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Type-I prenyl protease function is required in the male germline of Drosophila melanogaster.

Adolphsen K, Amell A, Havko N, Kevorkian S, Mears K, Neher H, Schwarz D, Schulze SR - G3 (Bethesda) (2012)

Bottom Line: The result is a male fertility defect, manifesting late in spermatogenesis.Our results also show that the ancestral type I prenyl protease gene in Drosophila is under strong purifying selection, while the more recent replicates are evolving rapidly.We propose that potential targets for the male-specific type I prenyl proteases include proteins involved in the very dramatic cytoskeletal remodeling events required for spermatid maturation.

View Article: PubMed Central - PubMed

Affiliation: Biology Department, Western Washington University, Bellingham, Washington 98225.

ABSTRACT
Many proteins require the addition of a hydrophobic prenyl anchor (prenylation) for proper trafficking and localization in the cell. Prenyl proteases play critical roles in modifying proteins for membrane anchorage. The type I prenyl protease has a defined function in yeast (Ste24p/Afc1p) where it modifies a mating pheromone, and in humans (Zmpste24) where it has been implicated in a disease of premature aging. Despite these apparently very different biological processes, the type I prenyl protease gene is highly conserved, encoded by a single gene in a wide range of animal and plant groups. A notable exception is Drosophila melanogaster, where the gene encoding the type I prenyl protease has undergone an unprecedented series of duplications in the genome, resulting in five distinct paralogs, three of which are organized in a tandem array, and demonstrate high conservation, particularly in the vicinity of the active site of the enzyme. We have undertaken targeted deletion to remove the three tandem paralogs from the genome. The result is a male fertility defect, manifesting late in spermatogenesis. Our results also show that the ancestral type I prenyl protease gene in Drosophila is under strong purifying selection, while the more recent replicates are evolving rapidly. Our rescue data support a role for the rapidly evolving tandem paralogs in the male germline. We propose that potential targets for the male-specific type I prenyl proteases include proteins involved in the very dramatic cytoskeletal remodeling events required for spermatid maturation.

No MeSH data available.


Related in: MedlinePlus

No changes in the principal stages of spermatogenesis in triple knockout vs. control genotypes. Phase contrast images of testes dissected from age- and condition-matched heterozygous or wild-type (A, C, E, G) and homozygous triple knockout (B, D, F, H) males. (A, B) Apical sections of testes from 10- to 15-day-old males, showing gonial and mitotic spermatogonia, elongated spermatids, and “waste bags,” spherical structures containing excess cytoplasm squeezed out of the spermatids during elongation. (C, D) Midsections of testes from 21-day-old males, showing meiotic spermatids, postmeitoic (round, phase light) spermatids, elongating spermatids, and onion stage mitochondria (nebenkern, phase dark). (E, F) Dissections releasing mature sperm from 3-day-old males. Note lesser amounts of sperm from triple knockout males. (G, H) Dissections releasing mature sperm from 8- to 10-day-old males. Note that although there is still less sperm in the triple knockout homozygote (H) than in the wild-type (G), at this point the triple knockouts are virtually completely sterile. Scale bars: 100 µM (A, B, E, F, G, H) and 50 µM (C, D).
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fig5: No changes in the principal stages of spermatogenesis in triple knockout vs. control genotypes. Phase contrast images of testes dissected from age- and condition-matched heterozygous or wild-type (A, C, E, G) and homozygous triple knockout (B, D, F, H) males. (A, B) Apical sections of testes from 10- to 15-day-old males, showing gonial and mitotic spermatogonia, elongated spermatids, and “waste bags,” spherical structures containing excess cytoplasm squeezed out of the spermatids during elongation. (C, D) Midsections of testes from 21-day-old males, showing meiotic spermatids, postmeitoic (round, phase light) spermatids, elongating spermatids, and onion stage mitochondria (nebenkern, phase dark). (E, F) Dissections releasing mature sperm from 3-day-old males. Note lesser amounts of sperm from triple knockout males. (G, H) Dissections releasing mature sperm from 8- to 10-day-old males. Note that although there is still less sperm in the triple knockout homozygote (H) than in the wild-type (G), at this point the triple knockouts are virtually completely sterile. Scale bars: 100 µM (A, B, E, F, G, H) and 50 µM (C, D).

Mentions: Triple knockout males are almost completely sterile, and there were no obvious defects in courtship and mating (Greenspan and Ferveur 2000). Because homozygous triple knockout males are fertile for only a narrow window early in their adult life, they can make functional sperm. But do they simply make less of it, or do they make larger quantities of defective sperm? To establish when during spermatogenesis type I prenyl protease function might be required, we carried out phase contrast microscopy to examine all the stages of spermatogenesis in homozygous triple knockout males and their heterozygous siblings or wild-type controls. Each testis is a long coiled tubular organ, with a stem cell niche at the apical tip. Stem cells divide asymmetrically to produce a gonial cell that undergoes four incomplete mitotic divisions, resulting in a group of 16 interconnected primary spermatocytes encased in a cyst composed of two somatic cells. The cysts of maturing spermatocytes gradually move down the length of the testes as the spermatocytes undergo meiosis, followed by dramatic remodeling events that lengthen the spermatocyte nuclei, coalesce the mitochondria, and build flagella. Ultimately, 64 meiotic products mature into full-length individualized spermatids (Fuller 1993; Zhao et al. 2010). As can be seen in Figure 5, there are no notable differences in any of the principle stages when homozygous triple knockout males are compared with their heterozygous siblings (or wild-type). In addition, there appeared to be no reduction in premeiotic cell types as the males aged, arguing against a stem-cell defect. We did notice that sperm spilled (by dissection) from the triple knockout testes was less abundant than sibling controls (Figure 5, E and F). Interestingly, the sperm abundance seemed unchanged, regardless of male age. Compare Figure 5, E and F, in which sperm was spilled by dissection in 3-day-old males, with Figure 5, G and H, where the same procedure was carried out on 8- to 10-day-old males. At this time point, the homozygous triple knockout males are virtually sterile, but their capacity for producing mature sperm appears to be unchanged.


Type-I prenyl protease function is required in the male germline of Drosophila melanogaster.

Adolphsen K, Amell A, Havko N, Kevorkian S, Mears K, Neher H, Schwarz D, Schulze SR - G3 (Bethesda) (2012)

No changes in the principal stages of spermatogenesis in triple knockout vs. control genotypes. Phase contrast images of testes dissected from age- and condition-matched heterozygous or wild-type (A, C, E, G) and homozygous triple knockout (B, D, F, H) males. (A, B) Apical sections of testes from 10- to 15-day-old males, showing gonial and mitotic spermatogonia, elongated spermatids, and “waste bags,” spherical structures containing excess cytoplasm squeezed out of the spermatids during elongation. (C, D) Midsections of testes from 21-day-old males, showing meiotic spermatids, postmeitoic (round, phase light) spermatids, elongating spermatids, and onion stage mitochondria (nebenkern, phase dark). (E, F) Dissections releasing mature sperm from 3-day-old males. Note lesser amounts of sperm from triple knockout males. (G, H) Dissections releasing mature sperm from 8- to 10-day-old males. Note that although there is still less sperm in the triple knockout homozygote (H) than in the wild-type (G), at this point the triple knockouts are virtually completely sterile. Scale bars: 100 µM (A, B, E, F, G, H) and 50 µM (C, D).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3362292&req=5

fig5: No changes in the principal stages of spermatogenesis in triple knockout vs. control genotypes. Phase contrast images of testes dissected from age- and condition-matched heterozygous or wild-type (A, C, E, G) and homozygous triple knockout (B, D, F, H) males. (A, B) Apical sections of testes from 10- to 15-day-old males, showing gonial and mitotic spermatogonia, elongated spermatids, and “waste bags,” spherical structures containing excess cytoplasm squeezed out of the spermatids during elongation. (C, D) Midsections of testes from 21-day-old males, showing meiotic spermatids, postmeitoic (round, phase light) spermatids, elongating spermatids, and onion stage mitochondria (nebenkern, phase dark). (E, F) Dissections releasing mature sperm from 3-day-old males. Note lesser amounts of sperm from triple knockout males. (G, H) Dissections releasing mature sperm from 8- to 10-day-old males. Note that although there is still less sperm in the triple knockout homozygote (H) than in the wild-type (G), at this point the triple knockouts are virtually completely sterile. Scale bars: 100 µM (A, B, E, F, G, H) and 50 µM (C, D).
Mentions: Triple knockout males are almost completely sterile, and there were no obvious defects in courtship and mating (Greenspan and Ferveur 2000). Because homozygous triple knockout males are fertile for only a narrow window early in their adult life, they can make functional sperm. But do they simply make less of it, or do they make larger quantities of defective sperm? To establish when during spermatogenesis type I prenyl protease function might be required, we carried out phase contrast microscopy to examine all the stages of spermatogenesis in homozygous triple knockout males and their heterozygous siblings or wild-type controls. Each testis is a long coiled tubular organ, with a stem cell niche at the apical tip. Stem cells divide asymmetrically to produce a gonial cell that undergoes four incomplete mitotic divisions, resulting in a group of 16 interconnected primary spermatocytes encased in a cyst composed of two somatic cells. The cysts of maturing spermatocytes gradually move down the length of the testes as the spermatocytes undergo meiosis, followed by dramatic remodeling events that lengthen the spermatocyte nuclei, coalesce the mitochondria, and build flagella. Ultimately, 64 meiotic products mature into full-length individualized spermatids (Fuller 1993; Zhao et al. 2010). As can be seen in Figure 5, there are no notable differences in any of the principle stages when homozygous triple knockout males are compared with their heterozygous siblings (or wild-type). In addition, there appeared to be no reduction in premeiotic cell types as the males aged, arguing against a stem-cell defect. We did notice that sperm spilled (by dissection) from the triple knockout testes was less abundant than sibling controls (Figure 5, E and F). Interestingly, the sperm abundance seemed unchanged, regardless of male age. Compare Figure 5, E and F, in which sperm was spilled by dissection in 3-day-old males, with Figure 5, G and H, where the same procedure was carried out on 8- to 10-day-old males. At this time point, the homozygous triple knockout males are virtually sterile, but their capacity for producing mature sperm appears to be unchanged.

Bottom Line: The result is a male fertility defect, manifesting late in spermatogenesis.Our results also show that the ancestral type I prenyl protease gene in Drosophila is under strong purifying selection, while the more recent replicates are evolving rapidly.We propose that potential targets for the male-specific type I prenyl proteases include proteins involved in the very dramatic cytoskeletal remodeling events required for spermatid maturation.

View Article: PubMed Central - PubMed

Affiliation: Biology Department, Western Washington University, Bellingham, Washington 98225.

ABSTRACT
Many proteins require the addition of a hydrophobic prenyl anchor (prenylation) for proper trafficking and localization in the cell. Prenyl proteases play critical roles in modifying proteins for membrane anchorage. The type I prenyl protease has a defined function in yeast (Ste24p/Afc1p) where it modifies a mating pheromone, and in humans (Zmpste24) where it has been implicated in a disease of premature aging. Despite these apparently very different biological processes, the type I prenyl protease gene is highly conserved, encoded by a single gene in a wide range of animal and plant groups. A notable exception is Drosophila melanogaster, where the gene encoding the type I prenyl protease has undergone an unprecedented series of duplications in the genome, resulting in five distinct paralogs, three of which are organized in a tandem array, and demonstrate high conservation, particularly in the vicinity of the active site of the enzyme. We have undertaken targeted deletion to remove the three tandem paralogs from the genome. The result is a male fertility defect, manifesting late in spermatogenesis. Our results also show that the ancestral type I prenyl protease gene in Drosophila is under strong purifying selection, while the more recent replicates are evolving rapidly. Our rescue data support a role for the rapidly evolving tandem paralogs in the male germline. We propose that potential targets for the male-specific type I prenyl proteases include proteins involved in the very dramatic cytoskeletal remodeling events required for spermatid maturation.

No MeSH data available.


Related in: MedlinePlus