Impact of Age and Fertility Status on the Consistency of Repeat Measurements of Sperm DNA Damage: A Single-center, Prospective, Dual Visit Study.

OBJECTIVE: To examine the relationship between abnormal routine semen parameters and DNA damage associated with advanced paternal age and infertility by controlling for common biases contested in the current literature. MATERIALS & METHODS: Prospective study using 151 semen samples collected from men aged 18-80 at 2 visits with 1-3 months intervals. Samples were collected from both infertile and general population controls. Conventional semen parameters were measured including volume, concentration and motility. Sperm DNA damage was measured using the %DNA Fragmentation Index (%DFI) and High DNA stainability (%HDS) using Sperm Chromatin Structure Assay (SCSA). Patients were then classified according to %DFI as normal (<18), intermediate (18-27), or high (>27). RESULTS: Significant correlation between all sperm parameters was seen between both visits regardless of age. DFI had the highest correlation between both visits (R2 = 0.77). Progressive motility, total motility and %DFI were significantly affected in men ≥50 years old when compared to men <35 and men 35-49 years old (P <.001). Forty-eight percent of men with intermediate %DFI changed category on their second visit, whereas men with high and low %DFI changed category in 15% and 9%, respectively. CONCLUSION: Sperm and SCSA parameters do not change significantly between 2 visits at 1-3 months intervals in the total population and after subgrouping. Men of advanced age have poorer sperm parameters and more DNA damage. Men with initially normal or elevated %DFI are unlikely to change DNA damage category. Older men are more likely to have sperm parameters and DNA damage vary on repeat semen analysis compared to younger men.

Interaction of milk proteins and Binder of Sperm (BSP) proteins from boar, stallion and ram semen.

BACKGROUND: Mammalian semen contains a family of closely related proteins known as Binder of SPerm (BSP proteins) that are added to sperm at ejaculation. BSP proteins extract lipids from the sperm membrane thereby extensively modifying its composition. These changes can ultimately be detrimental to sperm storage. We have demonstrated that bovine BSP proteins interact with major milk proteins and proposed that this interaction could be the basis of sperm protection by milk extenders. In the present study, we investigated if homologous BSP proteins present in boar, stallion and ram seminal plasma display a similar affinity for the milk proteins in order to assess whether the mechanism of sperm protection by milk for these species could be general. METHODS: Skim milk was incubated with seminal plasma proteins (boar, stallion and ram), chromatographed on a Sepharose CL-4B column and protein fractions were analyzed by immunoblotting. RESULTS: Boar, stallion and ram BSP proteins displayed affinity for a milk protein fraction (F1) mainly composed of α-lactalbumin, ß-lactoglobulin, and κ-casein. They also had affinity for another milk protein fraction (F2) composed mostly of casein micelles. However, stallion BSP showed higher affinity for the fraction (F1). CONCLUSIONS: These results further extend our view that the association of BSP proteins with milk proteins could be a general feature of the mechanism of mammalian sperm protection by milk to prevent detrimental effect of prolonged exposure of sperm to seminal plasma.

The major proteins of bovine seminal plasma interact with caseins and whey proteins of milk extender.

Milk has been used routinely as an extender for sperm preservation. Caseins, the major proteins in milk, are proposed to be the protective constituents of milk during sperm preservation. It is unclear whether the whey proteins in milk are also implicated in the protection of sperm. Our previous studies have shown that the major proteins of bovine seminal plasma (recently named as binder of sperm or BSP, which comprises BSP1, BSP3, and BSP5 proteins) mediate a continuous phospholipid and cholesterol efflux from the sperm plasma membrane that is detrimental for sperm preservation. In this study, we investigated whether the protective effect of milk could be due to an interaction between BSP proteins and milk proteins. The binding of BSP proteins to milk proteins was demonstrated by gel filtration chromatography. Milk was fractionated into three fractions: the first containing whey protein aggregates and kappa-casein, the second containing all milk proteins, and the third containing small peptides, salts, and sugars. BSP1 has a higher affinity for the milk proteins in the milk fractions as compared to BSP3 and BSP5. The binding of BSP proteins to milk proteins was further characterized by isothermal titration calorimetry. We demonstrated that BSP1 binds to caseins and the titration could be simulated with a Scatchard approach, leading to an affinity constant (K(a)) of 350 mM(-1) and a stoichiometric parameter for the association (n) of 4.5 BSP1 per casein. The association between BSP1 and alpha-lactalbumin was characterized by a K(a) of 240 mM(-1) and an n value of 0.8. These results indicate the existence of an interaction between BSP proteins and milk proteins that could be the origin of the protection of sperm during preservation in milk.

Induction of epididymal boar sperm capacitation by pB1 and BSP-A1/-A2 proteins, members of the BSP protein family.

A family of proteins designated BSP-A1, BSP-A2, BSP-A3, and BSP-30-kDa, collectively called BSP (bovine seminal plasma) proteins, constitute the major protein fraction of bull seminal plasma. BSP proteins can stimulate sperm capacitation by inducing cholesterol and phospholipid efflux from sperm. Boar seminal plasma contains one homologous protein of the BSP family, named pB1; however, its physiological role is still unknown. In the current study, we report a novel method to purify pB1 from boar seminal plasma by chondroitin sulfate B-affinity chromatography and reverse-phase-high performance liquid chromatography. We also studied the effect of pB1, BSP-A1/-A2, and whole boar seminal plasma on boar sperm capacitation. Boar epididymal sperm were washed, preincubated in noncapacitating medium containing pB1 (0, 2.5, 5, 10 or 20 microg/ml), BSP-A1/-A2 (0 or 20 microg/ml) proteins, or whole seminal plasma (0, 250, 500, or 1000 microg/ml), then washed and incubated in capacitating medium. Acrosomal integrity was assessed by chlortetracycline staining. The status of sperm capacitation was evaluated by the capacity of sperm to undergo the acrosome reaction initiated by the addition of the calcium ionophore, A23187. The pB1 and BSP-A1/-A2 proteins increased epididymal sperm capacitation as compared with control (sperm preincubated without proteins). This effect reached a maximum level at 10 microg/ml pB1 and at 20 microg/ml BSP-A1/-A2 (2.3- and 2.2-fold higher than control, respectively). Whole boar seminal plasma did not induce sperm capacitation. In addition, pB1 bound to boar epididymal sperm and was lost during capacitation. These results indicate that BSP proteins and their homologs in other species induce sperm capacitation in a similar way.