- Email: [email protected]
Functional analysis of LHβ knockout mice
Molecular and Cellular Endocrinology 269 (2007) 81–84
Review
Functional analysis of LH knockout mice T. Rajendra Kumar a,b,∗ a
Department of Molecular and Integrative Physiology, University of Kansas Medical Center, Kansas, KS 66160, USA Department of Pathology and Laboratory Medicine, University of Kansas Medical Center, Kansas, KS 66160, USA
b
Received 1 August 2006; received in revised form 12 October 2006; accepted 12 October 2006 Review based on the presentation at the Gonadal and Non-gonadal Actions of LH/hCG; University of Turku, Finland; 3–4 June 2005
Abstract LH and FSH act on gonadal cells to regulate steroidogenesis and gametogenesis. To model human reproductive disorders involving loss of LH function and to define LH-responsive genes, we produced knockout mice lacking the hormone-specific LH subunit. LH null mice are viable but demonstrate postnatal defects in gonadal growth and function resulting in infertility. Mutant males have decreased testes size, a block in Leydig cell differentiation, and a reduction in serum and intra-testicular testosterone levels. Furthermore, spermatogenesis is blocked at the round spermatid stage resulting in a total absence of the elongated spermatids. Mutant female mice are hypogonadal and demonstrate decreased levels of serum estradiol and progesterone. Ovarian histology reveals normal thecal layer, defects in folliculogenesis including many degenerating antral follicles and absence of corpora lutea. The defects in both sexes are not secondary to aberrant FSH regulation, since FSH levels were unaffected in null mice. Finally, the null mice can be pharmacologically rescued by exogenous hCG indicating that LH-responsiveness of the target cells is not irreversibly lost. Thus, LH null mice provide a useful model to study the consequences of an isolated deficiency of LH ligand in reproduction, while retaining normal LH-responsiveness in target cells. © 2007 Elsevier Ireland Ltd. All rights reserved. Keywords: Pituitary; Gonadotropins; Testis; Ovary; Knockout mice
Contents 1. 2. 3. 4. 5. 6.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Gene targeting at the LH locus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Phenotypes of LH null male mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Phenotypes of LH null female mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Rescue of LH null mice. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Future directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction Luteinizing hormone (LH) is a heterodimeric pituitary glycoprotein that shares a common alpha subunit (␣-GSU) with other ∗
Correspondence address: Department of Molecular and Integrative Physiology, 3011 WHE, Mail Stop 3043, 3901 Rainbow Blvd., University of Kansas Medical Center, Kansas City, KS 66160, USA. Tel.: +1 913 588 0414; fax: +1 913 588 0455. E-mail address: [email protected]. 0303-7207/$ – see front matter © 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.mce.2006.10.020
81 82 82 83 83 83 83 83
glycoprotein hormones of the pituitary and placenta including FSH, TSH and CG (Bousfield et al., 1994; Pierce and Parsons, 1981). In each case, the -subunit is receptor and hence hormone-specific. Human CG binds LH-receptors and elicits identical response to LH both in vitro and in vivo (Bousfield et al., 1994; Pierce and Parsons, 1981). LH receptor is a G-protein coupled seven transmembrane helices containing glycoprotein. LH receptors are expressed on Leydig cells of the testicular interstitium and the ovarian granulosa and theca cells (Themmen and Huhtaniemi, 2000). LH and FSH are both secreted from
82
T.R. Kumar / Molecular and Cellular Endocrinology 269 (2007) 81–84
gonadotropes and act in a coordinated fashion to regulate gonadal growth, differentiation, steroidogenesis and gametogenesis (Kumar and Matzuk, 2000). Our previous gain-of-function studies indicated that overexpression of hCG causes Leydig cell hyperplasia accompanied by other seminiferous tubule defects in the male and hemorrhagic and cystic ovaries with thecal expansion in the female (Matzuk et al., 2003). Gain-of-function LH phenotypes in humans include activating mutations in LHreceptors that result in male-limited precocious puberty (Matzuk et al., 2003; Themmen et al., 1998; Themmen and Huhtaniemi, 2000). In contrast, inactivating mutations in human LH receptor are more common and manifest in men as infertility often associated with Leydig cell hypoplasia (Huhtaniemi et al., 1999; Huhtaniemi, 2002; Matzuk et al., 2003; Themmen et al., 1998; Themmen and Huhtaniemi, 2000). Mutations in human LH encoding gene are very rare and only two cases of affected men have been reported (Valdes-Socin et al., 2004; Weiss et al., 1992). Phenotypic manifestations of loss of LH ligand function in the female are unknown. LH receptor null mice (LuRKO) have been generated (Lei et al., 2001; Zhang et al., 2001) but it is unknown to what extent these would mimic the loss of LH ligand function in vivo. There are no naturally occurring mutations at the LH locus in mice. To study the consequences of loss of LH ligand in vivo, and to delineate the functions of LH and FSH, we have produced LH null mice and compared them with the previously generated FSH null mice. 2. Gene targeting at the LH locus The single copy LH encoding gene is localized to mouse chromosome 7 (Kumar and Matzuk, 1995). We disrupted this gene in ES cells and subsequently generated viable and fertile heterozygous mice. Intercrosses between these heterozygous mice resulted in production of LH null mice. Pituitary immunofluorescence using LH- and FSH-specific antibodies, serum RIAs, Western blot analyses have all confirmed that we engineered a null mutation at the LH locus with LH deficiency and FSH levels were unaltered. These mutant mice were
infertile and demonstrated postnatal defects in gonadal growth and differentiation. 3. Phenotypes of LH null male mice The mutant males were hypogonadal and demonstrated reduced size testes and accessory glands, consistent with decreased serum and intra-testicular testosterone levels (Ma et al., 2004). Histological analysis of adult testes indicated insignificant interstitium containing very few and small size Leydig cells. Gene expression analyses confirmed an increase in fetal Leydig cell marker, thrombospondin-2, and reduction in many of the steroidogenic pathway enzymes (Ma et al., 2004). Serum assays demonstrated increased levels of the androgen precursor, androstenedione, indicating the presence of fetal Leydig cells in the mutant testes (Ma et al., 2004). To further characterize the morphology of Leydig cells, we analyzed 3hydroxysteroid dehydrogenase immunostained testes sections by confocal microscopy. In contrast to control sections that contained mostly mature Leydig cells, the mutant testes showed three distinct types of Leydig cells. Based on their characteristic morphology, these were classified as fetal-like, spindle shaped progenitors and small round immature Leydig cells (Zama et al., in preparation). There were no mature Leydig cells identified in the mutant testes interstitium. Further molecular analyses revealed that various markers normally expressed in progenitors and immature Leydig cells are unaffected in the absence of LH (Ma, X., Kumar, T.R., unpublished). Thus, these studies indicate that LH signaling is required for differentiation of both progenitors to immature and immature to mature adult-type Leydig cells. Because Sertoli cells are the major targets of androgen action within the testis, expression of Sertoli-specific markers was evaluated in the mutant testes. These studies identified that although expression of some markers (FSH receptor and inhibin ␣) was not affected, expression of inhibin A- and B-subunits and AMH was upregulated (Ma et al., 2004). To analyze the consequences of severely reduced testosterone on spermatogenesis, the mutant testes were analyzed histologically and expression of spermatogenesis markers assessed. The mutant testes consist
Fig. 1. Spermiogenesis defect in the absence of LH. Cross sections of testes from adult wild-type (WT) and LH knockout (KO) mice showing different stages of spermatogenesis. Note that in contrast to the WT section, no late stage spermatids and sperm are present in the mutant testis section. Thus, absence of LH leads to a spermiogenesis block. Bouin’s reagent-fixed sections were stained with a nuclear dye to visualize different cell morphologies. White arrows indicate late stage spermatids in the WT section whereas they indicate round spermatids in the KO section. Photographs taken at 40× magnification using an Olympus microscope.
T.R. Kumar / Molecular and Cellular Endocrinology 269 (2007) 81–84
of spermatogonia, spermatocytes (meiotic cells) and round spermatids (Ma et al., 2004). Confocal microscopy confirmed that at least eight different stages of tubules were identifiable in the mutant testes. The majority of the tubules showed type B spermatogonia, pachytene spermatocytes and round spermatids (Fig. 1). No late stage or elongated spermatids were observed consistent with an absence of histone H1-like linker protein, a late stage spermatid marker that was suppressed in the mutant testes (Ma et al., 2004). Additional defects in the mutant tubules include aberrant expression of various structural proteins and enhanced apoptosis (Zama et al., in preparation). Therefore, FSH alone is not sufficient to promote full spermatogenesis in the absence of LH and/or testosterone. Thus, the loss of LH leads to both somatic and spermatogenic cell defects, similarly seen in other models with a Sertoli cell-selective androgen receptor deletion (SCARKO) (Holdcraft and Braun, 2004a,b). LH mutants provide useful models for further studying somatic–germ cell interactions in the testis.
83
6. Future directions Our data clearly indicate that loss of LH action has more severe consequences in gonadal growth and differentiation and that FSH alone cannot compensate this loss. LH knockout mice closely phenocopy LuRKO mice and demonstrate LH signaling is not required for prenatal development. One advantage of our LH ligand knockout model is that we can pharmacologically rescue these null mice at defined time points and thus we will be able to define LH and steroid responsive genes in gonads. The absence of LH in these mice will also allow us now to test the role of LH in gonadal tumor-prone inhibin ␣ knockout mice (Kumar et al., 1996, 1999). Furthermore, LH null mice will be useful to develop novel double mutants lacking both FSH and LH but with an intact GnRH signaling system. Because GnRH analogs are often used to treat various gonadal cancers (Nagy and Schally, 2005; Weiss et al., 2002), these double LH/FSH mutant mice will enable us to determine the direct effects of GnRH, if any, on gonadal function.
4. Phenotypes of LH null female mice LH knockout females, similar to mutant males were hypogonadal and showed thin uterine horns (Ma et al., 2004). Histological analysis of the ovaries indicated absence of healthy antral, preovulatory follicles and corpora lutea, confirming impaired estrous cycles (Ma et al., 2004). Primary and secondary follicles appeared normal, whereas many antral follicles were abnormal containing degenerating oocytes. Despite these defects in granulosa cells and oocytes, a prominent thecal layer was obvious in follicles at different stages of progression (Ma et al., 2004). These observations suggest that differentiation of thecal layer was not impaired in the absence of LH signaling. However, expression of various thecal cell markers including many steroidogenic enzymes was significantly reduced (Ma et al., 2004). Consequently, serum progesterone and estradiol levels were decreased and the mutant uteri were hypoplastic consisting of a thin endometrial layer (Ma et al., 2004). These ovarian phenotypes in LH knockout mice are distinct from those of FSH knockout mice (Kumar et al., 1997) and provide a genetic evidence for distinct roles of FSH and LH during folliculogenesis. 5. Rescue of LH null mice Pharmacological rescue of both male and female LH knockout mice was achieved by short-term treatment of hCG. Testes and ovarian markers are upregulated following hormone replacement of the mutants with hCG indicating that responsiveness to LH is not irreversibly lost in target cells in the absence of LH (Ma et al., 2004). More recently, we have performed microarray analyses and compared large-scale gene expression profiles in the testes of LH null male mice treated with hCG for 48 h and testosterone for 7 days (Svojanovsky, S., Kumar, T.R., unpublished). Thus, LH knockout mice serve as a useful model to study LH- and steroid-responsive genes and for identifying to what extent steroids mediate the effects of LH in testis development and function.
Acknowledgments I thank Dr. Irving Boime for encouragement and support. Studies reported in this Review are supported in part by The Moran Foundation, Baylor College of Medicine, Houston, TX and The Hall Family Foundation, Kansas City, MO. I also thank the contributions of my colleagues, Xiaoping Ma, Yanlan Dong and Aparna Zama during various phases of this work. References Bousfield, G.R., Perry, W.M., Ward, D.N., 1994. Gonadotropins: chemistry and biosynthesis. In: Knobil, E., Neill, J.D. (Eds.), The Physiology of Reproduction. Raven Press, New York, pp. 1749–1792. Holdcraft, R.W., Braun, R.E., 2004a. Androgen receptor function is required in Sertoli cells for the terminal differentiation of haploid spermatids. Development 131, 459–467. Holdcraft, R.W., Braun, R.E., 2004b. Hormonal regulation of spermatogenesis. Int. J. Androl. 27, 335–342. Huhtaniemi, I., Jiang, M., Nilsson, C., Pettersson, K., 1999. Mutations and polymorphisms in gonadotropin genes. Mol. Cell. Endocrinol. 151, 89–94. Huhtaniemi, I.T., 2002. The role of mutations affecting gonadotrophin secretion and action in disorders of pubertal development. Best Pract. Res. Clin. Endocrinol. Metab. 16, 123–138. Kumar, T.R., Matzuk, M.M., 1995. Cloning of the mouse gonadotropin beta-subunit-encoding genes. II. Structure of the luteinizing hormone betasubunit-encoding genes. Gene 166, 335–336. Kumar, T.R., Matzuk, M.M., 2000. Gene knockout models to study the hypothalamus-pituitary-gonadal axis. In: Shupnik, M.A. (Ed.), Gene Engineering and Molecular Models in Endocrinology. The Humana Press, Totowa, NJ. Kumar, T.R., Palapattu, G., Wang, P., Woodruff, T.K., Boime, I., Byrne, M.C., Matzuk, M.M., 1999. Transgenic models to study gonadotropin function: the role of follicle-stimulating hormone in gonadal growth and tumorigenesis. Mol. Endocrinol. 13, 851–865. Kumar, T.R., Wang, Y., Lu, N., Matzuk, M.M., 1997. Follicle stimulating hormone is required for ovarian follicle maturation but not male fertility. Nat. Genet. 15, 201–204. Kumar, T.R., Wang, Y., Matzuk, M.M., 1996. Gonadotropins are essential modifier factors for gonadal tumor development in inhibin-deficient mice. Endocrinology 137, 4210–4216.
84
T.R. Kumar / Molecular and Cellular Endocrinology 269 (2007) 81–84
Lei, Z.M., Mishra, S., Zou, W., Xu, B., Foltz, M., Li, X., Rao, C.V., 2001. Targeted disruption of luteinizing hormone/human chorionic gonadotropin receptor gene. Mol. Endocrinol. 15, 184–200. Ma, X., Dong, Y., Matzuk, M.M., Kumar, T.R., 2004. Targeted disruption of luteinizing hormone beta-subunit leads to hypogonadism, defects in gonadal steroidogenesis, and infertility. Proc. Natl. Acad. Sci. U.S.A. 101, 17294–17299. Matzuk, M.M., DeMayo, F.J., Hadsell, L.A., Kumar, T.R., 2003. Overexpression of human chorionic gonadotropin causes multiple reproductive defects in transgenic mice. Biol. Reprod. 69, 338–346. Nagy, A., Schally, A.V., 2005. Targeting of cytotoxic luteinizing hormonereleasing hormone analogs to breast, ovarian, endometrial, and prostate cancers. Biol. Reprod. 73, 851–859. Pierce, J.G., Parsons, T.F., 1981. Glycoprotein hormones: structure and function. Annu. Rev. Biochem. 50, 465–495. Themmen, A.P., Martens, J.W., Brunner, H.G., 1998. Activating and inactivating mutations in LH receptors. Mol. Cell. Endocrinol. 145, 137–142.
Themmen, A.P.N., Huhtaniemi, I.T., 2000. Mutations of gonadotropins and gonadotropin receptors: elucidating the physiology and pathophysiology of pituitary-gonadal function. Endocr. Rev. 21, 551–583. Valdes-Socin, H., Salvi, R., Daly, A.F., Gaillard, R.C., Quatresooz, P., Tebeu, P.M., Pralong, F.P., Beckers, A., 2004. Hypogonadism in a patient with a mutation in the luteinizing hormone beta-subunit gene. N. Engl. J. Med. 351, 2619–2625. Weiss, J., Axelrod, L., Whitcomb, R.W., Harris, P.E., Crowley, W.F., Jameson, J.L., 1992. Hypogonadism caused by a single amino acid substitution in the beta subunit of luteinizing hormone. N. Engl. J. Med. 326, 179– 183. Weiss, J.M., Diedrich, K., Ludwig, M., 2002. Gonadotropin-releasing hormone antagonists: pharmacology and clinical use in women. Treat. Endocrinol. 1, 281–291. Zhang, F.P., Poutanen, M., Wilbertz, J., Huhtaniemi, I., 2001. Normal prenatal but arrested postnatal sexual development of luteinizing hormone receptor knockout (LuRKO) mice. Mol. Endocrinol. 15, 172–183.
Review
Functional analysis of LH knockout mice T. Rajendra Kumar a,b,∗ a
Department of Molecular and Integrative Physiology, University of Kansas Medical Center, Kansas, KS 66160, USA Department of Pathology and Laboratory Medicine, University of Kansas Medical Center, Kansas, KS 66160, USA
b
Received 1 August 2006; received in revised form 12 October 2006; accepted 12 October 2006 Review based on the presentation at the Gonadal and Non-gonadal Actions of LH/hCG; University of Turku, Finland; 3–4 June 2005
Abstract LH and FSH act on gonadal cells to regulate steroidogenesis and gametogenesis. To model human reproductive disorders involving loss of LH function and to define LH-responsive genes, we produced knockout mice lacking the hormone-specific LH subunit. LH null mice are viable but demonstrate postnatal defects in gonadal growth and function resulting in infertility. Mutant males have decreased testes size, a block in Leydig cell differentiation, and a reduction in serum and intra-testicular testosterone levels. Furthermore, spermatogenesis is blocked at the round spermatid stage resulting in a total absence of the elongated spermatids. Mutant female mice are hypogonadal and demonstrate decreased levels of serum estradiol and progesterone. Ovarian histology reveals normal thecal layer, defects in folliculogenesis including many degenerating antral follicles and absence of corpora lutea. The defects in both sexes are not secondary to aberrant FSH regulation, since FSH levels were unaffected in null mice. Finally, the null mice can be pharmacologically rescued by exogenous hCG indicating that LH-responsiveness of the target cells is not irreversibly lost. Thus, LH null mice provide a useful model to study the consequences of an isolated deficiency of LH ligand in reproduction, while retaining normal LH-responsiveness in target cells. © 2007 Elsevier Ireland Ltd. All rights reserved. Keywords: Pituitary; Gonadotropins; Testis; Ovary; Knockout mice
Contents 1. 2. 3. 4. 5. 6.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Gene targeting at the LH locus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Phenotypes of LH null male mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Phenotypes of LH null female mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Rescue of LH null mice. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Future directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction Luteinizing hormone (LH) is a heterodimeric pituitary glycoprotein that shares a common alpha subunit (␣-GSU) with other ∗
Correspondence address: Department of Molecular and Integrative Physiology, 3011 WHE, Mail Stop 3043, 3901 Rainbow Blvd., University of Kansas Medical Center, Kansas City, KS 66160, USA. Tel.: +1 913 588 0414; fax: +1 913 588 0455. E-mail address: [email protected]. 0303-7207/$ – see front matter © 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.mce.2006.10.020
81 82 82 83 83 83 83 83
glycoprotein hormones of the pituitary and placenta including FSH, TSH and CG (Bousfield et al., 1994; Pierce and Parsons, 1981). In each case, the -subunit is receptor and hence hormone-specific. Human CG binds LH-receptors and elicits identical response to LH both in vitro and in vivo (Bousfield et al., 1994; Pierce and Parsons, 1981). LH receptor is a G-protein coupled seven transmembrane helices containing glycoprotein. LH receptors are expressed on Leydig cells of the testicular interstitium and the ovarian granulosa and theca cells (Themmen and Huhtaniemi, 2000). LH and FSH are both secreted from
82
T.R. Kumar / Molecular and Cellular Endocrinology 269 (2007) 81–84
gonadotropes and act in a coordinated fashion to regulate gonadal growth, differentiation, steroidogenesis and gametogenesis (Kumar and Matzuk, 2000). Our previous gain-of-function studies indicated that overexpression of hCG causes Leydig cell hyperplasia accompanied by other seminiferous tubule defects in the male and hemorrhagic and cystic ovaries with thecal expansion in the female (Matzuk et al., 2003). Gain-of-function LH phenotypes in humans include activating mutations in LHreceptors that result in male-limited precocious puberty (Matzuk et al., 2003; Themmen et al., 1998; Themmen and Huhtaniemi, 2000). In contrast, inactivating mutations in human LH receptor are more common and manifest in men as infertility often associated with Leydig cell hypoplasia (Huhtaniemi et al., 1999; Huhtaniemi, 2002; Matzuk et al., 2003; Themmen et al., 1998; Themmen and Huhtaniemi, 2000). Mutations in human LH encoding gene are very rare and only two cases of affected men have been reported (Valdes-Socin et al., 2004; Weiss et al., 1992). Phenotypic manifestations of loss of LH ligand function in the female are unknown. LH receptor null mice (LuRKO) have been generated (Lei et al., 2001; Zhang et al., 2001) but it is unknown to what extent these would mimic the loss of LH ligand function in vivo. There are no naturally occurring mutations at the LH locus in mice. To study the consequences of loss of LH ligand in vivo, and to delineate the functions of LH and FSH, we have produced LH null mice and compared them with the previously generated FSH null mice. 2. Gene targeting at the LH locus The single copy LH encoding gene is localized to mouse chromosome 7 (Kumar and Matzuk, 1995). We disrupted this gene in ES cells and subsequently generated viable and fertile heterozygous mice. Intercrosses between these heterozygous mice resulted in production of LH null mice. Pituitary immunofluorescence using LH- and FSH-specific antibodies, serum RIAs, Western blot analyses have all confirmed that we engineered a null mutation at the LH locus with LH deficiency and FSH levels were unaltered. These mutant mice were
infertile and demonstrated postnatal defects in gonadal growth and differentiation. 3. Phenotypes of LH null male mice The mutant males were hypogonadal and demonstrated reduced size testes and accessory glands, consistent with decreased serum and intra-testicular testosterone levels (Ma et al., 2004). Histological analysis of adult testes indicated insignificant interstitium containing very few and small size Leydig cells. Gene expression analyses confirmed an increase in fetal Leydig cell marker, thrombospondin-2, and reduction in many of the steroidogenic pathway enzymes (Ma et al., 2004). Serum assays demonstrated increased levels of the androgen precursor, androstenedione, indicating the presence of fetal Leydig cells in the mutant testes (Ma et al., 2004). To further characterize the morphology of Leydig cells, we analyzed 3hydroxysteroid dehydrogenase immunostained testes sections by confocal microscopy. In contrast to control sections that contained mostly mature Leydig cells, the mutant testes showed three distinct types of Leydig cells. Based on their characteristic morphology, these were classified as fetal-like, spindle shaped progenitors and small round immature Leydig cells (Zama et al., in preparation). There were no mature Leydig cells identified in the mutant testes interstitium. Further molecular analyses revealed that various markers normally expressed in progenitors and immature Leydig cells are unaffected in the absence of LH (Ma, X., Kumar, T.R., unpublished). Thus, these studies indicate that LH signaling is required for differentiation of both progenitors to immature and immature to mature adult-type Leydig cells. Because Sertoli cells are the major targets of androgen action within the testis, expression of Sertoli-specific markers was evaluated in the mutant testes. These studies identified that although expression of some markers (FSH receptor and inhibin ␣) was not affected, expression of inhibin A- and B-subunits and AMH was upregulated (Ma et al., 2004). To analyze the consequences of severely reduced testosterone on spermatogenesis, the mutant testes were analyzed histologically and expression of spermatogenesis markers assessed. The mutant testes consist
Fig. 1. Spermiogenesis defect in the absence of LH. Cross sections of testes from adult wild-type (WT) and LH knockout (KO) mice showing different stages of spermatogenesis. Note that in contrast to the WT section, no late stage spermatids and sperm are present in the mutant testis section. Thus, absence of LH leads to a spermiogenesis block. Bouin’s reagent-fixed sections were stained with a nuclear dye to visualize different cell morphologies. White arrows indicate late stage spermatids in the WT section whereas they indicate round spermatids in the KO section. Photographs taken at 40× magnification using an Olympus microscope.
T.R. Kumar / Molecular and Cellular Endocrinology 269 (2007) 81–84
of spermatogonia, spermatocytes (meiotic cells) and round spermatids (Ma et al., 2004). Confocal microscopy confirmed that at least eight different stages of tubules were identifiable in the mutant testes. The majority of the tubules showed type B spermatogonia, pachytene spermatocytes and round spermatids (Fig. 1). No late stage or elongated spermatids were observed consistent with an absence of histone H1-like linker protein, a late stage spermatid marker that was suppressed in the mutant testes (Ma et al., 2004). Additional defects in the mutant tubules include aberrant expression of various structural proteins and enhanced apoptosis (Zama et al., in preparation). Therefore, FSH alone is not sufficient to promote full spermatogenesis in the absence of LH and/or testosterone. Thus, the loss of LH leads to both somatic and spermatogenic cell defects, similarly seen in other models with a Sertoli cell-selective androgen receptor deletion (SCARKO) (Holdcraft and Braun, 2004a,b). LH mutants provide useful models for further studying somatic–germ cell interactions in the testis.
83
6. Future directions Our data clearly indicate that loss of LH action has more severe consequences in gonadal growth and differentiation and that FSH alone cannot compensate this loss. LH knockout mice closely phenocopy LuRKO mice and demonstrate LH signaling is not required for prenatal development. One advantage of our LH ligand knockout model is that we can pharmacologically rescue these null mice at defined time points and thus we will be able to define LH and steroid responsive genes in gonads. The absence of LH in these mice will also allow us now to test the role of LH in gonadal tumor-prone inhibin ␣ knockout mice (Kumar et al., 1996, 1999). Furthermore, LH null mice will be useful to develop novel double mutants lacking both FSH and LH but with an intact GnRH signaling system. Because GnRH analogs are often used to treat various gonadal cancers (Nagy and Schally, 2005; Weiss et al., 2002), these double LH/FSH mutant mice will enable us to determine the direct effects of GnRH, if any, on gonadal function.
4. Phenotypes of LH null female mice LH knockout females, similar to mutant males were hypogonadal and showed thin uterine horns (Ma et al., 2004). Histological analysis of the ovaries indicated absence of healthy antral, preovulatory follicles and corpora lutea, confirming impaired estrous cycles (Ma et al., 2004). Primary and secondary follicles appeared normal, whereas many antral follicles were abnormal containing degenerating oocytes. Despite these defects in granulosa cells and oocytes, a prominent thecal layer was obvious in follicles at different stages of progression (Ma et al., 2004). These observations suggest that differentiation of thecal layer was not impaired in the absence of LH signaling. However, expression of various thecal cell markers including many steroidogenic enzymes was significantly reduced (Ma et al., 2004). Consequently, serum progesterone and estradiol levels were decreased and the mutant uteri were hypoplastic consisting of a thin endometrial layer (Ma et al., 2004). These ovarian phenotypes in LH knockout mice are distinct from those of FSH knockout mice (Kumar et al., 1997) and provide a genetic evidence for distinct roles of FSH and LH during folliculogenesis. 5. Rescue of LH null mice Pharmacological rescue of both male and female LH knockout mice was achieved by short-term treatment of hCG. Testes and ovarian markers are upregulated following hormone replacement of the mutants with hCG indicating that responsiveness to LH is not irreversibly lost in target cells in the absence of LH (Ma et al., 2004). More recently, we have performed microarray analyses and compared large-scale gene expression profiles in the testes of LH null male mice treated with hCG for 48 h and testosterone for 7 days (Svojanovsky, S., Kumar, T.R., unpublished). Thus, LH knockout mice serve as a useful model to study LH- and steroid-responsive genes and for identifying to what extent steroids mediate the effects of LH in testis development and function.
Acknowledgments I thank Dr. Irving Boime for encouragement and support. Studies reported in this Review are supported in part by The Moran Foundation, Baylor College of Medicine, Houston, TX and The Hall Family Foundation, Kansas City, MO. I also thank the contributions of my colleagues, Xiaoping Ma, Yanlan Dong and Aparna Zama during various phases of this work. References Bousfield, G.R., Perry, W.M., Ward, D.N., 1994. Gonadotropins: chemistry and biosynthesis. In: Knobil, E., Neill, J.D. (Eds.), The Physiology of Reproduction. Raven Press, New York, pp. 1749–1792. Holdcraft, R.W., Braun, R.E., 2004a. Androgen receptor function is required in Sertoli cells for the terminal differentiation of haploid spermatids. Development 131, 459–467. Holdcraft, R.W., Braun, R.E., 2004b. Hormonal regulation of spermatogenesis. Int. J. Androl. 27, 335–342. Huhtaniemi, I., Jiang, M., Nilsson, C., Pettersson, K., 1999. Mutations and polymorphisms in gonadotropin genes. Mol. Cell. Endocrinol. 151, 89–94. Huhtaniemi, I.T., 2002. The role of mutations affecting gonadotrophin secretion and action in disorders of pubertal development. Best Pract. Res. Clin. Endocrinol. Metab. 16, 123–138. Kumar, T.R., Matzuk, M.M., 1995. Cloning of the mouse gonadotropin beta-subunit-encoding genes. II. Structure of the luteinizing hormone betasubunit-encoding genes. Gene 166, 335–336. Kumar, T.R., Matzuk, M.M., 2000. Gene knockout models to study the hypothalamus-pituitary-gonadal axis. In: Shupnik, M.A. (Ed.), Gene Engineering and Molecular Models in Endocrinology. The Humana Press, Totowa, NJ. Kumar, T.R., Palapattu, G., Wang, P., Woodruff, T.K., Boime, I., Byrne, M.C., Matzuk, M.M., 1999. Transgenic models to study gonadotropin function: the role of follicle-stimulating hormone in gonadal growth and tumorigenesis. Mol. Endocrinol. 13, 851–865. Kumar, T.R., Wang, Y., Lu, N., Matzuk, M.M., 1997. Follicle stimulating hormone is required for ovarian follicle maturation but not male fertility. Nat. Genet. 15, 201–204. Kumar, T.R., Wang, Y., Matzuk, M.M., 1996. Gonadotropins are essential modifier factors for gonadal tumor development in inhibin-deficient mice. Endocrinology 137, 4210–4216.
84
T.R. Kumar / Molecular and Cellular Endocrinology 269 (2007) 81–84
Lei, Z.M., Mishra, S., Zou, W., Xu, B., Foltz, M., Li, X., Rao, C.V., 2001. Targeted disruption of luteinizing hormone/human chorionic gonadotropin receptor gene. Mol. Endocrinol. 15, 184–200. Ma, X., Dong, Y., Matzuk, M.M., Kumar, T.R., 2004. Targeted disruption of luteinizing hormone beta-subunit leads to hypogonadism, defects in gonadal steroidogenesis, and infertility. Proc. Natl. Acad. Sci. U.S.A. 101, 17294–17299. Matzuk, M.M., DeMayo, F.J., Hadsell, L.A., Kumar, T.R., 2003. Overexpression of human chorionic gonadotropin causes multiple reproductive defects in transgenic mice. Biol. Reprod. 69, 338–346. Nagy, A., Schally, A.V., 2005. Targeting of cytotoxic luteinizing hormonereleasing hormone analogs to breast, ovarian, endometrial, and prostate cancers. Biol. Reprod. 73, 851–859. Pierce, J.G., Parsons, T.F., 1981. Glycoprotein hormones: structure and function. Annu. Rev. Biochem. 50, 465–495. Themmen, A.P., Martens, J.W., Brunner, H.G., 1998. Activating and inactivating mutations in LH receptors. Mol. Cell. Endocrinol. 145, 137–142.
Themmen, A.P.N., Huhtaniemi, I.T., 2000. Mutations of gonadotropins and gonadotropin receptors: elucidating the physiology and pathophysiology of pituitary-gonadal function. Endocr. Rev. 21, 551–583. Valdes-Socin, H., Salvi, R., Daly, A.F., Gaillard, R.C., Quatresooz, P., Tebeu, P.M., Pralong, F.P., Beckers, A., 2004. Hypogonadism in a patient with a mutation in the luteinizing hormone beta-subunit gene. N. Engl. J. Med. 351, 2619–2625. Weiss, J., Axelrod, L., Whitcomb, R.W., Harris, P.E., Crowley, W.F., Jameson, J.L., 1992. Hypogonadism caused by a single amino acid substitution in the beta subunit of luteinizing hormone. N. Engl. J. Med. 326, 179– 183. Weiss, J.M., Diedrich, K., Ludwig, M., 2002. Gonadotropin-releasing hormone antagonists: pharmacology and clinical use in women. Treat. Endocrinol. 1, 281–291. Zhang, F.P., Poutanen, M., Wilbertz, J., Huhtaniemi, I., 2001. Normal prenatal but arrested postnatal sexual development of luteinizing hormone receptor knockout (LuRKO) mice. Mol. Endocrinol. 15, 172–183.