Effectively, treatment of 5-wk-old female mice with cabergoline, even for a short duration, restored the normal cyclicity and pregnancy success with normal timing of parturition. In contrast, the short-term cabergoline treatment of 3-month-old hCGβ+ females failed to rescue their reproductive function. This strongly suggests that the functional alterations of the hCGβ+ ovary at an early age are critical for the reproductive disturbances in adulthood.
In rodents, prolactin is an essential luteotrophic agent by maintaining the corpus luteum function and progesterone production during early pregnancy or pseudopregnancy (40
). The effect of prolactin in this process involves the stimulation of increased progesterone synthesis in response to pituitary LH by the up-regulation of Lhcgr
expression in luteal cells and by the inhibition of the expression of Akr1c18
, encoding the 20α-hydroxysteroid dehydrogenase enzyme that converts progesterone into a biologically inactive 20α-dihydroprogesterone (41
). It has been shown that the administration of hCG to PRLR−/−
mice stimulates their Lhcgr
expression but is unable to restore fertility due to persistently high expression of Akr1c18
, preventing thus the maintenance of sufficient progesterone levels to allow embryo implantation (44
). Our results showed that 2-month-old hCGβ+ females exhibited premature luteinization of the ovarian follicles and interstitial cells and the occurrence of hemorrhagic cysts. The abnormal ovarian structure of the 2-month-old hCGβ+ females was accompanied by elevated concentrations of prolactin, which apparently induced down-regulation of ovarian Akr1c18
mRNA expression (43
). Consequently, although the Lhcgr
expression was unchanged, the high hCG concentration was likely to induce the steroidogenic pathway, which, together with the reduced expression of Akr1c18
, would explain the elevated levels of circulating progesterone of these mice.
Our results showed that the pituitary-gonadal function appeared disturbed already in 2-month-old hCGβ+ females, and these alterations became more intense throughout life. The persistent stimulus of prolactin and hCG in 6-month-old hCGβ+ females provoked massive ovarian luteinization, with the entrapment of oocytes within luteinized follicles as a clear evidence of ovulation failure. These morphological changes were accompanied by a significant increase of ovarian Lhcgr
in concert with reduced Akr1c18
expression, which resulted in elevated levels of circulating progesterone. It is interesting that after a short-term cabergoline treatment at 5 wk of age, all alterations in the ovarian function of the hCGβ+ mice were prevented, in parallel with the reduction of progesterone and testosterone synthesis. These findings remark the importance of prolactin in triggering the ovarian defects in this model. There are several studies showing that dopamine agonist therapy reduces the incidence of ovarian hyperstimulation syndrome in women at risk (45
) and in hyperprolactinemic women with polycystic ovary syndrome undergoing assisted reproduction (48
). This effect seems to be due to a deregulation of the dopaminergic tone and dopamine D2
receptor signaling that affects the vascular permeability of the ovary (49
The influence of estradiol on prolactin secretion, lactotrope proliferation, and formation of prolactinomas is well known (51
). In hCGβ+ females, the development of prolactinomas is dependent on the ovarian function because ovariectomy prevents the hyperprolactinemia and pituitary adenoma formation, in the face of persistently elevated hCG production (23
). Conspicuously, the estradiol levels in the hCGβ+ mice are elevated only during a short period peripubertally, and thereafter they are indistinguishable from WT levels in later life (23
). The molecular mechanisms by which the dopamine agonists act on the pituitary gland have long been recognized (11
). Nevertheless, the mechanism to explain that a short treatment with cabergoline may persistently suppress the prolactin production and pituitary expansion, even several months after treatment deserves a special consideration.
In our previous report, we found that the elevated progesterone levels promote the growth of these estrogen-dependent tumors in hCGβ+ mice through activation of the tumorigenic cyclin D1/cyclin-dependent kinase 4/retinoblastoma protein/E2F1 signaling cascade (27
). We showed here that the gene expression of proliferating cell nuclear antigen (Pcna
), cyclin D1 (Ccnd1
, and the cell cycle regulator Cdkn2b
were not activated in 2-month-old hCGβ+ pituitaries, suggesting a nonproliferative processes involved in the increased pituitary size at this age. In contrast, these regulator factors were suppressed in cabergoline-treated 6-month-old hCGβ+ females and thus correlated with the blockade of pituitary expansion. In this regard, our results show that the cabergoline treatment was able to abolish the main proliferative stimulus responsible for the pituitary growth and tumor development. Based on our findings, we suggest that once the prolactin secretion was initially controlled by cabergoline, the massive luteinization of the ovary and the progesterone production were prevented. In this regard, lactotrope proliferation would be suppressed both by a direct action on the pituitary by increasing the dopaminergic tone and also by an indirect effect by reducing the progesterone-induced tumorigenic signaling pathways (27
Different studies carried out in patients with prolactinomas have shown controversies about the influence of prolactin and dopamine agonist therapies on the body weight (55
). In female rats, a direct relationship between prolactin and increased food intake and body weight was demonstrated, indicating that elevated prolactin regulates the energy balance, an effect that can be suppressed by bromocriptine (59
). A reduction in body weight gain was also shown in old PRLR−/−
female mice (61
). Our results showed that cabergoline treatment reduced the increased body weight of 2-month-old hCGβ+ females. In addition, long-term treatment with bromocriptine was efficient in preventing obesity of 6-month-old hCGβ+ females.
In summary, we have demonstrated that the primary cause of infertility in the hCGβ+ mice is the elevated level of prolactin. Hyperandrogenism, hyperprogesteronemia, acyclicity, and anovulation are all conditions triggered by prolactin deregulation that could be reversed by dopamine agonist treatment in the presence of persistently high hCG levels. We demonstrated that long-term bromocriptine treatment reversed the hyperprolactinemia of the hCGβ+ females. Cabergoline administration for a short time at the beginning of the reproductive age proved effective as a preventive treatment for hyperprolactinemia-associated reproductive dysfunctions in these mice. It will be interesting to ascertain whether such a situation can also occur in certain reproductive pathologies in humans. In this respect, the hCG hypersecreting mouse model contributes to a better understanding of the interplay of LH and prolactin in the regulation of ovarian function. Even though the role of prolactin in the ovarian function has species-specific features, recent data suggest a possible contribution of prolactin for the ovarian function and the initializing of human pregnancy (62
). It is possible that subtle changes in the secretion of prolactin are sufficient to affect the reproductive function also in humans.