PMC

Analysis of Fst mRNA and protein in the FST288-only mouse. A, Northern blot analysis of RNA from PMSG-treated WT and FST288-only ovaries. WT FST mRNA is at the expected 2.5-kb size, but FST RNA from FST288-only ovaries was 1.5 kb larger (see Materials and Methods and supplemental material for explanation). Note that total FST mRNA is reduced in FST288-only ovaries (stronger β-actin normalization band). B, Western blot analysis of follicular fluid from WT and FST288-only mouse ovaries after deglycosylation. In WT follicular fluid, three bands were detected, corresponding to FST315, FST303, and FST288 (,), whereas in follicular fluid from FST288-only females, only the FST288 was detected. This confirms that the genetic modification leads to production of only one FST isoform in FST288-only mice.

Analysis of Fst mRNA expression in WT and FST288-only mice using qPCR. A, Fst mRNA expression was reduced by about half in FST288-only ovaries (black bars) compared with WT ovaries (white bars). However, Fst expression in FST288-only testis is not different from WT testis. In contrast, Fst expression in FST288-only liver is about 4-fold greater than WT liver (n = 4–6 samples for each tissue type). B, Ovarian expression of Fst-related genes (Fstl3) or genes in the activin signaling pathway (activin A, activin B, SMAD2, and SMAD3) are not altered in FST288-only mice compared with WT mice. These results suggest that Fst expression is differentially regulated in different tissues, but expression of genes in the activin signaling pathway are not altered in ovaries.

Immunohistochemical localization of FST in WT and FST288-only ovary. FST was localized using a new polyclonal antibody to mouse FST315 in ovarian sections. A, FST staining was observed in oocytes, granulosa cells, parts of stromal cells, and ovarian surface epithelium. Note that nuclear staining was observed in many granulosa cells and oocytes. B, After preincubation of antibody with recombinant FST315, most staining in granulosa cells, oocytes, and surface epithelium is drastically reduced, although some weak staining in stroma remains. C, Even when FST antibody is deleted, some staining remains in the stroma. These results confirm the specificity of this antibody for FST. D–F, Enlargement of preovulatory follicle from A–C, respectively, providing details of cellular localization. G–I, FST staining in WT large antral, small antral, and primary follicle, respectively. J–L, FST staining in similar follicles from FST288-only ovaries. FST staining is much stronger WT ovaries (G–I) compared with FST288-only ovaries (J–L), and the nuclear staining is not observed in FST288-only mice, suggesting that both total amount and intracellular compartmentalization of FST is altered in FST288-only mice.

Reproductive phenotype of FST288-only females. A, Over an 8-month breeding period, FST288-only females have significantly smaller litters (black bar) compared with WT females mated with males of the same genotype (n = 7 and 5, respectively). B, The mean interval between litters was significantly longer in FST288-only females compared with WT females. C, Analysis of cumulative number of pups for each genotype for all breeding females. Due to smaller litters and longer intervals, the total number of pups produced is significantly smaller in FST288-only females at all ages. D, The number of embryos collected at 0.5 dpc after superovulation was reduced by nearly half (P < 0.01) in FST288-only females (black bars) compared with WT females (white bars). E, The number of embryos collected at 3.5 dpc after superovulation was similarly reduced by nearly half in FST288-only females compared with WT females, a ratio that held up even when embryos were subcategorized into blastocyst, morula, or degenerating. F, When the results from E were expressed as a percentage of the total number of embryos, the differences in each category disappeared. This indicates that FST288-only females ovulate fewer eggs per cycle, but their developmental potential is not different from WT embryos.

Analysis of follicle and germ cell numbers in FST288-only and WT females. Ovaries from FST288-only (▪) and WT (•) females were collected (n = 3–6) at ages shown and analyzed for follicle stage and number as described in methods. A, The total numbers of nonatretic tertiary and antral follicles per ovary is significantly smaller in FST288-only females at 100 and 250 d, whereas this number is not different at puberty (d 42). B, The numbers of secondary follicles per ovary are significantly greater in FST288-only females at 8.5 d of age compared with WT females, but at puberty and later, this difference is no longer detectable. C, The numbers of primary follicles per ovary are significantly greater in FST288-only females at 8.5 and 42 d of age compared with WT females, a difference that becomes undetectable by 100 d of age. D, The total numbers of germ cells (still in oocyte nests), primordial follicles, and early primary follicles per ovary are almost double at 8.5 d of age in FST288-only females compared with WT females. However, this difference is not detectable after puberty through accelerated loss from this pool of oocytes. In addition, at 250 and 400 d of age, there are significantly fewer primordial follicles in FST288-only females compared with WT females. E, Total numbers of germ cells and primordial follicles are shown. This plot does not include those primordial follicles showing signs of activation that were included in D. Again, follicle number is nearly double at 8.5 d of age in FST288-only ovaries compared with WT, but the number decreases more rapidly in FST288-only ovaries so that primordial follicle number is significantly smaller than WT by 250 d of age. F, The numbers of activated primordial-primary transition follicles are significantly greater at 8.5 and 42 d of age in FST288-only females compared with WT females. By 400 d of age, the number of activated follicles is significantly greater in WT compared with FST288-ovaries. This accelerated activation may account for the faster depletion of the primordial follicle pool in these mice as well as the earlier ovarian failure. *, P < 0.05; **, P < 0.01; ***, P < 0.001.