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,
From the * Glickman Urological and Kidney
Institute, Cleveland Clinic, Cleveland, Ohio; the
Research Service, Wade Park Cleveland Veterans
Affairs Medical Center, Cleveland, Ohio; The
Berman-Gund Laboratory for the Study of Retinal Degenerations, Harvard Medical
School, Massachusetts Eye and Ear Infirmary, Boston, Massachusetts; and the
Department of Biomedical Engineering, Lerner
Research Institute, Cleveland Clinic, Cleveland, Ohio.
| Correspondence to: Dr Margot Damaser, Department of Biomedical Engineering, The Cleveland Clinic, 9500 Carnegie Ave ND20, Cleveland, OH 44195 (e-mail: damasem{at}ccf.org). |
| Received for publication July 3, 2008; accepted for publication January 21, 2009. |
Our objective was to investigate the genitourinary defects and fertility of
the male lysyl oxidase-like 1 gene (Loxl1) knockout
(Loxl1–/–) mouse, with particular attention to
fecundity and testicular, epididymal, gubernacular, and penile histopathology,
which may lead us to a better understanding of the role of the
elastin-homeostasis gene, LOXL1, in male sexual development. Genital
morphometric evaluation of 6- to 9-month-old male
Loxl1–/– mice (n = 26) was compared with
C57Bl/6 controls (n = 24). Measurements included: body weight, scrotal
development, evidence of feminization (nipples or vaginal pouch), penile
malformations, anogenital distance, and absence/presence and size of perineal
bulge. Sperm production was estimated using a standardized technique. A
breeding program was conducted to determine how much of the infertility
observed in Loxl1–/– pairs was due to the male
factor. Finally, we performed histopathologic comparison of the genitourinary
organs of Loxl1–/– and control mice.
Loxl1–/– mice weighed less than their
age-matched C57Bl/6 counterparts (P < .001). Size-adjusted
perineal bulge was larger (P < .001), and resting location of the
gonads was higher intra-abdominally (P = .048) in the
Loxl1–/– mice. Estimates of daily sperm counts
revealed that the Loxl1–/– mice had lower
sperm production (P = .048). Loxl1–/–
males bred with control females demonstrated relative fecundity values
intermediate between Loxl1–/– pairs (lowest
fecundity) and control pairs (highest fecundity), suggesting a component of
male-factor infertility. No histologic differences were noted using
hematoxylin-eosin or specialized elastin staining of the gonads, gubernaculum,
and penis. Although further studies are warranted, these findings suggest a
subtle and likely multifactorial role of the LOXL1 protein in male sexual
development and fertility.
Key words: Mouse, elastin, penis, reproductive tract, semen analysis, testis, mouse model
Loxl1 knockout mice (Loxl1–/–) demonstrate enlarged alveoli, increased skin laxity, vascular abnormalities, uterine prolapse, and rectal prolapse (Liu et al, 2004). During breeding of Loxl1–/– mice for studies of pelvic organ prolapse in females, it was noted that Loxl1–/– males were subfertile. When their male genitalia were examined, the male mice demonstrated phenotypic features suggestive of impaired testicular descent, including a perineal bulge, less developed scrotal morphology, reduced anogenital distance (AGD), and poorly palpable or nonpalpable testicles. To date, no studies investigating the role of LOXL1 in male infertility and cryptorchidism have been reported in the literature.
The purpose of this project was to investigate the genitourinary defects and fertility of the male Loxl1–/– mice, with particular attention to fecundity and testicular, epididymal, gubernacular, and penile histopathology, with the expectation that this would lead to an improved understanding of the role of the elastin-homeostasis gene, LOXL1, in male sexual development.
Materials and Methods
This investigation consists of: 1) morphometric evaluation of male Loxl1–/– gonads and genital organs, 2) a breeding program to estimate the relative fecundity of male Loxl1–/– mice, and 3) histopathologic evaluation of the genitourinary organs of Loxl1–/– male mice.
Loxl1–/– Mouse Model and Animal Facilities
Loxl1–/– mice on a mixed C57Bl/6 and Sv129
background backcrossed to C57Bl/6 for several generations were maintained in
the Cleveland Wade Park Veterans Affairs animal research facility. Age-matched
control mice were male C57Bl/6 mice (Charles River Laboratories, Wilmington,
Massachusetts) and were housed in separate cages in identical environments.
All animals were maintained in a 12-hour light-dark housing facility at
22°C and were given free access to food and water. All breeding and animal
handling practices were implemented per established procedures within the
Cleveland Wade Park Veterans Affairs Animal Research Facility and were
conducted in accordance with the Institutional Animal Care and Use Committee.
Breeding to obtain experimental mice and breeding to maintain the colony were
identical.
Dissection Techniques
Adult males 6 to 9 months of age were used for this study. Intraperitoneal
ketamine and xylazine (100 mg/kg and 10 mg/kg, respectively) mixture was used
for anesthesia. Genitourinary morphometric measurements were obtained for male
Loxl1–/– (n = 26) and male C57Bl/6 (n = 24)
mice (see below). Prior to euthanasia, a midline incision was made from the
xiphoid to pubis, and the abdominal contents were carefully inspected with the
assistance of a dissecting microscope or x2.5 loupe dissecting lenses.
Fluid was collected from the vas deferens of live animals. Animals then were
euthanized using intracardiac pentobarbital (100 mg/kg). At necropsy, the left
testicle was removed for estimation of daily sperm production (see below). The
right testicle, epididymis, and vas deferens with gubernacular and posterior
abdominal wall attachments as well as the penis were removed for histologic
evaluation (see below).
Morphometric Measurements of Loxl1–/– Gonads and Genital Organs
The following variables were collected after induction of anesthesia and
prior to dissection: age (weeks); body weight (grams); scrotal development (1,
no rugae/pigmentation; 2, rugae with poor sac development and without evidence
of testis; and 3, descended testes in well-formed sac); evidence of
feminization (nipples or vaginal pouch); penile malformations; AGD (base of
the penis to the lumen of the anus); absence/presence of perineal bulge; and
size of perineal bulge. All measurements were made using digital calipers, and
the perineal bulge was characterized using a modified version of the Mouse
Pelvic Organ Prolapse Quantification system, a system originally designed for
female mice (Weislander et al,
2006). The height of the perineal bulge was measured in
millimeters from the point of insertion in the inner thigh to the maximal
height of the perineum (90° to the insertion of the thigh, posterior to
the genital tubercle). Perineal bulge and AGD were standardized to mass to
correct for differences in mass between the 2 groups.
After all measurements were completed, a midline incision was carefully made, and the abdominal contents were examined. Because mice are quadrapeds with open inguinal rings, their testicles move freely from scrotum to abdomen throughout their life. Therefore, the resting location of each testicle while the animal was under anesthesia was chosen as a surrogate variable for the average position of the testes during the resting state. If the testicle had to be pulled out of the scrotal sac, the resting position was considered "scrotal" in location. Testicles superior to the scrotum and inferior to the bladder were graded as "pelvic." When the testicles were at or superior to the level of the bladder, they were graded as "abdominal" in location. For grading purposes, each location was assigned an ordinal number: 1, scrotal; 2, pelvic; or 3, abdominal.
Semen Evaluation for Qualitative Motility Assessment
Semen evaluation was performed in 10 animals (5
Loxl1–/– and 5 C57Bl/6). A sperm suspension
was prepared by expressing the contents of the left epididymis into 100 µL
of Quinn Sperm Washing Medium (modified human tubal fluid [HTF] with 5.0 mg/mL
human albumin; SAGE In-Vitro Fertilization Inc, Trumbull, Connecticut)
maintained at 37°C. This suspension was immediately vortexed and then
observed using standard light microscopy. Motility was graded using a
qualitative motility assessment (A, straight, forward progression; B, not
straight but still progression in a forward direction; C, random motility; D,
immotile). A total of 75–100 sperm were graded per sample.
Breeding Program
A breeding program was conducted to characterize the type of infertility
(male factor, female factor, combined) that was responsible for the
subfertility noted in the Loxl1–/– pairs
relative to the C57Bl/6 pairs (Charles River Laboratories, Wilmington,
Massachusetts). Ten sexually mature (8–12 weeks old)
Loxl1–/– males were paired with ten
12-week-old C57B1/6 females (Charles River Laboratories) in single pairs over
a 3-month time period. All mice that were used for this portion of the study
were proven breeders, and therefore had successfully bred at least one time
prior to inclusion in this program. Careful breeding records were collected to
obtain the length of time of continuous pairing, date of deliveries, litter
size, and number of litters. Litters remained in the parent cages until
postnatal days 21–22. The relative fecundity was calculated using the
following equation: Relative fecundity = (productive mating frequency) x
(litter size) x (number of litters). The relative fecundity of each
breeding pair was calculated, and an average fecundity value was generated.
This value was then compared with average fecundity estimates measured in our
facility for C57Bl/6 mating pairs, Loxl1–/–
mating pairs, and Loxl1–/– female-C57Bl/6 male
mating pairs. Pairs were bred for a minimum of 3 months and a maximum of 15
months. Loxl1–/– females with severe (grade 3)
pelvic organ prolapse were excluded from fecundity calculations because the
pelvic organ prolapse may be a confounding factor impacting fecundity.
Evaluation of Sperm Production
The left testicle was harvested and cleaned of its investments using
microdissection techniques, and the tissue was immediately flash-frozen in
liquid nitrogen and maintained in a freezer at –80°C until
homogenization was performed. All specimens were weighed immediately prior to
homogenization. The left testicle was then suspended in 1 mL of saline
containing 0.05% Triton X-100 (Sigma-Aldrich, St Louis, Missouri) and 0.25
mol/L Thimerosol (Sigma-Aldrich) and homogenized for 3 minutes using a tissue
homogenizer (Power Gen 125; Thermo Fisher Scientific Inc, Waltham,
Massachusetts). Two aliquots of 20 µL were then placed on a Makler Counting
Chamber (MidAtlantic Diagnostics, Mt Laurel, New Jersey), and 3 randomly
selected areas were counted for each aliquot (Sohara et al, 2006). An average
count was generated by calculating an average of these 6 separate counts. The
cells that survived the homogenization procedure were step 14–16
spermatids. The number of spermatids counted was divided by testicular weight
to provide an average number of sperm per gram of testicular tissue. Then,
because developing spermatids spend 4.84 days in steps 14–16 of
spermatogenesis, the number of spermatids per gram was divided by 4.84 to
provide an estimate of daily sperm production (Sohara et al, 2006).
Histologic Analysis of Loxl1–/– Genitourinary Organs
The right testicle, epididymis, and right gubernacula were harvested en
bloc and fixed in formalin for 24 hours, then transferred to 70% alcohol. The
penis was degloved, amputated at the bladder neck, and fixed in a similar
manner. Tissues were microdissected prior to embedding, sectioning, and
staining. Sections of the testis with epididymis and gubernacular attachments
were formalin fixed and paraffin embedded for histologic evaluation using
hematoxylin and eosin (H&E), von Gieson, and Movat Pentachrome stains.
Statistics
Data are presented as mean ± SEM for each catagoric and continuous
measurement. Univariate statistical analysis was conducted using a
Mann-Whitney rank sum test and Sigma-Stat version 3.5 (Systat Software Inc,
Port Richmond, California) software. A P value less than .05 was
considered statistically significant.
Results
Morphometric Measurements
There were no significant differences in age between the
Loxl1–/– male mice (n = 26) and the C57Bl/6
male mice (n = 24). The Loxl1–/– males weighed
significantly less than their age-matched C57Bl/6 counterparts (P
< .001). None of the C57Bl/6 male mice had a visible perineal bulge on
exam, whereas 14 of 21 Loxl1–/– male mice had
prominent perineal bulges (Table
1). The mass-adjusted perineal bulge was significantly larger on
examination in the Loxl1–/– mice (P
< .001). Although the mass-adjusted AGD did not differ significantly
between the 2 groups, Loxl1–/– males
demonstrated smaller AGD, likely owing to their smaller body size
(Figure 1).
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No significant differences in scrotal development, penile morphology, or other markers of feminization were noted. Incidentally, 2 Loxl1–/– male mice had rectal prolapse on exam. The resting location of C57Bl/6 testicles (n = 24) was lower than the Loxl1–/– (n = 24) testicles (P = .048; Figure 1c).
Semen Evaluation for Qualitative Motility Assessment
Qualitative motility assessment of the
Loxl1–/– (n = 3) and C57Bl/6 (n = 3) mice
demonstrated sperm with forwardly progressive motility. No significant
differences were observed between the 2 groups.
Evaluation of Sperm Production
Estimated daily sperm production of the
Loxl1–/– male mice (59 x 106
± 3.57 SEM; n = 17) was significantly lower than the C57Bl/6 male mice
(87 x 106 ± 7.89 SEM; n = 14; P = .002;
Figure 2).
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Histologic Analysis
Histologic exam of the testicle, epididymis/gubernaculums, and penile
tissue was compared between Loxl1–/– mice and
C57Bl/6 mice using H&E, von Gieson (elastin), and Movat Pentachrome
staining (Figure 1d). Although
individual differences were seen in some of the elastin-containing
Loxl1–/– tissues, particularly in the region
of the penile tunica albuginea, consistent differences between the 2 groups
were not observed, and are therefore not presented.
Figure 3 demonstrates
representative micrographs of the corpora cavernosa at x4
(Figure 3a and c) and x40
(Figure 3b and d)
magnification, showing no gross differences by H&E staining.
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Discussion
Cryptorchidism is the most common of newborn anomalies, occurring in about 3% of full-term newborns and up to 30% of preterm newborns (Schneck and Bellinger, 2002). It has been associated with testicular cancer/carcinoma in situ and infertility (Schneck and Bellinger, 2002). Despite the frequency of cryptorchid testes, surprisingly little is known about the mechanisms that lead to testicular descent. Hormonal and cytokine signaling, intra-abdominal pressure, signaling from the genitofemoral nerve, and guidance from the gubernaculum are a few of the factors believed to influence testicular descent (Schneck and Bellinger, 2002). As a result, cryptorchidism is seen in intersex disorders (from aberrant hormonal signaling), defects of the abdominal wall (prune belly syndrome), and a number of other disorders and diseases, as well as idiopathically (Schneck and Bellinger, 2002).
Testicular descent is believed to occur in 2 distinct phases: intra-abdominal descent and inguinal/scrotal descent, with the intra-abdominal portion thought to be mediated by gubernacular guidance. The exact mechanism by which the gubernaculum participates in testicular descent is largely unknown. Intra-abdominal descent occurs between days 15.5 and 17.5 of gestation in mice (Zimmerman et al, 1999). Inguinal/scrotal descent occurs after gestation and is completed by approximately 3 weeks of life in the mouse. During inguinal/scrotal descent, the gubernacular bulb is believed to invaginate and be replaced by mesenchymal cells, which eventually coalesce into the cremaster muscle (Hutson et al, 1997; Yuan et al, 2006). In the human fetus, the presence of collagen and elastin, as well as striated muscle, increases during gestational weeks 15–29. This is consistent with the timeframe of testicular descent, suggesting that critical remodeling of this structure involving extracellular maintenance proteins facilitates testicular descent (Costa et al, 2002). During testicular migration, gubernacular connective tissue undergoes extensive remodeling and ultimately becomes an essentially fibrous structure rich in collagen and elastic fibers.
The LOXL1 gene (GC15P072005; 7 exons) is located at position 72 005 852 to 72 031 529 of human chromosome 15 (15q24) (Csiszar, 2001). Importantly, 60% of persons affected with Kartagener syndrome (immotile cilia syndrome) demonstrate a chromosomal abnormality that localizes to 15q24-25 (Geremek et al, 2006). In addition, a case report describing 2 male children with interstitial deletions of 15q24 describes patients with hypotonia, dysmorphic facial features, and eye abnormalities, as well as genitourinary defects, specifically microphallus and a small scrotal sac in one child and hypospadias in the other (Cushman et al, 2005). Another study localizes the 15q24 microdeletion syndrome to an area that includes the LOXL1 gene and describes 4 human male patients who present with joint laxity/scoliosis, developmental delay, growth retardation and low body weight, characteristic facial anomalies, eye abnormalities, anomalies of the hands and feet, hypospadias (3 of 4), hearing loss (2 of 4), and recurrent chest infections (2 of 4; Sharp et al, 2007). Some of these features are similar to the findings seen in the Loxl1–/– mice both in this study and in others (Liu et al, 2004; Liu et al, 2006), with growth retardation/low body weight, skin laxity, and alveolar enlargement. Other features (hearing loss, anomalies of the hands, feet, and eyes, and recurrent chest infections) remain to be investigated but could theoretically be related to extracellular matrix malfunction or degeneration. Although hypospadias has been reported in humans with microdeletion of 15q24, we did not observe this finding in Loxl–/– male mice, suggesting a difference in the role of this gene in penile development between species or, alternatively, a function of neighboring genes knocked out in the microdeletion syndrome but not in the Loxl1–/– mice. Although preliminary human studies evaluating the role of the LOXL1 gene in uterine prolapse have been published recently (Klutke et al, 2008), full characterization of fertility (males and females) and pelvic floor anatomy and function (especially in females) in humans with this microdeletion syndrome remains to be published.
This project incorporated multiple methodologies, all aimed at investigating how and where the LOXL1 gene may influence gonadal function, morphology, and fertility, perhaps through abnormal development or degeneration of the gubernaculum. We postulated that lack of LOXL1 may lead to abnormal gonadal development in utero and subsequent evidence of feminization (reduced AGD, a surrogate of hypospadias, and development of nipples) and gonadal dysgenesis. However, our findings only partially support this hypothesis. Our data suggest that the Loxl1–/– mice may demonstrate phenotypes suggestive of testicular maldescent, including the presence of a perineal bulge and higher resting position of the gonads. However, we did not find any additional morphologic changes suggestive of feminization or histologic differences on H&E or von Gieson (elastin) staining in either the gonads or external genitalia.
Our estimates of daily sperm count suggest that although histologic differences in the gonad may not be appreciable, the Loxl1–/– mice appear to have lower sperm production. This may, in part, account for the reduced breeding productivity we noted. Our breeding results suggest that the infertility initially observed in the Loxl1–/– pairs is likely combined male-female factor. Fecundity calculations for the female-factor and male-factor breeding pairs are intermediate between the control (highest fecundity) and Loxl1–/– (lowest fecundity) pairs. Although this study attempted to quantify breeding efficiency through formal fecundity calculations, one factor that we did not assess in our investigation is sexual performance, including mating practices, efficiency, and frequency. This would be quite difficult given the nature of mouse mating practices, which are generally nocturnal and short-lived. Our breeding program results do suggest that there is overall reduced breeding productivity and that at least some of this reduced productivity can be attributed to the male factor. Our gonadal evaluation points to one possible etiology: lower sperm production. However, given the lack of histologically apparent differences in the gonad, the effect of the LOXL1 gene in male sexual function is likely to be more subtle, and possibly multifactorial. Additionally, given its role in elastin repair and remodeling, the effect may be degenerative as opposed to congenital. This is consistent with the findings demonstrated in the female Loxl1–/– mice with aging (Liu et al, 2004; Liu et al, 2006).
We suspected that some of the fertility problems encountered may be due to functional problems of the sperm, given that 60% of patients with Kartegener syndrome demonstrate genetic abnormalities at the 15q24 locus. However, our investigation did not support this, because motility did not differ between the 2 groups. This may in part be due to the small number of motility assessments done and the extreme variability between specimens. Patients with Kartegener syndrome are generally infertile, with immotile or nonprogressively motile sperm (Afzelius and Eliasson, 1983). However, case reports of patients with motile sperm have been published (Conraads et al, 1992). The Loxl1–/– mice, on the other hand, demonstrated only subfertility, and semen analysis showed motile sperm that demonstrate adequate progression. Whether the gene may play a more subtle role in dynein arm structure or function remains to be determined through further ultrastructural investigation of the spermatozoa.
This study provides preliminary data regarding the potential role the LOXL1 gene may play in male mouse sexual function and/or fertility. It does, however, have several limitations. First, although all of the Loxl1–/– mice lacked the Loxl1 gene, these animals were on a mixed C57Bl/6 and 129Sv background, and therefore they were not genetically identical to each other. So, differences between the groups could also be attributed to heterogeneous factors in their genetic backgrounds. Differences between the groups, therefore, could be due to the null mutation in the Loxl1 gene, the mixed background, the effect of strain (129Sv and C57Bl/6), or the effect of the null mutation on the strain. Further studies are needed to identify the exact etiology of the described differences. In addition, reduced fertility is well described in genetic knockout mice as well as all inbred mouse strains (Silver, 1995). Furthermore, the determination of whether this effect is congenital or degenerative remains to be investigated through additional studies that evaluate the organs of the Wolffian system throughout the life cycle of the animal.
Conclusions
Loxl1 knockout male mice demonstrate perineal bulges, more
intra-abdominal resting locations of their testes, smaller body mass, reduced
estimated daily sperm counts, and male-factor infertility. This suggests a
subtle, possibly multifactorial, role for the LOXL1 gene in
fertility, although the specific mechanism(s) remains to be determined.
Further evaluation to determine the specific role of this gene in male sexual
differentiation and fertility should be aimed at localization of the gene
product using immunohistologic techniques and electron microscopy.
Acknowledgments
Technical support was provided by Mrs Linda Vargo. We are grateful for her expertise and commitment to our project.
Footnotes
Supported by the Society for Women in Urology (2007, Elisabeth Pickett Award), the Glickman Urological and Kidney Institute of the Cleveland Clinic, and the Rehabilitation Research and Development Service of the Department of Veterans Affairs.
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