Oocyte-specific overexpression of mouse bone morphogenetic protein-15 leads to accelerated folliculogenesis and an early onset of acyclicity in transgenic mice

Abstract

Whereas mutations in the bmp15 gene cause infertility in ewes and women due to defects in folliculogenesis, most defects in female mice lacking bone morphogenetic protein (BMP)-15 are confined to the ovulation process, supportive of the observation that functional mouse BMP-15 is barely detected in oocytes in vivo until after the LH surge. In addition, the mouse BMP-15 proprotein is not processed into the functional mature protein in transfected cells. However, a chimeric protein consisting of the human proregion, human cleavage site, and mouse mature region (termed hhmBMP-15) is processed and the mature protein secreted. To study the role of BMP-15 in folliculogenesis, we generated transgenic mice overexpressing hhmBMP-15, exclusively in oocytes during folliculogenesis and confirmed the overexpression of mouse BMP-15 mature protein. Immature transgenic mice exhibited accelerated follicle growth with decreased primary follicles and an increase in secondary follicles. Granulosa cells of immature mice displayed an increased mitotic index and decreased FSH receptor mRNA expression. Adult mice had normal litter sizes but an increased number of atretic antral follicles. Interestingly, aging mice exhibited an early onset of acyclicity marked by increased diestrus length and early occurrence of constant diestrus. These findings indicate the role of BMP-15 in vivo in promoting follicle growth and preventing follicle maturation, resulting in an early decline in the ovarian reserve of transgenic mice. Therefore, the lack of mouse BMP-15 during early folliculogenesis in the wild-type mice may be relevant to their polyovulatory nature as well as the preservation of ovarian function as the mice age.

Figure 1

Oocyte-specific overexpression of hhmBMP-15F. A, The loxP-flanked Neo resistance gene is expressed under control of the cytomegalovirus (CMV) enhancer and chicken β-actin promoter. The hhmBMP-15 transgene is composed of the human BMP-15 preproregion and mouse BMP-15 mature region. Recombination after Cre recombinase expression under control of the ZP3 promoter results in conditional overexpression of hhmBMP-15 exclusively in the oocytes. Primers C1 and H1 were used for PCR genotyping. SV40, Simian virus 40. B, PCR analysis of genomic DNA from tails of mice positive or negative for the hhmBMP-15^flox transgene, including the founder male. Positive control (pos) is the transgene plasmid. GAPDH, Glyceraldehyde-3-phosphate dehydrogenase. C, PCR analysis of genomic DNA from tails of offspring of the founder male. D, PCR analysis of genomic DNA from tails or oocytes of mice carrying the ZP3-Cre and/or hhmBMP-15F^flox transgene. E, Western blot analysis of wild-type or transgenic ovaries using antihuman BMP-15 (left panel) or anti-FLAG (right panel) antibodies. F, Immunohistochemical analysis of BMP-15 in 8-wk-old wild-type (WT) or transgenic (TG) ovaries using antihuman BMP-15 antibody.

Figure 2

Follicle development in wild-type (WT) and transgenic (TG) ovaries. A, Histology of ovaries from 25-d-old wild-type and transgenic mice. Ovaries were photographed at ×40 (left panels) magnification. The boxed areas are shown at ×100 magnification (right panels). B, Numbers of primordial, primary, and secondary follicles were determined in wild-type and transgenic ovaries at 25 d of age. Ovaries were serially sectioned and every fifth section was counted, and total follicle numbers were determined. These data represent the mean ± sem of combined results from analysis of four mice per age and genotype (*, P ≤ 0.05).

Figure 3

Follicle development in 5-month-old wild-type (WT) and transgenic (TG) ovaries. Numbers of primary and secondary follicles were determined in wild-type and transgenic ovaries at 5 months of age. Ovaries were serially sectioned and every fifth section was counted, and total follicle numbers were determined. These data represent the mean ± sem of combined results from analysis of four mice per age and genotype (*, P ≤ 0.05).

Figure 4

Mitotic index in secondary follicles of 25-d-old wild-type (WT) and transgenic (TG) ovaries. Ovaries were serially sectioned, and 50 sections from each mouse were photographed under ×400 magnification. A grid corresponding to 200 granulosa cells was overlayed and the number of mitotic events was counted. These data represent the mean ± sem of combined results from analysis of three mice per genotype (*, P ≤ 0.05).

Figure 5

Steady-state mRNA levels of FSHR in 25-d-old wild-type (WT) and transgenic (TG) mice. Total RNA was extracted from granulosa cells and subjected to real-time PCR to determine the mRNA levels of FSHR. Data are normalized to L19 mRNA levels and shown as a relative ratio to the mRNA level of wild-type controls. These data represent the mean ± sem of combined results from the analysis of three mice per genotype (*, P ≤ 0.05).

Figure 6

Antral follicle atresia in 5-month-old wild-type (WT) and transgenic (TG) ovaries. Ovaries were serially sectioned and total antral follicles (healthy and atretic) were counted and the percentage of atretic antral follicles was determined. These data represent the mean ± sem of combined results from analysis of three mice per genotype (*, P ≤ 0.05).

Figure 7

Diestrus length in wild-type (WT) and transgenic (TG) females. The stages of the estrous cycle were determined by daily vaginal smears. Estrous cycles were examined bimonthly for a minimum of three complete cycles in wild-type and transgenic females (n = 15 for each genotype) from 10 to 20 months of age or until the onset of constant diestrus. These data represent the mean ± sem of combined results (*, P ≤ 0.05).

Figure 8

Percentage of mice exhibiting constant diestrus. The stages of the estrous cycle were determined by daily vaginal smears. Estrous cycles were examined bimonthly for a minimum of three complete cycles in wild-type (WT) and transgenic (TG) females (n = 15 for each genotype) from 10 to 20 months of age.

Figure 9

Histological analysis of transgenic ovaries. Ovarian sections were stained with hematoxylin and eosin and photographed at ×40 magnification. A, Normal ovary from a cycling mouse exhibiting follicles of various developmental stages and corpora lutea. B and C, Ovaries from transgenic mice that had entered constant diestrus, exhibiting hemorrhagic (asterisk) and fluid-filled (arrowhead) cysts and a lack of developing follicles.