ABSTRACT
The aim of the study was to assess the cumulative effects of aging and Y-chromosome long arm deletion on sperm quality parameters. Motility, mitochondrial activity, and head morphology were evaluated for sperm of 3- and 12-month-old males from B10.BR-Ydel and B10.BR congenic mouse strains. The study revealed that quality and fertilizing potential of sperm produced by younger and older B10.BR males persist on similar levels, but worsen significantly with age of B10.BR-Ydel males. The findings imply that partial Yq deletions might be more harmful for spermiogenesis in advancing age and may be applicable to other species including humans.
Abbreviations: AZF: azoospermia factor; MSYq: male-specific region of the Y-chromosome long arm
Introduction
Late paternity is becoming popular in contemporary societies. Multiple studies investigating the influence of male age on reproductive functions have concluded that with advancing age semen quality and fertilization rates decline significantly. Among sperm quality parameters, motility and morphology were recognized as the most age-dependent [Eisenberg and Meldrum Citation2017; Herati et al. Citation2017]. These parameters are also under strong genetic control of the azoospermia factor (AZF) region of the Y chromosome long arm. Deletions within AZF are a common cause of male infertility or reduced fertility [Roberts Citation1998; Koh et al. Citation2010]. In the present study we examined the combined effects of increased age and Y-chromosome long arm deletion on sperm quality, using a unique model of two congenic mouse strains: B10.BR and B10.BR-Ydel. Animals of both strains have the same genotype, but while B10.BR males possess an intact Y chromosome, B10.BR-Ydel males suffer from a partial deletion of the male-specific region of the Y chromosome long arm (MSYq) which contains multicopy spermiogenic genes [Ellis et al. Citation2007; Soh et al. Citation2014]. Sperm motility, sperm mitochondrial activity, and sperm head morphology were assessed for 3-month-old and 12-month-old B10.BR-Ydel males and were compared with the analogous results obtained for 3-month-old and 12-month-old B10.BR males.
Results and discussion
B10.BR-Ydel males are the classic example of mice suffering from the partial MSYq deletion. They are fertile, but produce sperm of lower quality than their B10.BR counterparts. Numerous past studies revealed frequent morphological and ultrastructural abnormalities [Styrna et al. Citation1991a; Styrna et al. Citation1991b; Styrna et al. Citation2002], deterioration of movement parameters [Grzmil et al. Citation2007], difficulties in crossing uterotubal junction [Kotarska and Lenartowicz Citation2011], aberrant expression of surface receptors [Kotarska et al. Citation2015], and lower fertilization efficiency of B10.BR-Ydel spermatozoa [Xian et al. Citation1992; Styrna et al. Citation2002; Kotarska and Styrna Citation2012]. All these studies were focused, however, only on young adult individuals. The aim of the present study was to assess the effect of aging on quality of sperm produced by B10.BR-Ydel and control B10.BR males. shows that the percent of motile sperm is almost the same in ejaculates of 3-month-old B10.BR-Ydel and B10.BR males. This is in accordance with the former work which showed that although the deletion deteriorates properties of sperm movement, it does not negatively influence the total percentage of motile gametes produced by young B10.BR-Ydel males [Grzmil et al. Citation2007]. The percent of motile sperm in ejaculates of 12-month-old B10.BR males is very similar to the percent recorded for the young animals but is decreased significantly (p<0.05) in the case of 12-month-old B10.BR-Ydel males.
Figure 1. Percent of motile spermatozoa (A), percent of spermatozoa with active mitochondria stained with MitoTracker probe (B), and percent of spermatozoa with deformed heads (C) in semen of 3- and 12-month-old males from B10.BR and B10.BR-Ydel strains (means ± SEM, n=5-8 per group). Significantly different results (p<0.05) are indicated by different superscripts (a, b, and c). BR_3m: 3-month-old B10.BR males, BR-Ydel_3m: 3-month-old B10.BR-Ydel males, BR_12m: 12-month-old B10.BR males, BR-Ydel_12m: 12-month-old B10.BR-Ydel males.
illustrates mitochondrial activity of spermatozoa, directly associated with their ability of movement. The percent of sperm with active mitochondria is only slightly lower for 3-month-old B10.BR-Ydel males than for 3-month-old B10.BR males and equal with the percent characteristic for 12-month-old B10.BR males. But among spermatozoa produced by 12-month-old B10.BR-Ydel males, gametes with active mitochondria are only half as frequent as in samples derived from the three other experimental groups (p<0.01).
It is well established that Yq deletions cause numerous abnormalities of sperm head morphology [Styrna et al. Citation1991a; Styrna et al. Citation1991b; Styrna et al. Citation2002; Touré et al. Citation2004]. As is shown in , we recorded 75.3% of deformed sperm heads in samples derived from 3-month-old B10.BR-Ydel males and 19.6% in samples derived from 3-month-old B10.BR males. The 12-month-old B10.BR males produce slightly more deformed spermatozoa than young B10.BR animals but the difference was not statistically significant. A statistically significant (p<0.05) rise in the frequency of head abnormalities is characteristic for sperm of 12-month-old B10.BR-Ydel males compared with 3-month-old B10.BR-Ydel individuals.
In summary, our assessment of sperm motility, mitochondrial activity, and head morphology revealed that quality of gametes produced by young and older B10.BR males is practically the same, but worsens significantly with aging of B10.BR-Ydel males. Simultaneously, sperm count is similar for all examined groups of mice (data not shown). Additionally, we analyzed the breeding records of the two congenic strains from the last 10 years and counted litters sired within this period by males mated at the age of 3 or 12 months with a female (75 successive males of each category were evaluated). The total numbers of recorded litters were as follows: 70 for 3-month-old B10.BR males (mean litter size 6.03), 57 for 12-month-old B10.BR males (mean litter size 5.04), 60 for 3-month-old B10.BR-Ydel males (mean litter size 5.48), and only 27 for 12-month-old B10.BR-Ydel males (mean litter size 4.12). This clearly shows that the deterioration of sperm quality typical for aged B10.BR-Ydel male mice is associated with reduced fertilization rates. The 12-month-old B10.BR-Ydel males rarely become fathers although young males with the deletion have similar reproductive outcomes as the control, young and older B10.BR males.
All the above observations imply that natural processes of aging initiate or intensify some perturbations in spermiogenesis caused by the Y-chromosome long arm deletion. We have not yet discovered molecular mechanisms underlying the described regularity but we believe that it is worth further investigation, also in the context of human reproduction. The results of our study suggest that Yq deletions could be more harmful for sperm quality and their fertilizing potential in advancing age. This is another argument against too late fatherhood, consistent with many recent works showing a strict correlation between paternal aging and increased risk of sperm defects.
Material and methods
Animals and sperm collection
Experiments were performed on 3- and 12-month-old male mice from the congenic B10.BR/SgSn and B10.BR-Ydel strains maintained at the Institute of Zoology of the Jagiellonian University, Krakow. To avoid genetic divergence between the two strains, in each generation B10.BR-Ydel males were backcrossed to B10.BR females. Mice were kept under a 12 h light–dark cycle with free access to water and standard laboratory diet. The experiments were approved by the First Local Ethical Committee on Animal Testing in Krakow. Males in the appropriate age were killed by cervical dislocation. Sperm were collected from both vas deferens of each male into 0.2 ml of warmed to 37ºC M2 medium (Sigma-Aldrich, USA) and immediately subjected to further procedures.
Assessment of sperm motility
Sperm suspension of 10 μl was transferred onto a warmed slide, gently covered with a coverslip, and immediately examined with a light microscope. Two hundred successive spermatozoa of each sample were counted and classified as motile or immotile. Sperm of 5 to 7 individuals were analyzed for each strain and age point.
Assessment of sperm mitochondrial activity
Freshly collected sperm of individual males were incubated in 0.1 ml of M2 medium with MitoTracker Red CM-H2XRos probe (Molecular Probes, USA) in a concentration of 400 nM. After 40 min incubation (37ºC, 5% CO2), sperm were washed twice by centrifugation (300g, 5 min, 25ºC) with 1 ml of M2 medium. Sperm pellets were suspended in 50 µl of M2 medium. Small drops of sperm suspensions (10 μl) were smeared on slides, air-dried, mounted with Citifluor AF1 medium (Citifluor, USA), and covered with a coverslip. The preparations were examined with a Nikon Eclipse TS100F microscope equipped with an epifluorescence attachment, Nikon DS-Fi1c camera (Nikon Corporation, Japan), and a Semrock BrightLine TRITC-A-Basic filter set (Semrock, USA). On each slide, 200 successive spermatozoa were classified as having active mitochondria (stained midpieces exhibiting red fluorescence) or having non-functional mitochondria (midpieces not stained with the probe). Sperm of 5 individuals were analyzed for each strain and age point.
Assessment of sperm head morphology
Drops of sperm suspensions were smeared on slides, air-dried, fixed in a mixture of methanol with acetic acid (3:1), and stained with 1% aqueous solution of eosin Y (Sigma-Aldrich). On each slide, 200 successive spermatozoa were classified due to their head morphology as normal, slightly deformed, or severely deformed using previously described criteria [Styrna et al. Citation1991a; Styrna et al. Citation1991b]. Sperm of 5 to 8 individuals were analyzed for each strain and age point.
Statistical analysis
For statistical analyses all results in percentages were normalized using angular transformation. Differences between groups were evaluated with one-way ANOVA followed by the Bonferroni post-hoc test. Significance was assumed at p<0.05.
Declaration of interest
This work was supported by the Polish National Science Centre (grant number N N303 816840). The authors report no conflict of interest both financial and personal.
Additional information
Notes on contributors
Katarzyna Kotarska
Conceived and designed the experiments: KK, JS; Performed the experiments: KK, AD, MB; Analyzed the data: KK, ZP; Contributed reagents and materials: JS; Wrote the paper: KK.
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