A pair of related microRNAs highly expressed in the pituitary gland are essential to ovulation in mice, according to a study published last week by Osaka University researchers. And because this miRNA cluster also exists in humans, the findings may offer new clues about treating human infertility.
Given the growing body of data linking miRNAs to a host of biological processes, the investigators aimed to determine the small, non-coding RNAs’ impact on reproduction. To do so, they produced knockout mice lacking miR-200b, which was chosen because of its expression in mouse testes, and miR-429, which has the same seed sequence as miR-200b and is found in the same genomic vicinity.
While the double knockout had no effect on the testes or the fertility of male mice, the researchers found that it led to “greatly reduced fertility” in females, which seldom became pregnant after mating with heterozygous males. Successful coitus was determined by the formation of a vaginal plug.
According to the study, which appeared in Science, the pregnancy rate of the double-knockout female mice was at most 9 percent, compared with 85 percent in wild-type and heterozygous animals.
Additionally, the knockout mice that did become pregnant had markedly smaller litters and did not become pregnant again “even after another three months of pairing with males,” the investigators wrote.
To account for the possibility that the infertile phenotype was the result of unknown side effects that have been reported in some gene knockout experiments, the team created miR-200b- and miR-429-expressing transgenic mouse lines and crossed them with the double knockout line. Doing so returned fertility to the knockout mice, indicating that the sterility was directly caused by the miRNA deficiency.
Speculating that the miRNAs influence ovulation, the scientists attempted to collect oocytes from the oviducts of the female knockout mice on the day vaginal plugs were found, but were only able to do so for 2 out of 20 animals.
The researchers then prepared histology sections of the ovaries of the double knockout mice on the day of vaginal plug formation. Unlike sections from heterozygous mice, which showed many early corpora lutea — a mass of progesterone-secreting cells formed following ovulation — only about 10 percent of the knockout mice formed these structures, according to the Science paper.
Notably, when the knockout mice were superovulated through the administration of pregnant mare serum gonadotropin and human chorionic gonadotropin, they ovulated roughly the same number of oocytes as their heterozygous counterparts. And when these oocytes were fertilized in vitro, fertilization occurred at a normal rate and produced normal pups after the embryos were transplanted into wild-type foster mothers.
Further, the ovaries of the knockout mice were found to be structurally normal, and proper corpora lutea formation occurred in their superovulated mature ovaries.
Given that the miR-deficient mice can undergo normal oogenesis and ovulate with the addition of hormones, the findings suggested that impaired hormonal regulation was behind the animals’ infertility.
An examination of the hormone levels of the knockout mice showed a statistically significant decrease in serum luteinizing hormone, or LH, and serum progesterone levels in the females, but not the males — a sex-dependent phenotype the research team was not able to explain.
Since ovulation is controlled in mammals by hormones released by the hypothalamo-pituitary-ovarian axis, the team examined the expression of miR-200b and miR-429 in the ovary, hypothalamus, and pituitary gland, finding very high expression of both in the pituitary.
“Working on the hypothesis that these miRNAs might act in regulating pituitary function, we searched for their target mRNAs in public databases,” the research team wrote in Science. “Among the putative target mRNAs listed, the 3’-untranslated regions (UTRs) of both mouse and human Zeb1 and Zeb2 possess the highest number of loci that were complementary to the seed sequence of miR-200b and miR-429.”
Focusing on Zeb1, they found that the amount of its protein in the pituitary of the knockout mice increased, while remaining normal in the hypothalamus where the two miRNAs are not highly expressed.
“Given that the Zeb1 mRNA level in the pituitary was not altered by deficiencies in these miRNAs, the amount of [its protein] must be regulated by a posttranscriptional mechanism,” they speculated. In addition, the increased level of the protein in the knockout mice was lowered to near wild-type levels in the miR-200b and miR-429 transgenic mouse lines.
Additional experimentation with transgenic mouse lines confirmed that LH was suppressed as a result of increased ZEB1 expression, which itself resulted from the loss of miR-200b and miR-429 — a finding that the researchers believe may be applicable to human reproductive physiology given the presence of the miRNA cluster in humans.