Levels of the Retinoic Acid Synthesizing Enzyme ALDH1A2 are Lower in Testicular Tissue from Men with Infertility

Abstract

Objective

To determine if decreased testicular levels of enzymes necessary for retinoic acid biosynthesis were associated with male infertility, as retinoic acid is known to be necessary for spermatogenesis.

Design

Observational analysis of testicular tissue samples, sperm indices and serum hormone concentrations.

Patients

32 infertile men and 11 control men.

Setting

Two infertility centers in Chile.

Intervention

Measurement of the three enzymes necessary for retinoic acid biosynthesis, aldehyde dehydrogenase (ALDH) 1A1, 1A2 and 1A3, in testicular tissue by a novel LC/MS/MS peptide assay.

Main Outcome Measures

ALDH isozyme levels were compared by type of infertility and correlated with testicular germ cell numbers, sperm parameters, serum and intratesticular hormone concentrations.

Results

Men with infertility had significantly reduced levels of ALDH1A2, but not ALDH1A1 or ALDH1A3 in their testicular tissue compared to men with normal spermatogenesis. ALDH1A2 protein levels were strongly correlated with the number of germ cells on testicular biopsy.

Conclusions

These findings suggest that ALDH1A2 is the enzyme involved in retinoic acid biosynthesis in human germ cells. Further study of the relationship between intratesticular ALDH1A2 and male infertility is warranted to determine if men with infertility have a reduced ability to synthesize retinoic acid within their germ cells that could impair spermatogenesis.

Introduction

Infertility attributable to the male partner accounts for 30–40% of all cases of infertility (1). The most common form of male infertility involves some type of impairment in spermatogenesis. Unfortunately, over 75% of men with infertility from impaired spermatogenesis do not have a medically treatable cause, such as a gonadotropin deficiency (2). In such men, surgical sperm extraction from the testes coupled with in vitro fertilization or intra-cytoplasmic sperm injection offers some hope of fertility (3). However, these procedures are invasive, expensive, unsuccessful in some cases, and don’t address the underlying cause of infertility. Therefore, new insights into the etiology of male infertility are needed before more effective treatments can be developed.

The essential role of vitamin A (retinol) in spermatogenesis has been long appreciated, as vitamin A deficiency induces sterility secondary to a cessation of spermatogenesis (4). In vitamin A deficient rodents, the conversion of undifferentiated to differentiated spermatogonia is arrested. Spermatogenesis can be re-initiated in vitamin A deficient animals by the administration of either vitamin A (5) or retinoic acid (6), its active metabolite. Vitamin A is converted to retinoic acid in the germ and Sertoli cells via the activity of retinol and retinal dehydrogenases (7, 8). Retinoic acid then binds retinoic acid receptors to regulate gene transcription (9). In the testes, two retinoic acid receptors, α and γ, are present in Sertoli cells and developing germ cells (10–12), and deletion of either of these receptors in mice results in male infertility (13, 14). In addition, spermatogenesis is completely and reversibly suppressed by pharmacological inhibition of retinoic acid biosynthesis (15) or by the administration of retinoic acid receptor antagonists (16, 17), demonstrating that intratesticular retinoic acid biosynthesis and function are necessary for spermatogenesis.

Given the crucial role of retinoic acid in spermatogenesis, it seems possible that some men with “idiopathic” infertility may have intratesticular concentrations of retinoic acid below those necessary to initiate or maintain spermatogenesis. However, whether deficiencies of intratesticular retinoic acid contribute to infertility in men is unknown. In theory, poor dietary intake of vitamin A could lead to male infertility, but a nutritional cause of infertility seems highly unlikely for most men. More feasibly, a low intratesticular concentration of retinoic acid could occur in an infertile man due to impaired retinoic acid biosynthesis from vitamin A or increased catabolism of retinoic acid to inactive metabolites. Recently, our group observed that concentrations of intratesticular retinoic acid were significantly lower in men with sub-normal sperm quality as compared to men with normal sperm quality (18). However, the direct measurement of testicular retinoic acid concentrations is challenging given its low concentration in tissue, the corresponding need for large samples of testicular tissue, and the specialized collection techniques required given the photo-sensitivity of retinoic acid, which rapidly degrades with light exposure.

An alternative to the direct measurement of intratesticular retinoic acid is to measure the relative levels of the enzymes involved in retinoic acid biosynthesis. These proteins are more stable in frozen tissue than retinoic acid and provide insight into the capacity of the tissue to synthesize retinoic acid. The three isozymes of aldehyde dehydrogenase (ALDH) that are responsible for synthesizing retinoic acid are ALDH1A1, ALDH1A2 and ALDH1A3 (7, 19). These ALDH enzymes catalyze the final step of retinoic acid biosynthesis, from retinaldehyde to retinoic acid. Retinoic acid then induces the expression of Stra8, whose function is unknown, but is essential for the differentiation of A spermatogonia to A1 spermatogonia, as Stra8 knockout mice are infertile, exhibiting only type A spermatogonia on testes biopsies (20). In particular, ALDH1A2, which was first cloned and characterized in testicular extracts in 1996 (21), appears to be specifically expressed in the testes with much lower levels of expression in other tissues (19, 22). Indeed, work in mice suggests that ALDH1A2 localizes to germ cells within the seminiferous tubules, while ALDH1A1 and ALDH1A3 localize to Leydig and Sertoli cells (23).

Therefore, to better understand the synthesis of retinoic acid in the human testes in fertile and infertile men, we used a novel liquid chromatography/tandem mass spectrometric peptide assay to measure the level of ALDH1A1, 1A2 and 1A3 in testicular tissue from a well characterized cohort of men with infertility and controls (24). This cohort had frozen testicular tissue available as well as extensive information regarding testicular germ cell numbers and serum and intratesticular hormones concentrations, which allowed for correlation between these measurements and the levels of ALDH isozymes in the testes. As ALDH1A2 seemed most likely to be responsible for retinoic acid biosynthesis in developing germ cells, we hypothesized that testicular tissue from infertile men would have reduced levels of ALDH1A2 compared to testicular tissue from men with normal spermatogenesis.

Methods

Subjects

A subset of 43 men, from the original cohort, in whom testicular tissue was still available for measurement of ALDH1A1, 1A2 and 1A3 were included in this study. Inclusion criteria included azoospermia or oligozoospermia and an indication for testicular biopsy for sperm retrieval or diagnosis. The original workup and biopsies were performed at the Institute of Maternal and Child Research and at the Jose Joaquin Aguirre Hospital of the University of Chile during a 12-year period between 1999 and 2011. All infertile subjects underwent a complete physical examination, karyotype and an evaluation for Y-chromosome microdeletions. Subjects with abnormal karyotypes, hypogonadotropic hypogonadism, chronic disease or undergoing hormonal treatments were not included. The study was approved by the Institutional Review Boards of both hospitals in Chile where the biopsies were obtained and samples were anonymized prior to analysis of ALDH protein levels at the University of Washington, which also approved of this study.

Measurements

The seminal fluid analyses and hormone measurements were performed as described in the original manuscript (24). Histological analysis of testicular biopsy material was performed on a small piece of tissue fixed in Bouin’s solution and stained with hematoxylin-eosin, PAS, Masson’s (trichrome) or orcein staining. Each biopsy was classified into a definite histopathological type (normal, Sertoli-cell only, maturation arrest, or hypospermatogenesis) as described in the original report (24). Quantitation of germ cell numbers was performed on 20 seminiferous tubules, allowing for enumeration of the number of a given type of germ cell per tubule cross-section (25, 26).

The relative expression of ALDH1A1, 1A2 and 1A3 in testicular tissue was measured by LC-MS/MS using an ABSciex 5500 qTrap Q-LIT mass spectrometer, (Framingham, MA) coupled to an Agilent 1290 UHPLC (Santa Clara, CA). To identify unique peptides for each ALDH isoform, ALDH1A1, ALDH1A2 and ALDH1A3 were trypsin digested in silico using Skyline software (Seattle, WA, USA). All of the resulting peptides were screened against peptides from the human proteome downloaded from uniprot.org using Skyline and peptides used for relative quantification of each ALDH1a protein were chosen based on their predicted selectivity and solubility. Selectivity was determined using the SRM Collider to decrease redundancy with peptides that share similar fragmentation and retention characteristics. Peptides with reported sites of post translational modifications or mutations were excluded (27). For each peptide, 2 fragments were required for quantification.

For ALDH1A protein analysis, testicular tissues were homogenized using a Precellys 24 bead homogenizer and kept cold with liquid nitrogen using an advanced temperature controller Cryolys (Bertin Technologies, Villeurbanne France). Each tissue sample was homogenized in 500 µL 0.1% NaCl using 2 mL ceramic bead tubes (Omni International, Kennesaw, GA.) and centrifuged at 10,000g for 60 minutes to pellet cellular membranes, nuclei and organelles. The supernatant was collected and used for ALDH1a analysis. The samples of testicular supernatant were prepared for LC-MS/MS analysis as follows: 4 µL dithiothreitol (100 mM) and 10 µL of ammonium bicarbonate buffer (100 mM, pH 7.8) were added to 20 µL of sample at total protein concentration of 2 mg/ mL followed by 10 µL of 0.4% Rapigest (Waters). The samples were incubated at 56°C for 30 minutes, cool ed to room temperature and 4 µL of iodoacetamide (200 mM) was added. The samples were then incubated at room temperature in the dark for 20 minutes trypsin was added at a 1:25 trypsin:protein ratio and the proteins digested at 37 °C for 24 hours. The digestion was q uenched by addition of 30 µL chilled 50:50 acetonitrile:H₂O containing 0.1% formic acid and a peptide labeled at the N terminal Lysine or Arginine with C¹³ and N¹⁵ for ALDH1A1, ALDH1A2 and ALDH1A3 as internal standards. Samples were centrifuged at 3,000 g for 25 minutes at 4°C prior to analysis. The peptides were separated using a Supelco express C18 column (150 X 2.1 mm) with a 5 uM particle size at 40°C using a 16 minute gradient between H₂O + 0.1% FA and ACN + 0.1% FA. The peptides were quantified using peak area of the main MS/MS fragment of the analyte peptide and the ratio between the target peptide and the heavy labeled internal standard was calculated. The mean expression level of each ALDH1a enzyme in the control samples was calculated and the fold difference in the ALDH1A expression was determined from the peak area ratios of the individuals in each group. The relative expression levels in each group are reported as fold difference in comparison to the group of men with normal spermatogenesis

Statistical Analysis

Due to non-normal distributions, the relative protein levels of ALDH1A1, 1A2 and 1A3 in the testicular tissue are presented as medians and interquartile ranges. For comparison of ALDH isozymes levels between men with proven fertility and those with Sertoli-cell only, maturation arrest and hypospermatogenesis, a Kruskal–Wallis (non-parametric) ANOVA with a Wilcoxon rank-sum post-hoc test was used. Correlations between ALDH isozymes level and germ cell numbers were performed using Spearman’s technique. Linear regression was used to examine the relationship between ALDH levels, germ cell number, intratesticular and serum hormone levels, serum gonadotropins and age for hypothesis generation. Statistical analyses were performed using STATA (College Park, TX, USA). For all comparisons, an alpha of 0.05 was considered significant.

Results

Subject Characteristics and Hormone Concentrations

There was sufficient testicular tissue for measurement of ALDH isozymes levels on 43 men of the original cohort who also had undergone quantitation of testicular germ cell numbers as well as serum and intratesticular hormones measurements (Table 1). Of these 43 men, there were 11 normal men, nine of whom had obstructive azoospermia as their indication for testicular biopsy, 20 men with Sertoli-cell only syndrome, 8 men with maturation arrest and 4 men with hypospermatogenesis. There were no significant differences between the groups in terms of age, but a significant difference in ejaculate volume was noted, with the normal men being significantly reduced compared to the other groups, consistent with the fact that most of these men had obstruction as the cause of their infertility. The germ cell populations between the groups were markedly different, with the Sertoli-cell only group being significantly different than all of the other groups due to the absence of germ cells. The maturation arrest group had significantly larger numbers of spermatogonia and spermatocytes compared with the other groups (Table 1). The serum and intratesticular hormonal data is presented in Table 2. The normal group had a significantly greater serum testosterone, while the Sertoli-cell only group had significantly greater serum LH and FSH. There were no other significant differences between the groups in terms of serum or intratesticular hormones.

Table 1.

Demographics, ejaculated sperm characteristics, and testicular germ cell populations by testicular histology type of the 43 men included in the analysis. All values are expressed as median (25^th, 75^th percentiles).

	Testicular Histology
	Normal† (n=11)	Sertoli-Cell Only (n=20)	Maturation Arrest (n=8)	Hypospermatogenesis (n=4)	p-value
Age (years)	30 (29,37)	35 (32,39)	31 (29,33)	35 (31,37)	.32
Sperm Characteristics
Semen Volume (ml)	1.8 (0.5,2.7)**	3 (2.5, 3.0)	4 (3.8,4.8)	2.5 (0.7,6)	.01
Sperm Concentration (106/ml ejaculate)	0 (0,0.2)	0 (0,0)	0 (0,0.01)	0 (0,0)	.25
Total Sperm Count (106/ejaculate)	0 (0,0.3)	0 (0,0)	0 (0,0.04)	0 (0,0)	.15
Germ Cell Populations (per tubule cross-section)
Spermatogonia	11 (9,14)	0 (0,0)**	16 (10,18)*	6.8 (6.7, 7.0)	0.001
Spermatocytes	14 (13,15)	0 (0,0)**	19 (14,29)*	5.2 (4.9,8.6)	0.001
Spermatids (type a+ type b)	9 (5,13)*	0 (0,0)**	1.5 (0.3,2.5)	3.1 (0.8,4.4)	0.001
Spermatids (type c+ type d)	8 (7,9)*	0 (0,0)**	0 (0,0.9)	0.8 (0.7,1.6)	0.001

^†Nine of these men had obstructive azoospermia.

^**p<0.01 c.f. other groups;

^*p<0.05 c.f. other groups

Table 2.

Serum and intratesticular hormones by testicular histology type of the 43 men included in the analysis. All values are expressed as median (25^th, 75^th percentiles).

	Testicular Histology
	Normal (n=11)	Sertoli-Cell Only (n=20)	Maturation Arrest (n=8)	Hypospermatogenesis (n=4)	p-value
Testosterone (ng/ml)	4.5 (2.4,4.9)*	2.5 (2.0,3.2)	2.8 (2.6,3.6)	2.3 (2.2,3.0)	.03
FSH (IU/L)	3.8 (2.9,4.7)	18 (13.5,24)**	6.7 (3.6,13)	2.9 (2.0,6.6)	.001
LH (IU/L)	2.2 (1.3,3.3)	6 (4.3,8.3)**	4.1 (2.5,5.9)	4.6 (2.7,4.9)	.005
SHBG (nmol/L)	24 (14,45)	26 (19,31)	25 (18,31)	22 (12,23)	.76
Estradiol (pg/ml)	47 (42,53)	33 (28,40)	39 (29,51)	42(19,61)	.31
Intratesticular testosterone (ng/mg protein)	24 (13, 63)	41 (25, 88)	35 (19,50)	18 (13,24)	.30
Intratesticular estradiol (ng/mg protein)	167 (103, 269)	136 (67, 359)	214 (158,270)	81 (73,110)	.39

^*p<0.05 c.f. other groups;

^**p<0.01 c.f. other groups.

ALDH Protein Levels

The protein levels in the men, stratified by sub-type of infertility are shown in Figure 1. There were no significant differences in ALDH1A1 or ALDH1A3 protein levels between the groups. However, there were significant relative reductions in ALDH1A2 protein level observed in both the Sertoli-cell only group [0.53 (0.42, 0.67) units, p=0.0001], and in the hypospermatogenesis group [0.64 (0.42, 0.87) units, p=0.03], compared with control values 1.0 (0.95, 1.3) units, corresponding to a 36–47% reduction in this protein in the testes of the infertile men. There were no significant differences in ALDH1A2 protein level between the normal men and the men with maturation arrest, or between the sub-types of infertility. When all 32 infertile men were grouped together, the ALDH1A2 protein levels in the infertile men were significantly reduced by 36% compared to those in the eleven fertile men [(Normal; 1.0 (0.95, 1.3) units vs. all infertile men; 0.64 (0.46, 0.73) units, p=0.0004].

Figure 1.

Box and whiskers plots of ALDH1A1(A), 1A2 (B) and 1A3 (C) from testicular tissue of the men by histological subtype. Each box represents the median, 25^th and 75^th percentiles, with the upper and lower values represents by the bars, and outliers represented as open circles. Values are expressed relative to the mean of the group of normal men. Therefore, amounts of protein are comparable between groups of subjects within an assay, but not between proteins. **p<0.001 compared with normal men; *p<0.05 compared with normal men.

ALDH1A2 protein levels were highly significantly correlated with the number of each type of germ cell seen in the testicular biopsies (Figure 2A–2C). This correlation was similar for spermatogonia and spermatocytes, and was strongest between spermatids and ALDH1A2 protein levels (Figure 2C). When the subjects with Sertoli-cell only were excluded in a sensitivity analysis, the correlation between germ cell number and ALDH1A2 was no longer significant between ALDH1A2 and spermatogonia and spermatocytes, but remained significant between ALDH1A2 and both early (r=0.52, p=0.02) and advanced spermatids (r=0.64, p=0.0003). Testicular protein levels of ALDH1A1 and ALDH1A3 were not significantly correlated with germ cell numbers (not shown)

Figure 2.

Scatter plots of spermatogonia (A), spermatocytes (B) and early spermatids (C) vs. ALDH1A2 protein level from testicular tissue of the 43 study subjects. Correlations were performed using Spearman’s technique.

Predictors of ALDH1A2 Protein Level

As ALDH1A2 was the ALDH isozymes most strongly associated with increased numbers of germ cells on testicular histology, we sought to determine if any demographic or hormonal factors appeared to correlate with testicular ALDH1A2 protein levels. Of the examined factors, only serum LH and FSH were significantly correlated with testicular ALDH1A2 protein levels in linear regression analysis [FSH: −0.02 (95%CI −0.03 to −0.01); p=0.01] and [LH: −0.05 (95% CI −0.08 to −0.02; p=0.007], suggesting that higher gonadotropins were associated with lower levels of testicular ALDH1A2 in the group as a whole. When subjects with Sertoli-cell only syndrome were excluded from this analysis, the associations between FSH and LH and testicular ALDH1A2 protein level were no longer significant.

Discussion

Despite the demonstrated importance of intratesticular retinoic acid for spermatogenesis in experimental animals, there has been little work investigating the role of intratesticular retinoic acid in human spermatogenesis. In this study, for the first time, we demonstrate a significant association between the protein levels of one of the three enzymes known to synthesize retinoic acid and germ cell numbers in a cohort of men with infertility. Using a novel mass spectrometric peptide assay, we found strong associations between ALDH1A2 protein level and populations of germ cells in testicular tissue. In particular, men with Sertoli-cell only syndrome had significantly reduced levels of ALDH1A2 compared to men with normal spermatogenesis. Men with hypospermatogenesis also appear to have reduced testicular levels of ALDH1A2, but this reduction was not as great as that seen in Sertoli-cell only syndrome, possibly due to the presence of some germ cells in the men with hypospermatogenesis. Notably, protein concentrations of ALDH1A1 and ALDH1A3 were not different between normal men and men with infertility and did not correlate with germ cell populations from the testicular biopsies. These findings suggest that the reduction of germ cells observed in the testicular tissue from the infertile men is driving the observed difference in ALDH1A2 protein levels between the groups. From these observations, it seems likely that ALDH1A2 is the major isozyme of ALDH involved in retinoic acid biosynthesis within germ cells in the human testes. This conclusion is consistent with the data from mice, which localizes ALDH1A2 to murine germ cells using immunohistochemical staining (23).

Interestingly, the relationship between ALDH1A2 protein level and germ cells was stronger for the more advanced germ cell types. Indeed, the correlation between ALDH1A2 protein level and spermatids was the only one to persist when subjects with Sertoli-cell only syndrome were omitted from the analysis. This suggests a role for retinoic acid in spermiogenesis in man, in addition to its likely role in spermatogonial differentiation. Such a role has been demonstrated in mice in elegant studies performed by Wolgemuth and colleagues (11, 12). In one such study, germ cell stem cells from mice deficient for retinoic acid receptor-α were transplanted into germ-cell depleted wild-type testes. Although the transplanted germ cells were able to initiate spermatogenesis, probably via retinoic acid receptor-γ signaling, they failed to develop proper cellular associations during spermiogenesis and abnormal sperm formation was observed, demonstrating the need for retinoic acid signaling during spermiogenesis.

The absence of a correlation between serum and intratesticular hormones suggests that ALDH1A2 expression and function are not regulated by serum or testicular hormones. While some investigators have suggested an inter-relationship between androgens and retinoids (28, 29), the factors controlling testicular retinoic acid biosynthesis are not well understood. It is known that the effect of retinoic acid appears to be mediated in part by the induction of the 45 kD protein STRA8 (“stimulated by retinoic acid-8”) within the developing germ cells (20), but the molecular function of this protein remains to be elucidated. The signals regulating retinoic acid biosynthesis in humans will be the focus of future work.

It is unknown if deficiencies in retinoic acid production or function are a cause of human infertility. Recently, a case-control study identified polymorphisms in retinoic acid pathways that were associated with male infertility. Interestingly, a polymorphism which decreased STRA8 function significantly increased the risk of infertility by a factor of 2-fold. In contrast, a polymorphism that decreased the activity of Cyp26, the enzyme that catabolizes retinoic acid to inactive metabolites, (thereby increasing intratesticular retinoic acid) decreased the risk of infertility by 50% (30). Clearly, further study examining other associations between polymorphisms and mutations in proteins in the retinoic acid pathway and infertility is warranted.

Our study has several important limitations. Firstly, we cannot determine if the reduced protein levels of ALDH1A2 caused the reduction in germ cell number or if reduced germ cell numbers leads to reduced ALDH1A2 protein levels, although the latter possibility may be more likely. Secondarily, our sample size was only moderate. Ideally, larger studies examining the ALDH concentrations in infertile men can be performed in the future to determine the strength of this association. However, obtaining sufficient testicular tissue for this type of analysis is extremely challenging. The specimens used in this study were obtained over a 12 year period, and represent a unique and valuable cohort for this type of analysis given the extensive information available on germ cell population number and serum and intratesticular hormone concentrations. Another limitation of the study is that the control samples of testicular tissue came from men with obstructive azoospermia. Chronic obstruction can lead to vacuolations of Sertoli cells and may have affected the results of our study (31). Ultimately as an observational study, the significance of the findings is unknown. Additional study of the molecular mechanisms underlying testicular ALDH expression and RA synthesis will be required before the role of intratesticular retinoic acid in human infertility is understood.

In conclusion, for the first time, we have measured the levels of ALDH1A2 protein in normal human testicular tissue and tissue from men with infertility. We have demonstrated that ALDH1A2 protein level is significantly reduced in testicular tissue from infertile men and is strongly correlated with testicular germ cell populations, but not with serum or intratesticular hormone concentrations. Significantly, testicular levels of ALDH1A1 and ALDH1A3 did not differ in the infertile men and were not associated with germ cell populations in testicular tissue. Taken together, these findings suggest that ALDH1A2 is the most important ALDH isozyme for retinoic acid biosynthesis in human germ cells. Given the importance of retinoic acid in spermatogenesis in animal models, further study of intratesticular retinoic acid biosynthesis and ALDH1A2 levels in human spermatogenesis and male infertility is warranted.