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Female Reproductive Tract |
Departments of Pathology,3
Molecular and Cellular Biology,4
Molecular and Human Genetics,5 Baylor College of Medicine, Houston, Texas 77030
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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subunit are normal and fertile, both male and female transgenic mice overexpressing only the hormone-specific hCGß subunit are infertile. The hCGß subunit-expressing transgenic female mice progressively develop cystic ovaries, whereas the male transgenic mice are infertile but otherwise are not phenotypically discernible. In contrast, both the male and female transgenic mice coexpressing high levels of the hCG subunits (i.e., the hCG dimer) demonstrate multiple reproductive defects. The male transgenic mice have Leydig cell hyperplasia, very high levels of serum testosterone, reduced testis size, and dramatically enlarged seminal vesicles and are infertile and display overly aggressive behavior when caged with females. The female transgenic mice are also infertile, have elevated levels of serum estradiol, and progressively develop hemorrhagic and cystic ovaries with thecal layer enlargement and stromal cell proliferation and degenerating kidneys. These results suggest that the in vivo biological effects of ectopically expressed free hCGß subunit are distinct from those of the hCG dimer and are gender specific. These transgenic mice are useful models for studying the biology of free hCGß subunit, for further analyzing the gain of function effects of hCG during early Leydig cell development, and for studying the roles of hCG in ovarian and kidney pathophysiology and function.
female reproductive tract, male reproductive tract, ovary, pituitary, testis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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subunit that is noncovalently associated with a hormone/receptor-specific ß subunit to form biologically active heterodimers. The hCGß and the human LHß (hLHß) genes are organized into a multigene cluster on chromosome 19; these genes and their encoded polypeptide sequences share significant identity [2]. Furthermore, LH and hCG bind to identical receptors that are coupled to G-proteins in the gonads of both sexes. Structurally, hCGß is characterized by an O-glycosylated carboxy terminal peptide (CTP) that is not present in hLHß [3]. This CTP sequence confers an extended serum half-life to hCG or to other family members such as LH and FSH when genetically fused and expressed in vitro or in vivo [3, 4]. Accordingly, more potent analogs have been generated and clinically tested for their efficiency in various in vitro fertilization protocols [5]. The CTP is a critical determinant for polarized apical secretion of hCG in Madin-Darby canine kidney cells, in contrast to the basolateral secretion of LH, which lacks this sequence [6]. Human CG is a pregnancy-specific hormone; it is not normally synthesized in men. Although hCG expression from the placenta is detected during the early stages of pregnancy, with peak levels attained during the first trimester, free hCGß subunit is known to be expressed in many other tissues in women [7, 8]. Similarly, the LH/hCG receptor has a wide tissue expression pattern, including the uterus, brain, and duodenum [7, 8]. However, the biological significance of this expression is unknown. Moreover, free hCGß subunit is expressed at high levels in multiple cancers and has been a useful diagnostic marker at least in some cases [9, 10]. It is not known what function the free ß subunit exerts in this pathological condition. Similarly, elevated levels of pituitary gonadotropins are associated with ovarian cancer [11].
Our research group and others have used transgenic and knockout mouse technology to generate models for human diseases involving the gonadotropin-signaling cascade. Female FSHß and FSH receptor (FSH-R) knockout mice phenocopy human ovarian dysgenesis, whereas the male mutant mice have apparently normal fertility (FSHß knockouts) [12] or reduced fertility (FSH-R knockouts) [13, 14]. Human FSH-overexpressing male transgenic mice are infertile and have elevated testosterone levels. The female transgenic mice are also infertile and demonstrate symptoms similar to those of human ovarian hyperstimulation syndrome, including disrupted ovarian folliculogenesis leading to hemorrhage and cyst formation and accompanied by urinary tract abnormalities [15]. Male transgenic mice that overexpress an LHß-CTP analog are reportedly normal, whereas the female transgenic mice develop strain-dependent granulosa/stromal cell tumors, mammary tumors, pituitary adenomas, and defects in adrenal cortex development [16, 17]. Hypersecretion of LH in women causes polycystic ovarian syndrome, and this phenotype is attributed to a perturbed secretion of nonsteroidal ovarian hormones [18]. Two research groups have recently reported phenotypes of LH receptor (LH-R) knockout mice that phenocopy human LH-R-inactivating mutations [19, 20]. Typical features of these mutant mice include Leydig cell hypoplasia, reduced testosterone levels, and infertility in males. The female mice are infertile because of a blockade of ovarian folliculogenesis at the antral follicle stage. Activating mutations in the human LH-R gene cause male-limited precocious puberty, but women harboring the same mutation have normal fertility [21–24]. Clearly, these studies with mouse models and clinical cases suggest that the effects of gain and loss of function for gonadotropins are gender specific.
To distinguish the biological effects of free hCGß and hCG dimer in vivo and to analyze the consequences of overexpression of hCGß and hCG dimer in both male and female reproductive tract development and function, we used mouse metallothionein 1 (mMT-1) promoter to generate transgenic mice expressing either the hCGß subunit or both hCG subunits together in multiple tissues. Here, we report distinct gender-specific phenotypes of these transgenic mice.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The metallothionein (MT)-hCG transgene was constructed as described previously [15]. A 1.8-kilobase (kb) mMT-1 promoter was inserted upstream of the 3.6-kb hCGß gene [25]. The transgene fragments were released from the vector backbone with appropriate restriction enzyme digestions, purified using the GENECLEAN kit (Bio 101, Carlsbad, CA), and microinjectioned into fertilized eggs to produce transgenic mice [26].
Transgenic Mice
Independent lines of mice carrying either the MT-hCG transgene or the MT-hCGß transgene were separately generated by standard pronuclear injections into fertilized eggs from C57BL/6/C3H x ICR hybrid mice. Stable pedigrees of MT-hCG transgenic mice were obtained by crossing Southern blot-positive founder mice with control wild-type littermates. Additionally, mice expressing hCG heterodimer were generated by coinjection of both MT-hCG and MT-hCGß transgenes. All animal studies were conducted in accordance with the NIH guidelines for Care and Use of Experimental Animals, as approved by Baylor College of Medicine.
Southern Blot Analysis
For genotype analysis of founders and offspring, Southern blot analysis was performed on tail DNA using 32P-labeled probes as previously described [12, 15]. MT-hCG transgenic mice were screened as described previously [15]. The MT-hCGß transgene was detected with a probe made from 700 base pairs of exon 3 amplified by polymerse chain reaction (PCR) that does not cross-react with the endogenous mouse LHß gene sequence.
Northern Blot Analysis
Total RNA was extracted from different tissues of wild-type, MT-hCG, or MT-hCGß transgenic mice by the TRI-reagent (Leedo Medical Laboratories, Houston, TX) method [15]. RNA was denatured, separated on 1.4% agarose-formaldehyde gels, and transferred to nylon membranes. The membranes were hybridized with hCG or hCGß probes, washed, and exposed to autoradiographic film. Blots were then stripped and rehybridized with an 18S rRNA probe to check for equal loading of RNA [15].
Reverse Transcription PCR
Total RNA was isolated from multiple tissues of wild-type and transgenic mice and used in a reverse transcription (RT) reaction as previously described [15]. The first strand cDNA templates were used in a PCR with hCGß-specific exon 3 primers (forward: 5' ACC CGC GTG CTG CAG GGG 3'; reverse: 5' TTA TTG TGG GAG GAT CGG 3'). The amplified products were separated on 1.5% agarose gels and transferred to GeneScreen plus (Perkin Elmer, Boston, MA) membranes, and Southern blot analysis was performed using a hCGß-specific probe.
Hormone Assays
Mice under anesthesia were exsanguinated by closed cardiac puncture. Serum samples were collected and stored frozen at -20°C until further use. RIAs for LH and FSH were performed using National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) hormone assay kits according to the methods previously described [12, 15, 27] or by the standard protocols of the Ligand Assay and Analysis Core Laboratory (University of Virginia, Charlottesville, VA). Serum samples were analyzed for dimeric hCG by an ELISA using paired monoclonal antibodies or for hCGß free subunit using an IRMA kit (Diagnostic Systems Laboratories, Webster, TX), according to the manufacturer's instructions. The testosterone and estradiol assays were performed as described previously using solid phase RIA kits [12, 15, 27].
Histological Analysis
Testes were fixed in Bouin reagent overnight and rinsed several times in LiCO3-saturated 70% ethanol. Kidneys and ovaries were fixed in formalin. The tissues were processed and embedded in paraffin, and 4-µm sections were cut and stained with periodic acid-Schiff-hematoxylin reagents as previously described [12, 15, 27].
Statistical Anaylsis
Statistical analysis was performed with a Student t-test using a Microsoft Corp. (Redford, WA) Excel (version 6.0) software program. Differences were considered significant at P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and MT-hCGß Transgenes
We previously used a 1.2-kb fragment of the mMT-1 promoter to successfully overexpress various transgenes, including the hCG subunit, in multiple tissues of mice [15, 28]. Using the same promoter sequences, we produced transgenic mice that carry the hCGß subunit (Fig. 1, A–C). In addition, we generated mice that carry both the hCG and hCGß subunits by coinjecting the two transgenes into one-cell embryos. Southern blot analysis with specific probes identified transgenic founders that carried multiple copies of the hCGß subunit or both the and ß subunits together in low (
5–10 copies) and high (>50 copies) numbers. Stable pedigrees could be established for MT-hCG transgenic mice but not for transgenic mice that carried either the MT-hCGß subunit or both subunits.
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MT-hCGß Subunit Transgenic Mice Are Infertile and Demonstrate Gonadal Defects
All of the transgenic male (three of three) and female mice (three of three) that carried the MT-hCGß subunit were infertile when mated to either wild-type or hCG subunit transgenic mice over a period of 6 mo, suggesting that the free hCGß subunit, secreted from cells where it was produced, interfered with fertility in these mice. Expression of the hCGß subunit was found in multiple tissues of transgenic mice, similar to the expression of the hCG subunit [15], by RT-PCR assay (Fig. 1D) and Northern blot analyses [15] (data not shown). A dimer-specific immunoradiometric assay did not detect any hCG dimer in the serum of the hCGß subunit transgenic mice (data not shown). Although testes of the infertile MT-hCGß male transgenic mice appeared normal morphologically and histologically, ovaries from the female transgenic mice demonstrated a block in folliculogenesis. These ovaries did not contain antral follicles or corpora lutea, indicating that the mice did not cycle. In some cases, the female transgenic mice developed cysts and hemorrhages and enlargement of the uterine horns. These results demonstrate that the hCGß subunit-expressing mice are infertile, and the female transgenic mice have ovarian defects.
Low-Level hCG Dimer-Expressing Transgenic Mice Develop Progressive Infertility
Because the MT-hCGß-expressing transgenic mice were infertile, to study the gain of function effects of the hCG dimer, we coinjected both the MT-hCG and MT-hCGß transgenes into mouse embryos to generate hCG dimer-expressing transgenic mice. Male and female mice that carry both the transgenes in low copy numbers (5–10 copies) were fertile initially and produced offspring that carried both the transgenes. The litter sizes were normal, with an average of 8.3 ± 0.3 pups/litter (35 litters, five mating pairs), when compared with those from wild-type matings (8.3 ± 0.2 pups/litter, 104 litters, 12 mating pairs). However, these transgenic male and female mice progressively became infertile by 6–7 mo of age but displayed no other apparent differences. The gonads were normal, with no discernible evidence of histological abnormalities (data not shown). Thus, constitutive low-level expression of hCG causes progressive infertility of unknown etiology in both male and female transgenic mice.
High-Level hCG Dimer-Expressing Male Transgenic Mice Are Infertile and Develop Leydig Cell Hyperplasia
In contrast to the low-level hCG dimer-expressing mice, male founder mice that expressed high levels of hCG dimer were all (five of five) infertile. These male mice never mated with females (no vaginal plugs) of any genotype (wild-type or transgenic) over a period of 6 mo. They had very high levels of serum hCG dimer and consequently elevated testosterone levels (Table 1). As a result of the elevated levels of testosterone, these male transgenic mice demonstrated massive enlargement of fluid-filled seminal vesicles (Table 1 and Fig. 2, A and B). The testis size in these mice was reduced (Fig. 2, A and B), and histological analysis revealed significant Leydig cell hyperplasia (Fig. 2, F and H), with some tubules demonstrating a "Sertoli cell only" phenotype (Fig. 2G) characteristic of germ cell loss in mice and humans. Occasionally, many vacuolated tubules were also observed near the periphery of the tubules (Fig. 2, F and H). Serum LH and FSH levels were suppressed in these male mice compared with the wild-type controls (Table 1), presumably because of heightened steroid feedback (Table 1). These results suggest that high levels of hCG cause multiple male reproductive defects, including Leydig cell hyperplasia, seminal vesicle enlargement, and infertility.
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High-Level hCG Dimer-Expressing Female Transgenic Mice Are Infertile and Develop Polycystic Ovaries and Ovarian Thecomas
MT-hCG female transgenic mice that express high levels of the hCG dimer were all (four of four) infertile when mated to wild-type male mice. Serum analysis of these mice demonstrated elevated levels of estradiol (Table 1) consistent with the presence of enlarged uterine horns, compared with levels in wild-type mice (Table 1 and Fig. 3, A and G). These transgenic mice also displayed a dramatic ovarian phenotype by 6–7 wk of age, with massive hemorrhage and the presence of multiple cysts (Fig. 3, G–I). Histological analysis revealed multiple aberrant ovarian follicles with enlarged thecal cell layers (Fig. 3, J and K) and proliferating stromal tissue. Many of these stromal cells appeared multinucleated (Fig. 3K).
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Additional Phenotypes in High-Level hCG Dimer-Expressing Transgenic Mice
In addition to the gonadal phonotypes described above, the high-level hCG dimer-expressing mice demonstrated phenotypes in nongonadal tissues. Male mice overexpressing hCG dimer at high levels were behaviorally very aggressive; they were ferocious and displayed mutilating behavior when caged with either transgenic or nontransgenic control males or females. The aggressive behavior was noted only in males and not in females expressing hCG dimer, suggesting that the high testosterone was causal for this behavioral finding. Serum estradiol levels were not elevated in these male mice (Table 1), suggesting that the observed aggressive phenotype in male transgenic mice is not due to an estrogen effect.
In contrast to these behavioral defects in males, the female transgenic mice overexpressing hCG developed urinary tract defects, including marked enlargement of the kidneys and bladder coincident with the ovarian hemorrhage and cysts (Fig. 4, A–D) (data not shown). Histological analysis of the enlarged kidneys indicated total disruption of the glomerular architecture, and many cystic spaces suggested degeneration of the glomeruli (Fig. 4, D and E) compared with normal glomerular architecture in the wild-type mouse kidneys (Fig. 4C).
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Despite the above phenotypes resulting from prolonged hCG stimulation, the mammary and the adrenal glands in these mice were morphologically and histologically normal at all ages examined (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, MT-FSHß, and MT-FSH transgenic mice. High levels of expression of these transgenes was achieved using an mMT-1 promoter. The MT-1 promoter is activated very early during mouse embryogenesis and is ubiquitously expressed. It is also highly expressed in the liver. We used this same promoter (1.8 kb) to generate multiple MT-hCGß transgenic mice. Similar to our earlier results, we obtained mice strains for both low and high transgene copy numbers. The MT-hCGß transgene was expressed at high levels in multiple tissues, consistent with the high basal activity of the MT-1 promoter in multiple tissues of the mouse.
In contrast to the normal fertility of male and female MT-hCG and male MT-hFSHß transgenic mice, both male and female transgenic mice expressing MT-hCGß were infertile. This result is probably related to the intracellular assembly and secretion behavior of glycoprotein hormone free subunits. Free hFSHß is not efficiently secreted from cells, whereas free hCGß is efficiently secreted in vivo and in vitro [25, 29, 30]. Free hCGß subunit is secreted into serum in the MT-hCGß transgenic mice, interfering with endogenous LH binding to LH/hCG receptors on gonads and thus causing male infertility. There is some in vitro evidence to suggest that free hCGß subunit can bind LH/hCG receptors and block steroidogenesis [31, 32]. However it is not known what effects large amounts of hCGß alone can elicit when secreted from multiple tissues, including the testis. Male mice expressing only hCGß only are infertile even though hCGß acts as a weak agonist in LH/hCG receptor binding assays and in vitro its activity is only 1–2% that of the intact heterodimer. Contrary to the expected regressed testis phenotype, these male mice demonstrated apparently normal testis histology. The infertility in these male mice could be the result of quantitative and functional differences in spermatogenesis, although qualitatively all stages of spermatogenesis appear normal. In female transgenic mice expressing only hCGß, which were also infertile, additional ovarian cystic and hemorrhagic phenotypes may be caused by alteration of local factors or by a direct effect of locally produced hCGß subunit within the ovary (mMT-1 promoter is active in the ovary) [15]. Furthermore, the phenotypes of mice expressing only hCGß and those expressing the hCG dimer are distinct, suggesting that ubiquitous expression of hCGß does not cause bioactive production of hCG heterodimer by assembling with the mouse -glycoprotein subunit (GSU) in the pituitary, as has been recently reported in ubiquitin C-hCGß transgenic mice. Bioactive hCG dimer was not produced in these MT-hCGß transgenic mice, consistent with previous observations indicating MT-1 promoter is not active in pituitary gonadotrope cells [15].
Because the transgenic male and female mice expressing MT-hCGß subunit were infertile, we coinjected the individual subunit transgenes to generate mice expressing the hCG dimer. This strategy resulted in production of female mice that expressed low and high levels of hCG dimer. Although, initially normal and fertile, the low-level hCG dimer-expressing mice progressively became infertile by 5–6 mo of age. This finding suggests that sustained production of hCG from multiple tissues can cause infertility, although the exact mechanism remains to be elucidated. Similar production of low levels of FSH from the same promoter does not affect male or female fertility [15, 33]. However, in each case, it is difficult to compare levels of FSH and hCG, their receptor binding capacities, and their bioactivity in the target cells. It is also difficult to assess how the steroid and nonsteroidal gonadal hormone feedback mechanisms operate at the hypothalamus-pituitary level in each case.
High-level hCG dimer-expressing male mice had multiple reproductive defects. Most of these appeared to be secondary to elevated serum testosterone levels and included suppression of endogenous LH and FSH levels and greatly enlarged seminal vesicles. Similar to hFSH transgenic male mice, hCG-overexpressing male mice were infertile. The important difference between these two strains of mice is that testes were normal size in hFSH-overexpressing mice but were small in hCG-overexpressing male mice. Furthermore, unlike hFSH-overexpressing mice, these mice were highly aggressive and displayed abnormal sexual behavior. The phenotypes in hCG dimer-expressing male mice are in sharp contrast to normal fertility in transgenic male mice in which the expression of an LH analog (LHß-CTP) was targeted to the gonadotropes using a bovine glycoprotein hormone subunit promoter [34]. Similarly, these phenotypes are not present in male human patients who demonstrate precocious puberty due to constitutively active LH-R signaling [23]. The aggressive phenotype in hCG-overexpressing mice could have two causes: 1) high levels of testosterone are known to affect male behavior, and 2) local production of hCG in regions of the brain known to be important for aggressive and sexual behavior might cause alterations in LH/hCG receptor signaling. In transgenic mice that harbor a 36-kb cosmid transgene consisting of six hCGß genes driven by the homologous promoter, a different subset of CGß genes, CGß1 and CGß 2, normally not transcribed in human placenta, are abundantly expressed in the cerebral cortex [35]. Similarly, some evidence suggests that LH/hCG receptors and orphan receptors belonging to the glycoprotein hormone receptor family, the leucine-rich repeat-containing G protein-coupled receptors, are present within the hypothalamus and brain, respectively [36, 37]. The high levels of hCG in the brain of our hCG transgenic mice might be interfering with these signaling pathways, resulting in behavioral defects.
Another important testis phenotype of hCG-overexpressing mice is Leydig cell hyperplasia. Leydig cells develop as two distinct populations during testis development and are dependent upon LH stimulation. Because the MT-1 promoter is active very early during mouse embryogenesis, continuous production/stimulation by hCG (which binds the LH receptor) can result in Leydig cell hyperplasia, leading to the high testosterone production that we observed. The Leydig cell phenotype of hCG-overexpressing transgenic mice is consistent with previous observations from in vitro and in vivo studies, in which continuous injections of pharmacological doses of hCG for a prolonged time in rats and mice resulted in accelerated Leydig cell hyperplasia with a corresponding elevation in serum testosterone levels. Future studies could focus on determining the aberrant cell cycle events triggered by overexpression of hCG (continuously from embryo to adult stage) that lead to the Leydig cell phenotype in these mice.
Female transgenic mice that overexpress hCG demonstrated reproductive and urinary tract defects. These mice had very high serum estradiol levels and enlarged uterine horns. Similar to the LHß-CTP transgenic mice, these female mice were infertile and had hemorrhagic and cystic ovaries [38, 39]. In contrast to granulosa cell tumors that develop in a genetic strain-dependent manner in LHß-CTP female transgenic mice, hCG-overexpressing female mice developed thecomas with excessive interstitial cell proliferation, and the ovaries demonstrated disrupted folliculogenesis. Other similarities between these two strains of transgenic mice are the kidney defects that are specific to females. The renotropic activity of LH has been reported; however, the biological basis for this has not been explored [40, 41]. Transgenic female mice overexpressing hCG develop enlarged cystic kidneys with progressive degeneration of glomerular architecture (Fig. 4C). Prolonged hCG stimulation of the kidneys in these female transgenic mice might be causing the glomerular degeneration.
More recently, female transgenic mice overexpressing the hCGß subunit driven by a ubiquitin C promoter have been generated [42]. Unlike the 100% infertility that was observed with our MT-hCGß mice, two of the five founder ubiquitin C-hCGß transgenic mice were fertile. These mice had high levels of bioactive serum hCG (because of the heterodimeric assembly of the hCGß with the mouse -GSU in the pituitary), leading to increased secretion of ovarian steroids. Additional phenotypes in these mice resemble those seen in bovine glycoprotein hormone -LHß-CTP mice and include precocious puberty, pituitary (lactotrope) adenomas accompanied by hyperprolactinemia, and mammary adenocarcinoma. No kidney changes were reported. It is not known whether male transgenic mice expressing hCGß from the ubiquitin C promoter demonstrate any reproductive anomalies.
The distinct phenotypes observed in our hCG dimer-expressing male mice suggest that the extent of hCG overexpression may be critical for functional activity. Because MT-1 promoter is active very early during mouse development, and LH-Rs are expressed at this stage, we hypothesize that the defects we observed in our transgenic mice are a result of both abnormal morphogenesis and hyperaction of the hormone. Further, because the hCG-overexpressing founder male and female mice were all infertile, it was not possible to evaluate the progression of phenotypes with age. In future studies, we plan to use a tetracyclin on/off system to produce mice that express hCG in a temporal fashion and to evaluate the resultant phenotypes during multiple developmental stages.
We used the mMT-1 promoter to develop transgenic mice that constitutively expressed either hCGß subunit or hCG dimer in multiple tissues. The phenotypes of mice expressing only hCGß are distinct from those of mice expressing the hCG dimer. Furthermore, hCG dimer-expressing mice are similar to other existing gonadotropin mouse models and display additional unique reproductive, urinary tract, and behavioral phenotypes (Table 2), suggesting the importance of pituitary targeted versus ectopic expression of gonadotropic hormones in reproductive physiology. The hCG-overexpressing transgenic mice may be useful for further study of the mechanisms of Leydig cell hyperplasia and ovarian hyperstimulation syndrome accompanied by kidney defects.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: T. Rajendra Kumar, Department of Pathology, Baylor College of Medicine, Houston, TX 77030. FAX: 713 798 7505; tkumar{at}bcm.tmc.edu
Received: 3 December 2002.
First decision: 17 December 2002.
Accepted: 7 March 2003.
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REFERENCES |
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