Fetal Origin Programming of the Male Reproductive System

Abstract

The Developmental Origin of Health and Disease (DOHaD) theory, in which the prenatal environment is involved in the development of diseases after birth, has been widely accepted. This theory is widely accepted, and the involvement of the prenatal environment in the development of adult diseases (lifestyle diseases) is almost certain. As an extension of the DOHaD theory, the Testicular Dysgenesis Syndrome (TDS) hypothesis, which focuses specifically on diseases of the male reproductive system, proposes that environmental changes during the embryonic period are involved in the development of a number of diseases of the male reproductive system, such as hypospadias, cryptorchidism, low sperm count, and infertility. A few experimental studies were performed; however, the results have been limited and have not addressed the pathogenic mechanism of TDS. We have conducted research using a mouse model of maternal nutritional deprivation. In this study, under/hyponutrition during fetal life impairs testosterone production in the fetal testis and causes a decrease in sperm count after growth. Further studies elucidated that this may be due to oxidative stress-induced germ cell apoptosis caused by fetal testosterone depletion. The molecular biological background to the DOHaD theory is epigenetic modification, but very few studies have focused on epigenetic modification in TDS, which shares the same background as the DOHaD phenomenon. We will further discuss the contribution of epigenomic modifications in the development of TDS.

1. Introduction

The Developmental Origin of Health and Disease (DOHaD) theory that posits the prenatal environment is involved in the development of diseases after birth is widely accepted, and the involvement of the prenatal environment in the development of adult diseases has been established [1]. Furthermore, as an extension of the DOHaD theory, the Testicular Dysgenesis Syndrome (TDS) hypothesis, which particularly focuses on male reproductive system diseases, has been proposed by Professor Skakkebeak in Denmark [2, 3]. This hypothesis proposes that environmental changes during the embryonic period are involved in the development of a series of male reproductive system diseases, such as hypospadias, cryptorchidism, testicular cancer, decreased sperm count, and infertility. The pathogenesis of TDS is assumed to be reduced male hormone (testosterone) action due to testicular damage during the embryonic period [4].

2. Fetal growth restriction (FGR) and male reproductive system disorder

Nutrition and metabolism are essential factors when discussing the prenatal environment. Maternal undernutrition is one of the important causes of FGR [5]. It has been reported that FGR is strongly associated with mild phenotype disorder of sex development/differentiation including hypospadias [6, 7] and cryptorchidism [8, 9, 10]. Furthermore, a recent, large cohort study on the relationship between birth records and fertility has been reported from Denmark [11]. The study included more than 10,000 individuals born between 1984 and 1987, consisting of 5342 women and 5342 men. Of these, approximately 10% were born small for gestational age (SGA- a birth weight below the 10th percentile). This study found that there was a 55% increased risk for infertility in men born SGA compared with men born appropriate for gestational age (AGA), not in women. The following study from Sweden also showed that men born SGA or with low birth weight had a lower chance of becoming fathers than men born AGA or with normal birth weight [12]. In addition, an association between FGR and the development of TDS, which considers multiple male reproductive system diseases as a syndrome related to the fetal environment, has been reported by a human study [13]. Together, many epidemiological data from human studies have reported the significant relationship between FGR and wide-ranging male reproductive problems.

Here are several previous reports from basic studies using experimental animals. Maternal 50% food restriction during both gestation and lactation or lactation alone significantly reduced testicular growth in offspring, and also reduced circulating levels of FSH in rats [14]. In an experiment in which pregnant ewes were fed with 50% calory intake in early and late gestation, male lambs born from nutritionally restricted mothers showed a decrease in Sertoli cells in the testis at 10 months of age. Correspondingly, an excess response of FSH in the GnRH loading test was observed [15]. A study in piglets found that maternal calorie restriction during pregnancy reduced Sertoli cells, embryonic cells, and Leydig cells in male piglets born. Apoptotic cells were also found to be more in male piglets from calorie-restricted mothers [16]. Transcriptome analysis in the testes of male pigs revealed that maternal calorie restriction altered a group of genes involved in lipid metabolism, apoptosis, and cell proliferation. In rat offspring, maternal protein restriction during pregnancy reduces the testicular and epidydimal sperm count and affects fertility in the rat offspring [17, 18, 19].

Although these experiments provide scientific support for the association between maternal nutrition and the development of male reproductive problems after birth, basic data are still scarce, and the molecular biological background is in the process of being elucidated.

3. Importance of energy metabolism in various aspects of the testis

The testis is the organ responsible for spermatogenesis and the secretion of the major male hormone testosterone. Energy metabolism in the testis has been shown to be important for differentiation and development of testis, and maintenance of testicular function.

In mice, a key event in the early stages of testis differentiation is the activation of Sex-determining region Y (SRY) in pre-Sertoli cells in the gonadal ridges. At this time, testis-specific glycogen accumulates in the pre-sertoli cells. This serves to store an energy source for morphogenesis and hormone production during testis development [20]. Sertoli cell differentiation, a central event in testis formation, requires SRY expression and subsequent SRY-Box9 (SOX9) activation. Through glucose deprivation and metabolic rescue experiments in mice genital ridge cultures, it was demonstrated that an adequate supply of glucose was the most important environment for establishing SOX9 activation in testis differentiation [21]. Additionally, during the differentiation of fetal Leydig cells in mice, a number of genes involved in metabolic pathways, such as tricarboxylic acid cycle, glycolysis, and oxidative phosphorylation, are heavily expressed [22]. Turning to germ cells primordial germ cells (PGCs), the origin of germ cells are found to have very different energy metabolism from pluripotent stem cells (PSCs). Furthermore, for the differentiation of PSCs into PGCs, oxidative phosphorylation is essential [23]. Together, the unique energy metabolic system is important for establishing and maintaining PGC characteristics.

These results suggest that proper nutrition and metabolism play a crucial role in the growth and functioning of the testes. Therefore, it has been hypothesized that intrauterine malnutrition contributes to the emergence of “testicular dysgenesis syndrome (TDS),” which is primarily brought on by unfavorable environmental factors during fetal life and is linked to a number of reproductive abnormalities, such as hypospadias, cryptorchidism, and infertility (Figure 1).

Figure 1.

Maternal caloric restriction resulted in decreased sperm counts after birth (Fujisawa et al. [24]).

4. Intrauterine under/hyponutrition leads to male reproductive dysfunction

Previous studies have shown that maternal under/hyponutrition is thought to increase the chance of developing TDS in the human. However, the underlying mechanism(s) remain largely unknown despite some experimental studies. To clarify the underlying mechanism(s), we performed experimental studies using mice. We set two groups: control females (C-females) were given regular food ad libitum throughout the course of pregnancy while calorie-restricted females (R-females) received 50% of the C-females’ mean daily intake from 6.5 dpc. Then, we evaluated male reproductive results between 17.5-days post-coitum-old male mice delivered from C-females (C-fetuses) and those from R-females (R-fetuses) and between six-week-old male mice born to C-females (C-offspring) and those born to R-females (R-offspring) (Figure 2) [24].

Figure 2.

Maternal under/hyponutrition and male reproductive diseases associated with testicular dysgenesis in fetuses.

The external genitalia of the R-fetuses were morphologically normal. However, the anogenital distance (AGD) index (AGDI) calculated by dividing the AGD by the cube root of body weight was significantly shorter in the male R-fetuses than in the male C-fetuses. This indicates reduced exposure to androgen during the fetal period. Intratesticular testosterone levels were significantly low in the R-fetuses compared with the C-fetuses, in association with significantly reduced expressions of steroidogenic genes including Star, Cyp11a1, Cyp17a1, Hsd3b1, and Hsd17b3. In contrast, the testicular histopathological findings were comparable between C-fetuses and R-fetuses. Altogether, it is inferred that intrauterine under/hyponutrition compromises fetal testosterone production primarily via the hypofunction of fetal steroidogenic cells. Further, Nr5a1 (also known as Sf-1 and Ad4bp) and Insl3 expression levels were considerably lower in R-fetuses than in C-fetuses. The master gene known as Nr5a1 has been shown to up-regulate the expression of steroidogenic genes as well as Insl3 [25]. Interestingly, in addition to controlling INSL3 expression, NR5A1 also regulates the intracellular ATP and NADPH concentrations necessary for de novo steroid biosynthesis from acetyl-CoA [26, 27] and the generation of de novo cholesterol from acetyl-CoA [25]. Thus, compromised Nr5a1 gene expression might have played an important role in the development of reduced testosterone production in the fetal testis. On the other hand, Sox9 and Amh expressions were similar between the C-fetuses and the R-fetuses despite their expressions being up-regulated by Nr5a1 [28]. We speculate that Sox9 and Amh expressions are controlled by multiple genes, in cooperation with Nr5a1 [29, 30].

Furthermore, sperm count was significantly lower in the R-offspring than in the C-offspring at 6 weeks of age while the testicular size and sperm motility were comparable between the two groups. In addition, the number of the R-offspring’s TUNEL-positive cells—which are apoptotic cells—was noticeably larger than the C-offspring’s. Moreover, the number of tubules containing TUNEL-positive cells was much higher in the R-offspring than in the C-offspring, and the percentage of TUNEL-positive cells per 100 tubules was obviously high in the R-offspring. The examined sperms must have been generated during the perinatal period, when it is expected that the intratesticular testosterone in R-fetuses is still low, taking into consideration the length of spermatogenesis and movement from the testis to the cauda epididymis [31]. Given that it has been reported that testosterone deprivation leads to germ cell apoptosis [32], a low testosterone environment during the fetal period is likely to be associated with lower sperm count induced by cell apoptosis.

Microarray analysis on the testis in offspring at 6 weeks of age revealed more than 1000 genes that showed significant variation. Next, we picked two genes that showed up-regulation and eight genes that showed down-regulation that were reportedly important for spermatogenic activity. The R-offspring showed considerably up-regulated expressions of Notch1 and Esr2 and considerably down-regulated expressions of Amhr2, Dazl, Hormad1, Nr0b1 (also known as Dax1), Gja1, Stra8, and Inha, according to RT-qPCR. Notch1 and Esr2 were shown to be up-regulated, and it has been reported that Notch1 gain-of-function in germ cells causes spermatogenic failure in mice [33] and activation of Esr2 causes spermatocyte apoptosis and spermiation failure [34]. Amhr2, Dazl, Hormad1, Nr0b1, Gja1, Stra8, and Inha were revealed to be down-regulated. According to reports, AMHR2 is expressed in Sertoli cells as well as spermatocytes, and in humans, AMHR2 mutations are frequently linked to infertility [35, 36]; spermatogenic failure is linked to DAZL polymorphisms in humans, and male DAZL knockout (KO) animals exhibit spermatogenic failure [37, 38]; Hormad1 contributes to the production of synaptonemal complexes, and male and female mice with the Hormad1 KO mutation are infertile [39]; male Nr0b1 KO mice exhibit progressive spermatogenic failure and subsequent infertility, and Nr0b1 is essential for the development of the adrenal and reproductive systems [40, 41]; sertoli cells and germ cells are connected by the testicular gap junction protein Gja1, and Gja1 knockout male mice are infertile as a result of maturation arrest [42, 43]; Stra8 is solely expressed in germ cells, and Stra8 KO mice, both male and female, are unable to commence meiosis [44]; and male mice with Inha heterozygous KO have decreased spermatogenic activity and Inha is significantly expressed in Sertoli cells [45, 46]. Altogether, maternal under/hyponutrition is likely to alter expressions of multiple genes, which could exert an accumulative deleterious effect on spermatogenesis.

Of note, by microarray analysis at 6 weeks of age, the R-offspring showed lower expressions of Gstp1, Gpx1, Prdx1, and Prdx2 [32, 47, 48, 49, 50] with anti-oxidative stress and elevated expression of Nox4 mediating oxidative stress. While anti-apoptotic Bcl2l1 was upregulated and pro-apoptotic Bax and Bid and apoptosis-related protease Casp6 were down-regulated [51, 52, 53], this may be explained as the protective responses against the occurrence of apoptosis, probably induced by enhanced oxidative stress. The findings, along with the earlier data, such as the link between low testosterone and oxidative stress-induced apoptosis activation, indicate that TDS is an element of the clinical spectrum of DOHAD and that decreased fetal testosterone production is the main underlying cause of TDS development in intrauterine under/hyponutrition.

5. Epigenomic modifications as the biological basis for the TDS hypothesis

TDS can be considered part of the clinical spectrums of DOHaD theory. This theory proposes that epigenetic changes occurring during fetal and early neonatal period determine disease risk and health [51]. In fact, a number of epigenetic changes have been reported to occur during early life induced by nutritional conditions [52]. Thus, the pathophysiological background of TDS would contain epigenomic modification. Very few studies have focused on epigenomic modifications in TDS, which shares a common background with the DOHaD phenomenon. Recently the epigenome during gametogenesis is altered by factors such as nutritional environment and aging, and can cause multiple diseases.

For example, DNA methylation in sperm due to aging increases or decreases in specific genomic regions [53, 54]. Furthermore, the histone modification H3K4me3 in sperm acts as an apologetic sensor for folate deficiency and obesity [55] and H3K9me2 in spermatozoa is reduced by protein deficiency in [56]. Together, germline-specific epigenomic regulation mechanisms very likely link to metabolic status. Namely the “metabolic-epigenomic crosstalk”, in which intracellular metabolic changes lead to epigenomic changes, may function during gametogenesis. Furthermore, non-coding RNAs (ncRNAs), which regulate gene expressions and chromatin structure has been found to involve in the epigenetic program. Recently, environmental stressors such as environmental chemicals have been shown to induce TDS-like symptoms in the next generation through ncRNA-mediated epigenetic modifications in the germline of pups [57, 58]. These findings suggest that research focusing on the importance of epigenomic modification mechanisms as a pathogenic mechanism of TDS is expected to be developed in near future.