Overexpression of Mouse Follistatin Causes Reproductive Defects in Transgenic Mice

Qiuxia Guo, T. Rajendra Kumar, Teresa Woodruff, Louise A. Hadsell, Francesco J. DeMayo and Martin M. Matzuk

Departments of Pathology (Q.G., T.R.K., M.M.M.), Cell Biology (L.A.H., F.J.D., M.M.M.), and Molecular and Human Genetics (M.M.M.) Baylor College of Medicine Houston, Texas 77030
Departments of Medicine (T.W.) and Neurobiology and Physiology and The Center for Reproductive Sciences Northwestern University Chicago, Illinois 60611


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Follistatin is an activin-binding protein that can act as an activin antagonist in vitro. Follistatin also binds heparin sulfate proteoglycans and may function as a reservoir for activins in vivo. In the mouse, follistatin mRNA is first detected in the deciduum on embryonic day 5.5 and later in the developing hindbrain, somites, vibrissae, teeth, epidermis, and muscle. We have previously shown that follistatin-deficient mice have numerous embryonic defects including shiny, taut skin, growth retardation, and cleft palate leading to death within hours of birth. To further define the roles of follistatin during mammalian reproduction and development, we created gain-of-function mutant mice in which mouse follistatin is overexpressed. The mouse metallothionein (MT)-I promoter was placed upstream of the six-exon mouse follistatin (FS) gene. To distinguish wild-type and transgenic follistatin mRNA, the 3'-untranslated region of the mouse follistatin gene was replaced with the SV40 untranslated and polyA sequences. Three male and two female founder transgenic mice were produced, were fertile, and transmitted the transgene to offspring. Northern blot analysis demonstrated that the transgene mRNA was expressed at varying levels in the livers of offspring from four of five of the transgenic lines and was expressed in the testes in all five lines. In MT-FS line 4, which had the highest expression of the transgene mRNA in the liver, the transgene transcripts were also present in multiple other tissues. Phenotypically, the MT-FS transgenic lines had defects in the testis, ovary, and hair. Mice from MT-FS lines 7 and 10 had slightly decreased testis size, whereas mice from lines 4, 5, and 9 had much smaller testes and shiny, somewhat irregular, fur. Histological analysis of the adult testes from line 5 and 9 males showed variable degrees of Leydig cell hyperplasia, an arrest of spermatogenesis, and seminiferous tubular degeneration leading to infertility. Female transgenic mice from lines 4 and 9 had thin uteri and small ovaries due to a block in folliculogenesis at various stages. Many of the line 9 female mice eventually became infertile, and all of the line 4 female mice were infertile. Suppressed serum FSH levels were seen in only the line 4 transgenic male and female mice, the line with widespread expression of the transgene. Serum FSH levels were not significantly different in gonadectomized wild-type and line 5 transgenic male mice despite high levels of the follistatin transgene mRNA in the liver of these transgenic mice. These results suggest that follistatin exerts its effects at the levels of the gonads and pituitary as a local regulator of activin and possibly other transforming growth factor-ß family members.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Activins and inhibins are structurally related proteins belonging to the transforming growth factor-ß (TGF-ß) superfamily. The activins are ß:ß dimers (ßA:ßA, ßB:ßB, ßA:ßB) that share common ß-subunits with the heterodimeric {alpha}:ß inhibins. Activins and inhibins were initially recognized as gonadal peptides that stimulate or inhibit FSH production by the pituitary in adult mammals (1, 2, 3). More recently, two related mammalian activin subunit genes have been cloned: activin ßC and ßE (4, 5, 6). It is unclear whether dimers of activin ßC or ßE can regulate FSH synthesis or whether these subunits can dimerize with an inhibin {alpha} subunit. Similar to inhibin, follistatin, a glycosylated monomeric protein, suppresses pituitary FSH synthesis and secretion (7, 8, 9, 10). In 1990, Sugino and colleagues (7) demonstrated that follistatin binds to the ß-subunits of activin in a 1:1 molar ratio to form an inactive complex (8). In humans, rats, and pigs, the mature follistatin protein has two forms, follistatin-288 and follistatin-315, which are generated by alternative splicing of the follistatin mRNA (9).

Activins and follistatin are expressed in multiple tissues during mammalian development and are involved in diverse physiological and developmental processes (1, 2, 9, 11, 12, 13, 14). During rat development, activins can stimulate proliferation of Sertoli cells in the testis and regulate folliculogenesis in the ovary (3, 15, 16, 17). Studies in Xenopus laevis and Oryzias latipes have shown that activins are potent mesoderm-inducing factors (18, 19). Overexpression of follistatin in Xenopus laevis blocked activin’s effects on mesoderm formation and induced neural tissue directly (20). However, studies using knockout mice in our laboratory failed to demonstrate a role of activins, follistatin, or activin type II receptor in mesoderm formation or neural development. Mice deficient in activins can survive to birth but demonstrate craniofacial defects (21). Mice lacking follistatin have multiple defects, including musculoskeletal and dermatopathological abnormalities, and die within hours of birth (22). These defects are more widespread than those seen in activin- or activin receptor type II-deficient mice (21, 23).

In the liver, activin A has been reported to inhibit the initiation of DNA synthesis in rat hepatocytes in vitro and in vivo (24). Use of recombinant activin A in vitro or in vivo induces cell death via apoptosis in rat and murine hepatocytes (25, 26). Studies in our laboratory using mice deficient in both inhibin and activin receptor II (ActRII) have demonstrated that activins, secreted from gonadal tumors, signal through ActRII in hepatocytes to directly cause the hepatocellular necrosis seen in {alpha}-inhibin-deficient mice (27, 28). Intraportal or intravenous administration of follistatin can block the inhibitory effects of activins on hepatocytes, thereby accelerating liver regeneration in partially hepatectomized rats (29, 30).

The generation of mice lacking either inhibin or activin receptor type II has also confirmed that inhibins and activins are important regulators of pituitary FSH levels in vivo (23, 31). Mice lacking inhibin have elevated FSH levels (31) whereas mice lacking activin receptor type II (23) have suppressed FSH levels. Females lacking activin receptor type II are infertile due to a block at the antral follicle stage, whereas males lacking this activin receptor have small gonads and delayed fertility (23). However, since follistatin-deficient mice die at birth, similar analyses of the essential roles of follistatin in the regulation of FSH synthesis and secretion and its roles in adult reproductive physiology are impossible.

To further understand the function of follistatin in mammalian development and reproduction, we created transgenic mice overexpressing the wild-type mouse follistatin gene product using the mouse metallothionein (MT)-I promoter. In this report, we show that the follistatin transgene was expressed in multiple tissues leading to major defects in the reproductive tract and minor defects in hair formation. These studies demonstrate that follistatin plays an important role in reproductive physiology in vivo.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Generation of Mice Overexpressing Follistatin and Expression Analysis of the MT-FS Transgene mRNA
Transgenic mice overexpressing the wild-type mouse follistatin gene product using the mouse metallothionein-I promoter (32, 33) (Fig. 1Go) were created in this study. Five founder transgenic mice (three male and two female) were produced and were fertile. Southern blot analysis of genomic DNA from offspring from these founder mice demonstrated that all five lines transmitted the transgene. However, breeding of the line 4 founder male with wild-type mice demonstrated that the ratio between transgenic and wild-type mice was much lower than 50% (10 transgene positive offspring of 80 total). This suggested that the testes (i.e. germ cells) of this line 4 founder mouse were likely mosaic. As expected, the integrated transgene copy number was different between the various lines: line 9 had the highest copy number and line 4 had the lowest (Fig. 2Go and data not shown).



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Figure 1. The MT-FS Transgene Construct

A 1.8-kb mouse metallothionein-I (mMT-I) promoter fragment (32, 33) was fused to a 5.1-kb genomic fragment (2, 22) containing the six coding exons of the mouse follistatin gene. A 973-bp fragment containing the simian virus 40 (SV40) 3'-untranslated region and polyadenylation signal were placed downstream of the six coding exons in lieu of the 3'-UTR of the endogenous follistatin gene.

 


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Figure 2. Southern Blot Analysis of Transgenic Genomic DNA

Transgenic mice were identified by Southern blot analysis using the 973-bp SV40 3'-UTR/poly A fragment as a probe. Offspring from three of the five transgenic lines are shown from the same hybridization reaction. The order of lines with respect to number of transgene copies are approximated as follows: line 9 >= line 5 > line 10 > line 7 >= line 4.

 
Northern blot analysis was performed to analyze the expression of the MT-FS transgene in both reproductive and nonreproductive tissues. As shown in Fig. 3AGo, the transgene mRNA was expressed at varying levels in the livers of offspring from four of five of the transgenic lines and was expressed in the testes of all five of the lines. Since liver expression of the MT-FS transgene was highest in lines 4 and 5, multitissue Northern blot analysis was also performed on these two lines to determine whether the transgene was constitutively expressed in any tissues other than the liver and testes. The MT-FS transgene was also expressed in the skin and brain in both lines (Fig. 3BGo and data not shown). In line 4, there was additional expression in the heart, lung, spleen, stomach, kidney, and small intestine. Since overexpression of follistatin in Xenopus laevis causes defects in mesoderm formation and neurulation, we also checked whether the follistatin transgene was expressed during embryogenesis. By Northern blot analysis of total RNA, the MT-FS transgene was expressed in the livers of all line 5 mice examined at embryonic day 18.5 (E18.5) (data not shown). RT-PCR analysis of total RNA from E6.5 and E7.5 line 5 embryos also demonstrated the presence of the MT-FS transgene transcript (data not shown).



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Figure 3. Follistatin Transgene Expression in Adult Tissues

Total RNA (15 µg) extracted from various tissues of adult transgenic mice was subjected to Northern blot hybridization using the 973-bp SV40 3'- UTR/poly A fragment as a probe. Total RNAs from a wild-type mouse were used as negative controls. Equivalent loading of RNA was confirmed by hybridization with a ribosomal 18S RNA cDNA probe. A, Follistatin transgene expression in livers and testes from various transgenic lines. B, Follistatin transgene expression in multiple tissues from a line 4 transgenic mouse.

 
In other mammalian species, endogenous follistatin mRNA is alternatively spliced at the 3'-end, giving rise to two distinct protein forms (9, 13). To determine which mRNA and protein forms were expressed in wild-type and MT-FS transgenic mice, we performed PCR analysis of RNA derived from either wild-type ovary (the most abundant source of follistatin) or transgenic testis (line 7). Electrophoretic and Southern blot analysis of these PCR products demonstrated that both wild-type and transgene follistatin mRNAs exist as only one species, encoding the long 343-amino acid precursor form of the mouse protein (11) (data not shown).

Morphological and Histological Analyses
All of the MT-FS transgenic offspring from the five independent lines were viable and developed to adults. In contrast to overexpression of follistatin in Xenopus laevis, in which mesoderm and neurulation defects are observed, overexpression of follistatin during early mouse development did not result in any deleterious effects. Activins have been shown to have an inhibitory effect on GH production in vitro (1, 34), and therefore, the MT-FS transgenic mice and littermates were weighed weekly. Similar to activin ßB (35) or activin receptor type II knockout mice (23), adult female and male MT-FS transgenic mice did not demonstrate any statistically significant changes in body weight compared with controls (data not shown). Since the MT-FS transgene is highly expressed in liver, and since follistatin and activins are known to effect liver apoptosis and regeneration (24, 25, 26, 27, 28, 29, 30), we examined the transgenic livers morphologically and histologically. The liver weights of these transgenic male and female mice are not statistically different compared with the liver weights of wild-type littermate controls (data not shown). Histological analysis of livers from these transgenic lines also did not show any abnormalities (data not shown).

Newborn mice lacking follistatin (22) or activin ßA (23) have whisker defects. Prepubertal and adult MT-FS transgenic mice also had gross abnormalities in the hair. Mice from lines 4, 5, and 9 demonstrate shiny, somewhat irregular, fur (Fig. 4AGo) and could be distinguished grossly from their littermates as early as 3 weeks of age. Histological analysis of the skin, however, revealed no obvious cause for this phenotype (data not shown).



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Figure 4. Morphological, Histological, and Biochemical Analysis of Wild-Type and MT-FS Transgenic Male Mice

A, Gross analysis of the fur of a 4.5-month-old MT-FS line 4 transgenic mouse (bottom) and its littermate control (top). B, Gross analysis of the testes of a 6-week-old wild-type mouse (left) and a littermate MT-FS mouse (right). C, Testis weights of 6-week-old mice. Values shown are mean ± SEM. The number of mice used is shown in parentheses as follows: wild-type (15), line 7 (14), line 10 (11), line 5 (11), line 9 (13), and line 4 (3). D–G, Histology of testes from a wild-type adult mouse with normal Leydig (arrows) cells (high power, D), a 6.5-month-old line 9 transgenic mouse at low (E) and high (F) magnification, and a 6-month-old line 5 transgenic mouse (low power, G). Leydig cell hyperplasia (asterisks) and tubular degeneration (arrows or "t") are shown. H, Serum FSH levels of control and MT-FS adult male mice, the majority of which are at 42 days. Values shown are mean ± SEM. The number of male mice shown in parentheses used in the analyses are as follows: wild-type (5), line 7 (5), line 10 (4), line 5 (7), line 9 (5), and line 4 (2).

 
Since follistatin is known to function in the reproductive axis, we tested the fertility of these transgenic mice by intercrossing them to wild-type controls for up to 1 yr. Offspring from transgenic lines 7 and 10 showed normal fertility. However, transgenic male mice from lines 4, 5, and 9 [i.e., lines with highest expression of the transgene mRNA in the liver and testes (Fig. 3AGo)] and transgenic female mice from lines 4 and 9 demonstrated reproductive defects. All eight line 4 transgenic female mice failed to have offspring, demonstrating that line 4 female mice are infertile. Five of nine line 4 male mice were infertile whereas the other four of the line 4 male mice had two or three litters before they became infertile at approximately 4 months of age. Male and female mice from line 9 sired two or three litters before becoming infertile at approximately 4.5–5 months of age. Additionally, three of eight adult female line 4 mice died of unknown causes.

To determine the causes of the infertility in these follistatin transgenic mice, morphological and histological analyses were performed. Morphological studies showed that the follistatin transgenic male mice had smaller testes than wild-type controls. Testes from lines 7 and 10 were slightly decreased in size compared with control mice, whereas testes from line 4 were dramatically smaller compared with controls (Fig. 4BGo). Testes weights from all five transgenic lines of mice and littermate control mice at 6 weeks of age are shown in Fig. 4CGo. Testes weights inversely correlated with the follistatin transgene mRNA expression in the testes; in lines 4, 9, and 5, which had the highest expression of the MT-FS transgene mRNA in the testis (Fig. 3AGo), the testes were smaller compared with lines 7 and 10, which had the lowest mRNA expression. Histological analysis demonstrated Leydig cell hyperplasia and partial or total tubular degeneration in adult testes from lines 5 and 9 after 4 months of age. In contrast to the testes of wild-type mice (Fig. 4DGo), spermatogenesis was absent in these tubules, and only a few Sertoli cells remained (Fig. 4Go, E–G). Surprisingly, sections of testes from line 4 show fairly normal stages of spermatogenesis and normal levels of spermatozoa (data not shown) despite being infertile. As might be expected and consistent with their fertility, testes from lines 7 and 10 always show normal stages of spermatogenesis despite having slightly decreased testis size. To determine the testis cell type where the transgene was expressed, in situ hybridization was performed using the SV40 3'-untranslated region (UTR) and poly A probe. The MT-FS transgene mRNA signal appeared to be present at high levels in both spermatogonia and Sertoli cells. The signal was not detected in Leydig cells, elongating spermatids, round spermatids, and late pachytene and diplotene spermatocytes. Consistent with these findings, endogenous expression of MT-I mRNA is also expressed in Sertoli cells and spermatogonia in adult mouse testis (36).

Similar to the males, the transgenic female mice also demonstrated defects in the reproductive axis. Female mice from line 4 were infertile, and many of the female mice from line 9 eventually became infertile at 4 to 5 months of age. Figure 5AGo shows the morphological appearance of the ovaries and uteri of a line 4 female mouse (right) and its wild-type littermate (left) analyzed at 4.5 months of age. Compared with the wild-type controls, ovaries from the line 4 female were small, and the uterus was thin, similar to the GnRH-deficient hypogonadal (hpg) mouse (37, 38). Histological analysis of these transgenic mice demonstrated small ovaries and a block in folliculogenesis. Ovaries from a severely affected 4.5-month-old line 4 transgenic female demonstrated an early block in folliculogenesis before antral follicle formation (Fig. 5Go, C and D). In the least affected line 4 and 9 transgenic females, folliculogenesis progressed to the early secondary (antral) follicle stage (Fig. 5EGo); however, there were no tertiary follicles or corpora lutea present in these ovaries as compared with ovaries from wild-type mice (Fig. 5BGo). In addition, follicular atresia was present (Fig. 5EGo), similar to ovaries from FSH-deficient female mice (39). Ovaries from another line 4 female mouse demonstrated seminiferous tubule-like structures in the ovary (Fig. 5Go, F and G), similar to the ovaries from inhibin-deficient mice that have gonadal sex cord-stromal tumors (Refs. 2, 31, and 40; see Discussion). Most of the line 5 female mice had normal ovaries and were fertile even at later timepoints in adult development. However, ovaries from some of the line 5 females demonstrated abnormalities in folliculogenesis with one mouse having a block in folliculogenesis at the early primary follicle stage. In situ hybridization analysis of ovaries from line 4 female mice showed that the transgene mRNA was expressed weakly in stromal (interstitial) cells (data not shown).



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Figure 5. Morphology and Histology of the Reproductive Tract and Ovaries of Wild-Type and MT-FS Transgenic Female Mice

A, Gross analysis of the reproductive tract (uterus and ovaries) of a 4.5-month-old wild-type (left) and MT-FS transgenic line 4 mouse (right). B–G, Histological analysis of adult ovaries. B, Wild-type, mouse ovary (low power). Black arrow, Corpus luteum; white arrow, antral follicle. C and D, An ovary from the 5-month-old transgenic line 4 mouse shown grossly in panel A at intermediate (C) and high (D) power magnification, respectively. Note the small ovary and the arrest of folliculogenesis at the primary follicle stage. Arrows, Primary follicles. E, An ovary from a 9-month-old transgenic line 9 mouse. An atretic follicle is shown at the left (white arrow). F and G, An ovary from a 5.5-month-old female line 4 mouse at intermediate (F) and high (G) power magnification, respectively. Note the seminiferous tubule-like structures (S) with cells with nuclei (arrows) that resemble Sertoli cells.

 
Serum and Pituitary Analysis
Activins stimulate pituitary FSH synthesis and secretion in vitro (1, 2), and activin receptor type II-deficient mice have suppressed FSH levels (23). Follistatin has been shown to block activin action at the level of the pituitary in vitro (9). Since many of the ovary and testis defects in the follistatin transgenic mice were similar to FSH-deficient (39) and activin receptor type II-deficient (23) mice, we investigated whether any of the reproductive defects in our follistatin transgenic mice could be due to the suppression of FSH by follistatin. In the adult male mice, the FSH levels, as measured by RIA, were significantly suppressed only in the line 4 transgenic males compared with control mice (Fig. 4HGo). Decreased FSH levels were also observed in the adult transgenic line 4 females (24.3 ± 1.9 ng/ml.; n = 5), comparable to the suppressed FSH levels seen in activin receptor type II knockout mice (35.2 ± 5.2 ng/ml; Ref.23). Consistent with the morphological (i.e. thin uterus and small ovaries) and histological (i.e. block in folliculogenesis) findings and suppressed serum FSH, the line 4 female mice had serum estradiol levels below the sensitivity of the assay (<5 pg/ml). We also measured the serum FSH levels in gonadectomized adult wild-type and line 5 transgenic males. The results showed no significant difference in FSH levels between the gonadectomized MT-FS transgenic male mice (105 ± 13 ng/ml; n = 4) and nontransgenic controls (107 ± 8 ng/ml; n = 3). Neither "free" follistatin nor activin A were detected in the serum of the follistatin transgenic mice (41, 42). These data suggest that the major effects of overexpression of follistatin were at the level of the gonads (see Discussion).

The MT-FS transgenic male mice that displayed reproductive defects were also analyzed to determine whether there were any secondary abnormalities in steroid production. Since androgen biosynthesis in males occurs mainly in the interstitial Leydig cells, the serum testosterone level of transgenic male mice that display Leydig cell hyperplasia in the testis was determined by RIA. The results showed no significant difference in serum testosterone levels in these transgenic mice (1.7 ± 1.1 ng/ml; n = 7) compared with adult wild-type mice (1.9 ± 1.7 ng/ml; n = 5) consistent with grossly normal secondary sex organs (i.e. seminal vesicles, prostate). This suggests that the androgen biosynthesis pathway in these hyperplastic Leydig cells might be functionally suppressed or abnormal.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Follistatin is an activin binding protein that has been shown to antagonize the function of activin in a number of assays (7, 9) and may also modulate activin action in vivo at the cell surface (22, 43). In the present study, a "gain of function" mouse model was generated to further understand the function of follistatin in mammalian physiology. Despite the expression of the MT-FS transgene during early embryogenesis, and in contrast to mice lacking follistatin (22), mice overexpressing the follistatin transgene were viable and developed to adulthood. The most obvious finding in three of the follistatin transgenic lines with the highest transgene expression was shiny, irregular fur (Fig. 4AGo). This might have been expected based on our previous data that follistatin-deficient mice have shiny, taut skin, whisker defects, and aberrant keratin 6 expression in the interfollicular epidermis (22), and that activin ßA-deficient mice lack whiskers (21). Consistent with an important role of activins and follistatin in the skin, we have recently shown that radiolabeled activin A binding in the skin of follistatin-deficient embryos at E18.5 was absent compared with wild-type littermates (44). Taken together, these findings further suggest that follistatin may bind activins and act as an important reservoir for activins in the epidermis.

In adult rats, activin ßA and follistatin mRNA are expressed in Sertoli cells surrounding leptotene spermatocytes at stages IX–XI, and activin is a potent stimulator of spermatogonial and Sertoli cell proliferation (3, 17). Since the role of follistatin in adult reproduction could not be addressed by our conventional knockout of follistatin because the mutant mice died soon after birth (22), the present gain-of-function studies allowed us to ascertain the function of follistatin in mammalian reproduction. Overexpression of follistatin resulted in decreased testis size, seminiferous tubular degeneration, and an arrest of spermatogenesis in MT-FS lines 5 and 9. Although a similar decrease in testis size was seen in FSH-deficient (39) and ActRII-deficient mice (23), which have absent and decreased FSH levels, respectively, these knockout male mice were still fertile. Interestingly, the serum FSH levels of MT-FS transgenic mice were only suppressed in line 4 male mice that had the broadest expression of the transgene. This suggests that the decrease in testes size in the MT-FS transgenic mice is caused by follistatin acting locally to block activin, consistent with a hypothetical testis-specific activin knockout. However, it is unclear why these follistatin overexpressor mice do not mimic the phenotype of ActRII-deficient mice (23). One possibility is that activins continue to signal via ActRIIB in the absence of ActRII. Alternatively, follistatin may regulate the function of other TGF-ß family members. For example, follistatin-deficient mice (22) have skeletal defects reminiscent of BMP5-deficient mice, and high concentrations of follistatin can block the effects of BMP7 and BMP4 in vitro (45, 46). Adult bone morphogenetic protein 8b (BMP8b) knockout male mice demonstrate increased apoptosis of spermatocytes leading to germ cell depletion, tubule degeneration, and infertility (47). Thus, overexpression of follistatin may also block BMP8b to cause the infertility, and under physiological conditions, follistatin may normally play a role in maintenance of spermatogenesis by modulating the function of BMP8b in the mouse. In addition, Leydig cell hyperplasia was present in the testis of line 5 and 9 male mice. In Müllerian inhibiting substance (MIS, a member of TGF-ß family) knockout male mice or MIS/inhibin double-mutant male mice, Leydig cell hyperplasia and Leydig cell neoplasia, respectively, were observed (48, 49). Some aspects of the Leydig cell hyperplasia in our follistatin transgenic mice may be due to antagonism of MIS (i.e. follistatin overexpression may act functionally like an MIS null mutation).

Activin stimulates granulosa cell proliferation and follicular development in vitro (3, 16). Varying levels of infertility were observed in line 4 and 9 female mice that demonstrated thin uteri, small ovaries, and a block in folliculogenesis. Similar to the ovarian findings in mice lacking FSH [i.e. only primary follicles were observed (39)], folliculogenesis in many of the MT-FS transgenic female mice from these two lines is halted between the primary and secondary follicle stages, follicular atresia was present, and in most ovaries, no corpora lutea were seen. Interestingly, one line 4 female mouse exhibited Sertoli tubule-like structures (Fig. 5Go, F and G), similar to ovarian tumors from inhibin-deficient mice and inhibin-deficient ovaries transferred to the bursa of wild-type control mice (2, 31, 40). Since FSH levels were only suppressed in line 4 female mice, a local role of follistatin in the ovary may also be responsible for the ovarian defects by blocking activins and/or other TGF-ß family members. Mice deficient in the oocyte-specific protein growth differentiation factor 9 (GDF-9), a member of the TGF-ß family, demonstrate a block in folliculogenesis at the one-layer primary follicle stage (50). High levels of follistatin in our MT-FS transgenic mice may bind to GDF-9 and block its effects, thereby resulting in folliculogenesis defects similar to the GDF-9-deficient mice. Thus, if follistatin interacts with other members of the TGF-ß superfamily, this would suggest further promiscuity in the TGF-ß superfamily signaling pathways and give us important insight into human infertility and potential treatments.

We have shown that activin signaling through activin receptor type II directly causes the cancer cachexia-like syndrome in inhibin-deficient mice (27, 28). Other studies have demonstrated that activin inhibits hepatocyte proliferation and that follistatin accelerates liver regeneration (24, 25, 26, 29, 30, 51). Since the MT-FS transgene is highly expressed in the liver of lines 4 and 5, we will breed these mice to our inhibin-deficient mice to determine whether overexpression of follistatin can block this activin-mediated cancer cachexia-like syndrome. These future studies will be important to further understand the roles of follistatin in vivo.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Construction of the MT-FS Transgene
A 1.8-kb mouse metallothionein-I promoter fragment (32, 33) was placed upstream of the six coding exons of the mouse follistatin gene (5.1 kb, Refs. 2 and 20; Fig. 1Go). To distinguish the follistatin transgene mRNA from the endogenous follistatin mRNA, a 973-bp BamHI/ClaI fragment, which contains the simian virus 40 (SV40) untranslated (UTR) and polyA sequences, was placed in the 3'-untranslated region of the transgene. The plasmid containing the MT-FS transgene was digested with EcoRI and BamHI to release the transgene fragment, which was subsequently isolated using GENECLEAN (BIO 101, La Jolla, CA).

Generation of Transgenic Mice and Morphological and Histological Analysis
The MT-FS transgene DNA (a 7.9-kb linearized fragment) was microinjected into the pronucleus of fertilized eggs generated from C57BL/6/C3H x ICR hybrids (52). Microinjected eggs were transferred to the oviducts of foster mothers (ICR female mice, 6–7 weeks old). Twenty four mice were obtained from six foster mothers. Genomic (tail) DNA (~5 µg) from the founder mice and offspring was digested with XbaI, electrophoresed on 0.7% agarose gels, and subjected to Southern blot analysis using the SV40 3'- UTR and polyA probe as described above. All transgenic founder and F1 mice were intercrossed with wild-type or transgenic littermates beginning at 6 weeks of age. Fourteen, eight, and six breeding pairs of transgenic male or female mice from lines 4, 9, and 5, respectively, were set up for up to 1 yr to test the fertility of these mice. The number of offspring born and frequency of births were recorded. Transgenic mice and littermate controls were weighed weekly on the same day until 26 weeks of age. Ten pairs of testes from all five transgenic lines of mice and 11 pairs of testes from control mice were weighed at 6 weeks of age, and serum from these mice were also collected at the same time. Any mice that demonstrated infertility for >=3 months were analyzed morphologically and histologically. Testes were fixed in Bouin’s solution overnight and then transferred to 70% ethanol in saturated lithium carbonate, and rinsed daily for approximately 1 week. Other tissues were dissected and immediately fixed by immersion overnight in 10% neutral buffered formalin. The tissues were embedded in paraffin, and 5-µm sections were cut. Sections from ovaries and testes were stained with hematoxylin and periodic acid Schiff stain reaction. Sections from other tissues were stained with hematoxylin and eosin.

Serum and Pituitary Analysis
Serum and pituitary levels of LH and FSH in wild-type and MT-FS transgenic mice were analyzed by RIA using kits obtained from the National Hormone and Pituitary Distribution Program, National Institutes of Diabetes, and Digestive and Kidney Diseases as described previously by our group (23, 31, 35, 39, 50). Serum levels of testosterone and estradiol from control and transgenic mice were determined using Diagnostics Systems Laboratories, Inc. (Webster, TX) RIA assay reagents as previously described (2). Serum activin levels were determined by enzyme-linked immunosorbent assay (ELISA) (Serotec, Ltd., Raleigh, NC) as described (42). Serum collected from inhibin-deficient mice was used to validate the mouse activin assay. Native mouse activin A was diluted linearly and in parallel to the recombinant human activin A standard curve. All samples were run within one assay. The intraassay coefficient of variance for positively detected samples was 5.2%. All samples from PMSG-induced mice, control wild-type mice, and MT-FS transgenic mice were below the detection limit of the assay (0.078 ng/ml). Free follistatin was measured in a two-site ELISA assay format as described (41). The assay detects recombinant human follistatin 288; however, activin A interferes with the detection of follistatin in this assay. Follistatin was not immunodetected using this assay format in the serum of follistatin transgenic mice. The assay is sensitive and specific for human follistatin but may not detect mouse follistatin. This possibility cannot be tested without a mouse follistatin standard.

RNA Analysis
Total RNA was extracted from fresh tissues from both transgenic and wild-type mice using RNA STAT-60 reagent (Leedo Medical Laboratories, Houston, TX) according to the manufacturer’s protocol. For Northern blot analysis, 15 µg of total RNA were loaded on a 7.6% formaldehyde-1.2% agarose gel, and the RNA was transferred to Hybond-N (Amersham, Arlington Heights, IL) nylon membrane as described (50). The membrane was hybridized with the same SV40 UTR/poly A probe used for Southern blot analysis as described (50). For the quantitative control, the membrane was stripped and rehybridized with a mouse 18S ribosomal RNA cDNA probe. To determine whether the MT-FS transgene was expressed at early embryonic stages, total RNA from E6.5 or E7.5 embryos from timed matings of carrier transgenics was processed using the RNA STAT-60 reagent, and 10% of the total RNA was used for RT-PCR analysis. RT-PCR was performed using the Titan One Tube RT-PCR system (Boehringer Mannheim, Indianapolis, IN) according to the manufacturer’s protocol.

In Situ Hybridization Analysis
In situ hybridization was performed as described by Albrecht et al. (53). In brief, freshly dissected mouse testes and ovaries were fixed in Bouin’s and freshly prepared 4% paraformaldehyde-PBS, respectively. Testes samples were immersed for 3 h in Bouin’s and then transferred to 70% ethanol. After fixation, the tissues were paraffin embedded and sectioned at 5 µm. The SV40 3'-UTR and polyA riboprobe, labeled with [{alpha}-35S]UTP, was used to detect the follistatin transgene expression. Hybridization was carried out at 50–55 C with 5 x 106 cpm/slide riboprobe for 16 h in 50% deionized formamide, 0.3 M NaCl, 20 mM Tris HCl (pH 8.0), 5 mM EDTA, 10 mM NaPO4 (pH 8.0), 10% Dextran sulfate, 1x Denhardt’s reagent, and 0.5 µg/ml yeast RNA. A high-stringency wash was carried out in 0.1x sodium chloride-sodium citrate at 65 C. Dehydrated sections were exposed to X-OMAT film for 1–3 days. After film exposure, slides were dipped in NTB-2 Kodak emulsion and exposed for 1–10 days at 4 C. After the slides were developed and fixed, they were stained with hematoxylin or hematoxylin/eosin and mounted with permount for photography.

Sperm Parameters
Epididymides were dissected from 6- to 7-week-old male mice, and the sperm were allowed to disperse into M-2 medium upon incubation at 37 C for 15–30 min. A 1:10 dilution was used for hemocytometric counts and for motility testing, while a 1:20 dilution was used for viability testing using Eosin-Y (52, 54).


    ACKNOWLEDGMENTS
 
We thank Ms. Grace Hamilton and Mr. Bliss Walker for histological technical assistance, Ms. Julia Elvin for critical review of the manuscript, and Ms. Shirley Baker for help in manuscript preparation. We thank Dr. Debra Wolgemuth for interpretative analysis of in situ hybridization results on the transgenic testes. We thank Dr. Richard Palmiter for the gift of the metallothionein I promoter and Dr. Patrick Sluss for the follistatin antibodies.

These studies were supported by NIH Grants HD-32067 and CA-60651 (to M.M.M) and Reproductive Center Grant HD-07495.


    FOOTNOTES
 
Address requests for reprints to: Martin M. Matzuk M.D., Ph.D., Departments of Pathology, Cell Biology, and Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas 77030.

Received for publication May 30, 1997. Revision received October 2, 1997. Accepted for publication October 20, 1997.


    REFERENCES
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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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