Colony-Stimulating Factor-1 Plays a Major Role in the Development of Reproductive Function in Male Mice

Paula E. Cohen, Matthew P. Hardy and Jeffrey W. Pollard

Department of Developmental and Molecular Biology (P.E.C., J.W.P.), Department of Obstetrics and Gynecology (J.W.P.), Albert Einstein College of Medicine, Bronx, New York 10461,
Population Council and The Rockefeller University (M.P.H.), New York, New York 10021


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Colony-stimulating factor-1 (CSF-1) is the principal regulator of cells of the mononuclear phagocytic lineage that includes monocytes, tissue macrophages, microglia, and osteoclasts. Macrophages are found throughout the reproductive tract of both males and females and have been proposed to act as regulators of fertility at several levels. Mice homozygous for the osteopetrosis mutation (csfmop) lack CSF-1 and, consequently, have depleted macrophage numbers. Further analysis has revealed that male csfmop/csfmop mice have reduced mating ability, low sperm numbers, and 90% lower serum testosterone levels. The present studies show that this low serum testosterone is due to reduced testicular Leydig cell steroidogenesis associated with severe ultrastructural abnormalities characterized by disrupted intracellular membrane structures. In addition, the Leydig cells from csfmop/csfmop males have diminished amounts of the steroidogenic enzyme proteins P450 side chain cleavage, 3ß-hydroxysteroid dehydrogenase, and P450 17{alpha}-hydroxylase-lyase, with associated reductions in the activity of all these steroidogenic enzymes, as well as in 17ß-hydroxysteroid dehydrogenase. The CSF-1-deficient males also have reduced serum LH and disruption of the normal testosterone negative feedback response of the hypothalamus, as demonstrated by the failure to increase LH secretion in castrated males and their lack of response to exogenous testosterone. However, these males are responsive to GnRH and LH treatment. These studies have identified a novel role for CSF-1 in the development and/or regulation of the male hypothalamic-pituitary-gonadal axis.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Colony stimulating factor-1 (CSF-1) is a homodimeric growth factor that regulates the survival, proliferation, motility, and differentiation of cells of the mononuclear phagocytic lineage (1). The action of CSF-1 is mediated through a high-affinity cell surface receptor tyrosine kinase, CSF-1R, which is the product of the c-fms protooncogene (2) and which is present on all cells belonging to the mononuclear phagocyte lineage, including terminally differentiated tissue macrophages, osteoclasts, and microglia of the brain. The functions for CSF-1 were confirmed in vivo by observations in mice homozygous for the CSF-1 gene null mutation, osteopetrotic (csfmop, formerly op) (3, 4). Phenotypically, these mice lack CSF-1 and have significantly depleted numbers of macrophages in most tissues and very few osteoclasts, the latter resulting in the characteristic phenotype, osteopetrosis (5, 6, 7). Detailed studies on particular macrophage populations in the nullizygous mice indicated that those macrophages associated with trophic and/or scavenging functions during development are the most affected by the absence of CSF-1 whereas those macrophages associated with immune functions are relatively unaffected (7). In addition to its expression in mononuclear phagocytes, the CSF-1R is expressed in oocytes, decidual cells, and trophoblast in females (8, 9), suggesting additional roles for CSF-1 outside the hematopoietic system. Studies of reproductive function in csfmop/csfmop mice revealed female fertility defects characterized by poor ovulatory rates, small litters, and failure to lactate (10, 11), confirming the importance of CSF-1 in the female reproductive tract. These studies also revealed a hitherto unsuspected role for this growth factor in male fertility regulation.

The absence of CSF-1 in csfmop/csfmop male mice results in 90% lower serum testosterone (T) concentrations than wild type males (10) and is associated with low epididymal sperm numbers and reduced libido (10). In the testis, CSF-1 regulates the resident population of testicular macrophages (TMs) (12), which form an intimate structural relationship with neighboring steroidogenic Leydig cells (13, 14, 15). In csfmop/csfmop males, TM numbers are severely depleted throughout development (12) and this, together with previous reports of a regulatory role for TMs on Leydig cell function (for review see 16 , suggests that CSF-1 might act in a paracrine fashion, through TMs, to regulate steroidogenesis. However, because CSF-1, by acting through microglia, is involved in the development of functional neuronal processing in the brain (17), CSF-1 might also act at the level of the hypothalamus and pituitary to modulate the release of GnRH and/or the gonadotropins, LH and FSH, such that the LH-mediated regulation of Leydig cell function is disrupted in csfmop/csfmop males. The present studies were aimed at defining the molecular basis of the fertility defects in csfmop/csfmop males and demonstrate that this growth factor plays an essential role in the development and functioning of the hypothalamic-pituitary-gonadal axis.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Basal Steroidogenesis by Leydig Cells of csfmop/csfmop Males
To determine whether the lower serum T levels in csfmop/csfmop males are due to decreased testicular steroidogenesis, rather than to increased metabolic clearance, basal T synthesis by isolated Leydig cells was assessed and was found to be significantly lower in cells recovered from csfmop/csfmop than those from +/csfmop controls (Table 1Go). The 86.3% reduction in basal T synthesis by Leydig cells derived from csfmop/csfmop males is similar to the ~90% lower serum T observed in these animals (10), suggesting that the reduction in serum T is due directly to lowered testicular steroidogenic capacity. Similar T synthesis results were obtained from incubations of whole testis in vitro (data not shown).


View this table:
[in this window]
[in a new window]
 
Table 1. Testosterone Synthesis by Isolated Leydig Cells in Vitro

 
To examine the cause of the reduced steroidogenesis, the enzymatic activity of the steroidogenic enzymes in isolated Leydig cells was examined. Side chain cleavage (P450scc), 3ß-hydroxysteroid dehydrogenase (3ßHSD), P450 17{alpha}-hydroxylase lyase (P45017{alpha}), and 17ß-hydroxysteroid dehydrogenase (17ßHSD) activities are all significantly reduced in isolated Leydig cells from csfmop/csfmop males compared with +/csfmop Leydig cells (Table 2Go). P450scc catalyzes the conversion of cholesterol to pregnenolone in the mitochondria. Its activity in csfmop/csfmop Leydig cells is reduced to 18.1% of that found in heterozygote Leydig cells. The activity of 3ßHSD, which is responsible for the conversion of pregnenolone to progesterone, is reduced to 66.3% of normal. P45017{alpha} is a dual-activity enzyme responsible for the conversion of progesterone to 17{alpha}-hydroxyprogesterone through its hydroxylase activity and the subsequent formation of androstenedione through its lyase activity. The hydroxylase activity of P45017{alpha} is reduced to 51.3%, while its lyase activity is 80.5% of normal levels (Table 2Go). 17ßHSD, which completes the pathway by converting androstenedione to T, is also reduced in csfmop/csfmop Leydig cells to 40.6% of that seen in +/csfmop Leydig cells (Table 2Go).


View this table:
[in this window]
[in a new window]
 
Table 2. Enzyme Activities in Isolated Leydig Cells of +/csfmop and csfmop/csfmop Males

 
To determine the molecular basis for the reduced enzymatic activity in Leydig cells from csfmop/csfmop males, the protein and mRNA concentrations for P450scc, 3ß-HSD, and P45017{alpha} were measured, either in whole testis or in isolated Leydig cells, or both. Anti-P450scc antibody detected a single band of 52 kDa in testis, Leydig cells, ovary, and adrenal extracts but not in spleen (Fig. 1Go, A and B). This band corresponds to that detected in a commercial control sample of purified bovine mitochondria (see Materials and Methods; Fig. 1AGo). The concentrations of P450scc in whole testicular extracts of csfmop/csfmop males, when normalized to glyceraldehyde phosphodehydrogenase (GAPDH) protein concentration in three separate Western blots, was very significantly reduced (P < 0.0005, Mann Whitney test, n = 9) to 16.7% of wild type concentrations (Fig. 1AGo). These data were confirmed by examination of an isolated Leydig cell protein extract in which the level of P450scc enzyme was 14.7% of that found in heterozygote Leydig cells (Fig. 1BGo). Interestingly, however, mRNA expression of P450scc, as determined by ribonuclease protection assay (RPA) and normalized to GAPDH mRNA expression, was similar in the testes of csfmop/csfmop and +/csfmop males (Fig. 1CGo), indicating a posttranscriptional effect on the testicular content of this enzyme.



View larger version (45K):
[in this window]
[in a new window]
 
Figure 1. Analysis of P450scc Expression in the csfmop/csfmop Testis

A, Western blot analysis of whole testis protein extracts using a rabbit antibody raised against the bovine adrenal P450scc. Lanes 1 to 3, +/csfmop; lanes 4 to 6, csfmop/csfmop; lane 7, ovary; lane 8, adrenal; lane 9, spleen; lane 10, control purified porcine mitochondrial extract. Solid arrowhead indicates specific band corresponding to P450scc. *, Significant variation between genotypes (P < 0.0005, Mann Whitney test). B, Western blot analysis of isolated Leydig cells using the same antibody as in panel A. Lane 1, +/csfmop; lane 2, csfmop/csfmop; lane 3, C-csfmop/csfmop; lane 4, ovary; lane 5, spleen. Solid arrowhead indicates band corresponding to P450scc. C, Autoradiograph of a RPA to detect mRNA expression of P450scc and GAPDH mRNA in total RNA isolated from whole testis. Lane 1, P450scc probe (P1); lane 2, GAPDH probe (P2); lanes 3 to 5, +/csfmop; lanes 6 to 8, csfmop/csfmop; lane 9, ovary; lane 10, adrenal; lane 11, yeast. The protected fragment for P450scc is 250 bp and for GAPDH is 147 bp. In each panel (A-C), densitometric analyses (ImageQuant) for means of testes lanes are presented in the adjoining bar charts. Protein levels were standardized using GAPDH levels (for whole testis) or cell number (for isolated Leydig cells). Bars represent arbitrary units ± SEM].

 
The concentration of testicular 3ßHSD in csfmop/csfmop males was also found to be significantly reduced compared with that of +/csfmop males (Fig. 2Go, A and B). The specificity of the anti-3ßHSD antibody was confirmed using protein extracts from COS-1 cells expressing either isoform I or VI, both of which are expressed in testicular Leydig cells, while only isoform I is found in the adrenal gland (18). Both isoforms migrate at similar rates on 10% polyacrylamide gels, having only ~2 kDa difference in molecular mass (18), and both are detected by the antibody. This band migrates at the same rate as that detected in the steroidogenic tissues, but no comigratory band was detected in the spleen samples. The 3ßHSD concentration in whole testis extracts from csfmop/csfmop males, determined by densitometric analysis of four separate Western blots, is 43.1% of the +/csfmop level (Fig. 2AGo), a figure that is statistically significant (P < 0.05, Mann Whitney test, n = 12). In an extract of Leydig cells from csfmop/csfmop males, anti-3ßHSD antibody Western blotting detects only 14.8% the amount of 3ßHSD detected in a Leydig cell extract from +/csfmop males (Fig. 2BGo). Northern blot analysis of total RNA from +/csfmop and csfmop/csfmop testes detects a mRNA transcript of 1.6 kb whose concentration does not differ between genotypes (Fig. 2CGo). A similar band is detected in RNA isolated from ovaries and adrenal glands, but not spleen (Fig. 2CGo). These results were confirmed by a RPA using a probe specific to nucleotides 502–839 of the published mRNA sequence (data not shown).



View larger version (49K):
[in this window]
[in a new window]
 
Figure 2. Analysis of 3ßHSD Expression in the csfmop/csfmop Testis

A, Western blot analysis of whole testis protein extracts using a rabbit anti-human 3ßHSD antibody. Lanes 1 to 3, +/csfmop; lanes 4 to 6, csfmop/csfmop; lane 7, ovary; lane 8, adrenal; lane 9, spleen; lanes 10 and 11, COS-1 cell extracts expressing 3ßHSD isoform I and isoform VI. *, Significant variation between genotypes (P < 0.05, Mann Whitney test). B, Western blot analysis of isolated Leydig cells using the same antibody as in panel A above. Lane 1, +/csfmop; lane 2, csfmop/csfmop; lane 3, C-csfmop/csfmop; lane 4, ovary; lane 5, spleen. C, Northern blot analysis of total testicular RNA (20 µg) probed with a [32P]-labeled cDNA to 3ßHSD. Lanes 1 to 3, +/csfmop; lanes 4 to 6, csfmop/csfmop; lane 7, ovary; lane 8, adrenal; lane 9, spleen. Upper bands represent unprocessed RNA. In each panel (A-C), densitometric analyses (ImageQuant) for means of testes lanes are presented in the adjoining bar charts. Protein levels were standardized using GAPDH levels (for whole testis) or cell number (for isolated Leydig cells). Bars represent arbitrary units ± sem. In all cases, solid arrowheads indicate specific bands corresponding to 3ßHSD.

 
The concentration of P45017{alpha} protein is also reduced in whole testis extracts from csfmop/csfmop males to 90% of normal (Fig. 3AGo) which, although being only a 10% reduction, is statistically significant (P < 0.05, Mann Whitney test for the means of two Western blots, n = 6). The antibody against P45017{alpha} detects a band of 52 kDa in protein extracts from testis, Leydig cell, and ovary, but not in adrenal or splenic samples. In testis and to a lesser extent in spleen, the antibody also detects a major cross-reactive band of around 49 kDa. This band is not present in isolated Leydig cell extracts. The P45017{alpha} protein level in two isolated Leydig cell extracts from csfmop/csfmop males is reduced to 44.7% of that seen in heterozygote Leydig cells (Fig. 3BGo). However, P45017{alpha} mRNA concentrations are normal in total RNA extracts from csfmop/csfmop whole testis (Fig. 3CGo). Expression of the 1.7-kb P45017{alpha} message is also detected in ovarian total RNA but not in adrenal and spleen RNA (Fig. 3CGo). The normal expression of P45017{alpha} mRNA in csfmop/csfmop testes was confirmed by RPA (data not shown).



View larger version (54K):
[in this window]
[in a new window]
 
Figure 3. Analysis of P45017{alpha} Expression in the csfmop/csfmop Testis

A, Western blot analysis of whole testis using a rabbit antibody raised against porcine testicular P45017{alpha}. Lanes 1 to 3, +/csfmop; lanes 4 to 6, csfmop/csfmop; lane 7, ovary; lane 8, adrenal; lane 9, spleen; lane 10, control purified sheep microsomal extract. B, Representative Western blot analysis of isolated Leydig cells using the same antibody as in panel A above. Lanes 1 and 2, +/csfmop; lanes 3 and 4, csfmop/csfmop; lane 5, C-csfmop/csfmop; lane 6, ovary; lane 7, adrenal; lane 8, spleen; lane 9, control microsomal extract from sheep adrenal gland. C, Northern blot analysis of total testicular RNA probed with a 32P-labeled cDNA to P45017{alpha}. Lanes 1 to 3, +/csfmop; lanes 4 to 6, csfmop/csfmop; lane 7, ovary; lane 8, adrenal; lane 9, spleen. Upper bands represent unprocessed RNA. In each panel (A-C), densitometric analyses (ImageQuant) for means of testes lanes are presented in the adjoining bar charts. Bars represent arbitrary units ± SEM and, in the case of the Western blots, are standardized to GAPDH control levels. In all cases, solid arrowheads indicate specific bands corresponding to P45017{alpha}. *, Significant variation between genotypes (P < 0.05, Mann Whitney test).

 
Adrenal Steroidogenesis in +/csfmop and csfmop/csfmop Males
To test whether adrenal steroidogenesis is also disrupted in csfmop/csfmop males, levels of P450scc and 3ßHSD protein in the adrenal gland were measured by Western blot analysis. Comparison of these enzyme levels in the adrenal glands of csfmop/csfmop and +/csfmop males revealed no differences between genotypes (Fig. 4Go, A and B). Furthermore, serum corticosterone concentrations in csfmop/csfmop and +/csfmop males were also not significantly different (Fig. 4C).



View larger version (32K):
[in this window]
[in a new window]
 
Figure 4. Adrenal Steroidogenesis in csfmop/csfmop Males

A, Western blot analysis of adrenal gland protein extracts using anti-P450scc antibody. Lanes 1 to 3, +/csfmop adrenals; lanes 4 to 6, csfmop/csmop adrenals; lane 7, +/csfmop ovary; lane 8, +/csfmop testis; lane 9, +/csfmop spleen. B, Western blot analysis of adrenal gland extracts using anti-3ßHSD antibody. Lanes are the same as those described for panel A. In all cases, solid arrowheads indicate specific bands corresponding to 3ßHSD. C, Serum corticosterone concentrations (ng/ml ± SD) in +/csfmop and csfmop/csfmop males (n = 12 for both groups). No statistical difference between groups.

 
Leydig Cell Ultrastructure
To assess the structural consequences of the csfmop mutation on testicular interstitial cells, we prepared testes for electron microscopy. The gross morphology of csfmop/csfmop testes appears normal (10) (despite the severe depletion of TMs). However, csfmop/csfmop Leydig cells had striking ultrastructural irregularities. Tightly coiled membranous whorls are a normal feature of rodent Leydig cells (19) and are absent in other testicular cell types. These structures are readily observed in Leydig cells from +/csfmop males (Fig. 5AGo) and are limited to a small portion of the cytoplasmic volume. In csfmop/csfmop males, however, the membranous whorls occupy the majority of the Leydig cell cytoplasm, often occluding and displacing the nucleus (Fig. 5BGo). The increased size of the whorls observed in csfmop/csfmop Leydig cells appears to result from unravelling of the whorls and/or dilation of the intermembrane spaces. The membranes of the mitochondria also appeared to be disrupted, with the occasional loss of cisternal structures.



View larger version (112K):
[in this window]
[in a new window]
 
Figure 5. Electron Micrographs of Testicular Leydig Cells

Top, +/csfmop; middle, csfmop/csfmop; and bottom, C-csfmop/csfmop male mice. Bars are 1 µm in length. L, Leydig cell; M, macrophage.

 
Stimulation of Leydig Cell Steroidogenesis Using Gonadotropins and Intermediate Substrate (22R-Hydroxycholesterol) for Side Chain Cleavage Enzyme
Since androgen biosynthesis by testicular Leydig cells is regulated by pituitary LH, the low T output in csfmop/csfmop males might result from reduced Leydig cell sensitivity to LH. Therefore, LH-stimulated T synthesis by isolated Leydig cells derived from +/csfmop and csfmop/csfmop males was measured (Table 1Go). As expected, LH stimulated T synthesis by Leydig cells taken from heterozygote males by 13-fold. Similarly, LH stimulated basal T synthesis in csfmop/csfmop Leydig cells to a level that, although still significantly lower than control values, was factorially greater (65-fold) as a result of the lower basal T concentration. Similar responses were obtained in vivo by treating mice with a single dose of the LH analog, human chorionic gonadotropin (hCG), and measuring serum T concentrations 1 h later. In confirmation of previous data (10), basal serum T concentrations were reduced by ~90% in csfmop/csfmop males. Heterozygous males responded to hCG injection with an 11-fold increase in circulating concentrations of T (Table 3Go). Circulating T levels rose even more dramatically (147-fold) in csfmop/csfmop males to a concentration equivalent to that of hCG-stimulated control males (Table 3Go). Together, these results, confirmed by analysis of LH-stimulated T synthesis by whole testes in vitro (data not shown), demonstrate that the steroidogenic ability of csfmop/csfmop males can be enhanced greatly by gonadotropin treatment both in vitro and in vivo, indicating that a sufficient number of LH receptors are present to elicit steroidogenic responses comparable to that of wild type males.


View this table:
[in this window]
[in a new window]
 
Table 3. Effect of hCG Treatment on Serum T Concentrations

 
To distinguish between LH-stimulated and LH-independent events during the early stages of steroidogenesis, T synthesis by isolated Leydig cells was assessed in the presence of 22R-hydroxycholesterol (22R-CHOL), a steroid intermediate substrate for the enzyme P450scc. Addition of 22R-CHOL to Leydig cell cultures bypasses the early LH-regulated events involving P450scc, as well as cholesterol mobilization from the intracellular lipid stores (20), but does not bypass the requirement for P450scc activity. 22R-CHOL stimulation results in similar T output in control and csfmop/csfmop Leydig cell groups and is comparable to the LH response (Table 1Go). However, the level of stimulation in csfmop/csfmop Leydig cells is far greater than that of +/csfmop Leydig cells due to the differences in basal T synthesis between genotypes.

Hypothalamic-Pituitary Function in csfmop/csfmop Males
The steroidogenic failure in csfmop/csfmop males might be due to low circulating LH concentrations. For this reason, serum LH concentrations were measured by RIA. Basal serum LH in csfmop/csfmop males is ~90% lower than that of heterozygote males (Table 4Go). Treatment with the potent GnRH agonist, histerilin, at a dose shown to cause maximal stimulation (data not shown), increases serum LH by 35-fold in csfmop/csfmop males, compared with a 5-fold increase in +/csfmop males (Table 4Go). Despite this large stimulation, however, histerilin-treated LH levels remain significantly lower in csfmop/csfmop males than in heterozygote controls. Interestingly, at a dose of 1 ng histerilin per g body weight, csfmop/csfmop males displayed a 10-fold increase in serum LH. However, this dose was not sufficient to stimulate LH secretion in +/csfmop males, whose serum LH concentrations remained at basal levels. Thus at this dose, the serum LH concentrations were comparable between genotypes.


View this table:
[in this window]
[in a new window]
 
Table 4. Serum LH Concentrations in csfmop/csfmop Male Mice before and after Treatment with the GnRH Agonist, Histerilin, or 5 Days after Bilateral Orchidectomy

 
Serum FSH concentrations were also significantly reduced in csfmop/csfmop males (P < 0.001, Mann-Whitney test). However, this reduction was not as great as that for serum LH; serum FSH in +/csfmop males was 6.87 ± 1.07 ng/ml (mean of 30 samples ± SEM) compared with 2.19 ± 0.34 ng/ml for csfmop/csfmop males (10 samples), representing a 70% reduction in circulating FSH. Histerilin treatment, at a dose of 100 ng/g body weight, increased serum FSH concentrations by 4-fold in both +/csfmop and csfmop/csfmop males, to 24.19 ± 2.80 ng/ml (13 mice) and 7.84 ± 2.93 ng/ml (11 mice), respectively.

To investigate the negative feedback sensitivity of csfmop/csfmop males, heterozygote and homozygote mutant mice were subjected to either bilateral orchidectomy or sham surgery, and serum samples were obtained 5 days later. Serum LH concentrations in bilaterally orchidectomized +/csfmop males was 1.8-fold higher than in control males, as would be expected by release of negative feedback (Table 4Go), while sham-operated males showed no such stimulation (not shown). In contrast, bilateral orchidectomy had no effect on the serum LH concentrations in csfmop/csfmop males, indicating a loss of feedback sensitivity in these males. Furthermore, treatment with physiological concentrations of T postpubertally results in elevated, although not significant (P < 0.06), basal LH concentrations as compared with that of untreated csfmop/csfmop males. These effects of both castration and T treatment contradict the expectation that low serum T would increase, while high serum T would decrease, circulating LH concentrations via the classic negative feedback regulatory system. Taken together, these studies suggest that the pituitaries of csfmop/csfmop males are able to respond to GnRH by increasing gonadotropin output to a higher level, but that the hypothalamic-pituitary response to circulating androgen is disrupted in these mice.

Effect of CSF-1 on Reproductive Function in csfmop/csfmop Males
To investigate the effect of postnatal restoration of serum CSF-1 concentrations on male reproductive function, csfmop/csfmop males were treated with human recombinant CSF-1 (hrCSF-1) from day 2 of life (C-csfmop/csfmop). This regimen has been shown in previous studies to restore circulating concentrations of CSF-1 (7). Leydig cells obtained from CSF-1-treated csfmop/csfmop males produce significantly more T (~ 2.3-fold) than those from untreated csfmop/csfmop Leydig cells (Table 1Go), but their T synthesis is not restored to wild type levels, in line with the failure of hrCSF-1 treatment to restore circulating T concentrations in these mice (10). LH treatment of C-csfmop/csfmop Leydig cells results in stimulation of T synthesis to the same level of LH-stimulated heterozygote Leydig cells (Table 1Go), while 22R-CHOL treatment produces a level of T synthesis that is only 62.7% of the level of 22R-CHOL-stimulated +/csfmop Leydig cells, despite being 27-fold stimulated from basal levels (Table 1Go). Interestingly, however, Leydig cells isolated from CSF-1-treated C-csfmop/csfmop males contain increased, and often almost normal, protein levels for P450scc (74.2% of wild type level; Fig. 1BGo), 3ßHSD (93.9% of the +/csfmop level; Fig. 2BGo) and P45017{alpha} (68.5% of wild type levels; Fig. 3BGo). The recovery of steroidogenic enzyme proteins in C-csfmop/csfmop Leydig cells and testis is associated with the restoration of apparently normal Leydig cell ultrastructure in these mice (Fig. 5CGo).

To investigate the effect of CSF-1 treatment on hypothalamic-pituitary function in csfmop/csfmop males, serum LH concentrations were measured as described in Materials and Methods. While basal LH concentrations in untreated csfmop/csfmop males is 7.4% of the normal circulating concentration, CSF-1 treatment results in a significant increase (2.5-fold) in LH concentrations to 18.2% of normal, consistent with the increased basal synthesis of T by Leydig cells.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The osteopetrotic mouse mutation, a null mutation in the CSF-1 gene located on chromosome 3, results in a complete absence of systemic CSF-1 (3, 4). Cells of the mononuclear phagocytic lineage, including microglia, tissue macrophages, and osteoclasts, express the transmembrane tyrosine kinase CSF-1 receptor (the c-fms protooncogene product). Many populations of these cells are depleted in the nullizygous animals, including osteoclasts, the latter resulting in the characteristic osteopetrosis (5, 7, 21, 22). In addition, in the female reproductive tract the CSF-1 receptor is also expressed in oocytes, decidual cells, and trophoblast, and several studies have confirmed roles for CSF-1 in ovulation and during pregnancy (8, 11, 23, 24, 25). It was also observed that male csfmop/csfmop mice display fertility defects characterized by a reduction in libido and reduced sperm number (10). Such defects could be ascribed to the reduced circulating and intratesticular T concentrations in the mutant mice. The present studies have shown conclusively that this reduction in T concentration is due to reduced Leydig cell steroidogenic enzymic activity, with the most affected step being the conversion of cholesterol to pregnenolone catalyzed by P450scc. The reduced steroidogenesis was also associated with a reduction of greater than 90% in circulating LH concentration, while treatment of Leydig cells, testicular cultures, or intact mice with pharmacological doses of LH elevated T concentration and T synthesis to levels similar to those of wild type mice. Furthermore, serum LH concentrations themselves can be restored by treatment of csfmop/csfmop mice with a GnRH agonist. These data strongly suggest a defect in the hypothalamic-pituitary axis of csfmop/csfmop mice, a defect that was confirmed by our studies showing a loss of T-feedback sensitivity in these mice. Since cells of the mononuclear phagocytic lineage are the only cells in males that have been shown to express the CSF-1R, these profound defects in the hypothalamic-pituitary-gonadal axis show that mononuclear phagocytes play an important role in the establishment and functioning of this neuroendocrine axis.

The hypothalamus is the primary regulator of sexual function, liberating GnRH in a pulsatile fashion which, in turn, stimulates release of LH from the pituitary (26). The hypothalamus is rich in mononuclear phagocytic-lineage microglial cells (27) that express the CSF-1R and respond in culture to CSF-1 (17). Although hypothalamic populations of microglia have not been studied in csfmop/csfmop mice, in the retina the acquisition of these cells is delayed during development (7). Furthermore, adult csfmop/csfmop mice have intracortical processing problems associated with deficits in both excitatory and inhibitory neuronal firing, even though at a gross morphological level, anatomical abnormalities could not be detected in the cortical regions of the brain (17). In culture, CSF-1 is a trophic factor for embryonic neurons derived from many areas of the brain including the hypothalamus, provided that microglia are present in the cultures (17). Together, these data suggest that CSF-1 acting through the microglia plays an important developmental role in the establishment of brain function including hypothalamic function. However, these actions in the hypothalamus and pituitary must be fairly specific since adrenal steroidogenesis is unaffected, suggesting normal secretion of CRF by the parvocellular neurons and the subsequently normal release of ACTH from anterior pituitary corticotrophs.

While it is reasonable to conclude that the reduced steroidogenic capacity of csfmop/csfmop Leydig cells is due to the low circulating LH in adult mice, there are a number of paradoxical observations that suggest there might also be a local requirement for CSF-1 in the testis. Our data show that all the steroidogenic enzyme activities are reduced, with P450scc being the most depressed, consistent with the reduction in protein concentration observed. However, the mRNA concentrations for these enzymes are unaffected, suggesting that the deficit is posttranscriptional. This is contrary to expectation for a deficiency of LH, since this gonadotropin has been shown to regulate steroidogenic enzyme mRNA expression (28, 29). Furthermore, while the activity of P45017{alpha} is most affected by LH (30, 31), in the csfmop/csfmop mouse this enzyme is the least affected, whereas P450scc whose activity is relatively less modulated by LH (31), is the most affected by the csfmop mutation. Although, experimentally, the consequence of maintaining LH concentrations at ~10% in a pulsatile manner in wild type mice throughout life is unknown, the data suggest that there may also be local influences of CSF-1 in the testis interacting with the systemic regulation by LH. Interestingly, despite its statistical significance, the reduction in serum FSH in csfmop/csfmop males is not sufficient to cause any severe effects on the gross testicular morphology and seminiferous tubule cytology.

There is a large body of literature reporting both anatomical and physiological interactions between Leydig cells and TMs. The idea that TMs and Leydig cells are functionally coupled arose from the characterization of their unique structural relationship, together with the observations that vasectomy causes simultaneous destruction of both cell types (32), while pharmacological depletion of either cell type has severe consequences on the performance of the other cell type (33, 34, 35, 36). The appearance of adult Leydig cells during puberty occurs in an environment in which the TM population is already established (12, 15, 16, 37, 38) and is prevented when TMs are pharmacologically ablated before puberty (36). In adult males, the removal of TMs results in disruption of Leydig cell steroidogenesis (for review see 16 . In addition, TM-conditioned media, as well as cytokines that can be macrophage-derived (including tumor necrosis factor-{alpha} and interleukin-1), alter the steroidogenic function of Leydig cells in vitro (39, 40, 41, 42, 43). We have shown that CSF-1 is the major regulator of TMs, the only testicular cell type that expresses CSF-1R (10), and that this macrophage population is severely depleted in csfmop/csfmop mice throughout development. This depletion of macrophages in the CSF-1 nullizygous testis, therefore, is likely to have significant consequences for the functioning of Leydig cells. Consistent with this is the disruption of Leydig cell morphology in the mutant.

Interestingly, an increase in abundance and/or disruption of the Leydig cell membranous whorls has also been described in other testicular perturbation models, such as in experimentally cryptorchid rats (44) and rats with experimentally induced damage to the seminiferous tubules (45), which are also deficient in steroidogenesis (46, 47). The functional significance of these whorls remains unknown, but previous studies in wild type mice have demonstrated that the whorls are contiguous with the smooth endoplasmic reticulum (SER), suggesting they are regional modifications of their system (48). Since three of the testosterone-biosynthetic enzymes are intimately associated with the SER, the membrane disruptions seen in the csfmop/csfmop males might result in destabilization of these enzymes with the consequent reduction in protein content without significant effects on mRNA concentration. Interestingly, P450scc, the enzyme that is the most affected by the csfmop mutation, resides in the mitochondria and is therefore unaffected by the disruption of the endoplasmic reticular system. However, our preliminary ultrastructural analysis indicates that there might be similar, albeit more subtle, disruptions of the mitochondria in csfmop/csfmop Leydig cells. This idea that enzyme activities are perturbed as a result of physical disruption of Leydig cell architecture is supported by the observation that treatment of the nullizygous mice from birth with CSF-1 corrects both the TM population and Leydig cell morphology and restores steroidogenic enzyme protein levels to near wild type concentrations without completely correcting the steroidogenic deficiencies. It is interesting to note that, despite the remarkable ultrastructural defects in the Leydig cells of csfmop/csfmop males, they are still capable of responding acutely to hCG/LH treatment both in vitro and in vivo, suggesting, perhaps, that the total surface area of SER is not reduced in these cells, despite the obvious membrane disruption.

Androgen biosynthesis by testicular Leydig cells is required for normal reproductive function in adult males, as well as for the processes of sexual differentiation and puberty. During sexual differentiation, a surge of T secretion by Leydig cells of the fetal testis masculinizes the hypothalamic-pituitary axis (49). However, disrupting pituitary secretion of LH and FSH by a targeted mutation of their common {alpha}-chain does not affect gonadal development until after birth (50) despite the requirement for T during pituitary development, suggesting that neonatal and, perhaps early postnatal, Leydig cell T synthesis occurs independently of gonadotropins. During puberty, another surge of T secretion is required to initiate changes in the hypothalamus and pituitary, resulting in an alteration in the pattern of GnRH release from the hypothalamus (49). This resetting of the GnRH system at puberty results in maturation of the classic T-mediated negative feedback loop from the testis to the hypothalamic-pituitary axis and the subsequent onset of fecundity (49, 51, 52). Disruptions in T biosynthesis by Leydig cells as a consequence of the absence of TMs in csfmop/csfmop males during development might have a significant impact on the establishment of this feedback loop, in a manner observed in the nullizygous mice. Furthermore, Gaytan et al. (35) suggested that TMs produce a factor that directly affects hypothalamic-pituitary development. These observations, together with the physiological interactions between Leydig cells and TMs, suggest that the phenotype of csfmop/csfmop males might result from a complex mix of the actions of CSF-1 in the hypothalamus, pituitary, and testis during development.

All the reproductive defects described in this paper can be ascribed to the lack of CSF-1. However, restoration of circulating CSF-1 during the postnatal period only partially restores many of these deficits. This may be due to a requirement for embryonic or local synthesis of CSF-1 or, because CSF-1 is synthesized in a variety of forms (including cell surface, secreted, and secreted proteoglycanated forms), there may be a requirement for a form other than the soluble human recombinant form administered. Indeed, evidence has been obtained for both local and humoral actions of CSF-1 in regulating other mononuclear phagocytic populations (7).

These studies in the csfmop/csfmop mouse model provide compelling evidence that CSF-1 plays a fundamental role in the establishment and functioning of the hypothalamic-pituitary-gonadal axis, through its action on macrophages or microglia. This unique role for CSF-1/macrophage interactions in male reproductive function will have a significant impact on the understanding of the development of classic endocrine feedback systems and the involvement of immune cells in these processes.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Animals
Homozygous mutant (csfmop/csfmop) and heterozygous (+/csfmop) male mice were used at 12 weeks of age, and all studies were performed under the NIH guidelines for the care and treatment of experimental laboratory rodents. Animals undergoing CSF-1 treatment (C-csfmop/csfmop) were injected subcutaneously daily from day 2 of life with 12 µg of human recombinant CSF-1 (hrCSF-1; a generous gift of Chiron Corp., Emeryville, CA) in 0.05 ml 0.9% saline.

Bilateral orchidectomies were performed under methoxyflurane anesthesia (Metofane, Pittman-Moore Inc., Mundelein, IL). Sham-operated animals received similar surgical treatment without testicular excision. The mice were allowed to recover for 5 days before blood sampling.

Hormone Treatments and RIAs
To assess the effects of hCG treatment in vivo, heterozygote and homozygote mutant males were injected with 5 IU hCG per 10 g body weight (ip), and blood samples were obtained 1 h later for serum T measurement. For GnRH agonist experiments, histerilin was administered subcutaneously (at doses of 1, 50, or 100 ng per gram body weight in 0.9% saline solution containing 5% mannitol), 3 h prior to blood sampling. Sera from mice injected with histerilin or vehicle alone were analyzed by RIA for LH. Animals undergoing T treatment were given an subcutaneous Silastic implant containing T in arachis oil (0.6 mg/ml) from 10 weeks of age (T-csfmop/csfmop). Implants were constructed as previously described (53).

T concentrations were assessed by RIA using a commercially available kit (DSL Inc., Webster, TX). Inter- and intraassay coefficients of variance were 38.8% and 3.0%, respectively. LH RIAs were performed as previously described (54). Inter- and intraassay coefficients of variance were 26.9% and 6.7%, respectively.

Serum corticosterone concentrations were measured using a commercially available kit (Diagnostic Products Corp., Los Angeles, CA).

Leydig Cell Isolation and Treatment
Leydig cells were isolated as previously described (55) and subjected to 3 h of incubation at 34 C in the presence or absence of either LH (100 ng/ml) or 22R-hydroxycholesterol (22R-CHOL: 5 µM). For each isolation procedure, eight to ten age-matched males were used. For each tube, 100,000 cells were incubated in a total volume of 0.5 ml, and incubations were performed in quadruplet. Media T content was assessed by RIA.

Western Blotting
Soluble protein extracts from 50,000 Leydig cells or transfected COS-1 cells or from testis, adrenals, ovaries, and spleen samples were used for Western blotting as previously described (56). Steroidogenic enzyme proteins were detected using the Boehringer Mannheim chemiluminescence Western blotting kit (Indianapolis, IN) and antibodies raised in rabbits against bovine adrenal P450scc, human placental 3ß-HSD, and porcine testicular P45017{alpha}. Densitometric analyses were performed using ImageQuant software (Molecular Dynamics, Sunnyvale, CA). Whole testis Western blot densitometry represents the means (± SD) of at least three separate Western blots (and, therefore, at least nine different testis samples for each genotype). The quantification was shown to be linear within the range of protein concentrations used (data not shown), and all protein concentrations were normalized to GAPDH protein levels on the same blots. Additional controls were used for each specific antibody; for P450scc and P45017{alpha}, samples of purified bovine mitochondria or purified sheep microsomes (Oxygene Dallas Corp., Dallas, TX) were run at the same time as the testis samples, while for 3ß-HSD, cell extracts were prepared from COS-1 cells transfected with cytomegalovirus-driven expression vectors containing the entire cDNA for 3ß-HSD isoforms I or VI (see below). Each antibody showed specificity for its respective enzyme, as determined by antigen localization in other steroidogenic tissues (adrenal and ovary) and the absence of antigen in nonsteroidogenic tissue (spleen). The antibodies detected proteins of 52 kDa for P450scc, 46 kDa for 3ß-HSD, 52 kDa for P45017{alpha}, and 36 kDa for GAPDH.

All of the primary antibodies used were generous gifts from various sources: anti-P450scc and anti-P45017{alpha} (both from Dr. Dale Buchanan Hales, Department of Physiology and Biophysics, University of Illinois, Chicago, IL), anti-3ßHSD (Dr. Ian Mason, Department of Biochemistry and Obstetrics and Gynecology, Southwestern Medical School, Dallas, TX), and anti-GAPDH (Dr. Y. G. Yeung, Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, Bronx, NY).

Transient Expression of 3ßHSD Isoforms
Expression vectors containing the cytomegalovirus-driven full-length cDNA for 3ßHSD isoform I and 3ßHSD isoform VI were generously provided by Dr Anita Payne (Department of Obstetrics and Gynecology, Stanford University, Stanford, CA). Transient expression in COS-1 cells was obtained using the diethylaminoethyl-dextran method as described (57). Seventy-two hours after transfection, the cells were harvested, and soluble protein extracts were prepared as described above.

Measurement of Steroidogenic Enzyme Activity in Freshly Isolated Leydig Cells
With the exception of P450scc, steroidogenic enzyme activities were measured by incubation of purified Leydig cells with radiolabeled substrates and separation of products by TLC as described by O’Shaughnessy and Payne (58). Reaction mixture (0.5 ml) was prepared in Leydig cell medium that contained 1 µM substrate (1 µCi, 1 µM) in Leydig cell culture medium. The reaction mixture was maintained at pH 7.2. Reactions were initiated by adding to the reaction mixture an aliquot of 0.1 x 106 preincubated intact Leydig cell suspension, using endogenous cofactors. The reaction mixtures, conducted in triplicate, were maintained at 34°C in a shaking water bath for 30 min. With this incubation time, the conversion of substrates to products was linear with respect to cell number and time of incubation. Reactions were terminated by adding ice-cold ethyl acetate, and steroids were rapidly extracted. The organic layer was dried under nitrogen. The steroid residues were chromatographed on TLC plates, and radioactivity was measured with a scanning radiometer (System 200/AC3000, Bioscan, Inc., Washington, DC).

Activity of P450scc was determined by measuring the conversion of side-chain-labeled [26,27-3H]25-hydroxycholesterol to [3H]4-hydroxyl-4-methyl-pentanoic acid as previously described (59). 25-Hydroxycholesterol, rather than cholesterol, was used as substrate to measure the P450scc reaction, since it does not depend on active transfer to the inner mitochondrial membrane and has a high aqueous solubility. The reaction was performed in 0.5 ml of medium containing 0.1 x 106 cells and 1 µM substrate for 30 min. Blank incubations were performed with medium containing BSA (1.5 mg/ml) in place of cell suspension. At the end of the incubation, 0.1 ml 1 M NaOH was added followed by 0.5 ml phosphate buffer adjusted to pH 13. Nonmetabolized substrate was removed by extracting twice with chloroform. An aliquot was counted in a liquid scintillation counter. Counts from the blank incubation were subtracted from the counts in the test system.

[26,27-3H]25-hydroxycholesterol (specific activity, 81.9 Ci/mmol), [7-3H(N)]-pregnenolone (specific activity, 25 Ci/mmol), [1,2-3H(N)]-17{alpha}-hydroxyprogesterone (specific activity, 48.7 Ci/mmol), [1ß,2ß-3H(N)]-androstenedione (specific activity, 45.3 Ci/mmol), and [1,2,6,7-3H(N)]-testosterone (specific activity, 101 Ci/mmol) were purchased from Dupont-New England Nuclear (Boston, MA). [1,2,6,7-3H(N)]-Progesterone (specific activity, 92 Ci/mmol) was purchased from Amersham International PLC (Amersham, England). 25-Hydroxycholesterol, pregnenolone, progesterone, 17{alpha}-hydroxyprogesterone, androstenedione, and T were purchased from Sigma Chemical Co. (St. Louis, MO). TLC plates (Baker-flex/UV254) were obtained from J.T. Baker, Inc. (Phillipsburg, NJ).

Northern Blot Analysis
Probes for Northern blots were obtained by RT-PCR using pairs of oligonucleotide primers designed from the published sequences of genes encoding the following steroidogenic enzymes: mouse P45017{alpha}, 5'-CCA GAC GTG GTC ATA TGC ATG CCA-3' and 5'-GAT GAG CGT AGA CAG ATC TCG GGA-3' (60); and mouse 3ß-HSD, 5'-TGG TCT GAT CCA TAC CCA TAC AGC-3' and 5'-TGG TGC GGG GTG TCA TCT GAG ATG-3' (61). Aliquots of 20 µg total mRNA, isolated as previously described (62, 63), were separated by agarose gel electrophoresis and transferred to nitrocellulose membrane using routine methods. After a 3-h incubation in prehybridization buffer at 45 C, [32P]-labeled probes were allowed to hybridize to the blots overnight at 42 C, before high stringency washing at 55 C and exposure to autoradiographic film for appropriate lengths of time.

RPAs
For RPA, [32P]-labeled antisense riboprobes were prepared from cDNA templates cloned into the pCRII vector (InVitrogen Corp., San Diego, CA). The templates were prepared by RT-PCR using pairs of oligonucleotide primers designed from the published sequences of genes encoding the following steroidogenic enzymes: rat P450scc, 5'-CCG CTT TGC CTT TGA GTC CAT-3' and 5'-ACA CCC AGA ACT TCT ACT GGG-3' (64); mouse P45017{alpha} (see above); and mouse 3ß-HSD (see above). Aliquots of 20 µg total RNA were combined with approximately 105 cpm labeled riboprobe and analyzed by RPA using standard techniques. Consistent mRNA loading was ensured by the simultaneous hybridization to [32P]-labeled antisense probe transcribed from the pTRI-GAPDH-mouse antisense control template (Ambion, Austin, TX), which contains a fragment of the mouse glyceraldehyde 3-phosphate dehydrogenase housekeeping gene. Protected RNA fragments were separated on a denaturing 6% PAGE/8 M urea gel and visualized by exposing the dehydrated gel to Hyperfilm (Amersham Life Sciences Inc., Arlington Heights, IL) for appropriate periods of time.

Electron Microscopy
Male +/csfmop, csfmop/csfmop, C-csfmop/csfmop mice were given a single dose of heparin (130 mU per g body weight ip) and anesthetized 15 min later. Mice were perfused transcardially for 30 min with fixative (1.3% sym-collidine, 1.2% acrolein, 25% glutaraldehyde in 45 µM HCl) before removal of both testes. The testes were stored in fresh fixative at 4 C until processing by conventional methods.


    ACKNOWLEDGMENTS
 
The authors wish to acknowledge, with gratitude, the help of Dr. Ren-Shan Ge for performing the enzyme activity assays and Ms. Chantal Sottas for her excellent technical assistance. In addition, our thanks go to Drs. A. H. Payne, D. B. Hales, I. Mason, and Y. G. Yeung for providing reagents and antibodies for the current studies. LH reference preparation (AFP-7187B) and anti-rat LH antibody (NIDDK-rLH-s-11) were generously provided by the National Hormone and Pituitary Program. We also thank Drs. W. Bardin, F. Pixley, and A. Hapel for critical comments on this manuscript.


    FOOTNOTES
 
Address requests for reprints to: Jeffrey W. Pollard, Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10451.

This work was funded by the Population Council (M.P.H.) and by grants from the NIH: R29-HD32588 (to M.P.H.) and HD/AI 30280 (to J.W.P.), The Albert Einstein Core Cancer Grant P30-CA13330 (to J.W.P.), and Chiron Corporation (to J.W.P.). J.W.P. is a Monique Weill-Caulier Scholar.

Received for publication November 19, 1996. Revision received June 9, 1997. Accepted for publication July 24, 1997.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Stanley ER, Guilbert LT, Tushinski RJ, Bartelmez SH 1983 CSF-1 A mononuclear phagocyte lineage-specific hemopoietic growth factor. J Cell Biochem 21:151–159[Medline]
  2. Sherr CJ, Rettenmier CW, Sacca R, Roussel MS, Look AT, Stanley ER 1985 The c-fms proto-oncogene product is related to the receptor for the mononuclear phagocyte growth factor, CSF-1. Cell 41:665–676[Medline]
  3. Wiktor-Jedrzejczak W, Bartocci A, Ferrante Jr AW, Ahmed-Ansari A, Sell KW, Pollard JW, Stanley ER 1990 Total absence of colony-stimulating factor 1 in the macrophage-deficient osteopetrotic (op/op) mouse. Proc Natl Acad Sci USA 87:4828–4832[Abstract]
  4. Yoshida H, Hayashi SI, Kunisada T, Ogawa M, Nishikawa S, Okamura H, Sudo T, Shultz LD, Nishikawa S-I 1990 The murine mutation osteopetrosis in the coding region of the macrophage colony stimulating factor gene. Nature 345:442–444[CrossRef][Medline]
  5. Wiktor-Jedrzejczak W, Ahmed A, Szczylik C, Skelly RR 1982 Hematological characterization of congenital osteopetrosis in op/op mouse. J Exp Med 156:1516–1527[Abstract]
  6. Marks Jr SC, Lane PW 1976 Osteopetrosis, a new recessive skeletal mutation on chromosome 12 of the mouse. J Hered 67:11–18[Medline]
  7. Cecchini MG, Dominguez MG, Mocci S, Wetterwald A, Felix R, Fleisch H, Chisholm O, Hofstetter W, Pollard JW, Stanley ER 1994 Role of colony stimulating factor-1 in the establishment and regulation of tissue macrophages during postnatal development of the mouse. Development 120:1357–1372[Abstract/Free Full Text]
  8. Arceci RJ, Pampfer S, Pollard JW 1992 Expression of CSF-1/c-fms and SF/c-kit mRNA during preimplantation mouse development. Dev Biol 151:1–8[Medline]
  9. Arceci RJ, Shanahan F, Stanley ER, Pollard JW 1989 Temporal expression and location of colony-stimulating factor 1 (CSF-1) and its receptor in the female reproductive tract are consistent with CSF-1-regulated placental development. Proc Natl Acad Sci USA 86:8818–8822[Abstract]
  10. Cohen PE, Chisholm O, Arceci RJ, Stanley ER, Pollard JW 1996 Absence of colony stimulating factor-1 in osteopetrotic (csfmop/csfmop) mice results in male fertility defects. Biol Reprod 55:310–317[Abstract]
  11. Cohen PE, Zhu L, Pollard JW 1997 The absence of CSF-1 in osteopetrotic (csfmop/csfmop) mice disrupts estrous cycles and ovulation. Biol Reprod 56:110–118[Abstract]
  12. Pollard JW, Dominguez MG, Mocci S, Cohen PE, Stanley ER 1997 Effect of colony stimulating factor-1 (CSF-1) null mutation, osteopetrotic (csfmop), on the distribution of macrophages in the male reproductive tract. Biol Reprod 56:1290–1300[Abstract]
  13. Miller SC, Bowman BM, Rowland HG 1983 Structure, cytochemistry, endocytotic activity and immunoglobulin (Fc) receptors of rat testicular interstitial-tissue macrophages. Am J Anat 168:1–13[Medline]
  14. Niemi M, Sharpe RM, Brown RA 1986 Macrophages in the interstitial tissue of the rat testis. Cell Tissue Res 243:337–344[Medline]
  15. Hutson JC 1992 Development of cytoplasmic digitations between Leydig cells and testicular macrophages of the rat. Cell Tissue Res 267:385–389[Medline]
  16. Hutson JC 1994 Testicular macrophages. Int Rev Cytol 149:99–143[Medline]
  17. Michaelson MD, Bieri PL, Mehler MF, Xu H, Arezzo JC, Pollard JW, Kessler JA 1996 CSF-1 deficiency in mice results in abnormal brain development. Development 122:1–12[Abstract/Free Full Text]
  18. Abbaszade IG, Arensburg J, Park C-H, J, Kasa-Vubu JZ, Orly J, Payne AH 1997 Isolation of a new mouse 3ß-hydroxysteroid dehydrogenase isoform, 3ß-HSD VI, expressed during early pregnancy. Endocrinology 138:1392–1399[Abstract/Free Full Text]
  19. Christensen AK, Fawcett DW 1995 The fine structure of testicular interstitial cells in mice. Am J Anat 118:551–572
  20. De Kretser DM, Kerr JB 1994 The cytology of the testis. In: Knobil E, Neill JD (eds) The Physiology of Reproduction. Raven Press Ltd., New York, pp 1177–1290
  21. Wiktor-Jedrzejczak W, Gordon S 1996 Cytokine regulation of the macrophage (M{phi}) system studied using the colony stimulating factor-1-deficient op/op mouse. Physiol Rev 76:927–947[Abstract/Free Full Text]
  22. Wiktor-Jedrzejczak W, Urbanowska E, Aukerman SL, Pollard JW, Stanley ER, Ralph P, Ansari AA, Sell KW, Szperl M 1991 Correction by CSF-1 of defects in the osteopetrotic op/op mouse suggests local, developmental, and humoral requirements for this growth factor. Exp Hematol 19:1049–1054[Medline]
  23. Pollard JW, Stanley ER 1996 Pleiotropic roles for CSF-1 in development defined by the mouse mutation osteopetrotic (op). Adv Dev Biochem 4:153–193
  24. Pollard JW, Hunt JW, Wiktor-Jedrzejczak W, Stanley ER 1991 A pregnancy defect in the osteopetrotic (op/op) mouse demonstrates the requirement for CSF-1 in female fertility. Dev Biol 148:273–283[Medline]
  25. Arceci RJ, Pampfer S, Pollard JW 1992 Role and expression of colony-stimulating factor-1 and steel factor receptors and their ligands during pregnancy in the mouse. Reprod Fertil Dev 4:619–632
  26. Haisenleder DJ, Dalkin AC, Marshall JC 1994 Regulation of gonadotropin gene expression. In: Knobil E, Neill JD (eds) The Physiology of Reproduction. Raven Press Ltd., New York, pp 1793–1813
  27. Barron KD 1995 The microglial cell. A historical review. J Neurol Sci 134 [Suppl 1]:57–68
  28. Payne AH, Youngblood GL 1995 Regulation of expression of steroidogenic enzymes in leydig cells. Biol Reprod 52:217–225[Abstract]
  29. Scott IS, Charlton HM, Cox BS, Grocock CA, Sheffield JW, O’Shaughnessy PJ 1990 Effect of LH injections on testicular steroidogenesis, cholesterol side-chain cleavage P450 mRNA content and leydig cell morphology in hypogonadal mice. J Endocrinol 125:131–138[Abstract]
  30. Payne AH, Sha L 1991 Multiple mechanisms for regulation of 3ß-hydroxysteroid dehydrogenase/{Delta}5-{Delta}4-isomerase, 17{alpha}-hydroxylase/C17-20 lyase cytochrome P450, and cholesterol side-chain cleavage cytochrome P450 messenger ribonucleic acid levels in primary cultures of mouse Leydig cells. Endocrinology 129:1429–1435[Abstract]
  31. Payne AH, O’Shaughnessy PJ 1996 Structure, function and regulation of steroidogenic enzymes in the Leydig cell. In: Payne AH, Hardy MP, Russell LD (eds) The Leydig cell. Cache River Press, Vienna, IL, pp 259–284
  32. Geierhaas B, Bornstein SR, Jarry H, Scherbaum WA, Herrmann M, Pfeiffer EF 1991 Morphological and hormonal changes following vasectomy in rats, suggesting a functional role for Leydig cell-associated macrophages. Horm Metab Res 23:373–378[Medline]
  33. Wang J, Wreford NGM, Lan HY, Atkins R, Hedger MP 1994 Leukocyte populations of the adult rat testis following removal of the Leydig cells by treatment with ethane dimethane sulfonate and subcutaneous testosterone implants. Biol Reprod 51:551–561[Abstract]
  34. van Rooijen N, Sanders A 1994 Liposome mediated depletion of macrophages: mechanism of action, preparation of liposomes and applications. J Immunol Methods 174:83–93[CrossRef][Medline]
  35. Gaytan F, Bellido C, Aguilar E, van Rooijen N 1995 Pituitary-testicular axis in rats lacking testicular macrophages. Eur J Endocrinol 132:218–222[Medline]
  36. Gaytan F, Bellido C, Aguilar E, van Rooijen N 1994 Requirement for testicular macrophages in Leydig cells proliferation and differentiation during prepubertal development in rats. J Reprod Fertil 102:393–399[Abstract]
  37. Kerr JB, Sharpe RM 1985 Stimulatory effect of follicle-stimulating hormone on rat Leydig cells. A morphometric and ultrastructural study. Cell Tissue Res 239:405–415[Medline]
  38. Hardy MP, Zirkin BR, Ewing LL 1989 Kinetic studies on the development of the adult population of Leydig cells in testes of the pubertal rat. Endocrinology 124:762–770[Abstract]
  39. Xiong Y, Hales DB 1993 Expression, regulation and production of tumor necrosis factor-alpha in mouse testicular interstitial macrophages in vitro. Endocrinology 133:2568–2573[Abstract]
  40. Hales DB 1992 Interleukin-1 inhibits Leydig cell steroidogenesis primarily by decreasing 17alpha-hydroxylase/C17–20 lyase cytochrome P450 expression. Endocrinology 131:2165–2172[Abstract]
  41. Xiong Y, Hales DB 1993 The role of tumor necrosis factor-alpha in the regulation of mouse Leydig cell steroidogenesis. Endocrinology 132:2438–2444[Abstract]
  42. Hutson JC 1993 Secretion of tumor necrosis factor alpha by testicular macrophages. J Reprod Immunol 23:63–72[CrossRef][Medline]
  43. Xiong Y, Hales DB 1994 Immune-endocrine interactions in the mouse testis: cytokine-mediated inhibition of Leydig cell steroidogenesis. Endocrine J 9:1277–1283
  44. Kerr JB, Rich KA, De Kretser DM 1979 Alterations of the fine structure and androgen secretion of interstitial cells in the experimentally cryptorchid rat testis. Biol Reprod 20:409–422[Medline]
  45. Rich KA, Kerr JB, Krester DM 1979 Evidence for Leydig cell dysfunction in rats with seminiferous tubule damage. Mol Cell Endocrinol 13:123–135[CrossRef][Medline]
  46. Bergh A, Ason Berg A, Damber JE, Hammar M, Selstam G 1984 Steroid biosynthesis and Leydig cell morphology in adult unilaterally cryptorchid rats. Acta Endocrinol (Copenh) 107:556–562[Medline]
  47. Risbridger GP, Kerr JB, De Kretser DM 1981 Evaluation of Leydig cell function and gonadotropin binding in unilateral and bilateral cryptorchidism: evidence for local control of Leydig cell function by the seminiferous tubule. Biol Reprod 24:534–540[Medline]
  48. Carr I, Carr J 1962 Membranous whorls in the testicular interstitial cell. Anat Rec 144:143–147[Medline]
  49. Ojeda SR, Urbanski HF 1994 Puberty in the rat. In: Knobil E, Neill JD (eds) The Physiology of Reproduction, ed 2. Raven Press, Ltd., New York, pp 363–409
  50. Kendall SK, Samuelson LC, Saunders TL, Wood RI, Camper SA 1995 Targeted disruption of the pituitary glycoprotein hormone {alpha}-subunit produces hypogonadal and hypothyroid mice. Genes Dev 9:2007–2019[Abstract]
  51. Schally AV, Arimura A, Baba Y, Nair RM, Matsuo H, Redding TW, Debeljuk L 1971 Isolation and properties of the FSH and LH-releasing hormone. Biochem Biophys Res Commun 43:393–399[Medline]
  52. Amoss M, Burges R, Blackwell R, Vale W, Fellowes R, Guillemin R 1971 Purification, amino acid composition and N-terminus of the hypothalamic luteinizing hormone releasing factor (LRF) of ovine origin. Biochem Biophys Res Commun 44:205–210[Medline]
  53. Cohen PE, Milligan SR 1993 Silastic implants for delivery of oestradiol to mice. J Reprod Fertil 99:219–223[Abstract]
  54. Chandrashekar V, Bartke A, Wagner TE 1988 Endogenous human growth hormone (GH) modulates the effect of gonadotropin-releasing hormone on pituitary function and the gonadotropin response to the negative feedback effect of testosterone in adult male transgenic mice bearing human GH gene. Endocrinology 123:2717–2722[Abstract]
  55. Klinefelter GR, Kelce WR, Hardy MP 1993 Isolation and culture of leydig cells from adult rats. Methods Toxicol 3A:166–181
  56. Yeung YG, Berg KL, Pixley FJ, Angeletti RH, Stanley ER 1992 Protein tyrosine phosphatase-1C is rapidly phosphorylated in tyrosine in macrophages in response to colony stimulating factor-1. J Biol Chem 267:23447–23450[Abstract/Free Full Text]
  57. Abbaszade IG, Clarke TR, Park CHJ, Payne AH 1995 The mouse 3ß-hydroxysteroid dehydrogenase multigene family includes two functionally distinct groups of proteins. Mol Endocrinol 9:1214–1222[Abstract]
  58. O’Shaughnessy PJ, Payne AH 1982 Differential effects of single and repeated administration of gonadotropins on testosterone production and steroidogenic enzymes in leydig cell populations. J Biol Chem 257:11503–11509[Free Full Text]
  59. Georgiou M, Perkins LM, Payne AH 1987 Steroid synthesis-dependent, oxygen-mediated damage of mitochondrial and microsomal cytochrome P-450 enzymes in rat leydig cell cultures. Endocrinology 121:1390–1399[Abstract]
  60. Youngblood GL, Payne AH 1992 Isolation and characterization of the mouse P450 17{alpha}-hydroxylase/C17-20-lyase gene (Cyp17): transcriptional regulation of the gene by cyclic adenosine 3', 5'-monophosphate in MA-10 leydig cells. Mol Endocrinol 6:927–934[Abstract]
  61. Bain PA, Yoo M, Clarke T, Hammond SH, Payne AH 1991 Multiple forms of mouse 3ß-hydroxysteroid dehydrogenase/{Delta}5- {Delta}4 isomerase and differential expression in gonads, adrenal glands, liver, and kidneys of both sexes. Proc Natl Acad Sci USA 88:8870–8874[Abstract]
  62. Chirgwin JM, Przybyla AE, MacDonald RJ, Rutter WJ 1979 Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18:5294–5299[Medline]
  63. Saade G, London DR, Lalloz MRA, Clayton RN 1989 Regulation of LH subunit and prolactin mRNA by gonadal hormones in mice. J Mol Endocrinol 2:213–224[Abstract]
  64. Oonk RB, Krasnow JS, Beattie WG, Richards JS 1989 Cyclic AMP-dependent and -independent regulation of cholesterol side chain cleavage cytochrome P-450 (P-450 scc) in rat ovarian granulosa cells and corpora lutea. cDNA and deduced amino acid sequence of rat P-450 scc. J Biol Chem 264:21934–21942[Abstract/Free Full Text]