No abstract

Glutathione peroxidase 4 (GPX4) and arachidonic acid 15-lipoxygenase (ALOX15) are antagonizing enzymes in the metabolism of hydroperoxy lipids. In spermatoid cells and/or in the male reproductive system both enzymes are apparently expressed, and GPX4 serves as anti-oxidative enzyme but also as a structural protein. In this study we explored whether germ line inactivation of the Alox15 gene might rescue male subfertility induced by heterozygous expression of catalytically silent Gpx4. To address this question we employed Gpx4 knock-in mice expressing the Sec46Ala-Gpx4 mutant, in which the catalytic selenocysteine was replaced by a redox inactive alanine. Because homozygous Gpx4 knock-in mice (Sec46Ala-Gpx4) are not viable we created heterozygous animals (Sec46Ala-Gpx4) and crossed them with Alox15 knock-out mice (Alox15). Male Sec46Ala-Gpx4 mice, but not their female littermates, were subfertile. Sperm extracted from the epididymal cauda showed strongly impaired motility characteristics and severe structural midpiece alterations (swollen mitochondria, intramitochondrial vacuoles, disordered mitochondrial capsule). Despite these structural alterations, they exhibited similar respiration characteristics than wild-type sperm. When Sec46Ala-Gpx4 mice were crossed with Alox15-deficient animals, the resulting males (Sec46Ala-Gpx4+Alox15) showed normalized fertility, and sperm motility was reimproved to wild-type levels. Taken together these data suggest that systemic inactivation of the Alox15 gene normalizes the reduced fertility of male Sec46Ala-Gpx4 mice by improving the motility of their sperm. If these data can be confirmed in humans, ALOX15 inhibitors might counteract male infertility related to GPX4 deficiency.

Optimizing cryopreservation protocols for nondomestic felids contributes to the successful development of assisted reproduction techniques and genetic resource banking. In this study, we describe a simple cryopreservation procedure for African lion (Panthera leo) ejaculates, which was tested with different packaging options and different sperm numbers per dose. By applying urethral catheterization and electroejaculation, 17 ejaculates with greater than 20% motile and greater than 5% progressively motile sperm were collected. A lyophilized extender (a modified egg yolk-Tes-Tris-fructose-glycerol medium) was rehydrated and added in one step at ambient temperature (∼25 °C) to semen, which was prediluted in cell culture medium M199. After slow cooling of insulated samples to 15 °C in a refrigerator (4 °C), the samples were fast frozen over the surface of liquid nitrogen or in a dry shipper. Aliquots of 300 μL containing 20 × 10 sperm were frozen in cryovials and in 0.5-mL straws. Differences were observed in the total motility after thawing between vial (31.5 ± 14.1%) and straw freezing (20.1 ± 8.6%). However, the subpopulations of vital (22.7 ± 7.8% for vial and 19.8 ± 8.5% for straw) and progressively motile (10.0 ± 7.9% for vial and 10.0 ± 6.4% for straw) sperm after washing and 1 hour incubation at 38 °C were of similar magnitude, velocity, and linearity for both packaging options. After freezing of five ejaculates with 20, 60, and 100 × 10 sperm per dose, best results were achieved at the lowest concentration. In general, post-thaw results were highly variable (2.2% and 56.5% total motility) and not correlated to motility or morphology of the fresh semen. To further characterize semen quality, we assessed the protective potential of seminal fluid against oxidative stress, which might be challenged on freeze thawing. The capacity of seminal fluid to reduce radicals was measured in 10 semen samples by electron spin resonance spectroscopy and a spin-labeled fatty acid as a radical probe. Moreover, we determined the lysophosphatidylcholines (LPC) as potential lipid oxidation products in the sperm and erythrocytes of the males. Individuals with a high radical reduction capacity in the seminal fluid and a low LPC content in their erythrocytes showed a better cryosurvival of sperm. This is a first indication that seminal fluid may affect the freezing potential of African lion ejaculates.

To support conservation strategies in wild species, simple but highly reproducible procedures of sperm cryopreservation are required for an application under field conditions. We used epididymal sperm of the domestic cat to optimize a sperm freezing procedure for felid species, particularly questioning the demand for sperm cooling to 4°C. We equilibrated sperm during slow cooling to only 15 or 10°C in a Tes-Tris-fructose extender with final concentrations of 4.7% (v/v) glycerol and 10% (v/v) of the water-soluble fraction of hen's egg yolk (low-density lipoproteins). Subsequently, sperm were frozen over liquid nitrogen. Total and progressive motility (mean ± SD) after thawing was 60.7 ± 8.6% and 53.9 ± 9.6% in samples cooled to 15°C or 61.6 ± 9.5% and 55.3 ± 9.9% in samples cooled to 10°C. Therefore, a one-step addition of glycerol to sperm at room temperature together with the freezing extender, the use of cryovials (loaded with diluted sperm aliquots of 300 μl), an equilibration period of 40 min comprising slow cooling to 15°C at a rate of approximately -0.14 K/min before rapid freezing over liquid nitrogen, yielded satisfying results. Cooling, freezing and thawing rates were exactly characterized as a prerequisite for further optimization and to provide a repeatable protocol to other practitioners.

The Mongolian gerbil has been used as laboratory animal since 1935. Breeding gerbils as an isolated laboratory population for decades may have led to a domestication process whose effects include changes in brain size. Quantitative changes in testicular activity could be assumed. Comparative intraspeci®c measurements were performed in 34 adult males of the laboratory strain (LAB) and in males raised as offspring of wild Mongolian gerbils (WILD) caught in central Mongolia (F 1 , n = 16; F 2 , n = 17). LAB and WILD were examined in January. Testicular spermatozoa were counted, proportions of different cell types were analysed using DNA¯ow cytometry, and mitotic and meiotic activity was calculated from DNA histograms. Intratesticular testosterone concentrations were measured with an enzyme immunoassay. In the WILD, testicular activity was lower and varied more. The overall weight, the ef®ciency of spermatogenesis (sperm/g testis) and resulting total sperm per testis were signi®cantly less in offspring of wild gerbils. This corresponded with lower levels of haploid cells, total germ cell transformation of diploid cells to spermatids and meiotic transformation of spermatocytes to spermatids. The most profound difference was found in testicular testosterone concentration: the mean level was 405.7 41.2 ng/g testis in LAB vs 6.4 2.0 ng/g in WILD F 1 . All parameters changed in WILD F 2 generation compared with F 1 and diminished the differences with LAB. Differences between F 1 and F 2 were signi®cant for testis mass, testis/ body weight ratio, percentages of haploid cells and cells in G 2 /M phase, both germ cell transformations and testosterone concentration. The results suggest rapid, adaptive changes of male reproductive physiology in the early offspring generations from wild populations under laboratory breeding conditions. The breeding of Mongolian gerbils in the laboratory has in¯uenced the testicular function resulting in increased spermatogenic activity and highly stimulated testosterone production.