Mapping Major Gene QTL Controlling Induced Ovulation Rate in Mice
Jimmy L. Spearow, Karl Mogel, Wooje Lee, Marylynn Barkley
Section of Neurobiology, Physiology & Behavior, University of California at Davis, CA 95616
The genes controlling the common variation in ovarian function and fertility have yet to be identified. Previous studies have revealed major differences in ovarian function, especially ovarian response to gonadotropins between individual females of many outbred species, including women. While some young women show a very low follicular maturation, steroidogenic and ovulatory response to gonadotropins, other women show a elevated response to gonadotropins, ovulating as many as 70 eggs. While failure to respond is not compatible with induced fertility, hyperstimulation is associated with the potentially lethal ovarian hyperstimulation syndrome (Lee et al., 1998), as well as, with premature deliveries and the associated child mortality and disabilities. Since strains of mice and breeds of sheep differ dramatically in ovarian response to gonadotropins, much of the variation in this trait commonly found between individual females in many outbred mammalian species, is likely genetic. The identification of the genes controlling the common variation in ovarian response to gonadotropins would improve our ability to accurately prescribe the optimal hormonal dose for each hormone-response genotype in humans and other species, as well as, providing genetic markers for enhanced selection for improved reproductive performance in livestock species.
A screen of 16 strains of mice for hormone-Induced Ovulation Rate, i.e., Ovulation Rate, hormone Induced (ORI) (e.g., superovulation response to PMSG and hCG) revealed dramatic 6-fold differences in ORI between strains. A/J and AKR/J strain mice ovulated 9+1 eggs (Mean + SEM), other strains were intermediate, while C57BL/6J (B6) ovulated 54+2 eggs (p<0.00001)(Spearow, 1988). The strain difference in ORI between A/J and B6 involves a major genetic difference in the induction of follicle maturation by gonadotropins and follicular atresia (Spearow et al., 1991). Further studies showed that the hormone-Induced Aromatase Activity (AAI) is also 16 to 21-fold higher in granulosa cells from B6 than from A/J strain mice (Spearow et al., 1999). We hypothesize that the poor ovarian response to gonadotropins of A/J strain mice also contributes to the poor fertility and reproductive performance of the A/J strain.
Major Strain Differences in Induced Ovulation Rate. The 5 to 6-fold difference in ORI between A/J and B6 segregated in B6AF2 and (B6xA)xA N2 backcrosses as though this trait was controlled by the action of approximately 3.5 to 4 loci with major effects (Spearow, 1988; Spearow and Barkley, 1999). Quantitative Trait Loci (QTL) controlling differences in ORI (Gene name = Ovulation Rate Induced QTL; Gene symbol = Oriq) were first mapped in backcrosses (Spearow et al., 1999). The standard PMSG-hCG superovulation protocol usually generates two sets of oocytes, namely: 1) those matured by endogenous gonadotropins and ovulated promptly in response to PMSG (and found out of cumulus at autopsy the day after hCG); and, 2) those matured by PMSG and ovulated 12-14 h after hCG (and found in a viscous cumulus mass). A significant ORI QTL controlling the number of eggs in cumulus maps to chromosome (Chr) 6 in the vicinity of D6Mit316 according to composite interval analysis (Spearow et al., 1999). Other suggestive ORI QTL controlling eggs in cumulus were mapped to regions of Chr 2, 9 and X (Spearow et al., 1999). Linkage analysis also detected a significant QTL controlling eggs out of cumulus on Chr 10 in the vicinity of Estrogen Receptor alpha (Estra) (Spearow et al., 1999).
Mapping ORI QTLs in Recombinant Inbred Strains: Recombinant Inbred (RI) strain mice such as the current AxB,BxA RI strain set are ideal for mapping genes controlling phenotypes regulated by one or two loci with major effects (e.g., phenotypes controlled by 1 locus would segregate into 2 classes) (Nesbitt and Skamene, 1984). Thus, ORI was determined in 32 AxB,BxA RI strains and a QTL linkage analysis was conducted with MapManager QTX software (Manly et al., 2001). Parental strains differed 4.8-fold in the number of eggs in cumulus, with A/J mice ovulating 9.7 + 0.7 and B6 mice ovulating 45.9 + 2.2 eggs in cumulus (P<0.0001). AxB,BxA RI strains also differed 3.6 fold in the ORI trait (number of eggs in cumulus) (P<0.0001). QTL linkage analysis with MapManager QTX detected four ORI QTL regions on Chrs 19, 17, 3 and 18 (in decreasing order of effects) with genome-wide significant effects on ORI. Interval analysis showed that the most significant QTL (on Chr 19) accounted for >50% of the variance in this trait, with an additive effect of 7.6 eggs per B6 allele. This ORI QTL was highly significant on a genome-wide analysis (Genome wide p<0.001). The next most significant locus mapped to Chr 17 near H-2, confirming previous observations of a ORI QTL closely linked to H-2 (Spearow et al., 1991). The ORI QTL mapped to Chr 3 and 18 were also significant on a genome wide scan and accounted for >43% of the phenotypic variance in ORI. The additive effects of these QTL on ORI were >1.54 times the A/J standard deviation and 0.79 to 1.01 times the F1 standard deviation. Thus, the Chr 19 locus fully qualifies as a major gene, and Chr 17, 3 and 18 QTL qualify as major to moderate genes controlling ORI. While correction for individual loci somewhat reduced the effect of the other loci, suggesting interactions, these data show that approximately 4 loci with major effects on ORI are segregating between A/J and B6 in the RI strain set.
What can be described best as a “follicle maturation” QTL was also mapped. Parental strains differed 3.7-fold in the number of eggs matured by endogenous gonadotropins, ovulated by PMSG alone and found in cumulus, with A/J mice ovulating 2.0 + 0.6 and B6 mice ovulating 7.4 + 1.0 eggs “out of cumulus” (P< 0.0001). AxB,BxA RI strains differed 34-fold in number of eggs out of cumulus (P<0.0001). QTL linkage analysis of the ovulation response to PMSG alone detected four genome-wide significant regions of Chr 10, 6, 16 and 17. The QTL near Estrogen receptor alpha (Estra) on Chr 10 with an effect of 3 oocytes, was by far the most significant and accounted for 49% of the variance in this trait. The additive effects of this endogenous follicle maturation QTL on Chr 10 was 0.92 times the A/J standard deviation and 0.48 times the mean F1 standard deviation. Thus, the Chr 10 QTL qualifies as a moderate-to-major gene controlling follicle maturation by endogenous gonadotropins and ovulation by PMSG. The Chr 10 and Chr 6 follicle maturation QTL peaks overlap with that of similar follicle maturation and ORI peaks found in backcrosses (Spearow et al., 1999).
Mapping Reproductive Traits in Congenic strains: Congenic strains provide a powerful resource for reducing genetic noise and enabling detection of genes controlling complex traits (Belknap et al., 2001). We developed several A.B6 Oriq congenic strains of mice by starting with a B6AF1, repeatedly backcrossing for 10 generations to A/J and then intercrossing to form homozygotes (Spearow et al., 1999; Spearow, 2001). In the first set of congenics strains, in each generation selection was made for B6 microsatellite alleles in chromosomal regions flanking suggestive to significant ORI QTL mapped in (B6A)xA backcrosses (Spearow et al., 1999). Progeny testing A.B6 Chr 2 or 6 or X specific congenics as testcrosses to the large litter size selected inbred strain, S15 (i.e., on the S15XA genetic background) revealed significant to highly significant QTL in these Chr regions with additive effects of 2.4 to 4.2 eggs (Spearow, 2001). Whereas progeny testing on the A/J genetic background tends to show somewhat smaller effects of Oriq on Chr 2 and Chr 6 (P<0.01).
Additional preliminary breeding record data indicate highly significant differences in total number and the number of live pups born per dam per month between A/J parental, and different A.B6 Oriq congenic strains. The number of live pups born per dam per month (Mean + SEM) for A/J parental, A.B6-Oriq Chr2, A.B6-Oriq Chr6, and A.B6-Oriq Chr X congenic strains was 2.0+0.2, 4.2+0.5, 3.0+0.7; and 3.8+0.3, respectively (P<0.001). These data suggest that in untreated females these Oriq affect either cyclicity/breeding or maintenance of pregnancy. We hypothesize that alleles involved with the induction of follicle maturation and steroidogenesis during gonadotropin induced cycles may also regulate the ability of females under certain environmental conditions to mature a crop follicles, produce sufficient estrogens to generate a LH surge and then ovulate. Thus, ORI QTL are also likely to influence natural (spontaneous) ovulation rate and/or estrus cyclicity and therefore overall breeding and reproductive performance.
Collectively, these and other data from our laboratory suggest that loci on Chr 2, 6 and X are insufficient to account for the observed differences in ORI between A/J an B6 strain mice. These data suggest that additional loci elsewhere in the genome also regulate ORI, including possibly QTL with non-additive or epistatic effects. The finding of moderate to major gene ORI QTL controlling eggs in cumulus on Chr 19, 3, 17 and 18 and controlling follicle maturation by endogenous gonadotropins on Chr 10, 6, 17 and 16 in AxB,BxA RI strains is likely to account for the remaining loci with major effects on there reproductive traits. A.B6 congenics with B6 alleles in regions flanking several of these major gene ORI QTL are underdevelopment to provide powerful genetic resources to enable mapping and identification of these genes with major effects on ovarian function and fertility.
The finding of approximately 4 ORI QTL, i.e., genes, controlling the observed variation in ORI between strains of mice may explain the tremendous variation in induced ovulation rate commonly found between strains of mice and segregating in many outbred species. The identification of genes controlling ovarian response to gonadotropins and other fertility defects will enhance the development of diagnostics to enhance prescription of the optimal gonadotropin dose for each major hormone-response genotype; and, the development of fertility defect-specific pharmacological treatments which restore the desired level of gonadal and reproductive function. The identification of genetic markers for genes controlling ovarian response to gonadotropins is also likely to provide genetic markers for enhanced selection for improved reproductive performance in livestock species.
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