Morphology of ovaries in laron dwarf mice, with low circulating plasma levels of insulin-like growth factor-1 (IGF-1), and in bovine GH-transgenic mice, with high circulating plasma levels of IGF-1

Senescence is a physiological process related to changes in many tissues and organs, including dysfunction of the endocrine system, and among the first changes observed are a decrease in growth hormone (GH) and sex hormone levels in plasma. It is well documented that serum levels of GH decline with age in both mouse and human, and several murine models have been identified as potential models for the role of GH in aging [1‐3].

GH circulating in plasma stimulates liver secretion of somatomedin-C, also known as insulin-like growth factor -I (IGF-1), which affects the function of several organs, including the gonads. Studies in mice deficient in GH, its receptor GHR, IGF-I, or IGF-I receptor (IGF-1R) revealed that GH/IGF-I signaling is required for the normal rate of sexual development and maturation [4‐7]. GH and IGF-I act on all levels of the hypothalamic–pituitary–gonadal axis (HPG) and regulate the function of the reproductive system and mammary glands. Specifically, GH/IGF-I signaling affects (i) release of gonadotropin-releasing hormone (GnRH) and gonadotropins, (ii) expression of receptors for gonadotropins in ovarian granulosa cells and in Leydig cells in testes, and (iii) development of mammary glands. GH can also temporarily mimic the function of gonadotropins, as previously reported [4, 8, 9].

The GH/IGF-I axis is crucial for development and maturation of ovarian follicles. GH also controls early phases of follicle development and stimulates formation of secondary follicles and the development of granulosa and theca cells. GH initiates the growth of primordial follicles and supports the development of primary and secondary follicles. Silva et al. [9] suggest that GH is the survival factor for primary follicles and regulates differentiation of granulosa cells. GH also augments IGF-1 secretion by granulosa and theca cells, steroidogenesis in granulosa cells, and development and maturation of oocytes [9]. It has also been shown that GH is responsible for maintaining the sensitivity of granulosa cells to gonadotropins. On the other hand, gonad-derived sex steroids enhance the release of GH, and synergy between sex steroids and GH promotes development and maturation of the follicles [4, 6, 9‐11].

It is well known that IGF-I is crucial for fertility and IGF-I knockout mice have been reported to be sterile [12]. There are two sources of IGF-I that affect the function of ovaries i) IGF-1 released from liver into the circulating blood plasma in response to stimulation by GH and ii) IGF-1 locally produced in tissues, including the gonads. The IGF-I receptor (IGF-IR) has been reported to be present on granulosa cells in most mammals and is also expressed on rat oocytes [13]. In ovaries, the IGF-I/IGF-1R axis i) activates development of preantral follicles, ii) maintains the larger pool of small antral follicles, iii) stimulates the development of follicles, iv) selects dominant follicles, and v) stimulates steroidogenesis in theca cells and secretion of progesterone by large antral follicles [9, 14]. Danilovich et al. [15] demonstrated that bovine growth hormone (bGH)-expressing transgenic mice display a decrease in athreticpreantral follicles, which suggests that GH or IGF-I prevents apoptosis of granulosa cells.

The bioavailability of IGFs is regulated by a family of intrafollicular-expressed IGF binding proteins (IGFBPs) [13, 14, 16]. Wandji et al. [16] analyzed the expression of IGFBPs in different stages of development and atresia of ovarian follicles. The high levels of these proteins has been observed during early development of follicles, and the decrease in IGFBP level leads to an increase in IGF-1 bioavailability, which stimulates proliferation of granulosa cells and steroidogenesis [13, 14].

The importance of GH/IGF-I signaling in reproduction has been investigated in different experimental animals. As mentioned above, while IGF-I knockout (IGF-1-KO) mice are sterile, GH receptor-knockout (GHR-KO) Laron dwarf mice are fertile [4, 5, 12]. Laron syndrome is an inherited, recessive disorder related to GH resistance and is characterized by high plasma GH levels and severely reduced levels of plasma-circulating IGF-I. An animal model of human Laron syndrome [5] has been created by targeted disruption of the growth hormone receptor binding protein (GHR/BP) gene, which significantly impairs GH-mediated release of IGF-I from liver. Female Laron dwarf mice were reported to have delayed sexual maturation, which is evident by the advanced maternal age at first conception. While the luteinizing hormone (LH) response to stimulation by gonadotropin-releasing hormone (GnRH) and secretion of follicle stimulating hormone (FSH) secretion are reduced in these mice, the prolactin (PRL) level is increased. At the morphological level, a reduction in the numbers of preovulatory follicles and corpora lutea has been found [5, 10].

The influence of GH on the female reproductive system has also been studied in transgenic mice that overexpress bovine (b) bGH (bGHTg mice), which represent an opposite endocrine endocrine phenotype than the long-living murine mutants with reduced activity of the GH/IGF-1 axis (e.g., Laron dwarf mice). Increases in GH/IGF-1 signaling lead to increases in body mass, organomegaly, and reduction in adipose tissue. The lifespan of bGH mice is reduced[17‐20], puberty accelerated, the ovulation rate increased, and yet fertility is reduced proportional to the increase in plasma GH levels [21, 22].

Although, there are several reports about the physiological effects of GH/IGF-1 signaling on the murine reproductive system, including in Laron dwarf and bGHTg mice, studies on ovarian morphology have not been performed. Thus, the aim of this study was to compare ovarian morphology between 2-year-old Laron dwarf mice and 2-year-old WT littermates, as well as between 0.5-year-old bGHTg animals and similarly aged WT littermates. The age of Laron dwarf and bGHTg animals employed in this study corresponded to approximately the midpoint of their life span.

In our study we compared the morphology of the ovaries isolated from 2-year-old Laron mice (with low circulating plasma levels of IGF-1) and 0.5-year-old bGHTg mice (with high circulating plasma levels of IGF-1) with their normal age-matched littermates. The ages of Laron dwarf mutants employed in our studies were selected on the basis of their overall survival (_~4 years for Laron dwarf mice and _~1 year for bGHTg mice). Thus, Laron dwarf and bGHTg animals were investigated at approximately the midpoints of their respective lifespans.

Ovarian morphology of 2-year-old Laron dwarf mice exhibited significant differences compared with 2-year-old WT mice, which had all the signs of senescence. While ovaries in Laron dwarf mice were smaller, they showed a normal structure, including cuboidal epithelium on the surface and all types of ovarian follicles (primary, preantral, antral and Graffian follicles) in the cortex. However, we also observed some degenerative follicles and macrophages, which is unsurprising given the advanced age of these mice. The interstitial tissue was less pronounced compared with 2-year-old WT mice, and blood vessels and arteries were found in the central region of the medulla. Overall, the ovaries of Laron dwarf mice had a morphological structure similar to the structure of normal wild type mice at reproductive age [24, 25], which suggests no significant ovarian failure in these mice. In support of this conclusion, we observed that some 2-year-old Laron dwarf mice can became pregnant and deliver life off spring (unpublished data). The lower biological age of ovaries in Laron dwarf mice and their continued fertility are, likely due to delayed aging [26].

While we investigated ovaries from 2-year-old Laron dwarf mice, the ovaries from these animals have also been studied extensively at younger ages. For example, Slot et al. [6] reported that ovaries from 9-week-old Laron dwarf mice contain more primordial follicles than WT mice. Bachelot et al. [4] studied reproductive system morphology in 10-week-old Laron dwarf mice and found that, despite normal structure, the number of follicles in ovaries from these animals was reduced. Furthermore, the ovulation rate was reduced and was not increased after gonadotropin stimulation, which indicates an ovarian defect rather than deficiency in gonadotropins. The intraovarian expression of IGF-I mRNA was similar to that found in WT mice. Interestingly, 18-month-old mice were able to reproduce, which suggests prolonged ovarian function [4]. In other reports [5, 11], Laron dwarf females were found to be fertile, but the estrous cycle was irregular. The number of pre-ovulatory follicles and corpora lutea and the ovulation and implantation rates were also reported to be reduced [5, 11].

The origin of oocytes in ovaries at advanced ages is still somehow controversial. It has been demonstrated that ovaries even at advanced age contain a population of primitive embryonic-like stem cells that can potentially give rise to oocytes [27‐32]. These cells have been assigned different names in the literature and could be related as postulated by Bhartiya [28] and Virant-Klun [29] to a population of so-called very small embryonic-like stem cells (VSELs). In support of this hypothesis, our recent studies demonstrated that Laron dwarf mice have increased numbers of these cells in BM [33, 34]. Thus, it is important to see whether the number of VSELs is increased in the ovaries of Laron dwarf mice.

The changes that we observed in ovaries of 2-year-old WT littermates, such as a blurred border line between cortex and medulla, large degenerative antral follicles developing into cysts, small degenerative follicles admixed with interstitial tissue, an increase in interstitial tissue, hypertrophic corpora lutea, numerous inflammatory cells and macrophages, and, most important, a lack of ovarian follicles that are observed at reproductive age, were similar to those described in the literature [25, 35]. Laszczyńska et al. [36] observed similar changes in the ovaries of postmenopausal women.

In contrast to Laron dwarf mice, in bGHTG mice (with high circulating plasma IGF-1 levels) the ovarian and follicle dimensions were larger. This could be explained as a response to bGH, which leads to hypertrophy of several organs, including bones, skeletal muscle, heart, liver, and spleen [19, 20, 37]. Danilovich et al. [15] reported that high level of GH protects granulosa cells from apoptosis and reduces follicular atresia. Cecim et al. [38, 39] found that bGHTg mice show accelerated prepubertal somatic growth and sexual maturation, but that the mating and pregnancy rates are reduced. Similar changes in fertility were observed in normal mice exposed to a prolonged series of bGH injections [38, 39]. The suppression of female fertility in bGHTg mice is proportional to plasma GH level [21, 22], and the elevated bGH in plasma could therefore explain the reduced fertility due to luteal failure and reduced progesterone levels during early pregnancy. Since the injection of progesterone enhanced the rate of pregnancy in bGHTg mice, the observed luteal failure is probably caused by inadequate prolactin (PRL) secretion, as injections of PRL significantly increased pregnancy rates in transgenic female mice [38, 39].

One has also take into consideration that despite high level of IGF-1 in bGHTg mice, IGF-1 may not be unavailable to the ovaries due to the high circualting IGFBP1 level that has a high binding affinity for IGF-1 and could sequester it – thus reducing bio-availability of IGF-1 to various tissues [40]. This could explain the normal appearing ovarian morphology despite high levels of IGF-1 in bGHGTg mice at age of 6 month.

Overall, our observations indicate that decreased fertility in transgenic bGH mice is not caused by morphological abnormalities. We also did not observe malignant transformation of ovarian tissue; however, this could be explained by the relatively small group of mice involved in this study [41]. We expect to see more changes in bGHT mice at the age of approximately 1 year.

Morphological analysis revealed a lower biological age of ovaries isolated from 2-year-old Laron dwarf mice compared with ovaries from normal WT littermates at the same age. At the same time, no morphological features of accelerated aging were found in 0.5-year-old bGHTg mice.