Curative GnRHa treatment has an unexpected repressive effect on Sertoli cell specific genes

Background Follicle stimulating hormone and testosterone stimulate Sertoli cells to support germ cell function and differentiation. During mini-puberty, when gonadotropin (GnRH) stimulates increases in plasma luteinizing hormone (LH) and testosterone levels, gonocytes are transformed into Ad spermatogonia. In cryptorchidism, impaired gonadotropin secretion during mini-puberty results in insufficient LH and testosterone secretion, impaired gonocyte transition to Ad spermatogonia, and perturbed Sertoli cell proliferation. Treatment with a gonadotropin-releasing hormone agonist (GnRHa/Buserelin) induced gonocytes to differentiate into Ad spermatogonia and rescued fertility. The present study evaluated the impact of low LH secretion on Sertoli cell function by comparing differential gene expression data between testes with low LH that lacked Ad spermatogonia (Ad-) and testes that completed mini-puberty (Ad+). Furthermore, we analyzed changes in the transcription of selected Sertoli cell specific genes in response to GnRHa treatment. Results Ad- testes showed reduced expression of nine out of 40 selected Sertoli cell specific genes compared to Ad+ testes. GnRHa treatment repressed most of the Sertoli cell specific genes, including the inhibins, but it increased the expression of genes that regulate apoptosis (FASLG) and proliferation (GDNF). Conclusions Impaired-minipuberty with decreased LH and testosterone levels affected Ad and Sertoli cell development through positive and negative regulation of morphoregulatory and apoptotic genes. GnRHa treatment had a repressive effect on most Sertoli cell specific genes, which suggested that Sertoli cells underwent a cellular rearrangement. We propose that gonadotropin-dependent increases in FASLG and GDNF expression drove Sertoli cell proliferation and germ cell self-renewal and supported the transition of gonocytes to Ad spermatogonia, independent of inhibins.


Background
During prepuberty four types of Sertoli cells develop under hormonal control [1]: fetal Sertoli cells (Sf ), which are observed in the first months after birth; the Sa and Sb types, which are observed during childhood; and the Sc type, which is observed in puberty and throughout life. In the first three months after birth, activation of the hypothalamic-pituitary-gonadal (HPG) axis leads to a transient increase in gonadotropins and testosterone [2][3][4], termed mini-puberty. During mini-puberty, the number of Sertoli cells increases [5,6].
Within the seminiferous tubules, Sertoli cells possess receptors for testosterone and follicle stimulating hormone (FSH). Gonadotropin releasing hormone (GnRH) drives testosterone and FSH signaling. In Sertoli cells, this signaling promotes the production of factors that support germ cell function and differentiation. Sertoli cells secrete anti-Müllerian hormone (AMH), activins, inhibins, glial cell-derived neurotrophic factor (GDNF), KIT ligand (KITLG), and FAS ligand (FASLG). Prepubertal Sertoli cells express subunits of the active inhibin B [7], which counteracts the effect of activin A by suppressing FSH secretion in gonadotropes [8,9].
The testosterone-dependent transition of gonocytes into A dark (Ad) spermatogonia during mini-puberty is an essential process for fertility [10,11]. Prepubertal human testes with defective mini-puberty lacked the transition of gonocytes into Ad spermatogonia (Ad-) and showed a diminished number of Sf and Sb Sertoli cells [12,13]. Those findings were consistent with recent observations of significantly reduced plasma levels of testosterone, AMH, and inhibin B in boys with cryptorchidism [14]. The early postnatal increase in inhibin B during mini-puberty [15] is presumably due to the activation of the HPG axis [16], and it reflects the proliferation of Sertoli cells. Of interest, the early postnatal rise in inhibin B was correlated more closely with luteinizing hormone (LH) and testosterone levels than with FSH levels [17,18]. That finding raised the possibility that Sertoli cell proliferation in neonatal life might depend more on LH/testosterone than on FSH. Studies in the literature have reported equivocal findings of normal, decreased, and increased inhibin B secretion in boys with cryptorchidism [9,[19][20][21][22][23][24]. Cortes and colleagues found a positive correlation between inhibin B and Ad spermatogonia by analyzing 2-μm thick sections of paraffin embedded specimens [25]. However, a more precise analysis method that employed 1-μm semi-thin sections of Epon embedded specimens could not confirm the earlier report of a correlation between the number of Ad spermatogonia and plasma inhibin B levels [26].
Treatment with the gonadotropin-releasing hormone agonist (GnRHa), Buserelin, induced an increase in LH and testosterone [27][28][29] and rescued fertility in the majority of pathological cryptorchid testes. Buserelin induced expression of genes in the HPG axis [30] and genes involved in gonocyte transition to Ad spermatogonia [31]. Ultrastructural changes within Sertoli cells after GnRHa treatment included the presence of abundant lipid droplets, changes in nuclear form, and increased amounts of rough endoplasmic reticulum in the cytoplasm [27]. In a randomized double-blind, placebo-controlled study cryptorchid boys were treated with a low dose (20 micrograms) of GnRHa, given as a nasal spray, for a short period of 28 days. Boys treated with Buserelin had the highest number and the best maturation index of germ cells [32]. Furthermore, in 2007 it has been shown that patients treated with Buserelin and with a normal Leydig cell testosterone secretory capacity will have normal testicular histology and Ad spermatogonia. Those with a suboptimal Leydig cell testosterone secretory capacity will have a low Ad spermatogonia count and consequently poor prognosis for future fertility, despite successful surgery [29].
In this study, we analyzed the impact of low (hypogonadotropic) LH on differential expression of specific Sertoli cell genes between Ad-testes with impaired mini-puberty and Ad+ testes that completed mini-puberty. Furthermore, we studied the effect of GnRHa on Sertoli cells by analyzing differential Sertoli cell specific gene expression after GnRHa treatment in Ad-testes. Our findings extended our understanding of how Sertoli cells and their secreted factors, including inhibins, are involved in the gonocyte transition to Ad spermatogonia.

Study population and biopsy sample collection
We selected 15 patients with isolated cryptorchidism, based on histological results, and divided them into 2 groups. Seven belonged to the Ad− (lacking Ad spermatogonia) and 8 to the Ad+ (presenting Ad spermatogonia) group. The patients had a median age of 18.5 months (range 8-59 months). Data from Ad− bilateral cryptorchid boys treated with GnRHa (Buserelin) following the first orchidopexy (surgery) (4 patients) were retrieved from an ongoing randomized study. Initial biopsies revealed no Ad spermatogonia, indicating defective mini-puberty (Ad− group). The second testis was managed by orchidopexy and biopsied 6 months after the initial surgery. Thus, results from 21 biopsies were compared. Patients were age and ethnicity matched. RNA sequencing data from our two previous studies [30,33] were used to analyze manually selected genes expressed specifically in Sertoli cells.
A cryptorchid testis is defined as a testis localized outside of the scrotum and incapable of being brought into a stable scrotal position. All undescended testes in this study were located in the inguinal region. Testicular biopsies were taken at the time of orchidopexy. This sample was then subdivided, with one fragment fixed in glutaraldehyde for histological processing, while the other one was immediately immersed in RNAlater (ThermoFisher Scientific, Waltham, Massachusetts, USA) and stored at -25°C until further processing (for RNA extraction and RNA-sequencing).

Histological analyses
Biopsies were fixed in 3% glutaraldehyde in phosphatebuffered saline (PBS, pH 7.4) and then embedded in Epon resin. Semi-thin sections (1 μm) were cut using a Reichert Om-U3 ultramicrotome (Reichert AG, Vienna, Austria). Sections were mounted on glass slides, stained with 1% toluidine blue, and examined under a Zeiss Axioskop light microscope (Carl Zeiss Microscopy GmbH, Jena, Germany) with an integrated photo-camera. Biopsies were histologically examined by two of the authors (F.H. and D.D.), each with expertise in the interpretation of semi-thin sections of prepubertal testes.
During histological analyses, at least 100 tubular cross sections per biopsy were evaluated, with regard to their number of spermatogonia per tubule (S/T) and presence of Ad spermatogonia. In the prepubertal testes, Ad spermatogonia were identified according to the criteria first published by Seguchi and Hadziselimovic [34]. This type of germ cell has a typical halo in the nucleus, termed the rarefaction zone, and cytoplasm with a darker aspect in comparison to Ap or fetal spermatogonia. For each biopsy, at least 100 tubular cross sections were evaluated. On the basis of this evaluation, biopsies were categorized into 2 groups, Ad-(high infertility risk, HIR) and Ad+ (low infertility risk, LIR), dependent on their infertility outcome. The Ad-group included biopsies with S/T ≤0.2 and no Ad and the Ad+ group recorded S/T scores of >0.6 with Ad. Cryptorchid boys in the Adgroup had 8 times lower plasma LH (0.11 IU/L) compared to the Ad+ group (0.89 IU/L, p<0.009) indicating hypogonadotropic hypogonadism [26].
RNA preparation, sequencing, data analyses, and RNA expression levels The workflow from RNA isolation, through to purification, library preparation, sequencing, data analyses, and expression level analysis, has been previously described in detail [30,33].

Data and differential gene expression analyses
Determination of differentially expressed genes, statistical analyses and model design were described previously [30,33]. Only genes with at least one read per million, in at least two samples, were included. P values and fold-changes were calculated for the treatment factor and differentially expressed genes were defined as those displaying a false discovery rate (FDR) of less than 0.05. Raw data files are available at the Database of Genotypes and Phenotypes (dbGaP) with the accession number phs001275.v1.p1.

Results
We analyzed the expression data of 40 manually selected Sertoli cell expressing genes [35], whose expression or protein products are commonly used to identify or differentiate Sertoli cells from other testicular cells at various developmental stages. The results were described as fold change (FC) of gene expression, log 2-fold-change (LogFC) and false discovery rate (FDR) between the tested groups (Ad-vs. Ad+ and GnRHa treated vs. untreated) in Table 1. A positive LogFC indicated a gene upregulation and a negative LogFC a gene downregulation in Ad-testes relative to Ad+ testes or in GnRHa treated versus untreated testes (Table 1).
Nine of 40 Sertoli cell specific genes were differentially expressed in Ad-testes We found four genes that showed lower expression in Ad-testes compared to Ad+ testes: BMP6 with 1.7 fold-, DES with 2.8 fold-, FGF9 with 2.1 fold-, and SOX8 with 2.8 fold decreased expression ( Fig. 1 and Table 1). The differential expression values of the genes BMP6 [30], FGF9 [33], and SOX8 [30] between the Ad-and Ad+ group was described by us earlier.
We noted that 29 Sertoli cell specific genes were not significantly differentially expressed. Moreover, the expression levels of FASLG and GATA1 were not detectably different between the two groups.
We found that GnRHa treatment highly upregulated the gene expression of FASLG (fivefold) and GDNF (2.8 fold). The strongly upregulated RNA expression of GDNF after GnRHa treatment was described by us earlier [31]. Eighteen genes did not show significant differential RNA expressions, and GATA1 expression was not detected.

Discussion
Positive and negative regulators mediate LH-dependent Sertoli cell development Contrary to our expectation, only nine out of 40 Sertoli cell specific genes were differentially expressed in LHdeficient Ad-testes compared to Ad+ testes (Table 1). Inhibin B, a dimer of α and β subunits (INHA and INHBB), is mainly produced by Sertoli cells; it negatively regulates the release of pituitary FSH and antagonizes the stimulating action of activin A [36][37][38][39]. Of importance, no differential expression was observed in the single inhibin subunits, INHA, inhibin βA (INHBA), and INHBB, between the two studied groups. This finding supported the observation that plasma values for inhibin B were similar between Ad-and Ad+ testes [26]; moreover, this finding brings into question the hypothesis that inhibin B is essential for the transformation of gonocytes into Ad spermatogonia [25]. Alternatively, we propose that, during mini-puberty, LH and testosterone, but not inhibin B, drive the differentiation of gonocytes into Ad spermatogonia.
Bone morphogenetic protein 6 (BMP6) was reported to inhibit apoptosis and to influence the production of tight junction protein 1 (TJP1), GDNF, and KITLG in Sertoli cells [40]. Although we observed a downregulation of BMP6, the expression levels of TJP1, KITLG, and GDNF were not significantly (FDR>0.05) altered between the two groups.
Fibroblast growth factor 9 (FGF9) is a downstream effector of GATA4, ZFPM2, SRY, and SOX9 signaling pathway during male sex determination. FGF9 regulates Sertoli cell differentiation through FGFR2 signaling [41,42]. Both transcription factors, SOX8 and SOX9, can induce AMH expression, and it was suggested that these transcription factors might be redundant in testis differentiation [43,44]. In Ad-testes, FGF9 and SOX8 expression levels were reduced more than two-fold, but ZFPM2 and SOX9 were slightly increased (1.3 fold and 1.4 fold, respectively) ( Table 1, [30]); this result suggested that function of Sertoli cells was impaired in Ad-testes.
The cAMP response element binding protein 1 (CREB1) is a member of the family of transcription factors that are  [47]. We observed 1.1 fold increased CREB1 RNA expression in Ad-testes; however, we did not evaluate the activation state of CREB1 protein.
Alterations in gap junction protein alpha 1 (GJA1, also known as Connexin 43) expression were previously associated with different forms of spermatogenic impairment in men [48][49][50][51][52][53][54]. Furthermore, GJA1-based gap junctions have been reported to form a transverse and longitudinal intercellular communication network within seminiferous tubules, which ensures the synchronization of germ cell proliferation and differentiation [55]. However, the effect of an absolute fold change of 1.3 in GJA1 expression in Ad-testes remains unexplained.

GnRHa-treatment suppresses gene expression in Sertoli cells
GnRHa treatment caused downregulation of the cyto skeleton-related genes, KRT18, VIM, and KATNAL1, and the cell junction protein encoding genes, GJA1, TJP1, and CLDN11. This downregulation pointed to a structural remodeling process, both within Sertoli cells and in cell-cell connections between individual Sertoli cells and between Sertoli cells and germ cells.
Clusterin (CLU) was originally identified as an androgen/testosterone-repressed gene in the prostate [56] and reduced CLU protein levels in testis have been linked to male infertility [57,58]. In a recent report, it was demonstrated that CLU conferred protection against apoptosis and appeared to induce meiosis in male germ cells via VLDLR/LPR8 receptors in rats [59]. GnRHa treatment suppressed the expression of both these receptors (VLDLR logFC GnRHa -0.74 and LPR8 logFC GnRHa -0.76).
We suggest that the observed reduced levels of androgensensitive CLU expression resulted from GnRHa-induced increases in testosterone, and that this activity inhibited germ cells from entering premature meiosis.
Furthermore, we observed reductions in RNA expression of CDKN1B, CALB2, CTSL, CREB1, DMRT1 [31], and WT1. All these genes are involved in Sertoli cell function and differentiation. This finding suggested that GnRHa induced transcriptional changes in multiple target genes.
Cells that express AR in human testis include Sertoli cells, Leydig cells, peritubular myoid cells, arteriole smooth muscle cells, and vascular endothelial cells. Immunostaining methods for detecting AR protein showed rather poor expression in human prepubertal Sertoli cells, but AR expression increased after the age of four years [60][61][62][63]. Furthermore, AR expression is modulated by testosterone and FSH [64,65]. Therefore, the observed reduction in AR expression could be due to a GnRHa-dependent increase in testosterone in treated testes. Analyzing ARKO mice, Yue and coworkers compared expression levels of Ddx4, Amh, c-Kit, Mmp14/Mt1-mmp, Rhox5, Zbtb16, and Pou5f1 genes. They concluded that gonocyte transformation to A-type spermatogonia was androgen-independent in mouse and human. However, they did not analyze genes specific for Ad spermatogonia, like T/BRACHYURY, TERT, PAX7, DMRTC2, and FGFR3. Furthermore, Li et al. performed immunofluorescence histochemistry to detect the proliferation marker, Ki67, the Sertoli cell marker, AMH, and the germ-cell marker, DDX4. They did not observe any differences in gonocyte migration from the center to the tubular basement membrane, or in spermatogonial stem cell transformation [66]. Thus, they concluded that gonocyte transition to Ad spermatogonia and gonocyte migration towards the basement membrane were androgen-independent processes in both mouse and human. Although the expression of spermatogonial markers is conserved in species ranging from rodents to primates, species differences have been reported, including the succession of markers and their correlations with the differentiation state of spermatogonia (reviewed [67,68]). This species discrepancy was true for DDX4; in human, DDX4 was not localized in gonocytes, but its onset of expression was associated with the formation of spermatogonia [69]. Notably, there are different types of spermatogonia [34]. It is of paramount importance to realize that all spermatogonia are DDX4-positive cells, including spermatogonial stem cells (SSCs). In contrast to the findings of Li and co-workers, our results led us to conclude that, in infants with cryptorchidism and defective mini-puberty, testosterone is essential for the gonocyte-to-Ad spermatogonia transition; moreover, we concluded that testosterone had an effect after GnRHa treatment on expression of Sertoli cell specific genes, such as AR.
GnRHa treatment also had a negative effect on RNA levels of the inhibin-subunit encoding genes, INHA,  [7]. However, in pubertal and adult testis, INHBB is no longer expressed in Sertoli cells, but it continues to be expressed in germ cells and Leydig cells [7,70,71]. Serum inhibin B levels reflect Sertoli cell function [8,37]. Serum inhibin B levels were reported to be normal in boys with cryptorchidism, including patients in the high infertility risk group (HIR/Ad-) [7,21,26]. During mini-puberty, inhibin B levels are elevated. Earlier studies of gonadotropin treatment in prepubertal boys with cryptorchidism showed that gonadotropin stimulation increased serum inhibin B levels in an age-dependent manner [9,21]. In the present study, we did not evaluate inhibin B serum levels after GnRHa treatment, but the differential gene expression data of the single subunits (INHA, INHBA, and INHBB) pointed to gonadotropindependent transcriptional repression of both inhibin B and activin A. Importantly, upon treatment, although the expression of the protein encoding gene INHBA was repressed, the respective long non-coding RNA INHBA-AS1 (logFC GnRHa +2.02; [30]) was upregulated. Both INHBA and INHBA-AS1 were previously associated with dental caries in genome-wide association studies [72], and INHBA was postulated to play a role in early tooth development [73]. Our results suggested that INHBA-AS1 could be involved in the development of Sertoli cells. It was previously shown that activin A was involved, together with the germ-cell secreted protein, NODAL, in the upregulation of NANOS2 during germ cell differentiation. Both activin A and NODAL bind activin A receptor type 2 (ACVR2A/2B) and type I receptors (ACVR1B/1C), but NODAL requires concomitant binding of the transmembrane co-receptor, CFC1/CRIPTO1, for activation. After GnRHa treatment, ACVR2A was found to be downregulated (logFC GnRHa -0.60), and ACVR1C was upregulated (logFC GnRHa +0.85). The overall negative effect of GnRHa, except for its effect on ACVR1C, suggested that the treatment-induced Ad spermatogonial development in infants was independent of the activation of inhibin B and the activin A pathway in Sertoli cells [26]. This finding was inconsistent with the hypothesis that inhibin B might be important for gonocyte transformation into Ad spermatogonia [25].

FASLG, GDNF, and other genes with positive responses to GnRHa
FASLG expression was reported to be regulated by gonadotropin in human testis [74], and it was testosterone-dependent in rhesus monkeys [75]. With GnRHa treatment, we observed an increase in FASLG expression. This change could point to elevated apoptosis of Sertoli and germ cells; furthermore, it supported the notion that FASLG expression was dependent on testosterone and gonadotropin. Additionally, previous studies identified FASLG as target of matrix metallopeptidase 7 (MMP7), which proteolytically processes FASLG into a soluble form [76]. Interestingly, we also observed increases in MMP7 RNA levels upon GnRHa treatment (logFC GnRHa +2.52, FDR 0.0016), which suggested that soluble FASLG might be involved in GnRHa-induced germ cell development.
The influence of GDNF on the transition of gonocytes to spermatogonia in Ad-testes and the GnRHa dependence of GDNF expression was recently described [31]. GDNF was also reported to stimulate the proliferation of cultured, immature mouse Sertoli cells via its receptor subunit, NCAM, and the ERK1/2 signaling pathway [77]. Moreover, gonadotropin-dependent FSH stimulation was shown to induce increases in GDNF levels [78]; that interaction supported our findings that GDNF expression increased with GnRHa treatment. Furthermore, GDNF produced by peritubular myoid cells was reported to be testosterone-regulated and to be essential for repopulating the undifferentiated spermatogonial pool [79]. Therefore, a testosterone-dependent GDNF expression in Sertoli cells was likely, but a contribution of peritubular myoid GDNF RNA signaling to our data was also likely and has to be considered.
Sex hormone-binding globulin (SHBG/ABP), also known as androgen binding protein, is produced by Sertoli cells in most mammals. SHBG controls the availability of androgens in seminiferous tubules and in the epididymis (reviewed [80]). Transgenic mice that overexpressed rat SHBG developed progressive impairment of spermatogenesis [81]. Studies on human testis-specific SHBG revealed alternative splicing in exon 1. The Sertoli cellspecific SHBG transcripts included an exon 1 that lacked the secretion signal, and SHBG expression was repressed; the SHBG isoform with the other exon 1 was expressed in germ cells [82,83]. Based on those findings, we excluded SHBG as a Sertoli cell specific gene in the present study. Nonetheless, we would like to point out that SHBG expression was found four fold increased after GnRHa treatment (logFC GnRHa +2.00, FDR 0.0037). This finding supported the notion that SHBG expression was gonadotropin-dependent in the testis, which suggested that SHBG might be involved in the gonocyte transition to Ad spermatogonia.

Limitations and outlook
To get an indication of differential expression of specific Sertoli cell expressing genes we selected for this study testicular RNA sequencing data of 40 Sertoli cell expressing genes whose expression profiles and protein products are commonly used as Sertoli cell specifiers. However, a contribution of RNA signals of other testicular cells in this study could not be excluded. Furthermore, changes at the mRNA level do not necessarily mean changes at the protein level. Nonetheless, differential RNA levels as determined by RNA profiling are a good indication that a given gene is implicated in a certain process. Therefore, it is convincing that, despite the above mentioned limitations, the observed differential expressions reflect the pathological state in Ad-testes and is the consequence of GnRHa treatment. In future, protein and gene expression of single cells should be tackled.

Conclusion
This study showed that in Ad-testes with lower LH levels the expression of Sertoli cell specific genes, involved in cell morphology through complex gene networks, was affected positively and negatively. Based on these results, it is indicated that reduced LH and concomitantly reduced testosterone levels caused impaired Sertoli cell function and increased apoptosis.
GnRHa treatment mostly resulted in suppression of Sertoli cell specific genes. Based on our observations of differential gene expression of intermediate filaments and cell-cell junction proteins, we suggested that GnRHa triggered a restructuring process in Sertoli cells. Furthermore, we proposed that GnRHa induced GDNF and FASLG expression, which supported Sertoli cell proliferation and the gonocyte transition to Ad spermatogonia.
Finally, our results indicated that, during mini-puberty, LH and testosterone, but not inhibin B, were critical factors in driving the gonocyte transition to spermatogonia.