Abstract
Oestrogen plays an important role in testicular function. This study used mice null for oestrogen receptor α (ERα) or β (ERβ) to investigate which receptor mediates the effects of oestrogen within the testis. Groups of ERα knockout mice (αERKO) and ERβ knockout mice (βERKO) and wild-type littermates (n=5–8) were killed at 11 weeks post partum. One testis was fixed in Bouin’s fluid for stereology and the other frozen for testosterone measurement. Trunk blood was collected for testosterone RIA. The optical disector combined with the fractionator methodology was used to estimate Leydig, Sertoli and germ cell numbers. At all times, the knockout animals were compared with their wild-type littermates. The physical disector quantified cells stained immunohistochemically for the apoptotic marker active caspase-3 and Hoechst staining was used to identify nuclear fragmentation. The mean Leydig cell volume was measured using the point sampled intercept method. The Leydig cell number per testis was significantly increased in βERKO mice but not in αERKO mice. Plasma and testicular testosterone concentrations were increased in αERKO mice but no changes were observed in βERKO mice. Hypertrophic Leydig cell changes were observed in αERKO mice, and a decreased mean cell volume was seen in βERKO mice. No difference in Sertoli cell number per testis was observed in any of the groups. The spermatogonial cell number per testis was increased in βERKO mice. Immunohistochemistry identified increased numbers of active caspase-3-labelled germ cells per testis in αERKO mice but not βERKO mice. Hoechst staining supported these findings. There was significant germ cell loss in αERKO mice. This study suggests that ERβ may be involved in regulation of Leydig cell proliferation and testosterone production in the adult mouse testis.
Introduction
It is now evident that oestrogen plays an important role in male reproductive function (see reviews by Couse & Korach 1999, O’Donnell et al. 2001). Oestrogen is produced within the testis by the aromatisation of testosterone (Nitta et al. 1993) and has been shown to affect the major cell types within the organ. Administration of exogenous oestrogen during development has been shown to reduce Sertoli cell numbers in the adult (Atanassova et al. 1999) but the effect of endogenous oestrogen in this process is less clear. Oestrogen disrupts development of fetal Leydig cells (Delbes et al. 2005) and can also act directly on the Leydig cell to inhibit testosterone production (Zhai et al. 1996). Oestrogen has also been shown to inhibit gonocyte development in the fetal testis (Delbes et al. 2004) and in the adult to enhance spermatogenesis by inhibiting apoptosis of human post-meiotic germ cells (Pentikainen et al. 2000).
The effects of oestrogen are mediated by two distinct receptors: oestrogen receptor α (ERα) and β (ERβ), both of which are found within the testis. ERβ appears to be the more abundant being localised in the mouse to the Leydig and Sertoli cells as well as spermatogonia and elongated spermatids (Rosenfeld et al. 1998, Jefferson et al. 2000). ERα has been identified in adult Leydig cells (Zhou et al. 2002) and are thus the only cells in the mouse testis reported to express both ERs.
While the localisation of the ERs is becoming clearer, the biological functions mediated by each receptor are less well established. Studies of the ERα knockout mouse (αERKO) have demonstrated that although the testes of these animals begin to develop normally, by 20 days post partum there is dilation of the lumen of the seminiferous tubules and rete testis. A reduced fluid reabsorption in the efferent ducts (Hess 2000) results in testicular disruption and loss of germ cells in the tubules culminating in subsequent infertility (Lubahn et al. 1993). How these changes influence Sertoli cell numbers remains unknown. Male ERβ knockout mice (βERKO), on the other hand, are reported to have normal fertility, even though the βERKO females have reduced fertility when compared with their wild-types (Krege et al. 1998).
As well as abnormalities in the seminiferous epithelium the inactivation of ERα also affects testosterone production. αERKO mice exhibit increased follicle-stimulating hormone (FSH) levels and also have raised circulating concentrations of luteinizing hormone (LH; Lindzey et al. 1998) and testosterone (Akingbemi et al. 2003). Leydig cells in the αERKO mice are hypertrophic and have been shown to produce more testosterone than cells from wild-type mice (Akingbemi et al. 2003).
The effects of ERβ on the seminiferous epithelium and Leydig cells are less clear. Delbes et al.(2004) have demonstrated that ERβ may affect proliferation of gonocytes during development but found no effect on the number or testosterone production of fetal Leydig cells at 2 days post partum.
The aim of this study was to investigate the effects of loss of the ERβ receptor on cellular makeup of the testis in the adult mouse and to determine whether lack of ERα affects Sertoli cell number.
Materials and Methods
Animals
Adult αERKO and βERKO mice were kind donations from Prof. Allan Herbison (Department of Physiology, University of Otago). ERKO mice and wild-type littermates were generated by mating C57BL/6J heterozygous mice expressing the disrupted ERα (Lubahn et al. 1993) or ERβ gene (Krege et al. 1998). Genotyping of the offspring was performed on tail-tip DNA using PCR amplification (Couse et al. 2003).
Mice were housed under 12 h light:12 h darkness cycles and given food and water ad libitum. Experiments were performed with approval from the University of Otago Animal Ethics Committee in accordance with the New Zealand Animal Welfare Act of 1999.
Tissue collection
Male αERKO mice (n=8) and their wild-type littermates (n=8), and male βERKO mice (n=8) and their wild-type littermates (n=5) were killed at 11 weeks post partum by CO2 inhalation. The testes were removed and one fixed overnight in Bouin’s fluid and then stored in 70% alcohol until processing for histological analysis and the other flash-frozen in liquid nitrogen and stored at −80 °C. Trunk blood was collected and plasma and testicular testosterone concentrations measured by RIA (Yeung et al. 1988).
Cell number estimation using the optical disector
A fractionated sample combined with the optical disector has been shown to be a precise method to estimate testis cell number (Wreford 1995). Each testis was fractionated according to the smooth fractionation method (Gundersen 2002) into ~12 pieces from which four blocks were selected randomly. Each selected block was sectioned exhaustively into 30 μm sections and one section from each selected randomly and stained using the periodic acid Schiff (PAS) technique (Clermont & Perey 1957). Microscope images were captured on Spot analysis software (Diagnostic Instruments Inc., Sterling Heights, MI, USA) using a 100× oil free objective (NA 0.95) on an Olympus BX-51 microscope with a Spot RT colour camera attachment. A 100 mm×100 mm square reference frame (Howard & Reed 1998) was placed on the computer screen over the captured microscope images. The disector was imaged through the z-axis initially incorporating a 2 μm guard zone that randomly and systematically traversed the entire section. The optical disector method was used to count the nucleus of each cell (Gundersen 1986, Gundersen et al. 1988).
The testicular cells were identified by the following criteria (see Fig. 1): Leydig cells were located within the interstitial tissue and identified by their round to ovoid cell margins with a single identifiable nucleus. Sertoli cells were identified by their irregular tripartite nucleolus (Russell et al. 1990) and their location within the lower third of the seminiferous tubule. Spermatogonia were located adjacent to the basement membrane and did not exhibit PAS staining but rather a particulate appearance of their nuclei (Leblond & Clermont 1952). Spermatocytes were larger, contained clumped chromatin and meiotic chromatin. Spermatids had characteristic PAS staining of the acrosome in both round and elongating cells (Russell et al. 1990). On an average, 68±6 Leydig cells; 82±6 Sertoli cells; 48±3 spermatogonia; 549±165 spermatocytes; 1538±556 spermatids; 150±11 caspase-positive cells and 118±28 Hoechst-positive cells were counted in the disectors for each animal.
Point counting
Sections selected for stereology were also used to determine the tissue constituents in the αERKO and βERKO mice and their wild-type littermates. Six images from each animal were captured at 10× magnification, and using a randomly placed 20 mm grid the percentage (Vv) of seminiferous tubules and interstitial tissue was determined.
Measurement of apoptotic germ cells
To investigate whether there was an increased rate of regulated cell death in the βERKO mice during and after meiosis and to determine if the cellular loss seen in the αERKO was due to active caspase-3 activity, the number of cells exhibiting immunostaining for activated caspase-3 was compared between the knockout and wild-type mice using a method developed by Fenwick & Hurst (2002). Briefly, sections (5 μm) were deparaffinised in xylene and rehydrated through graded alcohols into distilled water. Antigens were unmasked by boiling for 20 min in 0.1 mol Tris–HCl buffer (pH 10) with 5% (w/v) urea (Shi et al. 1996). Peroxidase activity was quenched with 0.3% H2O2 (v/v) in methanol. Sections were blocked with a 1:200 dilution of donkey serum for 10 min and then incubated in rabbit anti-active caspase-3 polyclonal antibody overnight at 4 °C (Cell Signaling, Boston, MA, USA). Negative control sections were incubated with an equivalent concentration of rabbit immunoglobulin G (IgG). The sections were incubated in biotinylated anti-rabbit IgG (Amersham Pharmacia Biotech) at a dilution of 1:200 for 30 min and then streptavidin-biotinylated horseradish peroxidase complex (Amersham Pharmacia Biotech) at a dilution of 1:200 for 30 min. Immunoreactivity was visualized with 3-amino-9-ethylcarbazole (Sigma–Aldrich Co.) and counterstained with Harris Haematoxylin.
Slides were viewed under an Olympus BX-51 microscope. Images were captured using the Spot imaging system. The physical disector (Sterio 1984) was used with pairs of serial sections mounted on the same slide and with systematic random sampling across the sections. At least 100 immunopositive cells were scored within the disector reference frames.
Hoechst staining was used as another approach to study and count the numbers of apoptotic cells. Morphological criteria identified apoptotic cells with nuclear fragmentation, blebbing and chromatin condensation. Apoptotic cells stained with Hoechst were intensely bright and irregularly stained. Normal cells were lightly regularly stained and residual bodies remained unstained. Cells were scored as apoptotic if they exhibited a bright fluorescent staining as well as defined morphological criteria. Tissue from randomly selected blocks were cut (5 μm) and dewaxed. After equilibrating in PBS for 5 min, the Hoechst stain (H-3570, Molecular Probes, Eugene, Oregon, USA) was applied (10 μg/ml in PBS) for 10 min in the dark. The slides were washed in PBS for 5 min and then coverslipped using Vector-shield mounting media (H-1000, Vector Laboratories, Burlingame, CA, USA). The physical disector method was used to count Hoechst stained cells in a manner identical to the method used for active caspase-3 immunostaining.
Mean Leydig cell volume
It has previously been described that αERKO mice have hypertrophic Leydig cells and increased circulating testosterone levels (Akingbemi et al. 2003). In this study, the optical disector determined that there were changes in Leydig cell numbers in βERKO mice. To determine if there were changes in the Leydig cell volume of the knockout mice when compared with their wild-type littermates, the point sampled intercept method was used (Gundersen et al. 1988).
Using the same images that were randomly captured for Hoechst staining, the Leydig cell outlines were clearly identified due to their distinct morphology. A sine weighted frame was placed on the image and the intercept test grid was placed so that one of the intercept lines passed through the bottom left corner as well as a randomly selected number.
Using Spot analysis software and following the intercept grid as a guide, a line was drawn across the chosen cells. One hundred cell intercepts were measured for each animal.
Testosterone RIAs
Testosterone was extracted from plasma with ether according to the method of Pierantoni et al.(1984) and from tissue using the method of Fink et al.(2005). Testosterone was measured in the extracted tissue and plasma samples (Yeung et al. 1988). The interassay variation was 6.3% and the intraassay variation was 2.8% for the testicular testosterone and 7.6% for the plasma testosterone RIAs.
Statistical analyses
All statistical analyses were performed using GraphPad Prism 4.0 (GraphPad Software Inc., San Diego, CA, USA). Data are expressed as the mean±s.e.m.
When comparing the knockout animals with their wild-type littermates, the Student’s unpaired t-test was used to determine significance. The significance level was set at P<0.05. When determining significance across all groups, the ANOVA was used with a post hoc Bonferroni correction test with the significance level set at P<0.05.
The coefficient of error equation was used to test variability of the observations to ensure adequate sampling (Gundersen & Jensen 1987). This equation was used for the optical disector, the physical disector and point counting.
Results
No significant differences in body weight between the αERKO (t-test; P=0.42) or βERKO mice (t-test; P=0.77) and their respective wild-type littermates were seen. There was, however, a significant difference in body weight between the two groups of wild-type littermates (Bonferroni, P<0.001; Fig. 2a). Testis weight was significantly reduced in the αERKO mice when compared with the wild-type controls (t-test; P=0.0022; Fig. 2b) but no difference in testis weight was seen between the βERKO mice and their wild-type controls (t-test; P=0.24).
Histological sections from wild-type littermates of both the αERKO and the βERKO exhibited normal testicular histology with full spermatogenesis (Fig. 3). The testes of the αERKO male mice showed disrupted seminiferous tubules with a partial or complete loss of germ cells. In the most severely affected tubules, Sertoli cells were seen lining the otherwise empty tubules. The testes from the βERKO mice appeared to show a normal histology with complete spermatogenesis. However, within the interstitial tissue there appeared to be increased numbers of Leydig cells.
Stereology
When the volume contributed by seminiferous tubules and interstitial tissue was estimated using tissue point counting, no significant differences were seen between the αERKO and the βERKO mice and their respective wild-type littermates in the percentage of seminiferous tubules (92.9–93.9%) and the interstitial tissue (6.1–7.1%), despite the severe cellular loss in some but not all αERKO tubules.
The fractionator method and optical disector were used to evaluate the effect of loss of ERα and ERβ on the individual cell types within the testis. The number of Leydig cells per testis was significantly increased (t-test; P=0.0078) in βERKO mice when compared with their wild-type littermates (Fig. 4a) with ~40% more Leydig cells observed in the βERKO mice. No significant difference was observed in the number of Leydig cells per testis in the αERKO when compared with their respective wild-types (t-test; P=0.79). There were no differences seen in Sertoli cell numbers per testis in the βERKO (t-test; P=0.099) or the αERKO mice (t-test; P=0.27) when compared with their wild-type litter-mates (Fig. 4b).
A significant loss of all germ cells per testis was seen in the αERKO mice; spermatogonia (t-test; P=0.024), spermatocytes (t-test; P=0.0106) and spermatids (t-test; P=0.008; Fig. 5) compared to their wild-type littermates. Conversely, spermatogonial numbers per testis were significantly increased in the βERKO mice (t-test; P=0.033). No significant change in the number of spermatocytes per testis (t-test; P=0.43) or spermatids per testis (t-test; P=0.12) was observed in the βERKO mice when compared with the β wild-type animals.
In all groups, active caspase-3 was immunolocalised only to the spermatocytes of the testes (Fig. 6a–c). There were no significant differences in the number of cells per testis showing immunoreactivity for active caspase-3 cells (t-test; P=0.63) between the βERKO and wild-type mice. The numbers of active caspase-3 immunopositive cells per testis in the αERKO were, however, increased significantly (t-test; P=0.0008) when compared with their wild-type littermates (Fig. 7a).
Hoechst staining was used to strengthen the results seen in the active caspase-3, as this stain identifies apoptotic cells by nuclear morphology. Predominantly, spermatocytes were seen as Hoechst-positive but one or two round spermatids per section were also seen. Both cell types were included in the total number of apoptotic cells (Fig. 6d–f). Once more, there were no significant differences in the number of cells per testis, showing reactivity for Hoechst between the βERKO and wild-type mice (t-test, P=0.95). The numbers of Hoechst-positive cells per testis in the αERKO were significantly increased (t-test; P=0.046) when compared with their wild-type littermates (Fig. 7b).
Testosterone concentrations
Plasma and testicular testosterone concentrations (Fig. 8) were significantly increased (t-test; P<0.0001 plasma; P=0.0013 testes) in the αERKO mice when compared with their wild-type littermates. Despite the increased number of Leydig cells per testis in the βERKO mice there was no significant increase in testicular testosterone (t-test; P=0.81) or plasma testosterone (t-test; P=0.51) concentrations in these animals, when compared with the wild-type mice.
Discussion
This study confirms earlier reports that there is a major disruption of spermatogenesis in the adult αERKO mice (Lubahn et al. 1993) and that apparently testicular morphology is unaltered following loss of the ERβ (Krege et al. 1998). However, our data show that inactivation of the ERβ gene does affect the cellular composition of the testis (Table 1) and demonstrates that confining a study to qualitative assessment and sperm output may not provide an accurate picture of testicular morphology.
The results show that the number of adult Leydig cells per testis was unaffected by the inactivation of the ERα, however, loss of ERβ resulted in a 40% increase. This is in contrast with fetal Leydig cells where it has been shown that loss of neither ERα nor β affects the number of cells in the mouse 2 days post partum (Delbes et al. 2004, 2005). Fetal Leydig cells express ERα but have not, as yet, been shown to express ERβ (Jefferson et al. 2000), which may explain the lack of effect of ERβ on these cells. Our data suggest that activation of ERβ may normally have an inhibitory effect on the regulation of adult Leydig cell number.
As well as affecting Leydig cell number, oestrogen also affects the steroidogenic capacity of the Leydig cell. Studies using aromatase-deficient mice have shown that these animals have elevated circulating testosterone and LH and hyperplastic Leydig cells (Robertson et al. 1999). Our data confirm earlier studies that the inhibitory effect of oestrogen on testosterone production may be mediated by ERα since αERKO mice display similar changes to mice deficient of oestrogen (Oliveira et al. 2002). On the other hand, βERKO mice, despite having increased Leydig cells per testis, have normal plasma and testicular testosterone concentrations and circulating LH levels (Temple et al. 2003). At the cellular level these Leydig cells had a smaller volume suggesting a decrease in testosterone production by each individual cell. Since these changes occur in the presence of normal LH levels this suggests that activation of ERβ, unlike ERα, may normally mediate a direct stimulatory effect on testosterone production in the adult Leydig cell. Such an effect is not observed in fetal Leydig cells which lack ERβ (Delbes et al. 2004).
In the rat, there is evidence that administration of exogenous oestrogens and/or xenoestrogens during development may inhibit Sertoli cell number in the adult (Atanassova et al. 1999). The evidence for a role of oestrogen in Sertoli cell proliferation in the mouse is less clear. Sertoli cell numbers were unchanged in the aromatase-deficient mouse (Robertson et al. 1999). Similarly, no effect on Sertoli cell number per testis was seen in either the αERKO or the βERKO mice in the present study when compared with their respective wild-type littermates. This is perhaps surprising since ERα mRNA and protein are expressed in Sertoli cells and, while ERβ mRNA expression has yet to be demonstrated, the protein has been reported in these cells (Zhou et al. 2002). The reported effects of exogenous oestrogens on Sertoli cell number in the rat (Atannasova et al. 1999) may thus be a species specific effect or reflect the fact that Sertoli cell proliferation is only inhibited by supraphysiological levels of oestrogen. The latter is supported by Robertson et al.(2002) who demonstrated that a soy containing diet may decrease Sertoli cell number in the wild-type but not the aromatase knockout mouse.
The effects of the loss of the ERα receptor on germ cells have been previously described (Lubahn et al. 1993). Gonocyte number was found to be normal in the neonatal αERKO mice (Delbes et al. 2005) but spermatogenesis progressively became disrupted due to the increased tubular pressure relating to reduced fluid reabsorption in the efferent ducts (Hess 2000). Our findings of increased caspase-3-positive staining and fragmented pyknotic nuclei using the Hoechst stain confirm that perturbations of the efferent ducts increase the rate of apoptosis in germ cells.
The effects of inactivation of ERβ in the adult mouse have not previously been described. Delbes et al.(2004) have demonstrated that an increased number of gonocytes are present in the 2-day-old βERKO mice and suggested that this increase results from an increase in cell proliferation and decreased apoptosis. A similar increase in the number of spermatocytes per testis was observed in 11-week-old mice in this study. There were, however, no significant increases in the number of meiotic germ cells in these mice. The lack of an increased rate of germ cell apoptosis and the presence of normal circulating FSH concentrations in the βERKO mice (Temple et al. 2003) suggests that while there may be a larger pool of spermatogonia persisting in the adult mouse the number being recruited into spermatogenesis at any one time is not affected by the presence or absence of ERβ. Further studies are required to test this hypothesis.
Conclusion
In conclusion, this study demonstrates that in the mouse ERβ is involved in the regulation of adult Leydig cell number and may also affect the steroidogenic capacity of individual Leydig cells. In addition, the disrupted endocrine profile in the αERKO mouse resulted in hypertrophic Leydig cells and profound cellular loss through active caspase-3 activation in the spermatocytes.
Summary of the effects of loss on the testis of oestrogen receptor α (ERα) and β (ERβ) when compared with wild-type mice.
| Parameter | αERKO | βERKO |
|---|---|---|
| Testis weight | ↓ | = |
| Leydig cell number per testis | = | ↑68% |
| Leydig cell volume | ↑ | ↓ |
| Plasma testosterone | ↑ | = |
| Number of Sertoli cells per testis | = | = |
| Number of spermatogonia per testis | ↓28% | ↑38% |
| Number of spermatocytes per testis | ↓42% | = |
| Number of spermatids per testis | ↓45% | = |
| Germ cell apoptosis per testis | ↑ | = |
Histology of the testis. The testis cells are annotated inside the disector frame. Cells that appeared completely within the frame or intersect the dashed inclusion lines were counted. Cells that intersect the solid exclusion lines were not counted. L, Leydig; S, Sertoli; A, spermatogonia; C, spermatocytes. The round and the elongated spermatids are identified on the left and right respectively.
Citation: Reproduction 134, 2; 10.1530/REP-07-0025
Comparison of (a) body weight and (b) testicular weight between α and β oestrogen receptor knockout mice. Bars represent mean±s.e.m. αWT (α wild-type littermates) n=8, αERKO body weight n=8, αERKO testis weight n=7, βWT (β wild-type littermates) n=5, βERKO n=8. **P<0.005, ***P<0.001 when compared with wild-type mice.
Citation: Reproduction 134, 2; 10.1530/REP-07-0025
Haematoxylin- and eosin-stained sections of testis showing (a) seminiferous tubule disruption with severe cellular loss in the αERKO mice. In atrophic tubules, Sertoli cells are seen lining the basement membrane. (b) βERKO testes showing normal tubules with increased Leydig cell numbers. (c) Testis sections from the αERKO wild-type littermate’s and (d) from the βERKO wild-type littermates showed normal morphology. Bars=50 μm.
Citation: Reproduction 134, 2; 10.1530/REP-07-0025
The total number of (a) Leydig cells and (b) Sertoli cells per testis in α and β oestrogen receptor knockout mice. Bars represent mean±s.e.m. αWT n=8, αERKO n=8, βWT n=5, βERKO n=8. ** P<0.01 when compared with β wild-type mice.
Citation: Reproduction 134, 2; 10.1530/REP-07-0025
The total number of (a) spermatogonia, (b) spermatocytes and (c) spermatids per testis from α and β oestrogen receptor knockout mice. Bars represent mean±s.e.m. αWT n=8, αERKO n=8, βWT n=5, βERKO n=8. *P<0.05, **P<0.01 when compared with β wild-type mice.
Citation: Reproduction 134, 2; 10.1530/REP-07-0025
Immunohistochemistry for active caspase-3 (a–c). (a) Active caspase-3 apoptotic cells seen in the testis sections from αERKO mice. Testis sections from the βERKO mice (b) and both the αERKO and βERKO wild-type littermates (not shown) had only occasional active caspase-3 immunoreactive cells. Arrows indicate immunopositive cells. The rabbit IgG control (c) showed no immunopositive staining. Bars=50 μm. Hoechst stained testis sections (d–f) allowed identification of apoptotic cells using morphological criteria as indicated by arrows. (d) There was a significant increase in cell numbers in the testis sections from αERKO mice. Both the βERKO mice (e) and the wild-type littermates (f) showed only the occasional apoptotic cell. Bars=10 μm.
Citation: Reproduction 134, 2; 10.1530/REP-07-0025
(a) The total number of apoptotic spermatocytes indicated by active caspase-3 immunostained cells per mouse testis between the α and the β oestrogen receptor knockout mice. Bars represent mean±s.e.m. αWT n=8, αERKO n=8, βWT n=5, βERKO n=8. ***P<0.001 when compared with α wild-type mice. (b) The total number of apoptotic cells as indicated by Hoechst staining per testis of the α and βERKO mice and their respective wild-types. Bars represent mean±s.e.m. αWT n=3, αERKO n=3, βWT n=3, βERKO n=3. *P<0.05 when compared with α wild-type mice.
Citation: Reproduction 134, 2; 10.1530/REP-07-0025
Testosterone concentrations in (a) plasma (b) testis in the α and the β oestrogen receptor knockout mice. Bars represent mean±s.e.m. αWT n=8, αERKO n=8, βWT n=5, βERKO n=8. ***P<0.0001 when compared with wild-type mice.
Citation: Reproduction 134, 2; 10.1530/REP-07-0025
Mean Leydig cell volume in α and βERKO mice and their respective wild-types. Bars represent mean±s.e.m. αWT n=8, αERKO n=8, βWT n=5, βERKO n=8. ***P<0.001, *P<0.05 when compared with wild-type mice.
Citation: Reproduction 134, 2; 10.1530/REP-07-0025
The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.
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