Complement Factor B Deficiency Is Dispensable for Female Fertility but Affects Microbiome Diversity and Complement Activity

Abstract

Complement factor B (CFB) is a crucial component for the activation of the alternative pathway due to the formation of the C3 convertase with C3b, which further produces C3b to enhance the overall complement activity. Although Cfb is expressed not only in the immune tissues, but also in the reproductive tract, the physiological role of the alternative complement pathway in reproduction remains unclear. In this study, we addressed this issue by producing Cfb-knockout (KO) mice and analyzing their phenotypes. Sperm function, number of ovulated oocytes, and litter size were normal in KO mice. In contrast, the diversity of microbiomes in the gut and vaginal tract significantly increased in KO mice. Some serine protease activity in the serum from KO mice was lower than that of wild-type mice. Since the serum from KO mice showed significantly lower activity of the alternative complement pathway, CFB was found to be essential for this pathway. Our results indicate that although the alternative pathway is dispensable for normal fertility and development, it maintains the gut and vaginal microbiomes by suppressing their diversity and activating the alternative complement pathway.

Keywords: alternative pathway; complement factor B; gut microbiome; mouse fertility; vaginal microbiome.

Figure 1

Complement factor b (Cfb) expression analysis and generation of Cfb-KO mice. (a) Cfb mRNA expression in several tissues was analyzed by qPCR. n = 3. Data are expressed as means ± SE. (b) Immunoblotting of several tissue lysates with anti-CFB Ab and anti-GAPDH. GAPDH is an endogenous control. The densitometry analyses of band intensity normalized with GAPDH. (c) Schematic illustration of the wild-type (WT) and Cfb-KO (KO) allele of the Cfb gene. Black boxes mean exons. We designed gRNAs at exon 2 and exon 12, and KO allele deleted a 4 kbp long sequence in chromosome 17. (d) Sequences of PCR products with using Primer F/R in an WT and F1 mouse. gRNA sequences were found in WT allele, but not in F1 allele. Green characters indicated intron sequences between exon 1 and 2, and pink characters means exon 12 sequence. (e) Genomic DNA sequence at breakpoint junction in KO mice. Green and pink shading characters indicate the intron between exon 1 and 2 and the exon 12, respectively. (f) Efficiencies of i-GONAD treatment. (g) Representative image of WT and KO mice. (h) Genotyping of the F2 by using primer F and R. (i) Immunoblotting the serum from KO mice with anti-CFB Ab.

Figure 2

Fertility of KO mice. (a) A method for measuring mouse fertility. For the analysis of male fertility, cauda epididymal sperm were collected from male KO mice. After the incubation in TYH medium, sperm were subjected to sperm motility and concentration analysis. For female fertility, Cumulus-oocyte complexes were isolated from super-ovulated KO mice. The number of pups produced after mating was counted to determine the reproductive potential of both males and females. (b) Sperm motility during 3 h of incubation between WT mice (triangles) and KO mice (solid circles): * p < 0.05. n = 3. Data are expressed as means ± SE. (c) Rates of acrosome reacted sperm during 3 h of incubation between WT mice (triangles) and KO mice (solid circles): ** p < 0.01. n = 3. Data are expressed as means ± SE. (d) Epididymal sperm concentration: ns, not significant, n = 3. Data are expressed as means ± SE. (e) The number of ovulated eggs: ns, not significant, n = 3. Data are expressed as means ± SE. (f) Representative image showing an entire reproductive tract of estrous female. (g) Fecundity of KO mice: ns, not significant, n = 3. Data are expressed as means ± SE.

Figure 3

Characterization of gut and vaginal microbiota. (a) Stool samples and vaginal lavages were collected from identical mice at the estrous stage. (b) Relative abundance of Bacteroides in the gut microbiota: ns, not significant, n = 3. Data are expressed as means ± SE. (c) Relative abundance of Firmicutes in the gut microbiota: * p < 0.05, n = 3. Data are expressed as means ± SE. (d) Total alpha diversity (Chao 1) of the gut microbiota: * p < 0.05, n = 3. Boxes represent the interquartile range, lines indicate medians, and whiskers indicate the range. (e) Total alpha diversity (Shannon) of the gut microbiota: * p < 0.05, n = 3. Boxes represent the interquartile range, lines indicate medians, and whiskers indicate the range. (f) Relative abundance of Firmicutes in the vaginal microbiota: ns, not significant, n = 3. Data are expressed as means ± SE. (g) Relative abundance of Proteobacteria in the vaginal microbiota: ns, not significant, n = 3. Data are expressed as means ± SE. (h) Total alpha diversity (Chao 1) of the vaginal microbiota: * p < 0.05, n = 3. Boxes represent the interquartile range, lines indicate medians, and whiskers indicate the range. (i) Total alpha diversity (Shannon) of the vaginal microbiota: ns, not significant, n = 3. Boxes represent the interquartile range, lines indicate medians, and whiskers indicate the range.

Figure 4

Enzyme assay of serum from KO mice. (a) Experimental flow. Serum was collected from wild-type and KO mice and then incubated with various MCA substrates at 37 °C for 1 h. Released fluorescence was measured every 10 min for 1 h. (b) Substrates with strong enzyme activity (i), with moderate enzyme activity (ii), with weak enzyme activity (iii). * p < 0.05, *** p < 0.001, **** p < 0.0001. n = 3. Data are expressed as means ± SE.

Figure 5

Hemolysis assay of serum from KO mice. (a) Experimental flow. For measuring hemolytic activity of complement classical pathway, sheep erythrocyte was sensitized with rabbit IgG and then incubated with the serum from WT and KO mice. To measure that of complement alternative pathway, rabbit erythrocyte was incubated with the serum in the presence of EGTA. After incubation, the rates of hemolysis were calculated. (b) Classical pathway hemolytic activity: ns, not significant, n = 3. Data are expressed as means ± SE. (c) Alternative pathway hemolytic activity: * p < 0.05, n = 3. Data are expressed as means ± SE. (d) Schematic model of the relationship between innate immunity and the microbiota in female mice. The relationship between microbiota diversity and female fertility remains unclear.