Abstract
Mammals share common strategies for regulating reproduction, including a conserved hypothalamic–pituitary–gonadal axis; yet, individual species exhibit differences in reproductive performance. In this report, we describe the discovery of a species-restricted homeostatic control system programming testis growth and function. Prl3c1 is a member of the prolactin gene family and its protein product (PLP-J) was discovered as a uterine cytokine contributing to the establishment of pregnancy. We utilized mouse mutagenesis of Prl3c1 and revealed its involvement in the regulation of the male reproductive axis. The Prl3c1-null male reproductive phenotype was characterized by testiculomegaly and hyperandrogenism. The larger testes in the Prl3c1-null mice were associated with an expansion of the Leydig cell compartment. Prl3c1 locus is a template for two transcripts (Prl3c1-v1 and Prl3c1-v2) expressed in a tissue-specific pattern. Prl3c1-v1 is expressed in uterine decidua, while Prl3c1-v2 is expressed in Leydig cells of the testis. 5′RACE, chromatin immunoprecipitation and DNA methylation analyses were used to define cell-specific promoter usage and alternative transcript expression. We examined the Prl3c1 locus in five murid rodents and showed that the testicular transcript and encoded protein are the result of a recent retrotransposition event at the Mus musculus Prl3c1 locus. Prl3c1-v1 encodes PLP-J V1 and Prl3c1-v2 encodes PLP-J V2. Each protein exhibits distinct intracellular targeting and actions. PLP-J V2 possesses Leydig cell-static actions consistent with the Prl3c1-null testicular phenotype. Analysis of the biology of the Prl3c1 gene has provided insight into a previously unappreciated homeostatic setpoint control system programming testicular growth and function.
Introduction
The process of reproduction has elements of regulation that are well conserved among diverse mammalian species (Achermann & Jameson 1999, Matzuk & Lamb 2008). This is best illustrated by the hypothalamic–pituitary–gonadal (HPG) axis and the hormones used as messengers to control fertility (Achermann & Jameson 1999, Matzuk & Lamb 2008). Interestingly, reproductive function is also characterized by features that are seemingly unique to a species. These unique features represent a broad spectrum of biological processes associated with reproduction, including the regulation of gonadal size and function, gamete characteristics, the onset of puberty, sexual behavior, pregnancy, fecundity as well as many others (Bronson 1989). Physiological impacts of these species-specific traits can be robust and have profound effects on reproductive fitness. Thus, on a backbone of a highly conserved reproductive axis, there are prominent differences in reproductive performance among species.
The testis is regulated by a conserved homeostatic pathway involving the hypothalamus and pituitary (Desjardins 1978, Hedger & de Kretser 2000), yet, exhibits striking species-specific characteristics (Fawcett et al. 1973, Kenagy & Trombulak 1986, Short 1997). Within the conserved regulatory pathway, the hypothalamus and pituitary produce gonadotropin-releasing hormone (GnRH) and gonadotropins (luteinizing hormone, LH; follicle-stimulating hormone, FSH), respectively. GnRH regulates gonadotropin biosynthesis and secretion while LH and FSH act to regulate the growth and function of the testis (Hedger & de Kretser 2000, Matzuk & Lamb 2008). LH drives Leydig cell production of testosterone (Hedger & de Kretser 2000), which acts on an array of downstream targets to promote function of the entire male reproductive axis, including stimulating spermatogenesis, sperm maturation and transport and male sexual and social behaviors (Desjardins 1978). FSH acts together with testosterone to promote sperm production (McLachlan et al. 1996, Matzuk & Lamb 2008). Homeostasis is achieved through a negative feedback system of testicular hormone action at the hypothalamus and pituitary to modulate the output of GnRH and gonadotropins, respectively (Amory & Bremner 2001, Tilbrook & Clarke 2001). These conserved elements of the male reproductive regulatory pathway have been effectively modeled in the mouse (Matzuk & Lamb 2008), but do not account for the species diversity in testicular size and function in mammals (Kenagy & Trombulak 1986, Short 1997, Montoto et al. 2011, 2012). In some mammals, the size of the testis can represent as much as 8 percent of body mass, whereas in other mammals, the testis contributes to less than 0.1 percent of body mass (Kenagy & Trombulak 1986; e.g. Gerbilliscus afra, 8.4%; Mesocricetus auratus, 2.9%; Ovis aries, 0.9%; Mus musculus, 0.8%; Macaca mulatta, 0.7%; Bos taurus, 0.1%; Homo sapiens, 0.08%). Testis size correlates with contributions of Leydig cell and spermatogenic compartments and with their capacity to produce testosterone and sperm, respectively (Short 1997, Birkhead 2000, Simmons & Fitzpatrick 2012). Considerable species differences exist in sperm production. It has been proposed that sperm production relates to the mating system used by a species (monogamy vs polygyny vs polyandry), where sperm production and competition directly influence reproductive success (Short 1997, Birkhead 2000, Simmons & Fitzpatrick 2012). These extraordinary species differences in testis growth and function necessitate species-specific mechanisms modulating the function of the male reproductive axis. The origins of these species-specific reproductive traits are not yet fully appreciated.
In this report, we present the discovery of a rapidly evolving homeostatic control system contributing to the regulation of testis size and function in the mouse and provide insights into a mechanism employed to achieve species-specific reproductive traits.
Materials and methods
Animals and tissue collection
C57BL/6 mice and testes from M. spretus, M. caroli and M. pahari were purchased from The Jackson Laboratory. Testicular RNA samples from C57BL/6, 129S1/Svlmj, AKR/J, BALB/cByJ, BTBRT + tf/J, C3H/HeJ, CAST/EiJ, DBA/2J, FVB/NJ, KK/HIJ, NOD/LtJ, NZW/LacJ, PWD/PhJ, WSB/EiJ and SJL/J mouse strains were obtained from Dr Curtis Klaassen (University of Kansas Medical Center, Kansas City, KS, USA). CD-1 mice and Brown Norway (BN) rats were purchased from Charles River Laboratories International. Fischer 344 (F344) and Holtzman Sprague Dawley (HSD) rats were obtained from Harlan Laboratories (Indianapolis, IN, USA). Animals were housed in an environmentally controlled facility with lights on from 06:00 to 20:00 h and were allowed free access to food and water. Timed pregnancies were generated by cohabitation of male and female mice. The presence of a seminal plug in the vagina was designated day 0.5 of gestation. Tissues for histological analysis were frozen in dry ice-cooled heptane and stored at −80°C until processing. Tissue samples for RNA or protein extraction were snap frozen in liquid nitrogen and stored at −80°C until processing. Male mice were individually housed for two weeks prior to the collection of blood samples from the orbital sinus under isoflurane anesthesia.
Prl3c1-mutant mouse embryonic stem (ES) cells were produced by the NIH Knock-Out Mouse Project (KOMP) and obtained from the KOMP Repository (www.komp.org; VG10539). The University of Kansas Medical Center Transgenic and Gene-targeting Facility (Kansas City, KS, USA) injected the mutant ES cells into C57BL/6 blastocysts. Germline-competent chimeras were generated on C57BL/6 background, and the mutation was subsequently moved to a mixed background of C57BL/6 X CD-1. Genotyping was routinely performed on genomic DNA isolated from tail biopsies using PCR with forward primers specific for the wild type allele (5′ CAGGGAATTCTTTGATTTGC 3′) and mutant allele (5′ GCAATTGGACCGTTTGATCT 3′) and a common reverse primer (5′ ATCTGAGTTGCTGGCTTGGT 3′). Amplicons generated by the PCR reactions for the wild type and mutant alleles were 798 and 471 bp, respectively. All null mutant animals were generated on the mixed background using heterozygous × heterozygous breeding and wild type and null littermates were used in the experiments.
All experimentation with animals was performed in accordance with guidelines recommended by the National Institutes of Health (Bethesda, MD, USA). The University of Kansas Animal Care and Use Committee approved protocols for the care and use of animals.
Fertility tests
Beginning at 2–2.5 months of age, male and female mice were cohabited. Mutant and wild type males were housed with multiple wild type females; whereas, mutant and wild type females were paired with fertile wild type males. The presence of seminal plugs in the vagina was examined daily. Seminal plug-positive females were killed on day 9.5 of pregnancy and the number of viable conceptuses was counted.
Serum hormone measurements
Serum testosterone and LH levels were measured by radioimmunoassay at the University of Virginia Center for Research in Reproduction Ligand Assay and Analysis Core (Charlottesville, VA, USA). Details of the assay procedures can be found at http://www.medicine.virginia.edu/research/institutes-andprograms/crr/ligand-page. Serum FSH levels were measured using an FSH Milliplex MAP kit (Millipore).
Sperm quantification and assessment of sperm motility
Sperm preparations and quantifications were based on previously described procedures (Jimenez et al. 2010). The caudal portion of the epididymis along with vas deferens of adult mice were isolated and cut at various points with a razor blade to release sperm into pre-warmed modified Whitten’s Medium (100 mM NaCl, 4.7 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgSO4, 5.5 mM glucose, 0.8 mM Na pyruvate, 4.8 mM lactic acid, 20 mM HEPES and 1.7 mM CaCl2, pH 7.3). Following a 10-min incubation at 37°C, sperm in the supernatant were counted with a hemocytometer.
Assessment of sperm motility. Wild type and Prl3c1-null sperm were analyzed by CASA, using the Minitube Sperm Vision Digital Semen Evaluation system (version 3.5; Penetrating Innovations, Verona, WI, USA), as previously described (Jimenez et al. 2010, 2011).
Seminiferous tubule staging and testicular compartment enrichment
Seminiferous tubule staging was determined according to criteria presented by Hess (Hess 1990). Testicular interstitium and seminiferous tubule enrichments were prepared as previously described (Dufau & Catt 1975). Briefly, decapsulated testes were transferred to DMEM/F12 Medium (Sigma-Aldrich) supplemented with 2.5% fetal bovine serum (FBS, Sigma-Aldrich), 5% horse serum (Sigma-Aldrich), 15 mM HEPES and 0.25 mg/mL collagenase (type IV; Sigma-Aldrich, C-5138) and incubated at 37°C for 20 min with continuous rotation. After settling for 5 min at room temperature, supernatants were transferred to a new tube and left on ice. Pellets were resuspended in the same medium and incubated as described above for another 10 min. Supernatants were combined and centrifuged at 600 g for 8 min to collect Leydig cells. The enriched Leydig cell preparations were used as a source of RNA and genomic DNA.
Immunohistochemical and immunofluorescence staining
All histochemical staining was performed on 10 μm frozen sections. After fixation with Bouin’s fixative at 4°C, testis sections were incubated overnight at 4°C with antibodies against cytochrome P450 side-chain cleavage enzyme (CYP11A1; 1:300) (Roby et al. 1991), MKI67 (Santa Cruz Biotechnology; sc-15402, 1:100) or PLP-J (1:50) (Alam et al. 2008). Quantification of CYP11A1 immunostaining was obtained using the Automated Cellular Imaging System (San Juan Capistrano, CA, USA). Confocal images were obtained with a Nikon C1 plus confocal system equipped with 543-nm HeNe, 488-nm Ar and 405-nm diode lasers integrated into an Eclipse TE2000-U microscope with 20×/0.75 Plan Apochromat and 60×/1.40/0.21 oil Plan Apochromat objectives (Nikon Instruments). Image acquisition was performed using Nikon EZ C1 software.
RNA analysis
RNA was extracted from tissues or cells using TRI Reagent (Sigma-Aldrich) according to the manufacturer’s instructions. Reverse transcription and quantitative RT-PCR (qRT-PCR) were performed as previously described (Kent et al. 2010, Konno et al. 2010). Primer sequences used for RT-PCR and qRT-PCR are provided in Supplementary Table 1 (see section on supplementary data given at the end of this article).
Rapid amplification of 5′ cDNA ends (5′ RACE)
Transcription start sites for Prl3c1-v1 and Prl3c1-v2 were determined with RNA isolated from anti-mesometrial uterine decidual tissues (gestation day 7.5) and enriched Leydig cell preparations using a 5′ RACE kit (Roche, 03353621001), according to the manufacturer’s instructions. Primer sequences used in the 5′RACE: SP1-Rev568: 5′ CCACCTGTCAGGCTCGTTAT 3′; SP2-Rev518-495: 5′ GTAAAGCTTTTGCTCCCTCCAGAA 3′; SP3-Rev227-196: 5′ TGTGACGAGAAGAGGAAAGCAGATTGTCATAG 3′.
ChIP analysis
Decidual cell and enriched Leydig cell preparations were used as starting material for the ChIP analyses. Anti-mesometrial uterine decidual tissue was dissected from gestation day 7.5 implantation sites. Implantation sites were pooled from pregnant females. Immediately following dissection, the tissue was incubated in pre-warmed PBS supplemented with collagenase (type IV, 0.25 mg/mL, Sigma-Aldrich), trypsin (0.25 mg/mL, Invitrogen) and Dispase (2 units/mL, Worthington Biochemical, Lakewood, NJ, USA) with continuous rotation at 37°C for 30 min. Decidual cells were collected from the dispersed cell suspensions by centrifugation at 600 g. Pellets were then washed with cold PBS and used for crosslinking and lysis. ChIP analysis was performed using an antibody against RNA polymerase II (EMD Millipore; 05-623B) and the EZ-ChIP kit (EMD Millipore, 17-371) according to the manufacturer’s instructions. Sonication of decidual genomic DNA was achieved using Bioruptor 300 (Diagenode, Belgium) yielding DNA fragments ranging in length from 300 to 600 bp (Bioruptor 300 settings: power, high; sonication, 20 s on and 50 s off, 22 cycles). For the Leydig cell ChIP, freshly isolated enriched Leydig cells from testes were pooled. Cells were washed with cold PBS and the ChIP was performed using EZ-ChIP kit. Sonication of the enriched Leydig cell genomic DNA was achieved using Bioruptor 300 yielding DNA fragments ranging in length from 300 to 600 bp (Bioruptor 300 settings: power, high; sonication, 20 s on and 50 s off, 15 cycles). Primers used for the ChIP PCR analysis are provided in Supplementary Table 1.
DNA methylation analysis
DNA methylation was determined using bisulfite sequencing, direct sequencing and MultiNA-Combined Bisulfite Restriction Analysis (COBRA).
Bisulfite sequencing
Bisulfite PCR products were purified with the Wizard SV Gel and PCR Clean-up System (Promega). For direct sequencing, purified PCR products were used for sequencing reactions with primers used for bisulfite PCR. PCR products were cloned into pGEM-T Easy Vector (Promega). Up to 16 plasmid clones were amplified with the Templiphi DNA Amplification Kit (GE Healthcare) and sequenced. The data were processed using the Quantification Tool for Methylation Analysis (QUMA) website (http://quma.cdb.riken.jp/).
COBRA with MultiNA microchip
Genomic DNA (2–8 µg) was digested with EcoRV at 37°C overnight. The DNA was extracted with phenol/chloroform/isoamyl alcohol (PCI; 25:24:1), precipitated with ethanol and dissolved in TE. Five hundred ng of the EcoRV-digested DNA were bisulfite-converted using EZ DNA Methylation-Direct Kit (Zymo Research, Irvine, CA, USA) and eluted in M-Elution Buffer. One-fortieth of the DNA was used for PCR in 20 µL of a reaction containing 1× buffer, 1.5 mM MgCl2, 0.2 mM dNTP and 0.25 µM each of primers with BIOTAQ HS DNA Polymerase (Bioline, Taunton, MA, USA) under the following conditions: denaturation at 95°C for 7 min, and 43 cycles of 1 min at 94°C, 30 s at 58–61°C and 1 min at 72°C, followed by 10 min at 72°C. Primer sequences are listed in Supplementary Table 2. One-fifth of the PCR product was digested with HpyCH4IV or TaqI in 20 µL at 37°C overnight and analyzed with MultiNA Microchip Electrophoresis System (Shimadzu, Kyoto, Japan). The methylation level was calculated as previously described (Sato et al. 2010).
Cell culture
MA-10 mouse Leydig cells (a gift from Dr Mario Ascoli, University of Iowa, Iowa City, IA, USA) were cultured in Waymouth’s MB752/1 Medium (Life Technologies) supplemented with 15% horse serum (Sigma-Aldrich) and 20 mM HEPES. TM3 mouse Leydig cells (ATCC; CRL-1714) were cultured in DMEM/F12 Medium (Sigma-Aldrich) supplemented with 2.5% fetal bovine serum (FBS, Sigma-Aldrich), 5% horse serum and 15 mM HEPES. MLTC-1 mouse Leydig cells (ATCC, CRL-2065) were cultured in RPMI-1640 Medium (Life Technologies) supplemented with 10% FBS. COS-7 African green monkey kidney fibroblast-like cells (ATCC, CRL-1651) were cultured in DMEM Medium (Life Technologies) supplemented with 10% FBS. Cell number was assessed using MultiTox-Fluor Multiplex Cytotoxicity Assay (Promega, G9200), according to the manufacturer’s instructions.
Plasmids, transfection, subcellular fractionation and Western blotting
A Prl3c1-v1 cDNA was obtained from Open Biosystems (Thermo Scientific) and used as a template for PCR amplification of the coding sequence for Prl3c1-v2. Prl3c1-v1 and Prl3c1-v2 coding sequences were cloned into pCMV6-Entry (OriGene, Rockville, MD, USA) using XhoI and EcoRV restriction sites generating constructs with inframe C-terminal FLAG tags. The accuracy of both expression constructs was confirmed by DNA sequencing. Cells were transfected with an empty pCMV6-Entry vector or with vectors containing tagged Prl3c1-v1 or Prl3c1-v2 using Lipofectamine 2000 (Life Technologies). Cells were harvested for subcellular fractionation 48 h after transfection using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific, 78833) according to the manufacturer’s instructions. Transfection efficiencies for MLTC-1, MA-10, TM3 and COS7 cells were 72.2 ± 7.1, 62.0 ± 6.2, 70.0 ± 6.4 and 88.0 ± 4.2%, respectively. Western blotting procedures were performed as previously described (Deb et al. 1993). PLP-J V1-FLAG and PLP-J V2-FLAG were detected with antibody to the FLAG tag (Sigma-Aldrich, F3165, 1:2500). Antibodies against Histone H3 (Abcam; ab94817, 1:10,000) and GAPDH (EMD Millipore; MAB374, 1:7,500) were used as controls to monitor the integrity of the subcellular fractionation.
Statistical analysis
Differences between two groups were assessed by Student’s t test. Data are presented as the mean ± s.e.m. P values less than 0.05 are considered statistically significant.
Results
PRL family gene expansion and Prl3c1 gene disruption
Gene duplication represents a potential mechanism for acquisition of species-specific physiological traits (Ohno 1970, Kaessmann 2010). Several genes associated with reproduction have undergone expansions (Rawn & Cross 2008). Prolactin (PRL), a well-conserved hormone/cytokine best known for its actions on the reproductive tract and mammary gland (Freeman et al. 2000, Bachelot & Binart 2007), has undergone species-specific gene duplication (Soares 2004, Soares et al. 2007). In some species, the ancestral PRL served as a template for the birth of new genes (mouse, rat and cow), while in other species, exemplified by the human, cat and dog, only the PRL ortholog is present in the genome (Soares 2004, Soares et al. 2007). Mutagenesis experiments suggested that a driving force in the expansion of the mouse PRL family was associated with pregnancy-dependent adaptations to physiological stressors (Ain et al. 2004, Soares 2004, Alam et al. 2007, Soares et al. 2007). Mouse Prl contributes to the regulation of pregnancy and mammary gland development (Horseman et al. 1997), whereas Prl4a1 and Prl8a2 possess roles in adaptations to environmental challenges during pregnancy (Ain et al. 2004, Alam et al. 2007). Prl3c1 encodes a protein called PRL-like protein-J (PLP-J, also referred to as PRL3C1) and was originally identified in uterine decidua of the mouse and rat (Hiraoka et al. 1999, Ishibashi et al. 1999, Toft & Linzer 1999, Dai et al. 2000). PLP-J was shown to act as a heparin-binding cytokine contributing to growth regulation of cells within the uterus (Alam et al. 2008). These observations prompted the generation of mice possessing a null mutation at the Prl3c1 locus, with the expectation of a pregnancy-related phenotype. The entire coding sequence of Prl3c1 was replaced with an inframe β-galactosidase (LacZ) cassette (inserted at the ATG codon in the first exon) followed by a neomycin resistance cassette (Fig. 1A and B). The mutation was successfully transmitted through the germline.
Prl3c1-mutant mice exhibit an abnormal male reproductive tract phenotype. (A) Schematic of the mouse Prl3c1 locus and the targeting construct. (B) PCR analysis of genomic Prl3c1 locus from wild type (+/+), heterozygous (+/−) and null (−/−) animals. (C) Gross appearance of the scrotal area on postnatal day 21 of wild type (+/+) and Prl3c1-null (−/−) mice. (D) Gross appearance of adult testis of wild type and Prl3c1-null mice. (E) Testis-to-body weight ratio of wild type (2–2.5 months, n = 14; 4–5 months, n = 8) and Prl3c1-null (2–2.5 months, n = 20; 4–5 months, n = 15) mice. (F) CYP11A1 immunofluorescence staining of testis cross-sections from wild type and Prl3c1-null mice. (G) Quantification of CYP11A1 positive staining area/total cross-section area of wild type and Prl3c1-null testes, n = 5 testes per group. (H, I, J, K, L and M) MKI67 immunofluorescence staining of testis cross-sections from wild type and Prl3c1-null mice. Scale bar: 50 μm. *P < 0.05; **P < 0.001.
Citation: Journal of Endocrinology 234, 2; 10.1530/JOE-17-0250
Assessment of fertility
Prl3c1-null and wild type males exhibited similar fertility. All wild type males successfully impregnated wild type females (n = 13 males generated n = 28 seminal plug-positive females, leading to 28 pregnancies; 100% success rate). Prl3c1-null males also successfully impregnated wild type females (n = 12 males generated n = 24 seminal plug-positive females, leading to 19 pregnancies; 79% success rate). All wild type females (n = 13) effectively mated during cohabitation with wild type males, which led to 12 pregnancies (n = 12; litter size: 10.5 ± 0.4). The Prl3c1-null females were subfertile. Only 58% (7 of 12) mated during cohabitation with wild type males and only 57% of the seminal plug-positive females were pregnant on gestation day 9.5 (n = 4; litter size: 11.0 ± 3.9). In this report, we focus our analysis on the reproductive tract phenotype of Prl3c1-null male mice.
Prl3c1-null mice have an altered male reproductive tract phenotype
Mice possessing a null mutation at the Prl3c1 locus exhibited an unexpected male reproductive tract phenotype. At the time of weaning, Prl3c1-null male mice exhibited precocial development of the scrotal region (Fig. 1C). The scrotal anomaly was associated with large testes (Fig. 1D, E and Supplementary Table 3). Since body weights were significantly greater in the Prl3c1 mutant males, we have included both relative and absolute testis measurements (Fig. 1E and Supplementary Table 3). Within the testis, interstitial compartments from Prl3c1-deficient mice were significantly expanded and contained more MKI67-positive cells when compared to testes of wild type mice (Fig. 1F, G, H, I, J, K, L and M). Postnatal testis development is associated with an initial expansion and then a decline in Leydig cell progenitors (proliferative population) and a subsequent increase in adult Leydig cells (post-mitotic population) (Benton et al. 1995, Ge et al. 2005, Chen et al. 2010, Stanley et al. 2010). Most interestingly, several Leydig cell progenitor-associated transcripts remained elevated in the adult Prl3c1-null testis (Fig. 2A). Conversely, an indicator of adult Leydig cells, Hsd3b6, failed to exhibit the expected developmental increase in the adult Prl3c1 null testis and instead, Hsd3b1, another 3β hydroxysteroid dehydrogenase isoform with similar substrate specificity was elevated in the adult Prl3c1-null testis (Fig. 2B). Two transcripts encoding 5α reductase were decreased in Prl3c1-null testes (Fig. 2B). Other testicular transcripts encoding proteins participating in steroidogenesis were more modestly affected by Prl3c1 disruption (Fig. 2C). Indices of testicular function were also examined. Serum testosterone levels were elevated, androgen-responsive tissues were hyper-stimulated and sperm production was significantly greater in Prl3c1 null vs wild type male mice (Fig. 3 and Supplementary Table 3). Diameters of seminiferous tubules and the ratio of stage VIII seminiferous tubules to the total numbers of seminiferous tubules per cross-sectional area of the testis were similar in Prl3c1-null and wild type testis; however, the Prl3c1-null testes had significantly more seminiferous tubules per cross-section than did the wild type testis (wild type: 228.2 ± 7.2 vs Prl3c1 null: 347.7 ± 5.8, P < 0.001). Wild type and Prl3c1-null sperm exhibited similar patterns of motility. Seminal vesicle glands continued to grow as the Prl3c1-null mice aged and showed pathologic changes, including exhibiting regions of necrosis and hemorrhage (Fig. 3C and D). Overall, the evidence supported a hyperfunctionality of the Prl3c1-null testis driven by an alteration of the developmental maturation of Leydig cells. Prl3c1-mutant Leydig cells retain their ability to proliferate and functionally generate a hyperandrogenic condition.
Assessment of Leydig cell developmental states in wild type and Prl3c1-null mice. (A) qRT-PCR analysis of transcripts associated with the progenitor Leydig cell state in wild type postnatal day 15 (PND 15), adult (2.5–4 months) wild type and Prl3c1-null testes. PND15 represents a transition point from progenitor to adult Leydig cells. qRT-PCR analysis of transcripts for Prl3c1 (B) and transcripts encoding proteins involved in steroid hormone biosynthesis (B, Hsd3b, Srd5a2 and Srd5a3; C, Star, Cyp11a1, Cyp17a1 and Hsd17b3) in adult wild type and Prl3c1-null testes (n = 6; *P < 0.05, **P < 0.001).
Citation: Journal of Endocrinology 234, 2; 10.1530/JOE-17-0250
Measures of testicular function in wild type and Prl3c1 null mice. (A) Serum testosterone levels from wild type (n = 8) and Prl3c1-null (n = 10) male mice (3.5–4.5 months). (B) Gross appearance of seminal vesicles from 4- to 5-month wild type and Prl3c1-null mice. (C and D) Gross appearance of seminal vesicles from 11- to 12 -month wild type and Prl3c1-null mice. (E) Seminal vesicle-to-body weight ratio of wild type (2–2.5 months, n = 14; 4–5 months, n = 8) and Prl3c1-null (2–2.5 months, n = 20; 4–5 months, n = 15) mice. (F) Total sperm count of wild type and Prl3c1-null mice (n = 5 per genotype). Scale bar: 50 μm. *P < 0.05; **P < 0.001.
Citation: Journal of Endocrinology 234, 2; 10.1530/JOE-17-0250
A linkage between the Prl3c1-null phenotype and Prl3c1 gene expression was explored. In a survey of several tissues from wild type male mice, Prl3c1 transcripts were prominently detected in the testis and preferentially localized to the interstitial compartment within the testis, as was PLP-J immunoreactivity (Fig. 4A, B and C). LacZ activity corresponding to activation of the Prl3c1 gene in heterozygous Prl3c1-mutant testes was co-localized with CYP11A1 immunoreactivity, demonstrating Prl3c1 gene activation specifically within Leydig cells (Fig. 4D). Localization of Prl3c1 to Leydig cells of the mouse testis has been previously reported (Yang et al. 2014). Testicular Prl3c1 expression showed a developmental ontogeny similar to adult Leydig cell marker genes, including Hsd3b6 (Fig. 5).
Prl3c1 transcript and the PLP-J protein are expressed in Leydig cells of the testis. (A) Tissue survey (RT-PCR analysis) for Prl3c1 transcripts in various tissues from male C57BL/6 mice. (B) RT-PCR analysis for Prl3c1 transcripts in testicular interstitium and seminiferous tubule compartments. Insl3 and Dmrt1 were included as controls for the interstitium and seminiferous tubule compartments, respectively. (C) Immunohistochemical (IHC) staining for PLP-J on tissue sections from wild type testis. (D) The Prl3c1 locus is transcriptionally active in Leydig cells of mice with β-galactosidase (LacZ) knocked into the Prl3c1 locus. Left panel: LacZ reporter activity; middle panel: immunofluorescence staining for CYP11A1; right panel: merged image of LacZ and CYP11A1 staining. LacZ reporter activity was localized to CYP11A1 positive Leydig cells in mice. Scale bar: 250 µm.
Citation: Journal of Endocrinology 234, 2; 10.1530/JOE-17-0250
Ontogeny of Prl3c1 and steroidogenic enzyme transcripts in testes of wild type mice. (A) RT-PCR analysis of Prl3c1 transcript expression in testes from postnatal days 5 to 45 (n = 3 per day). Positive control (P.C.) is adult testis. Insl3 and Gapdh transcript analyses were used as internal controls. (B and C) qRT-PCR measurements of transcripts for Prl3c1 (B) and transcripts encoding proteins involved in steroid hormone biosynthesis (B, Hsd3b and Srd5a2; C, Hsd17b3, Hsd3b1, Srd5a3) in testes at postnatal days 15 (D15), 21 (D21), and 45 (D45) (n = 6, *P < 0.05, **P < 0.001, compared to D15).
Citation: Journal of Endocrinology 234, 2; 10.1530/JOE-17-0250
Collectively, the phenotype suggested that PLP-J expression by Leydig cells regulates programming of the setpoint for testicular growth and function. In the absence of a functional Prl3c1 gene, the setpoint for testicular size and functional output was increased.
The testicular homeostatic setpoint control system includes the pituitary
The normal core functions of the testis are driven by an efficient homeostatic interplay of hypothalamic and pituitary signals, which are responsive to the testicular output they control. Since LH is the primary driver of Leydig cell function and Leydig cells are hyperfunctional in the Prl3c1-null mouse, we examined the contributions of LH to the Prl3c1-null phenotype. Serum LH levels were higher in Prl3c1-null mice, as was the Lhb transcript concentration of the pituitary (Fig. 6A and B). Elevations in LH prompted serum FSH and Fshb transcript measurements. Serum FSH levels did not differ between the genetic groups; however, Fshb transcript concentrations were significantly lower in pituitaries from Prl3c1-null males (Fig. 6C and D). Thus, there is a potential contribution of the pituitary, and more specifically elevated circulating LH, to the regulation of the Leydig cell phenotype observed in the Prl3c1-null testis. The male HPG axis appears to be operating in a unidirectional mode in Prl3c1-null mice with a distorted setpoint for negative feedback modulation by gonadal testosterone.
The testicular homeostatic setpoint control system includes the pituitary. (A) Serum LH levels in wild type (n = 8) and Prl3c1 null (n = 10) adult male mice. *P < 0.05. (B) Lhb transcript levels in pituitaries of wild type (n = 5) and Prl3c1 null (n = 7) adult male mice analyzed by qRT-PCR. **P < 0.001. (C) Serum FSH levels in wild type (n = 18) and Prl3c1 null (n = 18) adult male mice. (D) Fshb transcript levels in pituitaries of wild type (n = 5) and Prl3c1 null (n = 7) adult male mice analyzed by qRT-PCR. **P < 0.001.
Citation: Journal of Endocrinology 234, 2; 10.1530/JOE-17-0250
The Prl3c1 gene is a template for two transcripts
Prl3c1 transcripts expressed in uterine decidua and testes were compared and determined to be distinct, each utilizing different first exons driven by alternative promoters (Fig. 7). Decidual and Leydig cell Prl3c1 transcription start sites and promoters were identified by 5′ RACE (Supplementary Fig. 1A and B) and chromatin immunoprecipitation (ChIP), using antibodies to RNA polymerase II (POL II; Fig. 7B), respectively. The two transcripts were operationally defined as Prl3c1-v1 and Prl3c1-v2 (Fig. 7C). The transcription start site for Prl3c1-v1 was located 46 bp upstream of the ATG for Prl3c1-v1, whereas the transcription start site for Prl3c1-v2 was located 136 bp upstream of the ATG for Prl3c1-v2 (Supplementary Fig. 1A and B). An upstream promoter directs Prl3c1-v1 transcription (Fig. 7B and C). The Prl3c1-v1 transcript contains an upstream exon defined as exon 1a and encodes a secreted protein previously characterized from the uterine decidua (Alam et al. 2008), which we refer to as PLP-J V1 (Fig. 7C). A second promoter, embedded within the first intron of the Prl3c1 gene, directs Prl3c1-v2 transcription (Fig. 7B and C). The Prl3c1-v2 transcript contains an alternate first exon defined as exon 1b and encodes a protein termed PLP-J V2 (Fig. 7C). Within the testis, the alternative promoter is operative in Leydig cells. This pattern of testicular Prl3c1-v2 expression is conserved in fifteen different mouse strains (Supplementary Fig. 1C).
The Prl3c1 gene is a template for two transcripts with distinct tissue expression patterns. (A) Prl3c1 transcript-specific RT-PCR analysis revealed tissue-restricted expression patterns for Prl3c1-v1 (decidua) and Prl3c1-v2 (testis). (B) ChIP-qPCR analysis for RNA POL II at regions proximal to Exons 1a of Prl3c1-v1 (V1) and 1b of Prl3c1-v2 (V2) in decidua and Leydig cells. Locations of primer sets used in ChIP-qPCR are depicted as arrowheads shown in panel C (n = 4, *P < 0.05 compared to IgG control). (C) Schematic representation of transcription start sites for Prl3c1-v1 (decidua) and Prl3c1-v2 (Leydig cells) as determined by 5′RACE (Supplementary Fig. 1A and B).
Citation: Journal of Endocrinology 234, 2; 10.1530/JOE-17-0250
Origin of the testicular homeostatic setpoint control system
Transposable elements are sources of genetic information that have contributed to the evolution of the mammalian genome (Kaessmann 2010, Rebollo et al. 2012, Chuong et al. 2017). They are vestiges of viral infections and exist as repetitive sequences dispersed throughout the genome (Kidwell & Lisch 2001, Rebollo et al. 2012). Although the host organism has defense mechanisms for silencing transposable elements; in some cases, these elements escape repression and contribute to the birth of new genes and regulatory information controlling gene transcription (Kidwell & Lisch 2001, Jern & Coffin 2008, Rowe & Trono 2011, Feschotte & Gilbert 2012, Rebollo et al. 2012, Chuong et al. 2017). Transposable element contributions to the regulation of reproduction have been described (Rebollo et al. 2012, Feschotte & Gilbert 2012, Emera & Wagner 2012, Chuong et al. 2017).
Comparison of mouse vs rat Prl3c1 loci established a connection between the testicular homeostatic setpoint control system and the insertion of a composite transposable element within the mouse Prl3c1 locus. The rat possesses a Prl3c1 ortholog, which is expressed in uterine decidua (Hiraoka et al. 1999, Ishibashi & Imai 1999, Toft & Linzer 1999, Dai et al. 2000) but not in testes (Fig. 8A). Closer inspection of the rat Prl3c1 gene revealed that it possesses a shorter first intron that lacks exon 1b, which is characteristic of the mouse Prl3c1-v2 transcript (Fig. 8B). The extended length of the first intron of mouse Prl3c1 was attributed to the insertion of a composite transposable element consisting of two parts of an LTR-type element referred to as RMER17D_MM separated by a LINE element (L1 Mus_3 end; Fig. 8C). The RMER17D_MM element is restricted in its genomic distribution. Of the 4977 RMER17D_MM insertion sites (80–100% sequence identity) within the mouse genome, 76.79% (3823 copies) are located within intergenic regions, 23.2% (1149 copies) in intronic regions and 0.01% (5 copies) within exons. Insertion of the composite transposable element into the first intron of the mouse Prl3c1 gene provides an alternative promoter and an alternative first exon (exon 1b), structures absent from the rat Prl3c1 locus.
Origin and species specificity of the testicular homeostatic setpoint control system. (A) RT-PCR analysis for Prl3c1 transcripts with primers targeting various exons of the rat Prl3c1 gene in testes of Fischer 344 (F344), Holtzman Sprague Dawley (HSD) and Brown Norway (BN) rats. Decidua tissue (HSD, gestation day 7.5) was included as a positive control. (B) PCR amplification of the first intron of the Prl3c1 gene from the mouse (C57BL/6) and rat (BN). Schematic diagram of exon-intron structure indicates the locations of the primers used for amplification. (C) Schematic comparison of the mouse and rat Prl3c1 genes reveals a longer first intron in the mouse due to the insertion of a composite transposable element (TE), which contains Exon 1b of Prl3c1-v2. (D) PCR amplification of the first introns of the Prl3c1 genes from M. musculus, M. spretus, M. caroli, M. pahari, and R. norvegicus. (E) Schematic representations of the first introns of the Prl3c1 genes from M. musculus, M. spretus, M. caroli, M. pahari, and R. norvegicus. Sequence identity comparisons are with the M. musculus sequence. (F) Phylogenetic tree based on the nucleotide sequences for the first introns of Prl3c1 genes from M. musculus, M. spretus, M. caroli, M. pahari, and R. norvegicus. (G) Primer validation for M. caroli and M. pahari. Schematic diagram indicates the locations of the two sequence-specific primer sets used for detection of Prl3c1 transcripts in testes from M. caroli and M. pahari. Primer sequences were based on partially sequenced coding regions of M. caroli (GenBank Accession No. KJ125427) and M. pahari (GenBank Accession No. KJ125428) Prl3c1 genes and validated by PCR amplification of corresponding Prl3c1 genomic regions with genomic DNA from these species. (H) RT-PCR analysis for Prl3c1 transcripts in testes of M. musculus, M. spretus, M. caroli, and M. pahari. Two sets of sequence-specific primers were used for M. caroli and M. pahari. (I) Annotated schematic of the first intron of Prl3c1 from M. musculus and M. caroli. Note the prominent differences in the organization of RMER17D in M. musculus vs M. caroli.
Citation: Journal of Endocrinology 234, 2; 10.1530/JOE-17-0250
Rapidly evolving testicular homeostatic setpoint control system
The rat is estimated to have diverged from the mouse approximately 25.2 million years ago (Mya) (Hedges et al. 2006). We reasoned that other Mus species sharing a closer evolutionary history with M. musculus (divergence from M. musculus: M. spretus, 1.8 Mya; M. caroli, 3.2 Mya; M. pahari, 7.9 Mya) (Guenet & Bonhomme 2003, Petkov et al. 2004, Keane et al. 2011) could provide additional insights into the evolutionary origin of this alternative Prl3c1 transcript. Nucleotide sequences for the first intron of the Prl3c1 gene corresponding to each of the four Mus species were determined and compared (Fig. 8D, E and F). M. musculus and M. spretus possess a virtually identical organization of exon 1a and intron 1, including insertion of the composite transposable element with exon 1b, and testis expression of Prl3c1-v2 but not Prl3c1-v1 (Fig. 8D, E, F, G and H) (Keane 2011). The extended intron 1 with components of the composite transposable element is also present in M. caroli (GenBank Accession No. KF767571); however, the organization of the composite transposable element shows marked differences, and this species lacks testicular expression of either Prl3c1-v1 or Prl3c1-v2 (Fig. 8D, E, F, G, H, and I). The more divergent M. pahari lacks the composite transposable element and does not express either Prl3c1-v1 or Prl3c1-v2 within its testes (GenBank Accession No. KF767572; Fig. 8D, E, F, G, and H). Consistent with the Leydig cell-static actions of PLP-J V2 in M. musculus, M. pahari lacks testicular Prl3c1 expression and interestingly possesses a proportionately larger interstitial compartment within its testis when compared to M. musculus or M. spretus testes (Montoto et al. 2012). Collectively, these data indicate that the Prl3c1 locus exhibited significant changes within a short evolutionary timeframe. Furthermore, comparisons of rodent amino acid sequences indicated that proteins encoded by the Prl3c1 gene have undergone positive selection (Chuong et al. 2010), a measure of a rapidly evolving gene.
Regulation of Prl3c1-v1 vs Prl3c1-v2 expression
A conserved method for controlling regulatory elements and defense against inappropriate activation of transposable elements is via DNA methylation (Rowe & Trono 2011). Consequently, we investigated DNA methylation throughout the mouse Prl3c1 locus, in uterine decidua (Prl3c1-v1 expression), enriched Leydig cells isolated from the adult mouse testis (Prl3c1-v2 expression) and liver (negative control) using bisulfite sequencing, direct sequencing and MultiNA-Combined Bisulfite Restriction Analysis (COBRA) (Yagi et al. 2008, Hayakawa et al. 2012). Collectively, the analyses indicated that DNA methylation status at the Prl3c1 locus was different in decidual cells vs Leydig cells. A region approximately 2 kb upstream of the Prl3c1-v1 transcription start site was hypomethylated in decidual cells and hypermethylated in Leydig cells, whereas the reciprocal was true for the proximal regulatory sequence immediately upstream of exon 1b, including parts of the composite transposable element (Supplementary Figs 2, 3 and 4). The results are consistent with the idea that tissue-specific DNA methylation at the Prl3c1 locus could contribute to the differential regulation of Prl3c1-v1 and Prl3c1-v2.
The two Prl3c1 transcripts encode proteins with distinct intracellular targeting and actions
Exon 1a of the Prl3c1 gene encodes the 5′ untranslated region (UTR) of the Prl3c1-v1 transcript and the initial portion of the signal peptide for the PLP-J V1 protein (Alam et al. 2007). In contrast, exon 1b is a noncoding exon, contributing solely to the 5′ UTR of the Prl3c1-v2 transcript. The common exon 2 contributes the translation initiation codon of the Prl3c1-v2 transcript. Prominent differences in the N-terminal portion of the PLP-J V1 and predicted PLP-J V2 proteins prompted a comparison of their intracellular distributions in mouse Leydig cell lines (TM3, MA-10 and MLTC-1) and COS7 cells following transfection of expression vectors encoding FLAG-tagged PLP-J V1 and PLP-J V2 proteins. Western blotting of nuclear and cytosolic lysates of transfected MLTC-1 and COS7 cells demonstrated that PLP-J V1 was preferentially localized to the cytoplasm, whereas PLP-J V2 was targeted predominantly to the nucleus (Fig. 9A and Supplementary Fig. 5A). Interestingly, part of the mechanism of action of PRL in T-lymphocytes requires its nuclear translocation (Clevenger et al. 1991). We next determined whether ectopic expression of PLP-J V1 or PLP-J V2 affected MLTC-1 Leydig cell behavior. PLP-J V2 inhibited MLTC-1 Leydig cell proliferation, whereas PLP-J V1 did not significantly affect cell number (Fig. 9B and C). PLP-J V2 also effectively inhibited TM3, MA-10 and COS7 cell proliferation (Supplementary Fig. 5B, C and D). TM3 cells possess features resembling Leydig cell progenitors, while MLTC-1 cells represent a more mature Leydig cell population (Supplementary Fig. 6A) (Mather 1980, Rahman & Huhtaniemi 2004). Ectopic expression of PLP-J V2 in MLTC-1 Leydig cells did not affect transcript levels for steroidogenic enzymes but did affect the expression of a subset of Leydig cell progenitor markers (Supplementary Fig. 6B, C and D). In contrast, ectopic expression of PLP-J V2 in TM3 Leydig cells stimulated Hsd3b1 and Hsd3b6 transcript levels, similar to LH treatment, but did not affect the expression of Leydig cell progenitor markers (Supplementary Fig. 6E, F and G). The absence of an effect of ectopic expression of PLP-J V1 on TM3 cell cycle or steroidogenesis has been previously reported (Yang et al. 2014). Thus, ectopic PLP-J V2 can alter Leydig cell behavior, but its actions are context dependent and influenced by the developmental state of the target Leydig cell population.
Intracellular distribution and distinct functions of PLP-J V1 vs PLP-J V2. FLAG-tagged PLP-J V1 and FLAG-tagged PLP-J V2 were ectopically expressed in MLTC-1 Leydig cells (1 µg/mL). Cells transfected with an empty vector were used as a control (1 µg/mL). (A) Subcellular fractionation followed by Western blotting for FLAG (Cyto, cytoplasm; Nu, nucleus). Western blotting for GAPDH and Histone H3 were used to monitor the integrity of the cytoplasmic and nuclear preparations, respectively. (B) Effects of ectopic expression of Prl3c1-v1 (left panel) or Prl3c1-v2 (right panel) on MLTC-1 Leydig cell numbers following 72 h of culture. Cells were cultured in 96 well plates with 0.1 mL of medium/well (n = 6, **P < 0.001 compared to empty vector control). (C) Representative images of MLTC-1 cells transfected with empty vector, Prl3c1-v1, or Prl3c1-v2 (1 µg/mL) following 72 h of culture.
Citation: Journal of Endocrinology 234, 2; 10.1530/JOE-17-0250
To summarize, the contributions of the composite transposable element resulted in the use of an alternative exon and the production of a protein targeted to the nucleus with Leydig cell-static actions. Such actions are consistent with the testiculomegaly and Leydig cell hyperplasia observed in Prl3c1-deficient mice (Fig. 1D, E, F, G, H, I, J, K, L and M).
Overview
The ‘big’ testis phenotype in Prl3c1-null mice is associated with a disruption in the HPG axis characterized by concomitant elevations in LH and testosterone and the consequences of their actions; a condition known as hypergonadotropic–hypergonadal–hyperandrogenism. Characterization of the Prl3c1-null phenotype led to the discovery of a homeostatic setpoint control system programming testicular growth and function.
Discussion
Male fertility is dependent upon the appropriate functioning of the HPG axis (Achermann & Jameson 1999, Hedger & de Kretser 2000, Matzuk & Lamb 2008). Each functional unit within the axis works as part of a homeostatic mechanism to precisely control core testicular functions, which include testosterone and sperm production. Two important features of the mammalian testis are evident: (1) there is a wide range of testis size and functional output across mammalian species; (2) within a species, there are constraints governing testicular size and function. Dysregulated growth and function of the male gonads have consequences. Deficiencies are associated with subfertility and infertility, whereas excesses have costs to adult male health, including androgen-driven hyperplasia that can lead to neoplasia, and potentially negative consequences on social structure (Short 1997, Hedger & de Kretser 2000, Mascaro et al. 2013). Species-specific regulatory pathway(s) controlling this elemental reproductive process are poorly understood. We have discovered a reproductive trait with its evolutionary origins associated with gene duplication and co-optation of transposable elements. In the mouse, the size of the testis and its capacity to produce testosterone and sperm are modulated by the recruitment of a composite transposable element to the Prl3c1 locus and its contribution to the regulation and biosynthesis of a unique protein. Prl3c1 is a gene within the expanded PRL family. This novel control system impacts the establishment of the homeostatic setpoint determining the size and function of the testis. Although the existence of this testicular homeostatic setpoint control system is restricted to the mouse, species-specific epiregulation of the male reproductive axis may represent a universal concept, contributing to each species’ reproductive success. Indeed, the testis is a common site for the expression of new and rapidly evolving genes with restricted phylogenetic distributions (Turner et al. 2008, Heinen et al. 2009, Chen et al. 2013). The widespread existence of species-specific modulation of the male reproductive axis evokes questions with broader implications. Are the growth and function of other organs governed by species-specific mechanisms? Are these species-specific mechanisms prone to dysregulation and do they represent potential therapeutic targets for treating disease? The existence of layers of control that are not conserved among species represents a challenge, as well as an opportunity to investigate physiological regulation and its relationship to disease.
Supplementary data
This is linked to the online version of the paper at http://dx.doi.org/10.1530/JOE-17-0250.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Funding
This work did not receive any specific grant from any funding agency in the public, commercial, or not-for-profit sector.
Acknowledgements
The authors thank Dr Jacques Tremblay (Laval University) and Drs Gustavo Blanco, T Rajendra Kumar, Gladys Sanchez and Ossama Tawfik (University of Kansas Medical Center) for their advice and suggestions regarding the execution of the experimentation. They also thank Dr Sumedha Gunewardena (University of Kansas Medical Center) for assistance with the bioinformatic analysis, Dr Vincent Lynch (University of Chicago) for advice with the RNA POL II ChIP analysis and Dr Curtis Klaassen for providing the testis samples used in the mouse strain survey. They are very appreciative of the valuable feedback and advice provided by Dr Gunter Wagner (Yale University), R Michael Roberts (University of Missouri), Dr Dixie Mager (University of British Columbia) and Dr Justin Blumenstiel (University of Kansas) during various stages of the research. This work was supported by a grant from the NIH HD055523 and a National Institute of Environmental Health Sciences postdoctoral fellowship to P B (T32-ES007079). The University of Virginia Center for Research in Reproduction Ligand Assay and Analysis Core is supported by the Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD)/National Institutes of Health (Specialized Cooperative Centers Program in Reproduction and Infertility Research (SCCPIR) grant U54-HD28934).
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