An Important Role of Pumilio 1 in Regulating the Development of the Mammalian Female Germline

Abstract

Pumilio/FBF (PUF) proteins are a highly conserved family of translational regulators. The Drosophila PUF protein, Pumilio, is crucial for germline establishment and fertility. In mammals, primordial folliculogenesis is a key process that establishes the initial cohort of female mammalian germ cells prior to birth, and this primordial follicle pool is a prerequisite for female reproductive competence. We sought to understand whether PUF proteins have a conserved role in mammals during primordial folliculogenesis and female reproductive competency. In mammals, two homologs of Pumilio exist: Pumilio 1 (Pum1) and Pum2. Here, we report that PUMILIO (PUM) 1 plays an important role in the establishment of the primordial follicle pool, meiosis, and female reproductive competency, whereas PUM2 does not have a detectable function in these processes. Furthermore, we show that PUM1 facilitates the transition of the late meiotic prophase I oocyte from pachytene to diplotene stage by regulating SYCP1 protein. Our study reveals an important role of translational regulation in mammalian female germ cell development.

Keywords: Pumilio; RNA binding protein; meiosis; primordial folliculogenesis; subfertility; translational regulator.

FIG. 1

Pum1^−/− females have subfertility due to a lower number of viable oocytes that are ovulated. A) Immunoblot analysis showing PUM1 and PUM2 protein levels in wild-type mouse ovaries (+/+) and Pum1^−/− ovaries (−/−) from 6- to 10-wk-old female mice. This immunoblot was performed three times with a total of 10 biological replicates. Glyceraldehyde phosphate dehydrogenase (GAPDH) used as loading control. Densitometric measurements showed no significant differences in PUM2 protein levels between the two genotypes. B) Litter sizes of wild-type (n = 5) and Pum1^−/− (n = 11) females singly housed with a wild-type stud male continuously collected over a minimum of 6 mo, revealing a 32% reduction in average litter sizes. **P < 0.01. Error bars represent SD. C) Litter size distribution of wild-type and Pum1^−/− females with advancing age. Error bars represent SD. Using linear regression analysis, Pum1^−/− litter size negatively correlated with age and significantly deviated from zero with a P < 0.05. No correlation was found with wild-type females. D) Total number of superovulated MII nondegenerate (Non-deg) and degenerate (Deg) oocytes per 6- to 7-wk-old wild-type (n = 14) and Pum1^−/− (n = 15) females. *P < 0.05. Total number of oocytes between the two groups was not statistically significant. E) Brightfield image of nondegenerate MII oocytes (no arrow) and degenerate MII oocytes (arrowhead). Bar = 50 μm. F) Total number of two-cell embryos obtained from wild-type (n = 6) and Pum1^−/− (n = 11) females naturally mated with wild-type stud male. *P < 0.05.

FIG. 2

PUM1 is involved in the establishment of the primordial follicle pool. A and B) Histological sections of 6-wk-old wild-type (+/+) and Pum1^−/− (−/−) ovaries, respectively, stained with hematoxylin/eosin. C) Total number of primordial, primary, and preantral follicles in 4-wk-old wild-type (n = 6) and Pum1^−/− (n = 6) littermate ovaries. *P < 0.05. D) Total number of primordial, primary, and preantral follicles in 6-mo-old wild-type (n = 5) and Pum1^−/− (n = 6) littermate ovaries. *P < 0.05. E) Total number of primordial, primary, and preantral follicles in 4-wk-old wild-type (n = 4) and Pum2^−/− (n = 5) littermate ovaries.

FIG. 3

PUM proteins are present during embryonic female gonadal development, and PUM1 is enriched, but not required, in oocytes for oogenesis. A) Immunoblot analysis of whole-ovary lysates from 13.5, 15.5, and 18.5 dpc female embryos (n = 10–15 per biological replicate) with anti-PUM1, anti-PUM2, and anti-GAPDH antibodies. This immunoblot was performed three times with new samples each time. Both PUM proteins are present in all developmental time points. B) Immunoblot analysis of lysates from 13.5-dpc FACS-sorted oocytes and somatic cells (n = 20–40) by anti-SSEA1 antibody. Per FACS sort, usually more than 20 000 oocytes and more than 60 000 somatic cells are sorted. Total protein lysate was loaded per experiment. The anti-SSEA-1 FACS-sorting was highly efficient at separating the oocyte and somatic cell compartment. GAPDH was used as loading control. C and D) The bar graph shows the summary of the densitometric measurements (mean ± SEM) of the immunoblots performed on four different FACS sorts done on four separate days. Paired Student t-test was performed. *P < 0.05. E) Total number of primordial, primary, and preantral follicles in ovaries of 4-wk-old-wild-type mice with vasa Cre (Wt + vasa); n = 6) and Pum1^−/flox with vasa cre mice (Pum1^−/f; + vasa cre; n = 6). Error bars represent SD.

FIG. 4

PUM1 is not required for PGC mitosis or to prevent excessive female germline apoptosis. A) Images of immunofluorescence staining with the germ cell marker VASA (green) on sections of 13.5-, 15.5-, and 18.5-dpc ovaries of wild-type (+/+) and Pum1^−/− (−/−) females. B) The total number of germ cells in 13.5-, 15.5-, and 18.5-dpc ovaries from sibling-matched wild-type (n = 5, 3, 3, respectively) and Pum1^−/− females (n = 5, 4, 3, respectively).

FIG. 5

PUM1 plays a role during female meiotic progression. A) Representative images of immunofluorescence staining of meiotic spreads with SYCP1 (red) and SYCP3 (green). Examples of each stage during mouse meiotic prophase I are shown: during the leptotene stage, the chromatin starts to condense into fine threads; at the zygotene stage, distinct chromosomes with tripartite SCs at homologous pairing sites are seen; at the pachytene stage, maximal shortening of the homologous paired chromosomes occurs; at the diplotene stage, homologous paired chromosomes start to separate; at the dictyate stage, decondensation of chromatin and formation of between two and four nucleoli occur. B) Distribution of meiotic prophase I stages in 15.5-dpc (n = 5), 18.5-dpc (n = 5), and PND1 (n = 4) ovaries in sibling-matched wild-type and Pum1^−/− females. A paired Student t-test was used for this analysis. *P < 0.05.

FIG. 6

Aberrant meiosis in Pum1^−/− females leads to reduction in primordial follicle formation postnatally. A and B) Histological sections of PND1 wild-type (+/+) and Pum1^−/− (−/−) ovaries, respectively, stained with hematoxylin/eosin. C) Total number of primordial follicles and oocytes without surrounding pregranulosa cells (naked oocytes) in wild-type (n = 5) and Pum1^−/− (n = 5) littermate ovaries, and Pum1^−/flox;vasa cre (n = 5) PND1 ovaries. *P < 0.05. D and E) Histological sections of PND5 wild-type (+/+) and Pum1^−/− (−/−) ovaries, respectively, stained with hematoxylin/eosin. *P < 0.05. F) Total number of primordial and primary follicles in wild-type (n = 8) and Pum1^−/− (n = 6) littermate ovaries, and Pum1^−/flox;vasa cre (n = 6) PND5 ovaries. *P < 0.05. G) Percentage of oocytes at 15.5 dpc, 18.5 dpc, PND1, and PND5 present in germ cell nests in wild-type (n = 3, 3, 5, 8, respectively) and Pum1^−/− (n = 4, 3, 5, 6, respectively) ovaries. At each developmental time point, Mann-Whitney test was performed, and no statistically significant difference was found.

FIG. 7

PUM1 down-regulates SYCP1 during late meiotic prophase I in mouse oocytes. A) Representative images of immunofluorescence staining with SYCP1 (red) and SYCP3 (green); examples of linear, punctate, and absent SYCP1 protein are shown. B) Total number of meiotic spreads with linear and nonlinear (sum of punctate and absent SYCP1 spreads) SYCP1 immunostaining in PND1 wild-type and Pum1^−/− littermate ovaries. *P < 0.05.