# Effects of Osteocalcin on Synthesis of Testosterone and INSL3 during Adult Leydig Cell Differentiation

Authors: Gulfidan Coskun; Leman Sencar; Abdullah Tuli; Dilek Saker; Mustafa Muhlis Alparslan; Sait Polat
Journal: International Journal of Endocrinology
 (2019)

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## Abstract

Proliferation and differentiation of adult Leydig cells are mainly completed in puberty. In many studies, apart from normal postnatal development process, it is widely indicated that, through administrating EDS, Leydig cell population is eliminated and regenerated. It is believed that osteocalcin released from osteoblasts, which is responsible for modulating bone metabolism, induces testosterone production in Leydig cells, independent of the HPG axis. In addition, INSL3 produced by Leydig cells, such as testosterone, plays a critical role in bone metabolism and is known to reflect the development process and functional capacities of Leydig cells. This study is aimed at investigating OC-mediated testosterone regulation and INSL3 synthesis during differentiation of adult Leydig cells that are independent of LH. For this purpose, male rats were divided into 2 groups: prepubertal normal rats and adult EDS-injected rats. Each group was divided into 4 subgroups in which GnRH antagonist or OC was applied. After adult Leydig cells completed their development, testicular tissue samples obtained from the sacrificed rats were examined by light-electron microscopic, immunohistochemical, and biochemical methods. Slight upregulation in 3βHSD, INSL3, and GPRC6A expressions along with the increase in serum testosterone levels was observed in groups treated with osteocalcin against GnRH antagonist. In addition, biochemical and microscopic findings in osteocalcin treated groups were similar to those in control groups. While there was no significant difference in the number of Leydig cells reported, the presence of a significant upregulation in INSL3 and GPRC6A expressions and the increase in serum testosterone and ucOC levels were observed. After evaluation of findings altogether, it is put forward that, for the first time in this study, although osteocalcin treatment made no significant difference in the number of Leydig cells, it increased the level of testosterone through improving the function of existing adult Leydig cells during normal postnatal development process and post-EDS regeneration. This positive correlation between osteocalcin-testosterone and osteocalcin-INSL3 is concluded to be independent of LH at in vivo conditions.


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## Body

## 1. Introduction

Sex steroid hormones are extensively known to be necessary for skeletal development and bone health throughout adult life, except for reproductive functions [1–3], which could set new hypotheses related to the effects of the bone on the synthesis and secretion of steroid hormones. The most common noncollagenous protein in the bone is osteocalcin in 2 forms: Gla-OC and Glu-OC, according to carboxylated (cOC) and uncarboxylated (ucOC) osteocalcin, respectively. While the active form of Glu-OC (ucOC) is used as hormone, the Gla-OC (cOC) contained in the bone matrix is biologically inactive. In in vivo and in vitro research studies, ucOC is shown to regulate male fertility by increasing testosterone production in Leydig cells that are independent of LH and inhibiting germ cell apoptosis. The specific ucOC receptor in Leydig cells is GPRC6A which also exists in the brain, heart, lung, spleen, kidney, skeletal muscle, adipose tissue, and pancreatic B cells, but not in the ovaries. In order to activate the steroidogenesis process, ucOC bypasses LHR and is connected to GPRC6A [4–9]. In addition, ucOC has been reported to induce pancreatic B-cell proliferation and insulin secretion, resulting in insulin sensitivity in the liver, muscle, and white fat [10–15]. Also, the orally administered Gla-OC form is reported to be decarboxylated in stomach acidity and converted into Glu-OC, resulting in increased serum ucOC and insulin levels [16].Another hormone that plays a critical role in bone metabolism is INSL3. INSL3 is produced by Leydig cells in the prenatal and the postnatal period in rodents. It has also been reported that INSL3, known to be regulated by long-term LH secretions during adulthood, is used as a marker of Leydig cell development and functions [17–23]. Besides, the reason of synthesis and regulation of INSL3 in the testis is still unclear.The populations of Leydig cells that differentiate prenatally and postnatally are identified as fetal and adult Leydig cells, respectively. In the fetus of rodents and humans, fetal Leydig cells produce testosterone and INSL3, which are needed for normal development of male external genitals and testis descent into the scrotum [24–30]. In contrast to fetal Leydig cells, adult Leydig cell differentiation is LH dependent. In rodents, Leydig cells complete their development in 4 distinct cellular stages which have different steroidogenic enzymes and mitotic activities. These steps are stem Leydig cells, progenitor Leydig cells, immature Leydig cells, and adult Leydig cells. Stem Leydig cells give rise to a progenitor cell type that further differentiates into immature Leydig cells followed by their maturation into adult Leydig cells. Adult Leydig cells produce androgens during the entire adult life span. Testosterone production by mature adult Leydig cells is required for the onset of spermatogenesis. Moreover, by expressing INSL3, adult Leydig cells play a role in germ cell viability and bone metabolism [25, 31–36].The development process of the adult Leydig cell population can also be mimicked in adulthood after EDS injection. EDS is an alkylating agent that specifically kills adult Leydig cells without affecting other testicular cell types. After one dose of intraperitoneal injection of 70 mg/kg of EDS, all adult Leydig cells were reported to have been destroyed within 4–7 days where within 7- to 10-week-old new adult Leydig cells proliferate from stem Leydig cells [37–41].Revealing paracrine factors and signal pathways that take part in differentiation of the adult Leydig cell population provides new approaches towards pharmacological and/or nutritional therapy, in impaired Leydig cell functions and in reduction in Leydig cell numbers for any reason [42]. In experimental studies, it is thought that ucOC could be a new therapeutic agent in regulating male fertility through increasing Leydig cell numbers and functions [4, 5]. Although the relationship between INSL3, testosterone, and osteocalcin has been reported in the previous study [43] in patients with Klinefelter syndrome, this is the first study in which the relationship between osteocalcin, testosterone, and INSL3 in Leydig cell development is reported.Therefore, this study is aimed at investigating the effects of orally administered Gla-OC on testosterone and INSL3 synthesis in adult Leydig cells during normal development and after EDS injection. In addition, the obtained light-electron microscopic, immunohistochemical, and biochemical findings are compared for LH levels.


## 2. Materials and Methods

Seventy-two Sprague Dawley male rats were divided into two groups, namely, Group 1 and Group 2. While 35-day-old rats were chosen for normal adult Leydig cell development, 12- to 13-week-old rats were preferred for post-EDS Leydig cell regeneration. In addition, 4 subgroups were formed within each group. Subjects were obtained from the Medical Sciences and Experimental Research and Application Center of Cukurova University (Adana, Turkey) and housed in well-ventilated polypropylene cages with food and tap water ad libitum. They were kept under controlled laboratory conditions of a normal light/dark cycle and normal temperature (25 ± 2°C) and were allowed to acclimatize for one week. After receiving the approval of Cukurova University’s Experimental Animal Ethics Committee (dated 28.04.2014), all experimental procedures were carried out according to the Universal Declaration of International Animal Rights.


### 2.1. Chemicals

In our study, cetrorelix acetate (450μg/kg/day) (Sigma C5249, USA) with less side effects as a GnRH antagonist was preferred [44], and osteocalcin (Gla-OC) protein (10 μg/kg/day) (Fitzgerald 30R-3286, USA) was administered to the subjects orally [16]. Furthermore, EDS, used for adult Leydig cell elimination in postpubertal subjects, was synthesized at the Chemistry Department of Science Faculty in Cukurova University (Adana-Turkey) as specified by Jackson and Jackson (1984) [45]. Firstly, 75 mg of EDS was dissolved in 0.5 ml of dimethylsulfoxide (DMSO) where 1.5 ml of distilled water was added to prepare 2 ml of EDS solution injected intraperitoneally into the rats as 2 ml per kg.


### 2.2. Surgical Procedure

To investigate the development of normal adult Leydig cells in the postnatal period, 18 adult female rats induced with 50 IU hCG and 30 IU PMSG for ovulation [46] were divided into 6 separate cages containing 3 female and 1 male rats. After 48 hours, 15 female rats, which had vitelline plug formation, were taken into separate cages to complete the 21-day gestation period. The born pups were kept in the same cages with their mothers without any application for 35 days. Thirty-two male pups of the 67 offsprings were allocated to 4 subgroups under the application period until postnatal day 63, as summarized in Figure 1:Group 1A: control group (n = 8). No application procedure was applied.Group 1B: cetrorelix treated group (n = 8).Group 1C: cetrorelix [44] and Gla-OC treated group (n = 8).Group 1D: Gla-OC treated group (n = 8).Figure 1
Group 1: postnatal normal adult Leydig cell development surgical procedure.To investigate the development of adult Leydig cells in EDS-injected animal models, from the 35th day to the 63rd day, after a single dose of EDS injection, 40 adult male rats were divided into 4 subgroups, as summarized in Figure2:Group 2A: EDS treated group (n = 16). Four days after EDS application, 8 rats in this group were sacrificed to research whether EDS eliminated Leydig cells. The remaining 8 rats survived for 63 days with no application except for a single dose of EDS.Group 2B: EDS and cetrorelix treated group (n = 8).Group 2C: EDS, cetrorelix, and Gla-OC treated group (n = 8).Group 2D: EDS and Gla-OC treated group (n = 8).Figure 2
Post-EDS regeneration of the adult Leydig cell surgical procedure.On the 63rd day of the postnatal normal developmental period and post-EDS application period, the differentiation of adult Leydig cells in all subjects was completed, and therefore, the 63rd day was selected as the sacrifice time of the subjects [31–33, 35].Testicular tissue samples obtained from the sacrificed experimental animals were taken into Bouin’s solution for light microscopic examination, 5% glutaraldehyde solution for electron microscopic examination, and 4% paraformaldehyde for immunohistochemical examination.LH, testosterone, and ucOC levels were measured by the blood serum samples, and testicular tissues were processed by light-electron microscopic examination and immunohistochemical staining for 3βHSD, INSL3, and GPRC6A. The data obtained were evaluated with appropriate statistical methods.


### 2.3. Light Microscopic Methods

In order to perform light microscopic examinations, one of each animal’s testes was fixed in Bouin’s solution for 8 hours and dehydrated for 6 hours by 50% alcohol and then by 70% alcohol. Tissue samples were washed under tap water and then dehydrated with an alcohol series (70%, 80%, 90%, 96%, and 100%). Thereafter, the tissues were kept in xylene for an hour in three separate containers at the same concentration. After xylene treatment, tissues were kept in molten paraffin at 60°C which was allowed to solidify at room temperature overnight. Paraffin-embedded tissue sections were cut by a microtome (Shandon Finesse 325, Thermo Scientific) to a thickness of 5μm. After staining with hematoxylin and eosin, tissue sections were examined under light microscope (Olympus BX53, Tokyo, Japan) [46].


### 2.4. Immunohistochemical Methods

The most commonly used marker to identify adult Leydig cells is 3βHSD [47–49]. INSL3 is an indicator of adult Leydig cell activity [17–19]. GPRC6A acts as an osteocalcin receptor in the testis [4, 5]. For 3βHSD, INSL3, and GPRC6A immunoreactivity analysis, testicular tissue specimens obtained from rats sacrificed on day 63 were fixed in 4% paraformaldehyde for 24 hours at room temperature. After routine tissue attachments with a Leica TP 1020 autotechnical device, paraffin sections with a thickness of 4 μm were taken from the tissue specimens embedded in paraffin. After being dewaxed in xylene and rehydrated in graded alcohol, 4 μm thick paraffin sections were treated with heat-induced epitope retrieval solution and put into a microwave irradiation for 10 min. Then, the sections were cooled off, washed in PBS, and incubated with 3% H2O2 to block endogenous peroxidase for 15 min at room temperature. After washing three times in PBS, the sections were blocked with blocking (IHC Kit ab93705, Abcam, MA, USA) for 15 minutes. The primary antibodies including anti-HSD3B1 (1 : 2000; anti-rat rabbit monoclonal antibody, ab150384, Abcam, MA, USA), anti-INSL3 (1 : 2000; anti-rat rabbit polyclonal antibody, ab65981, Abcam, MA, USA), and anti-GPRC6A (1 : 1000; anti-rabbit polyclonal antibody, NLS2576, Novus, Littleton, USA) were added before the samples were incubated overnight at 4°C. Negative control slides were prepared by omitting the primary antibodies. After washing them on the next day, the secondary antibody (IHC Kit, ab93705, Abcam, MA, USA) was added which was followed by staining with 3-amino-9-ethylcarbazole (AEC IHC Kit ab93705, Abcam, MA, USA) for 10 minutes and hematoxylin reagent for 3 minutes before dehydration. Images of the stained sections were analysed and photographed by light microscopy (Olympus BX53, Tokyo, Japan).


### 2.5. Scoring Assessment

For H-score assessment of each antibody, 10 fields were chosen randomly at ×400 magnification while the staining intensity in Leydig cells was scored as 0, 1, 2, or 3 corresponding to, respectively, the presence of negative, weak, intermediate, and strong staining. The total number of cells in each field and the number of cells stained at each intensity were counted. The average percentage of cells showing positive expression was calculated before the following formula was applied: IHC H-score = (% of cells stained at intensity category 1 × 1) + (% of cells stained at intensity category 2 × 2) + (% of cells stained at intensity category 3 × 3) [50]. The number of 3βHSD-positive Leydig cells was calculated regardless of the staining differences in each of the 10 random fields at ×400 magnification [51]. All evaluations were performed by two blinded histologists.


### 2.6. Electron Microscopic Methods

For electron microscopy, the other testis of each animal was fixed in 5% glutaraldehyde in a phosphate buffer (pH 7.2) for 24 hours before tissue pieces were postfixed in 1% osmium tetroxide. Thereafter, the tissue was dehydrated in graded ethanol, embedded in Araldite, and processed for electron microscopy. The stained sections were examined with a JEOL-JEM 1400 transmission electron microscope [46].


### 2.7. Serum Assays

Intracardiac blood samples were centrifuged at 3600 rpm for 5 minutes to obtain serum. Serum was kept in Eppendorf tubes at −20°C. Serum LH, testosterone, and Glu-OC levels were, respectively, measured through LH (CSB-E12654r, Cusabio), testosterone (CSB-E05100r, Cusabio), and Glu-OC (E-EL-R2477, Elabscience) ELISA kits in the ELISA Reader (Epoch) at the Medical Biochemistry Department of Cukurova University.


### 2.8. Statistical Analysis

The GraphPad Prism 5 (GraphPad Software Inc., USA) program is used to assess immunohistochemical scoring and biochemical data. Using the D’Agostino and Pearson omnibus normality test, data’s distribution normality was checked. One-way ANOVA by Tukey’s test was used to assess the differences between experimental and control groups based on immunoreactivity scores, Leydig cell numbers, and biochemical data. An unpairedt-test was used to compare the biochemical data of subjects that were sacrificed 4 days after EDS administration with the control groups. Moreover, P values less than 0.05 were considered significant.


## 2.1. Chemicals

In our study, cetrorelix acetate (450μg/kg/day) (Sigma C5249, USA) with less side effects as a GnRH antagonist was preferred [44], and osteocalcin (Gla-OC) protein (10 μg/kg/day) (Fitzgerald 30R-3286, USA) was administered to the subjects orally [16]. Furthermore, EDS, used for adult Leydig cell elimination in postpubertal subjects, was synthesized at the Chemistry Department of Science Faculty in Cukurova University (Adana-Turkey) as specified by Jackson and Jackson (1984) [45]. Firstly, 75 mg of EDS was dissolved in 0.5 ml of dimethylsulfoxide (DMSO) where 1.5 ml of distilled water was added to prepare 2 ml of EDS solution injected intraperitoneally into the rats as 2 ml per kg.


## 2.2. Surgical Procedure

To investigate the development of normal adult Leydig cells in the postnatal period, 18 adult female rats induced with 50 IU hCG and 30 IU PMSG for ovulation [46] were divided into 6 separate cages containing 3 female and 1 male rats. After 48 hours, 15 female rats, which had vitelline plug formation, were taken into separate cages to complete the 21-day gestation period. The born pups were kept in the same cages with their mothers without any application for 35 days. Thirty-two male pups of the 67 offsprings were allocated to 4 subgroups under the application period until postnatal day 63, as summarized in Figure 1:Group 1A: control group (n = 8). No application procedure was applied.Group 1B: cetrorelix treated group (n = 8).Group 1C: cetrorelix [44] and Gla-OC treated group (n = 8).Group 1D: Gla-OC treated group (n = 8).Figure 1
Group 1: postnatal normal adult Leydig cell development surgical procedure.To investigate the development of adult Leydig cells in EDS-injected animal models, from the 35th day to the 63rd day, after a single dose of EDS injection, 40 adult male rats were divided into 4 subgroups, as summarized in Figure2:Group 2A: EDS treated group (n = 16). Four days after EDS application, 8 rats in this group were sacrificed to research whether EDS eliminated Leydig cells. The remaining 8 rats survived for 63 days with no application except for a single dose of EDS.Group 2B: EDS and cetrorelix treated group (n = 8).Group 2C: EDS, cetrorelix, and Gla-OC treated group (n = 8).Group 2D: EDS and Gla-OC treated group (n = 8).Figure 2
Post-EDS regeneration of the adult Leydig cell surgical procedure.On the 63rd day of the postnatal normal developmental period and post-EDS application period, the differentiation of adult Leydig cells in all subjects was completed, and therefore, the 63rd day was selected as the sacrifice time of the subjects [31–33, 35].Testicular tissue samples obtained from the sacrificed experimental animals were taken into Bouin’s solution for light microscopic examination, 5% glutaraldehyde solution for electron microscopic examination, and 4% paraformaldehyde for immunohistochemical examination.LH, testosterone, and ucOC levels were measured by the blood serum samples, and testicular tissues were processed by light-electron microscopic examination and immunohistochemical staining for 3βHSD, INSL3, and GPRC6A. The data obtained were evaluated with appropriate statistical methods.


## 2.3. Light Microscopic Methods

In order to perform light microscopic examinations, one of each animal’s testes was fixed in Bouin’s solution for 8 hours and dehydrated for 6 hours by 50% alcohol and then by 70% alcohol. Tissue samples were washed under tap water and then dehydrated with an alcohol series (70%, 80%, 90%, 96%, and 100%). Thereafter, the tissues were kept in xylene for an hour in three separate containers at the same concentration. After xylene treatment, tissues were kept in molten paraffin at 60°C which was allowed to solidify at room temperature overnight. Paraffin-embedded tissue sections were cut by a microtome (Shandon Finesse 325, Thermo Scientific) to a thickness of 5μm. After staining with hematoxylin and eosin, tissue sections were examined under light microscope (Olympus BX53, Tokyo, Japan) [46].


## 2.4. Immunohistochemical Methods

The most commonly used marker to identify adult Leydig cells is 3βHSD [47–49]. INSL3 is an indicator of adult Leydig cell activity [17–19]. GPRC6A acts as an osteocalcin receptor in the testis [4, 5]. For 3βHSD, INSL3, and GPRC6A immunoreactivity analysis, testicular tissue specimens obtained from rats sacrificed on day 63 were fixed in 4% paraformaldehyde for 24 hours at room temperature. After routine tissue attachments with a Leica TP 1020 autotechnical device, paraffin sections with a thickness of 4 μm were taken from the tissue specimens embedded in paraffin. After being dewaxed in xylene and rehydrated in graded alcohol, 4 μm thick paraffin sections were treated with heat-induced epitope retrieval solution and put into a microwave irradiation for 10 min. Then, the sections were cooled off, washed in PBS, and incubated with 3% H2O2 to block endogenous peroxidase for 15 min at room temperature. After washing three times in PBS, the sections were blocked with blocking (IHC Kit ab93705, Abcam, MA, USA) for 15 minutes. The primary antibodies including anti-HSD3B1 (1 : 2000; anti-rat rabbit monoclonal antibody, ab150384, Abcam, MA, USA), anti-INSL3 (1 : 2000; anti-rat rabbit polyclonal antibody, ab65981, Abcam, MA, USA), and anti-GPRC6A (1 : 1000; anti-rabbit polyclonal antibody, NLS2576, Novus, Littleton, USA) were added before the samples were incubated overnight at 4°C. Negative control slides were prepared by omitting the primary antibodies. After washing them on the next day, the secondary antibody (IHC Kit, ab93705, Abcam, MA, USA) was added which was followed by staining with 3-amino-9-ethylcarbazole (AEC IHC Kit ab93705, Abcam, MA, USA) for 10 minutes and hematoxylin reagent for 3 minutes before dehydration. Images of the stained sections were analysed and photographed by light microscopy (Olympus BX53, Tokyo, Japan).


## 2.5. Scoring Assessment

For H-score assessment of each antibody, 10 fields were chosen randomly at ×400 magnification while the staining intensity in Leydig cells was scored as 0, 1, 2, or 3 corresponding to, respectively, the presence of negative, weak, intermediate, and strong staining. The total number of cells in each field and the number of cells stained at each intensity were counted. The average percentage of cells showing positive expression was calculated before the following formula was applied: IHC H-score = (% of cells stained at intensity category 1 × 1) + (% of cells stained at intensity category 2 × 2) + (% of cells stained at intensity category 3 × 3) [50]. The number of 3βHSD-positive Leydig cells was calculated regardless of the staining differences in each of the 10 random fields at ×400 magnification [51]. All evaluations were performed by two blinded histologists.


## 2.6. Electron Microscopic Methods

For electron microscopy, the other testis of each animal was fixed in 5% glutaraldehyde in a phosphate buffer (pH 7.2) for 24 hours before tissue pieces were postfixed in 1% osmium tetroxide. Thereafter, the tissue was dehydrated in graded ethanol, embedded in Araldite, and processed for electron microscopy. The stained sections were examined with a JEOL-JEM 1400 transmission electron microscope [46].


## 2.7. Serum Assays

Intracardiac blood samples were centrifuged at 3600 rpm for 5 minutes to obtain serum. Serum was kept in Eppendorf tubes at −20°C. Serum LH, testosterone, and Glu-OC levels were, respectively, measured through LH (CSB-E12654r, Cusabio), testosterone (CSB-E05100r, Cusabio), and Glu-OC (E-EL-R2477, Elabscience) ELISA kits in the ELISA Reader (Epoch) at the Medical Biochemistry Department of Cukurova University.


## 2.8. Statistical Analysis

The GraphPad Prism 5 (GraphPad Software Inc., USA) program is used to assess immunohistochemical scoring and biochemical data. Using the D’Agostino and Pearson omnibus normality test, data’s distribution normality was checked. One-way ANOVA by Tukey’s test was used to assess the differences between experimental and control groups based on immunoreactivity scores, Leydig cell numbers, and biochemical data. An unpairedt-test was used to compare the biochemical data of subjects that were sacrificed 4 days after EDS administration with the control groups. Moreover, P values less than 0.05 were considered significant.


## 3. Results

### 3.1. Light Microscopic Results

It was observed that seminiferous tubules and interstitium were in normal structure in the light microscopic examination of the testicular tissue sections of the control groups (Figures3(a) and 3(e)). In the testicular tissue sections of GnRH-antagonist-administered groups, spermatogenic arrest that occurred from spillage in the tubular epithelium and accumulation of immature spermatogenic cells in the tubule lumen was observed. Along with vacuolization, hyalinization was observed in the seminiferous tubule epithelium. The membrane propria was seen to be thick and irregular. In addition, large spaces were observed in the interstitial area because of shrinkage in the tubules and decrease in the number of Leydig cells (Figures 3(b) and 3(f)). Subjects administered with osteocalcin and GnRH antagonist showed less vacuolization in their testicular tissue sections. However, an immature spermatogenic cell accumulation in the tubule lumens, a decrease in the number of Leydig cells, and an edema in the interstitial area were in progress in spite of many tubules preserving the integrity (Figures 3(c) and 3(g)). Testicular tissue sections of subjects only administered with osteocalcin had a similar appearance to seminiferous tubules and interstitium of control groups. In addition to this, it was observed that the Leydig cells in the interstitial area were in normal structure and distribution (Figures 3(d) and 3(h)).Figure 3
Light microscopic view of the testicular tissue samples of postnatal subjects (Group 1) and EDS-administered subjects (Group 2) (H&E). (a) Seminiferous tubules (arrows) and interstitial area (∗) were observed to be normal in control subjects of Group 1 (Group 1A). (b) Vacuolization (arrowheads) in the seminiferous tubule epithelium, immature cell accumulation (+) in the tubule lumen, empty seminiferous tubules (arrows), and interstitial edema (∗) were seen in GnRH-antagonist-administered subjects of Group 1 (Group 1B). (c) Vacuolization (arrows) in seminiferous tubules, immature cell accumulation (+) in the tubule lumen, and interstitial edema (∗) were seen to continue in GnRH-antagonist-and-osteocalcin-administered subjects of Group 1 (Group 1C). (d) Seminiferous tubules (arrows) and the interstitium (∗) were normal in only-osteocalcin-administered subjects of Group 1 (Group 1D). (e) In control subjects of Group 2 (Group 2A), the seminiferous tubules (arrows) and the interstitium (∗) were shown to be normal. (f) Vacuolization (arrows) and multinucleated giant spermatogenic cells (arrowheads) in the seminiferous tubule epithelium and interstitial edema (∗) were observed in GnRH-antagonist-administered subjects of Group 2 (Group 2B). (g) In GnRH-antagonist-and-osteocalcin-administered subjects of Group 2 (Group 2C), immature cell accumulation (+) and interstitial edema (∗) were seen to continue in some seminiferous tubule lumens. (h) Seminiferous tubules (arrows) and interstitium (∗) were observed as normal in only-osteocalcin-administered subjects of Group 2 (Group 2D). Bar = 100 μm.


### 3.2. Immunochemical Results

As a Leydig cell marker, 3βHSD expression was observed to be high in adult Leydig cells located in the interstitium of testicular tissues of control group subjects (Figures 4(a) and 5(a)). However, 3βHSD expression significantly decreased in testicular tissue sections of the GnRH antagonist treated group (Figures 4(b) and 5(b)). Testicular tissue sections of subjects administered with osteocalcin together with GnRH antagonist showed a slight increase in 3βHSD immunoreactivity compared to those of subjects of only-GnRH-antagonist-administered groups (Figures 4(c) and 5(c)). Immunohistochemical examinations of testicular tissue sections of only osteocalcin treated subjects have shown similarity to the expression of 3βHSD Leydig cells of the control groups (Figures 4(d) and 5(d)).Figure 4
3βHSD, INSL3, and GPRC6A immunoreactivity in testicular tissue samples of postnatal subjects (Group 1). (a) High 3βHSD expression was observed in Leydig cells of control subjects (Group 1A). (b) 3βHSD expression decreased in Leydig cells of GnRH-antagonist-administered subjects (Group 1B). (c) A slight increase in 3βHSD expression was seen in Leydig cells of GnRH-antagonist-and-osteocalcin-administered subjects (Group 1C). (d) Intensive 3βHSD expression was observed in Leydig cells of only-osteocalcin-administered subjects (Group 1D). (e) High INSL3 expression was seen in Leydig cells of control subjects (Group 1A). (f) INSL3 expression decreased in Leydig cells of GnRH antagonist subjects (Group 1B). (g) A decrease in INSL3 expression seemed to continue in Leydig cells of GnRH-antagonist-and-osteocalcin-administered subjects (Group 1C). (h) High INSL3 expression was noticed in Leydig cells of only-osteocalcin-administered subjects (Group 1D). (i) High INSL3 expression was noted in Leydig cells of control subjects (Group 1A). (j) GPRC6A expression decreased in Leydig cells of GnRH-antagonist-administered subjects (Group 1B). (k) A decrease in GPRC6A expression seemed to continue in Leydig cells of GnRH-antagonist-and-osteocalcin-administered subjects (Group 1C). (l) High GPRC6A expression was observed in Leydig cells of only-osteocalcin-administered subjects (Group 1D). Bar = 50 μm.Figure 5
3βHSD, INSL3, and GPRC6A immunoreactivity in testicular tissue samples of EDS-administered subjects (Group 2). (a) High 3βHSD expression was reported in Leydig cells of control subjects (Group 2A). (b) 3βHSD expression decreased in Leydig cells of GnRH-antagonist-administered subjects (Group 2B). (c) A decrease in 3βHSD expression was seen to continue in Leydig cells of GnRH-antagonist-and-osteocalcin-administered subjects (Group 2C). (d) Intensive 3βHSD expression was observed in Leydig cells of only-osteocalcin-administered subjects (Group 2D). (e) High INSL3 expression was detected in Leydig cells of control subjects (Group 2A). (f) INSL3 expression decreased in Leydig cells of GnRH-antagonist-administered subjects (Group 2B). (g) A decrease in INSL3 expression was reported to continue in Leydig cells of GnRH-antagonist-and-osteocalcin-administered subjects (Group 2C). (h) High INSL3 expression was observed in Leydig cells of only-osteocalcin-administered subjects (Group 2D). (i) High INSL3 expression was seen in Leydig cells of control subjects (Group 2A). (j) GPRC6A expression decreased in Leydig cells of GnRH-antagonist-administered subjects (Group 2B). (k) A decrease in GPRC6A expression was seen to continue in Leydig cells of GnRH-antagonist-and-osteocalcin-administered subjects (Group 2C). (l) High GPRC6A expression was observed in only-osteocalcin-administered subjects (Group 2D). Bar = 50 μm.As a functional indicator of adult Leydig cells, INSL3 expression was observed to be high in Leydig cells of the testicular tissue sections of control group subjects (Figures4(e) and 5(e)). Because of continued Leydig cell dysfunction, a decrease in INSL3 expression in the testicular tissue sections of subjects administered with GnRH antagonist together with osteocalcin (Figures 4(f) and 5(f)) was similar to that of only-GnRH-antagonist-administered groups (Figures 4(g) and 5(g)). Expression of INSL3 also showed an increase under the effect of osteocalcin on testicular tissue sections of subjects administered with only osteocalcin (Figures 4(h) and 5(h)).Osteocalcin receptor GPRC6A expression showed high expression in adult Leydig cells located in the interstitium of testicular tissues of control group subjects (Figures4(i) and 5(i)). Because of the decrease in the number of Leydig cells, a decrease in GPRC6A expression was observed in subjects administered with GnRH antagonist (Figures 4(j) and 5(j)). The expression of GPRC6A was observed to have moderately increased in testicular tissue sections of subjects administered with GnRH antagonist together with osteocalcin (Figures 4(k) and 5(k)). On the contrary, in subjects administered with only osteocalcin, a significant rise in GPRC6A expression was observed in comparison with control groups (Figures 4(l) and 5(l)).In order to show whether adult Leydig cells were eliminated as a result of single-dose EDS administration or not, expressions of 3βHSD, INSL3, and GPRC6A were monitored in the testicular tissue sections of the subjects sacrificed 4 days after EDS, and none was found. That is, the absence of any expression in the interstitium was interpreted as that EDS effectively eliminates Leydig cells (Figure 6).Figure 6
No (a) 3βHSD, (b) INSL3, and (c) GPRC6A immunoreactivity was observed in the testicular tissue sections of the subjects sacrificed on the 4th day after EDS injection. Bar = 50 μm.
(a)
(b)
(c)


### 3.3. Scoring Results

According to the results of immunohistochemical scoring, 3βHSD, INSL3, and GPRC6A immunoreactivity was significantly lower in testicular tissue sections of Group 1B, Group 1C, Group 2B, and Group 2C subjects administered with GnRH antagonist and GnRH antagonist together with osteocalcin when compared to control groups. Results of 3βHSD, INSL3, and GPRC6A immunohistochemical scoring of testicular tissue sections of subjects administered with GnRH antagonist and osteocalcin did not show any statistically significant increase when compared to only-GnRH-antagonist-administered subjects. However, 3βHSD immunohistochemical scoring results of only-osteocalcin-administered subjects did not show any significant difference from the testicular tissues of subjects in control groups, whereas INSL3 and GPRC6A immunoreactivity scores of the same groups showed a significant increase when compared to the control groups. When ucOC levels of the GnRH-antagonist-administered and GnRH-antagonist-and-osteocalcin-administered groups were compared with those of osteocalcin-administered groups, a significant increase was found in GnRH-antagonist-and-osteocalcin-administered groups (Figure 7). In consideration of the number of 3βHSD-positive Leydig cells, it was seen that the number of Leydig cells markedly decreased in testicular tissue sections of subjects administered with GnRH antagonist. However, a slight increase in the number of 3βHSD-positive Leydig cells was found to be statistically insignificant in testicular tissue sections of subjects administered with GnRH antagonist and osteocalcin. It was noted that 3βHSD-positive Leydig cell numbers in testicular tissue sections of only-osteocalcin-administered subjects did not show any significant difference when compared to control groups (Figure 8).Figure 7
Statistical evaluation of 3βHSD (a), INSL3 (b), and GPRC6A (c) immunoreactivity scores of Leydig cells in testicular tissue sections of Group 1 and Group 2 subjects. ∗∗∗p<0.0001; ∗∗p<0.001; ∗p<0.01.
(a)
(b)
(c)Figure 8
Statistical evaluation of 3βHSD-positive Leydig cell numbers in testicular tissue sections of Group 1 (a) and Group 2 (b) subjects. ∗∗∗p<0.0001; ∗∗p<0.001; ∗p<0.01.
(a)
(b)


### 3.4. Electron Microscopic Results

Testicular tissue samples of the control group subjects maintained under normal laboratory conditions showed normal seminiferous tubules and interstitium, at the electron microscopic level (Figures9(a) and 10(a)).Figure 9
Electron microscopic view of testicular tissue samples of postnatal subjects (Group 1). (a) Membrane propria (MP), Sertoli cells (S), spermatocytes (Spt), spermatids (Spd), and nucleus (N) were seen to be normal in control subjects (Group 1A). Tight connections between the Sertoli cells (arrowhead) were observed as normal like the cytoplasmic bridges (arrows) between the spermatogenic cells. Leydig cells (LC) were seen to have normal tubular-type mitochondria (M) and abundant lipid droplets (L) in their cytoplasm. SER: agranular endoplasmic reticulum; bar = 2μm. (b) Thick and curved membrane propria (MP) and Sertoli cells (S) containing excessive and abnormal vacuolization (V) were indicated in GnRH-antagonist-administered subjects of Group 1 (Group 1B). My: myoid cells. (c) A decrease in the cytoplasmic volumes of some Leydig cells (LC) and an increase in the peripheral heterochromatin patches in the nucleus (N) were seen in testicular tissue samples of Group 1B. In the cytoplasm of some Leydig cells (LC), low electron density, dense agranular endoplasmic reticulum (SER) vacuolization, and intact mitochondria (M) were noted. Bar = 0.5 μm. (d) Relatively reduced curvature and thickness in the membrane propria (MP) were observed. Giant vacuoles (V) between Sertoli cells (S) and relatively normal spermatocytes (Spt) were seen in GnRH-antagonist-and-osteocalcin-administered subjects of Group 1 (Group 1C). (e) Decreased cytoplasmic volume in Leydig cells (LC) showed an increase in indentation and a decrease in heterochromatin content of the nucleus (N) in testicular tissue samples of Group 1C. In the cytoplasm, it was noted that agranular endoplasmic reticulum (SER) seemed to have vacuolization, but mitochondria (M) remained intact. Bar = 0.5 μm. (f) Sertoli cells (S) and spermatocytes (Spt) were also found to be normal in seminiferous tubules surrounded by normal membrane propria (MP) in only-osteocalcin-administered subjects of Group 1 (Group 1D). The tight junctions between the Sertoli cells (arrowhead) were normal. Ls: lysosome; bar = 1 μm. (g) Increased lipid droplets (L) were observed in the cytoplasm of the Leydig cells (LC), which have decreased heterochromatin content and indentation in the nuclei (N) of testicular tissue samples of Group 1D. Cap: capillary; bar = 1 μm.Figure 10
Electron microscopic view of testicular tissue samples of EDS-administered subjects (Group 2). (a) Membrane propria (MP), Sertoli cells (S), spermatocytes (Spt), and spermatids (Spd) were in normal structure in control subjects of Group 2 (Group 2A). Tight connections between the Sertoli cells (arrowhead) were seen in normal structure. Tubular-type mitochondria (M) and abundant lipid droplets (L) were observed in the cytoplasm of Leydig cells (LC). N: nucleus; bar = 2μm. (b) An increase in membrane propria (MP) total thickness and giant vacuoles (V) between Sertoli cells (S) and spermatocytes (Spt) was observed in GnRH-antagonist-administered subjects of Group 2 (Group 2B). Ls: lysosome; bar = 2 μm. (c) Low electron density and agranular endoplasmic reticulum (SER) vacuolization were seen in some Leydig cells (LC) in testicular tissue samples of Group 2B. Mitochondria (M) were intact. Bar = 0.5 μm. (d) In some Leydig cells, a decrease in the cytoplasmic volume and an increase in peripheral heterochromatin patches in the nucleus (N) were also observed in testicular tissue samples of Group 2B. Bar = 0.5 μm. (e) Thickening and irregularity in the membrane propria (MP) were seen to continue. A relative decrease in the volume of the vacuoles between Sertoli cells (S) and spermatocytes (Spt) was observed in GnRH-antagonist-and-osteocalcin-administered subjects of Group 2 (Group 2C). Bar = 2 μm. (f) A decrease in the cytoplasmic volumes of Leydig cells (LC) and a decrease in the amount of indentation in the nucleus (N) and in the content of heterochromatin were observed in testicular tissue samples of Group 2C. Bar = 0.5 μm. (g) Sertoli cells (S), spermatogonia (Spg), and spermatocytes (Spt) surrounded by membrane propria (MP) were seen to be normal in only-osteocalcin-administered subjects of Group 2 (Group 2D). Bar = 1 μm. (h) Numerous lipid droplets (L) were observed in the cytoplasm of Leydig cells (LC) containing the nucleus (N) in normal structure in testicular tissue samples of Group 2D. F: fibroblast; bar = 0.5 µm.Electron microscopic examination of testicular tissue samples of GnRH-antagonist-administered subjects showed thickening and irregularity in the membrane propria. Spermatogenic cells were not observed in many seminiferous tubules because of tight junction disruption between Sertoli cells. In addition, excessive SER vacuolization was observed in the Sertoli cell cytoplasm. Nuclei of some Sertoli cells showed an increase in electron density (Figures9(b) and 10(b)). The nucleus of Leydig cells in the interstitium was observed to have become pyknotic and displayed peripheral heterochromatin accumulations. In some Leydig cells, a decrease in cytoplasmic volume along with an increase in electron density was observed. On the contrary, in some of them, low electron density in the cytoplasm and intensive SER vacuolization were noted. All in all, in the cytoplasm of all Leydig cells, the integrity of the mitochondria was preserved and a significant reduction in lipid droplets was reported (Figures 9(c), 10(c), and 10(d)).In electron microscopic examination of testicular tissue samples of osteocalcin-and-GnRH-antagonist-administered subjects, it was seen that the thickening and irregularity of the membrane propria went relatively down in comparison with the subjects administered with only GnRH antagonist. In contrast to only-GnRH-antagonist-administered groups, there were not any empty seminiferous tubules. On the contrary, giant vacuoles in the cytoplasm of Sertoli cells were observed to be maintained (Figures9(d) and 10(e)). Although the number of indentations in the nuclei of the Leydig cells indicated an increase, the heterochromatin content of these nuclei decreased when compared to Group 1B. While mitochondria in the Leydig cell cytoplasm were in normal structure, vacuolization in SER cisternae was noted. In addition, it was determined that the cytoplasmic volumes of many Leydig cells declined and contained a few lipid droplets. However, in the Leydig cell cytoplasm, it was shown that SER and mitochondria retained their normal structure (Figures 9(e) and 10(f)).In the electron microscopic examination of testicular tissue samples of osteocalcin-and-GnRH-antagonist-administered subjects, it was seen that seminiferous tubules and interstitium were similar to those in the control groups. Sertoli and spermatogenic cells forming the seminiferous tubule epithelium were observed as normal. The tight junctions between the Sertoli cells were intact (Figures9(f) and 10(g)). In contrast to Group 1B and Group 1C, Leydig cell nuclei indicated less heterochromatin and indentation, while cytoplasmic volumes were in normal size and contained a large number of lipid droplets. As a result, Leydig cells were observed to preserve normal structure in terms of nuclei and cytoplasmic organelles (Figures 9(g) and 10(h)).


### 3.5. Biochemical Results

In order to determine whether adult Leydig cells were eliminated as a result of single-dose EDS administration or not, serum samples of the subjects sacrificed on the 4th day after EDS administration were compared with the serum samples of the untreated adult subjects. In the EDS group, serum testosterone and LH levels were found to be significantly reduced. However, a significant difference was not seen in the ucOC level of these subjects sacrificed on the 4th day after EDS when compared to the control group (Figure11).Figure 11
Statistical evaluation of serum testosterone (a), LH (b), and ucOC (c) levels of subjects sacrificed on the 4th day after EDS administration.∗∗∗p<0.0001; ∗∗p<0.001; ∗p<0.01.
(a)
(b)
(c)According to biochemical data, subjects administered with GnRH antagonist were found to have significantly low testosterone, LH, and ucOC levels in comparison to not only control groups but also osteocalcin-administered groups. In addition to this, testosterone and LH levels were significantly less in GnRH-antagonist-and-osteocalcin-administered groups when compared to the control groups and osteocalcin groups. On the contrary, a slight and statistically insignificant increase in testosterone levels was observed when compared to only-GnRH antagonist-administered groups, while LH levels of GnRH-antagonist-and-osteocalcin-administered groups were not found to be significantly different from those of only-GnRH-administered groups. No significant difference was found between ucOC levels of Group 1A and Group 1C. However, subjects in Group 1B had significantly lower ucOC levels than Group 1C subjects. Moreover, ucOC levels of each subgroup of Group 1 were found to be higher than those of Group 2. Although LH levels of only-osteocalcin-administered groups did not significantly differ from those of the control groups, the testosterone and ucOC levels of these groups were significantly higher than those of the control groups (Figure12).Figure 12
Statistical evaluation of serum testosterone (a), LH (b), and ucOC (c) levels in Group 1 and Group 2 subjects.∗∗∗p<0.0001; ∗∗p<0.001; ∗p<0.01.
(a)
(b)
(c)


## 3.1. Light Microscopic Results

It was observed that seminiferous tubules and interstitium were in normal structure in the light microscopic examination of the testicular tissue sections of the control groups (Figures3(a) and 3(e)). In the testicular tissue sections of GnRH-antagonist-administered groups, spermatogenic arrest that occurred from spillage in the tubular epithelium and accumulation of immature spermatogenic cells in the tubule lumen was observed. Along with vacuolization, hyalinization was observed in the seminiferous tubule epithelium. The membrane propria was seen to be thick and irregular. In addition, large spaces were observed in the interstitial area because of shrinkage in the tubules and decrease in the number of Leydig cells (Figures 3(b) and 3(f)). Subjects administered with osteocalcin and GnRH antagonist showed less vacuolization in their testicular tissue sections. However, an immature spermatogenic cell accumulation in the tubule lumens, a decrease in the number of Leydig cells, and an edema in the interstitial area were in progress in spite of many tubules preserving the integrity (Figures 3(c) and 3(g)). Testicular tissue sections of subjects only administered with osteocalcin had a similar appearance to seminiferous tubules and interstitium of control groups. In addition to this, it was observed that the Leydig cells in the interstitial area were in normal structure and distribution (Figures 3(d) and 3(h)).Figure 3
Light microscopic view of the testicular tissue samples of postnatal subjects (Group 1) and EDS-administered subjects (Group 2) (H&E). (a) Seminiferous tubules (arrows) and interstitial area (∗) were observed to be normal in control subjects of Group 1 (Group 1A). (b) Vacuolization (arrowheads) in the seminiferous tubule epithelium, immature cell accumulation (+) in the tubule lumen, empty seminiferous tubules (arrows), and interstitial edema (∗) were seen in GnRH-antagonist-administered subjects of Group 1 (Group 1B). (c) Vacuolization (arrows) in seminiferous tubules, immature cell accumulation (+) in the tubule lumen, and interstitial edema (∗) were seen to continue in GnRH-antagonist-and-osteocalcin-administered subjects of Group 1 (Group 1C). (d) Seminiferous tubules (arrows) and the interstitium (∗) were normal in only-osteocalcin-administered subjects of Group 1 (Group 1D). (e) In control subjects of Group 2 (Group 2A), the seminiferous tubules (arrows) and the interstitium (∗) were shown to be normal. (f) Vacuolization (arrows) and multinucleated giant spermatogenic cells (arrowheads) in the seminiferous tubule epithelium and interstitial edema (∗) were observed in GnRH-antagonist-administered subjects of Group 2 (Group 2B). (g) In GnRH-antagonist-and-osteocalcin-administered subjects of Group 2 (Group 2C), immature cell accumulation (+) and interstitial edema (∗) were seen to continue in some seminiferous tubule lumens. (h) Seminiferous tubules (arrows) and interstitium (∗) were observed as normal in only-osteocalcin-administered subjects of Group 2 (Group 2D). Bar = 100 μm.


## 3.2. Immunochemical Results

As a Leydig cell marker, 3βHSD expression was observed to be high in adult Leydig cells located in the interstitium of testicular tissues of control group subjects (Figures 4(a) and 5(a)). However, 3βHSD expression significantly decreased in testicular tissue sections of the GnRH antagonist treated group (Figures 4(b) and 5(b)). Testicular tissue sections of subjects administered with osteocalcin together with GnRH antagonist showed a slight increase in 3βHSD immunoreactivity compared to those of subjects of only-GnRH-antagonist-administered groups (Figures 4(c) and 5(c)). Immunohistochemical examinations of testicular tissue sections of only osteocalcin treated subjects have shown similarity to the expression of 3βHSD Leydig cells of the control groups (Figures 4(d) and 5(d)).Figure 4
3βHSD, INSL3, and GPRC6A immunoreactivity in testicular tissue samples of postnatal subjects (Group 1). (a) High 3βHSD expression was observed in Leydig cells of control subjects (Group 1A). (b) 3βHSD expression decreased in Leydig cells of GnRH-antagonist-administered subjects (Group 1B). (c) A slight increase in 3βHSD expression was seen in Leydig cells of GnRH-antagonist-and-osteocalcin-administered subjects (Group 1C). (d) Intensive 3βHSD expression was observed in Leydig cells of only-osteocalcin-administered subjects (Group 1D). (e) High INSL3 expression was seen in Leydig cells of control subjects (Group 1A). (f) INSL3 expression decreased in Leydig cells of GnRH antagonist subjects (Group 1B). (g) A decrease in INSL3 expression seemed to continue in Leydig cells of GnRH-antagonist-and-osteocalcin-administered subjects (Group 1C). (h) High INSL3 expression was noticed in Leydig cells of only-osteocalcin-administered subjects (Group 1D). (i) High INSL3 expression was noted in Leydig cells of control subjects (Group 1A). (j) GPRC6A expression decreased in Leydig cells of GnRH-antagonist-administered subjects (Group 1B). (k) A decrease in GPRC6A expression seemed to continue in Leydig cells of GnRH-antagonist-and-osteocalcin-administered subjects (Group 1C). (l) High GPRC6A expression was observed in Leydig cells of only-osteocalcin-administered subjects (Group 1D). Bar = 50 μm.Figure 5
3βHSD, INSL3, and GPRC6A immunoreactivity in testicular tissue samples of EDS-administered subjects (Group 2). (a) High 3βHSD expression was reported in Leydig cells of control subjects (Group 2A). (b) 3βHSD expression decreased in Leydig cells of GnRH-antagonist-administered subjects (Group 2B). (c) A decrease in 3βHSD expression was seen to continue in Leydig cells of GnRH-antagonist-and-osteocalcin-administered subjects (Group 2C). (d) Intensive 3βHSD expression was observed in Leydig cells of only-osteocalcin-administered subjects (Group 2D). (e) High INSL3 expression was detected in Leydig cells of control subjects (Group 2A). (f) INSL3 expression decreased in Leydig cells of GnRH-antagonist-administered subjects (Group 2B). (g) A decrease in INSL3 expression was reported to continue in Leydig cells of GnRH-antagonist-and-osteocalcin-administered subjects (Group 2C). (h) High INSL3 expression was observed in Leydig cells of only-osteocalcin-administered subjects (Group 2D). (i) High INSL3 expression was seen in Leydig cells of control subjects (Group 2A). (j) GPRC6A expression decreased in Leydig cells of GnRH-antagonist-administered subjects (Group 2B). (k) A decrease in GPRC6A expression was seen to continue in Leydig cells of GnRH-antagonist-and-osteocalcin-administered subjects (Group 2C). (l) High GPRC6A expression was observed in only-osteocalcin-administered subjects (Group 2D). Bar = 50 μm.As a functional indicator of adult Leydig cells, INSL3 expression was observed to be high in Leydig cells of the testicular tissue sections of control group subjects (Figures4(e) and 5(e)). Because of continued Leydig cell dysfunction, a decrease in INSL3 expression in the testicular tissue sections of subjects administered with GnRH antagonist together with osteocalcin (Figures 4(f) and 5(f)) was similar to that of only-GnRH-antagonist-administered groups (Figures 4(g) and 5(g)). Expression of INSL3 also showed an increase under the effect of osteocalcin on testicular tissue sections of subjects administered with only osteocalcin (Figures 4(h) and 5(h)).Osteocalcin receptor GPRC6A expression showed high expression in adult Leydig cells located in the interstitium of testicular tissues of control group subjects (Figures4(i) and 5(i)). Because of the decrease in the number of Leydig cells, a decrease in GPRC6A expression was observed in subjects administered with GnRH antagonist (Figures 4(j) and 5(j)). The expression of GPRC6A was observed to have moderately increased in testicular tissue sections of subjects administered with GnRH antagonist together with osteocalcin (Figures 4(k) and 5(k)). On the contrary, in subjects administered with only osteocalcin, a significant rise in GPRC6A expression was observed in comparison with control groups (Figures 4(l) and 5(l)).In order to show whether adult Leydig cells were eliminated as a result of single-dose EDS administration or not, expressions of 3βHSD, INSL3, and GPRC6A were monitored in the testicular tissue sections of the subjects sacrificed 4 days after EDS, and none was found. That is, the absence of any expression in the interstitium was interpreted as that EDS effectively eliminates Leydig cells (Figure 6).Figure 6
No (a) 3βHSD, (b) INSL3, and (c) GPRC6A immunoreactivity was observed in the testicular tissue sections of the subjects sacrificed on the 4th day after EDS injection. Bar = 50 μm.
(a)
(b)
(c)


## 3.3. Scoring Results

According to the results of immunohistochemical scoring, 3βHSD, INSL3, and GPRC6A immunoreactivity was significantly lower in testicular tissue sections of Group 1B, Group 1C, Group 2B, and Group 2C subjects administered with GnRH antagonist and GnRH antagonist together with osteocalcin when compared to control groups. Results of 3βHSD, INSL3, and GPRC6A immunohistochemical scoring of testicular tissue sections of subjects administered with GnRH antagonist and osteocalcin did not show any statistically significant increase when compared to only-GnRH-antagonist-administered subjects. However, 3βHSD immunohistochemical scoring results of only-osteocalcin-administered subjects did not show any significant difference from the testicular tissues of subjects in control groups, whereas INSL3 and GPRC6A immunoreactivity scores of the same groups showed a significant increase when compared to the control groups. When ucOC levels of the GnRH-antagonist-administered and GnRH-antagonist-and-osteocalcin-administered groups were compared with those of osteocalcin-administered groups, a significant increase was found in GnRH-antagonist-and-osteocalcin-administered groups (Figure 7). In consideration of the number of 3βHSD-positive Leydig cells, it was seen that the number of Leydig cells markedly decreased in testicular tissue sections of subjects administered with GnRH antagonist. However, a slight increase in the number of 3βHSD-positive Leydig cells was found to be statistically insignificant in testicular tissue sections of subjects administered with GnRH antagonist and osteocalcin. It was noted that 3βHSD-positive Leydig cell numbers in testicular tissue sections of only-osteocalcin-administered subjects did not show any significant difference when compared to control groups (Figure 8).Figure 7
Statistical evaluation of 3βHSD (a), INSL3 (b), and GPRC6A (c) immunoreactivity scores of Leydig cells in testicular tissue sections of Group 1 and Group 2 subjects. ∗∗∗p<0.0001; ∗∗p<0.001; ∗p<0.01.
(a)
(b)
(c)Figure 8
Statistical evaluation of 3βHSD-positive Leydig cell numbers in testicular tissue sections of Group 1 (a) and Group 2 (b) subjects. ∗∗∗p<0.0001; ∗∗p<0.001; ∗p<0.01.
(a)
(b)


## 3.4. Electron Microscopic Results

Testicular tissue samples of the control group subjects maintained under normal laboratory conditions showed normal seminiferous tubules and interstitium, at the electron microscopic level (Figures9(a) and 10(a)).Figure 9
Electron microscopic view of testicular tissue samples of postnatal subjects (Group 1). (a) Membrane propria (MP), Sertoli cells (S), spermatocytes (Spt), spermatids (Spd), and nucleus (N) were seen to be normal in control subjects (Group 1A). Tight connections between the Sertoli cells (arrowhead) were observed as normal like the cytoplasmic bridges (arrows) between the spermatogenic cells. Leydig cells (LC) were seen to have normal tubular-type mitochondria (M) and abundant lipid droplets (L) in their cytoplasm. SER: agranular endoplasmic reticulum; bar = 2μm. (b) Thick and curved membrane propria (MP) and Sertoli cells (S) containing excessive and abnormal vacuolization (V) were indicated in GnRH-antagonist-administered subjects of Group 1 (Group 1B). My: myoid cells. (c) A decrease in the cytoplasmic volumes of some Leydig cells (LC) and an increase in the peripheral heterochromatin patches in the nucleus (N) were seen in testicular tissue samples of Group 1B. In the cytoplasm of some Leydig cells (LC), low electron density, dense agranular endoplasmic reticulum (SER) vacuolization, and intact mitochondria (M) were noted. Bar = 0.5 μm. (d) Relatively reduced curvature and thickness in the membrane propria (MP) were observed. Giant vacuoles (V) between Sertoli cells (S) and relatively normal spermatocytes (Spt) were seen in GnRH-antagonist-and-osteocalcin-administered subjects of Group 1 (Group 1C). (e) Decreased cytoplasmic volume in Leydig cells (LC) showed an increase in indentation and a decrease in heterochromatin content of the nucleus (N) in testicular tissue samples of Group 1C. In the cytoplasm, it was noted that agranular endoplasmic reticulum (SER) seemed to have vacuolization, but mitochondria (M) remained intact. Bar = 0.5 μm. (f) Sertoli cells (S) and spermatocytes (Spt) were also found to be normal in seminiferous tubules surrounded by normal membrane propria (MP) in only-osteocalcin-administered subjects of Group 1 (Group 1D). The tight junctions between the Sertoli cells (arrowhead) were normal. Ls: lysosome; bar = 1 μm. (g) Increased lipid droplets (L) were observed in the cytoplasm of the Leydig cells (LC), which have decreased heterochromatin content and indentation in the nuclei (N) of testicular tissue samples of Group 1D. Cap: capillary; bar = 1 μm.Figure 10
Electron microscopic view of testicular tissue samples of EDS-administered subjects (Group 2). (a) Membrane propria (MP), Sertoli cells (S), spermatocytes (Spt), and spermatids (Spd) were in normal structure in control subjects of Group 2 (Group 2A). Tight connections between the Sertoli cells (arrowhead) were seen in normal structure. Tubular-type mitochondria (M) and abundant lipid droplets (L) were observed in the cytoplasm of Leydig cells (LC). N: nucleus; bar = 2μm. (b) An increase in membrane propria (MP) total thickness and giant vacuoles (V) between Sertoli cells (S) and spermatocytes (Spt) was observed in GnRH-antagonist-administered subjects of Group 2 (Group 2B). Ls: lysosome; bar = 2 μm. (c) Low electron density and agranular endoplasmic reticulum (SER) vacuolization were seen in some Leydig cells (LC) in testicular tissue samples of Group 2B. Mitochondria (M) were intact. Bar = 0.5 μm. (d) In some Leydig cells, a decrease in the cytoplasmic volume and an increase in peripheral heterochromatin patches in the nucleus (N) were also observed in testicular tissue samples of Group 2B. Bar = 0.5 μm. (e) Thickening and irregularity in the membrane propria (MP) were seen to continue. A relative decrease in the volume of the vacuoles between Sertoli cells (S) and spermatocytes (Spt) was observed in GnRH-antagonist-and-osteocalcin-administered subjects of Group 2 (Group 2C). Bar = 2 μm. (f) A decrease in the cytoplasmic volumes of Leydig cells (LC) and a decrease in the amount of indentation in the nucleus (N) and in the content of heterochromatin were observed in testicular tissue samples of Group 2C. Bar = 0.5 μm. (g) Sertoli cells (S), spermatogonia (Spg), and spermatocytes (Spt) surrounded by membrane propria (MP) were seen to be normal in only-osteocalcin-administered subjects of Group 2 (Group 2D). Bar = 1 μm. (h) Numerous lipid droplets (L) were observed in the cytoplasm of Leydig cells (LC) containing the nucleus (N) in normal structure in testicular tissue samples of Group 2D. F: fibroblast; bar = 0.5 µm.Electron microscopic examination of testicular tissue samples of GnRH-antagonist-administered subjects showed thickening and irregularity in the membrane propria. Spermatogenic cells were not observed in many seminiferous tubules because of tight junction disruption between Sertoli cells. In addition, excessive SER vacuolization was observed in the Sertoli cell cytoplasm. Nuclei of some Sertoli cells showed an increase in electron density (Figures9(b) and 10(b)). The nucleus of Leydig cells in the interstitium was observed to have become pyknotic and displayed peripheral heterochromatin accumulations. In some Leydig cells, a decrease in cytoplasmic volume along with an increase in electron density was observed. On the contrary, in some of them, low electron density in the cytoplasm and intensive SER vacuolization were noted. All in all, in the cytoplasm of all Leydig cells, the integrity of the mitochondria was preserved and a significant reduction in lipid droplets was reported (Figures 9(c), 10(c), and 10(d)).In electron microscopic examination of testicular tissue samples of osteocalcin-and-GnRH-antagonist-administered subjects, it was seen that the thickening and irregularity of the membrane propria went relatively down in comparison with the subjects administered with only GnRH antagonist. In contrast to only-GnRH-antagonist-administered groups, there were not any empty seminiferous tubules. On the contrary, giant vacuoles in the cytoplasm of Sertoli cells were observed to be maintained (Figures9(d) and 10(e)). Although the number of indentations in the nuclei of the Leydig cells indicated an increase, the heterochromatin content of these nuclei decreased when compared to Group 1B. While mitochondria in the Leydig cell cytoplasm were in normal structure, vacuolization in SER cisternae was noted. In addition, it was determined that the cytoplasmic volumes of many Leydig cells declined and contained a few lipid droplets. However, in the Leydig cell cytoplasm, it was shown that SER and mitochondria retained their normal structure (Figures 9(e) and 10(f)).In the electron microscopic examination of testicular tissue samples of osteocalcin-and-GnRH-antagonist-administered subjects, it was seen that seminiferous tubules and interstitium were similar to those in the control groups. Sertoli and spermatogenic cells forming the seminiferous tubule epithelium were observed as normal. The tight junctions between the Sertoli cells were intact (Figures9(f) and 10(g)). In contrast to Group 1B and Group 1C, Leydig cell nuclei indicated less heterochromatin and indentation, while cytoplasmic volumes were in normal size and contained a large number of lipid droplets. As a result, Leydig cells were observed to preserve normal structure in terms of nuclei and cytoplasmic organelles (Figures 9(g) and 10(h)).


## 3.5. Biochemical Results

In order to determine whether adult Leydig cells were eliminated as a result of single-dose EDS administration or not, serum samples of the subjects sacrificed on the 4th day after EDS administration were compared with the serum samples of the untreated adult subjects. In the EDS group, serum testosterone and LH levels were found to be significantly reduced. However, a significant difference was not seen in the ucOC level of these subjects sacrificed on the 4th day after EDS when compared to the control group (Figure11).Figure 11
Statistical evaluation of serum testosterone (a), LH (b), and ucOC (c) levels of subjects sacrificed on the 4th day after EDS administration.∗∗∗p<0.0001; ∗∗p<0.001; ∗p<0.01.
(a)
(b)
(c)According to biochemical data, subjects administered with GnRH antagonist were found to have significantly low testosterone, LH, and ucOC levels in comparison to not only control groups but also osteocalcin-administered groups. In addition to this, testosterone and LH levels were significantly less in GnRH-antagonist-and-osteocalcin-administered groups when compared to the control groups and osteocalcin groups. On the contrary, a slight and statistically insignificant increase in testosterone levels was observed when compared to only-GnRH antagonist-administered groups, while LH levels of GnRH-antagonist-and-osteocalcin-administered groups were not found to be significantly different from those of only-GnRH-administered groups. No significant difference was found between ucOC levels of Group 1A and Group 1C. However, subjects in Group 1B had significantly lower ucOC levels than Group 1C subjects. Moreover, ucOC levels of each subgroup of Group 1 were found to be higher than those of Group 2. Although LH levels of only-osteocalcin-administered groups did not significantly differ from those of the control groups, the testosterone and ucOC levels of these groups were significantly higher than those of the control groups (Figure12).Figure 12
Statistical evaluation of serum testosterone (a), LH (b), and ucOC (c) levels in Group 1 and Group 2 subjects.∗∗∗p<0.0001; ∗∗p<0.001; ∗p<0.01.
(a)
(b)
(c)


## 4. Discussion

In this study, it is determined that, during postnatal normal adult Leydig cell development or new adult Leydig cell regeneration after postpubertal EDS treatment, the positive effects of osteocalcin on testosterone and INSL3 synthesis are not completely independent of LH. Within 3-4 days following a single dose of EDS injection in adult rats, elimination in adult Leydig cells, decrease in testosterone production, and reduction in weights of the testis, epididymis, prostate gland, and seminal vesicle have been reported [37–39, 52–54]. In our study, 4 days after a single dose of 75 mg/kg EDS, no Leydig cell marker 3βHSD expression in the interstitium of testicular tissue samples obtained from injected postpubertal subjects was found. Furthermore, serum LH and testosterone levels of the same subjects were shown to decrease.As a medical hypophysectomy model, there are numerous studies about chronic GnRH antagonist applications that only inhibit GnRH release [55–61]. So, GnRH antagonist administration was preferred to determine whether the osteocalcin effects are LH dependent or not. In this research, LH suppression through administering a high dose of GnRH antagonist caused a significant decrease in serum LH and testosterone levels together with Leydig cell numbers when compared to normal subjects. In addition, there were significant degenerative changes in seminiferous tubules and interstitium due to impaired HPG axis. LH-controlled sex steroid hormone secretion has been reported to have a role in bone metabolism besides sexual maturation and reproductive functions in many studies [62–64]. Also, bone tissue has been found to regulate the synthesis and secretions of steroid hormones such as an endocrine organ as a result of clinical research studies and observations carried out within the last 10 years [4–6, 9]. In in vitro and in vivo studies, it has been put forward that LH and OC do not regulate each other; therefore, OC stimulates testosterone synthesis in Leydig cells via a second endocrine axis between the bone and the testis independently of the HPG axis [4]. This steroidogenesis process is activated by osteocalcin binding to GPRC6A, a specific receptor in Leydig cells. In our study, low levels of ucOC in subjects after GnRH antagonist application have shown that osteocalcin was adversely affected by decreasing testosterone levels because of suppression of LH. Moreover, this significant relationship between osteocalcin and testosterone has been shown not to be completely independent of LH in in vivo conditions. In this study, serum LH and testosterone levels of subjects administered with GnRH antagonist together with osteocalcin were reported to be significantly lower than those of control groups and only-osteocalcin-administered groups. However, there was no significant difference between LH levels compared to the GnRH antagonist groups, whereas serum testosterone and ucOC levels indicated a small increase. This nonsignificant increase in testosterone levels of subjects administered with GnRH antagonist together with osteocalcin showed that osteocalcin cannot fully tolerate decreased testosterone levels because of LH suppression by GnRH antagonist. Serum LH levels of subjects administered with only osteocalcin were similar to those of the control groups, while testosterone and ucOC levels were significantly higher than those of control groups. Whilst no significant difference was observed in terms of 3βHSD expression when testicular tissue sections were compared with control groups, it was observed that the expression of INSL3 and GPRC6A showed a significant increase because of exogenous osteocalcin administration. Immunohistochemical and biochemical findings suggest that osteocalcin does not cause considerable change in Leydig cell number; however, it may increase the function of current Leydig cells in only-osteocalcin-administered groups. In addition to the biochemical and immunohistochemical findings of only-osteocalcin-administered groups, light-electron microscopic examination showed seminiferous tubules and interstitium structure were found to be normal in testicular tissue samples.In parallel with LH secretions at puberty, the level of testosterone is known to increase, together with the rise in osteocalcin levels, because of the skeletal development [56, 65, 66]. So in this study, it was seen that the ucOC levels of the prepubertal subjects in all subgroups were higher than those of the postpubertal EDS-injected subjects. As reported by Mizokami and colleagues [16] and in consistent with this research, after Gla-OC oral administration, it was converted into the Glu-OC form and an increased serum ucOC level which is a hormone form. Comparing GnRH-antagonist-administered and GnRH-antagonist-and-osteocalcin-administered groups, a remarkable increase in ucOC levels was found in the Gla-OC-administered groups.INSL3 released from Leydig cells under LH control during adulthood regulates the functions of germ cells and Leydig cells by performing autocrine and paracrine effects as well as systemic effects [17, 21]. The positive relationship between LH and INSL3 has been shown in many studies in the literature over the last 15 years. In these studies, reduction in Leydig cell numbers and functions was shown to cause a decrease in INSL3 synthesis [67–72]. The INSL3 level is more sensitive than testosterone in determining Leydig cell damage and can be used clinically to determine androgen suppression [21]. Considering all these studies, the INSL3 concentration is thought to affect the terminal function capacities of the Leydig cells through creating a damage memory in cells during or after the development of the Leydig cells [17]. Although there are numerous studies showing a positive relationship between INSL3 and LH, only one study has been found in the literature investigating the association of INSL3 with osteocalcin. In the study published by Overvad et al. in 2014, researchers reported a positive correlation between INSL3 and osteocalcin by associating the decrease in bone mineral density with low INSL3 levels in patients with Klinefelter syndrome [43]. The findings of this study support the positive relation between LH, osteocalcin, and INSL3. In our study, because of LH suppression in the subjects administered with GnRH antagonist, an outstanding decrease in the expression of INSL3 and GPRC6A was observed in parallel with the decrease in ucOC and testosterone levels. In addition, in subjects administered with GnRH antagonist and osteocalcin, ucOC, INSL3, and GPRC6A expressions were reported to be significantly lower than those in control groups and only-osteocalcin-administered groups. It can be said that decreasing testosterone levels as a result of LH suppression decreases the level of ucOC in these groups, and therefore, there is a decrease in the expression of osteocalcin receptors GPRC6A and INSL3 which are positively associated with osteocalcin. On the contrary, it is determined that osteocalcin against severe loss of function observed in Leydig cells due to LH suppression does not cause any significant increase in the expression of INSL3, which is used as a function marker for Leydig cells. However, further studies are needed to answer the question of whether the decrease in INSL3 expression is due to LH suppression or indirectly sourced from the decrease in the osteocalcin level. Besides, the level of ucOC in subjects administered with GnRH antagonist together with osteocalcin showed a statistically insignificant increase when compared to only-GnRH-antagonist-administered subjects. This finding shows that INSL3 expression in LH control is also stimulated by osteocalcin such as testosterone when the LH release is suppressed. It was noted that osteocalcin caused a significant increase in the expression of INSL3 and GPRC6A by inducing the Leydig cell function in testicular tissue sections of the subjects administered with only osteocalcin. This increase in GPRC6A activity in only-osteocalcin-administered groups describes the significant increase in testosterone synthesis mediated by GPRC6A in the same groups. Furthermore, an increase in GPRC6A immunoreactivity may occur due to the fact that osteocalcin as a ligand increases its own receptor GPRC6A expression density or the number of receptors by using different signal pathways. On the contrary, it is a matter of discussion whether this increase in the testosterone level in the same groups stems from the effect of osteocalcin on increasing the number or activity of GPRC6A receptors or the increase in expression of INSL3.Although OC has been shown to stimulate testosterone synthesis independently of LH in many studies in in vitro conditions or using LHR−/− mice, no study has investigated the relationship of OC-testosterone in subjects undergoing LH suppression in in vivo conditions. In this study, whether OC was able to stimulate testosterone synthesis in patients who had an LH insensitivity problem or those who were diagnosed with hypogonadism due to LH deficiency was investigated. Based on these findings, although it was determined that OC stimulates testosterone synthesis moderately, the main effect was shown only in the subjects who had osteocalcin treatment without any LH suppression. According to the 3βHSD immunoreactivity scores and Leydig cell number analysis, osteocalcin is believed to increase the current Leydig cell function, which resulted in an increase in the level of testosterone, without any increase in the number of Leydig cells. Our study was the first to evaluate the relationship between osteocalcin and testosterone according to the Leydig cell numbers and Leydig cell functions during the development of adult Leydig cells. Additionally, in our study, the matter of whether OC could induce INSL3 synthesis in LH-suppressed subjects in the development process of adult Leydig cells like testosterone or not was investigated. It was determined that the expression of INSL3 such as testosterone increased by OC independently of LH, whereas in the subjects that did not undergo LH suppression, OC raised the Leydig cell function and INSL3 expression above the normal limits. Thus, this study is the first study to investigate the relationship between osteocalcin and INSL3 in the development process of adult Leydig cells.The most important advantage of this reciprocal functional relationship between osteocalcin and Leydig cell functions is bone health protection. However, in these conditions, unexpected side effects may occur in other systems. Therefore, there is a need for further studies to reveal the physiological relation between multiple tissues and organs such as nutrition, skeletal health, and reproduction. In recent years, except for environmental and genetic factors, the use of exogenous testosterone in men diagnosed with hypogonadism due to aging or testicular damage has been known to reduce long-term LH secretion and endogenous testosterone production. In addition to osteoporosis resulting from the decrease in bone mineral density, various side effects such as loss of muscle strength and sexual function, cardiovascular diseases, kidney failure, and prostate cancer have been reported [1, 2]. In order to eliminate all these side effects, it has been concluded that exogenous osteocalcin may be a new therapeutic route rather than testosterone replacement therapies, especially against primary hypogonadism or for improving existing fertility conditions.


## 5. Conclusion

When all the findings were evaluated together in this research, after oral administration of Gla-OC, a positive correlation between serum ucOC, testosterone, and INSL3 levels during the differentiation of adult Leydig cells has been put forward. However, this correlation was not identified completely independent of LH since it was observed to be indirectly affected by LH suppression. During the development of adult Leydig cells, even though ucOC has shown to have no change in the number of Leydig cells in the presence of LH, it had a positive effect on increasing the functional capacity of the current Leydig cells.



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*Source: 1041760-2019-08-28.xml*