Effects of short-term exposure to high-fat diet on histology of male and female gonads in rats

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Abstract

Obesity, which reaches an epidemic, is characterized by alterations in metabolic and hormonal profiles. Moreover, uncontrolled obesity may lead to development of diabetes type 2, which accounts for about 90% of all diabetic cases. In obesity, besides changes in metabolism, numerous co-morbidities are reported, e.g. disruptions of reproductive functions. Additionally, sex differences in development of this disease occur.

We hypothesized that short-term exposure to high-fat diet (HFD; containing 50% of total energy from fat) would alter histology of testes and ovaries, and thus contribute to reproductive disruptions in male and female rats. Adult rats were fed ad libitum with HFD for 6–7 weeks and its effects on histology of testes and ovaries (n = 4/sex and treatment group) were studied using hematoxylin-eosin staining followed by microscopic analysis and compared to control (laboratory chow fed) group.

We have found that in male rats fed with HFD there were: 1) decrease in diameter of seminiferous tubules due to smaller luminar diameter, and no change in epithelium height; 2) decrease in number of Sertoli cells; 3) no changes in number of spermatogonia and in percentage of semen in seminiferous tubules. In female rats exposed to HFD we have seen: 1) decrease in diameters of corpora lutea; 2) decrease in diameter of ovarian follicles types 7 and 8, but no changes in their number; 3) no changes in number of early primary follicles, primary follicles, and secondary follicles.

We concluded that relatively short-term exposure to HFD in rats leads to changes in histology of both testes and ovaries, thus affecting reproductive functions.

Introduction

Unhealthy diet with high concentration of fat and sugar, reduction in physical activity, and sedentary lifestyle are major factors which contribute to the development of obesity. This disease, which reaches an epidemic state, leads to alterations in metabolic and hormonal profiles, such as elevated levels of glucose, leptin, triglycerides, and cholesterol in blood. Obese patients may develop insulin resistance, and ineffective use of insulin produced by pancreas is often manifested as diabetes type 2 (diabetes mellitus - DM2) (Ramachandran, 2014). Moreover, DM2 is the most frequently occurring type of diabetes in human population (WHO, 2020) and accounts for over 90% of all diabetic cases (Smyth and Heron, 2006).

Among various secondary problems occurring in obese and DM2 patients there are cardiovascular and heart diseases (Della Vedova et al., 2016; Pandey et al., 2015; Perrone et al., 2016), systemic inflammation (Lumeng and Saltiel, 2011), and, of particular interest for the current paper, reproductive dysfunctions (Mahutte et al., 2018; Pasquali et al., 2007). In obese women, the most common manifestation of dysfunctions of reproduction is menstrual cycle irregularity (Castillo-Martinez et al., 2003; Polotsky et al., 2010). Additionally, obesity inhibits natural fecundity and prolongs the time to conception (Jensen et al., 1999; Zaadstra et al., 1993). Premature child births, miscarriages or infertility are also common consequences of this disease (Mahutte et al., 2018). In men, obesity is correlated with a reduction in fertility, and is often characterized by decrease in T level (Pitteloud et al., 2005). Obese men also have a high incidence of erectile dysfunction (Han et al., 2011; Pasquali, 2006). Disruptions of spermatogenesis and alterations in sperm parameters, such as sperm concentration, motility and morphology have been reported in obese patients (Glenn et al., 2019; Raad et al., 2017).

Several integrators mediate the link between energy intake and reproduction, and altered levels of leptin, ghrelin and glucagon-like peptide-1 are associated with obesity. Of particular interest, these hormones and their downstream signaling molecules regulate glucose and energy metabolism in peripheral organs, including the testis (Oliveira et al., 2017). For example, ghrelin, so called "hunger hormone" because it increases food intake, was shown to act as energy status sensor and at the levels of Sertoli cells to support spermatogenesis, and alterations in this hormone levels during metabolic imbalance affect male reproduction (Martins et al., 2016).

In animal models of obesity alterations in metabolic profile are similar to these reported in human (Fernandes et al., 2016). Of particular interest are studies which indicate that obesity leads to changes in histology of gonads in animals. In obese males decrease in number of Sertoli cells, reduction in number of Leydig cells (Miao et al., 2018), disruptions of seminiferous tubules (Demirci and Sahin, 2019; Yan et al., 2015), and reduction in sperm quality indices, such as concentration and motility (Crean and Senior, 2019; Demirci and Sahin, 2019; Merino et al., 2019; Nematollahi et al., 2019), were reported. In obese females lower number of primordial follicles and higher number of developing – primary and secondary follicles were found (Sohrabi et al., 2015; Wang et al., 2014). Additionally, in obese females no difference (Balasubramanian et al., 2012) or lower number of Graafian follicles (Sohrabi et al., 2015) and decrease in number of corpora lutea (Balasubramanian et al., 2012; Patel and Shah, 2018) were seen. Differences between studies may stem from variation in experimental paradigm, e.g. duration of exposure to diets, concentrations of fat and content of the diet. Moreover, to date (Fan et al., 2015; Miao et al., 2018), no comparison concerning HFD effects on gonads was done between males and females.

It is also well known that there are differences in development of obesity between men and women, with higher prevalence in women. Moreover, gender differences in glucose homeostasis and in response to oral glucose tolerance test were found. Women have lower levels of fasting glucose, but higher plasma glucose measured 2 h after glucose tolerance test (Mauvais-Jarvis, 2017). Similarly, gender differences were reported in prevalence of DM2, with higher percentage of diabetic men being reported before the age of puberty, and higher percentage of diabetic women after the menopause (Mauvais-Jarvis, 2017). This may indicate role of sex steroids in the development of this disease. Thus, there is a need to explore alterations, caused by metabolic imbalance, at the levels of gonads, which are major sources of sex steroids.

In the light of variable results regarding effects of HFD on histology of gonads and lack of studies, which emphasized comparisons of HFD effects between males and females, we conducted current studies. The purpose of this paper is to examine the effects of HFD-induced obesity on histology of gonads in male and female rats. We hypothesized that short-term exposure to HFD affects histology of gonads, and contributes to reproductive disruptions in male and female rats. To test the hypothesis at the level of testes we assessed: seminiferous tubules diameter (parameter used for the evaluation of spermatogenesis), epithelial height and luminal diameter, number of spermatogonia and Sertoli cells, and presence of sperm. In ovaries, effects of HFD on numbers of early primary follicles, primary and secondary follicles and corpora lutea, as well as diameter of follicles type 7 and type 8 (the latter are also known as Graafian or preovulatory follicles) were studied.

Section snippets

Materials and methods

Study was divided into two experiments: experiment 1 was performed on male and experiment 2 on female rats fed with HFD to induce obesity. In both experiments control animals (C) received standard laboratory chow.

Experiment 1. Induction of obesity in male rats

Forty-five male Wistar rats (200 ± 30 g) were obtained from licensed Animal Breeding Company in Brwinow, Poland. Animals were housed in cages under constant conditions of light/dark cycle 12/12 h and temperature (22 °C). Rats had ad libitum

Results

In the light of inconsistent data regarding effects of HFD on changes in histology of gonads, we conducted current study, in which the same diet (consisting of 50% calories delivered from fat) was given to both male and female rats. Such experimental design allowed us to study response to this dietary insult in both sexes and to look for possible sex-specific responses. Moreover, we were interested to find if relatively short-term exposure to HFD (6–7 weeks) would alter histology of ovaries and

Discussion

Our data is in agreement with other studies using longer exposure to high-fat diets, which induced obesity and caused reproductive dysfunctions. Similarly to current results, in other study in which rats were exposed to HFD for longer period of time it was found that HFD altered histology of testes (Demirci and Sahin, 2019). Using Johnsen scores as a measurement of quantitative description of spermatogenesis, it was revealed that obesity decreased this score compared to control rats. In

Conclusions

Our data indicate that negative effects of the diet on reproductive functions could be detected very early, only after 6–7 weeks of HFD exposure, and manifested as changes in histology of gonads. Thus, it seems that even relatively short exposure to HFD could have long-term consequences for reproductive functions in male and female rats. It is possible that alterations in testicular and ovarian morphology caused by HFD in rats are a consequence of hormonal imbalance reported in obesity. These

Authors statement

Julia Matuszewska: writing – original draft preparation, performing histological study and analysis

Kamil Ziarniak: writing-reviewing and editing, performing experiment 2

Monika Dudek: performing experiment 1

Paweł Kołodziejski: induction of obesity, performing ovariectomy

Ewa Pruszyńska-Oszmałek: induction of obesity, performing of orchidectomy

Joanna H. Śliwowska: conceptualization, provided input and assistance as needed, especially provided advice and input on experiments and on the resulting

Funding

The study was supported by National Science Center in Krakow, Poland (grant OPUS 2015/17/B/NZ4/02021 to JHS) and statutory funding 506-511-09-00 from Poznan University of Life Sciences

Declaration of Competing Interest

All authors declare no conflict of interest.

Acknowledgements

We would like to express our gratitude to Dr. Maciej Sassek for his help in performing experiments.

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