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BOR - Papers in Press, published online ahead of print July 14, 2004.
Biol Reprod 2004, 10.1095/biolreprod.104.031161
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BIOLOGY OF REPRODUCTION 71, 1605–1613 (2004)
DOI: 10.1095/biolreprod.104.031161
© 2004 by the Society for the Study of Reproduction, Inc.

Testis

Testicular Phenotype in Luteinizing Hormone Receptor Knockout Animals and the Effect of Testosterone Replacement Therapy1

Z.M. Lei2,3, S. Mishra4, P. Ponnuru4, X. Li4, Z.W. Yang5, and Ch.V. Rao3,4

Division of Research,4 Department of Obstetrics, Gynecology, and Women's Health, University of Louisville Health Sciences Center, Louisville, Kentucky 40292 Morphometric Research Laboratory,5 North Sichuan Medical College, Sichuan, China


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The LH receptor knockout model, developed in our laboratory, was used in determining what FSH alone can do in the absence of LH signaling and whether any of the testicular LH actions are not mediated by androgens. The results revealed that null animals contained smaller seminiferous tubules, which contained the same number of Sertoli cells, spermatogonia, and early spermatocytes as wild-type siblings. The number of late spermatocytes, on the other hand, was moderately decreased, the number of round spermatids was dramatically decreased, and elongated spermatids were completely absent. These changes appear to be due to an increase in apoptosis in spermatocytes. While the number of Leydig cells progressively increased from birth to 60 days of age in wild-type animals, they remained unchanged in null animals. Consequently, 60-day-old null animals contained only a few Leydig cells of fetal type. The age-dependent increase in testicular macrophages lagged behind in null animals compared with wild-type siblings. Orchidopexy indicated that –/– testicular phenotype was not due to abdominal location. Rather, it was mostly due to androgen deficiency, as 21-day testosterone replacement therapy stimulated the growth of seminiferous tubules, decreased apoptosis, and increased the number of late spermatocytes and round spermatids and their subsequent differentiation into mature sperm. The therapy, however, failed to restore adult-type Leydig cells and testicular macrophage numbers to the wild-type levels. In summary, our data support the concept that FSH signaling alone can maintain the proliferation and development of Sertoli cells, spermatogonia, and early spermatocytes. LH actions mediated by testosterone are required for completion of spermatogenesis, and finally, androgen-independent actions of LH are required for the formation of adult-type Leydig cells and recruitment of macrophages into the testes.

androgen, Leydig cells, LH receptor, luteinizing hormone, spermatogenesis, testis, testosterone


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The testes contain interstitium and seminiferous tubules. Interstitium is the steroidogenic compartment, which contains Leydig cells, macrophages, endothelial cells, and vascular smooth muscle [1]. Seminiferous tubuli form the spermatogenic compartment, which contains Sertoli cells, germ cells, and peritubular myoid cells [1]. The two compartments are morphologically and functionally distinct and yet they communicate with each other through the production of various hormones and other regulatory molecules. Leydig cells express luteinizing hormone (LH) receptors and produce testosterone in response to their activation [2, 3]. Sertoli cells contain androgen as well as follicle stimulating hormone (FSH) receptors, and their activation results in the support and nourishment of germ cells during spermatogenesis [4, 5].

Spermatogenesis is a highly specialized and efficient process, which results in the production of haploid mature spermatozoa from diploid spermatogonial stem cells. This complex and long chain of events consists of mitotic proliferation of spermatogonia, meiotic reduction division of spermatocytes, and morphological differentiation of haploid spermatids [6, 7]. Testosterone, produced as a result of LH stimulation of Leydig cells, regulates spermatogenesis [8, 9]. Increasing evidence suggests that estrogens, which are locally produced from the conversion of testosterone by aromatase present in Leydig, Sertoli, and germ cells, may also play a role [10–18]. Estrogens activate their {alpha} and ß receptors, which are also present in rete testis, efferent ducts, and Leydig cells (primarily {alpha}) and in germ cells (primarily ß) [19–23].

For the last several decades, the studies on hormonal regulation of spermatogenesis largely relied on animal models with hormonal ablations created by surgical, pharmacological, or immunological methods. Although these classical approaches have provided invaluable information, the interpretation of data was confounded by one or more of the following possible compensatory mechanisms: i) maternal gonadotropins during gestation, ii) residual and/or ectopic LH and FSH actions during adulthood, iii) alteration of multiple hormones, and iv) nonspecific and/or short lasting of hormonal ablations. Genetic inactivation of FSH ß subunit and FSH receptor mouse models have been used to study their roles in male reproduction [24, 25]. Generally, there is a paucity of information on what FSH alone can do in the absence of LH signaling and whether any of the testicular LH actions are not mediated by androgens. The LH receptor knockout animals, which have intact FSH signaling, were used for phenotyping the testes and testing the effect of testosterone replacement therapy (TRT) to provide insight into these issues.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
LHRKO Mice

Generation of LHRKO mice has previously been described [26]. Mice were maintained as required under the NIH guidelines for the Care and Use of Laboratory Animals. All studies have been approved by the Animal Care and Use Committee of the University of Louisville. Adult male and female heterozygous mice were mated to obtain wild-type (+/+), heterozygous (+/–), and homozygous (–/–) animals. The genotype was determined by Southern blotting with tail genomic DNA digested with Stu I and hybridized to [32P]-labeled DNA probe for the LH receptor gene as previously described [26]. The probe was designed to detect an 8-kilobase (kb) DNA fragment of the 5'-flanking region of a wild-type LH receptor gene and 10-kb mutant fragment, which included a neomycin gene used in the disruption strategy. Thus, a single Southern blot performed on each sample allowed unambiguous identification of all three genotypes. All animals were maintained on 12L:12D cycles with food and water provided ad libitum. Three to six 1-, 30-, and 60-day old mice of each genotype (day of birth was considered as Day 1) were used for each experiment.

Histology and Computerized Quantitative Morphometry

The animals were deeply anesthetized by intraperitoneal injection of sodium pentobarbital and the chest and abdominal cavities were opened by sagittal incision. After blood collection by cardiac puncture, the testes were excised, fixed in 10% buffered formalin and embedded in paraffin. Sections 5 µm thick were cut, stained with hematoxylin-eosin (H and E), and examined under bright-field microscope for quantification of morphological changes and for photography. Computerized Bioquant IV System (R and M Biometrics Inc., Nashville, TN) was used for determining morphological changes. Briefly, the sections were observed on a computer screen. Round or approximately round seminiferous tubules (the shortest to the longest axis ratio greater than 0.8) were randomly sampled for measurement of diameters and in 10 000 µm2 total area. For an objective determination of the number of spermatogenic and Sertoli cells, nuclei of various cell types were counted in seminiferous tubule cross sections covering a total area of 10 000 µm2. The results were expressed as cell number per seminiferous cross section. The number of Leydig cells was counted in several cross sections of interstitial areas covering 10 000 µm2 after immunostaining with 3ß-HSD antibody. The macrophage numbers were counted in interstitial areas of 10 000 µm2 after immunostaining with a specific antibody, F4/80.

Immunocytochemistry

This procedure was performed by an avidin-biotin immunoperoxidase method (Vector Laboratories, Inc., Burlingame, CA) [26, 27]. Briefly, deparaffinized testicular sections were treated for 10 min with 0.75% H2O2 in methanol to block endogenous peroxidase activity. All sections were incubated with primary antibodies overnight at 4°C. For immunostaining of macrophages, the sections were pretreated with 0.1% trypsin at 37°C followed by incubation with a 1:50 dilution of polyclonal antibody to mouse macrophage-specific antigen, F4/80 (Serotec Inc., Raleigh, NC). A 1:250 dilution of polyclonal antibody (Promega Corp., Madison, WI) was used to immunostain for active caspase-3. For immunostaining of fetal and adult-type Leydig cells, the sections were exposed to a 1:200 dilution of polyclonal antibody to 3ß-hydroxysteroid dehydrogenase (HSD) (a gift from Dr. Ian Mason at the University of Edinburgh, Edinburgh, Scotland). For immunostaining of adult-type Leydig cells, a 1:200 dilution of polyclonal antibody to 11ß-HSD (a gift from Dr. Matthew Hardy of the Population Council, New York, NY) was used. Polyclonal antibody to cyclin D3 was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA) and used in 1:200 dilution. Substitution of primary antibodies with nonspecific IgG served as procedural controls.

Testicular Apoptosis Assay

TdT-mediated dUTP Nick-End Label (TUNEL) staining was performed on formalin-fixed and paraffin-embedded sections using a DeadEnd colorimetric apoptosis detection system (Promega Corp.). The brown-colored nuclei of apoptotic cells in all the cross-sections of seminiferous tubules were visualized and counted under a bright-field microscope.

Orchidopexy Experiments

LHRKO homozygous male mice were anesthetized by intraperitoneal injection of sodium pentobarbital and a small vertical lower abdominal and a transverse scrotal incision were made in both the left and right sides. The right abdominal testis was brought down into the scrotum and fixed in place with two 5-0 chromic sutures approximating the tunica albuginea of the testis to the scrotal wall [28, 29]. The left testis of the same animal remained intraabdominal. The incisions were closed by stitches. Twenty-one days after the procedure, the animals were killed for collection of both left and right testes. The scrotal location of the right testis at the time of removal served to determine the effectiveness of the surgical procedure.

Testosterone Replacement Therapy (TRT)

Twenty-one-day time-release pellets containing 5 mg testosterone (Innovative Research America, Sarasota, FL) were subcutaneously implanted into null animals at 30 days of age, which corresponds to puberty when circulating testosterone levels normally begin to rise. The pellets represent a matrix-driven delivery system integrating the principles of diffusion, erosion, and concentration gradient resulting in a biodegradable matrix that effectively and continuously releases testosterone. Null mice for controls were implanted only with placebo pellets. The age of animals at the end of therapy corresponded approximately to the age (6–8 wk old) of sexual maturity and they were killed to recover testes and blood.

Measurement of Serum and Testicular Testosterone and Estradiol Levels

The total testosterone levels were measured by a Coat-A-Count radioimmunoassay (RIA) kit (Diagnostic Products, Los Angeles, CA) and estradiol by a third-generation RIA kit (Diagnostic Systems Laboratories, Inc., Webster, TX). The kit instructions were followed in the measurements. Supernatants obtained from centrifuging the testicular homogenates for 10 min at 10 000 x g were used without extraction for the measurement of testicular testosterone levels. Intratesticular estradiol levels, on the other hand, were measured after ethyl ether extraction of supernatants. The inter- and intra-assay coefficients of variations were less than 10% in the range of testosterone and estradiol levels found in the samples. The cross-reactivity was less than 1% for the other steroid hormones.

Statistical Analysis

The data presented are the means ± SEM. All results were analyzed by one-way ANOVA and Tukey multiple comparison post test using an Instat Version 3.06 program (Graphpad Software, Inc., San Diego, CA). A P value less than 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Serum Steroid Hormone Levels

While serum and testicular levels of testosterone are significantly lower, the estradiol levels are significantly higher in 60-day-old null animals as compared with wild-type siblings (Table 1).


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  		TABLE 1. Serum and intratesticular testosterone and estradiol levels in 60-day-old LHRKO mice

Morphological Analysis of the Seminiferous Tubules

The seminiferous tubules are somewhat smaller in null animals and contained hardly any lumen compared with wild-type siblings (Fig. 1B vs. Fig. 1A). Spermatogenic cells were found only through the round spermatid stage (Fig. 1B). The intertubular connective tissue in null animals contained very few Leydig cells compared with wild-type siblings (Fig. 1B vs. Fig. 1A).



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  		FIG. 1. Testicular morphology in 60-day-old +/+ and –/– animals with or without TRT. LT (D) indicates that left testis remained intraabdominal and RT (E) indicates that right testis of the same animal was moved into the scrotum by orchidopexy. The seminiferous tubules were smaller in –/– animals (B) and contained hardly any lumen compared with +/+ siblings (A). Spermatogenic cells were found only through the round spermatid stage (B) and the intertubular connective tissue contained very few Leydig cells in –/– animals (B) compared with +/+ siblings (A). The insets are higher magnification of the interstitial tissue area indicated by arrows. TRT restored spermatogenesis but not Leydig cells (C). Orchidopexy of –/– mice did not improve testicular morphology (E). Magnification (A–E = x150, all insets = x600

Quantification of morphological changes revealed that the diameters of seminiferous tubules and their area were lower in null animals than in wild-type siblings (Fig. 2, A and B). The number of Sertoli cells, which progressively increased from Day 1 through 60 days of age, was indistinguishable between null and wild-type animals (Fig. 2C). This finding is consistent with a recent report showing that Sertoli cell proliferation induced by FSH was independent of LH activity [30]. While the number of spermatogonia and early spermatocytes also remained indistinguishable between genotypes, the number of late spermatocytes moderately decreased and the number of round spermatids dramatically decreased in null animals compared with wild-type siblings (Fig. 2D). Development of spermatid in mouse comprises 12 distinct steps according to the changes in the shape of cell and the nucleus as well as the spread of the acrosome. Complete absence of spermatids undergoing cellular elongation or nuclear condensation in the null testes indicates that spermatogenesis does not progress beyond step 8. The arrest was also verified by periodic acid Schiff staining that labels the acrosome in spermatids and by immunocytochemistry with anti-cyclin D3 antibody, which strongly recognizes the elongated spermatids (data not shown).



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  		FIG. 2. The diameter of seminiferous tubules (A), tubular area (B), the number of Sertoli (C) and spermatogenic (D) cells in +/+ and –/– animals with or without TRT. The data presented in A, B, and D were from 60-day-old animals. *, P < 0.05; and **, P < 0.01 compared with wild-type siblings

Apoptosis in Testes

The results revealed that the number of tubules and the number of cells per tubule stained for apoptotic nuclei were higher in null animals than in wild-type siblings (Fig. 3, A–F). Counting revealed that the number of affected tubules and the number of affected cells per tubule increased in null animals compared with wild-type siblings (Table 2). The cells undergoing apoptosis were mainly found to be spermatocytes (Table 2). Other indices of increased apoptosis, such as active caspase-3 (Fig. 3, G–I) and Apaf-1 (data not shown), were also elevated in testes of null animals compared with wild-type siblings.



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  		FIG. 3. Germ cell apoptosis as determined by TUNEL staining (A–F) and by immunocytochemistry for active caspase-3 (G–I) in 60-day-old +/+ and –/– null animals with or without TRT. The numbers of apoptotic cells and active caspase-3 positive cells (H), indicated by arrows in –/– mice (B, E, and H), were higher than in +/+ siblings (A, D, and G), and TRT reversed these changes (C, F, and I). Magnification (A–C and G–I), x150; (D–F), x600


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  		TABLE 2. Germ cell apoptosis in 60-day-old LHRKO mice

Morphological Analysis of the Interstitium

As previously mentioned, testicular interstitium of 60-day-old null animals contained only a few Leydig cells and they appeared to be fetal-type based on their morphology. To distinguish them from adult-type Leydig cells and to track the changes in both cell types during the postnatal period, immunocytochemistry using 3ß-HSD antibody, which labels both fetal and adult-type cells and 11ß-HSD antibody, which only labels adult-type Leydig cells, was used [31, 32]. The 3ß-HSD immunostaining was similar between 1-day-old +/+ and –/– animals (Fig. 4, A and B). While this immunostaining increased in the testes of 60-day-old +/+ animals, it remained low in –/– animals (Fig. 4, C and D). Testes of 1-day-old +/+ and –/– animals did not immunostain for 11ß-HSD, indicating that the Leydig cells were fetal type (Fig. 4, F and G). While testes of 60-day-old +/+ animals immunostained for 11ß-HSD, the testes of null animals had no staining (Fig. 4, H and I), indicating that the Leydig cells in adult –/– animals are likely to be fetal type. Quantification of 3ß-HSD stained cells indicated that the number of Leydig cells increased with age in +/+ animals but not in –/– siblings (Fig. 5). Thus, the low number of Leydig cells present in testes of 30- and 60-day-old –/– animals are likely to be fetal type. Because newly formed immature adult-type Leydig cells do not contain 11ß-HSD [2], it is possible that null testes may also contain them.



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  		FIG. 4. Immunostaining for 3ß-HSD (A, B, and E–G) and 11ß-HSD (C, D, H, and I) in the testes of 1- and 60-day-old +/+ and –/– animals with or without TRT. 3ß-HSD-positive cells, indicated by arrows (B and F), were present in testicular interstitium of newborn (Day 1) and adult (Day 60) –/– mice. Their number in adult animals (F) was lower than in +/+ siblings (E), and TRT had no effect. Testes of adult +/+ mice contained only 11ß-HSD positive cells (H). Magnification (A–I), x300



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  		FIG. 5. The number of Leydig cells in the interstitium of 1-, 30-, and 60-day old +/+ and –/– animals with or without TRT. *, P < 0.01 compared with 1-day-old +/+ siblings; **, P < 0.01 compared with age-matched +/+ siblings; ***, P < 0.05 compared with 30-day-old +/+ siblings

The macrophages in testicular interstitium were identified by immunostaining with an F4/80 antibody. The results showed that, while there was little immunostaining in testes of 1-day-old +/+ and –/– animals, it progressively increased until 60 days, and this increase was greater in testes of +/+ animals than in –/– animals (Fig. 6). Quantification revealed that there was a progressive increase in the number of macrophages from Day 1 through Day 60 in +/ + animals, which lagged behind in –/– siblings (Fig. 7).



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  		FIG. 6. Immunostaining for F4/80, a macrophage marker, in 1-, 30-, and 60-day old +/+ and –/– animals with or without TRT. The macrophages, indicated by arrows in testicular interstitium, progressively increased in +/+ animals in an age-dependent manner (A, C, E, and F), and this increase was markedly altered in –/– siblings (B, D, and G), which could not be reversed by TRT (H). Magnification (A–E, G, and H), x150; (F), x300. All insets, x600



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  		FIG. 7. The number of testicular macrophages in the interstitium of 1-, 30-, and 60-day old +/+ and –/– animals with or without TRT. *, P < 0.01 compared with 1-day-old +/+ siblings; **, P < 0.01 compared with age-matched +/+ siblings; ***, P < 0.05 compared with 30-day-old +/+ siblings

Effect of Orchidopexy and TRT

The –/– testicular phenotype could be due to abdominal location, which exposes them to higher body temperature, which impairs spermatogenesis. Alternatively, it could be secondary to a testosterone deficiency. To determine the first possibility, scrotal orchidopexy was performed on 30-day-old null animals, wherein the right testis was moved into the scrotum while the left testis remained abdominal. Twenty-one days after the surgery, the morphology of the right testis remained the same as the left testis (Fig. 1), suggesting that abdominal location could not be responsible for –/– testicular phenotype. This left the possibility of testosterone deficiency, which was tested by placing 30-day-old null animals on 21-day TRT. The therapy restored testicular testosterone and estradiol as well as serum estradiol to that of wild-type levels (Table 1). Serum testosterone levels, on the other hand, were elevated by more than fourfold compared with wild-type animals, which is consistent with a previous report that showed that higher circulating levels were needed to restore testicular levels [6].

The TRT stimulated the growth of seminiferous tubules, which contained all stages of spermatogenic cells, including mature sperm in enlarged lumen (Figs. 1C; 2, A and B). The number of seminiferous tubules and number of cells per tubule affected by apoptosis returned to wild-type levels (Fig. 3; Table 2). As a result, the number of late spermatocytes and round spermatids increased (Fig. 2D) and elongated spermatids and mature sperm that could fertilize wild-type oocytes in vitro appeared [33]. While TRT was successful in reversing the above features, it failed to increase the number of either fetal or adult-type Leydig cells or the number of testicular macrophages to the levels of wild-type siblings (Figs. 4E, 5–7).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Mammalian testes contain two morphologically distinct and functionally interdependent compartments. They are primarily regulated by pituitary gonadotropins, FSH and LH. Previous studies using a variety of experimental approaches have revealed that, while FSH primarily regulates Sertoli cell functions, LH primarily regulates the functions of Leydig cells. The secretory products (androgens, their binding proteins, and a variety of growth factors, cytokines, chemokines, and other regulatory molecules) released from these cells act in an autocrine and paracrine manner to maintain testicular homeostasis [34–36]. The LH receptor knockout model developed in our laboratory was used in providing the insight into the issues concerning what FSH alone can do in the absence of LH signaling and whether all the testicular LH actions are mediated by testosterone. In this model, serum FSH levels are moderately elevated while testicular FSH receptor levels are not altered, suggesting that FSH signaling was intact [26]. The serum and testicular testosterone levels, on the other hand, were dramatically decreased, reflecting the loss of LH actions.

While wild-type and heterozygous animals were indistinguishable, null animals had a testicular phenotype that was not due to their abdominal location. It was due to testosterone deficiency, which was reflected by external features such as abdominal testes, micropenis, shorter perineal distance, and internal features such as small testes with arrested spermatogenesis and marked growth reduction in all accessory sex organs. This phenotype is similar to that seen in men harboring inactivating-type LH receptor gene mutations [37–40].

The histologic examination and quantification revealed that the size and area of seminiferous tubules decreased, which is probably due to the presence of fewer number of cells and obliteration of tubular lumen resulting from the spermatogenic arrest. The tubules, however, contained the same number of Sertoli cells, spermatogonia, and early spermatocytes as wild-type animals. The number of late spermatocytes, on the other hand, moderately decreased, the number of round spermatids dramatically decreased, and elongated spermatids were totally absent. These changes perhaps resulted from an increase in apoptosis in spermatocytes. The above testicular phenotype suggests that FSH signaling alone, perhaps in the presence of low testosterone levels, could maintain proliferation and development of Sertoli cells, spermatogonia and early spermatocytes and LH actions are required beyond this stage. As these actions are mediated by testosterone, it is then likely that testosterone replacement could stimulate the spermatogenic process to proceed to completion. Consistent with this possibility, TRT, which elevated testes and serum testosterone levels, decreased the apoptosis in spermatocytes, increased the number of late spermatocytes and round spermatids and the appearance of elongated spermatids and mature sperm, which could fertilize wild-type oocytes in vitro. The sperm concentrations were, however, lower, and whether this has anything to do with the loss of LH receptors in sperm cells is not known [41]. Nevertheless, testosterone-replaced animals remained infertile unless they were given a single testosterone injection during the neonatal period [33].

Leydig cells and macrophages are structurally and functionally interdependent in the testicular interstitium [42, 43]. There were no genotype differences in the number of Leydig cells and testicular macrophages in 1-day-old animals. As the animals aged, the number of both cell types increased in +/+ animals. In –/– siblings, while the Leydig cell number did not increase, the number of macrophages increased at a lower rate than in +/+ animals. The immunostaining for 3ß-HSD and 11ß-HSD revealed that the Leydig cells present in 1-day-old +/+ and –/– animals were fetal type. Leydig cells that increased with age in +/ + animals were adult type and Leydig cells that remained static in –/– animals were fetal type. These findings are consistent with previous reports [2, 44], which suggested that LH signaling is not required for the formation of fetal-type Leydig cells from mesenchymal precursors during the prenatal period and their maintenance during postnatal life. The LH signaling, on the other hand, is required for the formation of adult-type Leydig cells from precursors. It is also required for the migration of monocytes and their differentiation into macrophages in testes. The low testosterone levels present in testes, which probably reflect constitutive production by fetal-type Leydig cells, may be adequate for the low level macrophage recruitment, but certainly not for the formation of adult-type Leydig cells. Despite the defects, –/– testes supported the development of transplanted functional putative donor Leydig stem cells, indicating that the local environment was not substantially altered in the absence of LH signaling [45].

The dependence of adult-type Leydig cells formation and macrophage recruitment on LH signaling suggests that its actions could be mediated by androgens. To determine this possibility, the effect of TRT was examined. The results showed that TRT failed to restore their numbers to those of wild-type animals. These observations suggest that LH actions are not mediated by androgens. Instead, direct LH actions, in case of the formation of adult-type Leydig cells and perhaps nonsteroidal factors released from adult-type Leydig cells, which are absent in –/– animals, may be required for the recruitment of macrophages.

In contrast with LH receptor knockout animals, FSH receptor knockout and FSHß knockout animals had no dramatic testicular phenotype [24, 25]. Consequently, these animals were subfertile, suggesting that FSH actions are more important for qualitative and quantitative aspects rather than an absolute requirement for spermatogenesis. The testicular phenotype, in terms of a decrease in the number of late spermatocytes and round spermatids and absence of elongated spermatids in LH receptor knockout animals, is very similar to Sertoli cell-specific androgen receptor knockout animals [46, 47], confirming that androgen actions in Sertoli cells are required for their functional maturation to support the spermatogenic process through the maintenance and nourishment of germ cells. The general androgen receptor knockout [46, 48], however, resulted in a decrease in all the spermatogenic cell types, indicating that there are other sites of androgen action that are essential for normal testicular phenotype.

The reciprocal changes in serum and testicular testosterone and estradiol levels and their normalization, along with testes phenotype in testosterone-replaced null animals, suggest that the hormonal imbalance, rather than androgen deficiency alone, could have contributed to testicular phenotype. Although it is not completely clear why the testicular estradiol levels were so high when intratesticular testosterone was suppressed, it is possible that low testosterone levels could have favored a decrease in 5{alpha}-reductase increase in aromatase and/or a decrease in estrogen-specific sulfotransferase activities in the testis. Any one or all of the enzyme changes could potentially increase estradiol levels. It has become obvious that not only androgens but also estrogens regulate spermatogenesis through their cognate receptors. Increasing evidence has shown that an imbalance of androgens and estrogens could lead to the reproductive tract abnormalities and aberrant spermatogenesis [12–18, 49]. It is interesting to note that, while androgen receptors are not altered in –/– testes, ER{alpha} levels decreased and ERß levels increased. Previous studies reported that ER{alpha} knockout animals have testicular phenotype due to fluid reabsorption defects in the rete testes and efferent ducts and ERß knockout animals had no discernible testicular phenotype. Overall, these findings indicate a complicated relationship may exist between androgens and estrogens in maintaining testicular homeostasis.

In summary, our present study supports the concept that LH actions are more important than FSH in maintaining normal testicular phenotype. Androgens do not mediate the LH actions in the formation of adult-type Leydig cells and recruitment of macrophages into testes.


   ACKNOWLEDGMENTS
 
We gratefully acknowledge the technical assistance of Dr. Bingrui Xu, Mr. Fred Carman, Jr., and Mr. Mark Foltz.


   FOOTNOTES
 
1 Supported by NIH grant HD-40223. Back

2 Correspondence: Z.M. Lei, Department of Obstetrics, Gynecology, and Women's Health, 438 MDR Building, University of Louisville Health Sciences Center, Louisville, KY 40292. FAX: 502 852 0881; zhenmin.lei{at}louisville.edu Back

3 Correspondence: Ch. V. Rao, Department of Obstetrics, Gynecology, and Women's Health, 438 MDR Building, University of Louisville Health Sciences Center, Louisville, KY 40292. FAX: 502 852 0881; cvrao001{at}louisville.edu Back

Received: 23 April 2004.

First decision: 24 May 2004.

Accepted: 7 July 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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