Pattern of Messenger Ribonucleic Acid Expression of Tissue Inhibitors of Metalloproteinases (TIMPs) during Testicular Maturation in Male Mice Lacking a Functional TIMP-1 Gene¹

^c Department of Obstetrics and Gynecology, University of Kansas, Kansas City, Kansas 66160 ^d Department of Molecular and Cellular Biology, Roswell Cancer Institute, Buffalo, New York 14263 ^e Department of Obstetrics and Gynecology, University of Kentucky, Lexington, Kentucky 40536–0084

	ABSTRACT

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It has been proposed that proteolytic remodeling of the testicular extracellular matrix (ECM) plays a fundamental role in testicular development, morphogenesis, and spermatogenesis. Tissue inhibitor of metalloproteinase (TIMP)-1 regulates ECM turnover and has been reported to stimulate Leydig cell steroidogenesis. To assess the developmental changes in TIMP mRNA expression and the potential steroidogenic role of TIMP-1 in testicular physiology, an experiment was conducted that used male mice incapable of expressing the TIMP-1 gene product. TIMP-1-deficient and wild-type male mice (n = 6 to 15 per age group per genotype) were killed at 18, 21, 24, 27, 33, 41, and 49 days of age. Body weight, testis weight, serum total testosterone, and TIMP-1, -2, -3, and -4 transcript expression were determined. Northern analysis revealed the detection of TIMP-1 mRNA in wild-type males only. TIMP-1 mRNA levels (per 20 µg total RNA) were highest in 18- to 27-day-old male mice and decreased approximately 13-fold by Day 41. The pattern of TIMP-2 expression was similar between genotypes, with testicular levels of the 1.0-kilobase transcript increasing between Days 18 and 27 of age. The pattern of TIMP-3 transcript expression (per 20 µg total RNA) was similar between genotypes and decreased between Days 18 and 41 of age. When TIMP-3 mRNA levels were expressed on a per testis basis, TIMP-3 was seen to have increased throughout testicular development. TIMP-4 mRNA expression was undetectable by Northern analysis in all mice. No significant difference was detected in body weight or testis weight between genotypes, with the exceptions that 21-day-old TIMP-1 mutants had higher (p < 0.05) testis weights and lower (p < 0.05) serum total testosterone levels than age-matched wild-type males. It is concluded that each TIMP displays its own unique pattern of expression during the prepubertal period, suggesting that the various TIMPs may have specific roles in testicular development. The modest effect of TIMP-1 ablation on testosterone is interpreted to mean that TIMP-1 may function as a coregulator of basal testicular steroidogenesis; but overall, TIMP-1 appears to have little effect on testosterone production in mice lacking the TIMP-1 gene.

	INTRODUCTION

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It has been proposed that proteolytic remodeling of the testicular extracellular matrix (ECM) plays a fundamental role in testicular development, morphogenesis, and spermatogenesis. Interactions between cells of the testis and the ECM or its components regulate Sertoli cell differentiation, testicular cord formation, germ cell development, and the rate and pattern of Sertoli cell migration [1, 2]. Dynamic changes in the ECM involve a multitude of proteolytic enzymes and endogenous inhibitors whose coordinated activity dictates the rate and extent of ECM turnover [3, 4]. Proteinases such as the plasminogen activators [5–7] and matrix metalloproteinases [8–10] are believed to play a key role in these developmental processes. Furthermore, both classes of enzymes are produced by testicular cells in response to hormonal cues and cell-to cell-interactions [5–10], further demonstrating coordinated interaction of proteolytic enzyme expression with cellular and tissue development in the testis.

In addition to the proteinases, endogenous inhibitors of these enzymes may regulate the development and maturation of the male gonads. For example, it is postulated that tissue inhibitors of metalloproteinases (TIMPs), the endogenous inhibitors of matrix metalloproteinases, modulate ECM turnover and testicular development. Of the four currently identified TIMPs (referred to as TIMP-1 through TIMP-4), TIMP-1 and TIMP-2 have both been shown to be produced by testicular cells [10–12], although TIMP-3 and TIMP-4 have not been thoroughly explored in the testis. The members of the TIMP family range in size from 21 to 29 kDa and differ in their mode of action and selectivity for the various members of the metalloproteinase family. For example, in numerous tissues besides the testis, TIMP-1, TIMP-2, and TIMP-4 are secreted and found in the extracellular space whereas TIMP-3 is secreted and bound to the ECM [3, 4]. In addition to their purported role as proteinase inhibitors, TIMP-1 [13] and TIMP-2 [14] exhibit growth factor activity, and more recently, a TIMP-1-like protein has been shown to stimulate steroidogenesis by cultured Leydig cells [15]. These findings indicate that the TIMPs may be important regulators of testicular development and maturation not only at the level of tissue remodeling but also as a paracrine/autocrine regulator of testicular growth, differentiation, and steroidogenesis. To further examine the role of TIMP-1 in the reproductive endocrinology of the male gonads, the experiments reported here were conducted utilizing male mice incapable of expressing the gene product for TIMP-1.

	MATERIALS AND METHODS

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Animals

TIMP-1-deficient animals (referred to as TIMP-1 mutants) were generated by homologous recombination of a neo-containing gene-targeting vector in mouse embryonic stem cells [16]. Transmission of the mutant allele and the genotype of mice were determined by polymerase chain reaction analysis of the neo sequences in genomic tail DNA. TIMP-1 deficiency was confirmed at the transcript level by Northern analysis as previously described [17].

Animals were housed and maintained under the supervision of a licensed veterinarian. All animal procedures for these experiments were approved by the University of Kentucky Institutional Animal Care and Use Committee. Mice were maintained on a 14L:10D cycle. Pups were weaned at 21 days of age, at which time all pups were sexed. Male pups were ear notched for identification purposes, and a 1-cm segment of their tail (not including the keratinized tip) was removed and prepared for isolation of genomic DNA.

Tissue Collection

Homozygous wild-type (+/0) and mutant (-/0) males from the same litter were randomly assigned by litter size into the various age groups and were killed at 18, 21, 24, 27, 33, 41, and 49 days of age (n = 6 to 15 per age group per genotype). Male mice reach sexual maturity at about 45–50 days of age, and thus these ages allow us to examine TIMP-1 impact on testicular steroidogenesis and TIMP mRNA expression from the prepubertal to sexually mature stages of development. Animals were killed by decapitation, blood was collected for quantitation of serum total testosterone, and animal weight was recorded. Testes, heart, liver, lung, and dorsolateral peritoneal wall were then removed, cleaned of connexon, and weighed. All tissues were snap frozen in liquid nitrogen and stored at -75°C until analyzed for TIMP-1, TIMP-2, TIMP-3, and TIMP-4 mRNA content by Northern analysis.

Total RNA Isolation and Northern Blot Analysis

RNA was isolated by the method of Chomczynski and Sacchi [18] using an acid guanidinium thiocyanate-phenol-chloroform extraction procedure as modified by Nothnick and colleagues [19]. Testes were pooled from 2 to 3 animals to yield a total weight of at least 50 mg. Total RNA was isolated from testicular tissue by homogenization in 2 ml of denaturing solution (4 M guanidinium thiocyanate, 25 mM sodium citrate, pH 7.0; 0.5% sarcosyl; and 0.1 M ß-mercaptoethanol) followed by extraction with phenol:chloroform:isoamyl alcohol and reprecipitation in ethanol. The resulting pellet was washed in cold 75% ethanol (v:v) in diethylpyrocarbonate-treated water, vacuum dried, resuspended in 50–100 µl of diethylpyrocarbonate-treated water, and stored at -75°C until analyzed by Northern blotting.

Northern analysis was performed using 20 µg of total RNA per lane. Samples were electrophoresed through a 1% agarose gel containing 2.2 M formaldehyde and transferred to a nylon membrane (Nytran; Schleicher and Schuell, Keene, NH) as recommended by the manufacturer. The respective cDNA probe for mouse TIMP-1, TIMP-2, TIMP-3, and TIMP-4 (a generous gift from Dr. D. Edwards, University of Calgary, AB, Canada) was excised from its plasmid with the appropriate endonucleases and labeled using a random primers kit (Gibco BRL, Gaithersburg, MD). Each probe was labeled to a specific activity of approximately 1 x 10⁹ dpm/µg of DNA using [-³²P]dCTP (NEN-DuPont, Boston, MA). Filters were hybridized overnight according to the recommendations of the manufacturer. After probing for the various TIMPs, blots were hybridized for the transcript of the constitutively expressed 18S ribosomal protein using a rat 18S cDNA probe.

Serum Collection and Testosterone Quantitation

Upon decapitation, blood was collected into 1.5-ml Eppendorf tubes (Fisher Scientific, Pittsburgh, PA). Samples were centrifuged at 1875 x g for 15 min at 4°C; serum was removed and stored at -75°C until analyzed for testosterone concentrations. Serum testosterone concentrations were determined by a solid-phase ¹²⁵I RIA (Diagnostic Products Corp., Los Angeles, CA) previously validated in our laboratory [20]. All samples were analyzed in singlet (or duplicate when serum volumes were sufficient) using 50 µl of serum. The detection limit of the assay was 0.04 ng/ml, and the inter- and intraassay coefficients of variation were 7.8% and 5.2%, respectively.

Statistics

All TIMP Northern data were normalized to the relative expression of the 18S transcript. Data were expressed as the mean fold change (± SEM) per 20 µg of total RNA or per testis from the lowest level of transcript expression among the age groups. To accomplish this, the lowest level of transcript expression for each TIMP within each genotype was set equal to 1.0. The level of transcript expression for the other ages within that genotype was then expressed as the fold change from that value. To assess the changes in the pattern of expression for each TIMP within genotype among the various age groups, data were examined by one-way ANOVA [21] when the data were normally distributed or by the nonparametric Kruskal-Wallis test when data exhibited heteroscedasticity. To compare the pattern of mRNA expression of each TIMP between genotype for the same age group, data were analyzed by planned comparisons using two-sample t-tests for between-genotype analysis [21]. Data were expressed as the fold change (± SEM) in the TIMP mRNA/18S mRNA ratio in the TIMP-1 mutants compared to the appropriate age-matched wild-type males.

	RESULTS

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To assess the pattern of testicular TIMP-1 expression during maturation and to further confirm that knockout males were incapable of expressing the TIMP-1 gene product, Northern analysis of TIMP-1 was performed. Testicular TIMP-1 levels in the wild-type males, normalized per 20 µg of testicular RNA, were unchanged from Day 18 to Day 27 of age, with levels decreasing approximately 3-fold at Day 33 of age and 13-fold at Day 41 of age (Figs. 1 and 2A). When the TIMP-1 expression pattern was analyzed per testis (Fig. 2B), TIMP-1 mRNA levels were elevated at Days 24 and 27 before decreasing on Days 41 and 49. As expected, the TIMP-1 transcript was not detectable in testis of TIMP-1-deficient mice by Northern analysis using up to 20 µg of total RNA (Fig. 1), even with overdevelopment of the film (not shown). To confirm that the TIMP-1 gene disruption was not testis-specific, heart and lung samples (both positive for TIMP-1 mRNA expression) were analyzed from both TIMP-1 mutants and wild-type males of all ages. TIMP-1 transcript was detected in both tissue types from the wild-type males but not the mutants (data not shown), indicating that the disruption of the TIMP-1 gene was not testis-specific.

View larger version (83K): [in this window] [in a new window]   FIG. 1. Northern analysis of testicular TIMP mRNA expression in wild-type and TIMP-1-deficient male (i.e., mutant) mice. Mice were killed at various days of age, and testicular total RNA was isolated and analyzed for TIMP-1, TIMP-2, TIMP-3, and 18S mRNA content using 20 µg of total RNA as described in Materials and Methods. Northern analysis for TIMP-2 exhibited two transcripts of approximately 1.0 and 3.5 kb. The depicted blot is a representation of three separate experiments.

View larger version (20K): [in this window] [in a new window]   FIG. 2. Changes in testicular TIMP-1 mRNA in wild-type male mice during the prepubertal period. TIMP-1 levels were normalized to the 18S transcript expression. Data are expressed as the fold change in TIMP-1 mRNA/18S mRNA expression (mean ± SEM ) per 20 µg of total testicular RNA (A) or per testis (B) at the various days of age. A total of 3 separate experiments (n = 3) were performed. Values with different superscripts are significantly different (p < 0.05).

To characterize testicular expression of TIMP-2, TIMP-3, and TIMP-4 mRNA and to examine potential compensatory effects of deletion of the TIMP-1 gene product on expression of these TIMPs in the mutant mice, Northern analysis was performed. Testicular expression of the 1.0-kilobase (kb) transcript of TIMP-2 per 20 µg of total RNA was lowest in Day 21 wild-type males and increased at Day 27 of age (Figs. 1 and 3A). In the TIMP-1 mutant mice, testicular expression of the 1.0-kb transcript of TIMP-2 was lowest at Day 18, and the levels of mRNA from Days 21 to 49 of age displayed a pattern similar to that of the wild-type males (Figs. 1 and 3A). Comparison between the wild-type and TIMP-1 mutant males within the same age revealed no differences in the expression pattern of the 1.0-kb TIMP-2 transcript between the different genotypes. When the mRNA levels of the 1.0-kb transcript of TIMP-2 were analyzed per testis (Fig. 3B), a similar pattern of TIMP-2 mRNA expression was observed as when the data were analyzed per 20 µg of total RNA (Fig. 3A), with the exception of an increase at Day 49 in the TIMP-1-deficient mice.

View larger version (24K): [in this window] [in a new window]   FIG. 3. Changes in the testicular 1.0-kb TIMP-2 transcript in wild-type and TIMP-1-deficient (mutant) male mice during the prepubertal period. All TIMP-2 levels were normalized to the 18S transcript expression. Data are expressed as the fold change in TIMP-2 mRNA/18S mRNA expression (mean ± SEM ) per 20 µg of total testicular RNA (A) or per testis (B) at the various days of age. A total of 3 separate experiments (n = 3) were performed. Values with different superscripts are significantly different (p < 0.05) within genotypes.

Analysis of the expression of the 3.5-kb transcript of TIMP-2 per 20 µg of testicular RNA revealed no significant differences within the wild-type genotype across age, within the TIMP-1-deficient mice across age, or between the two different genotypes at each age (Figs. 1 and 4A). When the expression of the 3.5-kb transcript of TIMP-2 was analyzed per testis (Fig. 4B), mRNA levels in the wild-type males steadily increased between Days 18 and 33 to reach the highest levels at Day 49. In the TIMP-1 mutant mice, testicular expression of the 3.5-kb transcript of TIMP-2 per testis was similar to that for the wild-type males (Figs. 1 and 4B). Comparison between the wild-type and TIMP-1 mutant males within the same age revealed no differences in the expression pattern of the 3.5-kb TIMP-2 transcript between the different genotypes.

View larger version (21K): [in this window] [in a new window]   FIG. 4. Changes in the testicular 3.5-kb TIMP-2 transcript in wild-type and TIMP-1-deficient (mutant) male mice during the prepubertal period. All TIMP-2 levels were normalized to the 18S transcript expression. Data are expressed as the fold change in TIMP-2 mRNA/18S mRNA expression (mean ± SEM ) per 20 µg of total testicular RNA (A) or per testis (B) at the various days of age. A total of 3 separate experiments (n = 3) were performed. A) No differences were noted within genotypes or between genotypes throughout the prepubertal period. B) Values with different superscripts are significantly different (p < 0.05) within genotypes. No differences were noted between genotypes at the different days of age.

Testicular expression of the ECM-bound TIMP-3 per 20 µg of total RNA was highest in wild-type males 18 days of age, with levels decreasing with advancing age up to 41 days of age (Figs. 1 and 5A). Testicular TIMP-3 transcript expression within the TIMP-1 mutant genotype was similar to the expression pattern in the wild-type males and was not significantly different from that of the age-matched wild-type males at any day examined (Fig. 5A). In contrast to TIMP-3 expression per 20 µg of testicular RNA, TIMP-3 mRNA per testis increased between Days 18 and 33 in wild-type males, with no significant changes thereafter (Fig. 5B). In the TIMP-1 mutant mice, testicular expression of TIMP-3 per testis was similar to that of the wild-type males (Fig. 5B). Comparison between the wild-type and TIMP-1 mutant males within the same age revealed no differences in TIMP-3 mRNA levels between the different genotypes. Testicular TIMP-4 mRNA levels were nondetectable by Northern analysis using up to 20 µg of total RNA in both wild-type and TIMP-1-deficient male mice of all ages studied but was detectable in positive control tissue (peritoneum, data not shown).

View larger version (23K): [in this window] [in a new window]   FIG. 5. Changes in testicular TIMP-3 mRNA in wild-type and TIMP-1-deficient (mutant) male mice. All TIMP-3 levels were normalized to the 18S transcript expression. Data are expressed as the fold change in TIMP-3 mRNA/18S mRNA expression (mean ± SEM ) per 20 µg of total testicular RNA (A) or per testis (B) at the various days of age. A total of 3 separate experiments were performed (n = 3). Values with different superscripts are significantly different (p < 0.05) within genotypes. No differences were noted between genotypes throughout the prepubertal period.

Comparison of body weight between TIMP-1-deficient and wild-type males revealed no significant difference (p 0.05) in this parameter at any of the ages studied (Fig. 6A). In contrast, there was a minor but significant increase in testicular weight in 21-day-old TIMP-1 mutants as compared to their wild-type counterparts (Fig. 6B; p < 0.05). Males of all other ages showed no significant difference in testis weight between genotypes (Fig. 6B). Serum total testosterone was significantly lower (p < 0.05) in the 21-day-old TIMP-1 mutant mice compared to their wild-type littermates (Fig. 6C). No significant difference in serum total testosterone was detected between genotypes at all other ages.

View larger version (18K): [in this window] [in a new window]   FIG. 6. Changes in body weight, testis weight, and serum testosterone in wild-type and TIMP-1-deficient (mutant) male mice during the prepubertal period. Animal weight (A, n = 8–15) did not change between genotypes at the different days of age. Combined testis weight (B, n = 8–15) and serum testosterone (C, n = 4–6) were significantly different between wild-type and mutant male mice at 21 days of age. No differences were observed at any of the other days of age. *p < 0.05 by unpaired t-test between genotypes within age group.

	DISCUSSION

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This is the first study to characterize the pattern of TIMP expression in the testis of prepubertal to sexually mature mice. Furthermore, this study is unique in its examination of the role of TIMP-1 in testicular steroidogenesis using TIMP-1-deficient mice. The current findings demonstrate that each TIMP exhibits its own unique pattern of expression during the period of testicular maturation. This TIMP expression pattern is also influenced by the manner in which the mRNA levels are analyzed (i.e., per microgram of total testicular RNA or per testis). On a per microgram RNA basis, TIMP-1 mRNA levels were highest in 18- to 33-day-old wild-type mice and decreased approximately 13-fold in 41-day-old male mice, suggesting that TIMP-1 may exert its effect during early testicular growth and maturation. In contrast, TIMP-2 levels were higher in wild-type males at 27 and 33 days of age, suggesting that any effect of TIMP-2 on testicular maturation may be more evident at this time. Lastly, TIMP-3 transcript expression declined with increasing age in the wild-type males, suggesting that potential local effects mediated by this protein may diminish throughout testicular development.

Comparison of TIMP mRNA expressed per microgram of testicular RNA versus per testis showed differences in expression especially for the 3.5-kb transcript of TIMP-2 and TIMP-3. For example, when TIMP-3 mRNA was analyzed per microgram of RNA, there was a slow steady decline in TIMP-3 expression throughout testicular development. When these data were analyzed on a per testis basis, TIMP-3 mRNA levels increased. Messenger RNA expression is characteristically normalized to a per microgram RNA per sample basis to eliminate changes due to differences in sample weight. The present findings of an increase in overall testicular TIMP expression with development, however, may reflect an important proteolytic homeostatic mechanism as the testis matures. As with any of the various TIMPs, the possibility exists that TIMP-3 is compartmentalized within the testis to the Sertoli cells or peritubular cells and that measurement on a per microgram total RNA basis is diluted by testicular compartments that do not express TIMP-3. This may be particularly relevant in the present study, as there is a marked increase in the germ cell:Sertoli cell ratio during our sampling period [22] and both TIMP-1 and TIMP-2 have been localized to the Sertoli cells, Leydig cells, and peritubular cells, with the Sertoli cells being the major source of testicular TIMP-2 in the rat [10–12]. If TIMP-3 is localized to the Sertoli cell, its expression could be diluted by an increase in the number of germ cells, and expression per microgram of RNA would reflect a decrease in mRNA levels. Careful consideration of the cellular localization of the TIMPs, the changes in mRNA expression within certain cell types, and the changes in the relative cellular composition of the testis may provide insight into the role of the TIMPs in testicular development.

The present findings both support and contrast with previous developmental observations regarding testicular TIMP-2 expression. Grima and colleagues reported that the levels of the 1.3-kb transcript increased in rat testis collected at 20, 45, and 60 days of age whereas the expression of the 3.7-kb transcript decreased [12]. The present study showed that when TIMP expression patterns were examined more frequently, there was an initial increase in TIMP-2 expression of the 1.0-kb transcript, similar to the findings of Grima and coworkers for the expression of the 1.3-kb transcript [12]. However, the present study did not examine testicular expression at Day 60, a period when the 1.3-kb transcript was shown to be markedly elevated. In contrast to findings from this earlier investigation, expression of the 3.5-kb transcript in the current study showed no significant fluctuations over the 31-day sampling period. This difference in expression of the 3.5-kb transcript may be due in part to the species differences between the mouse and the rat or to differences in sampling frequency between the two studies.

To date there is limited information on testicular expression of TIMP-3 and TIMP-4. TIMP-3 differs in its mode of action from TIMP-1 and TIMP-2 in that, unlike these other TIMPs, which are secreted and found in the extracellular fluid, TIMP-3 is secreted and bound to the ECM [3, 4]. The present findings indicate that expression of this inhibitor tends to decrease with age as mice approach sexual maturity. The significance of TIMP-3 expression in testis remains to be determined. Initial identification of TIMP-4 revealed extremely low to undetectable levels of this inhibitor in human [23] and adult mouse testis [24]. Although the mechanism of TIMP-4 action has not been fully elucidated, it has inhibitory characteristics similar to those of TIMP-2 [25]. In the present study, TIMP-4 mRNA levels were undetectable in prepubertal mice testis by Northern analysis at all ages examined; these findings could be interpreted to mean that this particular TIMP has a limited role in early testicular development.

It has been proposed that testicular metalloproteinases and TIMPs regulate ECM deposition and remodeling during testicular development, morphogenesis, and spermatogenesis, thereby maintaining the integrity of the testis [8–10, 12, 26]. TIMPs have also been reported to have other cellular actions that may impact testicular function. For example, it has been suggested that the TIMPs act as growth factors on the basis of reports that TIMPs promote embryo growth and development [27], have erythroid potentiating action [28, 29], and stimulate cell growth in a variety of cells including endothelial cells, chondrocytes, epithelial cells, MCF7 adenocarcinoma cells, and lymphoma cells [13]. Thus, it is quite possible that the TIMPs may be involved in stimulation of early germ cell growth, cellular proliferation, and neovascularization during testicular development.

The TIMPs may regulate testicular steroidogenesis. A TIMP-1-like protein:cathepsin complex has been reported to stimulate testicular steroidogenesis [15]. In these studies, Boujrad and coworkers observed that Leydig cell pregnenolone production in vitro was stimulated in a dose-dependent manner up to 12-fold by a TIMP-1:procathepsin-L complex. The use of TIMP-1-deficient mice in the current study provides a unique experimental paradigm for examining the role of TIMP-1 in regulation of testicular steroidogenesis. The current findings, however, raise questions about the role of TIMP-1 in steroidogenesis. For example, TIMP-1 mRNA levels are highest in the wild-type males at 18–27 days of age. During this period in the TIMP-1 mutant mice, serum testosterone levels were significantly lower only in the 21-day-old males. Comparison of testosterone levels in males 33 days of age or older indicates that the pattern of steroid production is similar despite the fact that the TIMP-1 mutant mice do not express the TIMP-1 gene production. The possibility exists that TIMP-1 may play a role as a coregulator of early, basal testicular steroidogenesis in vivo along with other factors (in males 27 days of age or less). As the males begin to approach sexual maturity, testosterone production may become less reliant on TIMP-1 and may become more dependent on gonadotropins. Alternatively, one of the other TIMPs may be able to similarly stimulate steroidogenesis and may act in place of TIMP-1. Although no compensatory changes in expression of the other TIMPs was noted in the mutant mice, expression of one of the other TIMPs may be sufficient to substitute for TIMP-1. We were somewhat surprised to find that TIMP-1 ablation exhibited such a modest inhibition of steroidogenic activity. In view of this, we postulate that TIMP-1 has little effect on testicular testosterone production in vivo in mice lacking the TIMP-1 gene. We conclude from these studies that since the TIMP-1 mutant mice were fertile, either TIMP-1 is not prerequisite for overall germ cell development in the testis or its action can be substituted for by another mediator such as one of the other TIMPs. The changes in expression of the TIMPs during the prepubertal period are associated with specific actions in the testis that remain to be determined.

	ACKNOWLEDGMENTS

 
The authors would like to gratefully acknowledge the assistance of Ms. Carole Bryant in the performance of the RIAs and Ms. Sarah Wheeler in the preparation of the figures.

	FOOTNOTES

 
¹ This work was supported by NIH EY11279 (P.D.S.) and NIH HD23195 (T.E.C.).

² Correspondence: Thomas E. Curry, Jr., Department of Obstetrics and Gynecology, University of Kentucky Medical Center, Lexington, KY 40536–0084. FAX: (606) 323–1931; tecurry{at}pop.uky.edu

Accepted: March 25, 1998.

Received: October 14, 1997.

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