Abstract
Obesity and metabolic syndrome are the common causes of reproductive and fertility disorders in women. In particular, polycystic ovary syndrome, which is clinically characterized by hyperandrogenism, oligo/anovulation, and polycystic ovarian morphology, has been increasingly associated with metabolic disorders. However, given the broad interplay between metabolic and reproductive functions, this remains a field of intense research. In this study, we investigated the effect of monosodium l-glutamate (MSG)-induced obesity on reproductive biology of female rats. Newborn female rats were subcutaneously injected with MSG (4g/kg/day) or equiosmolar saline (CTR) each 2 days up to postnatal day (pnd) 10. On pnd 60, estrous cycle was evaluated using vaginal smears twice a day for 15 days, which showed MSG rats to be oligocyclic. Thereafter, animals were killed on estrous phase for blood and tissue collection. MSG rats had increased body mass, accumulation of retroperitoneal and visceral fat pads, and visceral adipocyte hypertrophy compared with CTR rats. MSG rats were also dyslipidemic and hyperinsulinemic but were normoglycemic and normoandrogenic. Ovarian morphology analysis showed that MSG rats had a two-fold decrease in oocyte count but a six-fold increase on ovarian follicular cysts, along with a higher number of total primordial and atretic follicles. Moreover, MSG rats had a four-fold increase in anti-Müllerian hormone immunohistochemical staining on antral follicles. Taken together, data presented here characterize MSG obesity as a unique model to study the metabolic pathways underlying reproductive disorders in the absence of overactivated hypothalamic–pituitary–gonadal axis.
Introduction
Obesity and metabolic syndrome (MetS) have escalating prevalence worldwide and have reached an epidemic proportion, affecting the world population regardless of ethnicity or Human Development Index (O’Neill & O’Driscoll 2015). The latest consensus defines MetS as the presence of three of the five metabolic dysfunctions: obesity/central obesity, insulin resistance, dyslipidemia mainly as hypertriglyceridemia, hypertension, and fasting hyperglycemia (Alberti et al. 2009). Of the abovementioned dysfunctions, obesity is of paramount importance in the development of MetS as it seems to precede other MetS features (O’Neill & O’Driscoll 2015). Concerning women’s health, obesity and MetS are of special importance as epidemiological studies have shown that obesity is more prevalent in women (Wells et al. 2012), increasing their mortality risk (O’Neill & O’Driscoll 2015) and predisposition for severe disturbances on reproductive physiology (Vryonidou et al. 2015).
The association between MetS risk factors, especially obesity and insulin resistance, and ovarian dysfunction has been reported in female mice (Wu et al. 2014b), rats (Sanchez-Garrido et al. 2015), and mini pigs (Newell-Fugate et al. 2014). Metabolic disturbances cause defects in the hypothalamic–pituitary–gonadal (HPG) axis via insulin and leptin feedback, which contributes to alter reproductive hormone levels (Watanobe 2002, Wu et al. 2014b). Obesity causes infertility in women, and even a modest 5% loss in body weight can lead to ovulation (Duval et al. 2015). Furthermore, experimental models to study the effects of obesity on ovarian microenvironment and its reproductive outcomes often present hyperandrogenism, longer and irregular estrous cycle, and polycystic ovaries (Shi et al. 2009, Newell-Fugate et al. 2014, Wu et al. 2014b). Such characteristics are hallmarks of polycystic ovary syndrome (PCOS), the main reproductive disorder associated with obesity and MetS (Rotterdam 2004). Nevertheless, the characterization of novel approaches to investigate the interplay between MetS and female reproductive disturbances remains a field of intense research.
On this matter, the monosodium l-glutamate (MSG)-induced obesity model, first described by Olney (1969), shows unique MetS-related characteristics. MSG administration to newborn rodents results in damage to the arcuate nucleus and median eminence of hypothalamus (Olney 1969, Olney & Sharpe 1969), decreasing growth hormone releasing hormone (GHRH) release, which impairs pulsatile growth hormone (GH) secretion by anterior pituitary somatosomes (Maiter et al. 1991). During adulthood, MSG rodents show stunted growth, mild obesity, increased fat accumulation, dyslipidemia, hyperleptinemia, hyperinsulinemia, and insulin resistance, which characterize it as a mild MetS model (Zhang et al. 2010, Franca et al. 2014, Miranda et al. 2014). Besides metabolic repercussions, MSG rodents show impaired reproductive parameters associated with dysfunctional HPG axis (Lamperti & Baldwin 1983, Dada & Blake 1985, Rose & Weick 1987, Franca et al. 2006). Albeit not being sterile, as initially suggested (Olney 1969), MSG female rodents exhibit reduced ovarian weight (Lamperti & Blaha 1976), longer estrous cycle (Nagasawa et al. 1974), delayed puberty (Lorden & Caudle 1986), and reduced fertility (Campos et al. 2008). However, ovarian morphology of MSG rodents has deserved little attention in the literature. Sasaki and Sano (1986) reported increased ovarian follicular atresia with no corpora lutea in MSG mice, whereas Bojanić et al. (2009) reported many atretic follicles with unspecific ovarian degeneration in MSG rats. More recently, it was shown that 75-day-old MSG mice presented an increased number of primary ovarian follicles, with no further analysis (Das & Ghosh 2011).
Notwithstanding the abovementioned features, there is scarce reports assessing the repercussions of metabolic and reproductive disturbances found in MSG animals on ovarian morphology. Therefore, in this study, we investigated the reproductive outcomes, particularly in the context of PCOS-like features, observed in MSG-induced obese female rats. To seek this purpose, morphometric, biochemical, and histological analyses were conducted in 75-day-old control and MSG female rats as an attempt to support the MSG obesity as a unique and affordable model to study how MetS may influence reproductive function in an animal without overstimulation of HPG axis.
Materials and methods
Animals
Pregnant Wistar rats were provided from the Animal Facility House of the Federal University of Maranhão, São Luís–MA, Brazil. After delivery, female newborns were used in this study. All animals were kept in a 12h light:12h darkness cycle, controlled temperature (22–24°C), with access to water and food ad libitum, in cages with up to four animals. Each group came from different mothers, with eight pups per litter. All procedures were performed in accordance with the National Council for the Control of Animal Experimentation (CONCEA) and approved by the Animal Care and Welfare Committee of the Federal University of Maranhão under ruling number 016/13.
Study procedure
All newborn animals received subcutaneous injections of MSG (4g/kg/day; Sigma-Aldrich; MSG group, n=7) or equiosmolar saline (CTR group, n=8) on alternate days, from postnatal day (pnd) 2 through 10 (Olney 1969). Animals were weaned at pnd 21. On pnd 60, the Lee Index ((body weight1/3 (g)/naso–anal length (cm))×1000) (Bernardis & Patterson 1968) was performed as a body mass index and vaginal smears were started to be collected. At pnd 73, animals were fasted for 8h and tail capillary blood was collected to assess glycemia (strip test Accu-check Active; Roche Diagnostics; 10mg/dL sensitivity and CV 2%), triglyceridemia (3mg/dL; CV 3.5%), and cholesterolemia (1.04 mg/dL CV 2%) (Labtest, Lagoa Nova, MG, Brazil). Oral glucose tolerance test (OGTT) was conducted in overnight fasted rats by administration of 4g/kg glucose and glycemia measurements 0, 15, 30, 60, and 120 min after gavage. Insulinemia was determined by radioimmunoassay with 125I (Genesis, São Paulo, SP, Brazil) as described previously (Ribeiro et al. 2015). Sensitivity was 0.1 ng/mL and intra-assay CV was 0.075%. To estimate insulin resistance, both the HOMA1-IR (Wallace et al. 2004) and the TyG (Simental-Mendia et al. 2008) indices were calculated. Rats were killed on estrous phase, between pnd 75 and pnd 80. Upon killing, the Lee Index was redone and animals were anesthetized with ketamine (70mg/kg) and xylazine (10mg/kg) (Syntec, Cotia, SP, Brazil). Blood was collected by aorta puncture, centrifuged, and serum stocked at −20°C until analysis. Ovaries, uterus, and retroperitoneal and gonadal (visceral) fat pads were weighted. Oviducts were collected bilaterally for fresh ex vivo oocyte count as described elsewhere (Bernuci et al. 2008). Ovaries and attached gonadal fat pads were fixed in 4% paraformaldehyde for 24h, and kept on 70% v/v ethanol for histological processing.
Estrous cycle assessment
Vaginal smears were taken twice a day for at least 15 consecutive days to determine the phases of the estrous cycle and cycles were deemed as regular, irregular, or prolonged estrous, as described previously (Marcondes et al. 2002, Connor et al. 2012). Briefly, regular cycles were typically 4–5 days long with characteristic transition between phases, irregular cycles presented discrepant phase transitioning and/or aberrant duration of phases, whereas cycles with two or more days of estrous were deemed prolonged estrous.
Ovarian morphology
The right ovary was embedded in paraffin and cut into 4µm sections. Six serial sections, 44µm apart each, were stained with hematoxylin–eosin (HE) and analyzed under light microscopy. Follicles containing an oocyte were classified according to Hirshfield and Midgley (1978): primordial follicles consisted of one layer, flattened cells surrounding an oocyte; primary follicles had one layer of cuboidal cells; secondary follicles had more than one layer of cuboidal granulosa cells; antral follicles had multiple layers of cuboidal granulosa cells and contained one or more antral spaces, cumulus oophorus, and theca layer may also have been evident. Healthy follicles had no deformation on any cell layers or pyknotic nuclei. Atretic follicles were characterized by shrinkage or collapsing, lacking the oocyte or presenting granulosa cells with at least two pyknotic nuclei (Brawer et al. 1986, Wright et al. 1999, Lara et al. 2000, Webber et al. 2003). Cystic follicles were considered large fluid-filled cysts with diminished granulosa cell layer (up to four layers) and thickened theca interna cell layer (Brawer et al. 1986, Lara et al. 2000, Manneras et al. 2007, Shi et al. 2009). Micrographs were taken at 40× magnification.
Immunohistochemistry
For immunohistochemical staining, 3-aminopropyltriethoxysilane (Sigma-Aldrich) slides were used. Sections were deparaffinized, blocked for endogenous peroxidase activity, washed with PBS, and immersed in 90°C citrate buffer. Slides were then blocked for non-specific binding in 5% v/v albumin–phosphate buffer. Primary antibody for anti-Müllerian hormone (AMH) (R&D Systems) was incubated at 1:400 dilutions at 4°C overnight. Slides were rinsed in PBS and incubated for 2 h at room temperature with secondary anti-goat antibody (R&D Systems) and then with streptavidin–biotin peroxidase complex (avidin biotin complex; Vector Laboratories, Burlington, ON, Canada). The peroxidase activity was developed with 3,3-diaminobenzidine tetrahydrochloride (Sigma-Aldrich) in distilled water and 0.03% v/v H2O2. After a final PBS wash, all sections were counterstained with hematoxylin. A negative control of primary antibody immunostaining is shown in Supplementary Fig. 1 (see section on supplementary data given at the end of this article). Three serial sections per animal were stained and analyzed under light microscopy. Follicles were divided into preantral (primordial, primary, and secondary) and antral. Micrographs were taken at 100× and 200× magnifications. Percentage of follicle area stained with AMH was calculated using ImageJ software 1.48v (National Institutes of Health, Bethesda, MD, USA; http://imagej.nih.gov.ij).
Measurement of adipocyte area
Gonadal fat pads were processed together with the right ovary and cut into 4µm sections. One hundred adipocytes were analyzed from each animal. All sections were at least 100µm apart to prevent multiple counts of the same adipocyte. Micrographs were taken at 200× magnification. The adipocyte area was calculated using AxionVision (AxioVs40x64 V 4.9.1.0; Carl Zeiss Microscopy GmbH). Subsequently, the frequency of adipocytes per similar size class with intervals of 500µm² was calculated.
Serum reproductive hormones
Steroidal hormones were extracted from serum before the assay. Briefly, serum was thawed on ice; diethyl ether 5v/v was added and thoroughly mixed. This mixture was centrifuged under 1000g for 3 min to separate phases. Upper layer (ether) was then stocked on a clean tube and was repeated twice for optimal extraction. A precision curve was run for a coefficient value of r²>0.96. Estradiol and testosterone assays (Roche Diagnostics GmbH) were performed on the same day. Sensitivity was 5pg/mL and 0.25ng/mL respectively. CV was 2.73% and <1% respectively. Luteinizing hormone (LH) serum levels were not extracted before the assay and were measured by enzyme-linked immunosorbent assay (ELISA) kit as per manufacturer’s instructions (Elabscience Biotechnology Co. Ltd, Wuhan, China). Sensitivity was 0.938mIU/mL and CV <10%.
Statistical analyses
The results are expressed as mean±s.e.m. on the tables and bar graphs. On the box plot graphs, the box shows the 25th and 75th percentiles and the middle line is considered the median. The whiskers indicate the upper and lower values not classified as statistical outliers. Student’s t-test was used for comparison and differences were considered significant at P<0.05.
Results
Morphometric parameters
Morphometric parameters were measured on pnd 75 for both CTR and MSG female rats (Table 1). Body weight did not differ between groups; however, MSG animals were shorter than CTR animals, which contributed to a higher body mass in MSG, as inferred from Lee Index calculation. Moreover, MSG rats presented higher white adipose tissue accumulation. Specifically, retroperitoneal and visceral fat pads were increased by almost 3- and 3.5-fold, respectively, compared with CTR rats. Interestingly, both uterus and ovaries were lighter in MSG rats, probably due to decreased GH serum levels, a hallmark of this model.
Morphometric parameters of CTR and MSG obese rats.
| CTR | MSG | |
|---|---|---|
| Morphometric parameters | ||
| Body weight (g) | 166.40±3.45 | 158.00±3.70 |
| Naso–anal length (cm) | 17.70±0.12 | 16.03±0.22** |
| Lee Index ((g1/3/cm)×1000) | 310.60±1.71 | 337.30±2.99** |
| Retroperitoneal fat pad (g/100 g BW) | 1.08±0.17 | 2.84±0.26** |
| Visceral fat pad (g/100 g BW) | 1.08±0.18 | 3.67±0.33** |
| Uterus (g/100 g BW) | 0.34±0.02 | 0.20±0.02** |
| Ovaries (mg/100 g BW) | 0.15±0.01 | 0.07±0.00** |
Results are mean±s.e.m. of n=5–7. **P<0.001 vs CTR.
Panels A and B in Fig. 1 show visceral fat attached to ovaries stained with HE for CTR and MSG respectively. The mean adipocyte area from each animal was two-fold increased in MSG animals compared with CTR animals (CTR 1551±223.70 vs MSG 3632±32.73µm2, P<0.001; Fig. 1C). Noteworthy, the histogram of adipocyte area distribution indicated that MSG animals had increased frequency of large adipocytes, shifting the curve to the right (Fig. 1D). These findings reiterate an imbalance on the metabolic aspects of MSG rats, which could impair reproductive functions on these animals.
Increased periovarian adipocyte area in MSG obese rats. (A and B) Representative sections of paraffin-embedded and hematoxylin–eosin-stained periovarian fat pads from CTR (A, n=5) and MSG (B, n=4) animals. (C) Mean adipocyte area demonstrated by box and whiskers plot illustrating median (central line), range (whiskers), and 25th and 75th percentiles (box) of at least 97 adipocytes per animal. (D) Adipocyte area distribution of CTR (open circles) and MSG (black circles) rats. ***P<0.001 compared with CTR by Student’s t-test. A full colour version of this figure is available at http://dx.doi.org/10.1530/JOE-15-0453.
Citation: Journal of Endocrinology 229, 2; 10.1530/JOE-15-0453
Serum metabolic and reproductive hormone profiles
Assessment of serum metabolic parameters, as shown in Fig. 2, revealed that MSG rats had lower glycemia (CTR 111.00±1.98 vs MSG 92.86±3.59mg/dL, P<0.01; Fig. 2A) despite higher triglyceridemia (CTR 75.53±8.07 vs MSG 106.80±8.17mg/dL, P<0.05; Fig. 2B), with no difference on cholesterolemia (CTR 53.11±3.06 vs MSG 59.30±2.04mg/dL; Fig. 2C). MSG animals were hyperinsulinemic, with roughly three times more fasting serum insulin levels than CTR animals (CTR 0.32± 0.07 vs MSG 0.89±0.2ng/mL, P<0.05; Fig. 2D). OGTT was performed on fasting animals without significant difference between groups (Fig. 2E; CTR 3451±836.60 vs MSG 5191±586.30; Fig. 2F). Insulin resistance was estimated by calculating both TyG (CTR 8.28±0.12 vs MSG 8.48±0.10; Fig. 2G) and HOMA1-IR (CTR 2.06±0.44 vs MSG 4.56±0.96, P<0.05; Fig. 2H) indices, which showed statistical difference only on the HOMA1-IR.
Serum metabolic profile of CTR and MSG animals. Fasted glycemia (A) measured by strip test. Fasted triglyceridemia (B) and cholesterolemia (C) determined by spectrophotometric assay kits. Fasted insulinemia (D) measured by radioimmunoassay. (E) Oral glucose tolerance test (OGTT) conducted in fasting CTR and MSG animals. (F) Area under the curve (AUC) of OGTT expressed no difference between groups. (G) TyG Index was calculated based on fasted glycemia and triglyceridemia: ln (fasting glycemia (mg/dL)×fasting triglyceridemia (mg/dL)/2). (H) HOMA1-IR index calculated based on fasted glycemia and insulinemia: (fasting insulinemia (μU/mL)×fasting glycemia (mmol/L)/22.5). All data expressed as box and whiskers plot: median (central line), range (whiskers), and 25th and 75th percentiles (box) with n=6–8. *P<0.05 vs CTR; ***P<0.001 vs CTR analyzed by Student’s t-test.
Citation: Journal of Endocrinology 229, 2; 10.1530/JOE-15-0453
Serum reproductive hormones of both groups are shown in Fig. 3. Surprisingly, no difference was found on serum testosterone (CTR 15.82±0.66 vs MSG 15.15±1.30ng/mL; Fig. 3A), estradiol (CTR 14.23±1.36vs MSG 15.63±2.46pg/mL; Fig. 3B), or LH (CTR 14.23±1.36vs 15.63±2.46 MSG mIU/mL; Fig. 3C) levels. In spite of the absence of reproductive hormone alterations, our data support MSG female rats suffer from MetS, which might affect other ovarian functions.
Serum reproductive hormones of CTR and MSG rats do not differ. All animals were on estrous upon blood collection. Serum testosterone (A) and estradiol (B) were extracted before the assay as described in the “Materials and methods” section. (C) Luteinizing hormone (LH) was measured without prior extraction. Data expressed as box and whiskers plot illustrating median (central line), range (whiskers), and 25th and 75th percentiles (box) with n=4–7. Student’s t-test analysis showed no difference between groups.
Citation: Journal of Endocrinology 229, 2; 10.1530/JOE-15-0453
Cyclicity, ovulation and ovarian morphology
Typically, CTR animals showed regular 4- to 5-day cycles, whereas MSG animals were oligocyclic, with less proestrus-to-estrus progression and longer metaestrus–diestrus phase (Fig. 4A). This was statistically translated by decreased regular (CTR 66.67±12.60 vs 4.76±4.76% MSG, P<0.05; Fig. 4B) and increased irregular (CTR 16.67±6.30 vs 54.76±12.58% MSG, P<0.05; Fig. 4B) cycles in MSG rats. There was no difference on prolonged estrous between groups (CTR 16.67±10.91 vs 40.48±14.14% MSG, P=0.19; Fig. 4B). Ovulation was evaluated by fresh ex vivo oocyte count. Our MSG rats showed a two-fold decrease in oocyte count compared with CTR (CTR 11.57±0.37 vs MSG 5.57±0.20, P<0.001; Fig. 4C). These results suggest that MSG rats have compromised fertility and reproductive function.
Hyperinsulinemic MSG animals exhibit irregular cycles and less ovulated oocytes. (A) Representative estrous cycles from three different CTR (top) and MSG (bottom) females taken twice a day from 60 to 75 pnd; P, proestrus; E, estrus; M, metaestrus; D, diestrus. (B) Estrous cycle classified as regular, irregular, or prolonged estrous according to the criteria exposed in the “Materials and methods” section; data presented as mean±s.e.m. (C) Mean fresh ex vivo oocyte count after ovulation on fallopian tubes from CTR and MSG rats demonstrated by box and whiskers plot illustrating median (central line), range (whiskers), and 25th and 75th percentiles (box). a P<0.05 vs regular CTR; b P<0.05 vs irregular CTR; ***P<0.001 vs CTR. All comparisons were analyzed by Student’s t-test. n=6–8 per group.
Citation: Journal of Endocrinology 229, 2; 10.1530/JOE-15-0453
Ovary sections were then analyzed under light microscopy (Fig. 5). Representative sections from CTR (Fig. 5A) and MSG (Fig. 5B) rats displayed striking differences between groups regarding follicular cyst count. Figure 5C shows increased total follicle count in MSG animals (CTR 79.00±16.44 vs MSG 136.30±7.13, P<0.05; Fig. 5C), greatly due to increased primordial follicle pool (CTR 53.50±12.31 vs MSG 91.80±9.12, P<0.05; Fig. 5D). MSG animals also had a three-fold increase in atretic follicle number when compared with CTR animals (CTR 4.25±1.70 vs MSG 12.20±2.56, P<0.05; Fig. 5E). Moreover, MSG animals had an astonishing six-fold increase on follicular cysts (CTR 0.75±0.48 vs MSG 6.20±0.37, P<0.01; Fig. 5F). Taken together, these reproductive data support PCOS-like characteristics in MSG animals.
Quantitative histological analysis of ovarian alterations in MSG obese rats. (A and B) Representative sections of paraffin-embedded and hematoxylin–eosin-stained ovaries from CTR (A) and MSG (B) rats; # in B indicates follicular cysts. (C, D, E and F) Ovaries from CTR (n=4) and MSG (n=5) rats were embedded with paraffin and cut into 4 µm sections. Every 11th section was stained with hematoxylin and eosin and analyzed for differential follicular counting according to the morphological pattern showed in each graph. Total follicles from CTR and MSG rats are demonstrated by box and whiskers plot illustrating median (central line), range (whiskers), and 25 and 75th percentiles (box). *P<0.05 vs CTR analyzed by Student’s t-test. A full colour version of this figure is available at http://dx.doi.org/10.1530/JOE-15-0453.
Citation: Journal of Endocrinology 229, 2; 10.1530/JOE-15-0453
AMH immunohistochemistry
As AMH hypersecretion by granulosa cells is involved in follicle maturation, ovarian immunohistochemistry was performed. Data in Fig. 6A, B, C and D show higher intensity of AMH staining on MSG antral follicles compared with CTR. The quantitative analysis showed no difference in the percentage of staining among preantral follicles (Fig. 6E). However, there was a four-fold increase in AMH staining on antral follicles of MSG rats (CTR 2.07±0.59 vs MSG 9.43±2.06, P<0.01; Fig. 6F). Despite this, there was no significant difference in total follicle staining (Fig. 6G). Altogether, these data are suggestive of a dysregulation on the granulosa cell layer with disrupted maturation of MSG ovarian follicles.
Immunohistochemical localization of anti-Müllerian hormone (AMH) in the ovaries of CTR and MSG obese rats. Ovarian sections from CTR (n=5) and MSG (n=4) rats were stained with AMH and analyzed three serial sections per animal as described in the “Materials and methods” section. (A, B, C and D) Representative sections of AMH staining on preantral and antral follicles are shown for CTR (A and B) and MSG (C and D) rats respectively. (E, F and G) Percentage of AMH staining on preantral (E), antral (F), and total (G) follicles area; mean percentage of staining in the ovaries from CTR and MSG rats are demonstrated by box and whiskers plot illustrating median (central line), range (whiskers), and 25 and 75th percentiles (box). **P<0.01 vs CTR by Student’s t-test. A full colour version of this figure is available at http://dx.doi.org/10.1530/JOE-15-0453.
Citation: Journal of Endocrinology 229, 2; 10.1530/JOE-15-0453
Discussion
This study strengthens the pivotal role of hyperinsulinemia and MetS-associated comorbidities in reproductive dysfunctions, resembling those present in PCOS. Subcutaneous injections of MSG to newborn female rats lead to hyperinsulinemia, although with unaltered serum testosterone, estradiol, or LH levels. MSG rats exhibited obesity, hypertriglyceridemia, and visceral adipocyte hypertrophy. Additionally, they had irregular estrous cycles, with oligo-ovulation, and increased number of ovarian follicular cysts and atretic follicles. Moreover, our MSG rats presented augmented AMH immunostaining on antral follicles and increased primordial follicle pool. Such characteristics suggest that MetS features, such as hyperinsulinemia, obesity, and adipocyte dysfunction, trigger reproductive deficits producing a PCOS-like phenotype despite the absence of hyperandrogenemia or increased LH levels. To the best of our knowledge, this report is the first to describe a MetS female model without overactivated HPG and one of the few to describe PCOS characteristics despite normoandrogenism. In tandem, our female MSG rat reveals itself to be an affordable model to study how MetS influences the reproductive system under no interference of increased serum HPG hormones.
Metabolic disturbances are highly prevalent among women diagnosed with PCOS and are considered to play an important role in the pathophysiology of this syndrome. Indeed, 50–80% of PCOS women are obese, whereas 44–70% are resistant to insulin (Dumesic et al. 2015). Obesity has been associated with more severe PCOS symptoms, whereas treatments directed to weight loss, such as exercise and anorexic drugs, significantly improve reproductive parameters. Numerous mechanisms correlating obesity and PCOS have been proposed, one of which consists in elevated insulinemia due to increased fat deposition, hypertriglyceridemia, and insulin resistance. It is important to notice that hyperinsulinemia has been proposed as a culprit for PCOS, and the concurrence of hyperinsulinemia/insulin resistance has higher prevalence in PCOS women, independent of body weight (Wu et al. 2014a, Dumesic et al. 2015).
Data presented herein show that our MSG rats developed MetS, translated by a rise in their Lee Index, accumulation of visceral and retroperitoneal fat pads, hypertriglyceridemia, and hyperinsulinemia. However, we found decreased serum glucose levels and unchanged OGTT in MSG rats. Data on insulin resistance were controversial, as TyG Index suggested that MSG rats have regular insulin sensitivity, whereas HOMA1-IR indicated the opposite. This could be explained by an exacerbated vagus nerve activity, a hallmark for MSG rodents, which causes insulin oversecretion independently from serum glucose levels and insulin resistance (Miranda et al. 2014). In fact, even 6-month male MSG rats showed lower glycemia than control (Liu et al. 2011), yet there is scarce data concerning glycemia measurements on MSG females. Therefore, despite increased HOMA1-IR index, 75-day-old MSG females are likely to have preserved insulin sensitivity, which needs to be addressed through molecular methods in future studies.
Another important metabolic finding of our MSG rats was adipocyte hypertrophy on visceral periovarian fat pads. The mean adipocyte area of MSG animals was two-fold higher than that of CTR animals, reflected by a right-sided shift in the histogram of adipocytes’ area. Adipocyte enlargement is related to hyperinsulinemia (Gao et al. 2015) and hyperleptinemia (Couillard et al. 2000). Leptin affects the reproductive physiology evoking gonadotropin-releasing hormone (GnRH) and LH secretion in rats (Watanobe 2002), while disturbing follicle maturation and ovulation (Escobar-Morreale & San Millan 2007). Of interest, hyperleptinemia has been described in MSG female rats as young as 30 days old (Franca et al. 2006), reaching a ten-fold increase in 120-day-old female rats (Moreno et al. 2006). Thus, adipocyte hypertrophy showed herein might be related to increased leptin levels, which contribute to a disruption of female reproductive function. In humans, it has been shown that PCOS women have a 25% increase in adipocyte diameter when compared with BMI-matched non-PCOS subjects (Manneras-Holm et al. 2011), advocating for a relationship between PCOS and adipocyte hypertrophy.
Assessment of reproductive hormone levels showed that testosterone, estradiol, and LH were unaltered in our MSG rats. Neonatal MSG administration causes destruction of neuronal bodies in arcuate nucleus and median eminence of hypothalamus, disrupting the hypothalamic–pituitary axis (Lamperti & Blaha 1976). However, it has been shown that axons and dendrites in passage through these nuclei are spared, maintaining a lower but preserved LH secretion (Dada & Blake 1985). Moreover, facing an insufficient GnRH release, LH levels are maintained by input from anterior hypothalamic nucleus (Lamperti & Baldwin 1983), as well as reorganization of other hypothalamic nuclei (Rose & Weick 1987). Subsequently, MSG rodents have been shown to exhibit low to normal levels of reproductive hormones (Nemeroff et al. 1981, Lamperti & Baldwin 1983, Dada & Blake 1985, Franca et al. 2006), despite their gonadal atrophy (Table 1) and possible peripheral aromatization of androgens by white adipose tissue, as shown in other animal obese models (Newell-Fugate et al. 2014).
It has been reported that LH is the main inductor of ovarian androgen production (Knudsen et al. 2010), whereas hyperinsulinemia contributes to roughly 20% of serum testosterone levels (Azziz et al. 2001, Norman et al. 2002, Creanga et al. 2008). In humans (Tosi et al. 2012) and animal MetS models (Newell-Fugate et al. 2014, Wu et al. 2014b), hyperinsulinemia is able to cause elevation of LH secretion with consequent hyperandrogenemia, as hypothalamic nuclei structures are intact. However, in our MSG female rats, hyperinsulinemia seems to be unable to increase LH secretion, given the hypothalamic damage caused by MSG injection, resulting in lower to normal testosterone production, as shown in Fig. 3. Therefore, considering the normoandrogenemia verified in our MSG rats and their dysfunctional HPG axis, reproductive disorders described herein might be directly consequent to metabolic disturbances, i.e., hyperinsulinemia, obesity, and adipocyte dysfunction.
Our MSG rats exhibited irregular estrous cycles, which reinforces previous findings (Nagasawa et al. 1974, Lamperti & Blaha 1976, Lorden & Caudle 1986, Campos et al. 2008) and supports the connection between MetS and reproductive disorders. The MSG rats had lighter ovaries when compared with CTR, explained by deficient GH pulsatile secretion. They were oligocyclic rather than acyclic, with a higher frequency of irregular cycles and two-fold less oocyte on their fallopian tubes after ovulation. Of note, one could hypothesize the oligocycles/oligo-ovulation in MSG animals to be due to increased prolactin levels; however, it is well documented that MSG rodents, especially females, exhibit unchanged prolactinemia (Clemens et al. 1978, Nemeroff et al. 1981, Franca et al. 2006). Nonetheless, we recognize this as a limitation in this report and suggest future studies to investigate the effect of MSG obesity on the prolactin feedback loop. Noteworthy, our MSG obese rats present disrupted estrous cycle and oligo-ovulation patterns similar to female obese mice (Wu et al. 2014b), rats (Manneras et al. 2007), and mini pigs (Newell-Fugate et al. 2014) with otherwise preserved HPG axis function, suggesting that the latter has a minor effect on MetS outcomes on estrous cycle and ovulation.
MetS and its highly associated reproductive disorder, PCOS, also cause anomalies on ovarian structures. For instance, PCOS women showed increased follicle count, primordial follicles, follicular cysts, and atresia (Rotterdam 2004, Shi et al. 2009, Walters et al. 2012). MetS female models, such as the genetically modified JCR:LA-cp rats, which has a malfunction on its leptin receptors, are also presented with PCO and follicle atresia (Shi et al. 2009), whereas the New Zealand obese mice showed increased primordial follicle counts and follicle atresia without PCO (Radavelli-Bagatini et al. 2011). Diet-induced obese mini pigs were also reported to have cystic ovarian follicles (Newell-Fugate et al. 2014). Our MSG rats showed increased number of primordial follicles when compared with CTR animals, an observation corroborated by previous findings that hyperinsulinemia promotes follicle maturation arrest (Franks et al. 2000). Moreover, MSG rats exhibited a robust increase in follicular cysts and atretic follicle counts, suggesting an impaired ovarian microenvironment. Atretic primary follicles were visualized only in the ovaries of MSG rats (Supplementary Fig. 2), which denote a severe impairment on follicle development. Of note, all the abovementioned models that present PCO also exhibit hyperandrogenism; therefore, the fact that our MSG female rats exhibited PCO without hyperandrogenism reinforces the role of hyperinsulinemia as an inductor of PCO per se, without the contribution of overactivated HPG axis.
Nonetheless, another important contributor to ovarian follicle development is AMH. Our MSG rats had increased AMH immunostaining on antral follicles, even though it had not affected total AMH staining when compared with CTR rats. Previous reports showed that AMH functions as an inhibitory growth factor in the ovary during early stages of folliculogenesis (Durlinger et al. 2002) and increases primordial follicle population (Kevenaar et al. 2006), which corroborates our findings of primordial follicle pool and follicular atresia in MSG rats. Intriguingly, an increase in AMH production specifically by antral follicles was also observed in PCOS women (Bhide et al. 2015). Thus, these data suggest a synergic role for hyperinsulinemia and AMH overproduction in the pathogenesis of impaired follicle development along with augmented follicular atresia and cysts.
Taken together, our reproductive data support a PCOS-like phenotype in MSG obese female rats, with the presence of some key diagnostic criteria, namely oligo-ovulation and PCO. However, one should be aware that these rats do not mimic all physiological processes seen in human PCOS and other animal models, particularly because of the hypothalamic basilar nuclei damage of MSG model with consequent stunted growth, a feature not described in PCOS women. Additionally, our MSG rats presented unchanged serum reproductive hormones, a feature found in a minority of PCOS women, but that deserves further investigation. Therefore, one should be cautious when translating data from our MSG female rats to humans.
In conclusion, our MSG obese female rats reiterate the fundamental role of hyperinsulinemia, obesity, and adipocyte dysfunction in reproductive disorders. To the best of our knowledge, there is no other rodent model presenting the characteristics described herein, which opens up the perspective of investigating in vivo how MetS affects reproductive biology without the interference of exacerbated HPG axis function.
Supplementary data
This is linked to the online version of the paper at http://dx.doi.org/10.1530/JOE-15-0453.
Declaration of interest
The authors declare that the research was conducted with no commercial or financial relationship that could be interpreted as a potential conflict of interest.
Funding
This study was funded by Fundação de Amparo à Pesquisa e ao Desenvolvimento Científico e Tecnológico do Maranhão–FAPEMA (Grant no. 00280/12 and scholarships to R S G, R O B, and J L L F) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior–CAPES (scholarship to C C V).
Acknowledgments
The authors are very grateful to Dr Deborah M Sloboda from the Department of Biochemistry and Biomedical Sciences, McMaster University, and Dr Farida Sohrabji from the Department of Neuroscience and Experimental Therapeutics, College of Medicine, Texas A&M University, for critical discussion of the data. The authors would also like to thank the superb technical support given by Tiago da Silva Teófilo from the Laboratory of Histology, Federal University of Maranhão, and Marco Antônio Sales dos Santos and Laboratório Gaspar for the support given during the hormone assays.
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