Docosahexaenoic acid supplementation fully restores fertility and spermatogenesis in male delta-6 desaturase-null mice.

Delta-6 desaturase-null mice (−/−) are unable to synthesize highly unsaturated fatty acids (HUFAs): arachidonic acid (AA), docosahexaenoic acid (DHA), and n6-docosapentaenoic acid (DPAn6). The −/− males exhibit infertility and arrest of spermatogenesis at late spermiogenesis. To determine which HUFA is essential for spermiogenesis, a diet supplemented with either 0.2% (w/w) AA or DHA was fed to wild-type (+/+) and −/− males at weaning until 16 weeks of age (n = 3–5). A breeding success rate of DHA-supplemented −/− was comparable to +/+. DHA-fed −/− showed normal sperm counts and spermiogenesis. Dietary AA was less effective in restoring fertility, sperm count, and spermiogenesis than DHA. Testis fatty acid analysis showed restored DHA in DHA-fed −/−, but DPAn6 remained depleted. In AA-fed −/−, AA was restored at the +/+ level, and 22:4n6, an AA elongated product, accumulated in testis. Cholesta-3,5-diene was present in testis of +/+ and DHA-fed −/−, whereas it diminished in −/− and AA-fed −/−, suggesting impaired sterol metabolism in these groups. Expression of spermiogenesis marker genes was largely normal in all groups. In conclusion, DHA was capable of restoring all observed impairment in male reproduction, whereas 22:4n6 formed from dietary AA may act as an inferior substitute for DHA.

Mice were single housed at weaning and received the diet until 4 months of age.

Male fertility
Fertility was evaluated by breeding single housed males with either a +/+ or +/ Ϫ female for 4 days at four different time points: 6, 9, 12, and 15 weeks of age; different females were used at each time point; at least 12 mating attempts were done per dietary group for each genotype. Copulatory behavior was confi rmed in all mice. The percentage of successful matings as indicated by pregnant females and viable litters was noted.

Tissue collection and histology
Animals were euthanized by carbon dioxide inhalation at 4 months of age. Left testis and left epididymis were removed and weighed; left testis was then frozen for HUFA and RNA analysis, while left epididymis was used for sperm collection from cauda. Right testis and epididymis were fi xed in Davidson's fi xative and transferred to 10% neutral buffered formalin after 24 h. Tissues were trimmed for paraffi n embedding. Sections were cut at 3 m and stained with hematoxylin and eosin for histological evaluation.

Sperm count and motility
The cauda epididymis was cut with a surgical blade, minced with small scissors, and placed in 2 ml of dmKBRT buffer at 37°C for 15 min. The dmKBRT buffer contained 120 mM NaCl, 2 mM KCl, 2 mM CaCl 2 , 10 mM NaHCO 3 , 0.36 mM NaH 2 PO 4 H 2 O, 1.2 mM MgSO 4 , 5.6 mM glucose, 1.1 mM Na pyruvate, 25 mM TAPSO, 18.5 mM sucrose, and 6 mg/ml BSA. The sperm cell suspensions were then observed using an inverted microscope to record sperm motility. Epididymal sperm counts were done by hemocytometer from epididymal sperm in 2 ml of dmKBRT buffer.

Gene expression
RNA was analyzed with a slight modifi cation of a method previously described ( 22 ). Testis was homogenized in TRIzol reagent (Invitrogen, Carlsbad, CA) and total RNA extracted. MultiScribe reverse transcriptase (Applied Biosystems, Foster City, CA), along with random hexamers, were used to synthesize cDNA. Real-time quantitative PCR, using SYBR Green fl uorescent dye (Applied Biosystems), was used to analyze RNA relative to a rRNA L7a. Oligonucleotides used for real-time quantitative PCR were mTISP69-F, 5 ′ -CGGACGCTCAGGTTAACTTGA -3 ′ , mTISP69-R,

Fatty acid extraction and GC-MS analysis
Total lipids were extracted from frozen testis according to the method of Folch, Lees, and Sloane Stanley ( 23 ). Very-long-chain ity ( 8 ). In addition to the presence of HUFAs in mammalian testis and spermatozoa, there are also very-long-chain polyunsaturated fatty acids (VLPUFAs) that contain C26-C38 hydrocarbon chains (9)(10)(11). These VLPUFAs are elongation products of the C20 and C22 chain HUFAs ( 12 ). These VLPUFAs are incorporated mainly into sphingomyelin and ceramides in the sperm head ( 9,10 ). These sphingolipids are suggested to be involved with capacitation of sperm ( 13,14 ).
In the study reporting discovery of essential fatty acids, testicular degeneration and a low breeding success rate were among the defi ciency symptoms in rats fed a fat-free diet ( 15 ). A later study reported that rats receiving a diet defi cient in all essential fatty acids had a lower epididymal sperm concentration ( 16 ). However, these previous studies were unable to demonstrate the essentiality of HUFAs for male fertility because these animals also had severe growth retardation and dermatitis. In order to deplete tissue HUFAs in these studies, the D6D enzyme substrates, linoleic acid and ␣ -linolenic acid, as well as all products were eliminated from the diet. However, linoleic acid is required for skin water barrier function ( 17 ). Thus, defi ciency of linoleic acid resulted in severe growth retardation and dermatitis, confounding the study into the physiological roles of HUFAs, including male fertility ( 15,18 ).
To overcome this confounding problem, we and others created mice with the D6D gene disabled ( 19,20 ). The D6D knockout ( Ϫ / Ϫ ) mouse is unable to synthesize HUFAs, thus allowing us to specifi cally create AA deficiency without depleting tissue linoleic acid or to create DPAn6 and DHA defi ciency without depleting tissue AA. The D6D-null mouse developed intestinal ulcers and severe dermatitis by 5 months of age despite an adequate supply of linoleic acid and ␣ -linolenic acid from diet ( 19 ). Moreover, the male Ϫ / Ϫ mouse became infertile before manifestation of dermatitis. Histology of the D6D Ϫ / Ϫ mouse revealed disrupted spermiogenesis, the last stage of spermatogenesis in which spermatids develop to spermatozoa ( 19,20 ). Although the essentiality of HUFAs in spermiogenesis and male fertility has been demonstrated by these studies, the specifi c role of each HUFA for spermatogenesis has not been elucidated. Thus, the objective of this study was to determine if dietary AA and DHA can restore spermatogenesis in the D6D-null mouse and to elucidate the role of these HUFAs in spermiogenesis.

DHA was required for sperm head elongation and fl agellum formation
Testis and epididymis weights did not differ among groups in either absolute weight or in percentage weight relative to body weight (data not shown). Testis histology showed all stages of spermatogenesis in +/+ and +/ Ϫ genotypes regardless of the dietary treatments ( Fig. 2A ). Consistent with our previous study ( 19 ), all animals of nonsupplemented Ϫ / Ϫ had disrupted spermatogenesis specifi cally at Step 9 of spermiogenesis, where round spermatids are elongated. Spermatogonia, spermatocytes, and round spermatids were present, while elongated spermatids and spermatozoa were absent ( Fig. 2B ). AA supplementation partially restored spermatogenesis ( Fig. 2C ), while DHA supplemented Ϫ / Ϫ show all stages of spermatogenesis from spermatogonia to spermatozoa ( Fig. 2D ).
All +/+ mice had spermatozoa in the lumen of the epididymis ( Fig. 3A ). Nonsupplemented Ϫ / Ϫ epididymis contained mostly sloughed round spermatids and spermatocytes, cells from an earlier stage of spermatogenesis than spermatozoa ( Fig. 3B ). A closer examination revealed that spermatozoa in the epididymis of Ϫ / Ϫ exhibited globozoospermia ( Fig. 3B , inset). Partial restoration of spermatogenesis by AA supplementation is indicated by a mix of mature spermatozoa, spermatocytes, and round spermatids present in the epididymal lumen ( Fig. 3C ). The DHA-supplemented Ϫ / Ϫ group presented only spermatozoa in epididymal lumen ( Fig. 3D ), the same as in the +/+ ( Fig. 3A ).

Expression of genes analyzed were largely unchanged in ؊ / ؊
Spermatogenesis did not proceed successfully beyond the round spermatid phase (Step 9 of spermiogenesis) in Ϫ / Ϫ males; therefore, we measured gene expression of late spermiogenesis markers in testis ( Table 1 ). There was a 45% decrease in sperizin (Znrf4, TISP69) RNA in Ϫ / Ϫ males of all dietary groups. Two other genes that encode sperm fl agellar proteins, Shippo1 (Odf3, TISP50) and A-kinase anchoring protein (Akap3), had a mild (20%) but statistically signifi cant decrease in RNA expression in Ϫ / Ϫ fed the nonsupplemented diet. Other spermiogenesis markers analyzed were transition protein 1 (Tnp1), cortactin (Cttn), and sperm fl agellum associated protein (Sfap1), all of which were normally expressed in Ϫ / Ϫ with highly unsaturated fatty acid methyl esters were prepared with a slight modifi cation of a method previously described ( 12 ). A mixture of pentadecanoic acid (15:0), heptadecanoic acid (17:0), heneicosanoic acid (21:0), pentacosanoic acid (25:0), and heptacosanoic acid (27:0) was added as an internal standard to the lipid extracts from the testis. The extracts were derivatized to fatty acid methyl esters (FAMEs) with HCl in methanol at 85°C overnight. After extracting with hexane, FAMEs were separated on TLC plates with hexane:ether (80:20) to remove cholesterol. Absolute ethanol was added to the scraped bands, which was then sonicated for 10 min. FAMEs were extracted with hexane after adding water. GC-MS analysis was performed as previously described ( 12 ).

Statistical analysis
Statistical analysis with Statview version 5.01 for Windows was conducted using one-way ANOVA with Fisher's PLSD post-test ( Table 1; Fig. 1B ), the Wilcoxon sum rank test ( Fig. 1A ), and the Student's t -test ( Table 2 ). Data are presented as mean ± SD; P < 0.05 was considered as statistically signifi cant.

DISCUSSION
In this study, we determined the effects of dietary AA and DHA on the fertility and spermatogenesis of the D6Dnull males. Supplementing 0.2% DHA alone was able to fully restore male fertility, spermiogenesis, sperm morphology, and sperm count in Ϫ / Ϫ males. This restoration occurred despite very low AA and near depletion of DPAn6, the major HUFA present in +/+ males. Considering the variable ratios of DPAn6 and DHA among species ( 24 ), DPAn6 and DHA may be interchangeable for sperm function; therefore, DPAn6 is dispensable for sperm function in mice as long as suffi cient DHA is present. It was a little unexpected that DHA supplementation alone can fully the nonsupplemented diet. These results suggest that a gene expression sequence still proceeds to the spermiogenesis stage in Ϫ / Ϫ .
The VLPUFAs (C у 26) in testis of all +/+ groups were elongation products of DPAn6 (26:5n6, 28:5n6, 30:5n6) and an elongation product of DHA (30:6n3), all of which were quantitatively minor ( Fig. 4A ). In nonsupplemented Ϫ / Ϫ , these VLPUFAs became undetectable, except  Yet to be elucidated is the mechanism underlying the loss of spermatogenesis in Ϫ / Ϫ and the remarkable restoration by dietary DHA. In monkey sperm, 99% of DHA is present in fl agella ( 6 ). Thus, the failure of spermatogenesis at a late stage of spermiogenesis may be at least in restore male reproduction and spermiogenesis because of proposed roles of AA as eicosanoid precursor in male reproductive function ( 7,8 ). Although the underlying mechanism is yet to be elucidated, there could be a functional redundancy that might compensate for the lack of eicosanoids. Alternatively, the residual AA present in the DHA-supplemented group might be suffi cient as a precursor of eicosanoids for male reproduction.
In AA-supplemented Ϫ / Ϫ mice, sperm counts and a breeding success rate were partially restored even though the testis DHA and DPAn6 were as low as the nonsupplemented Ϫ / Ϫ . This partial rescue is unlikely to be due to the restoration of testis AA in the AA-supplemented Ϫ / Ϫ , con-  Mean ± SD. Groups without a common letter differ by Fisher's PLSD after one-way ANOVA ( P < 0.05). nd, not determined; +/+ AIN, wild-type fed AIN93G diet; Ϫ / Ϫ AIN, knockout fed AIN93G; Ϫ / Ϫ AA, knockout with 0.2% AA supplementation; Ϫ / Ϫ DHA, knockout with 0.2% DHA supplementation. impairment of sterol metabolism. Globozoospermia is a rare form of infertility in humans, characterized by a rounded sperm head ( 25 ). The Jackson Laboratory lists 39 mutant mouse strains under globozoospermia (http:// www.jax.org), suggesting multiple causes of this abnormality. Mammalian sperm heads contain ceramides and sphingomyelins with high percentages of VLPUFAs ( 9,10 ). Thus, the loss of VLPUFA in nonsupplemented Ϫ / Ϫ may play a role in the impaired sperm head function and structure.
Another potentially important fi nding of this study is decreased CD in the lipid extract of nonsupplemented and AA-supplemented Ϫ / Ϫ and restoration by dietary DHA. Because of the paucity of literature on CD, it is unclear if CD is present in testis or if it is derived from sterols during sample processing, although presence of CD in cornea has been reported ( 26 ). Whichever the case, our data suggest an impairment of sterol metabolism in Ϫ / Ϫ that was restor ed by DHA supplementation. Several studies indi-part due to lack of DHA and DPAn6 for structural components of the fl agellar membrane phospholipids. However, the impairment of spermatogenesis is not limited to tail formation. It extends to globozoospermia and possible Mean ± SD. Groups without SD were pooled. +/+ AIN, wild-type fed AIN93G diet; Ϫ / Ϫ AIN, knockout fed AIN93G; Ϫ / Ϫ AA, knockout with 0.2% AA supplementation; Ϫ / Ϫ DHA, knockout with 0.2% DHA supplementation; nd, not detected. * P < 0.05, signifi cantly different versus +/+ AIN by Student's t -test. cate importance of sterols in spermatogenesis. Desmosterol (24-dehydrocholesterol), an intermediate metabolite of the last step of cholesterol synthesis, is not present in large quantity in most tissues. However, free desmosterol is the major sterol present in fl agella of monkey sperm followed by free cholesterol and cholesteryl esters ( 6,27 ). Furthermore, the major impairment in the hormonesensitive lipase-null mouse is a complete loss of spermatogenesis ( 28 ), a similar phenotype to our D6D-null mice. Because hormone-sensi tive lipase is the only esterase that can hydrolyze cholesteryl ester in testis, a loss of the enzyme resulted in accumulation of cholesteryl ester in Sertoli cells and loss of spermatogenesis ( 28 ), indicating essentiality of cholesterol in spermatogenesis. Further studies will be warranted to elucidate the role of DHA in sterol metabolism in testis. Sperizin is a protein highly expressed in spermatids and may act as ubiquitin ligase ( 29,30 ). A study reported complete abolition of sperizin RNA in the testis of D6D-null mice and suggested an arrest of the gene expression sequence at the spermiogenesis stage ( 20 ). However, in our study, sperizin showed only a mild decrease in the Ϫ / Ϫ animals of all three dietary groups, which displayed a drastic difference in spermiogenesis, excluding sperizin as the cause of impaired spermatogenesis. Moreover, several other markers specifi c to spermiogenesis showed largely normal expression in Ϫ / Ϫ animals fed different diets, including genes that encode proteins in sperm fl agella such as Shippo1 ( 31 ), Akap3 ( 32 ), and Sfap1 ( 33 ). Thus, our RNA analysis suggests that there is no general arrest of gene expression sequence at the spermiogenesis stage, although it is possible that expression of specifi c genes may be affected by DHA defi ciency.
In conclusion, this study demonstrated that DHA supplementation to D6D-null male mice restored spermatogenesis and fertility in the absence of DPAn6 and low AA in testis, while dietary AA was much less effective. The accumulation of 22:4n6 in the AA-supplemented Ϫ / Ϫ testis suggests that 22:4n6 may act as a lesser substitute for DPAn6 or DHA in spermiogenesis. CD was detected in testis lipid extracts from +/+ and DHA-supplemented Ϫ / Ϫ , whereas it greatly decreased in nonsupplemented and AA-supplemented Ϫ / Ϫ , suggesting impairment of sterol metabolism in the latter groups. The expression of spermiogenesis marker genes in Ϫ / Ϫ animals was largely normal. The mechanism underlying the loss of spermatogenesis in Ϫ / Ϫ and the rescue by dietary DHA is yet to be elucidated.
DHASCO and ARASCO oil were generously provided by Martek Biosciences (Columbia, MD). The authors acknowledge Kimberly Henry for technical assistance.