Overactivation of hedgehog signaling in the developing Müllerian duct interferes with duct regression in males and causes subfertility

in Reproduction

The influence of the hedgehog signaling pathway on reproduction was studied in transgenic mice in which a dominant active allele of the hedgehog signal transducer, smoothened (Smo), was conditionally expressed in the developing Müllerian duct and gonads through recombination mediated by anti-Müllerian hormone receptor 2-cre (Amhr2cre). Previous studies showed that development of the oviduct and uterus are abnormal in female Amhr2cre/+SmoM2 mice. In the current study, focusing on mutant males, litter size was reduced 53% in crosses with wild-type females. An extra band of undifferentiated tissue extended along each epididymis and vas deferens, a position suggesting derivation from Müllerian ducts that failed to regress fully. Hedgehog signaling was elevated in this tissue, based on mRNA levels of target genes. Amhr2 mRNA was dramatically reduced in the uterus of mutant females and in the extra tissue in the tract of mutant males, suggesting that AMHR2 signaling was inadequate for complete Müllerian duct regression. Spermatogenesis and sperm motility were normal, but testis weight was reduced 37% and epididymal sperm number was reduced 36%. The number of sperm recovered from the uteri of wild-type females after mating with mutant males was reduced 78%. This suggested that sperm transport through the male tract was reduced, resulting in fewer sperm in the ejaculate. Consistent with this, mutant males had unusually tortuous vas deferentia with constrictions within the lumen. We concluded that persistence of a relatively undifferentiated remnant of Müllerian tissue is sufficient to cause subtle changes in the male reproductive tract that reduce fertility.

Abstract

The influence of the hedgehog signaling pathway on reproduction was studied in transgenic mice in which a dominant active allele of the hedgehog signal transducer, smoothened (Smo), was conditionally expressed in the developing Müllerian duct and gonads through recombination mediated by anti-Müllerian hormone receptor 2-cre (Amhr2cre). Previous studies showed that development of the oviduct and uterus are abnormal in female Amhr2cre/+SmoM2 mice. In the current study, focusing on mutant males, litter size was reduced 53% in crosses with wild-type females. An extra band of undifferentiated tissue extended along each epididymis and vas deferens, a position suggesting derivation from Müllerian ducts that failed to regress fully. Hedgehog signaling was elevated in this tissue, based on mRNA levels of target genes. Amhr2 mRNA was dramatically reduced in the uterus of mutant females and in the extra tissue in the tract of mutant males, suggesting that AMHR2 signaling was inadequate for complete Müllerian duct regression. Spermatogenesis and sperm motility were normal, but testis weight was reduced 37% and epididymal sperm number was reduced 36%. The number of sperm recovered from the uteri of wild-type females after mating with mutant males was reduced 78%. This suggested that sperm transport through the male tract was reduced, resulting in fewer sperm in the ejaculate. Consistent with this, mutant males had unusually tortuous vas deferentia with constrictions within the lumen. We concluded that persistence of a relatively undifferentiated remnant of Müllerian tissue is sufficient to cause subtle changes in the male reproductive tract that reduce fertility.

Introduction

The hedgehog (HH) signaling pathway plays a crucial role in the development of diverse tissue and organ systems in the embryo and in the regulation of adult tissues (Ingham & McMahon 2001, Hooper & Scott 2005, King et al. 2008). It was of interest to determine the influence of this pathway on reproductive function. There are three HH ligands in mammals, sonic (SHH), desert (DHH) and Indian (IHH), which are secreted proteins. When a HH ligand binds to the membrane receptor patched (PTCH), it relieves inhibition by PTCH on the transmembrane signal transducer smoothened (SMO). The resulting activation of glioma-associated oncogene homolog transcription factors (GLI1, GLI2 and GLI3) leads to alterations in cell function (Huangfu & Anderson 2006).

To study the role of the HH signaling pathway in reproduction, we created mice with constitutive activation of the HH pathway in the gonad and reproductive tract. The mice express an allele of Smo, SmoM2, in which a point mutation prevents its negative regulation by PTCH, leading to constitutive activation of HH signaling. In Amhr2cre/+SmoM2 transgenic mice, SmoM2 is expressed after deletion of a floxed stop codon by the action of cre recombinase expressed from sequences inserted into the anti-Müllerian hormone receptor 2 (Amhr2) gene. We previously reported that this recombination event occurs in the developing reproductive tract and ovary of female Amhr2cre/+SmoM2 mice (Ren et al. 2009, Migone et al. 2012, Ren et al. 2012) and that the female mice are infertile. They fail to ovulate (Ren et al. 2009, Migone et al. 2016) and have abnormal development of the reproductive tract (Migone et al. 2012).

Our initial examination of male Amhr2cre/+SmoM2 mice suggested they are sub-fertile, prompting our thorough examination of the reproductive phenotype presented in the current report. HH signaling is known to play a role in male reproduction. DHH is produced after differentiation of somatic precursors into Sertoli cells in the embryonic testis (Cool & Capel 2009). Male Dhh-null mice are viable but infertile, lacking development of mature sperm (Bitgood et al. 1996). Testis cords in Dhh-null mice have disrupted basal laminae, beginning during embryonic development, likely due to altered development of peritubular myoid cells that are targets of HH signaling (Clark et al. 2000, Pierucci-Alves et al. 2001). A similar disruption of laminin distribution surrounding the seminiferous cords was observed in gonads from the tammar wallaby, after culture with an inhibitor of HH signaling (Chung et al. 2012). DHH induces differentiation of precursor cells surrounding the cords of the fetal testis into androgen producing fetal Leydig cells (Yao & Capel 2002, Yao et al. 2002). DHH is also necessary later in development for proliferation and differentiation of stem cells associated with the surface of the seminiferous tubules into adult Leydig cells (Chen et al. 2016). In some lines of Dhh-null mice, androgen production was apparently reduced, leading to feminization (Clark et al. 2000). Downstream targets of HH signaling are expressed in germ cells of the rodent testis and expression varies with the stage of the seminiferous epithelial cycle (Kroft et al. 2001, Szczepny et al. 2006, Makela et al. 2011). Inhibiting HH signaling in cultured fragments of mouse seminiferous tubules altered gene expression patterns, suggesting a role in gamete development (Szczepny et al. 2009). HH signaling also occurs in the epididymis, where SHH is the major ligand produced and other components of the pathway, Gli1, Gli3 and Ptch1, are expressed (Turner et al. 2004, 2006).

The objective of the current study was to determine the effect of constitutive activation of HH signaling in the developing gonads and reproductive tract on reproductive function in male Amhr2cre/+SmoM2 mice. Initial examination of the reproductive tract in Amhr2cre/+SmoM2-mutant males revealed the presence of extra tissue extending alongside the length of the epididymis and vas deferens that was not present in Amhr2+/+SmoM2 control mice. During embryonic development in both male and female mammals, the Müllerian duct develops as an invagination of the mesonephric epithelium that extends posteriorly along the length of the adjacent Wolffian duct. At the time of sexual differentiation, expression of anti-Müllerian hormone (AMH) by Sertoli cells in males initiates regression of the Müllerian duct through actions on AMHR2 while the absence of AMH expression during embryonic development in females allows the maintenance of the duct (Arango et al. 2008). The Müllerian duct primordium develops into the oviduct, uterus and upper part of the vagina in females and the Wolffian duct develops into the epididymis, vas deferens and seminal vesicles in males. An aim of this study was to investigate whether the unusual tissue present along the reproductive tract of Amhr2cre/+SmoM2 male mice represents Müllerian tissue that failed to fully regress.

Materials and methods

Generation of mice

Amhr2cre/+ mice, provided by Dr Richard Behringer (Jamin et al. 2002), and Gt(ROSA)26Sortm1(smo/EYFP)Amc/J mice (Jeong et al. 2004), purchased from The Jackson Laboratory, were mated to obtain Amhr2cre/+SmoM2 mutants and Amhr2+/+SmoM2 genotype-matched controls. Cre-mediated recombination was assessed in mice generated by mating Amhr2cre/+ mice with mice in which expression of a tdTomato transgene is blocked by an upstream loxP-flanked stop signal (Gt(ROSA)26Sortm1(CAG-tdTomato)HZE/J mice; The Jackson Laboratory). For fertility studies, CD-1 females were purchased from Charles River Laboratories. Mice were genotyped from tail DNA using protocols provided by The Jackson Laboratory. All animals were kept in accordance with the NIH Guide for the Care and Use of Laboratory Animals. Studies were approved by the Cornell University Institutional Animal Care and Use Committee.

Use of tdTOMATO reporter mice to determine the sites of Amhr2cre-mediated recombination

Reproductive tracts from Amhr2cre/+tdTomato mice and respective genotype-matched controls lacking cre were dissected the day after birth. Tracts were fixed in 2% paraformaldehyde for 4 h, rinsed in PBS and incubated for 2 h with Hoechst 33342 nuclear stain (2 µg/mL in PBS-0.1% Triton X-100) and placed between coverslips separated by 0.5 mm spacers. A Zeiss LSM 510 confocal microscope (Carl Zeiss Microscopy) was used for detection of tdTomato (excitation 561 nm and collection above 575 nm) and Hoechst (excitation 405 nm and collection at 420–480 nm).

Real-time RT-PCR analysis of gene expression

Total RNA was extracted from tissues obtained from mice the day after birth using an RNeasy Micro Kit (Qiagen), and reverse transcription performed using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). A StepOnePlus thermocycler (Applied Biosystems) was used to run mouse-specific PCR assays obtained from Applied Biosystems: Gli1, Mm00494645_m1; Ptch1, Mm00436026_m1; Hhip1, Mm00469580_m1; Amhr2, Mn00513847_m1; Hoxa9, Mm00439364_m1; Hoxa10, Mm00433966_m1; Hoxa11, Mm00439360_m1 and Hoxa13, Mm00433967_m1. Each assay included a standard curve prepared by serial dilution of an RNA pool. The RNA pool used was prepared from testis and epididymis of 1-day-old mice, except for the Amhr2 assay in which the pool was prepared from uteri of 25-day-old mice. Results were normalized to18S rRNA measured in each sample (assay 4319413E; Applied Biosystems).

Elongated spermatid count

Beyond stage 17 of spermatid development, the nuclei of elongated spermatids are resistant to destruction by detergents such as Triton X-100 (Robb et al. 1978, de Kretser & O’Donnell 2013). To determine the number of elongated spermatids, each testis was weighed, the capsule was removed and the decapsulated testis was placed in three volumes of 0.05% Triton X-100 in PBS and homogenized for ~20 s until evenly dispersed. Elongated spermatids were counted using a hemocytometer.

Caudal epididymal sperm count and characteristics

Sperm were collected from the caudal epididymides from 12-week-old (81–86 days of age) control and mutant mice as previously described (Marcello & Evans 2010, Chang & Suarez 2011). Briefly, both epididymides were dissected and placed under mineral oil on a Petri dish prewarmed and maintained at 37°C, and then cleaned of fat and connective tissue. Caudal epididymides were isolated, transferred into a 250 µL droplet of sperm capacitating media that was overlain with mineral oil and then minced with scissors. Sperm were allowed to disperse from the epididymides for 15 min in an incubator (37°C, 5% CO2). Total medium containing released sperm was transferred to a tube containing 200 µL of capacitating medium (Chang & Suarez 2011) and counted using a hemocytometer.

Aliquots of caudal sperm were evaluated for morphology, total and progressive motility, hyperactivation and capacitation at the time of collection (t = 0 h) and 2 h after incubation in capacitating media. Sperm morphology was assessed by determining the number of sperm that had abnormal characteristics such as bent mid-piece and agglutination from a total of 100 sperm. Total motility of sperm was assessed by imaging sperm on a 37°C heated stage of a Zeiss Axiovert 35 microscope; 100 sperm were scored and considered motile if they had flagellar activity. Progressive motility was determined using videotaped recordings of sperm and was characterized as the percentage of sperm that had a forward trajectory out of a total of 60–110 motile sperm observed. A sperm sample was designated to be hyperactivated if the majority of sperm had an asymmetrical pattern of flagellar movement and circular swimming pattern (Suarez & Osman 1987). Capacitation was assessed by the level of protein tyrosine phosphorylation (Visconti et al. 1995) on western blots of sperm lysates probed with protein phosphotyrosine antibody clone 4G10 (EMD Millipore) as previously described (Hung & Suarez 2012).

Percentage of seminiferous tubules in stages VII and VIII of spermatogenesis

The frequency of discrete stages in the cycle of the seminiferous epithelium, stages VII and VIII, were used to evaluate progression of spermatogenesis. Stages VII and VIII were identified by morphology as previously described (Oakberg 1956a,b) in hematoxylin and eosin-stained paraffin sections. Three cross-sectional planes (top, median and bottom) from each testis were used to quantify differences in stage VII and VIII seminiferous tubules between Amhr2cre/+SmoM2 mutant and Amhr2+/+SmoM2 control males.

Ejaculated sperm count

Adult 50- to 70-day-old wild-type C57BL/6Jx129S1 females were synchronized with injections of 5 IU eCG followed 48 h later by 5 IU hCG and were caged overnight with a Amhr2cre/+SmoM2-mutant or Amhr2+/+SmoM2 control male. Females with a copulatory plug 16 h after hCG were killed and the plug weighed. Each uterine horn was flushed using 200 µL PBS, and the number of sperm recovered was estimated using a hemocytometer. Sperm motility was assessed as described previously.

Statistical analysis

Differences in fertility parameters, sperm and elongated spermatid numbers and characteristics, testis and seminal vesicle weight, and percentages in stages VII and VIII of spermatogenesis between control and Amhr2cre/+SmoM2 mutant mice were analyzed by unpaired t-tests. Gene expression data were log transformed and analyzed by one-way ANOVA. The Student–Newman–Keuls procedure was used to compare individual means. Western blot was analyzed using gel analysis tools in ImageJ software. Signal intensity was calculated for each lane as the area under the curve of a plot of intensity, standardized against the signal intensity for tubulin, and expressed as a percent of the intensity of lane 1 (time = 0). Data were analyzed by t-test.

Results

Reduced fertility and gross differences in the reproductive tract of Amhr2cre/+SmoM2 males

A breeding trial was conducted in which young adult Amhr2cre/+SmoM2-mutant mice and genotype-matched Amhr+/+SmoM2 control mice were caged continuously with CD1 wild-type females over a four-month period. Mating to mutant males generated 53.2% fewer pups per litter than mating to control males (6.7 ± 0.6 vs 14.4 ± 1.6 pups/litter; P = 0.005, n = 6 mice), whereas the total number of litters produced over 4 months did not differ (4.7 ± 0.5 vs 5.0 ± 0.3 litters/4 months; P = 0.56).

Examination of the reproductive tract of Amhr2cre/+SmoM2-mutant males at 12 weeks of age revealed the presence of extra tissue extending alongside the length of the epididymis and vas deferens that was not present in Amhr2+/+SmoM2 control males (Fig. 1).

Figure 1
Figure 1

Epididymis and vas deferens of Amhr2cre/+SmoM2-mutant males and Amhr2+/+SmoM2 control males at 12 weeks of age. Extra tissue, indicated by brackets in panels B and D, is present alongside the length of the epididymis and vas deferens in mutant mice and is not present in controls. The size reference bar shown in panel D applies to all panels and represents 1 mm.

Citation: Reproduction 153, 4; 10.1530/REP-16-0562

Sites of SmoM2 expression in Amhr2cre/+SmoM2 males

In mice harboring the Amhr2cre allele, cre-mediated recombination has been reported to occur in the Müllerian duct at 12.5 days post coitus (dpc) (Jamin et al. 2002, Kobayashi et al. 2011) consistent with the normal timing of expression of Amhr2 (Arango et al. 2008). To confirm the location of Amhr2cre-mediated recombination in the male reproductive tract, mice with a floxed allele of the fluorescent reporter, tdTomato, were bred to Amhr2cre mice and tdTOMATO fluorescence examined in tracts of progeny mice on day 1 of age.

In Amhr2+/+tdTomato control mice lacking cre, tdTOMATO fluorescent signal was absent (Fig. 2, left panels A, C, E and G). tdTomato signal was also absent in liver, a tissue in which Amhr2 expression does not occur (not shown). In Amhr2cre/+tdTomato mice, tdTomato fluorescence was detected in the tubules of the epididymis and at low levels in the vas deferens (Fig. 2 panels B and D). Within the testis, cells outside the seminiferous tubules were fluorescent suggesting that they are Leydig cells (Fig. 2 panel F), whereas the presence of fluorescent signal within the tubules could not be not be definitively determined. As a control, examination of female Amhr2cre/+tdTomato mice at day 1 of age showed tdTomato fluorescence in the uterus consistent with previous findings that recombination is directed to the developing Müllerian duct (Jamin et al. 2002, Arango et al. 2005, Harman et al. 2011, Migone et al. 2012) (Fig. 2, panel H).

Figure 2
Figure 2

Sites of cre-mediated recombination in reproductive tracts and gonads of male and female Amhr2cre/+tdTomato mice on day 1 of age, determined by imaging of the tdTOMATO reporter protein by confocal imaging of whole-mount tissues. Tissues shown are epididymis (A and B), vas deferens (C and D), testis (E and F) and uterus (G and H). In panel F, arrows point to fluorescent Leydig cells. The size reference bar in panel H applies to all panels and represents 100 µm for panels A–F and 200 µm for panels G and H. Images are representative of analysis of 2–4 mice of each genotype.

Citation: Reproduction 153, 4; 10.1530/REP-16-0562

Increased signaling through the HH pathway in Amhr2cre/+SmoM2 males

It is possible that Amhr2cre-mediated expression of dominant active SMOM2 in the Müllerian duct during embryonic development in Amhr2cre/+SmoM2-mutant males prevented the normal regression of the duct, leading to the presence of extra tissue along the epididymis and vas deferens. To determine whether signaling through the HH pathway was increased in response to expression of dominant active SMOM2, relative levels of mRNA for genes that are known transcriptional targets of HH signaling (Gli1, Ptch1, Hhip1; (Marigo et al. 1996a,b, Chuang & McMahon 1999)) were measured in segments of the reproductive tract at day 1 of age (Fig. 3). Levels of mRNA for these genes in the testis, epididymis and vas deferens were not significantly different in mutants compared to controls. However, levels in the uterus were dramatically elevated in mutants compared to those in controls in agreement with our previous findings (Migone et al. 2012). In mutant males, levels of mRNA in the extra tissue abnormally attached to the epididymis and vas deferens were similar to levels in the uterus of mutant females (Fig. 3). This AMHR2CRE- induced increased expression of HH target genes is consistent with the derivation of the extra tissue attached to the reproductive tract of mutant males from Müllerian ducts.

Figure 3
Figure 3

Relative levels of mRNA for genes that are known transcriptional targets of HH signaling in reproductive tract tissues of Amhr2cre/+SmoM2-mutant males and Amhr2+/+SmoM2 control males on day 1 of age. Tissues analyzed are testis, epididymis (Epi), vas deferens (Vas), uterus and the extra tissue attached to the epididymis and vas deferens of Amhr2cre/+SmoM2-mutant mice. Data are mean ± s.e.m., based on RNA preparations from 4 mice. Within each panel, bars without a common superscript are significantly different (P < 0.05).

Citation: Reproduction 153, 4; 10.1530/REP-16-0562

Histological differences in the reproductive tract of Amhr2cre/+SmoM2 males

The vas deferens of adult (12-week-old) mutant and control mice appeared similar in histological sections. However, there was an extra-tubular structure extending along the length of the vas deferens in mutant mice that was not present in controls. It consisted of stromal tissue and lacked a lumen (Fig. 4, panels A, B, C, D and E). The histology of tubules of the epididymis appeared similar in mutant and control mice; however, extra tissue also extended along the length of the epididymis of mutant mice. This extra tissue lacked a lumen and had the appearance of vascularized stromal tissue, similar to the extra tissue seen along the vas deferens (Fig. 4, panels F, G, H, I and J).

Figure 4
Figure 4

Hematoxylin and eosin-stained sections of the reproductive tract of Amhr2cre/+SmoM2-mutant males and Amhr2+/+SmoM2 control males at 12 weeks of age. Extra tissue attached to the vas deferens and epididymis of mutant males is indicated by brackets in panels B and G respectively. Below each low power image (panels A, B, F and G) are higher power images of the regions outlined by boxes (panels C, D, E, H, I and J). The size reference bar in panel J applies to all panels; it represents 500 µm for panels A, B, F and G; 100 µm for panels C, D, E, H and I and 200 µm for panel J. Images are representative of analysis of 3 mice of each genotype.

Citation: Reproduction 153, 4; 10.1530/REP-16-0562

HH-induced alteration of AMHR2 signaling in the reproductive tract of Amhr2cre/+SmoM2 males

Amhr2cremice have been used in a number of studies to conditionally alter genes postulated to be required for regression of the Müllerian duct in males. This is possible because disruption of one allele of Amhr2 in Amhr2cremice by the inserted cre recombinase sequence does not have any effect on the development of the male reproductive tract or fertility. In Amhr2cre/+SmoM2-mutant males, the location of the extra tissue along the epididymis and vas deferens suggested that it was Müllerian-derived tissue that had failed to fully regress in response to elevated signaling through the HH pathway. Because AMHR2 is essential for regression of the Müllerian duct in response to AMH (Mishina et al. 1996), the relative levels of Amhr2 mRNA in the reproductive tract tissues were determined. Amhr2 mRNA levels were not different in testis, epididymis and vas deferens of control and mutant mice on day 1 of age. However, Amhr2 mRNA levels in the uterus were reduced 97% in mutant compared to that in control females and levels were similarly low in the extra tissue abnormally attached to the epididymis and vas deferens of mutant males (Fig. 5). Because levels of Amhr2 were dramatically reduced in the Müllerian-derived uterus of mutant females, the results are consistent with the possibility that low levels of Amhr2 in Müllerian tissue of mutant males contributed to its abnormal persistence.

Figure 5
Figure 5

Relative levels of mRNA for Amhr2 in reproductive tract tissues of Amhr2cre/+SmoM2-mutant males and Amhr2+/+SmoM2 control males on day 1 of age. Tissues analyzed are testis, epididymis (Epi), vas deferens (Vas), uterus and the extra tissue attached to the epididymis and vas deferens of Amhr2cre/+SmoM2-mutant mice. Data are mean ± s.e.m., based on RNA preparations from 4 mice. Bars without a common superscript are significantly different (P < 0.05).

Citation: Reproduction 153, 4; 10.1530/REP-16-0562

Homeobox genes in the reproductive tract of Amhr2cre/+SmoM2 males

Expression of genes within the homeobox family was examined because they are known to direct the anterior to posterior development of the male and female reproductive tracts into distinct morphological and functional regions (Hannema & Hughes 2007). Levels of mRNA for homeobox a (Hoxa) genes in the epididymis and vas deferens did not differ between mutant and control mice on day 1 of age (Fig. 6). However, expression varied with region of the tract as expected based on previous studies (Hannema & Hughes 2007); relative levels of Hoxa9 mRNA were similar in the epididymis and vas deferens, levels of Hoxa10 and Hoxa11 mRNA were higher in the vas deferens compared to that in the epididymis, and levels of Hoxa13 mRNA were higher in the vas deferens than those in the epididymis (Fig. 6).

Figure 6
Figure 6

Relative levels of mRNA for Hoxa genes in epididymis (Epi) and vas deferens (Vas) of Amhr2cre/+SmoM2-mutant males and Amhr2+/+SmoM2 control males on day 1 of age. Data are mean ± s.e.m., based on RNA preparations from 4 mice. Bars without a common superscript are significantly different (P < 0.05).

Citation: Reproduction 153, 4; 10.1530/REP-16-0562

Germ cell development in Amhr2cre/+SmoM2 males

Because Amhr2cre-mediated recombination and expression of dominant active SMOM2 occurred in Leydig cells (Fig. 2), it is possible that germ cell development was altered. In 12-week-old adult mice, testes weight was reduced 22% in mutants compared to that in controls (Fig. 7, panel A). The number of detergent-resistant elongated spermatids isolated per gram of testis did not differ in mutants and controls but based on the decreased weight of the testes, the total number per two testes was reduced 21% in mutants (Fig. 7, panels B and C). The number of sperm isolated from the caudal epididymides was also reduced 36% in mutants compared to that in controls (Fig. 7, panel D). The characteristics of sperm recovered from the epididymis were similar in mutant and control mice including total motility and forward motility, both immediately after collection and after capacitation for two hours (Table 1). Two hours after collection, sperm from both control and mutant mice showed qualitatively similar signs of hyperactivation and the extent of protein tyrosine phosphorylation in sperm lysates did not differ (lane intensity relative to time 0 was 194 ± 22% in sperm from control mice vs 238 ± 11% in sperm from mutant mice, P > 0.05) indicating that capacitation occurred normally (Fig. 8). In addition, analysis of histological sections through the testis showed that progression of seminiferous tubules to late stages of spermatogenesis, stages VII and VIII, did not differ between control mice (6.0% and 6.4% respectively) and mutant mice (5.6% and 5.1% respectively; P = 0.67 and 0.17 respectively; n = 3 mice from each genotype). An endpoint of androgen action, the weights of the seminal vesicles, did not differ between control and mutant mice (0.24 ± 0.01 and 0.23 ± 0.01 g respectively; P = 0.42, n = 8 mice of each genotype). Taken together, the reduction in sperm number in mutant males does not appear to be due to differences in the process of spermatogenesis but rather to the decrease in the weight of the testes.

Figure 7
Figure 7

Testis weight, the number of elongated spermatids in the testis, and the number of sperm in the cauda epididymis were determined in 12 week old Amhr2cre/+SmoM2-mutant males and Amhr2+/+SmoM2 control males (A–D). Data are mean ± s.e.m. of 5–10 mice. Within each panel, bars without a common superscript are significantly different (P < 0.05).

Citation: Reproduction 153, 4; 10.1530/REP-16-0562

Figure 8
Figure 8

Western blot analysis of protein tyrosine phosphorylation in lysates of epididymal sperm. Lysates from 1 × 106 sperm were resolved by 12% SDS-PAGE. Lysate in lane 1 was from an Amhr2+/+SmoM2 control male processed immediately after collection (non-capacitated). Lysates in lanes 2–7 were from sperm collected after 2 h incubation in capacitation medium either from three different control mice (lanes 2–4) or from three different Amhr2cre/+SmoM2-mutant mice (lanes 5–7). The blot was stripped and re-probed for β-tubulin as a loading control (bottom panel).

Citation: Reproduction 153, 4; 10.1530/REP-16-0562

Table 1

Motility of epididymal sperm in Amhr2+/+SmoM2 control and Amhr2cre/+SmoM2-mutant mice.

GenotypePercent at 0 haPercent at 2 h capacitationa
TotalbProgressivecTotalbProgressivec
Amhr2+/+SmoM27996.17590.2
Amhr2cre/+SmoM27996.87590.6
t-test (P)0.970.430.870.81

motility was assessed at the time of collection (0 h) and at 2 h after incubation in capacitation media (n = 6 mice/genotype).

Total motility was the percentage of the sperm with flagellar activity (100 sperm/mouse assessed).

Progressive motility was the percentage of motile sperm that were moving in a forward trajectory (60–110 sperm/mouse assessed).

Sperm transport along the male reproductive tract in Amhr2cre/+SmoM2 mice

The patency of the vas deferens was tested by infusion of trypan blue into the lumen. The vas deferens was unusually convoluted in mutant mice compared to its straight trajectory in controls and had regions in which much reduced trypan blue was observed within the lumen (Fig. 9, panels A, B and C). Whole mount phase-contrast images revealed a uniform diameter of the lumen of the vas deferens in control mice at positions both close to the connection with the epididymis and close to the connection with the seminal vesicle (Fig. 9, panels D and E). In contrast, these regions of the vas deferens in mutant mice had constrictions, which reduced the diameter of the lumen (Fig. 9, panels F and G).

Figure 9
Figure 9

Altered structure of the vas deferens in Amhr2cre/+SmoM2-mutant mice compared to Amhr2+/+SmoM2 control mice. Patency of the vas deferens was tested by injection of trypan blue into the vas deferens of control (panel A) and mutant mice (panels B and C). White arrowheads (panel C) point to regions lacking trypan blue. Whole mount phase contrast images of vas deferens from control and mutant mice at the epididymal end and at the seminal vesicle end are shown in panels D–G. White arrows in panels D–F point to the lumen of the vas deferens full of sperm while in panel G the arrow points to a region in which the lumen is constricted. Arrowheads in panel F point to areas of constriction in the lumen of the vas deferens. The size reference bar in panel G applies to all panels and represents 1.0 mm in panels A–C and 1.5 mm in panels D–G. Images are representative of analysis of 3–4 mice at 12–16 weeks of age.

Citation: Reproduction 153, 4; 10.1530/REP-16-0562

It is possible that reduced fertility of Amhr2cre/+SmoM2 male mice is due to impaired transport of sperm through the constricted lumen of the tortuous vas deferens, leading to decreased numbers of sperm in the ejaculate. To test this, sperm were flushed from the reproductive tracts of wild-type females that had been superovulated, caged overnight with mutant or genotype-matched control males and that displayed copulatory plugs in the morning. The number of ejaculated sperm recovered from the uteri was 78% less following mating with a mutant male compared to a control male (0.23 ± 0.03 vs 1.0 ± 0.06 million sperm; P < 0.001, n = 5 mice). The percentage of recovered sperm that were motile was similar for controls and mutants (50.3 ± 3.5% and 50.3 ± 3.1% respectively; P = 0.99, n = 5 mice). Copulatory plugs generated by mating with control and mutant males had grossly similar consistency upon dissection, were firmly adhered within the vagina and did not differ in weight (0.06 ± 0.01 and 0.06 ± 0.01 g respectively; P = 0.84, n = 5 mice). Therefore, a major difference between Amhr2cre/+SmoM2 mutant and control males is that the number of sperm in the ejaculate is reduced.

Discussion

Amhr2cre/+SmoM2 male mice are sub-fertile, generating half the number of pups per litter in matings with wild-type females. Mutant mice had extra tissue extending along the length of each epididymis and vas deferens that was not present in control mice. Several lines of evidence indicate that this extra tissue is derived from Müllerian ducts that failed to regress normally. The location of the extra tissue is consistent with the position of persistent Müllerian tissue in males of other genetically altered mouse strains (Behringer et al. 1994, Mishina et al. 1996, Parr & McMahon 1998, Jamin et al. 2002, Orvis et al. 2008, Tanwar et al. 2010b, Kobayashi et al. 2011). Amhr2 is known to be expressed in the developing Müllerian duct beginning at 12.5 days post coitus (dpc) and the Amhr2cre allele has been shown to direct recombination in Müllerian-derived tissues (Jamin et al. 2002, Arango et al. 2008, Orvis et al. 2008, Tanwar et al. 2010b, Kobayashi et al. 2011). We confirmed that cre-mediated recombination had occurred in the uterus of Amhr2cre/+tdTomato reporter mice by day 1 of age. In addition, levels of mRNA for genes that are transcriptional targets of HH signaling, Gli1, Ptch1 and hedgehog-interacting protein (Hhip) were elevated in the uterus of mutant compared to those in control females and were similarly elevated in the extra tissue attached to the epididymis and vas deferens of mutant males. These findings provide evidence that the extra tissue attached to the reproductive tract of mutant males is derived from Müllerian tissue. In addition, the data are consistent with overactivation of HH signaling leading to the persistence of this Müllerian tissue in males.

AMH is secreted by Sertoli cells of the testis beginning at 11.5 dpc, whereas in females, expression does not begin until after birth in granulosa cells. AMH acts on AMHR2 in the Müllerian duct to induce regression of the duct beginning around 14.5 dpc (Arango et al. 2008). Deletion of both alleles of either Amh or Amhr2 in mice results in persistence of the Müllerian duct in males but causes no detectable abnormality in the reproductive tract of female mice (Behringer et al. 1994). Overactivation of HH signaling in Amhr2cre/+SmoM2 mice was associated with very low levels of Amhr2 mRNA both in the uterus of females and in the extra tissue attached to the reproductive tract of males. This finding is consistent with the possibility that Müllerian ducts of Amhr2cre/+SmoM2-mutant males may have failed to fully regress because of dramatically reduced Amhr2 expression.

A number of factors influence the expression or activity of AMHR2 in the Müllerian duct and thereby regulate duct regression. AMH, like other ligands in the transforming growth factor-β family, acts by binding to a type II receptor that forms a heteromeric complex with a type I receptor that is required for downstream signaling through receptor SMADS. The type II receptor used by AMH is AMHR2. Conditional gene knockout studies showed that both bone morphogenetic protein receptor (BMPR1A) and activin receptor (ACVR1) can function redundantly as type I receptors mediating the action of AMH, with deletions leading to persistent Müllerian ducts in males (Jamin et al. 2002, Orvis et al. 2008). Additional conditional gene knockouts showed that SMAD5 is the receptor SMAD that preferentially mediates AMH-induced regression of the Müllerian duct (Orvis et al. 2008). Expression of Amhr2 in the mesenchyme of the Müllerian duct is dependent on the expression of Wnt7a in the ductal epithelium, and Müllerian ducts persist in male Wnt7a-null mice (Parr & McMahon 1998). Downstream of AMHR2 signaling, β-catenin is activated in the mesenchyme of the Müllerian duct; conditional deletion of β-catenin in mice caused retention of the Müllerian duct in males (Kobayashi et al. 2011). The importance of AMH signaling for duct regression is exemplified by the fact that most human patients with persistent Müllerian duct syndrome had mutations in either the Amh gene (45%) or the Amhr2 gene (39%) (Picard & Belville 2002).

The persistent Müllerian tissue in Amhr2cre/+SmoM2 males is a relatively undifferentiated solid mass of stromal tissue that runs alongside the epididymis and vas deferens. As for other mouse models in which the Müllerian duct persists in males, the extra tissue can be easily separated from the male tract by cutting common connective tissue between them (Behringer et al. 1994, Mishina et al. 1996, Jamin et al. 2002, Orvis et al. 2008). However, in other reported mouse models with persistent Müllerian ducts, there is greater differentiation of the duct along the female pathway. In male mice null for Amh and Amhr2, the persistent Müllerian tissue develops into a differentiated female tract including coiled oviducts and a uterus with distinct luminal epithelium, glands and endometrial and muscle layers (Behringer et al. 1994, Mishina et al. 1996). Similarly, Wnt7a-null males have persistent Müllerian ducts that develop into clearly identifiable oviducts and uterus; however, in both males and females, differentiation of the Müllerian duct is altered, generating a uterus that is deficient in glands and has altered stromal components. These findings demonstrate that although WNT7A is essential for Müllerian duct regression in males, it is also required for normal differentiation of the duct in females (Parr & McMahon 1998). Male mice with conditional deletion of Acvr1 and Bmpr1a develop oviducts and uterus, but uterine stromal tissue is reduced compared to that in wild-type females (Jamin et al. 2002, Orvis et al. 2008). Our data show that the retained Müllerian tissue in male Amhr2cre/+SmoM2 mice is undifferentiated. However, the Müllerian primordia in this strain has the potential to develop along the female pathway as in female Amhr2cre/+SmoM2 mice, all components of the reproductive tract are formed including poorly coiled oviducts and uterine horns that lack glands and have altered luminal epithelium (Migone et al. 2012). Taken together, our findings suggest that partial regression of the Müllerian duct occurs in Amhr2cre/+SmoM2-mutant males, perhaps due to reduced levels of Amhr2 mRNA, generating remnant tissue that cannot fully develop along the female pathway.

The vas deferens of Amhr2cre/+SmoM2-mutant males was unusually tortuous and had localized regions of luminal constriction. This phenotype was associated with a 78% reduction in the number of sperm recovered from the female reproductive tract after mating. The fact that the number of sperm in the epididymides was reduced 36% may have also contributed to the reduction of sperm in the ejaculate. Other mouse models with persistent Müllerian tissue were infertile, largely due to failure of sperm to exit the male tract. For example, in male mice null for either Amhr2 or Amh, the development of the persistent Müllerian duct into a highly differentiated female tract in some way prevents the normal passage of sperm, and this causes infertility (Behringer et al. 1994, Mishina et al. 1996). A similar failure of the vas deferens to make the appropriate connection at its distal end due to the presence of a persistent Müllerian duct was reported for mice with targeted deletion of Wnt7a although morphological details were not provided (Parr & McMahon 1998). Although sperm passage was not completely blocked in Amhr2cre/+SmoM2-mutant males, the approximately 5-fold reduction of sperm number in the ejaculate compared to the 1.6-fold reduction of sperm in the caudal epididymis suggests that blockage of the vas deferens contributed to subfertility.

The persistent Müllerian tissue attached to the male reproductive tract in Amhr2cre/+SmoM2 mice may have caused the vas deferens to develop with unusual tortuosity and luminal constrictions that impeded the transport of sperm. However, it was also possible that increased HH signaling in the developing male reproductive tract contributed to altered morphology and subfertility. Examination of the tdTOMATO reporter protein indicated Amhr2cre-mediated recombination in the tubules of the epididymis and low-level signal in the vas deferens on day 1 of age. Although levels of mRNA for HH target genes (Gli1, Ptch1, Hhip) were uniformly slightly elevated in epididymis and vas deferens of mutant compared to those in control mice on day one of age, these moderate differences were not statistically significant. This suggests that either expression of SMOM2 did not increase HH signaling in these tissues or that any potential change in the levels of mRNA for HH target genes during embryonic development may have normalized by day 1 of age. Hoxa genes were examined because they are known to play a role in determining the morphological and functional identity of distinct regions of the male and female reproductive tracts (Hsieh-Li et al. 1995, Benson et al. 1996, Hannema & Hughes 2007). Hoxa9, 10, 11 and 13 did not differ in their pattern of expression in control and mutant mice in the epididymis and vas deferens. Taken together, our results, combined with previous findings in other mouse models with persistent Müllerian ducts, suggest that the presence of Müllerian tissue impairs the development of the male reproductive tract and prevents normal exit of sperm.

The presence of the fluorescent reporter protein tdTOMATO in interstitial cells outside the seminiferous tubules in testis of 1-day-old Amhr2cre/+tdTomato mice suggests that Amhr2cre-mediated recombination occurred mainly in Leydig cells consistent with previous studies using the Amhr2cre allele (Boyer et al. 2008, Tanwar et al. 2010a). However, on day 1, levels of mRNA for HH target genes, Gli1, Ptch1 and Hhip, were not significantly elevated in testis of Amhr2cre/+SmoM2 mutants compared to those in controls. This result suggests that any overactivation of HH signaling that may have occurred during the development of testis in mutant mice had normalized by day 1 of age. DHH influences differentiation of fetal Leydig cells and is also essential for establishing the new population of adult Leydig cells by stimulating proliferation and differentiation of stem cells located on the surface of seminiferous tubules and along blood vessels, a process that occurs during the first several weeks of life in rats (Chen et al. 2016). Although testis weight was reduced 22% percent in Amhr2cre/+SmoM2-mutant mice compared to that in controls, seminal vesicle weight and mating behavior did not differ suggesting that androgen production by Leydig cells was not substantially altered. Previous work showed that Amhr2cre-mediated recombination was low to non-detectable in Sertoli cells of newborn mice (Tanwar et al. 2010a), and this finding is consistent with the lack of definitive tdTOMATO signal within the seminiferous tubules of 1-day-old Amhr2cre/+SmoM2-mutant mice. However, Amhr2cre-mediated recombination was reported to be prominent in Sertoli cells of adult mice (Tanwar et al. 2010a). It is possible that overactivation of HH signaling in Sertoli cells of Amhr2cre/+SmoM2-mutant mice accelerated their differentiation, before the completion of replication cycles that take place during the first two weeks of life (reviewed in Holsberger et al. 2003) and thereby reduced testis size.

In Amhr2cre/+SmoM2-mutant males, the number of elongated spermatids per gram of testis was not altered, but testis weight and the total number of spermatids produced was reduced, and this likely contributed to the reduced numbers of sperm in the epididymides. However, the proportion of the seminiferous tubules in stages VII and VIII and multiple aspects of sperm function were normal in mutant mice including motility, hyperactivation and capacitation. These findings suggest that subfertility of Amhr2cre/+SmoM2-mutant males was not due to a direct effect of overactivation of HH signaling on the development of germ cells.

In summary, overactivation of the HH signaling pathway in the developing Müllerian duct of Amhr2cre/+SmoM2 mice caused the persistence of Müllerian tissue in males. Levels of Amhr2 mRNA in the uterus of mutant females was dramatically reduced relative to genotype-matched controls and was similarly low in the persistent tissue present along the reproductive tract in mutant males, suggesting that abnormal persistence of Müllerian tissue in males may have been mediated by inhibition of signaling through AMHR2. The persistence of a relatively small amount of undifferentiated Müllerian tissue within the male reproductive tract and the associated changes in tract morphology contributed to a reduction in the number of sperm in the ejaculate. These findings demonstrate that relatively subtle changes in the male tract associated with persistent Müllerian tissue have the potential to remain undetected but can cause reduced fertility.

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 was supported by the Eunice Kennedy Shiver National Institute of Child Health and Human Development grant R03HD057648 (to S M Q), Research Initiation Award 0547373 from the National Science Foundation ADVANCE Institutional Transformation to Cornell University (to S M Q) and the Center for Vertebrate Genomics, Cornell University (to S M Q).

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Figures

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    Epididymis and vas deferens of Amhr2cre/+SmoM2-mutant males and Amhr2+/+SmoM2 control males at 12 weeks of age. Extra tissue, indicated by brackets in panels B and D, is present alongside the length of the epididymis and vas deferens in mutant mice and is not present in controls. The size reference bar shown in panel D applies to all panels and represents 1 mm.

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    Sites of cre-mediated recombination in reproductive tracts and gonads of male and female Amhr2cre/+tdTomato mice on day 1 of age, determined by imaging of the tdTOMATO reporter protein by confocal imaging of whole-mount tissues. Tissues shown are epididymis (A and B), vas deferens (C and D), testis (E and F) and uterus (G and H). In panel F, arrows point to fluorescent Leydig cells. The size reference bar in panel H applies to all panels and represents 100 µm for panels A–F and 200 µm for panels G and H. Images are representative of analysis of 2–4 mice of each genotype.

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    Relative levels of mRNA for genes that are known transcriptional targets of HH signaling in reproductive tract tissues of Amhr2cre/+SmoM2-mutant males and Amhr2+/+SmoM2 control males on day 1 of age. Tissues analyzed are testis, epididymis (Epi), vas deferens (Vas), uterus and the extra tissue attached to the epididymis and vas deferens of Amhr2cre/+SmoM2-mutant mice. Data are mean ± s.e.m., based on RNA preparations from 4 mice. Within each panel, bars without a common superscript are significantly different (P < 0.05).

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    Hematoxylin and eosin-stained sections of the reproductive tract of Amhr2cre/+SmoM2-mutant males and Amhr2+/+SmoM2 control males at 12 weeks of age. Extra tissue attached to the vas deferens and epididymis of mutant males is indicated by brackets in panels B and G respectively. Below each low power image (panels A, B, F and G) are higher power images of the regions outlined by boxes (panels C, D, E, H, I and J). The size reference bar in panel J applies to all panels; it represents 500 µm for panels A, B, F and G; 100 µm for panels C, D, E, H and I and 200 µm for panel J. Images are representative of analysis of 3 mice of each genotype.

  • View in gallery

    Relative levels of mRNA for Amhr2 in reproductive tract tissues of Amhr2cre/+SmoM2-mutant males and Amhr2+/+SmoM2 control males on day 1 of age. Tissues analyzed are testis, epididymis (Epi), vas deferens (Vas), uterus and the extra tissue attached to the epididymis and vas deferens of Amhr2cre/+SmoM2-mutant mice. Data are mean ± s.e.m., based on RNA preparations from 4 mice. Bars without a common superscript are significantly different (P < 0.05).

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    Relative levels of mRNA for Hoxa genes in epididymis (Epi) and vas deferens (Vas) of Amhr2cre/+SmoM2-mutant males and Amhr2+/+SmoM2 control males on day 1 of age. Data are mean ± s.e.m., based on RNA preparations from 4 mice. Bars without a common superscript are significantly different (P < 0.05).

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    Testis weight, the number of elongated spermatids in the testis, and the number of sperm in the cauda epididymis were determined in 12 week old Amhr2cre/+SmoM2-mutant males and Amhr2+/+SmoM2 control males (A–D). Data are mean ± s.e.m. of 5–10 mice. Within each panel, bars without a common superscript are significantly different (P < 0.05).

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    Western blot analysis of protein tyrosine phosphorylation in lysates of epididymal sperm. Lysates from 1 × 106 sperm were resolved by 12% SDS-PAGE. Lysate in lane 1 was from an Amhr2+/+SmoM2 control male processed immediately after collection (non-capacitated). Lysates in lanes 2–7 were from sperm collected after 2 h incubation in capacitation medium either from three different control mice (lanes 2–4) or from three different Amhr2cre/+SmoM2-mutant mice (lanes 5–7). The blot was stripped and re-probed for β-tubulin as a loading control (bottom panel).

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    Altered structure of the vas deferens in Amhr2cre/+SmoM2-mutant mice compared to Amhr2+/+SmoM2 control mice. Patency of the vas deferens was tested by injection of trypan blue into the vas deferens of control (panel A) and mutant mice (panels B and C). White arrowheads (panel C) point to regions lacking trypan blue. Whole mount phase contrast images of vas deferens from control and mutant mice at the epididymal end and at the seminal vesicle end are shown in panels D–G. White arrows in panels D–F point to the lumen of the vas deferens full of sperm while in panel G the arrow points to a region in which the lumen is constricted. Arrowheads in panel F point to areas of constriction in the lumen of the vas deferens. The size reference bar in panel G applies to all panels and represents 1.0 mm in panels A–C and 1.5 mm in panels D–G. Images are representative of analysis of 3–4 mice at 12–16 weeks of age.

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