Intraovarian Activins Are Required for Female Fertility
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0888-8809/07/$15.00/0 Molecular Endocrinology 21(10):2458–2471 Printed in U.S.A. Copyright © 2007 by The Endocrine Society doi: 10.1210/me.2007-0146 Intraovarian Activins Are Required for Female Fertility Stephanie A. Pangas,* Carolina J. Jorgez,* Mai Tran, Julio Agno, Xiaohui Li, Chester W. Brown, T. Rajendra Kumar, and Martin M. Matzuk Departments of Pathology (S.A.P., M.T., J.A., X.L., M.M.M.), Molecular and Human Genetics (C.W.B.), Molecular and Cellular Biology (M.M.M.), and Program in Developmental Biology (C.J.J., M.M.M.), Baylor College of Medicine, Houston, Texas 77030; and Department of Molecular and Integrative Physiology (T.R.K.), The University of Kansas Medical Center, Kansas 66160 Downloaded from https://academic.oup.com/mend/article/21/10/2458/2738443 by guest on 30 September 2021 Activins have diverse roles in multiple physiologi- are subfertile, B/A double mutant females are cal processes including reproduction. Mutations infertile. Strikingly, the activin A and B/A-defi- and loss of heterozygosity at the human activin cient ovaries contain increased numbers of func- receptor ACVR1B and ACVR2 loci are observed in tional corpora lutea but do not develop ovarian pituitary, pancreatic, and colorectal cancers. Func- tumors. Microarray analysis of isolated granulosa tional studies support intraovarian roles for ac- cells identifies significant changes in expression tivins, although clarifying the in vivo roles has re- for a number of genes with known reproductive mained elusive due to the perinatal death of activin roles, including Kitl, Taf4b, and Ghr, as well as loss A knockout mice. To study the roles of activins in of expression of the proto-oncogene, Myc. Thus, in ovarian growth, differentiation, and cancer, a tis- contrast to the known tumor suppressor role of sue-specific knockout system was designed to ab- activins in some tissues, our data indicate that activin late ovarian production of activins. Mice lacking A and B function redundantly in a growth stimu- ovarian activin A were intercrossed to Inhbb ho- latory pathway in the mammalian ovary (Molecular mozygous null mice to produce double activin Endocrinology 21: 2458–2471, 2007) knockouts. Whereas ovarian A knockout females CTIVINS, HOMODIMERS OR heterodimers of Aor growth of both human breast cancer cells (7) and prostate AB-subunits, were identified for their roles in posi- cancer cells (8). Mutations in ACVR1B have been observed tively regulating pituitary FSH synthesis and secretion in human pituitary tumors (9) and pancreatic cancers (10). (1–3). The -subunits also dimerize with the inhibin Likewise, ACVR2 mutations and loss of heterozygosity ␣-subunit to produce inhibins [inhibin A (␣:A) and in- have been seen in pancreatic (11), colorectal (12), and pros- hibin B (␣:B)] that negatively regulate FSH. The A- and tate cancers (13). These studies suggest that the activin B-subunits, encoded by the Inhba and Inhbb genes, signaling pathway functions as a tumor suppressor path- respectively, are produced in multiple tissues during em- way in some tissues to block cell growth and stimulate bryonic and postnatal development and have diverse differentiation. physiological effects. Mice lacking activin A (homozy- Inhba and Inhbb transcripts are prominently expressed gous Inhba null) die at birth secondary to craniofacial by granulosa cells of large preantral and antral follicles (14). defects, whereas homozygous Inhbb null mice are viable In vitro studies have identified multiple intraovarian roles for but exhibit eyelid closure and nursing defects (4–6). activins in granulosa cells including proliferation, potentia- Several studies indicate that activin signaling compo- tion of FSH action, and modulation of steroidogenesis (re- nents, including the receptors ACVR1B (also known as viewed in Refs. 15 and 16). To study the in vivo roles of activins, we produced an ovarian Inhba conditional knock- ALK4) and ACVR2 (also known as ACTR2B), are critical for out (A cKO). We also generated the A cKO in the Inhbb growth inhibition. For example, activins inhibit in vitro null background mice to produce mice lacking all ovarian First Published Online July 3, 2007 activins (double mutant mice; herein designated as B/A * S.A.P. and C.J.J. contributed equally to this work. dKO). In contrast to extragonadal roles of these proteins as Abbreviations: BMP, Bone morphogenetic protein; cKO, tumor suppressors, activins play redundant roles to block conditional knockout; CL(s), corpus luteum/corpora lutea; Ctgf, connective tissue growth factor; CV, coefficient of vari- terminal differentiation of granulosa cells, leading to dos- ation; dKO, double mutant mice; Gapd, glyceraldehyde age-dependent fertility defects. 3-phosphate dehydrogenase; Ghr, GH receptor; hCG, human chorionic gonadotropin; 20␣-HSD, 20␣-hydroxysteroid de- hydrogenase; HSD, honestly significant difference; Lhcgr,LH receptor; Mmp2, matrix metalloproteinase 2; PMSG, preg- RESULTS nant mare serum gonadotropin; PRL, prolactin; qPCR, quan- titative PCR. Generation of Ovarian Activin A Knockout Mice Molecular Endocrinology is published monthly by The Endocrine Society (http://www.endo-society.org), the tm3Zuk foremost professional society serving the endocrine A floxed Inhba allele was generated (Inhba ; community. herein called Aflox) for subsequent tissue-specific de- 2458 Pangas et al.•In Vivo Roles of Activins in the Ovary Mol Endocrinol, October 2007, 21(10):2458–2471 2459 letion of the Inhba gene in vivo (Fig. 1A). Exon 2 was BϪ/Ϫ; Aflox/Ϫ, and BϪ/Ϫ; Aflox/Ϫ;Amhr2cre/ϩ fe- floxed because it encodes the entire mature domain of males. Neither Aϩ/Ϫ nor Bϩ/Ϫ heterozygous mice the protein and a conditional exon 2 deletion would had fertility defects (5, 17). Double mutant females mimic the original null allele (Inhbatm1Zuk) (17). Aflox/ϩ with only one remaining A-subunit allele (BϪ/Ϫ; mice were intercrossed to produce Aflox/flox homozy- Aϩ/Ϫ or BϪ/Ϫ;Aflox/Ϫ) had severe fertility defects gous mice, which were viable and fertile and obtained (Table 1), indicating that activin dosage plays an im- at the expected Mendelian frequency. To verify the portant role in ovarian function. Of the 16 mice that presence of loxP sites and their ability to recombine in were studied over 6 months, three of five BϪ/Ϫ; vivo, we crossed the Aflox mice to EIIa-cre transgenic Aflox/Ϫ, and three of 11 BϪ/Ϫ;Aϩ/Ϫ female mice mice, which express cre recombinase in multiple tis- were infertile. In the mutant lines containing a single  sues, including germ cells (18). Southern blot analysis -subunit allele, the average number of litters per Downloaded from https://academic.oup.com/mend/article/21/10/2458/2738443 by guest on 30 September 2021 using a 5Ј probe demonstrated the presence of the month was significantly reduced (89–91%), and the various Inhba alleles, including the recombined de- number of pups per litter was 87–90% lower than the leted (A⌬) (data not shown). Because Aflox/Ϫ;EIIa-cre controls (Table 1). Finally, female mice lacking all in- newborn mice demonstrate the neonatal lethality seen traovarian activin A and B alleles (BϪ/Ϫ;Aflox/Ϫ; in the Inhba homozygous null mice (data not shown), Amhr2cre/ϩ) were infertile (Table 1). Thus, although recombination likely produced a null allele. activin A appears to be functionally dominant [be- Anti-Mu¨ llerian hormone receptor-cre (Amhr2cre/ϩ) cause activin A deficiency results in subfertility vs. knock-in mice were used to generate recombination of normal fertility for activin B deficiency (5)], dosage of Aflox in ovarian granulosa cells (19–21). Aϩ/Ϫ; the activin A and B alleles is important for normal Amhr2cre/ϩ mice were mated to Aflox/flox mice and all fertility. four genotypes were recovered at the expected Men- To assess whether ovulation defects were present in delian frequency. Recombination efficiency of the the activin-deficient females, 3-wk-old females were Aflox locus was determined by Southern blot analysis superovulated, mated and oocytes collected from ovi- of granulosa cells derived from cre-negative Aflox/Ϫ ducts. Four groups of mice were analyzed: wild-type, (control) mice and Aflox/Ϫ;Amhr2cre/ϩ (experimental) Aflox/Ϫ, Aflox/Ϫ;Amhr2cre/ϩ, BϪ/Ϫ;Aflox/Ϫ, and mice (Fig. 1B). Recombination of the floxed allele in BϪ/Ϫ;Aflox/;Amhr2cre/ϩ. There were no significant granulosa cells from female littermates varied from differences in the number of oocytes released after 60% to 99% (Fig. 1B). Variation in recombination and ovulation, except in mice deficient for all -subunits expression differences are likely the result of mo- (Table 2). Two of four female mice deficient for all sacism in recombination, as has been seen in other activin alleles produced no oocytes. conditional knockout mice using Amhr2-cre mice (21, 22). Loss of the A transcript was confirmed by North- Enhanced Corpus Luteum (CL) Formation in ern blot analysis (Fig. 1C), and no compensatory up- Activin-Deficient Ovaries regulation of the activin B-subunit was found in Aflox/Ϫ;Amhr2cre/ϩ ovaries (Fig. 1D). Because the To determine the causes of subfertility/infertility in the -subunit is also required for formation of inhibin, we activin-deficient mice, ovaries were analyzed at 3 and measured serum inhibin levels in the Aflox/Ϫ; 8 months of age. At 3 months of age, there were no Amhr2cre/ϩmice. The average serum inhibin A level in gross differences in appearance or size of the ovaries the A-deficient mice is significantly reduced from the between Aflox/Ϫ;Amhr2cre/ϩ and control mice control values, and six of nine females had undetect- (Aflox/Ϫ) (data not shown). The only visible histological able levels. Loss of dimeric inhibin A was not due to difference at 3 months of age was the presence of reductions in inhibin ␣-subunit mRNA expression be- ovarian follicles containing more than one oocyte cause ovaries and granulosa cells of mutant mice [50% occurrence in the experimental mice vs.