Seminal fluid metabolome and epididymal changes after antibiotic treatment in mice

Abstract

Paternal environment can induce detrimental developmental origins of health and disease (DOHaD) effects in resulting offspring and even future descendants. Such paternal-induced DOHaD effects might originate from alterations in a possible seminal fluid microbiome (SFM) and composite metabolome. Seminal vesicles secrete a slightly basic product enriched with fructose and other carbohydrates, providing an ideal habitat for microorganisms. Past studies confirm the existence of a SFM that is influenced by genetic and nutritional status. Herein, we sought to determine whether treatment of male mice with a combination of antibiotics designed to target SFM induces metabolic alterations in seminal vesicle gland secretions (seminal fluid) and histopathological changes in testes and epididymides. Adult (10- to 12-week-old) National Institutes of Health (NIH) Swiss males (n = 10 per group) were treated with Clindamycin 0.06 mg/kg day, Unasyn (ampicillin/sulbactam) 40 mg/kg day and Baytril (enrofloxacin) 50 mg/kg day designed to target the primary bacteria within the SFM or saline vehicle alone. Fourteen-day antibiotic treatment of males induced metabolomic changes in seminal vesicles with inosine, xanthine and l-glutamic acid decreased but d-fructose increased in glandular secretions. While spermatogenesis was not affected in treated males, increased number of epididymal tubules showed cribriform growth in this group (7 antibiotic-treated males: 3 saline control males; P = 0.01). Antibiotic-treated males showed more severe cribriform cysts. Current findings suggest antibiotic treatment of male mice results in seminal fluid metabolome and epididymal histopathological alterations. It remains to be determined whether such changes compromise male reproductive function or lead to DOHaD effects in resulting offspring.

Figure 1

Diagram of where the testes and epididymis were grossly sectioned. Section (A) included the testes, efferent ductules and caput epididymis. Section (B) permitted examination of the cauda epididymis.

Figure 2

Volcano plot after exclusion of the five outlier samples that includes the remaining seven replicates in the CUB antibiotic group and eight replicates in the saline vehicle control group. The red circles indicate that the metabolites are significantly different between two groups (P < 0.05) while the black ones are not statistically different.

Figure 3

Metabolome data from CUB antibiotic-treated (red) and saline control (green) males. (A) 3D PCA score plot for metabolome data from CUB antibiotic treated (red) and saline control (green) males that show overall metabolomics profile of the two groups separated especially on the PC2 axis. (B) Partial least squares discriminant analysis (PLS-DA) plot of the samples with outliers removed. The red triangle represents samples treated with antibiotics while the green cross represents saline controls. (C) Heat map based on the top differentially expressed metabolites between CUB antibiotic and saline-treated males.

Figure 4

Select metabolites (A-D) that were differentially expressed in the seminal fluid in CUB antibiotic-treated males versus saline control males. *P ≤ 0.05.

Figure 5

Purine degradation and urate metabolism pathway. The circled metabolites were decreased in the seminal fluid of males treated with the CUB antibiotic protocol regimen. Purines require glutamic acid for their original synthesis.

Figure 6

Histological section of testes and epididymides from saline control and CUB antibiotic-treated males. (A) Testis from saline control male showing normal spermatogenesis, Stage VII. Elongated spermatids (ES) line the epithelium with tails extending into the lumen. Round spermatids (RS) with normal acrosomal formations, as seen with periodic acid-Schiff’s staining (PAS) are found in the central region of the epithelium (PS, pachytene spermatocytes). (B) Testis from CUB antibiotic-treated male, late Stage VII. Normal elongated spermatids (ES) line the epithelium as in the control. Round spermatids (RS) with normal acrosomal formations are present, as well as pachytene spermatocytes (PS). (C) Proximal corpus epididymis from a control male. Note the concentration of sperm in the lumen (Lu) and a columnar epithelium consisting of principal (P), apical (A) and basal (B) cells. Long microvilli (Mi) extend into the lumen. (D) Proximal corpus epididymis from a CUB antibiotic-treated male showing cribriform cystic changes (arrows) in the epithelium. These abnormal formations appear as large vacuoles in the basal region that develops into a cystic luminal space, surrounded by shortened epithelial cells. Sperm are found in the epididymal lumen (Lu). A mitotic figure (Mit) is seen in the basal region. (E) Proximal corpus epididymis from a control male showing a rare cribriform cyst in the epithelium. This cyst (Cy) appears less developed than in the treated samples, because there is a sparse amount of secretory material and microvilli are lacking within the cystic lumen (Bm, basement membrane; Lu, lumen of the epididymis). (F) CUB antibiotic-treated proximal corpus epididymis. This his higher magnification of an area from D, showing more well-developed cribriform cysts (Cy), with some showing epithelial cells underneath on the basement membrane (Bm), as well as a well-developed ‘bridge’ of epithelial cells (Br). These cysts have well-defined secretory material (S) in the cystic lumen and the lining epithelial cells show microvilli (Mi) extending into both the cyst as well as the lumen (Lu) of the epididymis. Bars = 20 μm.