PMC

Presence of HSF1 during embryonic prophase I did not rescue maturation defects and female infertility in conditional Hsf1 knockout. (A) Histograms show the number of pups obtained by litter produced by females of the indicated genotype. Zp3 Cre; Hsf1^loxP/loxP females did not produce any offspring. (B) GV oocytes were cultured during 16 h, and a similar in vitro MI block was observed in constitutive Hsf1^−/− (n = 427) and conditional Zp3 Cre; Hsf1^loxP/loxP (n = 296) oocytes compared to WT (n = 541) oocytes. (C) Representative images of meiotic spindles observed in both types of mutant oocytes (blue, TO-PRO-3-stained DNA; green, α-tubulin).

Mechanisms involved in HSF1-dependent MI block. (A and B) Cold treatment. Meiotic spindles lacked cold-stable microtubules (A), and misaligned chromosomes (B) were more frequent in mutant (Hsf1^−/− and Zp3 Cre; Hsf1^loxP/loxP) (n = 73) oocytes compared to WT (n = 55) oocytes. (C and D) Spindle assembly checkpoint (SAC) inhibition by MspI-IN-18 in HSF1-deficient oocytes. (C) Percentage of oocytes resuming meiosis after treatment with DMSO (n = 108 oocytes) or DMSO plus Mps1-IN-18 (n = 194 oocytes); (D) representative images of MI (i), polar body extrusion (ii), and degenerated oocyte (iii).

HSF1 deficiency leads to defects in central element of the synaptonemal complex (SC). (A) Immunostaining for SYCP3 and SYCE1 performed on 17.5-dpc Hsf1^+/− (control) (n = 111) and Hsf1^−/− (n = 107) oocytes (n = 3 fetuses). (B) Synaptonemal complex length measured after SYCP3 staining was significantly increased in Hsf1^−/− (n = 53) versus Hsf1^+/− (n = 56) oocytes (13.88 ± 0.14 μm and 12.28 ± 0.19 μm, respectively). Measurements were performed using ImageJ software (). (C) SYCE1 intensity was measured from images as shown in panel A using ImageJ software () and plotted relative to the Hsf1^+/− value arbitrarily set to 1. (D) Western blot analysis of SYCE1 expression in 17.5-dpc ovaries and fully grown (GV) adult oocytes (control for equal loading, α-tubulin). ***, P < 0.001.

Recombination and the DNA repair process are altered in 17.5-dpc HSF1-deficient oocytes. (A) Diagram illustrating MSH4-related functions and indicating endpoint analyses performed on meiotic prenatal oocytes. MSH4 is included in transformed nodules (TN) which emerge from early nodules (EN) and subsequently—for some of them—evolve as recombination nodules (RN) associated with MLH1 (). MSH4 foci are analyzed in panels B, C, and D. DNA repair progression is assessed with γH2AX staining as the hallmark for DSB (see panels B and E). (B) SYCP3 (red), MSH4 (green), and γH2AX (blue) immunostaining performed on Hsf1^+/− (control) and Hsf1^−/− oocyte spreads at 17.5 dpc (n = 50 for each genotype). (C) The number of MSH4 foci calculated per oocyte was significantly different in Hsf1^−/− oocytes compared to that in Hsf1^+/− oocytes (number of foci, 96.5 ± 14 versus 270 ± 40, respectively). (D) SYCP3 (red) and MLH1 (green) immunostaining on pachytene oocytes showed that there was no difference between the number of late recombination nodules between Hsf1^−/− (24.61 ± 1.12 foci) and Hsf1^+/− (24.15 ± 1.46 foci) oocytes (n = 25). (E) The area covered by γH2AX staining was different in Hsf1^−/− oocytes from that in Hsf1^+/− oocytes (γH2AX area, 142 ± 11 versus 28.5 ± 3 μm²). **, P < 0.01; ***, P < 0.001.

Schematic model illustrating HSF1 as a regulator of the meiotic gene network. The HSF1 transcriptome analyzed in GV oocytes identified a group of genes listed in the “chromosome and chromatid cohesion” category. Genes with the name written in boldface were shown to be bound by HSF1 (). We found that HSF1 is regulating those genes from the beginning of the meiosis process (prenatal prophase I: L, Z, P, D stages). HSF1-dependent genes are listed in boxes (rectangles) colored in a similar way to their functions (circles). These different meiotic functions have been found to be mechanistically linked, as indicated by the arrows. Numbers in parentheses refer to the following papers: 1, Novak et al. (); 2, Zickler (); 3, Barbero (); and 4, Kan et al. (). Thus, HSF1 contributes to the coordinated expression of meiotic genes acting in a coordinated way. Question marks indicate some yet unexplained observations: e.g., role of genes such as Msh4 (participating in a DNA repair/recombination function) or Syce1 (contributing to synaptonemal complex) in adult oocytes. Finally, further analyses of the related functions pointed out a series of anomalies linked to the absence of HSF1 (HSF1 knockout [HSF1ko]) during the prenatal or postnatal/adult phases of meiosis. In conclusion, HSF1 deficiency affects meiotic gene expression and functions.

Distinct expression and function of Hsf1 and Hsf2 in adult murine oocytes. (A) RT-qPCR analysis of Hsf1 and Hsf2 transcripts in various tissues and cell types showed that mouse fully grown (germinal vesicle [GV]) oocytes are characterized by a very high level of Hsf1 transcripts in comparison to a lower level of Hsf2 mRNAs. The level of transcripts from the gene encoding ribosomal protein S16 was used as an internal reference to normalize the level of Hsf1 and Hsf2 mRNAs, respectively. Experiments were performed with tissues, oocytes, and embryos collected from n = 4 animals. Bars represent means ± SEM. (B) In vitro meiotic maturation of GV oocytes demonstrated that Hsf2^−/− oocytes, in contrast to Hsf1^−/− oocytes, normally reach metaphase II (MII). Bars represent the mean percentages of oocytes at the GV stage, metaphase I (MI), and MII following 16 h of culture (WT, n = 541 oocytes; Hsf1^−/−, n = 427 oocytes; Hsf2^−/−, n = 120 oocytes). (C) Comparison of HSF1- and HSF2-dependent transcriptome in GV oocytes. The number of genes differently expressed (≥1.5-fold; P < 0.05) in Hsf1^−/− and Hsf2^−/− oocytes in comparison to WT ones is indicated in circles colored in red or blue when they are upregulated in Hsf1^−/− and Hsf2^−/− oocytes, respectively, or in circles colored in green or yellow when they are downregulated in Hsf1^−/− and Hsf2^−/− oocytes, respectively. The number of genes regulated by both HSF1 and HSF2 is written in the area of the intersecting circles.

Postnatal deletion of Hsf1 (Zp3 Cre; Hsf1^loxP/loxP) partially rescues the defects observed in MI chromosomal structure. (A) RT-qPCR performed on adult fully grown oocytes obtained from Zp3 Cre; Hsf1^loxP/loxP females revealed that HSF1 target genes are as affected as those in the constitutive knockout. Relative quantities of mRNA were normalized against the quantity of the ribosomal S16 transcripts and their relative expression levels were compared to those of the WT sample, which was arbitrarily given the value 1. Bars represent means ± SEM from at least three independent experiments performed with oocytes collected from several females (n = 3). (B) Representative images of MI chromosome shown at two different magnifications (TO-PRO-3 staining). (C) Chromatid length, measured from images as shown in panel B by ImageJ software, was similarly increased in Hsf1^−/− (n = 103) and Zp3 Cre; Hsf1^loxP/loxP (n = 108) oocytes compared to WT (n = 112) oocytes. (D) MI chromosomes (as shown in panel B) were classified as bivalent, univalent, or single (number of analyzed chromatids, WT, n = 880; Hsf1^−/−, n = 1,494; Zp3 Cre; and Hsf1^loxP/loxP, n = 1,032). (E) Proportions of unified, adjacent, and separated sister centromeres in WT, Hsf1^−/−, and Zp3 Cre; Hsf1^loxP/loxP oocytes (number of centromeres analyzed per genotype, n = 100). *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Postnatal deletion of Hsf1 (Zp3 Cre; Hsf1^loxP/loxP). (A) Schematic representation of Hsf1 genomic sequence and associated protein domains. The constitutive Hsf1 knockout mouse line was made by insertion of Neo cassette in place of exons 3 to 6 (), whereas the conditional deletion leads to deletion of the exons 2 to 4, which correspond to a large part of the DNA binding domain of HSF1. (B) Phases of prenatal prophase I (L, leptotene; Z, zygotene; P, pachytene; D, diplotene) and adult female meiosis (GV, germinal vesicle; GVBD, germinal vesicle breakdown; MI, metaphase I; PBE, polar body extrusion; MII, metaphase II) illustrated with the profile of Hsf1 deletion in the Hsf1^−/− (constitutive) and Zp3 Cre; Hsf1^loxP/loxP (conditional) lines. (C) RT-qPCR was performed as in . Ovaries collected from Zp3 Cre; Hsf1^loxP/loxP female embryos (17.5 dpc) exhibited the same level of expression of HSF1 target genes as that in control samples. Bars represent means ± SEM from at least three independent experiments performed with ovaries collected from several fetuses (n = 5). (D) SYCP3 immunostaining was used to compare the different prophase I stages (L, Z, and P) in 17.5-dpc Hsf1^+/+; Hsf1^loxP/loxP (control, n = 128) and Zp3 Cre; Hsf1^loxP/loxP (n = 111) oocytes. Bars represent means ± SEM from experiments performed with several fetuses (n = 3). There was no difference in prophase I stages between Zp3 Cre; Hsf1^loxP/loxP and control Hsf1^+/+; Hsf1^loxP/loxP oocytes.

Abnormal prenatal prophase I in Hsf1^−/− oocytes. (A) Schematic representation of prenatal prophase I stages (L, leptotene; Z, zygotene; P, pachytene; D, diplotene). (B) RT-qPCR experiments showed that HSF1-dependent genes ( and D) are downregulated in 17.5-dpc Hsf1^−/− samples. RT-qPCR results were normalized against the ribosomal S16 transcript level, and the normal- ized expression level was compared to the Hsf1^+/− sample, which was arbitrarily given the value 1. Bars represent means ± SEM from at least three independent experiments performed with ovaries collected from several fetuses (n = 4). (C) SYCP3 immunostaining was used to compare prophase I progression at 17.5 dpc and 1 dpp. Representative images of observed stages at 17.5 dpc are presented in the upper panels. The percentage of zygotene, pachytene, and diplotene stages was calculated for each sample: 17.5 dpc for Hsf1^+/−, control (n = 209), and Hsf1^−/− (n = 224) oocytes and 1 dpp for Hsf1^+/− (control, n = 228) and Hsf1^−/− (n = 253) oocytes (graph, lower panel). Bars represent means ± SEM from experiments performed with several siblings (n = 4 to 17.5 dpc for embryos or −1 dpp for pups). Prophase I stages were differently represented in HSF1-deficient oocytes according to chi-square test (P < 0.001). (D) RT-qPCR experiments performed on 17-day-old testes show no changes in the expression of selected HSF1-dependent genes. Data are presented as in panel B. Bars represent means ± SEM from at least three independent experiments performed with testes collected from several males (n = 3). (E) Spreads of male germ cells were prepared from testes at 17 days postpartum. Analysis of Hsf1^−/− (n = 350) and Hsf1^+/− (n = 318) spermatocytes did not reveal any difference between the two genotypes (n = 3 males). **, P < 0.01; ***, P < 0.001.

HSF1 regulates meiotic genes. (A) Molecular functions of HSF1-dependent genes identified in the enriched biological process entitled “cell cycle” (see Table S1 in the supplemental material). (B) RT-qPCR validation of HSF1 target genes listed under the “chromosome and chromatid cohesion” process (see Table S2 in the supplemental material). RT-qPCR results were normalized against the ribosomal S16 transcript level, and the normalized expression level was compared to the WT sample, which was arbitrarily expressed as 1. Bars represent means ± SEM of at least three independent experiments performed with oocytes collected from several females (n = 5). (C) Schematic representation of the 5′ region of HSF1-dependent genes (Syce1, Stag2 and -3, and Msh4) showing the respective positions of HSE sites within the 10 kb upstream of the transcription start to be analyzed in panel D. (D) ChIP experiments performed with fully grown (GV) oocytes revealed that HSF1 and HSF2 bind distinct HSEs (see Tables S2 and S3 in the supplemental material). HSE numbers refer to panel C. HSEs indicated in panel C but not present in panel D correspond to a ChIP-negative sample or HSEs not bound. The HSP90α sample was included as a positive control (, ). Nonspecific antibody (NS) was used as a negative control, and acetylated histone H4 (AcH4) was used as an indicator of transcriptionally active promoters. *, P < 0.05; **, P < 0.01; ***, P < 0.001.