Genetic variation in physiological sensitivity to estrogen in mice.

Genetic variation in susceptibility to endocrine disruption by estrogenic agents was examined in juvenile male mice. Mice were implanted with increasing doses of estradiol (E2) at 3 weeks of age and reproductive responses were determined 3 weeks later. Greater than 16-fold differences in susceptibility to the disruption of reproductive development by E2 were detected between strains of mice. CD-1 was much more resistant to the inhibition of testes weight, vesicular gland weight and spermatogenesis by increasing doses of E2. Spermatid maturation was eliminated by low doses of E2 in unselected strains such as C17/Jls and C57BL/6J. In contrast, widely used, large litter size selected CD-1 mice showed little or no inhibition in spermatogenesis even in response to 16-fold higher doses of E2. Testicular sulfotransferase activity (EST) per gram body weight was 3.5-fold higher in untreated CD-1 than in B6 strain males. This suggests that genetic differences in testicular EST activity may play a critical role in the detoxification of estrogens. These and other findings emphasize the need to identify and study genetic variation in sensitivity to estrogen in laboratory animal models used to assess the risk of xenobiotic estrogen exposure.

Reassessment of models used to test xenobiotics for oestrogenic potency is overdue.

Product safety bioassays need to include data from animals with susceptible genotypes or the potential for environmental compounds to disrupt reproductive development in hormonally sensitive populations may be greatly underestimated. The continued use of resistant animal models is likely to result in allowable releases of toxic levels of oestrogenic agents that could differentially disrupt reproductive development and function of sensitive genotypes, leading to reproductive failure and loss or extinction of susceptible individuals, populations and species. Rather than ignoring the role of genetic differences in susceptibility to oestrogenic agent-induced carcinogenicity and endocrine disruption, government agencies should support efforts to identify the genetic mechanisms involved in these responses, and to screen for and develop strains of mice and rats which are sensitive to the induction of genotoxicity/carcinogenicity as well as the inhibition of reproductive development and function by oestrogenic agents. Such sensitive strains would be even more optimal for testing chemicals for endocrine disruptor activity.

Physiological stresses increase mouse short interspersed element (SINE) RNA expression in vivo.

The possible functionality of short interspersed elements (SINEs) is investigated by assaying the effects of physiological stress on their RNA polymerase-III-directed transcriptional expression in vivo. B2 RNA is expressed at moderately high levels in all mouse tissues investigated, namely liver, spleen, kidney and testis. B1 RNA is expressed in testis but is nearly undetectable in the other tissues. Following hyperthermic shock, the amounts of B1 and B2 SINE RNAs transiently increase in all tissues by as much as 40-fold in certain cases. The kinetics of these increases resemble those of heat shock protein mRNAs. An acute dose of ethanol also transiently increases the abundance of B1 and B2 RNA in liver, showing that other physiological stresses increase SINE RNA expression. The constitutive expression of B2 RNA in all tissues and tissue-specific differences in expression of B1 RNA imply that these transcripts serve a normal physiological function(s). Moreover, increased SINE RNA expression is a vital response to stress and by the criterion of their inducibility, mammalian SINEs behave like regulated cell stress genes.

Genetic control of hormone-induced ovulation rate in mice.

The nature of genetic differences in ovarian responsiveness to gonadotropins was examined in mouse strains and subspecies. Hormone-induced ovulation rate (HIOR) differed 5-fold between Mus musculus strains A/J (10.3 +/- 1.6 eggs in cumulus) and C57BL/6J (B6) (47.3 +/- 2.5 eggs in cumulus), and 6-fold among Mus spretus lines and crosses. Subspecies differed up to 10-fold in HIOR (Mus spretus/Ros: 4.8 +/- 1.0 eggs in cumulus versus B6). An additional experiment examined the genetics of HIOR in crosses. The number of eggs ovulated in response to equine chorionic gonadotropin (CG)/human CG averaged 8.4 +/- 0.9 in A/J, 40.7 +/- 1.7 in B6, 33.9 +/- 1.6 in B6AF1, and 20.2 +/- 0.3 in (B6xA)xA backcrosses. The 5-fold genetic differences in hormone-induced ovulation rate between Mus musculus strains A/J and B6 segregated in backcrosses as though they were controlled by the action of approximately 3 loci with major effects. This study demonstrates genetic variation in HIOR both within and between mouse subspecies, and provides confirmation that genetic differences are a major source of variation in the regulation of ovarian responsiveness to gonadotropins.

Mapping genes that control hormone-induced ovulation rate in mice.

The present study mapped quantitative trait loci (QTL) that control 6-fold genetic differences in hormone-induced ovulation rate (HIOR) between C57BL/6J (B6) (HIOR = 54) and A/J strain mice (HIOR = 9). (The gene name is Ovulation Rate Induced [ORI] QTL and the gene symbol is Oriq.) QTL linkage analysis was conducted on 167 (B6xA)xA backcross mice at 165 loci. Suggestive B6 ORI QTL that control the number of eggs in cumulus mapped, as follows, near: Cyp19 and D9Mit4 on chromosome (Chr) 9 (Oriq1); D2Mit433 on Chr2 (Oriq2); D6Mit316 on Chr6 (Oriq3); DXMit22 on ChrX (Oriq4) and were associated with a 2.7, 2.7, 2.6, and 4.2 egg increases in HIOR, respectively. Oriq3 was significant (LOD = 3.45) based on composite interval mapping. QTL linkage analysis of the number of eggs matured by endogenous gonadotropins and ovulated by eCG mapped a significant Oriq5 to Chr 10 and suggestive Oriq to Chr 6, 7, and X. These data provide the first molecular genetic markers for reproductive QTL that control major differences in ovarian responsiveness to gonadotropins. These and closely linked syntenic molecular markers will enable a more accurate prediction of ovarian responsiveness to gonadotropins and provide selection criteria for improving reproductive performance in diverse mammalian species.

Genetic variation in susceptibility to endocrine disruption by estrogen in mice.

Large (more than 16-fold) differences in susceptibility to disruption of juvenile male reproductive development by 17beta-estradiol (E2) were detected between strains of mice. Effects of strain, E2 dose, and the interaction of strain and E2 dose on testes weight and spermatogenesis were all highly significant (P < 0.0001). Spermatid maturation was eliminated by low doses of E2 in strains such as C57BL/6J and C17/Jls. In contrast, mice of the widely used CD-1 line, which has been selected for large litter size, showed little or no inhibition of spermatid maturation even in response to 16 times as much E2. Product safety bioassays conducted with animals selected for fecundity may greatly underestimate disruption of male reproductive development by estradiol and environmental estrogenic compounds.

Regulation of mouse liver microsomal esterases by clofibrate and sexual hormones.

Carboxylesterase activity was measured using six different substrates in microsomal preparations from female and ovariectomized female mice in order to evaluate the effects of female sex hormones on esterase expression. With three of the substrates (alpha-naphthyl acetate and esters 2 and 3), esterase activity was the same in both groups; however, with the others (rho-nitrophenyl acetate and esters 1 and 4), there was a small increase in activity in ovariectomized females, compared with intact females. Castration of males followed by treatment with testosterone caused only transient increases in activity for four of the substrates (alpha-naphthyl acetate and esters 1, 2, and 3) and no change in activity for the other two (rho-nitrophenyl acetate and ester 4). Treatment of male and female mice with the peroxisome proliferator clofibrate, with or without testosterone, resulted in increased hydrolysis of alpha-naphthyl acetate and rho-nitrophenyl acetate, but little change for the other substrates. Clofibrate also induced alpha-naphthyl acetate and rho-nitrophenyl acetate hydrolysis in castrated males, but clofibrate and testosterone administrated together resulted in significant increases of activity with all substrates, which were greater than the additive effects of the two compounds administered separately. These results indicate that clofibrate causes significant alterations in the regulation of esterase activity, whereas sex hormones only cause small changes. However, it would seem that testosterone can synergize the effect of clofibrate in castrated males, resulting in higher levels of activity than with clofibrate alone. Finally, an overall increase in esterase activity might be due to a large increase in the activity of a few esterases or to a small increase in many esterases. Enzyme staining of native polyacrylamide gels reveals that the latter is true, with the majority of esterases present in mouse liver microsomes being induced to a small degree by clofibrate.

Differential regulation of soluble epoxide hydrolase by clofibrate and sexual hormones in the liver and kidneys of mice.

Soluble epoxide hydrolase (sEH) activity was measured in the liver and kidneys of male, female, and castrated male mice in order to evaluate sex- and tissue-specific differences in enzyme expression. sEH activity was found to be higher in liver than in kidneys. Activity increased with age in the liver of females, males and castrated males, but only in males did activity in the kidneys increase. There was greater activity in both the liver and kidneys of adult males than females. This sexual dimorphism was more pronounced in the kidneys (283% higher) than in the liver (55% higher). Castration of males led to a decrease in activity in both organs, but it had a greater effect on renal activity (67% decrease) than on hepatic activity (27% decrease). Treatment of castrated mice with testosterone led to an increase in sEH activity of 400% in kidneys and 49% in liver compared with surgical controls. These results suggest differential regulation of sEH by testosterone in kidneys and liver. Ovariectomized female mice had renal and hepatic activities approximately 30% greater than control females. Feeding mice with the hypolipidemic drug clofibrate produced stronger induction of sEH in liver than in kidneys. Testosterone treatment, however, caused greater induction in kidneys than in liver of females and castrated males and had no effect in either kidneys or liver in males. When given together, the effects of these two compounds appeared to be additive in both liver and kidneys. Results from western blot showed that the increase in sEH enzyme activity in kidneys is correlated with an increase in sEH protein. These results suggest that clofibrate and testosterone independently regulate sEH activity in vivo, and that kidneys and liver respond differently to clofibrate and testosterone.

Genetic variation in plasma androgens and ovarian aromatase activity during mouse pregnancy.

Genetic variation in fetal survival, maternal plasma androgen levels, and ovarian aromatase activity was examined mid (Day 9) and late gestation (Day 16) in strains of mice that differ in reproductive performance (A/J, C57BL/6J, C8/JIs, C17/JIs, and S15/JIs). At both gestational stages, females selected for large litter size (S15/JIs) carried more fetuses than any of the other strains examined. Particularly at midpregnancy, S15/JIs females also maintained higher plasma levels of androstenedione and testosterone relative to both control strains, C8/JIs and C17/JIs. Consistent with previously reported changes in peripheral estrogen levels during mouse pregnancy, aromatase activity was higher on Day 16 than on Day 9. This study demonstrates genetic variation in fetal survival that is correlated with increased maternal androgen levels. A stage-specific gestational increase in aromatase activity occurs in several strains of mice and is associated with elevated plasma estrogen during the second half of pregnancy.

Chromosomal mapping and expression levels of a mouse soluble epoxide hydrolase gene.

The chromosomal location of a murine soluble epoxide hydrolase gene was determined using in situ mapping, restriction fragment length polymorphism (RFLP) and simple sequence length polymorphism (SSLP) analysis. In situ hybridization to mouse metaphase chromosomes using a soluble epoxide hydrolase cDNA probe showed that soluble epoxide hydrolase maps at band D of chromosome 14. An RFLP found between Mus castaneus (CAST) and Mus musculus (MEV) was used to map the soluble epoxide hydrolase gene in CAST x MEV intersubspecific testcross progeny to 14 cM from the Np-1 locus on mouse chromosome 14. SSLP markers were then used to confirm the location of soluble epoxide hydrolase at 14.0 +/- 3.7 cM distal to Np-1 and 19.2 +/- 4.3 cM proximal to D14Mit7. This region of mouse chromosome 14 is homologous with human chromosomes 8, 13 and 14. Enzyme assays and immunoblotting results suggest significant quantitative differences in expression of soluble epoxide hydrolase among three mouse strains. Northern blotting analysis showed that soluble epoxide hydrolase mRNA levels were correlated with the relative level of soluble epoxide hydrolase enzyme activity and soluble epoxide hydrolase protein in all three mouse strains.

The effect of H-2 region and genetic background on hormone-induced ovulation rate, puberty, and follicular number in mice.

We have examined the effects of major histocompatibility (H-2) haplotypes and genetic background (all loci other than the H-2 region) on hormone-induced ovulation rate in congenic strains of mice. In comparison with the H-2a haplotype, the H-2b haplotype increased hormone-induced ovulation rate 92% on the A/J (A) genetic background. However, H-2 haplotype did not affect hormone-induced ovulation rate on the C57BL/10J (C57) genetic background. The H-2b-linked gene(s) increased hormone-induced ovulation rate on the A/J genetic background largely by (1) enhancing the maturation of follicles in response to pregnant mare's serum gonadotrophin (PMSG) and (2) altering the stages of follicular development which can be induced to ovulate in response to human chorionic gonadotrophin (hCG). The observed effects of H-2 on hormone-induced ovulation rate were not explained by differences in the timing of puberty, the number of follicles present in untreated females, or the incidence of follicular atresia. The effect of genetic background on hormone-induced ovulation rate was much greater than was the effect of the H-2 region. We found that hormone-induced ovulation rate was five- to six-fold higher on the C57 genetic background than on the A genetic background. The C57 genetic background increased hormone-induced ovulation rate by (1) enhancing the induction of follicular maturation in response to gonadotropins and (2) by reducing the incidence of follicular atresia.

Major genes control hormone-induced ovulation rate in mice.

The present study examined the magnitude of genetic variation, mode of inheritance and number of loci controlling major genetic differences in hormone-induced ovulation rate in mice. Mice were injected with 5 i.u. PMSG at 28 days of age and 5 i.u. hCG at 30 days, and hormone-induced ovulation rate was determined from counts of oviducal eggs in cumulus the next morning. Six-fold genetic differences in induced ovulation rate were detected amongst strains, ranging from a low mean (+/- s.e.) value of 8.8 (+/- 0.9) in A/J up to 53.5 (+/- 2.2) in C57BL/6J. The number of ova differed significantly amongst strains and amongst F1 crosses (P less than 0.0001): 70% of the variation in hormone-induced ovulation rate was amongst strains. Of 9 F1 crosses examined, 4 showed positive heterosis, 3 showed no heterosis and 2 showed negative heterosis for hormone-induced ovulation rate. Analysis of parental, F1 and F2 generations revealed that the induced ovulation rate of A/J and C57BL/6J mice differed due to the action of about 3 or 4 loci, and A.SW/SnJ and SJL/J mice differed due to the action of about 2 to 3 loci. Analysis of recombinant inbred strains formed from A/J and C57BL/6J confirmed that these strains differed due to the action of a small number of loci. This study demonstrates the existence of a small number of major genes controlling hormone-induced ovulation rate in young mice.

Characterization of genetic differences in hormone-induced ovulation rate in mice.

Major genetic differences in hormone-induced ovulation rate were not explained by strain differences in age at puberty, by maturation and ovulation of follicles by endogenous gonadotrophins, or by differential responses to gonadotrophins at different ages. The major genetic differences in hormone-induced ovulation rate were explained by strain differences in ovarian responsiveness to exogenous gonadotrophins.

Development of a sensitive enzyme-linked immunosorbent assay for cattle, sheep, rat, and mouse luteinizing hormone.

This manuscript reports the development of a rapid, sensitive, and specific-enzyme linked immunosorbent assay (ELISA) suitable for measuring luteinizing hormone (LH) in cattle, sheep, rats, and mice. The LH ELISA used #15 anti-LH serum-coated 96-well plates and peroxidase-labeled bovine luteinizing hormone (bLH). Bovine LH labeled with peroxidase by the periodate method was stored at -15 degrees C for over 20 mo without appreciable loss of activity. With bLH-B5 used as the standard diluted in assay buffer, the LH ELISA had a sensitivity of 79.6 +/- 31 pg/ml, and a 50% displacement point of 359 +/- 69 pg/ml. With rat LH-RP-2 (rLH) used as the standard diluted in assay buffer, the LH ELISA had a sensitivity of 102 pg/ml and a 50% displacement point of 531 pg/ml. The LH ELISA was highly specific for LH from several mammalian species. This LH ELISA was seven times more sensitive for bLH than a radioimmunoassay (RIA) with comparable sample volumes. The LH ELISA was validated for measurement of LH in buffer, tissue culture media, and sera. Depending on the sensitivity desired, the LH ELISA can be conducted in 3 to 48 h, produces no hazardous waste, and can easily be automated. Use of this LH ELISA offers improvements in speed, convenience, economy, sensitivity, and safety over comparable RIA procedures. The LH ELISA was also conducted with other monoclonal and polyclonal antibodies to LH. The LH ELISA can be performed with other LH antisera, provided the antisera has a high affinity and specificity for LH and can be uniformly coated on 96-well plates.

The effects of curing and cooking on the detection of species origin of meat products by competitive and indirect ELISA techniques.

The purposes of this study were: (1) to develop and compare competitive and indirect enzyme linked immunosorbent assay (ELISA) methods for detecting species specific albumins in meat samples and (2) to examine the effect of curing and cooking on the identification of species origin of meats. Commercially obtained rabbit anti-pig serum albumin (anti-PSA) and rabbit anti-sheep serum albumin (anti-SSA) were affinity purified, and used to develop competitive and indirect ELISA procedures for PSA and SSA. The competitive ELISA procedures showed the lowest cross-reactivity with related serum albumins. Both ELISA procedures were capable of detecting as little as 5% pork or sheep in beef. Curing resulted in little or no inhibition in the ability of ELISA procedures to detect pork or sheep in beef. Cooking completely eliminated the ability of the competitive PSA ELISA to detect pork in beef, and of both SSA ELISA procedures to detect sheep in beef. Cooking also greatly reduced, but did not eliminate, the ability of the indirect PSA ELISA to detect pork in beef. Curing and cooking essentially eliminated the ability of the PSA ELISA procedure to detect pork in beef. Curing and cooking resulted in a 70 to 74% decrease in the signal of sheep meat in SSA ELISA procedures. These results demonstrated that competitive and indirect ELISA procedures are capable of determining the species origin of raw and cured meat. Heating raw or cured meats greatly reduced, but did not always eliminate, the ability of ELISA procedures to detect species origin of meats.

Changes in the kinetics of follicular growth in response to selection for large litter size in mice.

The present study examined a randomly selected control line (C) and a large litter size-selected line of mice (S1) to determine changes in the kinetics of follicular growth that have occurred in response to selection for large litter size. For each follicle type (FT), the number of healthy and atretic follicles, length of components of granulosa cell cycle, follicular growth rate, and follicular flux were determined microscopically from serially sectioned ovaries of Lines C and S1 mice. Selection for litter size significantly increased the number of small and medium, and some large follicle-size classes. While selection for litter size did not change the overall incidence of atresia at proestrus, it did decrease the incidence of atresia in the large Type 7 follicles by 19%. Selection for litter size also increased ovarian weight at proestrus. Selection for litter size increased the rate of growth through FT 3a, 5a, and 5b, and reduced the time required for follicles to grow from primordial to Graafian follicles from 39.1 days in Line C to 33.4 days in Line S1. Selection for large litter size also increased the flux of follicles through follicle Types 3a to 5b by 72%, and through follicle Types 6 and 7 by 21%. Genetic variation was found in many aspects of the kinetics of follicular growth.

Genetic variation in spontaneous ovulation rate and LH receptor induction in mice.

Selection for litter size (Line S1) and for post-weaning body weight gain (Line G) increased spontaneous ovulation rate in mature females by 69 and 73%, respectively, over that of randomly bred control mice (Line C). Inbreeding from S1 mice with selection for litter size produced highly inbred lines with elevated ovulation rates. Inbreeding from Line C mice produced a 21% divergence among lines, but did not depress the mean ovulation rate. Crosses of these lines revealed little heterosis in ovulation rate. LH receptors were induced by treating females from 22 days of age with diethylstilboestrol for 4 days and FSH for 2 days. The in-vitro binding of 125I-labelled hCG per microgram DNA decreased 56% in response to selection for litter size and increased 57% in response to selection for body weight gain, indicating high susceptibility of this trait to genetic change. Inbreeding from Line C mice produced a 135% divergence amongst lines, but did not depress the mean LH receptor induction. Body weight had significant effects on ovulation rate and LH receptor induction. These results show that selection for litter size and for rapid post-weaning body weight gain increases ovulation rate, but we suggest that different mechanisms are involved in these responses.