To generate pituitary Kiss1 receptor (Kiss1R) knockout (PKiRKO) mice, we crossed Kiss1r heterozygous (fl/wt) female mice (Novaira et al., 2014) with αGSU transgenic (αCre+/-) male (Naik et al., 2006; Brothers et al., 2010) mice. The αGSU transgenic (αCre+/-) effectively deletes floxed genes specifically in pituitary gonadotrophs and thyrotropes (Brothers et al., 2010). F1: female mice (Kiss1R fl/wt; αCre+) and male mice (Kiss1R fl/fl; αCre-) were crossed to produce PKiRKO mice (Kiss1R fl/fl; αCre+). Litter mates (Kiss1r fl/wt; αCre- and Kiss1r fl/fl; αCre-) were used as controls (referred as WT). DNA was extracted as described previously (Wolfe et al., 2008). Genotyping primers were designed to detect the presence of αCre and the floxed allele, WT allele, or knockout allele of Kiss1r: P1 sense (located in exon 1) and P3 antisense (located in exon 3). PCR of tail genomic DNA using primers P1 and P3 will amplify a 2096-bp amplicon to indicate the floxed Kiss1r allele and 1882-bp amplicon to indicate the WT allele. Genomic DNA obtained from the pituitary using primers P1 and P3 will amplify a 1120-bp amplicon if the sequence between the LoxP sites is excised indicating a KO allele. Primer set for αCre is αCre F: GCC​ACC​ACC​GCC​CTG​CTT​AAG​TAA; R: GCC​ACC​ACC​GCC​CTG​CTT​AAG​TAA); for Kiss1r is P1: CTG​GTC​GGA​AAC​TCA​TTG​GT; P3: AGA​GTG​GCA​CAT​GTG​GCT​TG.

Adult male and female mice (>2 months old) were used in this study. All animal studies were carried out in accordance with National Institutes of Health guidelines on animal care regulations and were approved by the Animal Care and Use Committee of the Johns Hopkins University. Mice were maintained under constant conditions of light and temperature (14: 10 h light⁄dark cycle; 22 C) and were fed a normal chow and water ad libitum.

RNA was extracted from the pituitary, liver, ovaries, uterus, testes, epididymis, adipose tissue and from two hypothalamic fragments encompassing the arcuate and the anteroventral periventricular nucleus (AVPV) (Quennell et al., 2011), using TRIzol reagent (Ambion life technologies, Carlsbad CA, United States ), according to the protocol provided by the supplier. 1µg of RNA was reverse transcribed to cDNA using an iScript cDNA kit (Bio-Rad Laboratories). Real-time qPCR was performed to determine the presence and relative expression levels of Kiss1r mRNA in the various tissues. Real-time qPCR was performed in duplicate using SYBR Green Master Mix (Bio-Rad Laboratories) and the CFX Connect qPCR machine (Bio-Rad Laboratories). Kiss1r primer sequences are Sense: CTG​CCA​CAG​ACG​TCA​CTT​TC; antisense, 5-ACA​TAC​CAG​CGG​TCC​ACA​CT. For each primer, PCR efficiency was determined by measuring a 10-fold serial dilutions of cDNA and reactions having 95 and 105% PCR efficiency were included in subsequent analyses. Relative differences in cDNA concentration between WT and PKiRKO mice were then calculated using the comparative threshold cycle (C_t) method. To compare the difference of Kiss1r expression in the same tissue between WT and PKiRKO, a △C_t was calculated to normalize for internal control using the equation: C_t (Kiss1R)—C_t (18S). △△C_t was calculated: △C_t (PKiRKO) -△C_t (WT). Relative Kiss1R mRNA levels were then calculated using the equation fold difference = 2^△△C_t.

Preputial separation (PPS) in males was assessed daily beginning after postnatal day 21 by determining whether the prepuce could be manually retracted with gentle pressure. PPS is testosterone dependent and thus is an indicator of activation of the reproductive axis in males. Puberty in rodents is dependent on body weight (Baker, 1985) hence, Body weight (BW) of PKiRKO and wild type were assessed once a week in prepuberty (day 21) through adulthood (day 49). Anogenital distance is testosterone dependent and was determined at 8 weeks of age. Wet testicular weights were determined in freshly dissected mice at 2 months of age. In females, age at vaginal opening and first estrus are two markers of puberty onset and were assessed for daily in mice after 21 days of life until achieved. Vaginal smears were obtained daily over a period of 14 consecutive days in 6- to 11-week-old mice and cellular morphology examined under microscope. BW was also recorded at the age of PPS and first estrus.

Blood samples were collected from submandibular vein (Wu et al., 2014; Wang et al., 2019) between 9:00 and 10:00 a.m. and basal levels of serum LH and FSH were measured. LH and FSH were measured using a Milliplex MAP immunoassay (Mouse Pituitary panel; Millipore, Massachusetts) on a Luminex 200IS platform (Luminex Corporation). The assay detection limit for LH was 0.012 ng/ml and for FSH was 0.061 ng/ml. The intra-assay and interassay coefficients of variation (CV) for LH and FSH were between 5 and 9%. Testosterone levels were measured by radioimmunoassay by the University of Virginia Ligand Assay Core (Charlottesville, Virginia) and the intra-assay CV was 5% and inter-assay CV was 9%.

To determine whether male PKiRKO mice were fertile relative to controls, fertility was examined in WT and PKiRKO mice using a rotating mating protocol. 3 WT and 4 PKiRKO male mice were housed individually with proven fertile WT female mice for 14 consecutive days and then were separated. If a pregnancy ensued, 1 week after female gave birth, a new male was inserted. Males were rotated among the proven females. To assess fertility in females, a similar strategy was used. Females were housed individually with proven fertile WT male mice for 14 consecutive days and then separated. If a pregnancy ensued, 1 week after female gave birth, a new male was inserted, rotating among the seven proven males. The duration of the fertility study was 4 months with four rotations for each mouse.

Data were analyzed by two tailed unpaired student t tests (parametric) with normality of residuals test Shapiro-Wilk (W)) using Prism software (GraphPad Software, Inc., La Jolla, CA). All results are expressed as mean ± SEM (standard error of the mean). p < 0.05 was defined as statistically significant.

PKiRKO (Kiss1R^fl/fl; αCre⁺) mice were generated by Cre recombinase mediated excision of exon 2 of the Kiss1r, resulting in an attenuated Kiss1r gene in pituitary, as shown in the schematic diagram of Figure 1A. The PCR product of genotyping indicates the homozygous floxed-Kiss1r alleles (2096 bp) and WT alleles (1882 bp) (Figure 1B); both bands are present in the heterozygous floxed-Kiss1r mouse schematized by Novaira HJ et al. (Novaira et al., 2014). Shown in Figure 1C Lane 1 is a WT αCre-male mice, followed by two αCre + male mice in lanes 2 and 3 showed by PCR of genotyping.

qPCR demonstrated a reduction in Kiss1r mRNA by 88% (Figure 1D) and 64% (Figure 1D) in the pituitary of male and female PKiRKO mice (n = 8), respectively, compared with WT mice (n = 8). In contrast, no difference in Kiss1r mRNA was observed between PKiRKO and control animals in other tissues including the hypothalamus, adipose tissue, liver, or gonads (p > 0.05, n = 4) (Figure 1D). There was more Kiss1r mRNA detected in the pituitaries of female PKiRKO mice.

Body weight (BW) was not different between male or female PKiRKO and WT mice (Figure 2A). In males, PPS (preputial separation) is an androgen dependent process that serves as an external sign of male puberty onset (Marty et al., 2001). No significant difference was observed age of PPS between the WT and PKiRKO groups ((28.1 ± 0.6 vs. 27.8 ± 0.6 days of life), Figure 2B), or achieved anogenital distance (Figure 2C). The age of vaginal opening and first estrus (Figure 2D), and anogenital distance (Figure 2E) was not different between female WT and PKiRKO groups. The BW at age of pubertal achievement (PPS at day 28 in males (BW: 14 ± 1 vs. 15 ± 1 g) and first estrous at day 35 in females (BW: 18 ± 1 vs. 16 ± 1 g) was not significantly different between WT and PKiRKO animals. In sexually mature mice, gonad weight is a marker of reproductive tissue function. Gonad weights were comparable between PKiRKO and WT male and female mice (Figure 3 A and B). Estrous cycling pattern was similar between PKiRKO and WT mice (Figure 3C); percent time spent at each cycle stage was not significantly different. Testes (Figure 3D) or ovary (Figure 3E) histology was not different between PKiRKO and WT mice. The morphology of seminiferous tubules were comparable between WT and PKiRKO male mice (Figure 3D). The number of sperm in the seminiferous tubules was not statistically different between WT and PKiRKO mice (data not shown).

Basal serum LH levels in PKiRKO males (1.8 ± 0.4 vs. 1.1 ± 0.2 ng/ml) or females (0.5 ± 0.1 vs. 0.6 ± 0.1 ng/ml) were not significantly different relative to WT mice, while FSH levels (10.2 ± 1.8 vs. 6.0 ± 1.9 ng/ml) were significantly lower in PKiRKO males (Figure 3F). Testosterone levels were not significantly different between WT and PKiRKO mice (Figure 3G).

PKiRKO male and female mice demonstrated a normal ability to produce offspring, as shown in Figure 4 A-C. Female WT mice bore their first litter with a similar latency after introduction to PKiRKO males or WT males ((22.0 ± 0.8days, n = 4 (PKiRKO) vs. 22.3 ± 0.6 days, n = 3(WT); p > 0.05)) (Figure 4A). Female WT mice had the same number of litters with PKiRKO and WT sires ((3.8 ± 0.5, n = 4 (PKiRKO) vs. 3.7 ± 0.6, n = 3 (WT); p > 0.05)) (Figure 4B) and a similar number of pups per litter ((7.4 ± 1.4, n = 4 (PKiRKO) vs. 9.3 ± 1.1, n = 3 (WT); p > 0.05)) (Figure 4C). Similar results were found between PKiRKO females and WT females. Female WT and PKiRKO mice bore their first litter with a similar latency after introduction to WT males ((22.4 ± 0.1 day, n = 6 (PKiRKO) vs. 22.6 ± 0.1 day, n = 7(WT); p > 0.05)) (Figure 4A, right panel). Female WT and PKiRKO mice had the same number of litters with WT males ((3.3 ± 0.2, n = 6 (PKiRKO) vs. 3.7 ± 0.2, n = 6 (WT); p > 0.05)) (Figure 4B, right panel) and a similar number of pups per litter ((8.3 ± 0.6, n = 6 (PKiRKO) vs. 9.7 ± 1.0, n = 6 (WT); p > 0.05)) (Figure 4C, right panel).