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. 2017 Jan 5;8(1):e2532.
doi: 10.1038/cddis.2016.414.

Globozoospermia and lack of acrosome formation in GM130-deficient mice

Feng Han  1   2 , Chunyi Liu  3 , Lianjun Zhang  1   2 , Min Chen  1   2 , Yang Zhou  1   2 , Yan Qin  1   2 , Yaqing Wang  3 , Min Chen  1 , Shuguang Duo  1 , Xiuhong Cui  1 , Shilai Bao  3 , Fei Gao  1   2
Affiliations

Globozoospermia and lack of acrosome formation in GM130-deficient mice

Feng Han et al. Cell Death Dis. .

Abstract

Globozoospermia is a common reproductive disorder that causes male infertility in humans, and the malformation or loss of acrosomes is the prominent feature of this disease. Although the acrosome is thought to be derived from the Golgi apparatus, the detailed molecular mechanisms remain unclear. GM130 is a cis-side localized Golgi matrix protein,whereas the physiological functions of this protein remain elusive. Here we showed that inactivation of GM130-caused male infertility in mouse model. The primary defects were the absence of acrosomes, round sperm heads, and aberrant assembly of the mitochondrial sheath, which comprise the characteristic features of human globozoospermia. Further investigation indicated that loss of GM130 did not affect the secretion of pro-acrosomic vesicles, whereas the vesicles failed to fuse into a single large acrosome vesicle. Co-localization of the adaptor protein complex AP1 and trans-Golgi network (TGN) protein TGN46 was disrupted, suggesting that the malformation of acrosomes is most likely due to the defect in the sorting and coating of Golgi-derived pro-acrosomic vesicles. Thus, the GM130-deficient mouse provides a valuable model for investigating the etiology of human globozoospermia.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Decreased sperm count and abnormal sperm heads in GM130-deficient mice at 2 months of age. (a) The size of the testes from GM130−/−mice was smaller than that of control mice. (b) The sperm number per mouse was reduced ~60% in the GM130-deficient males. The data are represented as the mean±S.E.M. of three independent experiments (N=6). The morphology of the seminiferous tubules and sperm from control and GM130−/−mice was examined by H&E staining. The seminiferous tubules were grossly normal in GM130−/−mice (d) compared with the control mice (c). Normal sperm with crescent-shaped heads were observed in both testes (e, arrowheads) and epidydimes (g, arrowheads) of control mice. The sperm heads in both testes (f, arrowheads) and epidydimes (h, arrowheads) of GM130-deficient mice were in round-shaped
Figure 2
Figure 2
Defects of morphogenesis and motility in GM130-deficient sperm from 2-month-old mice. The morphology of sperm was examined by immunohistochemistry and immunosenesence. Acrosomes were labeled with anti-Afaf antibody in control testes (a, arrowheads), and no Afaf signal was detected in GM130-deficient testes (b, arrowheads). (c) Single sperm image indicated the morphology of control and GM130-deficient sperm. (d) Acrosome-specific protein SP56 (red) was detected in the control sperm, but not in the sperm from GM130−/−mice. (e) Mitotracker-positive mitochondrial sheath (red) was observed in the mid-piece of control sperm tails, but not in the tails of GM130-deficient sperm. In contrast, Mitotracker-positive mitochondria (red) were located in the sperm head and surrounded the nucleus. The motility of sperm from control and GM130−/−mice was analyzed by CASA assay. The percentages of motile (f) sperm were substantially reduced in the GM130−/−mice compared with the control mice (*P<0.05 versus control). No progressive sperm were noted in GM130−/−testis (g)
Figure 3
Figure 3
Defect of spermiogenesis was observed in GM130−/flox; Stra8-Cre mice at 2 months of age. The morphology of the seminiferous tubules and sperm was exmained by H&E staining and immunosenesence. The seminiferous tubules were grossly normal in the GM130−/flox; Stra8-Cre mice (b) compared with the control mice. (a) Acrosomes were labeled with anti-Afaf antibody in control testes (c, arrowheads), whereas no Afaf signal was detected in the sperm of the GM130−/flox; Stra8-Cre mice (d, arrowheads). Normal sperm with crescent-shaped heads were observed in the epididymides (e, arrowheads) of the control mice. The sperm heads in the epididymides (f, arrowheads) of the GM130−/flox; Stra8-Cre mice were round. (g) single sperm image indicated the morphology of control and GM130−/flox; Stra8-Cre sperm. (h) Acrosome-specific protein SP56 was identified in the control sperm, but not in the sperm of the GM130−/flox; Stra8-Cre mice. (i) Mitotracker-positive mitochondrial sheath was observed in mid-piece of control sperm, but not in the tails of sperm obtained from the GM130−/flox; Stra8-Cre mice. In contrast, Mitotracker-positive mitochondria were located in the sperm heads and surrounded the nuclei
Figure 4
Figure 4
Ultrastructural analysis of spermatogenic cells from control and GM130-deficient mice at 2 months of age. The ultrastructure of sperm was exmianed by TEM. (a) In the control mice, the nuclei of the mature sperm was elongated and covered with acrosome. (b) The nucleus of the sperm from the GM130−/−mice did not elongate and remained round in the maturation phase. A cluster of mitochondria was also observed close to the nucleus. A single Golgi apparatus and several large Golgi-derived vesicles (black arrows) were identified in the control spermatids (c). The Golgi apparatus was fragmented into several small pieces, and numerous small Golgi-derived vesicles (black arrows) were observed in the GM130-deficient spermatids (d). Acrosome granules (c, arrowheads and e, arrow) were attached to the nuclear envelopes at the Golgi phase in the control sperm. An electron dense acrosome matrix (f, arrow) and a thin layer of acrosomal sac (g, arrowheads) were also identified in the GM130-deficient sperm. Mi, Mitochondria; Nu, Nucleus; Acr, Acrosome; G, Golgi apparatus
Figure 5
Figure 5
Immunofluorescence of Golgi-specific proteins. The expression of Golgi-specific proteins in both the control and GM130-deficient testes was examined via immunofluorescence. GM130 protein was detected in the germ cells of control testes (a, red), but not the GM130−/−testes (b). Golgin84 protein was identified in both control (c, white arrowheads) and GM130−/−testes (d, white arrowheads). However, the Golgin84-positive punctae in the GM130−/−testes were smaller than the control testes. TGN46 protein was also identified in both control (e, white arrowhead) and GM130−/−(f, white arrowhead) testes, and the TGN46-positive punctae in the GM130−/−testes were also smaller than the control testes
Figure 6
Figure 6
Immunofluorescence of P115, Grasp65 and Keratin5. The expression of GM130-interacting proteins and the acrosome-nucleus interacting protein was examined via immunofluorescence. P115 (a, white arrowhead) and Grasp65 (c, white arrowhead) proteins were detected in the germ cells of control testes, whereas both P115 (b) and Grasp65 (d) proteins were absent in the GM130−/−testes. Keratin5 was expressed in the spermatids of both the control (e, white arrows) and GM130−/−(f, white arrows) testes
Figure 7
Figure 7
Co-localization of TGN46 and AP1 was disrupted in 2 months GM130-deficient spermatids. The expression of AP1 and TGN46 was examined by immunofluorescence. TGN46 (d, red) and AP1 (e, green) proteins were detected in the germ cells of control testes, and these two proteins were completely co-localized (f, inset, white arrow). TGN46 (a, red) and AP1 (b, green) proteins were also detected in the GM130-deficient germ cells, whereas the TGN46 and AP1-positive punctae were substantially smaller than those in the control germ cells, and a substantial portion of the AP1-positive punctae were not co-localized with TGN46 (c, inset, white arrow)
Figure 8
Figure 8
Expression of globozoospermia-related and spermiogenesis-associated proteins. The expression of globozoospermia-related proteins (GOPC and PICK1) was not altered in the GM130−/−germ cells. The expression of vesicle trafficking-related proteins (AP1, CLATHRIN and VAMP2) was also not altered in the GM130−/−germ cells. The CDC42 protein level was significantly increased in the GM130-deficient germ cells
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