Hypoxia Inducible Factor 1 Alpha Is Expressed in Germ Cells throughout the Murine Life Cycle

Abstract

Pluripotent stem cells of the early embryo, and germ line cells, are essential to ensure uncompromised development to adulthood as well as species propagation, respectively. Recently, the transcription factor hypoxia inducible factor 1 alpha (Hif1α) has been shown to have important roles in embryonic stem cells; in particular, regulation of conversion to glycolytic metabolism and, as we have shown, maintenance of functional levels of telomerase. In the present study, we sought to assess whether Hif1α was also expressed in the primitive cells of the murine embryo. We observed expression of Hif1α in pre-implantation embryos, specifically the 2-cell stage, morula, and blastocyst. Robust Hif1α expression was also observed in male and female primordial germ cells. We subsequently assessed whether Hif1α was expressed in adult male and female germ cells. In the testis, Hif1α was robustly expressed in spermatogonial cells, in both juvenile (6-week old) and adult (3-month old) males. In the ovaries, Hif1α was expressed in mature oocytes from adult females, as assessed both in situ and in individual oocytes flushed from super-ovulated females. Analysis of Hif1α transcript levels indicates a mechanism of regulation during early development that involves stockpiling of Hif1α protein in mature oocytes, presumably to provide protection from hypoxic stress until the gene is re-activated at the blastocyst stage. Together, these observations show that Hif1α is expressed throughout the life-cycle, including both the male and female germ line, and point to an important role for Hif1α in early progenitor cells.

Fig 1. Hif1α expression in preimplantation embryos.

(A) Hif1α staining in two-cell stage embryo and morula (top) and negative control images without primary antibody (bottom). (B) Co-staining of OCT4 and Hif1α in inner cell mass of blastocyst (top) and negative control images without primary antibodies (bottom). Scale bar: 30μm.

Fig 2. Hif1α expression in 9.5dpc primordial germ cells.

(A) Whole-mount staining of Hif1α and GFP in Oct4-GFP embryo (lateral view). Scale bar: 100μm. (B) Hif1α and GFP staining of FACS-sorted PGCs. Scale bar: 20μm.

Fig 3. Hif1α expression in 15.5dpc germ cells.

(A) Sections of male (top) and female (bottom) gonads from 15.5 dpc Oct4-GFP embryos showing Oct4-GFP and Hif1α expressions in germ cells. Scale bars: 50μm (male) and 20μm (female). (B) FACS-sorted male (top) and female (bottom) 15.5dpc germ cells showing Oct4-GFP and Hif1α expressions. Scale bars: 100μm (male) and 50μm (female).

Fig 4. Hif1α expression in neonatal and adult testis.

(A) Section of testis from 5-day old (P5) male new born pups showing Hif1α expression in MVH+ gonocytes within the seminiferous tubules (top) and negative control images without primary antibodies (bottom). Scale bar: 50μm. (B) Western blot analysis of Hif1α expression in P5 testes (left) compared to extract of the adult brain sub-ventricular zone (SVZ) (right). Loading control (β-Actin) is shown below. (C) Section of adult (3 month old) testis showing Hif1α expression in spermatogonia. Scale bar: 30μm. (D) Western blot analysis of whole adult testis. HEK293 cells treated with DFX were used as a positive control and intestinal tissue was used as a negative control. Loading control (β-Actin) is shown below.

Fig 5. Hif1α expression in neonatal and adult ovary.

(A) Section of P5 ovary showing MVH expression in all oocytes and Hif1α expression only in small oocytes (primary follicles; top). Negative control images without primary antibodies (bottom). Scale bar: 50μm. (B) Section of adult ovary showing absence of Hif1α expression in primary follicles detected with MVH staining (top). Negative control without primary antibody (bottom). Scale bar: 50μm. (C) Section of adult ovary showing Hif1α expression in mature primary oocyte (arrow). Negative control without primary antibody (bottom). Scale bar: 100μm. (D) Image of a Metaphase II oocyte from superovulated three-month-old female showing Hif1α expression. Scale bar: 50μm.

Fig 6. Transcription of Hif1α during early stages of development.

RT-PCR analysis of Hif1α transcription in 3.5 dpc blastocysts, 9.5 dpc PGCs, whole testes tissue, mature oocytes, and hypoxic-cultured MEFs. Hif1α PCR products are shown in the upper row with HPRT single-copy gene control reactions in the bottom row, cDNA reactions are shown with complementary noRT reactions for each gene and sample (left and right respectively).