Rethinking progesterone regulation of female reproductive cyclicity

Abstract

The progesterone receptor (PGR) is a ligand-activated transcription factor with key roles in the regulation of female fertility. Much has been learned of the actions of PGR signaling through the use of pharmacologic inhibitors and genetic manipulation, using mouse mutagenesis. Characterization of rats with a null mutation at the Pgr locus has forced a reexamination of the role of progesterone in the regulation of the female reproductive cycle. We generated two Pgr mutant rat models, using genome editing. In both cases, deletions yielded a null mutation resulting from a nonsense frame-shift and the emergence of a stop codon. Similar to Pgr null mice, Pgr null rats were infertile because of deficits in sexual behavior, ovulation, and uterine endometrial differentiation. However, in contrast to the reported phenotype of female mice with disruptions in Pgr signaling, Pgr null female rats exhibit robust estrous cycles. Cyclic changes in vaginal cytology, uterine histology, serum hormone levels, and wheel running activity were evident in Pgr null female rats, similar to wild-type controls. Furthermore, exogenous progesterone treatment inhibited estrous cycles in wild-type female rats but not in Pgr-null female rats. As previously reported, pharmacologic antagonism supports a role for PGR signaling in the regulation of the ovulatory gonadotropin surge, a result at variance with experimentation using genetic ablation of PGR signaling. To conclude, our findings in the Pgr null rat challenge current assumptions and prompt a reevaluation of the hormonal control of reproductive cyclicity.

Keywords: PGR; progesterone; rat; reproductive cycles.

Fig. 1.

Phenotypic characterization of Pgr^Δ136E1 null female rats. (A) Temporal assessment of vaginal opening in wild-type (+/+) and Pgr^Δ136E1 null (−/−) female rats (n = 50/genotype). (B and C) Fertility tests and litter sizes from wild-type males mated to wild-type and Pgr^Δ136E1 null female rats and Pgr^Δ136E1 null males mated to wild-type females. (n = 6/mating scheme). (D) Sexual behavior in wild-type and Pgr^Δ136E1 null female rats. The ratio of female lordosis behavior to male mounting was quantified (n = 6/genotype; Movie S1). (E–J) Effects of exogenous gonadotropins on ovulation (E), ovarian weight (F), gene expression (G), and hematoxylin and eosin-stained paraffin-embedded ovarian tissue sections (H–J) in wild-type and Pgr^Δ136E1 null female rats (n = 6/genotype). (J) Trapped oocyte within an unruptured follicle. (K–M) Examination of artificial decidualization in wild-type and Pgr^Δ136E1 null female rats. (K) Schematic presentation of hormone treatments (E2, estradiol; P4, progesterone). (L) Gross responses of uterine tissue to a deciduogenic stimulus. (M) Quantification of uterine horn weights from nonstimulated (Nonstim) and stimulated (Stim) uterine horns (n = 6/genotype). (N and O) Mammary gland development in hormonally treated wild-type and Pgr^Δ136E1 null female rats. (P) Examination of acute uterine responses to progesterone in wild-type and Pgr^Δ136E1 null rats. Progesterone responsive transcripts were monitored by quantitative RT-PCR (qRT-PCR) (n = 6/group; C, vehicle; P, progesterone). Results are presented as mean ± SEM. Asterisks or different letters above bars signify differences between means (P < 0.05).

Fig. S1.

Zinc finger nuclease targeting of exon 1 within the rat Pgr locus. (A) Schematic representation of the rat Pgr gene and the zinc finger nuclease target site within exon 1 (NC_005107.3). (B) DNA sequence analysis showing a 136-bp deletion within the Pgr locus, resulting in a deletion within exon 1 (Δ136E1; Pgr^Δ136E1), leading to a premature stop codon and a null mutation. (C) Offspring were backcrossed to wild-type rats, and heterozygous mutant rats were intercrossed to generate homozygous mutants. Wild-type (+/+), heterozygous (+/−), and null (−/−) mutations were detected by PCR. (D) Western blot analysis of PGRA and PGRB protein species in wild-type and Pgr^Δ136E1 null uterine tissues. Note that PGR proteins were not detected in uterine tissues of Pgr^Δ136E1 null uterine tissues. (E) Heterozygous intercrossing generated a similar ratio of male and female offspring at an expected Mendelian ratio (n = 6 litters). (F and G) Body weights of male (12 wk; n = 6) and female (8 wk; n = 50) rats did not differ significantly between wild-type and Pgr^Δ136E1 null rats.

Fig. S2.

Immunohistochemical analyses of PGR expression in tissues from wild-type (+/+) and Pgr^Δ136E1 null (−/−) rats. (A and B) uterus; (C and D) mammary gland; (E and F) preoptic area/anteroventral periventricular nucleus (POA/AVPV); (G and H) ventromedial hypothalamus (VMH); (I and J) ovary. (Scale bar, 100 μm.)

Fig. S3.

Follicular development and corpus luteum formation in ovaries from adult wild-type (+/+) and Pgr^Δ136E1 null (−/−) rats. Ovaries were collected from 8-wk-old female rats during proestrus of the estrous cycle. Tissues were fixed, paraffin embedded, sectioned, and stained with hematoxylin and eosin. Note the presence of an unovulated oocyte trapped within a corpus luteum (see arrow) from a Pgr^Δ136E1 null rat.

Fig. 2.

Reproductive cyclicity in wild-type and Pgr^Δ136E1 null female rats. (A) Representative estrous cycle profiles of wild-type (+/+) and Pgr^Δ136E1 null (−/−) female rats. Estrous cycles were monitored for 7 wk by daily inspection of vaginal cytology (D, diestrus; P, proestrus; E, estrus). The graphs also indicate when males were introduced. Red points indicate the presence of sperm in the vaginal lavage. (B) Cyclic changes in hormone concentrations in wild-type (Upper) and Pgr^Δ136E1 null (Lower) female rats. Blood was collected from 8–10-wk-old females at 0800 h on the first day of diestrus (D; n = 6/genotype), proestrus (P8; n = 6/genotype), and estrus (E; n = 6/genotype), and also at 2000 h on proestrus (P20; n = 14/genotype). Serum LH, FSH, estradiol, and progesterone were measured. (C) Cyclic changes in uterine weight in wild-type and Pgr^Δ136E1 null rats. Uteri were collected from 8-wk-old female rats, weighed (n = 6/group), and analyzed histologically (Fig. S4). (D and E) Cyclic changes in activity patterns in wild-type and Pgr^Δ136E1 null rats. Representative activity patterns during estrous cycles (D) and quantification of relative activity during each stage of the estrous cycle (E; n = 9/genotype) Results are presented as mean ± SEM. Different letters above bars signify differences between means (P < 0.05).

Fig. S4.

Cyclic changes in uterine morphology and estrous cycle length of wild-type (+/+) and Pgr^Δ136E1 null (−/−) female rats. (A) Uteri were collected from 8-wk-old female rats at diestrus, proestrus, and estrus stages of the estrous cycle. (Left) Gross images of uteri from wild-type and Pgr^Δ136E1 null rats. The tissues were fixed, paraffin embedded, sectioned, and stained with hematoxylin and eosin (H&E). (Middle and Right) Images of the stained tissue sections. (B) Estrous cycle length was determined by assessing vaginal cytology from wild-type and Pgr^Δ136E1 null rats (n = 50 rats/genotype). The asterisk indicates a significant difference between wild-type and Pgr^Δ136E1 null rats (P < 0.05). (C) Progesterone administration (2 mg) at diestrus day 1 extended 4-d to 5-d cycles in wild-type females, but not in Pgr^Δ136E1 null females.

Fig. 3.

Cyclicity in progesterone treated wild-type and Pgr^Δ136E1 null female rats. Progesterone (P4) pellets (400 mg/rat) were implanted s.c. into cyclic wild-type and Pgr^Δ136E1 null female rats. Cyclicity was monitored by daily inspection of vaginal cytology for 14 d. (A) Vaginal cytology profiles for three representative wild-type and three representative Pgr^Δ136E1 null female rats are presented (D, diestrus; P, proestrus; E, estrus). (B and C) Quantification of the number of days in each stage of the estrous cycle and the number of cycles during the 14-d test period is presented (n = 6/genotype). Results are presented as the mean ± SEM. Asterisks indicate significant differences between wild-type and Pgr^Δ136E1 null female rats (P < 0.05).

Fig. 4.

Phenotypic characterization of Pgr^ΔE3 null female rats. (A) Examination of acute uterine responses to progesterone in wild-type (+/+) and Pgr^ΔE3 null (−/−) rats. Progesterone-responsive transcripts were monitored by qRT-PCR (n = 6/group). Results are presented as the mean ± SEM. Different letters above bars signify differences between means (P < 0.05). (B) Representative estrous cycle profiles of Pgr^ΔE3 null females. Estrous cycles were monitored for 7 wk by daily inspection of vaginal cytology (D, diestrus; P, proestrus; E, estrus). The graphs also indicate when males were introduced. Red points indicate the presence of sperm in the vaginal lavage.

Fig. S5.

CRISPR/Cas9 targeting of exon 3 within the rat Pgr locus. (A) Schematic representation of the rat Pgr gene and the guide-RNA target site within exon 3 (NC_005107.3). (B) DNA sequence analysis showing a 984-bp deletion within the Pgr locus, resulting in deletion of exon 3 (ΔE3) and leading to a premature stop codon and a null mutation. (C) Offspring were backcrossed to wild-type rats, and heterozygous mutant rats were intercrossed to generate homozygous mutants. Wild-type (+/+), heterozygous (+/−), and null (−/−) mutations were detected by PCR. (D) Western blot analysis of PGRA and PGRB protein species in wild-type and Pgr^ΔE3 null uterine tissues. Note that PGR proteins were not detected in uterine tissues of Pgr^ΔE3 null uterine tissues.

Fig. 5.

The effects of mifepristone on the LH surge. Wild-type (+/+) and Pgr^Δ136E1 null (−/−) female rats were monitored for cyclicity by daily inspection of vaginal cytology. At 1330 h on proestrus, animals were treated with vehicle (sesame oil) or mifepristone (6 mg/kg). Animals were killed at 2000 h on proestrus and blood collected for measurement of serum LH concentrations. Sample sizes: wild type-vehicle, n = 12; wild type-mifepristone, n = 14; Pgr^Δ136E1 null-vehicle, n = 8; Pgr^Δ136E1 null-mifepristone, n = 8. Results are presented as the mean ± SEM. The asterisk indicates a significant difference between the vehicle and mifepristone treatments (P < 0.05).